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Liposomal Vitamin C
How to Make LVC :
https://www.youtube.com/watch?v=7ipZ9bDvt6k
How to Make Liposomal C - The Proper Way
Aaron Murakami
https://www.youtube.com/watch?v=b3lazZRyW9c
https://www.youtube.com/watch?v=4edcwF4fxOQ
https://www.youtube.com/watch?v=ejMSZOV7gMA
https://www.youtube.com/watch?v=DuZcOMhQehk
https://www.youtube.com/watch?v=uDO6j2sUAVs
https://www.youtube.com/watch?v=gfiig6mxw6Q
ES2105973
Liposomal composition for cellular regeneration of the skin.
Liposomal composition for cellular regeneration of the skin, consisting of a suspension of liposomes with a size of 75 to 300 mm which encapsulate each of the active principles glycolic acid, vitamin C and vitamin E. The composition comprises: Content of active principle Liposomal glycolic acid 5.0-30.0% 0.100-0.600% Liposomal vitamin C 5.0-30.0% 0.250-1.500% Liposomal vitamin E 0.0025-0.0100% Excipient made up to 100 ml
This invention relates to a composition that regenerates the intercellular tissue of the epidermis that stimulates the natural renewal capacity of the skin.
The outermost layer of the skin, called the stratum corneum, forms the permeable epidermal barrier that regulates the water content of the skin.
Morphologically, the stratum corneum is made up of two well-differentiated structures: corneocytes and the intercellular matrix.
Corneocytes are anucleated cells with a high keratin content.
They originate in the stratobasal layer of the epidermis through a differentiation process called cornification.
They are removed by detachment from the surface of the skin, through the natural process of peeling, and are replaced by new cells.
The peeling or removal of dead cells from the stratum corneum is the natural process of constant renewal of the epidermis.
With the passage of time or in prematurely aged skin, this process slows down; so that the cohesion of these dead cells increases and their elimination is slower, the regeneration capacity of young skin decreases, the thickness of the stratum corneum increases, and it is lost. elasticity and moisturizing and wrinkles appear.
It would therefore be convenient to have a means to compensate for this decrease in epidermal functioning.
Logically, such a medium should provide the natural structural constituents of the skin, non-allergens and contributors to increasing its flexibility, such as glycolic acid, procollagen vitamins and restorative principles.
Alpha-hydroxy acids (A.H.A.) or fruit acids are organic compounds of natural origin, characterized by having in their molecule a carboxyl function and a hydroxyl group in an adjacent position. Their properties are derived from the presence and position of these two functions.
They come from natural products, mainly fruit, sugar cane and milk.
Glycolic acid is the alpha-hydroxy acid with the smallest molecular size, containing two carbon atoms.
Its small size facilitates skin absorption and improves effectiveness.
The A.H.A. Applied topically at low concentrations, they act in the innermost layers of the stratocornea, reducing the cohesion of the corneocytes by interfering with the ionic bonds and hydrogen bonds that hold them together. As a consequence, the external corneocytes detach in a way that is imperceptible to the naked eye.
They restore the skin's natural capacity for renewal.
The result of this reactivation is an epidermis of young cells and a decrease in the thickness of the stratum corneum.
The skin is much more elastic, fine, silky and velvety, the face is firmed and illuminated, small furrows and expression lines disappear, and wrinkles are attenuated.
This manifest regenerative effect is further enhanced by the rejuvenating action of A.H.A. since they stimulate the biosynthesis of glycosaminoglycans, collagen, elastin and reticulin, essential components of the dermis and responsible for eradicating small wrinkles.
By facilitating the progressive elimination of worm accumulations on the skin surface, A.H.A. also have a lightening and depigmenting effect on dark spots.
They are beneficial for any type of skin, even oily and seborrheic skin, since their exfoliating effect eliminates obstructions that prevent the correct drainage of the pilosebaceous follicle, the source of comedone formation.
It also reduces acne marks.
They vitalize the skin, they are not toxic, irritating, teratogenic, or photosensitizing.
Vitamin C is a water-soluble substance that, when applied to the skin, has the following effects:
Anti-radical: protects cell membranes from free radical attacks.
Anti-inflammatory and skin decongestant.
Procollagen effect: estimates the synthesis of collagen and glycosaminoglycans.
Regulates the acid-base balance of the skin.
Vitamin E is a fat-soluble vitamin essential for the human body. It is involved in numerous metabolic and functional processes as well as in the care and protection of the skin.
From a dermatological point of view, its activity is based on its extraordinary antioxidant properties, so that it protects cellular structures from the harmful effect of free radicals generated in countless physiological and biological processes and mainly responsible for cellular aging.
Vitamin E thus contributes to protecting and maintaining the integrity of cell membranes and the stability of epidermal structures, preventing aging and cellular deterioration.
Vitamin E strengthens the skin's defenses and makes it resistant to environmental aggressions, including ultraviolet rays.
Vitamin E also has an anti-inflammatory effect that provides rest and comfort to the skin.
It has been proven, however, that the simple application of the active ingredients indicated above would imply the use of large quantities of them and this would not translate into optimal effects either since a good part of the active ingredients would not be absorbed into the skin and, therefore , would not reach the lower layers of the skin, so its action would be partial and superficial, not exempt from toxic effects for the outermost layers of the skin.
It has now been discovered that the application of liposomal techniques for the transport of the aforementioned active ingredients results in a high moisturizing and restoring efficiency of the skin's natural capacity to maintain its optimal level of regeneration.
