rexresearch.com

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Christopher Rinsch, et al.
Pomegranate Anti-Aging ( Urolithin-A )

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Youtube.com : Pomegranates reveal its powerful anti-aging secret

Ecole Polytechnique Fédérale de Lausanne : Pomegranate Reveals its Powerful Anti-aging Secret

US8933217 : COMPOUNDS, COMPOSITIONS AND METHODS FOR PROTECTING BRAIN HEALTH IN NEURODEGENERATIVE DISORDERS

US2012164243 : Compositions and methods for improving mitochondrial function and treating neurodegenerative diseases and cognitive disorders

US2016000753 : Enhancing Autophagy or Increasing Longevity by Administration of Urolithins or Precursors Thereof

US2015183758 : Process-Scale Synthesis of Urolithins

WO2015097231 : PRODRUGS OF UROLITIHNS AND USES THEREOF

Pomegranate Patents : Extraction & Cultivation

Pomegranate Cultivation

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https://www.youtube.com/watch?v=Lf1vCyfaosE

Pomegranates reveal its powerful anti-aging secret

École polytechnique fédérale de Lausanne (EPFL)

Scientists have managed to prove pomegranates anti-aging potential: intestinal bacteria transform a molecule contained in the fruit with spectacular results. Although tests in humans are still underway, scientists have already published the initial promising results from animal studies in the journal Nature Medicine.

Nature Medicine, July 2016
'Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents'

Dongryeol Ryu, Laurent Mouchiroud, Pénélope A Andreux, Elena Katsyuba, Norman Moullan, Amandine A Nicolet-dit-Félix, Evan G Williams, Pooja Jha, Giuseppe Lo Sasso, Damien Huzard, Patrick Aebischer, Carmen Sandi, Chris Rinsch & Johan Auwerx

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http://www.biosciencetechnology.com/news/2016/07/pomegranate-reveals-its-powerful-anti-aging-secret
http://actu.epfl.ch/news/pomegranate-finally-reveals-its-powerful-anti-agin/
Press kit (photos, video, article, TV B-roll with 3D animation): http://bit.ly/pomegranate2016

Pomegranate Reveals its Powerful Anti-aging Secret

by

Ecole Polytechnique Fédérale de Lausanne

Are pomegranates really the superfood we've been led to believe will counteract the aging process? Up to now, scientific proof has been fairly weak. And some controversial marketing tactics have led to skepticism as well. A team of scientists from EPFL and the company Amazentis wanted to explore the issue by taking a closer look at the secrets of this plump pink fruit. They discovered that a molecule in pomegranates, transformed by microbes in the gut, enables muscle cells to protect themselves against one of the major causes of aging. In nematodes and rodents, the effect is nothing short of amazing. Human clinical trials are currently underway, but these initial findings have already been published in the journal Nature Medicine.

As we age, our cells increasingly struggle to recycle their powerhouses. Called mitochondria, these inner compartments are no longer able to carry out their vital function, thus accumulate in the cell. This degradation affects the health of many tissues, including muscles, which gradually weaken over the years. A buildup of dysfunctional mitochondria is also suspected of playing a role in other diseases of aging, such as Parkinson's disease.

One molecule plays David against the Goliath of aging

The scientists identified a molecule that, all by itself, managed to re-establish the cell's ability to recycle the components of the defective mitochondria: urolithin A. "It's the only known molecule that can relaunch the mitochondrial clean-up process, otherwise known as mitophagy," says Patrick Aebischer, co-author on the study. "It's a completely natural substance, and its effect is powerful and measurable."

The team started out by testing their hypothesis on the usual suspect: the nematode C. elegans. It's a favorite test subject among aging experts, because after just 8-10 days it's already considered elderly. The lifespan of worms exposed to urolithin A increased by more than 45% compared with the control group.

These initial encouraging results led the team to test the molecule on animals that have more in common with humans. In the rodent studies, like with C. elegans, a significant reduction in the number of mitochondria was observed, indicating that a robust cellular recycling process was taking place. Older mice, around two years of age, showed 42% better endurance while running than equally old mice in the control group.

Human testing underway

Before heading out to stock up on pomegranates, however, it's worth noting that the fruit doesn't itself contain the miracle molecule, but rather its precursor. That molecule is converted into urolithin A by the microbes that inhabit the intestine. Because of this, the amount of urolithin A produced can vary widely, depending on the species of animal and the flora present in the gut microbiome. Some individuals don't produce any at all. If you're one of the unlucky ones, it's possible that pomegranate juice won't do you any good.

For those without the right microbes in their guts, however, the scientists are already working on a solution. The study's co-authors founded a start-up company, Amazentis, which has developed a method to deliver finely calibrated doses of urolithin A. The company is currently conducting first clinical trials testing the molecule in humans in European hospitals.

Darwin at your service: parallel evolution makes good dinner partners According to study co-author Johan Auwerx, it would be surprising if urolithin A weren't effective in humans. "Species that are evolutionarily quite distant, such as C elegans and the rat, react to the same substance in the same way. That's a good indication that we're touching here on an essential mechanism in living organisms."

Urolithin A's function is the product of tens of millions of years of parallel evolution between plants, bacteria and animals. According to Chris Rinsch, co-author and CEO of Amazentis, this evolutionary process explains the molecule's effectiveness: "Precursors to urolithin A are found not only in pomegranates, but also in smaller amounts in many nuts and berries. Yet for it to be produced in our intestines, the bacteria must be able to break down what we're eating. When, via digestion, a substance is produced that is of benefit to us, natural selection favors both the bacteria involved and their host. Our objective is to follow strict clinical validations, so that everyone can benefit from the result of these millions of years of evolution."

The EPFL scientists' approach provides a whole new palette of opportunities to fight the muscular degeneration that takes place as we age, and possibly also to counteract other effects of aging. By helping the body to renew itself, urolithin A could well succeed where so many pharmaceutical products, most of which have tried to increase muscle mass, have failed. Auwerx, who has also published a recent discovery about the anti-aging effects of another molecule in the journal Science, emphasizes the game-changing importance of these studies. "The nutritional approach opens up territory that traditional pharma has never explored. It's a true shift in the scientific paradigm."

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US8933217
COMPOUNDS, COMPOSITIONS AND METHODS FOR PROTECTING BRAIN HEALTH IN NEURODEGENERATIVE DISORDERS

Aspects of the invention relate to compounds, extracts and compositions thereof, and methods of using of the same, to treat neurodegenerative disorders and/or improve brain health. In certain embodiments, said compounds are pomegranate flavonoids.

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US2012164243  
Compositions and methods for improving mitochondrial function and treating neurodegenerative diseases and cognitive disorders
 

Provided are compositions comprising compounds or precursors to compounds which may be used for a variety of therapeutic applications including, for example, treating and/or preventing a disease or disorder related to reduced or inadequate mitochondrial activity, including aging or stress, diabetes, obesity, and neurodegenerative diseases. The compounds relate generally to urolithins and precursors thereof, including but not limited to ellagitannins and urolithin A. In certain embodiments the compositions are presented in or as food products or nutritional supplements. These same compounds and compositions can also be used advantageously in generally healthy individuals to increase or maintain metabolic rate, decrease percent body fat, increase or maintain muscle mass, manage body weight, improve or maintain mental performance (including memory), improve or maintain muscle performance, improve or maintain mood, and manage stress.

BACKGROUND OF THE INVENTION

[0002] Ellagitannins are monomeric, oligomeric, and polymeric polyphenols that are abundant in some fruits, berries and nuts, such as pomegranates, raspberries, strawberries, black raspberries, walnuts and almonds. The fruits and berries are widely consumed fresh and as beverages, such as juice, and these have been reported to promote health.

[0003] In commercial fruit juice processing methods, ellagitannins, which are particularly abundant in some fruit peels, are extracted in large quantities into the juice. Ellagitannins belong to the chemical class of hydrolyzable tannins, which release ellagic acid upon hydrolysis. In vitro studies have suggested that ellagitannins, at concentrations in the range of 10-100 micromolar (μM), have potential anti-oxidant, anti-atherogenic, anti-thrombotic, anti-inflammatory, and anti-angiogenic effects. Fruits may have different ellagitannins that are predominant, for example, in fruit juice prepared from pomegranate, the predominant ellagitannin is punicalagin [2,3 hexahydroxydiphenoyl-4,6-gallagylglucose], which occurs as a mixture of isomers. The reported potent anti-oxidant properties of pomegranate juice have been attributed to the high content of punicalagin isomers, which can reach levels >2 g/L of juice. Ellagitannins have also been identified as the active anti-atherogenic compounds in pomegranate juice. It has also been suggested that pomegranate ellagitannins and pomegranate fruit extracts inhibit the proliferation of human cancer cells and modulate inflammatory sub-cellular signaling pathways and apoptosis. See, for example, Seeram et al. (2005) J Nutr Biochem. 16:360-7; Adams et al. (2006) J Agric Food Chem. 54:980-85; Afaq et al. (2005) Photochem Photobiol. 81:38-45; Afaq et al. (2005) Int J Cancer. 113:423-33. Pomegranate fruit extract has also been reported to reduce prostate tumor growth and prostate serum antigen (PSA) levels in athymic nude mice implanted with CWR22Rv1 prostate cells. Malik et al. (2005) Proc Natl Acad Sci. 102:14813-8.

[0004] Unfortunately, for the most part ellagitannins are poorly absorbed by the human gut. However, a number of metabolites derived from ellagitannins are absorbed by the human gut, including certain metabolites ultimately formed in the gut by commensal microorganisms (i.e., intestinal microflora).

[0005] Ellagitannins release ellagic acid under physiological conditions in vivo, and ellagic acid is then gradually metabolized by the gut microflora in the intestine to produce urolithin D, urolithin C, urolithin A (UA) and urolithin B (UB). Once the metabolites are absorbed, they undergo glucuronidation and once in the liver, they are further metabolized to produce glucuronides, and/or sulfates, to give a combination of metabolites secreted in the bile.

[0006] Urolithins are metabolites of ellagic acid, punicalagin (PA), punicalin (PB), tellimagrandin (TL), and other ellagitannins (Cerda, Espin et al. 2004; Cerda, Periago et al. 2005). Ellagic acid (EA) is abundant in pomegranate juice (Gil, Tomas-Barberan et al. 2000). The ellagitannin tellimagrandin (TL) has been previously isolated and characterized before from pomegranate and other plants (Tanaka, Nonaka et al. 1986; Tanaka, Nonaka et al. 1986; Satomi, Umemura et al. 1993). Structural formulas for UA, PA, PB, EA, and TL are presented in FIG. 1.

[0007] Considerable efforts have been made to understand the mechanism of metabolic disorders, neurodegeneration and cognitive decline, so as to better design treatment modalities including those based on natural products. One of the key observations has been therole of declining mitochondrial energy production, corresponding with increased oxidative stress and apoptosis, plays a significant role in degenerative diseases and the process of aging. A variety of degenerative diseases have now been shown to be caused by mutations in mitochondrial genes encoded by the mitochondrial DNA (mtDNA) or the nuclear DNA (nDNA). Importantly, somatic mtDNA mutations accumulate with age in post-mitotic tissues in association with the age-related decline in mitochondrial function and are thought to be an important factor in aging and senescence. Inherited diseases can result from mtDNA base substitution and rearrangement mutations and can affect the CNS, heart and skeletal muscle, and renal, endocrine and hematological systems.

[0008] Mitochondria generate most of the cellular energy by oxidative phosphorylation (OXPHOS), and they produce most of the toxic reactive oxygen species (ROS) as a by-product. Genetic defects that inhibit OXPHOS also cause the redirection of OXPHOS electrons into ROS production, thus increasing oxidative stress. A decline in mitochondrial energy production and an increase in oxidative stress can impinge on the mitochondrial permeability transition pore (mtPTP) to initiate programmed cell death (apoptosis). The interaction of these three factors is believed to play a major role in the pathophysiology of degenerative diseases and the aging process, which affects all tissues of the body.

[0009] In the normal brain, optimal cognitive function mainly relies on the activity and communication between neurons, highly complex cells able to convey electric signals and elicit chemical neurotransmission. Neuronal function depends on long and complex cellular processes that can extend over centimeters or even meters to connect neurons or target cells, and can make more than 100,000 synaptic contacts. As such, neurons are highly dependent on energy supply and, therefore, are exposed to oxidative stress damage. Cognitive function is dependent on a careful balance of intracellular signaling that takes place within a complex network of neurons. Optimal cognitive function can be impaired by numerous factors such as aging, cellular stress, chronic stress, and neurodegenerative disorders. Cognitive decline may be characterized by a decrease in performance in thinking, learning, memory, alertness, and/or impaired psychological skills, as well as by depression and anxiety.

[0010] Mitochondrial function has also been shown to be important in metabolic disorders. Diabetes and obesity have been correlated with compromises in mitochondrial function. It has been suggested that the coupling efficiency in mitochondria, or the proportion of oxygen consumption necessary to make ATP, is related to levels of obesity, with high coupling efficiency possibly resulting in higher deposition of fat stores (Harper, Green et al. 2008). In diabetes, recent work has suggested that mitochondrial dysfunction is a cause of insulin insensitivity in myocytes and adipocytes, as a result of insufficient energy supply or defects in the insulin signaling pathway (Wang, Wang et al. 2010).

SUMMARY OF THE INVENTION

[0011] The invention relates to compositions comprising compounds or precursors to compounds which may be used for a variety of therapeutic applications including, for example, treating and/or preventing disease or disorders related to reduced or inadequate mitochondrial activity, including aging or stress, diabetes, obesity, and neurodegenerative diseases. These same compounds and compositions can also be used advantageously in generally healthy individuals to increase or maintain metabolic rate, decrease percent body fat, increase or maintain muscle mass, manage body weight, improve or maintain mental performance (including memory), improve or maintain muscle performance, improve or maintain mood, and manage stress….

[0140] Remarkably, the inventors have discovered that certain compounds derived from ellagitannins are useful in the treatment and prevention of physiological and psychological manifestations of stress, including oxidative stress. Without meaning to be tied to any particular mechanism of action, it is believed that the compounds exert beneficial effects on mitochondria, promoting and restoring crucial mitochondrial functions and counteracting stress-induced mitochondrial dysfunction. These same compounds have been discovered, in accordance with the instant invention, to be useful in the treatment and prevention of any of a variety of conditions, diseases, and disorders related to mitochondrial dysfunction including, without limitation, neurodegenerative diseases and cognitive disorders, metabolic disorders including insulin resistance, mood disorders, and anxiety disorders.

[0141] Ellagitannins (ETs) are polyphenols included within the so called “hydrolyzable tannins” in which hexahydroxydiphenic acid forms diesters with sugars (most often β-D-glucose). ETs can occur as complex polymers reaching molecular weights up to 4000 and higher. These polymers can be hydrolyzed with acids or bases to yield ellagic acid (EA), which can be used indirectly to quantify ETs. EA in turn is a source of additional metabolic products including urolithins.

[0142] Many plant species containing ellagitannins have been used for the treatment of diseases, particularly in Asia (Okuda et al., 2009). These include Agrimonia pilosa (agrimoniin), Camelia japonica (camelliatannin A), Cornus officinalis (cornussin A), Geranium thunbergii (geraniin), Geum japonicum (gemin-A), Liquidambar formosana (casuarictin), Mallotus japonicus (mallotusinic acid), Oenothera erythrosepala (oenothein B), Punica granatum (pomegranate) (granatin B), Rosa rugosa (rugosin), and Terminalia chebula (chebulinic acid), among others. The main uses of these medicinal plants have been associated to their antioxidant, anti-diarrheic, anti-microbial, and immunomodulatory activities.

[0143] Ellagitannins are also present in significant amounts in many berries, including strawberries, red and black raspberries (Zafrilla et al., 2001), blueberries, and blackberries. Ellagitannins have also been found in apples, cherries, cloudberries, cranberries, currants, grapes, lime, mango, pineapple, pomegranate, prune, rhubarbs. Serrano et al. (2009) Mol Nutr Food Res. 53:S310-29. The ellagitannin rubusuaviin C can be isolated from the leaves of the Chinese sweet tea Rubus suavissimus S. Lee. Ellagitannins have also been identified in appreciable amounts in nuts, including walnuts (Fukuda et al., 2003), pistachios, cashew nuts, chestnuts, oak acorns (Cantos et al., 2003) pecans (Villarreal-Lozoya et al., 2007) and peanuts.

[0144] They are also abundant in pomegranates (Gil et al., 2000), and muscadine grapes (Lee and Talcott, 2002) and are important constituents of wood, particularly oak (Glabasnia and Hofmann, 2006). Ellagitannins can be incorporated into food products, such as wines, and whiskey, through migration from wood to the food matrix during different aging processes. Ellagic acid has also been found in several types of honey and it has been proposed as a floral marker for heather honey (Ferreres et al., 1996). Free ellagic acid and different glycosidic derivatives are also present in these food products, including glucosides, rhamnosides, arabinosides and the corresponding acetyl esters (Zafrilla et al., 2001).

[0145] A number of studies have shown that the ellagitannin content of several food products can be quite high (Table 1). For example, a glass of pomegranate juice (200 mL) can provide as much as 1 g of ellagitannins and ellagic acid together, a raspberry serving (100 g raspberries) around 300 mg, a strawberry serving 70 mg, and four walnuts some 400 mg of ellagitannins.

[0146] Representative dietary ellagitannins include punicalagin of pomegranate, sanguiin-H-6 of strawberry and raspberry, and pedunculagin of walnuts. All of these release ellagic acid upon hydrolysis, although other metabolites can also be produced and are distinctive of individual ellagitannins (e.g., gallagic and ter-gallagic acids)…

Example 1

Preparation of Functional Extracts from Pomegranate Compounds

[0336] The pomegranate extracts described in this application containing specific molecules were prepared using an extraction procedure based on adsorption of polyphenols in a standard polymer adsorption-based column as described. For the preparation of the extracts 31008 and 1108 derived from pomegranate juice, pomegranates were juiced using a standard juicing and manufacturing process and adsorbed onto a polymeric chromatographic resin as pure juice. The resin Amberlite XAD-16 (Rohm & Haas) was packed into semi-preparative columns and loaded with the extracted juice. The column was washed with water to remove the sugars until completion (Brix levels were below 0.1%). The polyphenols were eluted with 100% ethanol. The remaining ethanol was evaporated under vacuum to produce a concentrated extract containing 4.5 g of total polyphenol per liter as determined using the Folin assay for total polyphenol content. Extract 1011 was prepared in a similar manner as extract 31008 and 1108, but the liquid extract was then spray dried utilizing a spray dryer to produce a final powder extract. Utilizing HPLC-MS for the identification of compounds, extract 31008, 1108, and 1011 were found to contain the molecules punicalagin, punicalin, tellimagrandin, and pedunculagin.

[0337] The extract 71109 derived from the pomegranate husk was prepared by manually separating the husk from the pomegranate arils pulp, followed by pressing with a manual fruit press. To extract the maximal amount of polyphenols, the cake/pomace of pressed pomegranate parts were soaked in water consecutively for several periods of time (5 minutes) in order to increase extraction efficiency. The extracted pomegranate solution was clarified by centrifugation before being adsorbed onto the polymeric chromatographic Amberlite XAD-16 resin (Rohm & Haas), packed in semi-preparative columns, and loaded with the water extracted from pomegranate husk. The column was washed with water to remove the sugars until completion (Brix levels were below 0.1%). The polyphenols were eluted with 100% ethanol. The remaining ethanol was evaporated under vacuum to produce a concentrated extract containing 17.1 g of total polyphenol per liter as determined using the Folin assay for total polyphenol content. This technique is a modification of methods known in the art as described by several published methods for purification of polyphenols from various plants and berries. Tuck, K. L. and P. J. Hayball (2002) “Major phenolic compounds in olive oil: metabolism and health effects.” J Nutr Biochem 13(11):636-644; and Schieber, A., P. Hilt, et al. (2003) “A new process for the combined recovery of pectin and phenolic compounds from apple pomace.” Innovative Food Sci. Emerging Technol. 4:99-107.

[0338] For the preparation of Extract 61109, an aqueous extract of the pomegranate was fractionated utilizing centrifugal partition chromatography. The isolation fractions were lyophylized to produce extract 61109, highly enriched in punicalagin (>90%).

Purification of Punicalagin

Preparation of Extract

[0339] Extract from pomegranate was dissolved in 16 mL of the organic/aqueous phase mixture (1:1) and filtered on a Teflon filter (0.45 μm).

[0000] Separation of Punicalagin from Extract Using Centrifugal Partition Chromatography

[0340] Separation of punicalagin from pomegranate extract was achieved by utilizing Centrifugal Partition Chromatography CPC. The CPC apparatus was a FCPC® 1000 apparatus provided by Kromaton Technologies (Angers, France) that is fitted with a rotor of 1000 mL capacity. The solvents were pumped by a 4-way binary high-pressure gradient pump. The samples were introduced into the CPC column via a high pressure injection valve (Rheodyne) equipped with a 20 mL sample loop. The effluent was monitored with a diode array detection (DAD) detector equipped with a preparative flow cell. Fractions were collected by a fraction collector. The separation steps were conducted at room temperature.

[0341] To accomplish the extraction, the stationary phase was first introduced into the column in the ascending mode without rotating, and mobile phase was then pumped through the stationary phase until an equilibrium stage was reached. Then, the rotation speed was increased from 0 to 1000 rpm and the mobile phase was pumped into the column at a flow-rate of 20 mL/min. After injection of 10 g of pomegranate extract, fractions of 20 mL were collected every minute. The content of the outgoing organic phase was monitored by online UV absorbance measurement at λ=260 nm.

[0342] An elution-extrusion procedure was used to recover all the compounds from the column: after a classical elution of 100 min, the mobile phase was replaced by the stationary phase used as mobile liquid, until all volume contained (1000 mL) was pushed out the column. A fraction containing punicalagins (mixture of A and B isomers) with 94-97% chromatographic purity was obtained between 51 and 63 minutes of elution, and a second fraction with a chromatographic purity of 85-88% was obtained between 64 and 79 min.

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Enhancing Autophagy or Increasing Longevity by Administration of Urolithins or Precursors Thereof
US2016000753

Disclosed are methods, compounds, and compositions useful for increasing autophagy and promoting longevity. The methods, compounds, and compositions relate to urolithins and urolithin precursors and use thereof. Certain urolithins are represented by Formula I, while certain urolithin precursors are represented by Formula IV. The urolithin may be urolithin A, urolithin B, urolithin C, or urolithin D. The urolithin precursor may be ellagic acid or an ellagitannin. The methods include in vivo, ex vivo, and in vitro uses of the compounds and compositions.

BACKGROUND

[0002] Autophagy is a lysosomal degradation pathway in both animals and plants that is essential for development, differentiation, homeostasis, and survival. In animals, autophagy serves principally as an adaptive mechanism to protect organisms against diverse pathologies, including infection, cancer, neurodegeneration, heart disease, and aging. The repertoire of routine housekeeping functions performed by autophagy includes elimination of defective proteins and organelles, prevention of the accumulation of abnormal protein aggregates, and elimination of intracellular pathogens. The autophagy pathway is uniquely capable of degrading entire organelles, such as mitochondria, peroxisomes, and endoplasmic reticulum.

[0003] Multiple reports indicate that proteins required for autophagy induction, such as sirtuin 1, have reduced expression in aged tissues; levels of autophagy have been shown to diminish with age. Reduced levels of autophagy have also been associated with obesity, diabetes, cancer, neurodegenerative diseases, cardiovascular disease, osteoarthritis, and age-related macular degeneration.

[0004] A number of compounds that stimulate autophagy have been identified, including rapamycin, resveratrol, metformin, spermidine, and glucosamine.

[0005] Urolithins are ellagitannin- and ellagic acid-derived metabolites produced, e.g., by mammalian colonic microflora, including human colonic microflora. Urolithins are known to exhibit anti-oxidant activity.

SUMMARY OF THE INVENTION

[0006] An aspect of the invention is a method of increasing autophagy in an animal, comprising the step of administering to an animal in need thereof an effective amount of a urolithin or a precursor thereof, thereby increasing autophagy in the animal.

DETAILED DESCRIPTION OF THE INVENTION

Overview

[0193] Autophagy is a process by which cells degrade their own components, recycling amino acids and other building blocks that can be reused. Such degradation is performed by lysosomal acidic hydrolases. It is a tightly regulated process that plays an important role in normal cell growth, development, and homeostasis, helping to maintain a balance between the synthesis, degradation, and subsequent recycling of cellular products. It is a major mechanism by which starving cells can reallocate nutrients from less-essential processes to more essential processes.

[0194] During nutrient starvation, increased levels of autophagy lead to the breakdown of non-vital components and the release of nutrients, ensuring that vital processes can continue. Mutant yeast cells that have a reduced autophagic capability rapidly perish in nutrient-deficient conditions. A gene known as Atg7 has been implicated in nutrient-mediated autophagy, and studies in mice have shown that starvation-induced autophagy was impaired in Atg7-deficient mice. Komatsu M et al. (2005) J Cell Biol. 169:425-434.

[0195] Autophagy degrades damaged organelles, cell membranes, and proteins. The failure of autophagy is thought to be an important factor in the accumulation of cell damage and, therefore, aging.

[0196] Three types of autophagy can be distinguished, depending on the pathway along which cellular components are delivered to lysosomes: macroautophagy, microautophagy, and chaperone-mediated autophagy (CMA).

Macroautophagy

[0197] Macroautophagy involves the degradation of long-lived proteins and whole cellular organelles through a multistep process (FIG. 1). Macroautophagy begins with the formation of a double-layered isolation membrane (phagophore) around the molecules and/or organelles to be degraded. The phagophore engulfs cytosolic components and seals around the content, forming an autophagosome. Eventually, the autophagosome fuses with a lysosome, evolving into an autophagolysosome (or autolysosome), wherein lysosomal hydrolases digest the cargo. Microautophagy involves the direct sequestration of cytosolic components through invaginations or armlike projections of the lysosomal membrane. Microautophagy may serve for the turnover of long-lived proteins; however, the significance and regulation of this type of autophagy remain poorly understood. Finally, chaperone-mediated autophagy is a highly selective process devoted to the degradation of soluble cytosolic proteins.

[0198] The microtubule-associated protein 1A/1B-light chain 3 (LC3), a mammalian homolog of the yeast Atg8, is a soluble protein with a molecular mass of approximately 17 kDa which is distributed ubiquitously in mammalian tissues and cultured cells. It is processed immediately after its synthesis by Atg4B, a cysteine protease, that exposes the C-terminal glycine residue (LC3-I). During autophagy, autophagosomes engulf cytoplasmic components, including cytosolic proteins and organelles. Concomitantly, a cytosolic form of LC3 (LC3-I) is conjugated to phosphatidylethanolamine (PE) to form and LC3-PE conjugate (LC3-II), which is recruited to autophagosomal membranes (FIG. 1).

[0199] p62, also known as sequestosome-1, was identified as a novel partner of the atypical protein kinase Cs (aPKCs) and is a ubiquitiously expressed cellular protein. p62 is known to have domains that interact with and bind to ubiquitinated proteins, and it has been identified as a component of inclusion bodies observed in human diseases, especially neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis) as well as in liver diseases. p62 also has been identified as an LC3 interacting protein and it has been demonstrated that an 11 amino acid sequence in the mouse p62 serves to recognize the LC3 protein. As seen in FIG. 1, LC3 binds to p62 and transports it (and any ubiquitinated proteins or cell components bound to it) into the autophagosome, where it is degraded. Consequently, one of the hallmarks of autophagy is an increase in the ratio of LC3-II/LC3-I with a concomitant decrease in the level of cellular p62.

[0200] Of the three types of autophagy described, macroautophagy is the best characterized in mammalian cells. Starvation is the strongest stimulus of macroautophagy. During nutrient deprivation, macroautophagy breaks down cellular components, generating amino acids, fatty acids, and carbohydrates, which can be harnessed for energy production and for the synthesis of essential cellular molecules. Macroautophagy is also involved in specific cytosolic rearrangements during embryogenesis and postnatal development. Furthermore, macroautophagy is induced during viral or bacterial infections, in hypoxia, and under various stress conditions, including radiation exposure and increased reactive oxygen species (ROS) generation. In these circumstances, macroautophagy is essential for the maintenance of cell homeostasis by its promotion of the removal of damaged components. Indeed, impairments in macroautophagy induce premature aging and shorten the lifespan in several organisms, including C. elegans, yeast, and Drosophila. Hars E S et al. (2007) Autophagy 3:93-95; Matecic M et al. (2010) PLoS Genet. 6:e1000921; Lee J H et al. (2010) Science 327:1223-1228. Conversely, upregulation of macroautophagy is proposed to be a major mechanism underlying the lifespan-extending properties of calorie restriction. Toth M L et al. (2008) Autophagy 4:330-338; Morselli E et al. (2010) Cell Death Dis. 1:e10.

[0201] More than 35 Atg (AuTophaGy-related) proteins have been identified in yeasts and mammals; however, the precise role each Atg protein plays during autophagy is not yet fully established. As illustrated in FIG. 1, the process of macroautophagy can be divided into discrete steps, namely, induction and nucleation, expansion, fusion, and degradation. The induction phase is mediated by the ULK1-Atg13-FIP200 kinase complex. The regulation of the nucleation stage, which consists of the recruitment of Atg proteins to the phagophore assembly site, is not yet completely understood. However, the vacuolar protein sorting-34 (Vps34), a class III phosphatidylinositol-3-kinase (PI3K), is required for this step. Vps34 associates with Beclin1, the mammalian homologue of yeast Atg6, and subsequently recruits Atg14 and Vps15 (p150) to the preautophagosomal structure. The elongation and expansion of the phagophore membrane require two ubiquitin-like conjugation systems involving Atg12 (conjugated to Atg5) and Atg8/microtubule-associated protein 1 light chain-3 (LC3, conjugated to phosphatidyl ethanolamine), along with other Atg proteins such as Atg9 and Atg16. The fusion of the autophagosome with a lysosome relies on canonical cellular fusion machinery that consists of the Rab-SNARE (Soluble N-ethylmaleimide-sensitive factor Attachment protein REceptor) system and requires the presence of lysosomal membrane-associated protein-2 (LAMP-2) and the UV radiation resistance-associated gene (UVRAG). Finally, the digestion of the cargo is accomplished by lysosomal hydrolases, followed by the transportation of degraded components into the cytoplasm by lysosomal efflux transporters such as Atg22.

[0202] With regard to the regulation of macroautophagy, mTOR, the mammalian target of rapamycin, is considered to be a major checkpoint, linking the cellular nutritional state with the level of ongoing autophagy. Under nutrient-rich conditions, mTOR is active and inhibits the ULK1-Atg13-FIP200 complex required for the induction of macroautophagy. Energy deprivation leads to mTOR inactivation and stimulation of AMP-activated protein kinase (AMPK), which both induce macroautophagy. AMPK functions as an energy-sensing kinase and is activated by increases in the cellular AMP to ATP ratio. Under such circumstances, AMPK promotes autophagy by directly activating ULK1 and by relieving the mTOR-mediated inhibition of macroautophagy.

[0203] Macroautophagy can be selectively directed toward the removal of particular targets, e.g., peroxisomes (pexophagy), endoplasmic reticulum (reticulophagy), intracellular lipids (lipophagy), ribosomes (ribophagy), and intracellular pathogens (xenopathy). Likewise, mitochondria can be selectively targeted for degradation via macroautophagy (mitophagy).

Mitophagy: A Specialized Form of Macroautophagy

[0204] Mitophagy is a highly selective process that can promote the elimination of dysfunctional or unnecessary mitochondria. Wang K et al. (2011) Autophagy 7:297-300. The loss of mitochondrial membrane potential (Δψm) represents a major trigger of mitophagy. Indeed, laser-induced photo damage of selected mitochondria inside living hepatocytes results in the rapid dissipation of Δψm, followed by the quick removal of depolarized mitochondria through mitophagy. In addition, oxidative damage can lead to the formation of asymmetrical daughter mitochondria characterized by different Δψm, with autophagy specifically targeting mitochondria with lower Δψm. Apart from the degradation of damaged mitochondria under stress conditions, mitophagy is essential for mitochondrial turnover in the basal state and during cell differentiation, such as the maturation of reticulocytes into mature red blood cells.

[0205] Investigations into the molecular regulation of mitophagy have unveiled several mitophagy-specific proteins. Parkin and Pink1 are believed to play important roles in the selective degradation of damaged mitochondria, at least under certain circumstances. Parkin is a cytosolic E3-ubiquitin ligase that is selectively recruited to dysfunctional mitochondria and assists in their removal by mitophagy. Narenda D (2008) J Cell Biol. 183:795-803. Pink1 is imported into healthy mitochondria through a Δψm-dependent process and is degraded by the presenilin-associated rhomboidlike (PARL) protease. Matsuda N et al. (2010) J Cell Biol. 189:211-221. The dissipation of Δψm results in the accumulation of Pink1 on the mitochondrial surface, leading to the recruitment of Parkin, which ubiquitinates outer membrane proteins, including the voltage-dependent anion channel (VDAC). It is proposed that ubiquitin-tagged mitochondria are targeted directly to autophagic vacuoles through the interaction of ubiquitinated proteins with the autophagosomal marker LC3 (Atg8). In addition, Parkin can ubiquitinate the inner mitochondrial membrane and apoptosis regulator protein B-cell lymphoma-2 (Bcl-2), thereby de-repressing Beclin1.

[0206] Recent evidence also suggests that the opening of the mitochondrial permeability transition pore (mPTP) may be required for the selective removal of damaged mitochondria. Opening of the mPTP causes a sudden increase of the inner membrane permeability to solutes with molecular weight up to 1500 Da. This results in mitochondrial depolarization, activation of the mitochondrial ATPase (i.e., ATP synthase operating in reverse), and swelling and rupture of the outer membrane. The loss of Δψm subsequent to permeability transition targets individual mitochondria for degradation. The loss of Δψm and the activation of macroautophagy are prevented by cyclosporin A, an inhibitor of the mPTP component cyclophilin D. Furthermore, starvation fails to induce macroautophagy in cyclophilin D-deficient murine cardiomyocytes, whereas autophagy is enhanced even under fed conditions in cardiac cells from mice overexpressing cyclophilin D. The nicotinamide adenine dinucleotide (NAD)-dependent deacetylase sirtuin-3 (SIRT3) appears to be critically involved in the control of mPTP by modulation of cyclophilin D.

[0207] Similar to the mPTP, the apoptotic proteins Bnip3 (Bcl-2 and adenovirus E1B 19-kDa-interacting protein-3) and Nix (Nip3-like protein X) are thought to trigger selective mitophagy through mitochondrial depolarization. Moreover, Bnip3 may induce mitophagy by competitively disrupting the inhibitory interaction between Bcl-2 and Beclin1. Finally, Nix associates with mitochondrial membranes and directly interacts with LC3 (Atg8).

[0208] Although the molecular regulation of mitophagy has not yet been completely elucidated, the mTOR/AMPK pathway is proposed to be a major checkpoint. AMPK, in addition to stimulating mitochondrial removal through autophagy, enhances the activity of sirtuin-1 (SIRT1) and its downstream target PGC-1α, resulting in stimulation of mitochondrial biogenesis. Hence, through the activity of AMPK, mitophagy and mitochondrial biogenesis are coordinately regulated, maintaining a healthy and functional pool of mitochondria in the cell.

[0209] Lipophagy is a recently recognized alternative pathway of lipid metabolism in which intracellular lipid droplet triglycerides and cholesterol are taken up by autophagosomes and delivered to lysosomes for degradation by acidic hydrolases, thereby releasing free fatty acids. Lipophagy, therefore, functions to regulate intracellular lipid stores, cellular levels of free lipids, such as fatty acids, and energy homeostasis.

[0210] Xenophagy is a recently recognized mechanism of defense against various types of intracellular pathogens, including Mycobacterium tuberculosis, Salmonella typhimurium, Legionella pneumophila, Brucella species, Chlamydia species, Coxiella burnetti, Listeria monocytogenes, Shigella flexneri, Rickettsia species, Mycobacterium marinum, Burkholderia species, and Francisella tularensis.

[0211] Microautophagy involves lysosomes directly engulfing cytoplasm by invagination, protrusion, or septation of the lysosomal limiting membrane.

Chaperone-Mediated Autophagy

[0212] Chaperone-mediated autophagy (CMA) concerns only those proteins that have a consensus peptide sequence that can be recognized by the binding of a hsc70-containing chaperone/co-chaperone complex. The CMA substrate/chaperone complex then moves to the lysosomes, where the CMA receptor lysosome-associated membrane protein type-2a (LAMP-2A) recognizes it. The protein is unfolded and translocated across the lysosome membrane assisted by the lysosomal hsc70 on the other side. Thus, CMA substrates are translocated across the lysosomal membrane on a one-by-one basis, whereas in macroautophagy and microautophagy the substrates are engulfed or sequestered in bulk. Moreover, CMA degrades only certain proteins and not organelles.

Exemplary Therapeutic Indications for Increased Autophagy

[0213] Compounds, compositions, and methods of the invention can be used to treat and prevent any of the following therapeutic indications for increased autophagy.

[0214] Autophagy Protects Organisms from Metabolic Stress

[0215] Nutrient deprivation, growth factor depletion, and hypoxia can induce metabolic stress leading to the induction of autophagy and to the generation of free amino acids and fatty acids. These can be recycled in a cell-autonomous fashion and be used for 1) de novo synthesis of proteins important in the stress response, and 2) fueling the TCA cycle to maintain ATP function. The importance of this process is demonstrated in the inability of mice and C. elegans with deficiencies in the ATG proteins important for autophagy to resist starvation. Thus, a critical role for autophagy is the mobilization of intracellular energy resources to meet cellular and organismal demand for metabolic substrates.

[0216] Induction of Autophagy for Treatment of the Heart

[0217] Cardiomyocyte function and survival rely critically on the presence of basal levels of cardiomyocyte autophagy. Autophagic recycling of damaged cellular components in nutrient-rich conditions constitutes a major means of protein and organelle quality control, ridding the cell of defective (e.g., misfolded or oxidized) proteins and dysfunctional organelles. This fact is highlighted by the observation that abrogation of autophagic pathways in adult heart by conditional inactivation of either the Atg5 or Atg7 genes triggers rapid-onset cardiac hypertrophy, left ventricular dilation, and diminished cardiac output.

[0218] Danon disease, a condition marked by severe and progressive myopathy, stems from defective fusion of autophagosomes with lysosomes. In early cardiac development, Atg5 disruption provokes in utero defects and embryonic lethality. At the other end of the age spectrum, age-related declines in the efficiency of autophagic clearance likely contribute to progressive accumulation of defective proteins and organelles which ultimately lead to functional deterioration over time. Normal aging is associated with loss of cardiac function mainly due to impaired relaxation during diastole. Varying formulations of caloric restriction (CR) can prolong lifespan and improve LV diastolic function; the underlying mechanisms are believed to be the induction of autophagy. Together, these facts highlight the vital housekeeping role for cardiomyocyte autophagy as a mechanism of protein and organelle surveillance and quality control.

[0219] Autophagy can Improve Skeletal Muscle Function in Setting of Muscular Atrophy

[0220] Skeletal muscle adapts its capacity to levels of load and utilization. A central aspect of this adaption is the regulation of fiber remodeling through degeneration or regeneration of muscle fibers.

[0221] In the absence of muscle activity, muscular atrophy occurs, resulting in decreased muscular capacity. This atrophy has been shown to occur due to increased levels of oxidative stress in disused muscle. Attenuation of this oxidative stress could lead to decreased atrophy.

[0222] The autophagy process, and in particular mitophagy are important in clearing damaged mitochondria and reducing the effects of increased oxidative stress on muscle functional capacity. Failure of the autophagy process has been shown to be an important contributing factor to muscle disuse atrophy, by failing to remove damaged mitochondria. This decrease in mitochondria turnover leads to an accumulation of dysfunctional organs and ensuing muscle damage.

[0223] Preserving Autophagy Function During Aging can Improve Sarcopenia

[0224] Skeletal muscle atrophy and impaired muscle strength represent an important health issue and may occur as a consequence of immobilization, disuse, injury, starvation, and aging. In particular, advanced age is ineluctably accompanied by the loss of muscle mass and strength. This condition, known as sarcopenia of aging, has significant effects on individual health and impacts the severity of frailty. Moreover, poor muscular strength is highly predictive of disability and mortality, and general weakness often results in the loss of independent living, thereby affecting individual quality of life and imposing a high burden on healthcare expenditure. Aside from aging, skeletal muscle can undergo significant atrophy following disuse.

[0225] Sarcopenia is characterized by a gradual loss of muscle proteins. The size of stable post-mitotic tissues, such as skeletal and cardiac muscles, is regulated by protein turnover, and skeletal muscle is influenced by a balance between protein synthesis and degradation and the turnover of contractile proteins. A key factor influencing the development of sarcopenia is the imbalance between the rates of protein synthesis and degradation. Protein degradation in skeletal muscle cells is essentially mediated by the activity of two highly conserved pathways: the autophagic lysosomal pathway and the ubiquitin-proteasome pathway.

[0226] Recent studies have shown that the impaired autophagy seen in ATG7 null muscles is characterized by muscle atrophy, weakness, and features of myofiber degeneration. Consequently, autophagy has been found to be essential for myofiber maintenance and for the clearance of damaged proteins and altered organelles.

[0227] Autophagy, which is activated when skeletal muscle is under nutritional stress (such as metabolic stress), plays a role in the catabolic condition and in the degradation of macromolecules and organelles. Catabolic pathways are accelerated during exercise to supply energy and substrates to the muscle for continuation of contractions. It has been well established that the rates of amino acid (relatively small) and glucose oxidation are increased during endurance exercise, and increased energy consumption is likely required to induce autophagy. It has been shown that autophagy is required for myofiber maintenance and for the clearance of damaged proteins and altered organelles.

[0228] Mild exercise has been shown to improve muscle function and decrease the decline in muscle function observed in sarcopenia. These positive benefits are at least in part due to an exercise induced improvement in the autophagy process. In aging mice, the autophagy proteins LC3-II, Beclin-1, ATG7, and MuRF-1 significantly decrease with age in muscle. However, mice undergoing a training regimen during the aging process show a significantly attenuated decrease in these autophagy proteins. In overweight older women, mild exercise has been shown to increase the transcript levels of the autophagy regulators LCB3, Atg7, and LAMP-2 and thus improve the autophagy process. Thus, preservation of autophagy may play an important role in skeletal myocyte homeostasis and optimal mitochondrial turnover in aged muscle.

[0229] An age-related attenuation of autophagy has been shown and results in a diminished efficiency of protein degradation and the clearance of damaged organelles. A decrease in proteolytic activity has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms.

[0230] Improving Autophagy as a Therapeutic Target for Muscle Degenerative Diseases

[0231] Muscular dystrophies are a group of genetic, hereditary muscle diseases characterized by defects in muscle proteins. These defects result in progressive skeletal muscle damage accompanied by myofiber necrosis and chronic local inflammation, leading to substitution of myofibers by connective and adipose tissue. In Duchenne muscular dystrophy (DMD), the most severe form of these diseases, the continuous and progressive skeletal muscle damage leads to complete paralysis and death of patients, usually by respiratory and/or cardiac failure.

[0232] The therapeutic protocols currently in use, based on corticosteroid administration, provide some delay in the progression of the disease, but they are associated with severe side effects. Therapies that substitute corticosteroids or at least may act as corticosteroid-sparing drugs are thus being actively pursued, and biological mechanisms relevant to skeletal muscle homoeostasis are explored, in order to identify new targets.

[0233] Autophagy is emerging as an important process that limits muscle damage. Inhibition/alteration of autophagy contributes to myofiber degeneration leading to accumulation of abnormal organelles. Mutations that inactivate Jumpy, a phosphatase that counteracts the activation of VPS34 for autophagosome formation and reduces autophagy, are associated with a centronuclear myopathy. This observation suggests that unbalanced autophagy is pathogenic in muscle degeneration. Likewise, hyperactivation of Akt as a consequence of muscle-specific deletion of the mammalian target of rapamycin (mTOR) leads to inhibition of autophagy and to a muscle phenotype resembling the one observed in muscular dystrophy. The validity of autophagy modulation as a therapeutic strategy has been shown in a mouse model of Ulrich myopathy characterized by defective autophagy and accumulation of dysfunctional organelles. Forced reactivation of autophagy in these animals yielded a beneficial therapeutic response.

[0234] In vivo and ex vivo analyses have shown that autophagy is defective in both the human (DMD) and mouse (mdx) muscular dystrophy and that such defect contributes to the pathogenesis of the disease. Muscle biopsies from DMD patients have been shown to have significantly lower levels of LC3 II and significant accumulation of p62, a protein known to be incorporated into autophagosomes and efficiently degraded, with respect to tissues from control, non-affected individuals.

[0235] A low protein diet has been shown in mice to lead to a prolonged induction of autophagy. In mice with DMD fed a low protein diet, an induction of autophagy leads to an improvement and management in the disease progression. Significant improvements in muscle function have been observed with an improvement of whole body tension, reduced muscle fibrosis, decreased collagen disposition, reduced accumulation of damaged organelles and reduced apoptosis of muscle fibers.

[0236] This demonstrates that induction of autophagy is an important homoeostatic mechanism that is disrupted in dystrophic muscles and indicates that novel therapeutic approaches aimed at reactivating autophagy can serve as a valuable strategy to reduce muscle damage in DMD.

[0237] Autophagy Protects the Liver from Oxidative Stress and Disease

[0238] During liver diseases such as cancer and cirrhosis, the liver can undergo tissue hypoxia. This process has been shown to induce an autophagy process, which if inhibited resulted in increased apoptosis of liver cells.

[0239] In α1-antitrypsin deficiency, the most common genetic cause of human liver disease, there is significant chronic inflammation and eventual carcinogenesis. In this disease, a point mutation occurs in al-antitrypsin Z (ATZ) leading to improperly folding and accumulation of aggregates. Deletion of ATG5 in hepatic cell lines lead to an accumulation of the mutant ATZ protein, demonstrating the important role for autophagy in reducing the impact of liver disease.

[0240] Autophagy is Important in Limiting Ischemic Reperfusion Injury

[0241] With advancing age, patients are more likely to acquire primary and secondary hepatic malignancies that are amenable to surgical resection and transplantation. Though the elderly patients may be treated surgically, the aged liver has significantly decreased reparative capacity following ischemia and reperfusion injury associated with these operations.

[0242] Ischemic preconditioning is the only promising strategy for improving the outcome of liver surgery, but its beneficial effects are limited to young patients. To date, no therapeutic strategy can suppress the age-dependent ischemia and reperfusion injury.