Liposomes are spherical vesicles whose interior is occupied by an aqueous cavity and whose envelope consists of a variable number of bimolecular layers of the phospholipid or sphingolipid type of natural origin.
Its size is variable, depending on the number of layers it contains, although the optimal size is 100-200 nm and the unilamellar structure, that is, a single bimolecular layer.
The liposome membrane has a composition very similar to that of cell membranes.
Because they are biologically compatible with the body's cells, they do not cause dermatological or tolerance problems, nor toxicity, they are totally safe.
The main property of liposomes is their ability to simultaneously encapsulate water-soluble substances and fat-soluble substances.
So that water-soluble substances are included, along with water, in the aqueous spaces inside the spheres, while any fat-soluble molecule, added to the solvent during the formation of the vesicle, is incorporated into the lipid bilayer.
They transport both types of substances in an aqueous medium.
When liposomes are applied to the surface of the skin, they quickly penetrate through the intercellular spaces to the innermost layers of the stratocornea and in some cases to the dermis.
Due to their chemical composition, analogous to the cell membrane, they come into contact with the cells and slowly release the encapsulated active ingredients, so that they progressively diffuse as the concentration of active ingredient in the external phase decreases.
This osmotic mechanism prolongs the action of the encapsulated substances and increases their effectiveness, while protecting the encapsulated molecules so that they reach the place of action intact.
Its main components, phospholipids and water, are natural constituents of the skin.
So, liposomes guarantee the supply of water to the epidermis and dermis, while preventing its loss due to the regenerative action of the epidermal barrier provided by phospholipids.
This results in increased hydration and flexibility of the skin.
These properties are added to those provided by the active ingredients carried in the liposome.
Without the targeted technology of liposomes, a much larger amount of active ingredient would be needed and its penetration into the skin would not be as effective since a good part, especially in hydrophilic molecules, would not be absorbed.
It is considered that the absorption of liposomed active ingredients in the skin is multiplied by a factor of 5 to 10 in the epidermis and 10 to 15 in the dermis, in relation to the same molecules without liposomes.
Considering the same effectiveness of action, the presence of liposomes allows the amount of active ingredient to be reduced, also reducing its possible side effects.
Consequently, and in accordance with the invention, a suspension composition is provided composed of liposomes that serve as vehicles for the transport of the active ingredients: glycolic acid, vitamin C and vitamin E, whose liposomes, when applied to the skin, penetrate through the interstices of the intercellular matrix, transporting glycolic acid, vitamin C and vitamin E throughout the stratum corneum and the epidermis, releasing them in contact with the cells.
Specifically, the invention provides a skin regenerating composition composed of a suspension of liposomes with a size of 75 to 300 nm that encapsulate each of the active ingredients: glycolic acid, vitamin C and vitamin E, characterized in that it comprises:
Content in active ingredientLiposomed glycolic acid5.0-30.0%0.100-0.600%Liposomed vitamin C5.0-39.0%0.250-1.500%Liposomed vitamin E0.0025-0.0100%Excipient c.s.p. 100ml
The following example of preparation of the liposomal composition of the invention is offered below, whose example must be considered only illustrative and in no way limiting the scope of the invention.
Example
Pharmaceutical grade soy lecithin (phosphatidylcholine content: at least 92%) is dissolved in ethanol (96%, DAB 10), thus preparing solution I.
Next, and depending on the desired final concentration, the lipophilic agent (vitamin E) is dissolved in solution I.
Also depending on the desired final concentration, the hydrophilic agents (sodium salt of ascorbic acid and sodium salt of glycolic acid) are dissolved in water and, if necessary, an amphiphilic surfactant is added in a concentration of less than 1.4%, thus preparing solution II.
All these stages are carried out under aseptic conditions.
Finally, solutions I and II are combined, mixed thoroughly, diluted to the final concentration with sterile water, adjusted to the desired pH value and filtered under sterile conditions.
According to the skin absorption tests carried out with these liposomes, the skin absorption, in extent and depth, and therefore the effectiveness of an active ingredient is 10 to 15 times greater when administered in liposomal form than when applied without liposomes.
In this way, low doses of active ingredients administered in liposomes achieve high efficiency of action, while preventing toxicity and cellular "accustomization" due to excess product.
It is also important to highlight the beneficial effect of phospholipids, components of liposomes, in the hydration and regeneration of the skin, which potentializes the effectiveness of the active ingredients.
WO2024144526
ANTI-AGEING LIPOSOMAL AND ETHOSOMAL FACIAL CARE FORMULAS
The present invention relates to a gel-type skin care product which enables to deliver retinol and vitamin C into the skin upon being encapsulated within vesicular delivery systems and to create an anti-ageing effect.
Background of the Invention
The skin consists of three main layers: epidermis, dermis and hypodermis. Epidermis is the outermost layer of the skin and it is further divided into four layers in itself as well. These layers are stratum corneum (SC), stratum granulosum, stratum spinosum and stratum basalis.
Startum basale is the living and germinative layer of the epidermis. This single-row layer consists of keratinocytes which are basic cells; melanocytes which are dendritic cells in different proportions according to the region where they are located, Langerhans cells in a proportion between 1-4%, cells such as migratory macrophages and lymphocytes. Keratinocytes are formed by means of mitosis in the stratum basalis and undergo a change by differentiating until they reach the SC where they will transform into comeocytes. Cells, which progressed to the granular layer and the SC, have become cells being surrounded by a cornified membrane and comprising keratohyalin. Epidermal lipids are located among these cells. The unique structure and composition of the SC forms the basis of its barrier function. With its strong barrier characteristic, the SC creates a tissue fragment which limits the penetration of active substances from the skin and diffusion into the skin following a topical application. By means of the acid mantle layer created by sebum and intercellular lipids (pH 4-6 depending on the location), SC also helps to protect the skin by controlling the number of microorganisms.