[0243] A reduction in autophagy has been observed in the old cells subjected to a severe stress such as ischemia followed by reperfusion. Studies have shown that by overexpression of autophagy genes in aged livers of mice, autophagy was increased and hepatocyte cell survival was increased after ischemia and reperfusion. Consequently, defective autophagy has been shown to be a causal mechanism for the age-dependent hepatic reperfusion injury and that enhancement of autophagy has been demonstrated to offer therapeutic benefit and reducing age-mediated liver ischemia reperfusion injury.

[0244] Autophagy in Intestinal Epithelial Cells as a Therapeutic Target

[0245] The intestinal epithelium interfaces directly with a diverse community of bacteria that includes benign commensals, opportunistic pathogens, and overt pathogens, and consequently is the first line of defense against bacterial invasion of host tissues. One means that the epithelial cells employ to defend themselves includes secreting antimicrobial proteins. Unfortunately, there are some intestinal pathogens, including Salmonella tyhpimurium or opportunistically invasive commensal bacteria, such as Enterococcus faecalis, which can avoid this first line of defense and enter the epithelial cells.

[0246] Autophagy has been shown to be essential for the recognition and degradation of intracellular pathogens, acting as an innate barrier to infection. In cell culture, autophagy has been shown to limit the replication of certain bacterial species.

[0247] It has been shown via genetic studies of inflammatory bowel disease (IBD) that autophagy plays an important role in the intestinal immune homeostasis. IBD is a chronic inflammatory disease of the intestine that arises from dysregulated interactions with resident microbiota.

[0248] Recently, it has been shown that polymorphisms in genes in the autophagic pathway are linked to Crohn's disease (CD). Crohn's disease is a chronic form of IBD that can affect any part of the gastrointestinal system, but is usually found in the colon or terminal ileum. The average onset is at 27 years of age in humans, and is usually present throughout the normal lifespan of the individual. It is characterized by severe colitis, strictures, and perianal fistulas, typically requiring surgery.

[0249] The chronic inflammatory process characteristic of CD requires the intensive interaction between intestinal epithelial cells and immune competent cells. In CD, there is an exaggerated immune response to the intestinal microbiota, characterized by an abnormal increase in Th17 cells, which play a major role in autoimmunity, and a down-regulation of Treg cells important for controlling the immune response.

[0250] It has recently been shown that intestinal epithelial cell autophagy is essential for mammalian intestinal defense against invasive bacteria. Autophagy in the epithelial cells protects against the dissemination of invasive bacteria. Following oral infection with the invasive pathogen Salmonella typhimurium as well as Enterococcus faecalis, mouse epithelial cells activate autophagy as a consequence of exposure to these pathogens. Autophagy was also shown to be critical to limit the extra-intestinal spread of S. typhimurium. This indicates that autophagy is a key epithelial cell-autonomous mechanism of antibacterial defense that protects against dissemination of intestinal bacteria.

[0251] The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the administration to and the treatment of individuals with inflammatory bowel disease (IBD) or Crohn's disease (CD) and in need of increasing the levels of autophagy in their in the epithelial cells of the intestine in order to treat either IBD or CD.

[0252] Autophagy is Important in Aging Cardiac Muscle

[0253] The effects of autophagy induction on improved outcome for ischemic injury and muscle maintenance makes it especially relevant for cardiac muscle maintenance and protection from injury. Cardiac muscle undergoes progressive decline in mitochondrial function, similar to that observed in skeletal muscle, resulting in an increase in reactive oxygen species, as well as an increase in the accumulation of defective organelles. The clearance of these damaged organelles by autophagy is important for the maintenance of cardiac muscle function. As autophagy decreases with age, promoting autophagy can serve to protect cardiac muscle function.

[0254] Cardiac muscle is also strongly exposed to ischemic episodes during cardiac infarcts. The level of cardiac muscle damage that these ischemic episodes produce is strongly dependent on the ability of the cells to mount an effective autophagy response to clear damaged organelles. In aged animals, a defective autophagy response leads to an increase in cardiac muscle damage after ischemic events. Thus, promotion of autophagy during these acute events could serve to protect cardiac muscle from damage.

[0255] Autophagy is Important in the Inflammatory Process

[0256] Due to the role of autophagy in clearing defective organelles, a defect in this process leads to a buildup of cellular debris and the induction of apoptosis. Autophagy also plays an important role in defending the organism against microbial pathogens by inducing their degradation. Additionally, autophagy plays an important role in the trafficking events that activate innate and adaptive immunity.

[0257] The autophagic removal of apoptotic corpses is critical for preventing danger signals that could lead to an inflammation response. In an impaired autophagy response, where apoptotic clearance is not efficient the resulting induction of inflammation, could overcome tolerance to self-antigens leading to autoimmune diseases such as systemic lupus erythematosus. Thus, induction of autophagy could serve to decrease inflammatory responses and the development of autoimmune diseases.

[0258] Applications of Autophagy for Treatment of Disorders of the Liver

[0259] A number of features of hepatocytes and the liver as a whole make this organ particularly dependent on autophagy. The liver is rather unique in its regenerative properties as while hepatocytes are normally in a quiescent state, they retain the ability to quickly enter the cell cycle when there is a loss of liver tissue due to injury or surgical removal. The lack of cell turnover makes hepatocytes particularly vulnerable to the effects of impaired autophagy, as cells having long lives accumulate high levels of damaged organelles, protein aggregates, etc. that are normally cleared by autophagy. This leads to cellular injury and potentially to transformation.

[0260] Hepatocellular Lipid Metabolism

[0261] The liver serves as the second largest repository of stored lipids in the body after adipose tissue. Hepatocytes are a major cellular storehouse for neutral lipids in the form of triglycerides (TGs) and cholesterol esters contained in specialized organelles termed lipid droplets (LD). Autophagy mediates the breakdown of intracellular LD stores through the process of lipophagy. This enables the hepatocytes to rapidly mobilize their lipid stores in times of metabolic need. The loss of hepatocyte autophagy leads to a marked increase in hepatic TG and cholesterol content, indicating that lipophagy limits lipid accumulation by the liver in vivo. Also, lipophagy controls cellular energy homeostasis by providing free fatty acids (FFA) from the breakdown of TGs, which subsequently drives mitochondrial β-oxidation and cellular ATP generation. It has been shown that the autophagosomal protein LC3, critical for autophagosome membrane formation, associates with LDs.

[0262] Autophagy Protects Against Hepatic Diseases

[0263] SERPINA1/al-anti-trypsin deficiency (ATD) is the most common genetic cause of human liver disease in children. This disease is caused by homozygosity for the SERPINA1/al-antitrypsin Z allele SERPINA1-Z, a point mutation, which renders the hepatic secretory glycoprotein SERPINA1 prone to misfolding, polymerization, and aggregation. The mutant SERPINA1-Z protein accumulates in hepatocytes and the levels of SERPINA1 found in the blood and body fluids are reduced to 10-15% of those normally observed. Accumulation of mutant SERPINA1-Z in the endoplasmic reticulum (ER) of hepatocytes leads to liver damage by a gain-of-function. It has been shown that intracellular degradation of SERPINA1-Z aggregates and polymers involves the autophagic pathway.

[0264] The drug carbamazepine, known to induce autophagy, was recently shown to be effective in cell based and mouse model of ATD. Carbamazepine increases autophagic degradation of SERPINA1-Z in cultured cells and when provided orally to the PiZ mouse model of ATD, it reduced the hepatic load of SERPINA1-Z. Additionally, inducing autophagy reduced hepatic fibrosis. Consequently, drugs enhancing autophagy are attractive candidates for improving the liver disease that develops in some patients with ATD.

[0265] The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals with ATD and in need of increasing the levels of autophagy in their liver and hepatocytes in order to reduce liver toxicity.

[0266] Autophagy Protects Against Nonalcoholic Fatty Liver Disease

[0267] Nonalcoholic fatty liver disease (NAFLD) is an important component of the metabolic syndrome together with obesity and diabetes. NAFLD encompasses a spectrum of hepatic abnormalities that range from simple fatty liver or steatosis, to fatty liver with hepatocellular injury and inflammation, which is known as nonalcoholic steatohepatitis (NASH). NAFLD is now the most prevalent liver disease in the USA and accounts for about 75% of all chronic liver diseases.

[0268] The most important role of autophagy in fatty liver disease could be to regulate the process of excessive lipid accumulation. In fact, mice with a hepatocyte-specific knockout of Atg7, a protein required for autophagy, consuming a high-fat diet led to a marked increase in liver TGs and cholesterol content, showing that autophagy defects can induce hepatic steatosis. When considering NASH, while its exact causes are unknown, free fatty acid (FFA)-induced lipotoxicity has been implicated in the mechanisms of hepatocellular injury of this disease. Evidence points to the fact that hepatocyte autophagy renders the cells more resistant to injury from FFA.

[0269] Autophagy is an attractive therapeutic target for the treatment and prevention of both NAFLD and NASH. Therapeutic intervention to increase autophagy may reverse not only the hepatic manifestations of NAFLD, including hepatocellular steatosis and injury, but also some of the underlying metabolic abnormalities of the disease via its effects on insulin resistance. Additionally, treatment by increasing autophagy may prevent common end-stage complications of NAFLD, such as hepatocellular carcinoma.

[0270] The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals with NAFLD and in need of increasing the levels of autophagy in their liver and hepatocytes in order to treat these conditions.

[0271] Autophagy Protects Against Alcoholic Liver Disease

[0272] Alcoholic liver disease (ALD) is a major cause of chronic liver disease, and like NAFLD, has a wide spectrum of pathogenic features, that range from steatosis to sever acute alcoholic hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma.

[0273] Autophagy has been shown to play a role in treating ALD. For example, induction of autophagy by administration of rapamycin significantly suppresses acute alcohol-induced steatosis. Also, a common feature of chronic alcohol abuse is the formation of hepatic protein aggregates known as Mallory-Denk bodies, which are cytosolic inclusion bodies enriched with Krt8/keratin 8 and Krt18 and proteins that include ubiquitin. Rapamycin treatment significantly reduces the number of Mallory-Denk bodies in proteasome inhibitor-treated KRT8 transgenic mice.

[0274] Consequently, enhancing hepatic autophagy is an attractive target for improving alcohol-induced liver disease. The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals with ALD and in need of increasing the levels of autophagy in their liver and hepatocytes in order to treat this condition.

[0275] Autophagy Protects Against Drug-Induced Liver Injury

[0276] Most drugs are metabolized and detoxified in the liver, making the liver the principal target for drug damage. Liver injury due to drugs is a common cause for the withdrawal of approved drugs on the market, and it is thought that drug-induced hepatotoxicity is responsible for more than half of acute cases of liver failure. Acetaminophen, also known as paracetamol and N-acetyl-p-aminophenol (APAP), is a widely used antipyretic and analgesic drug and is also the most common source of severe drug-induced hepatotoxicity. At therapeutic levels APAP is safe, but overdosing leads to toxicity mainly due to its reactive metabolite, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI can deplete hepatic stores of glutathione (GSH), an intracellular antioxidant. Following the depletion of GSH, NAPQI is known to react with cellular proteins as well as mitochondrial proteins to form protein adducts. These APAP-induced mitochondrial protein adducts can then lead to mitochondrial damage and subsequent necrosis.

[0277] When autophagy is enhanced with rapamycin, APAP-induced necrosis is significantly inhibited, both in cultured primary hepatocytes and in the livers of mice. Treatment with rapamycin two hours after APAP administration has been seen to significantly improve APAP-induced liver injury, even though APAP metabolism and hepatic GSH depletion have already occurred. This is particularly important as patients at risk for hepatotoxicity from an acute APAP overdose do not receive medical care until they are past the metabolic phase. Consequently, pharmacologic intervention targeting an enhancement of autophagy holds a potential therapeutic benefit for individuals with a risk of APAP hepatotoxicity following an overdose.

[0278] The present invention provides the know how-to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals at risk of hepatotoxicity due to drug side effects and in need of increasing the levels of autophagy in their liver and hepatocytes in order to treat or prevent the potential drug toxicity.

[0279] Autophagy is Important in Limiting Ischemia/Reperfusion Injury

[0280] Ischemia/reperfusion (I/R) injury is a causal factor contributing to morbidity and mortality. The vulnerability of the liver to I/R injury is a major obstacle to liver resection and transplantation surgery where reperfusion after sustained ischemia is unavoidable during hepatectomy and vascular reconstruction. Mitochondrial dysfunction is known to be one of the critical downstream events that lead to I/R-mediated cell death.

[0281] Autophagy clears abnormal or dysfunctional mitochondria to ensure an optimal cellular function and survival. With impaired or insufficient mitophagy, cells accumulate damaged mitochondria, which subsequently leads to uncontrolled ROS formation, mitochondrial DNA mutation, energetic failure, and ultimately cell death. Consequently, the failure of mitophagy to remove a small number of damaged mitochondria during I/R can have a significant impact on hepatocellular function and viability. Mitophagy is essential for hepatic function and survival following I/R injury.

[0282] While minimizing I/R injury plays an important role in the outcome of transplanted young livers, aged livers are even more susceptible to negative impact of I/R injury. In the case of aged livers, hepatocytes fail to respond to the I/R stress and upregulate their endogenous protective autophagy response. Similar to young livers following prolonged ischemia, aged livers after short-term ischemia accumulate dysfunctional mitochondria, undergo mitochondrial permeability transition, and lose their viability soon after reperfusion.

[0283] Methods of enhancing autophagy, including pre-ischemia nutrient depletion and over expression of pro-autophagy genes ATG7 or BECN1, lead to the suppression of the mitochondrial permeability transition and increases hepatocyte survival following reperfusion.

[0284] This indicates that treatments with agents that induce autophagy in the liver will offer protection during a situation of I/R and help to minimize cellular injury. Such treatments are applicable in situations of the transplantation of both young and aged livers. Treatments may involve: (i) pre-treatments of the liver tissue ex vivo by perfusion of the liver with a solution that contains an inducer of autophagy; (ii) treatment of the liver donor with an autophagy inducer; or (iii) treatment of the liver recipient prior to, during the operation and/or immediately after the surgical intervention. Of course, these treatment modalities may be applied individually or in any combination (for example: 1 and 2; 2 and 3; 1 and 3; 1, 2, and 3).

[0285] The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals and their livers, that may be at risk of I/R injury. These compounds may be provided orally or parenterally to the donor or recipient, or provided in a preconditioning solution that may be applied to the resected liver tissue.

[0286] Autophagy and Osteoarthritis

[0287] Osteoarthritis (OA) is the most common aging-related joint pathology and is characterized by degradation of cartilage extracellular matrix (ECM) and reduced cartilage cellularity. Changes in the articular cartilage appear to be critical in OA initiation and progression. Chondrocytes are the only cell population of adult articular cartilage. The capacity of the adult articular chondrocytes to regenerate the normal cartilage matrix architecture is limited and declines with aging, due to cell death and abnormal responsiveness to anabolic stimuli. Articular cartilage is characterized by a very low rate of cell turnover and it has been shown that autophagy play an important role in chondrocyte cellular function and survival. In fact, autophagy is a constitutively active and protective process for the maintenance of cartilage homeostasis. Studies have shown both in joint aging and OA in humans and in mice that there is a reduction in the expression of autophagy regulators, which was accompanied by an increase in chondrocyte apoptosis. Compromised autophagy is thought to contribute to the development of OA. It has been shown that treatment with the compound rapamycin, a known inducer of autophagy, has been able to increase activation of LC3 in cartilage in an animal model of OA and consequently reduce the severity of articular cartilage degradation. In the present invention, urolithins and their precursors have been shown to increase the levels of autophagy in tissues following oral consumption, making them ideal candidates for the treatment and reduction of the severity of osteoarthritis in young and aging humans and mammals.

[0288] Metabolic Syndrome, Diabetes, and Obesity

[0289] Compounds and methods of the invention are useful in the treatment and prevention of metabolic syndrome, type 2 diabetes mellitus, and obesity. As used herein, the term “metabolic syndrome” refers to a combination of medical disorders that, when occurring together, increase the risk of developing cardiovascular disease and diabetes. It affects one in five people in the United States and prevalence increases with age. Some studies have shown the prevalence in the United States to be an estimated 25% of the population. In accordance with the International Diabetes Foundation consensus worldwide definition (2006), metabolic syndrome is central obesity plus any two of the following:
Raised triglycerides: >150 mg/dL (1.7 mmol/L), or specific treatment for this lipid abnormality;
Reduced HDL cholesterol: <40 mg/dL (1.03 mmol/L) in males, <50 mg/dL (1.29 mmol/L) in females, or specific treatment for this lipid abnormality;
Raised blood pressure: systolic BP>130 or diastolic BP>85 mm Hg, or treatment of previously diagnosed hypertension; and
Raised fasting plasma glucose: (FPG)>100 mg/dL (5.6 mmol/L), or previously diagnosed type 2 diabetes.

[0294] Autophagy and Neurodegenerative Diseases

[0295] In neurodegenerative diseases, brain tissue accumulates autophagosomes, demonstrating an increase in autophagy, which in model organisms has been shown to have a protective effect. It plays an important role in clearing the misfolded proteins that accumulate as a result of several neurodegenerative diseases. These include proteins that have polyQ repeats as seen as in Huntington's diseases and spinocerebellar ataxia, mutant α-synucleins involved in Parkinson's as well as tau aggregates.

[0296] Knockdown of ATG genes important in autophagy in C. elegans resulted in increased aggregate formation and toxicity of PolyQ proteins. In Alzheimer's disease the autophagy process is impaired as a result of a defect in autophagosomal maturation that could be an important reason for aggregate accumulation. By contrast, autophagy induction by rapamycin in both Drosophila and mouse models of polyQ disease protected these animals from neurotoxicity. These results demonstrate that autophagy induction can have a protective role in neuronal cells against neurodegeneration.

[0297] The development of neurodegenerative diseases in patients implies that autophagy can reach a saturation point in which the ability to degrade mutant protein aggregates is exceeded. Thus, promotion of autophagy could help in delaying the onset of neurodegeneration disease.

[0298] Cognitive Disorder

[0299] Compounds and methods of the invention are useful for treating a cognitive disorder. As used herein, a cognitive disorder refers to any condition that impairs cognitive function. In one embodiment, “cognitive disorder” refers to any one or more of delirium, dementia, learning disorder, attention deficit disorder (ADD), and attention deficit hyperactivity disorder (ADHD). In one embodiment, the cognitive disorder is a learning disorder. In one embodiment, the cognitive disorder is attention deficit disorder (ADD). In one embodiment, the cognitive disorder is attention deficit hyperactivity disorder (ADHD).

[0300] Compounds and methods of the invention are useful for improving cognitive function, even in the absence of a cognitive disorder. As used herein, “cognitive function” refers to any mental process that involves symbolic operations, e.g., perception, memory, attention, speech comprehension, speech generation, reading comprehension, creation of imagery, learning, and reasoning. In one embodiment, “cognitive function” refers to any one or more of perception, memory, attention, and reasoning. In one embodiment, “cognitive function” refers to memory.

[0301] Methods for measuring cognitive function are well known and can include, for example, individual or battery tests for any aspect of cognitive function. One such test is the Prudhoe Cognitive Function Test. Margallo-Lana et al. (2003) J Intellect Disability Res. 47:488-492. Another such test is the Mini Mental State Exam (MMSE), which is designed to assess orientation to time and place, registration, attention and calculation, recall, language use and comprehension, repetition, and complex commands. Folstein et al. (1975) J Psych Res. 12:189-198. Other tests useful for measuring cognitive function include the Alzheimer Disease Assessment Scale-Cognitive (ADAS-Cog) (Rosen et al. (1984) Am J Psychiatry. 141(11):1356-64) and the Cambridge Neuropsychological Test Automated Battery (CANTAB) (Robbins et al. (1994) Dementia. 5(5):266-81). Such tests can be used to assess cognitive function in an objective manner, so that changes in cognitive function, for example in response to treatment in accordance with methods of the invention, can be measured and compared.

[0302] Protein Misfolding and Aggregation

[0303] These diseases and disorders, which are collectively referred to herein as “amyloid-related diseases”, fall into two main categories: (a) those which affect the brain and other parts of the central nervous system; and (b) those which affect other organs or tissues around the body.

[0304] Examples of amyloid-related diseases which fall under these two categories are listed in the following two sections; however, many other examples of rare, hereditary amyloid-related diseases are known which are not included here, and additional forms of amyloid-related disease are likely to be discovered in future.

[0305] Neurodegenerative Diseases Associated with Amyloidosis

[0306] Many different neurodegenerative diseases are associated with the misfolding and aggregation of a specific protein or peptide in a particular part of the brain, or elsewhere in the central nervous system, depending on the specific disease. Examples of such diseases follow.

[0307] Various forms of Alzheimer's disease (AD) as well as Down's syndrome, hereditary cerebral hemorrhage with amyloidosis (HCHWA, Dutch type), cerebral amyloid angiopathy, and possibly also mild cognitive impairment and other forms of dementia are associated with the aggregation of a 40/42-residue peptide called β-amyloid, Aβ(1-40) or Aβ(1-42), which forms insoluble amyloid fibers and plaques in the cerebral cortex, hippocampus or elsewhere in the brain, depending on the specific disease. Alzheimer's disease is also associated with the formation of neurofibrillary tangles by aggregation of a hyperphosphorylated protein called tau, which also occurs in frontotemporal dementia (Pick's disease).

[0308] Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) are associated with the aggregation of a protein called α-synuclein, which results in the formation of insoluble inclusions called Lewy bodies. Huntington's disease (HD), spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease), dentatorubral pallidoluysian atrophy (DRPLA), different forms of spinocerebellar ataxia (SCA, types 1, 2, 3, 6 and 7), and possibly several other inheritable neurodegenerative diseases are associated with the aggregation of various proteins and peptides that contain abnormally expanded glutamine repeats (extended tracts of polyglutamine). Creutzfeldt-Jakob disease (CJD), bovine spongiform encephalopathy (BSE) in cows, scrapie in sheep, kuru, Gerstmann-Straussler-Scheinker disease (GSS), fatal familial insomnia, and possibly all other forms of transmissible encephalopathy are associated with the self-propagating misfolding and aggregation of prion proteins.

[0309] Amyotrophic lateral sclerosis (ALS), and possibly also some other forms of motor neuron disease (MND) are associated with the aggregation of a protein called superoxide dismutase.

[0310] Familial British dementia (FBD) and familial Danish dementia (FDD), respectively, are associated with aggregation of the ABri and ADan peptide sequences derived from the BRI protein.

[0311] Hereditary cerebral hemorrhage with amyloidosis (HCHWA, Icelandic type) is associated with the aggregation of a protein called cystatin C.

[0312] Systemic Diseases Associated with Amyloidosis

[0313] In addition to the neurodegenerative diseases listed above, a wide variety of systemic ageing-related or degenerative diseases are associated with the misfolding and aggregation of a particular protein or peptide in various other tissues around the body (i.e., outside of the brain). Examples of such diseases follow.

[0314] Type II diabetes mellitus (also known as adult-onset diabetes, or non-insulin dependent diabetes mellitus) is associated with the aggregation of a 37-residue peptide called the islet amyloid polypeptide (IAPP, or “amylin”), which forms insoluble deposits that are associated with the progressive destruction of insulin-producing β cells in the islets of Langerhans within the pancreas.

[0315] Dialysis-related amyloidosis (DRA) and prostatic amyloid are associated with the aggregation of a protein called β2-microglobulin, either in bones, joints and tendons in DRA, which develops during prolonged periods of hemodialysis, or within the prostate in the case of prostatic amyloid.

[0316] Primary systemic amyloidosis, systemic AL amyloidosis and myeloma-associated amyloidosis are associated with the aggregation of immunoglobulin light chain (or in some cases immunoglobulin heavy chain) into insoluble amyloid deposits, which gradually accumulate in various major organs such as the liver, kidneys, heart and gastrointestinal (GI) tract.