Dermis is a layer which has a thickness of 1-4 mm and consists of fibrous proteins such as collagen, elastin, fibrinolectin and glycosaminoglycans, salts and water. In contrast to the epidermis, the dermis has an extensive vascular network and also lymph vessels and nerve tissue and nerve endings. Although they are epidermal structures, appendages of the skin such as hair follicles, sebaceous glands and sweat glands are also structures located in the dermis.
The hypodermis, which is known as subcutaneous tissue, acts as a support layer for epidermis and dermis. In addition to the dermal layer, the hypodermis is also a part of the tissue from which some skin problems originate, cellulite for example.
Since human skin has a characteristic of the stratum corneum barrier, which is the uppermost layer of the epidermis, the outer surface of the epidermis layer; it has a task of maintaining the haemostatic balance between the external environment and the body. The amount of moisture contained in the upper and the lower skin and the transepidermal water loss are affected by external factors. As a result of aging caused by both internal and external factors, the water loss of the skin increases, the elastic structure of the skin deteriorates and fine and coarse wrinkles begin to occur.
The most important cause of ageing based on external influences is exposure to sunlight, particularly UVA. The matrix, which is the main structure of the subcutis, consists of proteins such as chondrotin sulphate, dermatan sulphate, keratin sulphate, heparan sulphate and heparin produced by fibroblasts, and proteoglycans, glycoperoteins, glycosaminoglycans, water and hyaluronic acid. A flexible structure is also formed by means of fibrous protein structures such as collagen and elastin distributed within the matrix. Matrix proteins and fibrous structures deteriorate and their amounts reduce during aging. Smoking, sleep disorders, stress, air pollution, gases arising from traffic pollution such as NO2, in particular sun rays (especially UV rays, blue light band), are the external factors affecting skin ageing.
Decrease of protein synthesis, impairment in protein transcription, telomere shortening in time, glycosylation, DNA damage, lipid peroxidation, apoptosis are effective internal processes included in the aging process.
Anti-ageing products ensure that more dermal proteins are produced and enzymes such as collagenase, elastase and hyaluronidase -which enable to break down dermal proteins- are inhibited by basically increasing the activity of fibroblasts in the dermis. In the upper skin (epidermis), they enable to the epidermis to become thick and be renewed by increasing the proliferation of keratinocyte.
There are different routes for transition of active substance through the skin when topical products are applied on the skin. 99% of the products applied through the skin pass through the skin by transdermal route. 1% of the products pass through hair or sebaceous follicles and less than 0.1% through sweat glands, into the skin. Lipophilic components can pass through sebaceous glands, whereas polar components can pass through sweat glands in small amounts. The passage of the active substance from the outer layer of the skin, namely the SC, is the most important step of transdermal delivery. The passage of the active substance from the epidermis to the lower layers by means of passive diffusion is the second step. The passage of the active substance from the epidermis to the dermis through diffusion is an important penetration step. Topical products are not expected to take part in the systemic circulation, they are expected to be locally effective. The SC is the main barrier of the skin and while active molecules pass through it by means of passive diffusion, other factors play an important role as well; for example the size of the molecule and lipid solubility are important. Nanocapsules, which are one of the new vesicular delivery systems with a structure very similar to the structure of the cell membrane, are structures which enable the passage of the active substance from the SC.
Liposomes and ethosomes can be cited as an example of vesicle delivery systems that support the passage of products into the skin. Liposomes consist of an aqueous phase located innermostly in the structures and one or more lipid layers and form a vesicle. Ethosomes are vesicular delivery systems like liposomes and they are known as flexible liposomes and the aqueous phase included inside the vesicle consists of a mixture of water and alcohol.
Although currently used anti-ageing products comprise some vesicular delivery systems, both lipid-soluble and water-soluble active substances are not included in these vesicles together. In the state of the art, there are no anti-ageing cream formulations which comprises a vesicular delivery system containing a combination of retinol and vitamin C in the same liposome or ethosome.
Therefore, there is need for a facial care gel which enables to deliver retinol and vitamin C into the skin upon being encapsulated within vesicular delivery systems together and to create an anti-ageing effect.
The Korean patent document no. KR20150005928, an application included in the state of the art, discloses a cosmetic composition.
Summary of the Invention
An objective of the present invention is to realize a skin care gel which enables to deliver retinol and vitamin C under the skin upon being encapsulated within vesicular delivery systems and to create an anti-ageing effect. Another objective of the present invention is to realize an anti-ageing skin care gel which can deliver both hydrophilic and lipophilic active ingredients by using biocompatible vesicular systems.
Detailed Description of the Invention
The inventive skin care gel for creating an anti-ageing effect upon being encapsulated within vesicular delivery systems comprises:
Phosphatidylcholine which is used in the preparation of vesicular delivery systems; water which is used as a solvent; vitamin E used which is used as an antioxidant agent;
Retinol which enables to increase the synthesis of collagen and elastin in photo-aged skin, increases the synthesis of proteins and glycosamine glycones composing the dermis matrix, and also has anti-aging effects by inhibiting metalloproteinase enzymes;
Vitamin C which protects the skin against the negative effects of the sun, acts as a cofactor for the hydroxylase of prolyl and lysyl -key enzymes that cross-link and stabilize collagen fibres so as to perform collagen biosynthesis- stabilizes pro-collagen messenger RNA that regulates Type I and III collagen synthesis by directly activating transcription factors in collagen synthesis, and creates an anti-aging effect; phenoxyethanol which is used as a preservative; and a formulation of acrylate cross polymer which is used as a gel -forming agent if a gel-shaped end product is to be made.
In one embodiment of the invention, a gel having an anti-wrinkle effect is obtained by encapsulating the said gel formulation within a vesicle delivery system that is a liposome. In another embodiment of the invention, a gel with an anti -wrinkle effect is obtained with the vesicular delivery system obtained by adding ethanol, that acts as a solvent, into the said gel formulation and then encapsulating it in ethosomes.
The content of the gel prepared by encapsulating the inventive liposome within a vesicle delivery system comprises 1-10% phosphatidylcholine, 50-90% water, 0.01-3% vitamin E, 0.001-0.5% retinol, 0.1-5% vitamin C, 0.2-1% phenoxyethanol and 0.2-5% acrylate cross polymer.
The content of the gel prepared by encapsulating the inventive ethosome within a vesicle delivery system comprises 1-10% phosphatidylcholine, 50-80% water, 15- 50% ethanol, 0.01-3% vitamin E, 0.001-0.5% retinol, 0.1-5% vitamin C, 0.2-1% phenoxyethanol and 0.2-5% acrylate cross polymer.
All of the components included in the inventive gel content, except water, are taken into a container and then mixed with a mechanical mixer at optimum 300 RPM. Thereafter, it is mixed for another 5-30 minutes by adding water into the mixture slowly at 30°C. Finally, the size of the vesicles is reduced by circulating the mixture for 1-5 times with a microfludizer and the vesicular concentrate is added into the final product such as gel, cream with emulsion consistency and then is made ready by being mixed until it becomes homogeneous.
The inventive gel content provides an anti-aging product which will eliminate the effects that cause signs of aging and ensure reconstruction of epidermis and dermis, by encapsulating the combination of retinol and vitamin C -that is topically applied- within vesicular systems in the form of liposomes or ethosomes and passing it through the outer layer of the skin without being damaged at the highest efficiency and then reaching the epidermis and dermis layers.
The inventive gels are effective when they are used by being applied regularly to persons with skin surface wrinkles, particularly before sleeping at night, after cleansing. If desired, they can also be used in combination with other day care products.
When retinol and Vitamin C are encapsulated within an ethosome or liposome vesicle delivery system and then their penetration into the desired layer of the skin is ensured, the vesicles can exhibit their effectiveness at the highest efficiency by opening in the layer they reach.
Encapsulation of the inventive gel (cream) content into an ethosome vesicle delivery system facilitates the delivery of both hydrophilic and lipophilic active ingredients into deep skin layers. In general, the hydrophilic active ingredients included in the gel remain in the aqueous core; while the amphiphilic and lipophilic active ingredients interact intensively with the lipid bilayer of the vesicle. The capability to incorporate higher concentrations of ethanol into the ethosomes provides the active ingredients with better solubility in ethanol to be delivered subcutaneously. In addition, since ethosomes can be smaller in size and also can be deformed due to their elastic structure, their subcutaneous passage becomes easier. Their small size is another advantage. They increase the bioavailability of active substances by being very good delivery systems due to their structure and size advantages.
Encapsulation of the inventive gel content into the liposome vesicle delivery system enables to deliver high amounts of active substance into the skin layers through the skin by passing the SC barrier. Providing bioavailability of both hydrophilic and lipophilic substances via transdermal route enables to obtain a better dose/effect ratio, to perform a controlled drug release, to increase penetration and to penetrate into the skin more by means of its cationic nature.
RO138170
HYDROSCAR - HYBRID HYDRO-EMULSION WITH HEALING ACTION
The invention relates to a process for preparing a functionalized dermato-cosmetic product with healing action on cutaneous wounds to be used in healing process mediation therapy. According to the invention, the process consists of the following stages: a. formulation of a liposomal suspension incorporating cannabidiol (CBD) prepared by solubilization of 10% soy lecithin in 50% hemp oil, under continuous stirring, incorporating 3% CBD, 0.5% glycerin, 0.1% vitamin E and water, with suspension homogenization and sonication, in inert atmosphere, b. formulation of a hydro-emulsifying base by heating, to 70°C, a phase A consisting of 1...10% marine collagen, 0.5% glycerin, 0.3% usual rheological agents and 61.7% purified water, including phase B which consists of liposomal suspension with CBD and an emulsified phase C consisting of 0.5% hyaluronic acid-cholesterol conjugate, 0.1% vitamin E and 0.9% usual preservative, from which a biopolymeric hydrogel-type product results, representing a non-invasive life improving alternative for patients with inflammatory background.
Current state of knowledge
The skin is a multi-functional organ and an important part of the innate immune system, which fulfills multiple roles, through which it contributes to the body's homeostasis: protective, sensory barrier function, regulation of temperature and blood pressure, vital metabolic processes (Abdallah, Mijouin, and Pichon 2017; Wickett and Visscher 2006) Taking into account the fact that the skin can be damaged by a multitude of chemical agents, thermal, mechanical factors, or as a result of the reactions they generate, the epidermal regeneration process is a versatile one that ensures the restoration the barrier function of the skin to avoid blood loss and/or infection and restore mechanical and physiological properties.
The healing of the epidermis after skin injuries is thus an imperfect process, which inevitably leads to the formation of scars as the skin restores its integrity (Galliot et al. 2017; Sidgwick, McGeorge, and Bayat 2015b).