[0317] Reactive systemic AA amyloidosis, secondary systemic amyloidosis, familial Mediterranean fever, and chronic inflammatory disease are associated with the aggregation of serum amyloid A protein, which forms insoluble amyloid deposits that accumulate in major organs such as the liver, kidneys and splee. Senile systemic amyloidosis (SSA), familial amyloid polyneuropathy (FAP) and familial amyloid cardiomyopathy (FAC) are associated with the misfolding and aggregation of different mutants of transthyretin protein (TTR), which form insoluble inclusions in various organs and tissues such as the heart (especially in FAC), peripheral nerves (especially in FAP) and gastrointestinal (GI) tract. Another form of familial amyloid polyneuropathy (FAP, type II) is associated with the aggregation of apolipoprotein AI in the peripheral nerves. Familial visceral amyloidosis and hereditary non-neuropathic systemic amyloidosis are associated with misfolding and aggregation of various mutants of lysozyme, which form insoluble deposits in major organs such as the liver, kidneys and spleen.

[0318] Finnish hereditary systemic amyloidosis is associated with aggregation of a protein called gelsolin in the eyes (particularly in the cornea).

[0319] Fibrinogen α-chain amyloidosis is associated with aggregation of the fibrinogen A α-chain, which forms insoluble amyloid deposits in various organs, such as the liver and kidneys.

[0320] Insulin-related amyloidosis occurs by the aggregation of insulin at the site of injection in diabetics.

[0321] Medullary carcinoma of the thyroid is associated with the aggregation of calcitonin in surrounding tissues.

[0322] Isolated atrial amyloidosis is associated with the aggregation of atrial natriuretic peptide (ANP) in the heart.

[0323] Various forms of cataract are associated with the aggregation of y-crystallin proteins in the lens of the eyes.

[0324] Autophagy and Endothelial Cell Function and Associated Diseases

[0325] Endothelial Cell Dysfunction

[0326] Global endothelial cell dysfunction occurs in several diverse diseases such as diabetes, hypertension, chronic kidney disease, and atherosclerosis. In these diseases endothelial cell dysfunction is thought to occur as a result of stress-induced premature senencense (SIPS). SIPS is characterized by subverted autophagy and lysosomal dysfunction, with the accumulation of autolysosomal vacuoles.

[0327] Endothelial cell dysfunction also occurs as a result of aging with an increased incidence of cardiovascular diseases. This increase in cell dysfunction correlates with a decrease in autophagy. In older humans, expression of autophagy markers in arterial endothelial cells was impaired by 50% (P<0.05) and was associated with a 30% (P<0.05) reduction in arterial endothelium-dependent dilatation (EDD). Similarly, in C57BL/6 control mice aging was associated with a 40% decrease (P<0.05) in arterial markers of autophagy and a 25% reduction (P<0.05) in EDD, demonstrating that impaired autophagy is a cause of age-related arterial dysfunction.

[0328] In old mice, treatment with the autophagy-enhancing agent trehalose restored expression of autophagy markers, rescued NO-mediated EDD by reducing oxidative stress, and normalized inflammatory cytokine expression. The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals having health conditions linked to endothelial cell dysfunction and in need thereof.

[0329] Endothelial Cell Injury

[0330] Endothelial cell injury can occur as a result of disease processes such as sickle cell anemia or thalassemia in which pathologically high levels of heme and iron release can occur. Severe skeletal muscle damage, as well as cardiac ischemia injury results in the release of the heme protein, myoglobin, which also results in endothelial cell injury. This damage to the vascular endothelial cells can lead to vascular dysfunction and an increase in cardiovascular complications. Endothelial cell injury caused by heme toxicity is associated with a progressive decrease in endothelial cell mitochondrial membrane potential, leading to apoptosis.

[0331] Micro- and macro-vascular complications are commonly seen in diabetic patients, and endothelial dysfunction contributes to the development and progression of the complications. Abnormal functions in endothelial cells lead to the increase in vascular tension and atherosclerosis, followed by systemic hypertension as well as increased incidence of ischemia and stroke in diabetic patients. Mitochondrial dysfunction appears to be central to the vascular endothelial dysfunction. Enhanced mitochondrial fission and/or attenuated fusion leads to mitochondrial fragmentation and disruption of the endothelial physiological function. Abnormal mitochondrial biogenesis and disturbance of mitochondrial autophagy increase the accumulation of damaged mitochondria, such as irreversibly depolarized or leaky mitochondria, and facilitate cell death. Augmented mitochondrial ROS production and Ca<2+> overload in mitochondria not only cause the maladaptive effect on the endothelial function, but also are potentially detrimental to cell survival.

[0332] Endothelial cell injury can also result from cardiac procedures such as angioplasty, bypass surgery, and valve replacement. Upregulation of autophagy should lead to a reduction in the injury to the associated endothelial cells.

[0333] Strategies that increase autophagy would have clear therapeutic potential. The present invention provides the know-how to use compounds that include urolithins and their precursors as enhancers of autophagy for the treatment of individuals having health conditions linked to endothelial cell injury resulting from disease processes such as diabetes, sickle cell anemia, or thalassemia, as well as protecting endothelial cells from the more acute effects of severe muscle injury.

[0334] Autophagy and Cancer

[0335] Autophagy and cancer have similar regulatory pathways, with several tumor suppression genes such as PTEN, TSC1 and TSC2 leading to the upstream inhibition of TOR signaling, leading to the stimulation of autophagy. Additionally, the autophagy protein, Beclin1 has been identified as a tumor suppressor deleted in many human cancers. These results demonstrate that autophagy plays an important role in tumor suppression.

[0336] Aging

[0337] By far the greatest risk factor for neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), is aging. Mitochondria have been thought to contribute to aging through the accumulation of mitochondrial DNA (mtDNA) mutations and net production of reactive oxygen species (ROS). Although most mitochondrial proteins are encoded by the nuclear genome, mitochondria contain many copies of their own DNA. Human mtDNA is a circular molecule of 16,569 base pairs that encodes 13 polypeptide components of the respiratory chain, as well as the rRNAs and tRNAs necessary to support intramitochondrial protein synthesis using its own genetic code. Inherited mutations in mtDNA are known to cause a variety of diseases, most of which affect the brain and muscles—tissues with high energy requirements. It has been hypothesized that somatic mtDNA mutations acquired during aging contribute to the physiological decline that occurs with aging and aging-related neurodegeneration. It is well established that mtDNA accumulates mutations with aging, especially large-scale deletions and point mutations. In the mtDNA control region, point mutations at specific sites can accumulate to high levels in certain tissues: T414G in cultured fibroblasts, A189G and T408A in muscle, and C150T in white blood cells. However, these control-region “hot spots” have not been observed in the brain. Point mutations at individual nucleotides seem to occur at low levels in the brain, although the overall level may be high. Using a polymerase chain reaction (PCR)-cloning-sequencing strategy, it was found that the average level of point mutations in two protein-coding regions of brain mtDNA from elderly subjects was ̃2 mutations per 10 kb. Noncoding regions, which may be under less selection pressure, potentially accumulate between twice and four times as many. The accumulation of these deletions and point mutations with aging correlates with decline in mitochondrial function. For example, a negative correlation has been found between brain cytochrome oxidase activity and increased point-mutation levels in a cytochrome oxidase gene (COI).

[0338] Net production of ROS is another important mechanism by which mitochondria are thought to contribute to aging. Mitochondria contain multiple electron carriers capable of producing ROS, as well as an extensive network of antioxidant defenses. Mitochondrial insults, including oxidative damage itself, can cause an imbalance between ROS production and removal, resulting in net ROS production. The importance to aging of net mitochondrial ROS production is supported by observations that enhancing mitochondrial antioxidant defenses can increase longevity. In Drosophila, overexpression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD) and methionine sulfoxide reductase prolongs lifespan. This strategy is most successful in short-lived strains of Drosophila, and has no effect in already long-lived strains. However, it has recently been shown that overexpression of catalase experimentally targeted to mitochondria increased lifespan in an already long-lived mouse strain.

[0339] Improving Activity During Aging

[0340] Activity in animals is driven largely by circadian rhythm and is synchronized to the environment. Disruption of the circadian rhythm or a desynchronization with the environment can lead to increase in nighttime wakefulness or daytime naps.

[0341] Normal aging is accompanied by declining locomotor activity, altered circadian rhythms, as well as altered sleep and food intake patterns. These effects lead to a decrease in alertness and vigilance decreases in the elderly, leading to an increase in nighttime wakefulness, as well as an increase in daytime naps. Activity patterns can also be disrupted in a similar way by disease, such as Alzheimer's Disease.

[0342] Age-dependent changes in activity rhythms are also observed in other animals, for example, rats, hamsters, mice and dogs, with an increase in fragmentation and a decrease in synchronization with the environment. These age-dependent circadian disruptions have been linked to the degeneration of the suprachiasmatic nucleus of the hypothalamus. Sleep disruption in both humans and rodents have been shown to contribute to age-dependent cognitive dysfunction.

[0343] The increasing disruption of circadian rhythms is accompanied by a gradual decline in motor activity with age in several species, including humans, mice, monkeys, and dogs. Of particular note, the daytime activity of senior dogs (>10 years of age) declines as compared to young and middle-aged dogs. These changes in activity can be monitored by devices intended to measure activity, for example, by means of an accelerometer or by employing motion sensing cameras.

[0344] Many of the disruptions seen in aging as a result of decreased activity are also observed in younger populations where cultural trends have resulted in decreased activity, accompanied with increased caloric intake, leading to an obesity epidemic. The resulting caloric imbalance has led to an increase in several disease conditions such as type 2 diabetes, colon cancer, and metabolic syndrome, as well as mental health issues. Several prospective cohort studies and meta-analysis in humans have shown that physical inactivity is associated with an elevated risk for the development of metabolic syndrome, type 2 diabetes, hypertension, coronary artery disease, stroke, and cardiovascular disease.

[0345] Both humans and animals, particularly dogs, would benefit from the present invention and its ability to improve activity both during the youth and aging periods of life.

[0346] In one embodiment, the urolithin or precursor would increase activity of the recipient, human or animal. In yet another embodiment, the increase in activity is an increase by 1% to 100%. For example, the activity may be increased by 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. In certain embodiments, the increase in activity is an increase of 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 40-45%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, and 95-100%.

[0347] In one embodiment, treatment by urolithin or a precursor thereof would increase activity and lead to a reduction in risk of metabolic syndrome. In one embodiment, treatment by urolithin or a precursor thereof would increase activity and lead to a reduction in risk of type 2 diabetes. In one embodiment, treatment by urolithin or a precursor thereof would increase activity and lead to a reduction in risk of hypertension, coronary artery disease, stroke, and cardiovascular disease. In one embodiment, treatment by urolithin or a precursor thereof would increase activity and improve cognitive function.

[0348] Mood Disorders

[0349] Compounds and methods of the invention are useful for treating a mood disorder (also known as an affective disorder). As used herein, a “mood disorder” refers to a disturbance in emotional state, such as is set forth in the Diagnostic and Statistical Manual of Mental Disorders, published by the American Psychiatric Association. Mood disorders include but are not limited to major depression, postpartum depression, dysthymia, and bipolar disorder. In one embodiment, the mood disorder is major depression.

[0350] Compounds and methods of the invention are useful for treating or preventing a stress-induced or stress-related mood disorder….

[0351] Compounds and methods of the invention are useful for treating an anxiety disorder. As used herein, an “anxiety disorder” refers to a dysfunctional state of fear and anxiety, e.g., fear and anxiety that is out of proportion to a stressful situation or the anticipation of a stressful situation…

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Process-Scale Synthesis of Urolithins
US2015183758

Disclosed is a method of preparing a urolithin, or an intermediate or analog thereof, having a dibenzo[b,d]pyran-6-one core. The method is especially advantageous for the large-scale preparation of urolithins or intermediates or analogs thereof. The method may optionally include the preparation of a urolithin, or an intermediate or analog thereof, as a pharmaceutically acceptable salt.

BACKGROUND

[0002] Pomegranate (Punica granatum) fruits have been used for centuries in folk medicine. They are consumed fresh and as juice, both of which are excellent sources of ellagitannins and ellagic acid. Ellagitannins (ETs) are polymeric polyphenols abundant in some fruits and nuts such as pomegranates, raspberries, strawberries, black raspberries, walnuts and almonds. Despite numerous reports of the biological properties and human health benefits of ETs, knowledge of their bioavailability, pharmacokinetics, disposition and metabolic fate in humans is limited. Commercially-produced pomegranate juice contains gallagyl-type ellagitannins, including punicalagin isomers (1500-1900 mg/L), undefined hydrolyzable tannins (400-500 mg/L), and ellagic acid and its glycosides (120-260 mg/L). Gil et al. J. Agric. Food Chem. 2000, 48, 4581-4589. Punicalagins, ellagitannins in which gallagic and ellagic acids are linked to a glucose molecule, are abundant in pomegranate peel. Punicalagin isomers and ellagic acid derivatives are not present in the aril juice, but during industrial juice processing they are extracted from the husk and membrane surrounding the arils and released in large quantities into the juice. The fruit arils of pomegranates contain other polyphenols, such as anthocyanins, responsible for the fruit's bright ruby-red color. Ellagitannins belong to a group of compounds known as hydrolyzable tannins, which release ellagic acid (EA) upon hydrolysis.

[0003] Unfortunately, ellagitannins are typically poorly absorbed by the human gut. However, a number of metabolites derived from ellagitannins are absorbed by the human gut, including certain metabolites ultimately formed in the gut by commensal microorganisms (i.e., intestinal microflora). Ellagitannins release ellagic acid under physiological conditions in vivo, and ellagic acid is then gradually metabolized by the gut microflora in the intestine to produce the urolithins. Once the metabolites are absorbed, they are further metabolized to produce urolithin glucuronides and/or sulfates. There is growing evidence that urolithins have potent antioxidant, anticancer, and anti-hyperproliferative activity. See US 2011/0065662; US 2012/0164243; and US 2014/0018415; all of which are incorporated by reference.

[0004] Although urolithins are derived from ETs present in certain foods (e.g., pomegranates), the consumption of these foods does not always lead to sufficient bioavailability of the therapeutic metabolites. Specifically, certain individuals, referred to herein as non-producers, fail to produce detectable amounts of the metabolites after consumption of ET-containing foods (e.g., pomegranate juice). Even among individuals who are producers, there is a great deal of variation (from very low to very high) in the amount of urolithin metabolites produced. Furthermore, any FDA-approved therapeutic use of urolithins would require a reliable and standard dosing regimen; that is, a known dose of a fully-characterized compound or compounds. It would thus be necessary to administer one or more selected urolithins directly to patients in need thereof.

[0005] In light of the therapeutic promise of urolithin compounds, a tremendous need exists for a safe, economical, reliable, and scalable synthesis approach to the urolithins. A reliable source of multi-kilo and commercial quantities of urolithin compounds will allow their further clinical development, with the ultimate goal of exploiting their full therapeutic potential.

SUMMARY OF THE INVENTION

[0006] One aspect of the present invention is a method for the preparation of urolithin compounds or intermediates useful in preparing such compounds or analogs thereof. Certain methods of the present invention include a copper-catalyzed coupling of two urolithin precursor fragments to form a coupling product (Method A). Certain methods of the present invention include demethylating one or more phenolic methoxy groups of a urolithin intermediate (Method B). In certain embodiments, Methods A and B are performed sequentially, but not necessarily in that order, to yield a urolithin or analog thereof. The present invention is improved over previous methods for producing the same or similar compounds (e.g., in terms of cost, yield, purity of the resulting product(s), catalyst loading, safety profile, reaction time, temperature, or amount/type of solvent used)...

DETAILED DESCRIPTION

[0009] As mentioned above, ellagitannins generally are not absorbed in the gut. Rather, they release ellagic acid (EA) in the gut, which is only poorly absorbed in the stomach and small intestine. EA is largely metabolized by unidentified bacteria in the intestinal lumen to produce urolithins. Urolithins are putative metabolites produced by human (or animal) gut microflora from ellagic acid, punicalagin (PA), punicalin (PB), tellimagrandin (TL), and other ellagitannins through a series of chemical modifications grouped into several pathways, giving rise to many known urolithins. In terms of chemical structure, urolithins are dibenzopyran-6-one derivatives with varying hydroxyl substitution patterns. The processing of ellagic acid begins with the loss of one of the two lactones present in ellagic acid (lactonase/decarboxylase activity), and is followed by optional removal of one or more hydroxyl groups (dehydroxylase activities) and optional further reactions including methylation and glycosylation.

[0010] More specifically, microbial metabolism of ellagic acid starts in the small intestine, and the first metabolites produced retain four phenolic hydroxyls (urolithin D, four hydroxyl groups; see FIG. 1); these are further metabolized along the intestinal tract to remove hydroxyl units leading to urolithin C (three hydroxyls), urolithin A (two hydroxyls) and B (one hydroxyl) in the distal parts of the colon. The absorbed metabolites are conjugated with glucuronic acid (one or two units), and/or methylated to form methyl ethers (e.g., when ortho-dihydroxyl groupings are present). Urolithin A and B conjugates are the main metabolites detected in plasma and urine, although some trihydroxy derivatives (hydroxyl-UA) or EA-dimethyl ether glucuronide have also been detected in smaller amounts. The tetrahydroxy-urolithins, trihydroxy-urolithins, and EA derivatives generally are not detected in peripheral plasma, but they are absorbed in the small intestine and they are transported to the liver where they are further metabolized and excreted with bile to the small intestine, establishing an enterohepatic circulation that is responsible for the relatively long life of urolithins in plasma and urine.

[0011] Over the last twenty years many papers have appeared on the biosynthesis, isolation, and biological activity of tannins, especially ellagitannins. Access to pure ellagitannins by isolation from natural sources may be cumbersome and yield only relatively small quantities of pure natural products. See, for example, Okuda et al. (1982) Chem. Pharm. Bull. 30: 4230-4233; Okuda et al. (1982) Chem. Pharm. Bull. 30: 4234-4236. Methods are known for total synthesis of many ellagitannins. See, for example, Khanbabaee, K., Strategies for the synthesis of ellagitannins, In: Chemistry and Biology of Ellagitannins, Ed. S. Quideau, World Scientific Publishing, Singapore, 2009, pp. 152-202, including references cited therein.

[0012] The development of a process-scale synthesis of urolithins required substantial innovation. A useful process-scale synthesis must be efficient, cost-effective, and reproducible. Further, all starting materials and reagents must be reliably available in bulk, or able to be produced on site in a safe and economical fashion. The exacting regulatory standards for low impurity levels and overall safety of the process create additional challenges to development.

[0013] An Ullmann coupling is frequently used to couple the two phenyl rings present in all of the urolithin compounds. Unfortunately, the coupling routinely gives rise to product that is unacceptably contaminated with copper. The product also varies in color from batch to batch, from yellow to dark purple. Some residual copper may be removed by column chromatography; however, in process scale syntheses it is highly desirable to avoid column chromatography, due to its expense and large waste stream. Remarkably, improvements were made to a problematic Ullmann coupling. By drastically reducing the amount of copper catalyst, the isolated Ullmann coupling product consistently contained <1 ppm residual copper, and was off-white to light yellow in color. Moreover, the need for column chromatography was avoided.

[0014] A second improvement relates to a demethylation reaction. As discussed further in the Examples, the hydroxyl groups present in urolithins are often protected as methyl, ethyl, or alkyl ethers. Functionalizing the hydroxyl groups as ethers also allows access to a variety of more lipophilic and potentially more-bioavailable urolithin analogs. To allow access to the natural urolithins, demethylation/dealkylation of the ethers must be performed. This transformation has frequently been accomplished on similar substrates with BBr3 (boron tribromide), a chemical reagent associated with various hazards and drawbacks. Remarkably, it was discovered that the powerful Lewis acid AlCl3 (aluminum trichloride) can bring about the desired transformation in greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 85%, or even greater than 88% yield. Subsequent hydrolysis of the excess AlCl3, filtration, and a recrystallization provided the pure demethylated product containing <17 ppm aluminum...

[0061] In certain embodiments, the copper-containing catalyst is selected from the group consisting of copper powder, copper-bronze couple, CuSO4 hydrate, anhydrous CuSO4, Cu(acac)2, CuCl, CuCl2, CuBr, CuBr2, CuI, Cu2O, CuO, CuOTf, CuCN, and mixtures thereof….

[0065] In certain embodiments, the amount of copper-containing catalyst is at least a trace amount but less than 0.05 equivalents relative to either formula II or formula III.

[0071] In certain embodiments, the amount of copper-containing catalyst is at least a trace amount but less than 0.0001 (1×10<−4>) equivalents relative to either formula II or formula III.

[0072] In certain embodiments, the aqueous alkaline solvent comprises LiOH, NaOH, KOH, CsOH, Na2CO3, CaCO3, or Cs2CO3.

[0073] In certain embodiments, the aqueous alkaline solvent comprises NaOH or KOH.

[0074] In certain embodiments, the method is conducted at a temperature from about 20° C. to about 180° C.

&c...

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PRODRUGS OF UROLITIHNS AND USES THEREOF
WO2015097231

The invention provides compounds of formula (I) or salts thereof, wherein: A, B, C, D, W, X, Y and Z are as defined in the specification, and at least one of A, B, C, D, W, X, Y and Z is OR1; each R1 being independently H or C(=O)R2, and at least one R1 group being C(=O)R2; where each R2 is selected from: CHR3NHR4, where R4 is H and R3 is a group selected from CH3, CH2CH(CH3)2, CH(CH3)CH2CH3, CH2Ph, CH2-3-(1H-indole), CH2CH2SCH3, CH2OH, CHOHCH3, CH2SH, CH2SeH and CH2PhpOH, wherein said R3 group can optionally be substituted by one or more groups selected from halogen, cyano, nitro, ORA or C1-C4 alkyl; or R3 and R4 together with the C and N atoms to which they are attached form a 5-membered heteroalkyl ring, wherein said heteroalkyl ring can optionally be substituted by one or more groups selected from halogen, cyano, nitro, ORA or C1-C3 alkyl, wherein RA is C1-C4 alkyl optionally substituted with one or more halogen, cyano or nitro groups. The compounds are effective pro-drugs for urolithins and they enable the ready delivery of urolithins to the site in the digestive tract where they can be absorbed into the body.

Urolithin A Urolithin B

Urolithins have been proposed as treatments of a variety of conditions related to inadequate mitochondrial activity, including obesity, reduced metabolic rate, metabolic syndrome, diabetes mellitus, cardiovascular disease, hyperlipidemia, neurodegenerative diseases, cognitive disorder, mood disorder, stress, and anxiety disorder; for weight management, or to increase muscle performance or mental performance. See WO2012/088519 (Amazentis SA). In WO2007/127263 (The Regents of the University of California), the use of urolithins for the treatment of various neoplastic diseases is described.