Prolonged wound healing or the body's excessive responses to injury prevent normal healing, leading to scarring that requires careful management.
Therefore, one of the main reasons for rapid intervention at the level of skin wounds in order to heal them is to restore the barrier function and prevent infections.
These interventions must take into account the complex interactions between a multitude of cells and mediators.
In this context, stimulating the rapid transition from the inflammatory to the proliferative stage of wound repair is the main goal that the HydroSCAR product addresses in order to provide solutions with clinical value.
It is known from the specialized literature that superficial, small and clean wounds are usually associated with a short duration of the hemostatic and inflammatory phase, with the formation of the fibrin plug that closes the wound, with the elimination of minor amounts of cellular debris.
Intervention as quickly as possible at the affected site reduces the complications associated with infections and the formation of microbial biofilms, especially taking into account the fact that re-epithelialization begins already a few hours after injury by activating keratinocytes that are redirected to the wound bed made up of fibrin, fibronectin and vitronectin (A , A, and G 2007; WC, and MW 2013; Sorg et al. 2017 of repair is performed from the superficial layers to the base with the goal of rapid wound closure to prevent further fluid loss or infection.
The prerequisite for effective epithelization is the presence of an adequate extracellular matrix that facilitates keratinocyte migration.
Starting from these structural specificities of the skin, the cosmetics industry constantly aims at the creation of new and effective products, endowed with an improved biological activity and with the controlled release of some active substances at the level of the skin.
The design and formulation of dermato-cosmetic products is currently experiencing an extensive transformation, especially thanks to modern formulation techniques, supported by complex studies on the bioavailability of active principles, but also due to conceptual changes regarding the physiological effects of cosmetics, similar to those expected from a medicine .
The formulation of a cosmetic product must be optimized so that the active principle reaches the therapeutic target in the concentration required for maximum effectiveness, but with minimal absorption on the skin.
Formulation technology, especially that of regenerative cosmetics, approaches this field almost similarly to the formulation of topical pharmaceutical products, the art of formulation being an approach with a pronounced interdisciplinary applicative character, which appeals to principles of physics, chemistry-physics, colloid chemistry, analytical chemistry and technology.
The success of a new formulation consists in establishing an optimal balance between the creativity in the formulation and the applied scientific bases (the multiple interactions that occur in the processes of absorption, distribution and metabolism of the active principles, the concentrations with cytotoxic effect of the active principles established by in vitro tests, etc.
(Wiechers et al. 2004). These new technologies control the kinetics of target release and the duration of therapeutic activity (Patravale and Mandawgade 2008b; Vikas et al. 2018).
In recent years, the research and development of advanced regenerating cosmetic formulations has focused on the creation of controlled release transport systems.
These types of formulations, known as target transport systems, have several advantages compared to conventional preparations. The ability to control release active principles confer improved efficacy, reduced toxicity and increased target group compliance (Watkins et al. 2015). Targeted release allows controlling the place, time, frequency and cadence of active substance release. Among the controlled release systems are colloidal systems (Patravale and Mandawgade 2008a; Algin Yapar, Evren, and Yapar n.d. ; Puglia and Bonina 2012) similar to those addressed by the present project.
The topical and transdermal application of the active principles must be safe and non-toxic, so as not to cause irritation.
The preservation of the active principles is essential during the formulation, storage and application of the final products, because they can be unstable and sensitive to variations in temperature, pH, light and in the presence of oxidizing agents. Thus, encapsulation is a necessity for protecting the active ingredients in relation to external factors and for mediating their targeted and controlled release (Ammala 2013).
The problem that the invention solves, technical solutions, advantages
The use of product bases such as biopolymeric hydrogels (with collagen and hyaluronic acid) is currently considered one of the most effective strategies in mediating the healing process.
Such products have the advantage of favoring the healing of wounds by maintaining optimal values of humidity and the permanence of micromolecular species with a physiological role.
In addition, collagen hydrogels, for example, represent effective matrices for the controlled and easy incorporation of numerous pharmacologically active compounds, with the aim of obtaining (multi)functional products intended for the effective treatment of conditions or side effects involving the denaturation of the epidermal barrier.
Encapsulation is a technique by which an active agent (in our case cannabidiol) is immobilized in another substance (coating - liposome).
In this way, particles are obtained whose dimensions cover a wide spectrum, starting from a few nanometers and up to a few millimeters, being adapted to the purpose of the encapsulation and the substances used.
Until now, in the databases of the European Medicines Agency (EMA) and the Food and Drug Administration (FDA), only 14 types of liposomal products have been authorized for therapeutic use.
It should be noted that this list excludes generic drugs, lipid complexes (eg Abelcet, Amphotec and Onpattro) and liposomal products nationally authorized in Europe.
Doxil (Doxorubicin HCl Liposome Injectable) was the first liposomal product approved by the FDA in 1995. Of these marketed products, 43% were approved before 2000, and 57% were approved before 2010. Although the focus of systemic therapies aimed at the applicability of liposomes se focuses mainly on cancer therapy, however, there is scientific interest in other areas as well, such as infection therapy, anesthesia, vaccines, lung diseases, and photodynamic therapy. The dosage forms accepted by the EMA and the FDA mainly focus on sterile suspensions and lyophilized powders whose routes of administration include intravenous infusion, intramuscular and intrathecal injection, epidural, local infiltration, and oral inhalation (Liu, Chen, and Zhang 2022).
In recent years, many discoveries have been made about how cannabinoids such as cannabidiol (CBD) could benefit the skin.