International patent publication no. WO2014/004902 (derived from application PCT/US2013/48310) discloses a method of increasing autophagy, including specifically mitophagy, in a cell, comprising contacting a cell with an effective amount of a urolithin or a pharmaceutically acceptable salt thereof, thereby increasing autophagy, including specifically mitophagy, in the cell. Administration may be to a subject having a disease or condition selected from metabolic stress, cardiovascular disease, endothelial cell dysfunction, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, a 1 -antitrypsin deficiency, ischemia/reperfusion injury, inflammation, aging of the skin, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Huntington's disease, Parkinson's disease, amyotrophic lateral sclerosis, age-related macular degeneration, mitochondrial diseases (including for example poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction sometimes learning disabilities, and dementia as a result of mitochondrial disease), muscle diseases; sporadic inclusion body myositis (sIBM), cancer, cognitive disorder, stress, and mood disorder…

Treatments using compounds of the invention

As mentioned above, the invention provides compounds of formula (I) or (la) or salts thereof for use in the treatment of a disease or condition selected from the group consisting of metabolic syndrome, reduced metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease (NAFLD), Nonalcoholic steatohepatitis (NASH), drug-induced liver injury, drug-induced cravings, anaemia disorders, a 1 -antitrypsin deficiency, ischemia/reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, Huntington's disease, anxiety disorder, ulceration, amyotrophic lateral sclerosis, mitochondrial diseases (including for example poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction sometimes learning disabilities, and dementia as a result of mitochondrial disease. Further diseases related to mitochondrial dysfunction include: Diabetes mellitus and deafness (DAD); Leber's hereditary optic neuropathy (LHON); Leigh syndrome (subacute sclerosing encephalopathy); neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); myoneurogenic gastrointestinal encephalopathy (MNGIE); Myoclonic Epilepsy with Ragged Red Fibers (MERRF);

Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke- like symptoms (MELAS); and mtDNA depletion), sporadic inclusion body myositis (sIBM), and cancer, cognitive disorder, stress, and mood disorder; for improving cognitive function; for weight management; or to increase muscle or mental performance. The compounds of formula (I) or (la) or salts thereof are particularly suitable for use in improving muscle function, muscle strength endurance and muscle recovery.

In particular, the invention provides compounds of formula (I) or (la) or salts thereof for use in the treatment of a disease or condition selected from the group consisting of metabolic syndrome, reduced metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, drug-induced cravings, anaemia disorders, a 1 -antitrypsin deficiency, ischemia/reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, anxiety disorder, ulceration, amyotrophic lateral sclerosis, and cancer, cognitive disorder, stress, and mood disorder; for improving cognitive function; for weight management; or to increase muscle or mental performance.

The invention further provides compounds of formula (I) or (la) or a salt thereof for use in the treatment of a disease or condition selected from the group consisting of metabolic stress, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, a 1 -antitrypsin deficiency, ischemia/reperfusion injury, inflammatory bowel disease, Crohn's disease, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and cancer. The invention further provides compounds of the formula (I) or (la) or salts thereof for increasing autophagy or mitophagy in a cell. For example, the autophagy or mitophagy may be in embryonic stem cells, induced pluripotent stem cells, adult stem cells, differentiated cells, blood cells, hematopoietic cells, epithelial cells, exocrine cells, endocrine cells, connective tissue cells, adipose cells, bone cells, smooth muscle cells, striated muscle cells, nerve cells, sensory cells, cardiac cells, hepatic cells, gastric cells, intestinal cells, pulmonary cells, epidermal (i.e. skin) cells (including keratinocytes and fibroblasts), kidney cells, and germ cells. It may thus for example treat or prevent a disease or condition selected from the group consisting of metabolic syndrome, reduced metabolic rate, metabolic stress, cardiovascular disease, sarcopenia, muscle degenerative disease, Duchenne muscular dystrophy, alcoholic liver disease, nonalcoholic fatty liver disease, drug-induced liver injury, drug-induced cravings, anaemia disorders, a 1 -antitrypsin deficiency, ischemia/reperfusion injury, inflammation, inflammatory bowel disease, Crohn's disease, obesity, metabolic syndrome, type II diabetes mellitus, hyperlipidemia, osteoarthritis, neurodegenerative disease, Alzheimer's disease, Parkinson's disease, anxiety disorder, ulceration, amyotrophic lateral sclerosis, and cancer, cognitive disorder, stress, and mood disorder; or it can assist with weight management, or increase muscle or mental performance.

Amongst the neurodegenerative diseases, there may specifically be mentioned AIDS dementia complex, Alzheimer's disease, amyotrophic lateral sclerosis, adreno leukodystrophy, Alexander disease, Alper's disease, ataxia telangiectasia, Batten disease, bovine spongiform encephalopathy (BSE), Canavan disease, corticobasal degeneration, Creutzfeldt- Jakob disease, dementia with Lewy bodies, fatal familial insomnia, frontotemporal lobar degeneration, Huntington's disease, Kennedy's disease, Krabbe disease, Lyme disease, Machado-Joseph disease, multiple sclerosis, multiple system atrophy, neuroacanthocytosis, Niemann-Pick disease, Parkinson's disease, Pick's disease, primary lateral sclerosis, progressive supranuclear palsy, Refsum disease, Sandhoff disease, diffuse my elino clastic sclerosis, spinocerebellar ataxia, subacute combined degeneration of spinal cord, tabes dorsalis, Tay-Sachs disease, toxic encephalopathy, transmissible spongiform encephalopathy, and wobbly hedgehog syndrome. In one embodiment, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, and Parkinson's disease. In one embodiment, the neurodegenerative disease is Alzheimer's disease. An aspect of the invention is in improving cognitive function. In one embodiment, the cognitive function is selected from the group consisting of perception, memory, attention, speech comprehension, speech generation, reading comprehension, creation of imagery, learning, and reasoning. In one embodiment, the cognitive function is selected from the group consisting of perception, memory, attention, and reasoning. In one embodiment, the cognitive function is memory.

An aspect of the invention is in the treatment of cognitive disorder. In one embodiment, the cognitive disorder is selected from the group consisting of delirium, dementia, learning disorder, attention deficit disorder (ADD), and attention deficit hyperactivity disorder (ADHD). In one embodiment, the cognitive disorder is a learning disorder. In one embodiment, the cognitive disorder is attention deficit disorder (ADD). In one embodiment, the cognitive disorder is attention deficit hyperactivity disorder (ADHD).

An aspect of the invention is in the treatment of stress-induced or stress-related cognitive deficit. An aspect of the invention is in the treatment of a mood disorder. In one embodiment, the mood disorder is selected from the group consisting of depression, postpartum depression, dysthymia, and bipolar disorder. In one embodiment, the mood disorder is depression. In one embodiment, the mood disorder is dysthymia.

An aspect of the invention is in the treatment of stress-induced or stress-related mood disorder, e.g., dysthymia. An aspect of the invention is in the treatment of an anxiety disorder. In one embodiment, the anxiety disorder is selected from the group consisting of generalized anxiety disorder, panic disorder, panic disorder with agoraphobia, agoraphobia, social anxiety disorder, obsessive-compulsive disorder, and post-traumatic stress disorder. In one embodiment, the anxiety disorder is generalized anxiety disorder. In one embodiment, the anxiety disorder is post-traumatic stress disorder. An aspect of the invention is in the treatment of stress-induced or stress-related anxiety.

An aspect of the invention is in enhancing muscle performance. In one embodiment, the muscle performance is selected from the group consisting of strength, speed, endurance and recovery. In humans, muscle function generally declines with age starting during the third decade of life; the decline generally accelerates after age 65. An aspect of the invention is thus in maintaining muscle performance during the aging process. The enhancement of muscle performance may be as part of the use of the compounds in sports nutrition, in aiding healthy aging (for example from age 45 to 65), and in slowing the rate of muscle decline in those aged over 65 (pre-frail)

An aspect of the invention is in the treatment of a muscle or neuromuscular disease. In one embodiment, the muscle or neuromuscular disease is a myopathy. Ine one embodiment, the muscle or neuromuscular disease is sarcopenia. In one embodiment, the muscle or neuromuscular disease is sporadic inclusion body myositis (sIBM). In one embodiment, the muscle or neuromuscular disease is a muscular dystrophy. In one embodiment, the muscle or neuromuscular disease is Duchenne muscular dystrophy.

An aspect of the invention is in the treatment of mitochondrial disease. For example, a subject may require treatment of poor growth, loss of muscle coordination, muscle weakness, visual problems, hearing problems, heart disease, liver disease, kidney disease, gastrointestinal disorders, respiratory disorders, neurological problems, autonomic dysfunction sometimes learning disabilities, and dementia as a result of mitochondrial disease. Further diseases related to mitochondrial dysfunction include:

Diabetes mellitus and deafness (DAD); Leber's hereditary optic neuropathy (LHON); Leigh syndrome (subacute sclerosing encephalopathy); neuropathy, ataxia, retinitis pigmentosa, and ptosis (NARP); myoneurogenic gastrointestinal encephalopathy (MNGIE); Myoclonic Epilepsy with Ragged Red Fibers (MERRF); Mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like symptoms (MELAS); and mtDNA depletion.

Amongst cancers, there can specifically be mentioned solid tumours, for example prostate cancer, pancreatic cancer and colon cancer.

The effective amount of the compound will vary depending upon the manner of administration, the age, body weight, and general health of the subject. Factors such as the disease state, age, and weight of the subject may be important, and dosage regimens may be adjusted to provide the optimum response. A therapeutically effective amount of the compound may for example range from about 0.001 to 30 mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, more preferably about 0.1 to 20 mg/kg body weight, and even more preferably about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. Treatment may be by way of a single treatment or, preferably, can include a series of treatments. In one example, a subject is treated with the compound in the range of between about 0.1 to 20 mg/kg body weight, once per week for between about 1 to 10 weeks, preferably between 2 to 8 weeks, more preferably between about 3 to 7 weeks, and even more preferably for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of the compound may increase or decrease over the course of a particular treatment...

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Pomegranate Patents

Extracts --

ORAL FORMULATIONS FOR COUNTERACTING EFFECTS OF AGING
US2016067291
An oral formulation as described herein can comprise pomegranate extract, panax ginseng extract, and c. sinensis, where each is present in an amount effective to counteract and/or prevent effects of aging in a subject when administered to the subject. The effects of aging can include age-related changes in gene expression.

POMEGRANATE FRUIT POLYPHENOL COMPOSITION AND METHODS OF USE AND MANUFACTURE THEREOF
US9056083
A pharmaceutical composition with an active pharmaceutical ingredient including a pomegranate fruit polyphenol extract. The pomegranate fruit polyphenol extract includes at least about 3% combined punicalagin A and punicalagin B by weight, less than about 5% ellagic acid and their derivatives by weight, and less than about 1% anthocyanins by weight.

Extracting method for effective components of pomegranate leaves and obtained product
CN105232594
The invention discloses an extracting method for effective components of pomegranate leaves and an obtained product. The method comprises the steps that pomegranate leaves are thoroughly washed and kneaded with salt; the pomegranate leaves are dried through microwaves at 40-50 DEG C after kneading until the water content is lower than 10%; the dried pomegranate leaves are refrigerated for 5-10 min at minus 15 DEGC and then refrigerated for 20-30 min at minus 5 DEG C; the refrigerated pomegranate leaves are put into a heating container, compacted, subjected to heat preservation for 0.5-1 h at 50-60 DEG C and then heated and boiled, distilled-off components are collected, and the distilled-off components are aqueous extraction of the pomegranate leaves. The method is simple, easy to implement, clean and efficient, the pretreatment measures of kneading with salt, microwave drying and low-temperature refrigerating are adopted, more effective components can be extracted, and it is proved through tests that the obtained aqueous extraction of the pomegranate leaves has good antimicrobial and anti-inflammatory effects and can serve as raw materials of anti-inflammation medicine.

Preparation method of pomegranate extracting liquid
CN105147546
The invention provides a preparation method of pomegranate extracting liquid. The preparation method comprises the following steps: squeezing fresh whole pomegranates to obtain fresh pomegranate juice and fruit residues; adding water into the obtained fruit residues and carrying out continuous countercurrent extraction to obtain pomegranate residue liquid; mixing the obtained fresh pomegranate juice and the obtained pomegranate residue liquid; filtering to obtain the pomegranate extracting liquid. According to the preparation method provided by the invention, a process of combining spiral squeezing and a packing auger type continuous countercurrent extraction method is adopted, and the processes of peeling off the pomegranates, processing and the like are omitted, so that the comprehensive utilization time of the pomegranates is shortened and the yield of effective components is improved; the preparation method makes important contributions to operation simplification, energy saving, cost saving and improvement of the stability of the extracting liquid.

Preparation method of whole pomegranate seed powder
CN105053877
The invention discloses a preparation method of whole pomegranate seed powder. Peeled mature pomegranate fruits are processed into the whole pomegranate seed powder through the steps of carefully selecting pomegranate fruits as raw materials, performing color protection on whole pomegranate seeds, mashing the whole pomegranate seeds after the color protection so as to obtain pomegranate fruit paste, quenching and tempering the pomegranate fruit paste, and spraying and drying the quenched and tempered pomegranate fruit paste. The whole pomegranate seed powder prepared by the preparation method disclosed by the invention is rich in nutrition, high in retention rate of bioactive components, good in reconstituability, short in rehydration time, and favorable of storage and transportation; besides, the shelf life of the whole pomegranate seed powder can be prolonged; the defects of large loss of active components and low utilization rate of raw materials existing in the fruit powder which is prepared by spraying and drying fresh pomegranate juice or concentrated juice thereof are overcome, and a new way for processing and utilizing pomegranates is developed.

Cultivation --

Special pomegranate tree liquid organic fertilizer and preparation method thereof
CN105237184
The present invention provides a special pomegranate tree liquid organic fertilizer preparation method, which comprises: dissolving potassium dihydrogen phosphate, potassium nitrate and urea in pure water to achieve a saturated solution, and mixing to obtain a solution A; dissolving zinc sulfate, active boron, ferrous sulfate, manganese sulfate, copper sulfate and ammonium molybdate in pure water to achieve a saturated solution, and mixing to obtain a solution B; dissolving a surfactant and a composite aid in pure water to achieve a saturated solution, and mixing to obtain a solution C; dissolving amino acid original powder and potassium fulvate in pure water to achieve a saturated solution, and mixing to obtain a solution D; mixing the solution B and the solution C to obtain a first mixed solution; mixing the solution D and the solution A to obtain a second mixed solution; and mixing the first mixed solution and the second mixed solution, and adding potassium carbonate so as to obtain the special pomegranate liquid organic fertilizer. The present invention further provides the special pomegranate tree liquid organic fertilizer prepared through the preparation method. The special pomegranate tree liquid organic fertilizer of the present invention has the simple and easy ratio adjustment advantage, and is the green organic fertilizer.

Composite fertilizer for increasing pomegranate yield
CN105237091
The invention provides a composite fertilizer for increasing pomegranate yield. The composite fertilizer comprises the following effective components in parts by mass: 60-80 parts of an organic fertilizer, 5-10 parts of urea, 10-15 parts of monopotassium phosphate, 10-18 parts of calcium superphosphate and 15-25 parts of plant ash. Due to adoption of the composite fertilizer, organic matters in soil can be supplemented, micro-ecological environment can be improved, and the defect that the fertilizer efficiency of an organic fertilizer is slow to take into effect can be made up. Meanwhile the yield of crops can be increased. As the organic fertilizer is cheap and easily available, the cost of the composite fertilizer is also lowered.

Cultivation method of pomegranate trees and alliumjaponicurn regel at hilly arid area and application
CN105052652
The invention, which belongs to the technical field of agriculture growing, discloses a cultivation method of pomegranate trees and alliumjaponicurn regel at a hilly arid area and application. According to the method, a natural rain path is improved and a cistern is built; a terraced filed at a hilly arid area is modified into wave-shaped farmland; a hole bottom and the ground are covered by plastic films and pomegranate trees are planted in a seepage-proof and moisture preservation mode, and alliumjaponicurn regel is planted also in an intercropping mode; and then filed intercropping management is carried out on the pomegranate trees and alliumjaponicurn regel. According to the invention, the provided method is superior to the existing method obviously in terms of cultivation management; the yield can be improved; the gains can be increased on the condition that the management cost is not increased; and objectives of water saving, labor saving, and high yield and benefit increasing can be achieved. The method and application are suitable for cultivation of pomegranate trees and alliumjaponicurn regel at a hilly arid area.

Method for cultivating pomegranate seedlings in saline-alkali soil
CN105028107
The invention discloses a method for cultivating pomegranate seedlings in saline-alkali soil. The method comprises three steps for improving the saline-alkali soil, namely 1, phosphorus gypsum powder is applied to the saline-alkali soil for improving the saline-alkali soil; 2, organic fertilizer, mushroom residue and oil cakes are applied to the saline-alkali soil for improving the saline-alkali soil; 3, wild sweet potatoes are planted in the saline-alkali soil for improving the saline-alkali soil. By means of the method, the soil structure of the saline-alkali soil can be improved effectively, the soil pH can be reduced effectively, the saline-alkali soil is made to be suitable for the growth of the pomegranate seedlings, and therefore the pomegranate seedlings can be cultivated on the saline-alkali soil. The method has a remarkable effect especially for improving the moderate saline-alkali soil, the saline-alkali soil can be improved completely, the pomegranate seedlings can be cultivated by utilizing the saline-alkali soil, and the remarkable economic benefit is obtained.

Pomegranate fresh-keeping method
CN104970096
The invention provides a pomegranate fresh-keeping method which includes the steps of (1) collecting matured pomegranates, selecting health fresh fruits of the pomegranates; (2) coating the surface of the fresh fruits of the pomegranates with a film of a chitosan solution being 0.5-1.0% in mass concentration; (3) naturally drying the pomegranate fresh fruits coated with the chitosan; (4) packaging the naturally-dried pomegranate fresh fruits respectively into PE bags on which a plurality of holes are formed; and (5) storing the pomegranates at 4-6 DEG C under the relative humidity being 90-95%. The pomegranate fresh-keeping method can prolong preservation time of the fresh fruits of the pomegranates, allows off-season consumption, can increase the economic benefit of the pomegranates, is simple in process, is convenient to carry out, and is controllable in preservation scale.

High yield cultivating method of pomegranates
CN104969814
The invention relates to the technical field of plant planting, particularly to a high yield cultivating method of pomegranates. The high yield cultivating method comprises the following steps of: (1) preparing a seedling raising matrix; (2) raising seedlings by cutting of short shoots, (3) transplanting the seedlings; (4) managing the transplanted seedlings; and (5) managing the seedlings at a fruiting period and seedlings after pomegranate fruits are picked up, wherein a culturing matrix is weighed in parts by weight: 30-50 parts of poultry dung, 20-40 parts of turf, 10-30 parts of distillers' grains, 12-24 parts of vermiculite, 8-16 parts of perlite, and 2-5 parts of micro elements. Carbendazol is used for disinfecting the seedling raising matrix, and then the seedlings are raised, wherein the consumption of the carbendazol is 90-120g/m<3>. Through the high yield cultivating method of pomegranates, disclosed by the invention, the survival rate is high, the growing trend of the plants is quick, neat and consistent, the cycle of fruit setting is short, the yield is high, and the economic returns are considerable.

Leaf fertilizer special for pomegranate
CN104961607
he invention relates to a leaf fertilizer special for pomegranate. The leaf fertilizer is composed of raw materials including, by weight, 1-20 parts of isoleucine, 8-38 parts of proline, 1-18 parts methionine, 1-20 parts of cysteine, 8-30 parts of asparaginate, 0.1-0.8 part of glutamine, 10-100 parts of water, 0.8-2 parts of salicylic acid, 1-28 parts of abscisic acid and 8-20 parts of ammonium bicarbonate. The leaf fertilizer promotes crop growth, reduces the pesticide application amount, and is low in raw material cost and capable of preventing insect disease.

Formula of organic inorganic high-efficiency and safe bulk blended fertilizer specially used for pomegranate tree
CN104945036
The invention discloses a formula of an organic inorganic high-efficiency and safe bulk blended fertilizer specially used for a pomegranate tree. The formula is obtained by scientifically preparing according to a 'wooden barrel law' principle of balanced fertilization and a fertilizer requirement law that the pomegranate tree needs nitrogen fertilizer all the time from germination to a fruit swelling period and needs more phosphorus and potassium from a flowering period to a harvesting period, when every 1000kg of the bulk blended fertilizer is produced, 400kg of urea, 200kg of monoammonium phosphate, 200kg of potassium chloride, 150kg of organic fertilizer and 50kg of calcium, magnesium and phosphorus fertilizer are used, stirred and blended, so that the organic inorganic high-efficiency and safe bulk blended fertilizer, specially used for the pomegranate tree, meeting the requirements that ratio of three elements, namely nitrogen, phosphorus and potassium is 23:9:12, total nutrient accounts for 44%, medium trace element accounts for 4% and organic matter, amino acid and humic acid account for 6% is prepared. The formula of the organic inorganic high-efficiency and safe bulk blended fertilizer specially used for the pomegranate tree contains all the nutrients such as macro elements (nitrogen, phosphorus and potassium), medium elements (calcium, magnesium and sulphur) and trace elements (copper, iron, zinc, manganese, molybdenum, chlorine and boron) as well as organic matter, amino acid and humic acid and has the advantages of high efficiency, safety, low cost and strong pertinence.

Seedling-raising substrate preventing pomegranate seedling root rot
CN104926553
The invention provides a seedling-raising substrate preventing pomegranate seedling root rot, and relates to the technical field of pomegranate seedling raising. The substrate is prepared from the raw materials of, by weight, 30 parts of sandy soil, 15 parts of humus, 10 parts of wheat bran, 8 parts of soybean meal, 12 parts of corn flour, 1.5 parts of raw gypsum, 10 parts of vermiculite, 12 parts of diatomaceous earth, 6 parts of plant ash, 18 parts of fermented mud, 5 parts of edible fungus strain, 3 parts of quispualis indica, 2.5 parts of cyrtomium fortunei, 3 parts of radix euphorbiae lantu, 7 parts of chinaberry seed, 15 parts of golden cypress, 10 parts of ash bark, 4 parts of alum, 6 parts of fructus cnidii, and 2 parts of honeysuckle flower. The substrate has the advantages of high propagation speed, high germination rate, and effective pest and disease inhibition performance. With the substrate, pomegranate seedling root rot can be reduced, and a good condition for the seedlings to thrive is provided. With the substrate, transplanting survival rate is high. The seedling-raising substrate has good breathability, and is suitable for pomegranate seedling growth. The substrate is rich in nutrients. With the substrate, pomegranate seedling growth speed is high.

Bio-enzyme organic compound fertilizer for pomegranate
CN104926468
The invention relates to a bio-enzyme organic compound fertilizer for pomegranate. The bio-enzyme organic compound fertilizer is characterized by comprising the following components in parts by weight: 16-20 parts of serine protease, 36-66 parts of thiol protease, 16-31 parts of isoleucine, 16-31 parts of proline, 16-31 parts of aspartic protease, 16-31 parts of isoaspartic protease, 2-6 parts of protease, 7-16 parts of vinyl ethyl ether, 7-16 parts of ethyl glycollate and 6-10 parts of solanum nigrum. The bio-enzyme organic compound fertilizer has the advantages that the ecological agriculture target is realized, and large-scale agriculture is on the way to virtuous cycle.

Seed treating fluid for preventing pomegranate seedlings from rotted roots
CN104926429
The invention provides a seed treating fluid for preventing pomegranate seedlings from rotted roots, and relates to the technical field of seedling growing of pomegranate. The seed treating fluid is prepared from the following raw materials in parts by weight: 1500g of fermentation liquor, 1.2mg of auxin IBA, 3g of radix stemonae, 2g of cyrtomium fortune, 1.5g of radix euphorbiae lantu, 2.5g of Chinaberry seeds, 3g of golden cypress, 2g of roots of Chinese pulsatilla and 3.5g of honeysuckle flowers, wherein the fermentation liquor is a liquid prepared by fermenting 45 parts of pig manure, 280 parts of water, 15 parts of corn stalks and 15 parts of tobacco stems in a fermentation tank. The seed treating fluid is high in reproduction speed, high in germination rate, capable of effectively restraining plant diseases and insect pests and reducing rotted root conditions of the pomegranate seedlings and providing a condition for the seedlings to grow strongly and healthily; the provided seedling cultivation medium is good in air permeability, suitable for the pomegranate seedlings to grow and rich in nutrient, so that the pomegranate seedlings are high in growth speed; the treating fluid for treating pomegranate seeds has germination accelerating effect on seeds, and can be used for restraining the seed surfaces from growing mildew and reducing occurrence of plant diseases and insect pests, so that the survival rate of the pomegranate seedlings is high.