This knowledge led the scientific community to go one step further and investigate the healing potential of CBD (Palmieri, Laurino, and Vadala 2019). Cannabinoids or phytocannabinoids, e.g. (CBD), cannabichromene (CBC), cannabigerol (CBG), cannabinol (CBN) and delta (9)-tetrahydrocannabinol (Δ9-THC), are the active constituents of the Cannabis sativa plant and manifest their activity by binding to certain receptors: the cannabinoid receptors CB1 and CB2 (fig 2) (found in neurons, glial cells and, respectively, in spleen tissue (Van Sickle et al. 2005; Munro, Thomas, and Abu-Shaar 1993), are part of the class of G proteins (proteins that bind guanine nucleotides) and their activation results in the inhibition of adenylate cyclase activity (Di Marzo, Bifulco, and De Petrocellis 2004; Iversen 2003; Baker et al. 2003). As just one of the many active compounds that cannabis produces, CBD has entered the mainstream in recent years due to its lack of intoxicating, adverse effects.
Also, since it is possible to extract CBD from industrial hemp, it is not subject to the regulations regarding prohibited, psychoactive substances. The compound works by acting on the endocannabinoid system (ECS), responsible for regulating a wide range of biological functions. It is present in most human tissues, including the skin, stimulating the growth and development of cells and being involved in the regulation of the inflammatory response. The ECS appears to have an important, although little known, role in skin health and wound healing. Current research suggests that CBD could play a role in improving wound healing and reducing scar formation, as well as reducing inflammation (Palmieri, Laurino, and Vadala 2019; Toth et al. 2019; Zurier and Burstein 2016) being a non-invasive alternative to improve the quality of life of patients with an inflammatory background.
Another aspect that the invention aims to solve is related to the transdermal bioavailability of cannabidiol.
Transdermal administration avoids the effect of primary metabolism that is associated with the oral route and thus could increase the bioavailability of CBD compared to the oral route. However, other factors may decrease absorption, such as the possibility of local irritation and low skin penetration of chemicals that are highly lipophilic, such as CBD, which is estimated to have a log Kow/log P of about 6 (Tabboon, Pongjanyakul, et Limpongsa 2022). Several studies have demonstrated that the topical route of CBD administration represents a significant therapeutic tool; CBD plasma concentrations were observed in animal models after the application of a transdermal gel (Liput et al. 2013; Lodzki et al. 2003; Paudel et al. 2010); CBD has been proposed for several therapeutic indications related to the skin (Sophie A. Millar et al. 2018; Sophie Anne Millar et al. 2020); for its ability to modulate the skin's inflammatory response in several dermatological conditions, including psoriasis, atopic dermatitis and acne (Bruni et al. 2018; Sophie Anne Millar et al. 2020; Sheriff et al. 2020).
Cosmetic-grade products are commercially available from many manufacturers (Kiehl's, Josie Maran, Ildi Pekar, etc.), but for medical use (especially in the treatment of scars), there are not as many options available. According to the FDA (Food and Drug Administration), to date, no drug containing CBD has met the applicable FDA requirements to be legally marketed for over-the-counter use (“U.S. Food and Drug Administration,” n.d.). To bridge the gap between science and clinical treatment, there is an unmet clinical need for effective treatments for skin scarring, particularly to address the inflammation, pruritus, dryness, and redness commonly cited by patients as the factors most affecting them. (Sidgwick, McGeorge, and Bayat 2015a).
Example 1.
Technological operations necessary to obtain the HydroSCAR product a) Formulation of the liposomal suspension incorporating CBD
The cannabidiol used was provided by the Absolute Essential Oils production company, it being obtained from a controlled culture of Cannabis sativa (THC values < 0.2%), the Futura 75 variety, through an extraction, decarboxylation, purification and concentration process that led to obtaining a product of similar purity to the analytical standards.
The protocol for obtaining the liposomal suspension uses in the formulation as a base 10% soy lecithin, dissolved in 50% hemp oil, at 30°C, under continuous stirring (min. 1000 rpm).
This is followed by the incorporation of 3% CBD by continuous stirring at high mixing speed (4000 rpm), glycerin (0.5%), vitamin E (0.1%) and water.
The resulting suspension was homogenized at high mixing speeds for one hour at 30°C. This was followed by sonication for 10 minutes at 20 kHz (1 minute sonication with 1 minute break).
The reactions took place in the inert atmosphere, to avoid oxidation for 30 h.
b) Formulation of the conjugate consisting of hyaluronic acid and cholesterol
The synthesis of the conjugate formed from hyaluronic acid (HA) and cholesterol (Chol) was made using Cholesterol (Cho) from Sigma-Aldrich, sodium hyaluronate 93% - Lipo™ Hyaluronic acid - commercially available product, having a molecular weight of 1.1 - 2.2 x 10<sup>6</sup>Daltons) from Vantage Specialty Chemicals™ and distilled water.
The sample preparation process consisted of making stock solutions of hyaluronic acid and cholesterol in distilled water at a concentration of 10mg/mL. Homogenization was achieved using an ultrasound bath, each solution was sonicated for one minute, with a 2-minute break between each sonication step, the whole process being repeated three times. Three different concentrations of cholesterol were prepared (25μΜ, 20μΜ and 5μΜ respectively), the final volume of the prepared samples was 0.5mL with a final hyaluronic acid concentration of 5mg/mL. The reactions took place in an inert atmosphere to avoid oxidation for 30 h. The samples were subjected to lyophilization in order to obtain the complexation products.
c) Formulation of the hydro-emulsifying base
The hydro-emulsifying base was obtained by heating to 70°C the components of phase A consisting of marine collagen (1-10%), glycerin (0.5%), rheological agents (0.3%) and purified water (61.7%).