Special long-acting slow-release fertilizer for pomegranate
CN104909921
The invention discloses a special long-acting slow-release fertilizer for pomegranate, which is composed of the following components in parts by weight: 6-10 parts of diethylaminoethyl cellulose, 5-9 parts of naphthyl diisocyanate, 4-8 parts of butylated hydroxytoluene, 6-11 parts of dimethylphenyl phosphate, 10-20 parts of polyethyleneglycol, 3-7 parts of BCG, 2-8 parts of Albizzia julibrissin, 1-5 parts of borneol, 3-8 parts of red paeonia, 3-5 parts of reed root, 2-7 parts of asafetida, 6-8 parts of fried shallot, 8-14 parts of alanine, 1-2 parts of serine, 1-4 parts of acyl peptide hydrolase and 2-4 parts of slow-release agent. The special long-acting slow-release fertilizer for pomegranate can supply nutritional ingredients required by pomegranate for a long time, and enhances the soil fertility due to the addition of the microbial inoculant and organic fertilizer.

Cultivation method for pomegranate seedlings
CN104855214
The invention provides a cultivation method for pomegranate seedlings, relating to the technical field of pomegranate-seedling. The method comprises following steps: selecting seeds fully and completely filled with grains, putting and soaking the seeds into a treating fluid for 8 hours till the seeds are completely soaked; sowing soaked seeds onto a seeding bed. The seeding substrate laid on the seeding bed is 25-centimeter thick. Soil is covered to the thickness of 3 centimeters. Suppressing and watering operation are performed after sowing. The depth of water soaking into the seeding substrate is below the seeds for 2 to 3 centimeters. The cultivation method for the pomegranate seedlings has following beneficial effects: the cultivation method is featured by high in reproduction speed and germination capacity and is capable of effectively inhibiting diseases and insect pests and provides a condition for thriving seedlings with high survive rate after transplantation; the seeding substrate has fine air permeability and is suitable for growth of growing-fast pomegranate seedlings; the treating fluid used for treatment of pomegranate seeds has pregermination effect upon the seeds; mycete of seed surfaces is avoided; occurrences of diseases and insect pests are reduced; and high survive rate of the pomegranate seedlings is obtained.

Pomegranate blooming-stage cultivation method
CN104429778
The invention provides a pomegranate blooming-stage cultivation method which comprises the following steps: (1) trimming flowers: clearing away bell-shaped flowers, keeping tubular flowers and gourd-shaped flowers, arranging four foliage branches around each fruit branch with bell-shaped flowers, and spraying a growth regulator at an early blooming stage and a full blooming stage; (2) trimming fruit: clearing away the third batch of fruit, keeping the first batch of fruit and the second batch of fruit, and clearing away and deeply burying diseased fruit, wormed fruit and malformed fruit; (3) topdressing: in the full-bearing period, applying urea to resulting trees and young trees, spraying leaf fertilizer to leaves, and conducting watering, intertillage for loosening the soil and weed uprooting after topdressing; (4) trimming branches: girdling the branches which are more than 8 cm in thickness, and cutting off too dense branches germinating in spring and twigs germinating in summer; (5) pollination: releasing honeybees at the early blooming stage and the full blooming stage, gathering pollen-flying fruitless flowers every day, removing the sepals and the petals of the fruitless flowers to expose anthers, and point-pollinating open flower stigmas; (6) disease and pest control. The pomegranate blooming-stage cultivation method can improve the quality and the yield of fruit.

Cultivation method of polyploid pomegranate
CN103392606
The invention relates to a cultivation method of polyploid pomegranate. The cultivation method comprises following steps: selecting branched flower buds and branches which grow on the top of a diploid plant, inducing the flower to generate polyploid pollen by dropwise adding chemical materials into the flower buds, carrying out hybridizations between induced male flowers and normal female flowers or between induced female flowers and normal male flowers so as to obtain polyploid hybrid fruits, culturing the hybrid seeds in the hybrid fruits so as to obtain polyploid plants, and culturing the polyploid plants in a tissue cultivation mode so as to obtain polyploid pomegranate plants. The cultivation method overcomes the shortage that a large amount of time is consumed by the conventional chromosome reduplication methods. The cultivation method utilizes the fact that the pollen and ovule cells in the reproductive organs are haploids, namely 1n chromosome, during the pomegranate blossom period, uses chemical materials to process the pollen and ovule cells to induce chromosome doubling, thus largely reduces the time of chromosome doubling process, and has the advantages of available materials, simple operation, and high success rate.

Preparation method for high-yield cultivation fertilizer for pomegranate trees
CN102807427
The invention relates to a preparation method for high-yield cultivation fertilizer for pomegranate trees. The high-yield cultivation fertilizer consists of the following components in percentage by weight: dry cow manure, dry chicken manure, dry duck manure, phosphate fertilizer, zinc fertilizer, composite fertilizer, bone meal, benomyl, procymidone, triazolone, Bordeaux liquid, broad-spectrum bactericide and midothane wettable powder. The high-yield cultivation fertilizer has low cost, readily available materials and a simple preparation method. The growth requirements of the pomegranate trees can be met by a large number of beneficial microorganisms and organic fertilizer generated in the fertilizer. The pomegranate has large volume, sufficient weight, high growth speed, delicious taste and rich nutrients. The yield of the pomegranate trees is increased and the defects that the pomegranate has small volume, light weight and low growth speed and is in illness easily are overcome.

Method for grafting pomegranate in summer
CN102763563
A method for grafting pomegranate in summer relates to the technical field of pomegranate cultivation, and comprises the steps of selecting appropriate rootstocks, cutting wood and cions, cutting the rootstocks, grafting the scions and the rootstocks and then binding membranes outside the well grafted cions and rootstocks. The method provided by the invention has the benefits that the restriction on the pomegranate grafting season is broken, so that the pomegranate can be grafted and reproduced even in summer, and the problem of difficulty in grafting the pomegranate in summer is effectively solved. The method is simple to operate, improves the survival rate and ensures both production increase and income increase of the pomegranate.

Frame type cultivation method for pomegranates
CN102715098
The invention discloses a frame type cultivation method for pomegranates and belongs to the field of fruit cultivation. The method comprises adopting sandy loam to serve as a matrix of nursery, enabling plant line spacing for cultivating fixed planting to be 2m*3m, enabling one column to be erected between every two pomegranate trees, respectively drawing steel wires with the heights of 0.7-0.9m and 0.9-1.1m between adjacent columns, enabling two steel wires which are 0.9-1.1m high to be horizontally arranged and respectively distributed on two sides of the columns. An arbitrary pomegranate can be arranged in a quadrilateral area enclosed by a steel wire, branches can be bound according to the need, the problem that the branches are bent due to fruiting can be solved, or attractiveness of the pomegranates is improved by binding modeling. The frame type cultivation method is simple and practical, effectively controls growing density of the pomegranates and guarantees sunshine supply.

Method for extracting natural flocculation active ingredients from pomegranate bark and method for preparing flocculating agent
CN105129948
The invention relates to a method for extracting natural flocculation active ingredients from pomegranate bark and a method for preparing a flocculating agent. The method for extracting the natural flocculation active ingredients from the pomegranate bark includes the steps that 1, the pretreated pomegranate bark and ultra-pure water or distilled water or cooled boiling tap water are mixed according to the mass-volume ratio of 1:33.33-1:2.78; 2, particle residues in leaching liquor are filtered away to obtain a water extracting solution of the pomegranate bark. The method for preparing the flocculating agent by extracting the natural flocculation active ingredients from the pomegranate bark includes the steps that 1, the pretreated pomegranate bark and the ultra-pure water or the distilled water or the cooled boiling tap water are mixed according to the mass-volume ratio of 1:33.33-1:2.78; 2, the particle residues in the leaching liquor are filtered away to obtain the water extracting solution of the pomegranate bark; 3, 0.5-10 mL of the water extracting solution of the pomegranate bark is taken and diluted to 30 mL through the ultra-pure water or the distilled water or the cooled boiling tap water, and then the final flocculating agent is obtained.

Cultivation process for organic pomegranate
CN102726268
The invention discloses a cultivation process for an organic pomegranate. The cultivation process comprises field planting preparing, field planting, field drying, trimming and pruning, field managing, irrigating and filling water, bagging for vegetables and fruits, and disease and insect controlling. The cultivation process has a rational management specification, can rationally number and record milpa, plants, fertilization, irrigation, disease and insect control, pesticide usage time, harvesting date, planting variety, planting number, planting method, and the like; on the disease and insect control aspect, agricultural control, physical control and biological control are combined to effectively prevent diseases and insects, and a self-made organic fertilizer is adopted to fertilize, so that the fertilizer cost is saved, and the pollution of the organic fertilizer material to the environment is also reduced.

DESCRIPTION

The present invention discloses a process for organic pomegranate cultivation, including planting preparation, planting, will be dry, plastic trim, field management, irrigation and drainage water, fruit thinning and bagging pest control. The present invention is a reasonable management practices in the cultivated land, plants, fertilization, irrigation, pest prevention, pesticide use of time and date of harvest, cultivation, cultivation quantity, cultivation methods so that a reasonable number is recorded; in pest control on, the principle of agricultural control, physical and biological control combination, can effectively control pests and diseases; the use of homemade organic fertilizer in fertilization, saving fertilizer costs, but also reduce the pollution of organic fertilizer raw materials to the environment.

Background technique

Pomegranate pomegranate pomegranate families genus are deciduous shrubs or small trees, crown plexiform natural round shape, Roots brown, barren and more resistant to drought, fear of waterlogging, water demand growth season very much. Ripe pomegranate rind color Bright red or pink, often split, exposing the glistening jewel-like as grain, sweet and juicy, very loved by the people. Pomegranate cultivation but extensive management, low yields, such as fertilizer can not keep up, trim or not to trim unreasonable, Pest control is not timely unreasonable.

SUMMARY

Object of the present invention to overcome the deficiencies of the prior art, there is provided a process for the organic cultivation of pomegranate.

To achieve the above object, the present invention adopts the following technical scheme:

An organic pomegranate cultivation techniques, including planting preparation, planting, will be dry, plastic trim, field management, Row irrigation, fruit thinning and bagging pest control, in particular:

Planting Preparation: Setting line spacing and spacing according to terrain, pull dig planting pits, and then mixing organic fertilizer pit Soil, backfill compaction;

Colonization: straighten out the seeds, cut long roots, implanted pit backfill and fine soil, light pinch back mention seed Fill, do a tree plate along seed around, planting pouring enough water, cover and cover;

Set dry: after spring planting of seedlings after germination of new branches, cut the main branch, culture Celi branches, forming crown;

Plastic trim: Maintain the natural ecological crown, layered trimming, sawing off excess closely spaced branches, combined Pull support, vertical pressure, the crown form a variety of forms;

Field management: implementation of number management and fertilization management;

Irrigation and drainage water: Shupan ahead, irrigation, opened at the top of the cover Shupan irrigation, irrigation cover On the cover;

Thinning bagging: fruit be firmly secured, timely thinning, thinning Been dense fruit, put the fruit fruit bags;

Pest control: the agricultural control, physical control and biological pest control were combined.

Further technical solution is the spacing of 3 to 3.5 meters, the spacing of 2 to 3 meters, the Planting hole 70 cm deep wide 60㎝, if cultivated for the slopes, the planting of 100 to 120 per acre, if cultivated land To the ground, then planted per acre from 50 to 70, the use of organic manure pit every 50 kg.

Further technical solution, said Shupan 40㎝ wide, 20 cm deep, the covering comprises a plastic film, Straw in one or two.

Further technical solution is the second cut in the main branch refers to Jude Biao cut in 30 to 40 centimeters Go to the main branch, the branch training Celi 2-3.

Further technical solution is the use of weak individual hierarchical pruning prune and cultivate young branches empty chamber, Use topping foster long put crown, formed results Zhizu; crown is too large, heavy shears retracted, do not cut in line with scissors In addition, do not cut scissors yin yang, upright branches cut, do not cut the level of its side branches, cut overlapping interference sticks, do not cut the branches empty chamber Principle; the variety of forms including hollow tower, fan, umbrella shape.

Further technical solution is that the number of project management including planting, the plants, fertilization, irrigation Water, anti-pest, pesticide use time and date of harvest, cultivation, cultivation quantity, cultivation methods.

Further technical solution is the fertilization management projects include promoting fertilizers and reminders fruit fertilizer, promote the Fertilizers pomegranate harvest in late September, the organic fertilizer 800 ~ 1500kg / mu, homemade bio-organic fertilizer 200~ 300kg / acre, the pro-fertilization methods fertilizers are digging peripheral crown 40 to 50 cm wide and deep radial Ditch, promoting fertilizers and soil pit mixed uniformly applied into the trench, irrigation 2kg casing tramping; the fertilizer in the fruit catalyst 5 In late May or early June after the fruit firmly secured, reminders fruit fertilizer applied, depending on the vigor fertilization, the results of the amount of self-administered Health Organic fertilizer was 120 ~ 200kg / acre.

Further technical solution, said irrigation per plant 150 ~ 200kg / times, 25 to 30 days watering once.

Further technical solution is the fruit firmly secured in late May to mid-June on, the thinning Holding fruit leaves more than 300 to 400 leaves / fruit, fruit from 30 to 40 cm / fruit.

Further technical solution that includes the agricultural control fertilization and irrigation, thinning, drainage groove, Pruning, remove leaves and dig garden pests, including the use of physical control of the black light trap phototaxis Moths, insect repellent planting insecticidal chrysanthemum, including the protection of natural enemies of the biological control, promote the use of biological Pesticides, botanical pesticides, mineral pesticides pest control...

Example 1

An organic pomegranate cultivation techniques, including planting preparation, planting, will be dry, plastic trim, field management, Row irrigation, fruit thinning and bagging pest control, in particular:

Planting Preparation: Setting line spacing and spacing according to terrain, pull dig planting pits, and then mixing organic fertilizer pit Soil, backfill compaction;

Colonization: straighten out the seeds, cut long roots, implanted pit backfill and fine soil, light pinch back mention seed Fill, do a tree plate along seed around, planting pouring enough water, cover and cover;

Set dry: after spring planting of seedlings after germination of new branches, cut the main branch, culture Celi branches, forming crown;

Plastic trim: Maintain the natural ecological crown, layered trimming, sawing off excess closely spaced branches, combined Pull support, vertical pressure, the crown form a variety of forms;

Field management: implementation of number management and fertilization management;

Irrigation and drainage water: Shupan ahead, irrigation, opened at the top of the cover Shupan irrigation, irrigation cover On the cover;

Thinning bagging: fruit be firmly secured, timely thinning, thinning Been dense fruit, put the fruit fruit bags;

Pest control: the agricultural control, physical control and biological pest control were combined.

Further technical solution is the spacing of 3 m, the spacing is 2 meters, width of the planting pit 60Cm 70 cm deep, if cultivated for the slopes, the 110 acre planted, if the ground for cultivation, the per acre Planting 63, the organic fertilizer (composting manure) pit per 50 kg.

Further technical solution, said Shupan 40㎝ wide, 20 cm deep, the covering comprises a plastic film, Straw in one or two.

Further technical solution is to cut off the main branch of the second refers to the main Jude Biao cut 30 centimeters Branch, the branch training two Celi.

Further technical solution is the use of weak individual hierarchical pruning prune and cultivate young branches empty chamber, Use topping foster long put crown, formed results Zhizu; crown is too large, heavy shears retracted, do not cut in line with scissors In addition, do not cut scissors yin yang, upright branches cut, do not cut the level of its side branches, cut overlapping interference sticks, do not cut the branches empty chamber Principle; the variety of forms including hollow tower, fan, umbrella shape.

Further technical solution is that the number of project management including planting, the plants, fertilization, irrigation Water, anti-pest, pesticide use time and date of harvest, cultivation, cultivation quantity, cultivation methods.

Further technical solution is the fertilization management projects include promoting fertilizers and reminders fruit fertilizer, promote the Fertilizers in late September after the harvest pomegranate, organic manure (composting manure) 800kg / acre, homemade bio Organic fertilizer 200kg / acre (10 kg or organic fertilizer / plant, bio-organic fertilizer made 2 kg / plant), the pro-flower Fertilization fertilizer is a peripheral crown dig deep 40㎝ wide radial grooves, after promoting fertilizers and soil mix evenly applied into the pit Trench, irrigation 2kg casing tramping; the fertilizer is urging fruit in late May or early June fruit firmly secured, Shi reminders fruit fertilizer, depending on the vigor fertilization, the results of the amount of applied homemade bio-organic fertilizer 120kg / acre (or self-administered Health Of organic fertilizer 1.5kg / strain).

Further technical solution, said irrigation per plant 150kg / times, 25 days irrigation once.

Further technical solution is the fruit firmly secured in late May to mid-June on, the thinning Holding more than 300 fruit leaves leaf / fruit, fruit from 30 cm / fruit.

Further technical solution that includes the agricultural control fertilization and irrigation, thinning, drainage groove, Pruning, remove leaves and dig garden pests, including the use of physical control of the black light trap phototaxis Moths, insect repellent planting insecticidal chrysanthemum, including the protection of natural enemies of the biological control, promote the use of biological Pesticides, botanical pesticides, mineral pesticides pest control.

Example 2

An organic pomegranate cultivation techniques, including planting preparation, planting, will be dry, plastic trim, field management, Row irrigation, fruit thinning and bagging pest control, in particular:

Planting Preparation: Setting line spacing and spacing according to terrain, pull dig planting pits, and then mixing organic fertilizer pit Soil, backfill compaction;

Colonization: straighten out the seeds, cut long roots, implanted pit backfill and fine soil, light pinch back mention seed Fill, do a tree plate along seed around, planting pouring enough water, cover and cover;

Set dry: after spring planting of seedlings after germination of new branches, cut the main branch, culture Celi branches, forming crown;

Plastic trim: Maintain the natural ecological crown, layered trimming, sawing off excess closely spaced branches, combined Pull support, vertical pressure, the crown form a variety of forms;

Field management: implementation of number management and fertilization management;

Irrigation and drainage water: Shupan ahead, irrigation, opened at the top of the cover Shupan irrigation, irrigation cover On the cover;

Thinning bagging: fruit be firmly secured, timely thinning, thinning Been dense fruit, put the fruit fruit bags;

Pest control: the agricultural control, physical control and biological pest control were combined.

Further technical solution is the spacing of 3.5 m, the spacing is 3 m, the planting pit 60㎝ deep 70 cm wide, if cultivated for the slopes, the 100 acre planted, if the ground for cultivation, each Planting 50 acres, the organic fertilizer (composting manure) pit per 50 kg.

Further technical solution, said Shupan 40㎝ wide, 20 cm deep, the covering comprises a plastic film, Straw in one or two.

Further technical solution is to cut off the main branch of the second refers to the main Jude Biao cut 40 centimeters Branch, the branch training Celi 3.

Further technical solution is the use of weak individual hierarchical pruning prune and cultivate young branches empty chamber, Use topping foster long put crown, formed results Zhizu; crown is too large, heavy shears retracted, do not cut in line with scissors In addition, do not cut scissors yin yang, upright branches cut, do not cut the level of its side branches, cut overlapping interference sticks, do not cut the branches empty chamber Principle; the variety of forms including hollow tower, fan, umbrella shape.

Further technical solution is that the number of project management including planting, the plants, fertilization, irrigation Water, anti-pest, pesticide use time and date of harvest, cultivation, cultivation quantity, cultivation methods.

Further technical solution is the fertilization management projects include promoting fertilizers and reminders fruit fertilizer, promote the Fertilizers in late September after the harvest pomegranate, organic manure (composting manure) 1500kg / mu, homemade bio Organic fertilizer 300kg / acre (15 kg or organic fertilizer / plant, bio-organic fertilizer made 3 kg / plant), the pro-flower Fertilization fertilizer is a peripheral crown dig deep 40㎝ wide radial grooves, after promoting fertilizers and soil mix evenly applied into the pit Trench, irrigation 2kg casing tramping; the fertilizer is urging fruit in late May or early June fruit firmly secured, Shi reminders fruit fertilizer, depending on the vigor fertilization, the results of the amount of applied homemade bio-organic fertilizer 200kg / acre (or self-administered Health Of organic fertilizer 2.5kg / strain).

Further technical solution, said irrigation per plant 200kg / times, 30 days irrigation once.

Further technical solution is the fruit firmly secured in late May to mid-June on, the thinning Holding more than 400 fruit leaves leaf / fruit, fruit from 40 cm / fruit.

Further technical solution that includes the agricultural control fertilization and irrigation, thinning, drainage groove, Pruning, remove leaves and dig garden pests, including the use of physical control of the black light trap phototaxis Moths, insect repellent planting insecticidal chrysanthemum, including the protection of natural enemies of the biological control, promote the use of biological Pesticides, botanical pesticides, mineral pesticides pest control.

Example 3

An organic pomegranate cultivation techniques, including planting preparation, planting, will be dry, plastic trim, field management, Row irrigation, fruit thinning and bagging pest control, in particular:

Planting Preparation: Setting line spacing and spacing according to terrain, pull dig planting pits, and then mixing organic fertilizer pit Soil, backfill compaction;

Colonization: straighten out the seeds, cut long roots, implanted pit backfill and fine soil, light pinch back mention seed Fill, do a tree plate along seed around, planting pouring enough water, cover and cover;

Set dry: after spring planting of seedlings after germination of new branches, cut the main branch, culture Celi branches, forming crown;

Plastic trim: Maintain the natural ecological crown, layered trimming, sawing off excess closely spaced branches, combined Pull support, vertical pressure, the crown form a variety of forms;

Field management: implementation of number management and fertilization management;

Irrigation and drainage water: Shupan ahead, irrigation, opened at the top of the cover Shupan irrigation, irrigation cover On the cover;

Thinning bagging: fruit be firmly secured, timely thinning, thinning Been dense fruit, put the fruit fruit bags;

Pest control: the agricultural control, physical control and biological pest control were combined.

Further technical solution is the spacing of 3 m, the spacing is 2 meters, width of the planting pit 60Cm 70 cm deep, if cultivated for the slopes, the 120 acre planted, if the ground for cultivation, the per acre Planting, the organic fertilizer (composting manure) pit per 50 kg.

Further technical solution, said Shupan 40㎝ wide, 20 cm deep, the covering comprises a plastic film, Straw in one or two.

Further technical solution is to cut off the main branch of the second refers to the main Jude Biao cut 30 centimeters Branch, the branch training two Celi.

Further technical solution is the use of weak individual hierarchical pruning prune and cultivate young branches empty chamber, Use topping foster long put crown, formed results Zhizu; crown is too large, heavy shears retracted, do not cut in line with scissors In addition, do not cut scissors yin yang, upright branches cut, do not cut the level of its side branches, cut overlapping interference sticks, do not cut the branches empty chamber Principle; the variety of forms including hollow tower, fan, umbrella shape.

Further technical solution is that the number of project management including planting, the plants, fertilization, irrigation Water, anti-pest, pesticide use time and date of harvest, cultivation, cultivation quantity, cultivation methods.