Followed by the inclusion in phase A of phase B - liposomal suspension with CBD. Phase C consisting of the hyaluronic acid-cholesterol conjugate (0.5%), vitamin E (0.1%) and preservative (0.9%) was emulsified at 70°C, then cooled to 30°C and incorporated into the base (A/B).
Example 2.
Demonstration of antimicrobial properties
The quantitative determination of the antimicrobial activity was carried out by the microdilution method.
Bacterial suspensions of standardized strains of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Candida albicans, Klebsiela pneumoniae, Staphylococcus epidermidis and Streptococcus agalactiae were made from young cultures of 18-24 h, grown on Müller-Hinton medium and adjusted to a density of 1 .5*10<sup>8</sup>UFC/mL, using the 0.5 McFarland nephelometric standard.
Binary dilutions were obtained in a liquid medium (Müller-Hinton broth), and the minimum inhibitory concentration (MIC) is the minimum compound concentration capable of inhibiting microbial growth, macroscopically assessed by the absence of turbidity of the culture medium, after seeding and 24h incubation.
The final volume in each well was 150μL, and the volume of microbial suspension 15μL. The absorbance was measured at 620 nm. In order to evaluate the influence of the HidroSCAR product and its main active ingredient - CBD, on the development of microbial biofilms, the following materials and experimental methods were used: the microbial cells represented by the strains Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739 and Candida albicans ATCC 10321 were cultured in the presence of nutrient broth samples, were incubated at 37 °C for 24 hours. The plates containing the samples of interest were then emptied and washed twice with A.F.S; followed by fixing the adhered cells with methanol for 5 minutes. The methanol solution was removed by inversion; followed by staining the adhered cells with 1% alkaline crystal violet solution for 15 minutes. The staining solution was removed and the wells were washed under running tap water. The formed microbial biofilms were resuspended in acid
33% acetic acid (by bubbling), and the intensity of the colored suspension was evaluated by measuring the absorbance at 490 nm.
Example 3.
Demonstration of biocompatibility in vitro on cellular models
The evaluation of the degree of biocompatibility was carried out with the help of the MTT and LDH tests. The MTT test is a viability test that allows the quantitative evaluation of live cells in culture.
The compound MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] is permeable to living cell membranes.
After metabolizing the MTT compound, formazan crystals soluble in isopropanol are formed. It results in a solution (purple color) with an optical density that can be read at 550 nm. For the MTT assay, the rest of the culture medium was removed from the 24-well osteoblast assay plate. The surface was washed with PBS to remove any traces of fetal bovine serum, which inhibited the MTT compound. A 1 mg/ml MTT solution was prepared and each sample was incubated in the presence of 1 ml MTT solution for 4 h at 37oC and 5% CO2. To be able to read the results, the formed formazan crystals were solubilized with isopropanol. The resulting purple solution was read on a spectrophotometer at 550 nm. The intensity of the color is directly proportional to the number of living cells in the sample. For the LDH assay, the rest of the culture medium was removed from the 24-well osteoblast assay plate. The surface was washed with PBS, then the LDH solution was added according to the instructions in the kit and the samples were incubated for 30 minutes at 37oC with 5% CO2.
The resulting deep red solution was read using a spectrophotometer (Elisa reader) at 492 nm.
Example 4.
Evaluation of the release of nitric oxides in the cellular environment
Quantitative determination of nitrites and nitrates released in the cellular environment was performed on human fibroblasts, HDF line. Cells cultured as previously described (cytotoxicity test) were treated with 3 concentrations of HidroSCAR product and CBD for 24 hours.
The supernatant was collected and used for nitrite and nitrate determination using the Griess Nitric Oxide Reagent Phase Assay Kit (Thermo Scientific, Waltham, MA, USA) following the manufacturer's instructions.
Example 5.
Determination of immunotoxicity and capacity to stimulate cell proliferation
Example 6.
Evaluation of the induction of apoptosis and necrosis
Determination of early and late apoptosis was performed by flow cytometry using FACS Alexa cytometer and an Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit (Thermo Scientific, USA).
In total, 10,000 cells were analyzed per measurement.
Data were analyzed using FlowJo 10.0.7 software (Treestar Inc., Ashland, USA).
HEK-293 T cells were cultured in DMEM medium supplemented with 10% FBS under standard cell culture conditions (37°C, 5% CO2).
Cell count was assessed using a hemocytometer.
Trypsin/EDTA (0.05%, Invitrogen) was used to detach cells from the flask for either passaging, heat shock induction, or flow cytometry.
To induce apoptosis, cells were exposed to collagen scaffolds at room temperature (22°C) for a period of 24 hours.
HEK-293 T cells (3 × 106) were stained using the Alexa Fluor® 488 Annexin V/Dead Cell Apoptosis Kit according to the manufacturer's instructions.
The stained cells were diluted in annexin V binding buffer. The suspended cells were used to perform the flow cytometry assay.
Example 7.
Demonstration of cell proliferation and migration capacity - wound healing
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JP2022063034
VITAMIN C ABSORPTION PROMOTER AND COMPOSITION FOR VITAMIN C SUPPLY
To provide a new functional material that uses plant extract to help the absorption of vitamin C, which is a bioavailable substance.SOLUTION: The present invention discloses: a vitamin C absorption promoter containing Dracocephalum moldavica extract as an active ingredient; or a composition for vitamin C supply containing Dracocephalum moldavica extract and vitamin C, wherein vitamin C contained in the composition may be provided in at least one form selected from (1) ascorbic acid and/or a salt thereof, (2) ascorbic acid coated with oil and fat and/or a salt thereof, and (3) liposomal ascorbic acid and/or a salt thereof. The composition may be food and drink.