Further technical solution is the fertilization management projects include promoting fertilizers and reminders fruit fertilizer, promote the Fertilizers in late September after the harvest pomegranate, organic manure (composting manure) 800kg / acre, homemade bio Organic fertilizer 200kg / acre (10 kg or organic fertilizer / plant, bio-organic fertilizer made 2 kg / plant), the pro-flower Fertilization fertilizer is a peripheral crown dig deep 40㎝ wide radial grooves, after promoting fertilizers and soil mix evenly applied into the pit Trench, irrigation 2kg casing tramping; the fertilizer is urging fruit in late May or early June fruit firmly secured, Shi reminders fruit fertilizer, depending on the vigor fertilization, the results of the amount of applied homemade bio-organic fertilizer 120kg / acre (or self-administered Health Of organic fertilizer 1.5kg / strain).

Further technical solution, said irrigation per plant 150kg / times, 25 days irrigation once.

Further technical solution is the fruit firmly secured in late May to mid-June on, the thinning Holding more than 300 fruit leaves leaf / fruit, fruit from 30 cm / fruit.

Further technical solution that includes the agricultural control fertilization and irrigation, thinning, drainage groove, Pruning, remove leaves and dig garden pests, including the use of physical control of the black light trap phototaxis Moths, insect repellent planting insecticidal chrysanthemum, including the protection of natural enemies of the biological control, promote the use of biological Pesticides, botanical pesticides, mineral pesticides pest control.

Although the invention herein with reference to the explanatory embodiment of the present invention has been described, but not limited to System of the present invention, it should be appreciated that those skilled in the art can design many other modifications and embodiments, These modifications and embodiments will fall within the scope and spirit of the principles disclosed herein within. more specifically, In the present application disclosure, the drawings and claims, the component parts of the subject combination arrangement and / Or layout many variations and modifications. In addition to variations and modifications in the component parts and / or layout of For skilled in the art, the use of the present invention will be apparent.

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Pomegranate Cultivation

  

http://nhb.gov.in/report_files/pomegranate/POMEGRANATE.htm

POMEGRANATE PRODUCTION TECHNOLOGY

Agro-climatic requirements

Pomegranate grows well under semi-arid conditions and can be grown upto an altitude of 500 m. above m.s.l.. It thrives well under hot, dry summer and cold winter provided irrigation facilities are available. The tree requires hot and dry climate during fruit development and ripening. Pomegranate tree is deciduous in areas of low winter temperature and an evergreen or partially deciduous in tropical and sub-tropical conditions. It can tolerate frost to a considerable extent in dormant stage, but is injured at temperature below - 110 C.

Well drained, sandy loan to deep loamy or alluvial soils is suitable for cultivation.

Varieties Cultivated
 
Important pomegranate varieties cultivated in India are Alandi or Vadki, Dholka, Kandhari, Kabul, Muskati Red, Paper Shelled, Spanish Ruby, Ganesh (GB I), G 137, P 23, P 26, Mridula, Aarakta, Jyoti, Ruby, IIHR Selection, Yercaud 1 and Co 1.

Land Preparation

Land is prepared by ploughing, harrowing, leveling and removing weeds.

Planting

Planting Material

Pomegranate is propagated vegetatively by cuttings, air layering or gootee.

Planting season

Air layering is usually done during the rainy season and also in November-December.  Planting is usually done in spring (February-March) and July-August in sub-tropical and tropical regions respectively.

Spacing

High density planting is adopted in temperate regions. A spacing of 5-6 m. in northern India and also in the plains of Deccan plateau is usually followed.  High density planting with a spacing gives 2-2.5 times more yield than that obtained when the normal planting distance of 5 X 5 m. is adopted. Farmers have adopted a spacing of 2.5 X 4.5 m. Closer spacing increases disease and pest incidence.

Planting Method

Square system of planting is mostly adopted. Planting distance is decided on the basis of soil type and climate. A spacing of 4-5 m. on marginal and very light soils is recommended.

Pits of 60 X 60 X 60 cm. size are dug (at a spacing of 5 cm. in square system) about a month prior to planting and kept open under the sun for a fortnight. About 50 g. of 5% BHC or carbaryl dust is dusted on the bottom and sides of the pits as a pre-caution against termites. The pits are filled with top soil mixed with 20 kg. farmyard manure and 1 kg. super phosphate. After filling the pit, watering is done to allow soil to settle down. Cuttings/air layers are then planted and staked. Irrigation is provided immediately after planting.

Nutrition

The recommended fertilizer dose is 600-700 g. N, 200-250 g. P2O5 and 200-250 g. K2O /tree/year. Application of 10 kg. farmyard manure and 75 g. ammonium sulphate to 5 year old tree annually is adequate, whereas application of 50 kg. farmyard manure and 3.5 kg. oil cake or 1 kg. sulphate of ammonia prior to flowering is ideal for healthy growth and fruiting.  The time of application is December/January for ambe bahar, May/June for Mrig bahar and October/November for hasthe bahar.

The basal dose of farmyard manure @ 25-40 cart-loads /ha. besides the recommended doses of N, P and K should be applied to non-bearing trees in 3 split doses coinciding with growth of flushes during January, June and September. Fruiting should be encouraged from fourth year onwards. Nitrogenous fertilizer is applied in two split doses starting at the time of first irrigation after bahar treatment and next at 3 weeks interval, whereas full dose of P and K should be applied at one time. These should be applied in a shallow circular trench below tree canopy not beyond a depth of 8-10 cm. After application, fertilizers are covered with top soil and irrigated.

Irrigation

First irrigation is provided in case of mrig bahar crop in the middle of May followed by regular irrigation till the monsoon sets in. Weekly irrigation in summers and that during winters at fortnightly intervals is recommended. The check basin system of irrigation is usually followed.

Drip Irrigation

The average annual water requirement through drip irrigation is 20 cm. Drip irrigation helps to save 44% on irrigation and 64% when sugarcane trash mulch is used. It also helps to increase the yield by 30-35%.

Training

Plants are trained on a single stem or in multi-stem system. Since the crops trained on single stem training system are more susceptible to pests viz. stem borer and shoot hole borer, the other system is more prevalent in the country.

Pruning

Pruning is not much required except for removal of ground suckers , water shoots, cross branches , dead and diseased twigs and also to give shape to the tree. A little thinning and pruning of old spurs is done to encourage growth of new ones.

Inter-cropping

Inter-cropping with low growing vegetables, pulses or green manure crops is beneficial. In arid regions, inter-cropping is possible only during the rainy season, whereas winter vegetables are feasible in irrigated areas.

Regulation of bearing

Pomegranate plants flower and provide fruits throughout the year in central and southern India.  Depending on patterns of precipitation, flowering can be induced during June-July (mrig bahar), September-October (hasta bahar) and January-February (ambe bahar). In areas having assured rainfall where precipitation is normally received in June and continues upto September, flowering in June is advantageous; where monsoon normally starts in August, flowering during August is beneficial. Areas having assured irrigation potential during April-May, flowering during January can be taken and where monsoon starts early and withdraws by September induction of flowering in October is possible. Considering comparable yields, prices and irrigation needs it is recommended that October cropping could be substituted for January flowering.

Plant Protection Measures

Insect Pests

Insect pests mostly observed are fruit borer, mealy bugs, aphids, white fly and fruit sucking moths. Spraying with dimethoate , deltamethrin or malathion etc. depending upon the type of pest infestation has been found to be effective in most cases.

Diseases

The main diseases reported are leaf spot and fruit rot. Application of Mancozeb (2g./l.) during rainy season in case of the former and application of Kavach (2g./l) and Carbendazim/Thiophanate methyl/Baycor/Benomyl (1g./l.) during September/October in case of the latter has been found to be effective in most cases.

Disorders

Fruit cracking is a serious disorder. This physiological disorder observed in young fruits is due to boron deficiency and that in fully grown fruits is mainly due to moisture imbalances. Tolerant varieties viz. Bedana Bose and Khog may be cultivated and in other cases spraying with calcium hydroxide soon after fruit set has been found to be beneficial.

Harvesting  and Yield

Pomegranate being a non-climacteric fruit should be picked when fully ripe. Pomegranate plants take 4-5 years to come into bearing.  Harvesting of immature or over mature fruits affects the quality of the fruits. The fruits become ready for picking 120-130 days after fruit set. The calyx at the distal end of the fruit gets closed on maturity. At maturity, the fruits turn yellowish-red and get suppressed on sides.

POST HARVEST MANAGEMENT

Grading

Fruits are graded on the basis of their weight, size and colour. The various grades are super, king, queen and prince-sized. Besides that, pomegranates are also graded into two grades- 12A and 12 B. Fruits of 12-A grade are generally preferred in southern and northern region.

Storage

Fruits can be stored in cold storage upto 2 months or 10 weeks at a temperature of 50 C. Longer storage should be at 100 C and 95% RH to avoid chilling injury and weight loss.

Packing

The size of packages varies according to the grade of the fruits. Corrugated fibre board boxes are mostly used. In a single box, 4-5 queen sized fruits, 12 prince sized and some of 12-A and 12-B grades may be packed. The white coloured boxes having 5 plies are generally used for export purpose, whereas red-coloured ones having 3 plies are used for domestic markets. The red coloured boxes are cheaper than white coloured ones. The cut pieces of waste paper are generally used as cushioning material.

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http://homeguides.sfgate.com/pomegranate-tree-cultivation-58444.html

Pomegranate Tree Cultivation

by

Jill Kokemuller

The pomegranate (Punica granatum) is a deciduous fruiting plant that grows as a tree or a shrub in U.S. Department of Agriculture plant hardiness zones 7 through 11. Pomegranates grow about 10 feet tall in shrub form or 20 feet tall as a tree. They are summer blooming, with red-orange flowers, round red fruit and dark green foliage.

Soil and Sun

Pomegranates will grow in sand and heavy clay soils, although the best results come from loamy soils. Sandy soils will decrease fruit production, while clay soils will result in paler fruit. Pomegranates are not tolerant of alkaline soils, needing a more acidic soil pH between 5.5 and 7.0. Peat moss or sulfur will make soil more acidic while dolomite lime will raise soil pH if your soil is outside the optimal range. Full sun is necessary to enable the plant to produce fruit.

Planting

Pomegranates should be planted as early as possible after the last spring frost to give them time to harden for the winter. Work soil before planting to loosen, and add amendments such as peat moss, compost or other organic matter to increase moisture and nutrients. Space shrubs 6 to 9 feet apart and trees 15 to 18 feet apart. Planting holes should be at least twice the width of the root ball and backfilled with the soil surface lower than the crown of the tree or shrub.

Water and Fertilizer

Weekly watering will keep the soil moisture adequate for fruit production. The trees need 50 to 60 inches of water per year. During late summer and fall especially, keep soil consistently moist, watering when the top inch of soil dries. Pomegranates are drought tolerant, but without sufficient water may not have much of a yield. Young trees need about 3 ounces of nitrogen applied in late winter and early spring. When the tree matures, nitrogen should be increased to 1/2 to 1 pound of nitrogen split between the two application times. Pomegranates do not benefit from extra potassium or phosphorus, although occasional zinc sprays may be necessary.

Pruning

The pomegranate will naturally grow as a bushy shrub-type plant. To train it into a tree form, choose the strongest trunk and remove the others, pruning off any suckers that form. At planting, cut plants back to 24 to 30 inches tall. Cut branches back 40 percent the following winter. In subsequent years, prune suckers and water sprouts as needed. An annual winter pruning will keep the tree or shrub neat and healthy. Remove dead, damaged or diseased branches, and cut individual branches back to an outfacing bud to shape the tree.

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http://homeguides.sfgate.com/protecting-pomegranates-bugs-30843.html

Protecting Pomegranates From Bugs

by

Amanda Flanigan

A native of Iran and India, the pomegranate (Punica granatum) is a shrub or small tree that grows in U.S. Department of Agriculture Plant Hardiness Zones 7 through 10. Pomegranates produce leather-skinned, unusually shaped fruit and showy blooms in hues of orange and red. This sun-loving fruit is generally easy to care for but may develop problems caused by bugs and insects. Reduce pest damage by protecting the pomegranate from bugs.

Pomegranate Bugs

According to the University of California, aphids are a widespread, serious problem affecting many plants including pomegranates. Aphids considered the most damaging to the plant and cotton aphids and pomegranate aphids, although they are usually only damaging in heavy infestations. Mealy bugs can infest pomegranates. These small, white-colored bugs have a cottonlike appearance and suck the juices from the foliage. Other bugs that can infest pomegranates are scales, whiteflies, leafrollers, thrips, beetles and various insect larvae. Leaf wilting, discoloration, curling, browning, yellowing and fruit damage are all signs of pest infestation on pomegranates.

Treatment

Most pests that feed on or damage pomegranates can be controlled without chemicals by introducing lady beetles, lacewings, beneficial parasites and predatory insects. These beneficial bugs eat the problematic pests and keep them under control. Applying chemical pesticides can throw off the balance of beneficial bugs and increase the numbers of unwanted bugs leading to a pest problem. Chemical pesticides should be used only as a last resort. Instead, use less toxic products, such as Neem oil, insecticidal soaps and horticultural oil.

Prevention

If your pomegranate is healthy, it will handle pest and diseases with few problems. Encourage a healthy pomegranate by keeping the ground under the plant free from debris. Fallen plant matter -- leaves, twigs, fruit and branches -- invites unwanted pests, fungus and diseases to the pomegranate and increases the possibility of problems. A water-stressed plant is likely to develop pest and disease problems, so water young pomegranates during dry periods. After they are established, pomegranates tolerate drought conditions. During the pomegranates’ first and second springs, apply about 2 to 4 ounces of all-purpose nitrogen fertilizer to the soil. After the second spring, fertilizer is not generally needed. However, applying organic compost around the pomegranate every year protects its roots and adds extra nutrients to the soil.

Considerations

The pomegranate is generally a pest- and disease-free plant that rarely sees anything more than cosmetic damage caused by pests. However, hungry deer have been known to feed on the pomegranate causing foliage and fruit damage. If deer are a potential problem, use commercial deer deterrents that keep the creatures away from the pomegranates without harming them. Water the pomegranates in the morning hours to allow the foliage to dry before the sun sets and decrease the chance of fungal diseases.

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http://homeguides.sfgate.com/natural-insect-sprays-pomegranate-trees-54288.html

Natural Insect Sprays for Pomegranate Trees

by

Amanda Flanigan

Producing brightly colored blooms and unusually shaped fruit, pomegranate trees (Punica granatum) are a stunning addition to your garden. These India and Iran native fruit trees grow in U.S. Department of Agriculture plant hardiness zones 8 through 11. Like other fruit trees, insects can attack the pomegranate, requiring immediate action to reduce potential damage. Chemical pesticides are a common tactic to combat these insects, but they can cause more harm than good. These toxic insecticides kill the unwanted pests as well as beneficial insects. This can lead to an even worse infestation than before. Instead, use natural insect sprays to protect the pomegranate tree from pests.

Pomegranate Insects

Aphids are a serious problem affecting pomegranates with pomegranate aphids and cotton aphids being the two species causing the most damage in heavy infestations, notes University of California Division of Agriculture and Natural Resources. Other insects that infest pomegranate trees include mealybugs, scale insects, whiteflies, thrips, leafroller, beetles and the larvae of various butterflies and moths. A common sign of an insect infestation is discoloration, yellowing and wilting of the leaves or damage to fruit.

Insecticidal Soap

Containing fatty acids which break down quickly and -- once dry -- losses the insect-controlling abilities, insecticidal soap is a more natural option to pest control on pomegranate trees. It controls an array of unwanted insects, including aphids, scales, whiteflies and leafhoppers. For insecticidal soap to effectively control pests, it must come into contact with the insects and should only be applied when the temperatures are between 40 and 90 degrees Fahrenheit, on a calm day.

Homemade Oil Spray

A homemade oil spray consists of 6 tablespoons of canola oil mixed with 1/4 cup of mild dish soap and 2 gallons of water. Never use dish soap that contains fragrances, degreasers or bleach. This natural insect spray controls various soft-bodied pests -- such as mites, scales and aphids -- that feed on the leaves of the pomegranate tree. Only use this spray when temperatures won’t reach 85 F and thoroughly coat the tops and undersides of the leaves with the concoction.

Bacterium Insecticide

Bacterium insecticide, such as Bacillus thuringiensis strain Kurstaki, targets the larvae of butterflies and moths that feed on various parts of the pomegranate tree. This nontoxic insecticide is applied as a foliar spray and poisons the larvae when they consume it, causing them to stop feeding. After a few days, the larvae die of starvation. Bacillus thuringiensis doesn’t harm predatory and beneficial insects and is safe to use around people and animals. It is nontoxic to bees, birds and fish.

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http://homeguides.sfgate.com/pomegranate-tree-problems-48471.html

Pomegranate Tree Problems

by

Teri Silver

Pomegranates (Punica granatum L.) include more than 500 known cultivars and produce medium-sized, leathery fruits containing tangy, juicy berries. Pomegranates grow in U.S. Department of Agriculture plant hardiness zones 7 to 11. The trees are relatively easy to grow and maintain, notes Clemson University Extension, but they are susceptible to fruit rots, mold, pests and wood damage. Pollination failure and inadequate sunlight may keep pomegranate trees from producing flowers and fruits.

Molds

Gray mold (Botrytis cinerea) causes harvested pomegranates to decay at a faster than normal rate. The pathogen develops spores on flower petals, which remain in the blooms until the fruits have ripened. It creates a gray coating of fungal spores that live in the fruit tissue. Pomegranates that are stored in damp, humid conditions exhibit the gray-coated surface as they decay. Blue-green mold (Penicillium spp.) may develop on pomegranate trees but it usually appears when fruits are stored. Symptoms are wet areas on fruit skins and bluish-green powdery mold. Protecting pomegranate fruits from gray or blue-green mold is not easy or economical, notes University of California Extension, but removing old fruit and dead branches will help reduce fungal spore production.

Fruit Rots

Pomegranate trees are susceptible to rots caused by pathogens that develop during flowering and fruit development. Alternaria fruit rot (Alternaria alternate) grows inside the fruits, causing them to become stunted and discolored. Rain and overly saturated soil cause the fungus to grow within the fruit. Pathogens live on dead plant and fruit debris during the tree’s dormancy. Aspergillus fruit rot (Aspergillus niger) is similar to Alternaria -- the fungus grows inside the flowering, growing fruits after rainfall. Fruit skins become pale but not dramatically different. Insects also damage diseased pomegranates; pest control may useful.

Pests

Pomegranate trees attract pomegranate butterflies (Virachola isocrates), which deposit larvae on flower buds and fruits. Caterpillars emerge, entering a bud through the flower’s calyx. Borers can cause major destruction to the tree’s fruit crop. In early summer, stem borers such as Pleuroplaconema or Ceuthospora phyllosticta lay eggs. The larvae cause twig dieback by digging into fruits and seeds -- trees may die if left untreated. Pomegranate tree leaves also attract mealybugs, scale insects, thrips, mites and whitefly. Deer enjoy chewing through foliage on pomegranate trees. Sawdust at the base of the tree indicates termite damage.

Leaf and Fruit Spots

Like many deciduous trees, pomegranate foliage develops leaf blotch and fruit spot. Infected leaves are pale green or yellow and have small reddish brown spots that turn to black. Premature leaf drop is possible. Applying copper fungicide three times a year helps to alleviate leaf spotting. Blight (Colletotrichum gloesporioidesl, Pseudocercospora punicae, Curvularia lunata and Cercospora punicae) creates water-soaked spots on pomegranate foliage, causing leaves to drop prematurely. Useful fungal treatments include Topsin-M, Sulfex, Difolatan and Dithane-M, advises the National Horticulture Board. Cercospora fruit spot (Cercospora spp.) features irregular brown to black lesions on pomegranates. Although it is best to destroy diseased fruits, Dithane or Captan may control the fungus, notes the NHB.

About Pomegranate Trees

Most pomegranate cultivars are deciduous trees that grow best in full sun and well-draining soil with a pH of 5.5 to 7.0. Fertilizing with a 10-10-10 mixture in March and July will help fruit production. Pomegranate trees can tolerate cold snaps but may be damaged if temperatures consistently dip below 10 degrees Fahrenheit. Most cultivars are drought-resistant. Light pruning encourages new growth, flowers and fruits. Most cultivars are self-pollinating but some varieties must be cross-bred to produce fruit. Planting suitable varieties close together helps trees develop pollinated blooms.

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http://homeguides.sfgate.com/kind-dormant-spray-can-spray-pomegranate-trees-55156.html

What Kind of Dormant Spray Can You Spray on Pomegranate Trees?

by

Amanda Flanigan

Pomegranate trees (Punica granatum) are an interesting addition to your backyard orchard, producing showy flowers and oddly-shaped edible fruit, growing in U.S. Department of Agriculture plant hardiness zones 7 through 10. Pomegranates benefit from sprays which act as a protective cover applied during the dormant period to eliminate diseases and insects that may be overwintering on the tree and to protect the pomegranate tree from future insect, fungal and bacterial infestations.

Horticultural Oil

Horticultural oil is made from refined petroleum and can be used anytime throughout the year. Horticultural oil works by smothering overwintering, soft-bodied pests. It controls scales, mealybugs, aphids, mites and their eggs but doesn’t treat fungal or bacterial diseases. Horticultural oil can also control certain caterpillar species, such as leafrollers and tent caterpillars, that overwinter as eggs. For horticultural to work effectively, the insects must be entirely coated with the oil, so to ensure the pests are controlled, you must apply a liberal amount of the spray to the pomegranate trees. Most horticultural oils have a low toxicity to people, mammals and beneficial predator insects and dissipate quickly, leaving little to no residue.

Homemade Dormant Oil

Homemade dormant oil consists of inexpensive and less toxic ingredients than commercial insecticides and allows you to adjust the recipe depending on how much or how little oil spray you need. In a large pot, bring one-half gallon of mineral oil, one-quarter gallon of water and one-half pound of oil-based soap to a boil, mixing continuously until the ingredients are thoroughly combined. This concentrated solution is then diluted at a ratio of 1 part solution to 20 parts water. Homemade dormant oil must be used immediately, since the ingredients have a tendency to separate. You should apply the homemade dormant oil on a day when temperatures are above 40 degrees Fahrenheit, thoroughly coating the pomegranate tree with the liquid.

Fixed Copper Fungicide

Applications of copper fungicide during the fruit tree’s dormant period will help prevent the spread and reduce infection of various fungal and bacterial diseases. Copper fungicides can prevent future leaf spot and fruit spot infections when applied during the pomegranate’s dormancy period. Depending on the disease you are trying to prevent, several applications of the fungicide may be required to protect newly emerged flowers and shoots or due to an abundance of rain.

Considerations

Dormant sprays are applied when the pomegranate trees enter their dormant stage after the tree has lost its leaves. Typically, fruit trees are sprayed with dormant oil in late November until the bud swell stage in February or March. Refrain from applying dormant sprays after flower buds open, as doing so increases the chance of damaging the pomegranate fruit. A University of California Agriculture and Natural Resources Master Gardener's Tip Sheet recommends applying dormant sprays after the rainy or foggy weather ends and never apply during periods of rain, fog or during freezing temperatures.

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http://homeguides.sfgate.com/companion-plants-pomegranates-39139.html

Companion Plants for Pomegranates

by

Nicki Wolf

Pomegranates, a fruit popular in Spanish and Middle Eastern cuisine, can add beauty and function to a garden. The tall, broad tree grows in U.S. Department of Agriculture hardiness zones 7 through 10, and produces edible fruit in the fall and orange-red flowers through the summer. You can help ensure healthy pomegranate trees in your yard by including companion plants nearby -- the right plantings attract beneficial insects, repel predators and enhance the look of your trees.

Herbs

The pomegranate requires bees for pollination; without proper pollination, your tree will not produce fruit. A variety of herbs can attract bees, including dill, cilantro, parsley and mint. Basil, thyme and summer savory also attract bees. It can be especially helpful to allow herbs to flower, because introducing a spectrum of color into your garden keeps bees returning to the area.

Flowers

Planting a bed of lavender flowers near a pomegranate tree attracts bees, as will beds with cosmos, coreopsis, zinnias and sunflowers. Many flowers help repel insects harmful to pomegranates as well. Aphids can inflict damage this fruit tree, resulting in rotten spots on fruit, blossom drop and ideal conditions for sooty mold infestation. Choose flowers that repel aphids, such as nasturtiums. You may also opt for flowers that attract beneficial insects that eat aphids, such as ladybugs and lacewings. These insects are especially attracted to daisies and Queen Anne's lace. Flowers may also be used to bring out the color of pomegranates -- purple passion vine flowers contrast, and jasmine's white flowers offset the pomegranate's orange-red flowers.

Vegetables

There are several vegetables you can plant near a pomegranate tree that make good companions and help you maintain a truly edible garden. Fennel and leaf celery draw ladybugs and lacewings, both of which will eat the aphids that might damage your pomegranate tree.

Fruit

Melons and berries attract bees, another insect beneficial for the pomegranate. Planting a fruit garden near your tree helps ensure proper pollination. Opt for a variegated Calamondin orange as a companion plant to the pomegranate for a visually stunning and edible fruit orchard. The orange blossoms also help to attract bees.

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http://homeguides.sfgate.com/kind-soil-pomegranates-40936.html

What Kind of Soil for Pomegranates?

by

Richard Corrigan

Pomegranate (Punica granatum L.) grows as a large shrub or small tree, and is known for its red, pulpy fruit. Though it is native to arid parts of Asia and the Middle East, pomegranates can also be grown in the United States, where they thrive in U.S. Department of Agriculture plant hardiness zones 8 to 10. Though they are highly adaptable, but planting pomegranates in the right type of soil is essential to a healthy fruit crop.

Soil Types

Pomegranates are able to adapt to a variety of soils, ranging from acidic sandy loam to alkaline calcareous soils and everything in between. In India, they have been known to grow in rocky gravel. The only soil that will not support pomegranates is heavy clay, because soils with excessive clay tend to present drainage problems.

Irrigation and Drainage

Drainage is one of the most important things to consider when you choose a location to grow pomegranate. Pomegranates demand well-drained soil, and they will not fare well in a location where the ground becomes wet and waterlogged. Irrigation is usually not necessary in most areas because pomegranates can withstand significant periods of drought. Fruiting can be diminished in drought conditions, so watering may be helpful if you are growing pomegranate primarily for its fruit.

Soil pH

Neutral to slightly acidic soil is best for pomegranate. They will still survive under considerably more acidic or alkaline conditions, but a pH range of 5.5 to 7.0 is best. If necessary, you can raise the pH of your soil by adding ground agricultural limestone, or lower it by incorporating some form of sulfur. A soil test kit, available at most garden stores, will tell you the pH as well as the nutrient content of your soil.

Fertilization

Pomegranates are fairly well adapted to growing in poor-quality soils, but moderate nutrient levels are better for fruit production. You can fertilize your trees with a small application of some form of nitrogen fertilizer during their first two springs. Fertilizer is usually not necessary after that, though adult trees can still benefit from a fresh layer of organic mulch applied annually.

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http://homeguides.sfgate.com/protect-pomegranates-squirrels-28618.html

How to Protect Pomegranates From Squirrels

by

B. Sinclair

Pomegranates are easy to grow and flourish in U.S. Department of Agriculture hardiness zones 7 through 10. Grown as a shrub or small tree, the plant produces nearly round fruit with red, leathery, tough skin that protects sacs filled with juicy, edible ruby seeds. Although relatively free of insect and pest problems, squirrels can pose a problem by eating the fruit before you have a chance to harvest it. Take protective measures to ensure you’ll have a chance to savor the thirst-quenching pomegranate fruit.

Cover the fruit with sandwich-size paper bags and seal with a twist tie. Check the fruit every few days for ripening. Pomegranates mature five to seven months after blooming. Harvest when the fruit makes a metallic sound when tapped.

Cover the pomegranates with netting made of 1/4- to 1/2-inch mesh plastic. For pomegranates grown as shrubs, build a frame out of PVC piping around the plant and lay the netting over it so that it doesn’t actually touch the shrub. Drape the netting over pomegranate trees and tie it around the trunk of the tree so squirrels can’t get inside.

Cut trees back and keep them away from roof overhangs, telephone lines or anything else squirrels can use to hop on your pomegranates. Also, consider letting your dog have full run of the yard. They are usually a good deterrent to squirrels.

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http://homeguides.sfgate.com/grow-pomegranate-trees-containers-43843.html

How to Grow Pomegranate Trees in Containers

by

Diane Watkins

Pomegranate trees (Punica granatum) are especially suited for growing in containers. The dwarf trees are easier to care for than a full-size tree, while still producing a good harvest of fruit. Full-size trees grow up to 20 feet tall. Dwarf varieties such as "State Fair" grow to be about 5 feet tall and "Nana" is only 2 to 3 feet tall when fully mature. They are suitable for outdoor growing in U.S. Department of Agriculture plant hardiness zones 7b to 10. Bring them indoors for the winter in colder climates. Expect your tree to produce fruit the second or third year after planting.

Allow the soil to dry slightly before watering your pomegranate tree. Water thoroughly, saturating the soil. Allow excess water to draing through the drain holes.

Fertilize the pomegranate tree in November, February and May with an ammonium sulfate fertilizer. Apply approximately 1 ounce per foot of the tree's height around the edge of the pot, keeping the fertilizer away from the trunk.

Prune the tree to remove dead and diseased branches and branches that rub or cross fruit or other branches. Shape the tree to a pleasing size and shape. Remove excess suckers, leaving a few only where you want new branches.

Place the pot in a place that gets direct sun for most of the day. Bring the tree indoors whenever temperatures drop below 40 degrees Fahrenheit. While trees in the ground can withstand temperatures much lower, container-grown trees are more susceptible to cold damage.

Dust the tree with sulfur in June and again in July if mites are present. Control aphids by spraying them with soapy water.

Bring the container indoors in the fall, increasing the time spent indoors by one or two hours daily. Transitioning the tree this way gives it time to adjust to lower light levels. Reverse the process in the spring, bringing it outdoors for a longer period each day.

Check the tree every spring for signs that the roots are becoming pot-bound. Repot the tree in a larger pot before the roots have a chance to out grow the pot. Place 1 inch or more of organic potting mix in the bottom of a pot that is at least 2 inches larger than the original container. Remove the pomegranate from its container and place it in the center of the pot. Fill in with soil around the outside. Tamp the soil down, making sure the trunk remains exposed.

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http://ressources.ciheam.org/om/pdf/a42/00600264.pdf

Cultivation of Pomegranate

by

A Blumenfeld

SUMMARY -- This paper describes various aspects of pomegranate cultivation. Tradition, consumption and production target, recommended soil, fertilisation ...

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http://www.yourarticlelibrary.com/cultivation/pomegranate-plant-cultivation-in-india/24715/

Pomegranate Plant Cultivation in India

By

Samiksha S

Botany Name: Punica Granatum L

Family: Punicaeae

Pomegranate is one of the esteemed table fruit. Its fruits are known for their sweetness and fine blend of acidity.

Its juice is cool and refreshing with medicinal value. The hardy nature of pomegranate makes it the choicest kitchen garden fruit. Highly acidic fruits with 7-8% acidity are used to make ‘anardana’ (daru).

Origin:

Pomegranate is native to Iran. It is commercially cultivated in Afgharustan, Baluchistan, Iraq, Spain, Russia, India, Pakistan, Myanmar, Chiria and Japan.

Area and Production:

In India, pomegranate production is 8.07 lacs MT from an area of 109.2 thousand hactare and Maharashtra state contributed a share of 75.09 per cent to the total India production of pomegranate. The major growing states one Maharashtra, Kaniataka, Gujarat, Andhra Pradesh. Chhatisgarh, Himachal Pradesh, Nagaland, Orissa, Rajasthan and Tamil Nadu.

Uses:

The sweet fruits are used as table purpose and acidic for making ‘anardana’ a condiment used for ‘chatru’ making. Juice is prepared and preserved for use. Wine can be prepared from sweet coloured juice. Sodium citrate/citric acid is prepared from very acidic fruits. Pomegranate shoot bark is used for body slimming.

The root or rind (peel) of the fruit is used to control dysentery, diarrhoea and worm killing in the intestines. Every part of the pomegranate plant is used for one or the other human disease. Dyes are prepared from flower petals. The edible portion (grains) of fruit contain upto 78 percent water, 5% fibre, 1.6% protein, 16-18% carbohydrates (sugars). Each 100 gram of fruit flesh may contain calcium 10 mg, phosphorus 70 mg. and sufficient quantity of iron, riboflavin and vitamin C.

Botany:

It grows as a shrub but can be trained on modified leader system as a small tree. Pomegranate is evergreen as well as deciduous. Shrub trained plants remain smaller than single stemmed trained as trees. It is hardy tree and can live over 40 years. The leaves have small petioles and are oval to lanceolate in shape. The shoots have thorns which originate deep from the wood. It bears very beautiful red coloured flowers.

Flowers may be sohtary, axillary or appear in clusters on short spurs. The calyx is persistent and tubular with 5-7 petals inserted in calyx. Ovary have many locules. Pomegranate fruit is special fruit botanically. The edible portion is aril juicy covering of seed. Both types of seed are present is different cultivars. The hard seeded and soft seeded cultivars.

Soil and Climate:

Pomegranate thrives well in semiarid conditions. It can adapt wide range of soil and climatic conditions. Deep loam to sandy loam soils are considered ideal. It can tolerate alkaline/ saline soils with 9.0 pH having lower EC than 0.5 mm hos/cm. It can grow in light soils but with assured irrigation. It requires hot and dry summers with cool winters.

It is tolerant to frost and freeze fairly well very high temperature in summers and too low in winters encourage fruit cracking. Pomegranate have both types of cultivars, i.e. some are deciduous in winter and others are evergreen. The tree requires hot and dry climate for the production of high quality pomegranates. The evergreen cultivars do not shed leaves in very cold winters.

Cultivars:

Mostly pomegranate cultivars originated as seedling selections. Some promising cultivars have also been developed through controlled hybridization. The recommended cultivars are area specific for example Kandhari and Nabha for north plains, Jaloreseedless and Jodhpur white in Rajasthan, Velludu in Tamil Nadu, Dholka in Gujrat, Basse-in-seedless, Bhagawa for Karnataka and Phule Arakta, Ganesh in Maharashtra.

Some of the promising cultivars are discussed below :

Ganesh: A seedling selection from Alandi made by Dr. G.S. Cheema at Pune. Plants bear profusly and regularly. It is evergreen bush and precocious. Its fruits are of medium size, with yellow coloured peel with pink blush. Average fruit size 350 g. Arils are white, transparent with little pink tings. Seeds are soft and can be eaten along with arils. The juice percentage on the basis of grain weight is 80% or more. The TSS of juice varies from 13% to 16% with very Uttle acidity (0.3 to 0.5%). Tree can bear Ambe, Mrig and Hast bahars. One can opt for any bahar as per requirement of the area.

Under North Indian conditions, it is beneficial to take Ambe-bahar, which ripens in August. To improve the fruit size, flower buds appearing after 15th April should be manually removed. Fruit yield is 12-15 tonnes per hectare.

Kandhari: Trees are deciduous, vigorous and upright growing. It is regular bearer with good yield per tree. It bears only Ambebahar (April-May flowering). Fruits are of big size with yellow coloured peel and red splash. Average fruit weight is 370 g Arils are light pinkish to deep pinkish, with semihard seeds, depending upon the dry weather and prevailing temperature. The grains have 77 percent juice on the basis of weight of grains. The juice have 13-15% TSS and well blended acidity (0.5 to 0.6%).

To get well-sized fruits of high quality, remove the flower bud appearing after 25th of April manually. There is a considerable variation in yield from area to area. However, on an average it can yield 10-12 tonnes per hectare.

Nabha: Tree growth is just like Kandhari and is also deciduous in nature. The rind of the fruit remain yellow in colour. The fruit size becomes larger than kandhari if thinning is done. The average fruit weight is 350 g. The grains are of white colour, with hard seeds. The grains have 75 percent juice with 13% TSS and 0.5% acidity. Yield 10 tonnes per hectare.

Assam Local: Under North Indian conditions tree grows upto 3 m high and 1.5 m spread. Tree is healthy. The fruits remain free from fungal/bacterial attack during the rainy season. It also bear only one crop. Average fruit weight is 220 g. The arils are small, hence juice percentage on the basis of weight of grains in 47 with 15% TSS and 0.6% acidity. Average yield per hectare only 7 tonnes.

Jyothi: Trees are dwarf and evergreen. Under North Indian conditions also it bear three crops, i.e. Ambe, Mrig and Hast bahars. Due to very low temperature in January the fruits of mrig bahar crack. Therefore it is beneficial to get only ambe bahar. Fruits remains small in size. Average fruit weight is 200 g. The rind develops a typical red colour with pinkish-reddish coloured arils. The juice percentage is 75 on the basis of grains weight. The juice have 17% TSS with very low acidity 0.3%. Average yield 8 tonnes per hectare (ambebahar only).

P.A.U Selection: It originated as a chance seedling from Ganesh and was evaluated along with other thirty cultivars at Ludhiana. The plants are as vigorous as Ganesh and evergreen. The flesh characters resemble Ganesh, but seeds are little hard. It bears big-sized fruits like kandhari. Average fruit weight 450 g. Juice percentage on the basis of grain weight is 71% with 13% TSS and 0.7 acidity. Average yield 8 tonnes per hectare.

Mridula: It is a soft seeded evergreen cultivar and it is cross between Gul Shah Red x Ganesh. It also bear three crops, but it is better to get Ambe-bahar only. The plants remain dwarf than Ganesh. The fruits have red coloured rind. The grains are also blood red in colour. If thinning is done in Ambe-bahar the average fruit weight is 240 grams. The juice is 78 percent on the basis of grain weight the TSS of juice 17-18% with only 0.3% acidity.

Basse-in-Seedless: As the name shows the grains are seedless. The plants are as vigorous as Ganesh and are evergreen in nature. The fruits are prone to fungal/bacterial attack during rains. The average fruit weight in ambebahar is 340g. The grains are light pinkish as of Ganesh. The juice is 80 percent with 14% TSS and 0.4% acidity.

Ruby: The plants are slow growing, evergreen and bear all the three crops. It is a hybrid from a 3 way cross between Ganesh X Kabul X Yercand and Gulsha Rose Pink. It bears red coloured fruits of small size with red coloured arils. Average fruit weight is 200 g. Juice percentage 80 with 15% TSS and 0.3% acidity.

Some other important cultivars are, G-137 a clonal selection from Ganesh; P-23 and P-26 seedling selections from Muscat; IIHR selection, it is a selection from open pollinated seedlings; Yercand-1 and Co-1.

Propagation:

Pomegranate should not be propagated through seeds, due to cross pollination, considerable variation occurs in the seedlings. Some workers say that pre-conditioning of shoots during June-July by girdling or etiolation increases the level of root promoting cofactors, which help in rooting of cuttings. The maturity of shoots used for cuttings plays a great role in the rooting of cuttings. For getting healthy and precocious plants, pomegranate should be propagated through hard wood cuttings.

One season old cuttings should be prepared during first week of December. The length of cuttings should be 20-25 cms. Treat the lower ends of these cuttings with 100 ppm of IBA (Indole butyric acid) solution for 24 hours, before planting in the nursery beds for rooting. After 10 to 15 days of planting, the beds may be applied 5 litres of chloropyriphos solution @ 10 ml/L. to one square meter of the bed. Repeat this treatment 20 days after to control white ants attack. Chloropyriphos solution should be applied with a shower over the cuttings slowly so that it reaches the rootzone apply light irrigation after the treatment.

The cuttings should be irrigated lightly at an interval of 15-10 days from December to March and 7 days after wards. Aphid attacks the newly emerging foliage, which should be controlled by spraying Rogor @ 2 ml/L. of water or Nuvacuron (monocrotophos) @ 1 ml/L. More than 80 percent cuttings root and produce healthy plants. Deciduous cultivars root better than the evergreens. To improve rooting of cuttings in evergreen cultivars take only one crop, i.e. ambebahar remove flowers from June onward.

Planting:

Pomegranate should be planted on square system of planting at a distance of 5m x 5m apart. Ever green cultivars may be planted at 4m x 4m distance. Prepare 1 meter diameter and 1 m deep pits one month prior to planting of pomegranate.

Thus one need 400 to 625 plants for planting an hectare. The best time for planting of pomegranate is December-January. The evergreen cultivars should be planted in December. Young plants are attacked by white ants in the pits also. So apply one litre solution of chloropyriphos @ 10 ml/L. of water after two months of planting to each plant.

Training:

Normally no importance is given to the training of pomegranate plants. Pomegranate is left as such to grow as a bush which is not desirable. Plants can be trained as single stemed tree (e.g. Kandhari etc.) and 2 to 4 stemed bushes (evergreen). Multi stemed trained plants create problems later on as the stems inter mingle with each other.

Pomegranates should be trained as single stemed to get strong scaffold system. No branch should be allowed to develop from ground level to 30 cm of trunk height. Head back the leader at 1 m height to force the scaffolds to develop. Select only 4-5 well placed scaffolds on all sides of the main trunk.

If the plants are trained on two or three stems, then the plant it self develop a balanced scaffold system. Remove only intermingling branches from the stems. It has been recorded that deciduous cultivars perform better as single stemed trees and evergreens with 2 to 3 stems.

Pruning:

Pomegranate do not require annual pruning. The fruit is borne on short spurs as well as in leaf axils and shoot apex. Remove criss-crossing and dried branches. Some branches may be headed back during December by removing 30 percent of the growth to encourage fresh growth.

Crop Regulation:

Pomegranate is a precocious bearer. Plants start bearing in the 3rd year of planting. It continues to bear for 30 years. The evergreen cultivars flower through out the year and bear three crops. To obtain good quality of fruits and high yield. Select only one bahar as suited to the area. Remove the flowers manually for rest the bahars. Deciduous cultivars only bear Ambe bahar (flowering April-May). The evergreens flower in Ambe bahar as well as Mrig bahar (July) and Hast bahar (October-November).

The inflorescence is cyme, due to abscission of side buds and persistence of central flower, flowering appears to be solitary. Keep only one fruit at a place to get well-sized fruits. The flowers should be manually removed after mid-April when sufficient number of fruits have already set. On a mature tree select 80-100 fruits only.

Pollination:

Pomegranate bears three types of flowers i.e. pure male with rudimentary ovary; hermaphrodite with medium style and hermaphrodite with well developed style. The percentage of these flowers varies from cultivar to cultivar and bahar to bahar. There is no shortage of pollen and pollinating agents. Sufficient number of fruits set from hermaphrodite (pin) flowers. Pollen is available at noon and stigma remains receptive for 2-3 days. Both cross and self pollination take place.

Irrigation:

Apply irrigation just after transplanting the plants in December. Light and frequent irrigations should be given at an interval of 20 to 10 days from January to May and at weekly interval from May to end July. No irrigation may be given if rains come in. Increase the interval of irrigation. After the harvest of fruit in August September. The interval of irrigation may be a month. Deciduous cultivars may not be applied any irrigation during December-January when leaf fall starts.

Intercropping:

Pomegranate have short juvenile period. Plants are also planted closely and remain bushy and spreading in nature, hence, no intercrop should be grown. When planting distance is more than 4m x 4m then intercrops can be grown for the first two to three years. Growing of vegetables and pulses should be preferred over rabi crops and fodder crops. If wheat is to be grown prepare separate irrigation system for irrigation to pomegranate plants during March-April.

Manuring and Fertilization:

Manuring should be done as per nature of evergreen or deciduous. If crop is not regulated then trees bear two crops and evergreen three crops Accordingly, fertilizer requirements increase as ^e crops in a year. As it has been recommended to take e-bahar in North India, to get good quality of fruit, fertilizer doses may be applied.

Add farm yard manure, super phosphate and muriate of potash in December-January. Mix well in plant basins. Urea should be split into two parts. Apply half in February at sprouting and half in May after fruit set.

However, different fertilizer doses are given in different areas. In general a higher dose has been recommended in literature i.e. 1.5 kg urea, 1 kg superphosphate and 400 grams of muriate of potash per mature plant. This dose may hold good where more than one crop per year is taken.

Weed Control:

Pomegranate is closely planted, hence use of a tractor to inter-cultivate is not desirable. Weeds should be manually removed by hoeing the basins once in December at the time of
fertilization and then again in May. The left out places may have some weeds/grass growing. If need be spray Grammoxone (paraquat) @ 6 ml/L of water during July. Keep the nozzle of spray close to the weed growth to avoid drift of the chemical.

Harvesting and Marketing:

Pomegranate is non climacteric fruit hence should be harvested when fully ripe. Pomegranate fruits become ready for harvesting after 4-5 months of fruit set. As the flowering continues for over two months, in the same way fruit ripening and harvesting also continue for a month or so. The fruits are harvested at full maturity, when rind has developed a typical colour of the respective cultivar. At this stage calyx at the tips dries up. Fruits should be harvested with the help of secateurs.
Grading:

Fruits are graded on the basis of fruit weight and size. For big markets following grades are followed:

Super sized : Weight more than 750g, withoutspots.
King sized : 500g – 750g without spots.
Queen sized : 400g – 500g without spots.
Prince sized  300g – 400g well coloured.
12-A: 250 – 300g without spots.
12-B : 250 – 300g with some spots.

Marketing:

Fruits are packed as per their grade. CFB boxes are used for packaging and fruits are cushioned with paper strips/cut pieces. The fruit is transported to the markets. For small markets fruits are packed in wooden boxes of 5 to 10 kg size.

The suitable temperature for cold storage of pomegranate fruits ranges between 5-10°C, with 90-95% relative humidity. Fruits stored at lower temperatures 0-3°C develop chilling injury.
Physiological Disorder:

Fruit Cracking:

Fruit cracking usually occurs in fruits of mrig and hast bahars. It is more serious in arid zone. Cracked fruits get affected by fungii and fruits become unfit for consumption. Mrig bahar fruits crack due to wide variation in humidity and soil moisture. Prolonged drought in ambe-habar causes peel hardening and when rain comes or irrigation is applied the arils sweet and peel cracks. Some cultivars are more prone to fruit cracking. Hast bahar fruits crack due to low temperature in December and January.

Cracking can be controlled by supplying irrigation at regular intervals, this will reduce the fluctuations in soil moisture. Planting of wind break around the orchard also help in the reduction of fruit cracking. Spray of borax @ 0.1 to 0.2 percent, spray of GAj 30-40 days before fruit ripening @ 15-20 ppm checks cracking of fruits. Cultivars Ganesh, Kandhari and PAU selection show little cracking in ambe bahar fruits.

Internal Break Down of Arils:

Blackening or dis-colouration of arils in ripened fruits is a serious malady throughout the pomegranate growing areas. All cultivars are affected by this disorder. No cause or remedial measure can be suggested at this stage. The fruit should be harvested at ripening stage without keeping it on the tree for more time than required.

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