Vitamin C is a water-soluble vitamin that was historically discovered as a preventive factor for scurvy, and recent research has shown that it has various biological functions, such as immune-boosting, anti-stress, antioxidant, anti-cancer, anti-allergic, anti-viral, and antibacterial effects (see Non-Patent Documents 1 and 2).
Vitamin C is also known to play an important role in the biosynthesis of collagen, which is widely distributed in connective tissues such as skin, bones, and tendons, as well as in various tissues and organs (see Non-Patent Document 3). Furthermore, it is known to have the effect of suppressing melanin production (see Non-Patent Document 3). Vitamin C is also listed as a nutritional component in functional foods, and its functional claims include that it helps maintain healthy skin and mucous membranes and is a nutrient with antioxidant properties (food labeling standards based on the Food Labeling Act).
On the other hand, focusing on such an important role of vitamin C, Patent Document 1 discloses a nutritional composition for regulating the bioavailability of vitamin C. Furthermore, Patent Document 2 discloses a vitamin C transporter production promoter.
Akira Murata, "Various actions and mechanisms of action of vitamin C" Nippon Nogeikagaku Kaishi (1990) Vol.64, No.12, pp.1843-1845.
"Natural Medicine Database" (http://jahfic.or.jp/news/new-book-nmcdj-2017) J. M. Pullar, et al., "The Roles of Vitamin C in Skin Health" Nutrients 2017, 9, 866; doi:10.3390/nu9080866.
Patent Publication No. 2008-533078 International Publication No. 2007/094312
If new functional ingredients that aid in the absorption of bioavailable vitamin C were provided, consumers would have a wider range of choices.
In addition, if the active ingredient is a natural product or its primary processed product, the risk of side effects is thought to be low.
Therefore, an object of the present invention is to provide a new functional material that utilizes a plant extract to aid in the absorption of bioavailable vitamin C.
In order to achieve the above-mentioned object, the inventors conducted various studies and discovered that an extract of dragon's head has the effect of promoting the absorption of vitamin C, which led to the completion of the present invention.
That is, in a first aspect, the present invention provides a vitamin C absorption enhancer containing an extract of dragon's head as an active ingredient.
The vitamin C absorption enhancer of the present invention preferably has the effect of increasing the expression level of vitamin C transporter.
In the vitamin C absorption enhancer of the present invention, the vitamin C transporter is preferably SVCT2.
The vitamin C absorption enhancer of the present invention preferably has the effect of increasing the amount of vitamin C permeation in an absorbency evaluation system using a Caco-2 cell layer.
In a second aspect, the present invention provides a vitamin C supplement composition comprising an extract of dragon's head and vitamin C.
In the vitamin C supplement composition of the present invention, the vitamin C is preferably provided in at least one form selected from (1) ascorbic acid and/or a salt thereof, (2) oil-coated ascorbic acid and/or a salt thereof, and (3) liposomal ascorbic acid and/or a salt thereof.
In the vitamin C supplement composition of the present invention, the vitamin C is preferably provided in the form of (1) ascorbic acid and/or a salt thereof, (2) oil-coated ascorbic acid and/or a salt thereof, and (3) liposomal ascorbic acid and/or a salt thereof.
In the vitamin C supplementation composition of the present invention, it is preferable that the vitamin C is contained in an amount of 5 to 2,000 parts by mass, calculated as ascorbic acid, per 1 part by mass of the dragon head extract in terms of the dry matter content.
The vitamin C supplement composition of the present invention preferably contains, as a single intake amount, 100 to 2000 mg of vitamin C calculated as ascorbic acid, and 1 to 20 mg of the dragon's head extract calculated as dry matter.
The vitamin C supplement composition of the present invention is preferably in the form of a food or drink.
In a third aspect, the present invention provides use of an extract of dragon's head for the manufacture of a vitamin C absorption enhancer.
In a fourth aspect, the present invention provides the use of an extract of dragon's head and vitamin C for the manufacture of a vitamin C supplement composition.
In a fifth aspect, the present invention provides a method for promoting vitamin C absorption, which comprises administering an extract of dragon's head together with vitamin C.
According to the present invention, a new functional material can be provided that utilizes an extract of dragon's head to aid in the absorption of bioavailable vitamin C.
1 is a graph showing the results of examining the mRNA expression level of vitamin C transporter (SVCT2) in Caco-2 cells when each test sample was applied to the cells in Test Example 1.
1 is a graph showing the results of investigating the amount of ascorbic acid that permeated through a Caco-2 cell layer when each test sample was applied to the cell layer in Test Example 2.
This is a graph showing the results of investigating the change in blood vitamin C levels when each test meal was ingested in Test Example 3, and is a graph showing the results of measuring serum ascorbic acid concentrations 0, 1, 2, 3, 4, 6, 8, or 10 hours after ingesting each test meal before the start of the test or 4 weeks after the start of the test.
This is a graph showing the results of investigating the change in blood vitamin C levels when each test meal was ingested in Test Example 3, and is a graph showing the results of calculating the area under the concentration-time curve (AUC) from the results in Figure 3.
In this specification, the term "dragon head" has the same meaning as that generally understood by those skilled in the art, and specifically refers to a plant of the Lamiaceae family, Dracocephalum genus (scientific name: Dracocephalum moldavica, also known as Hozakimu Sharindou).
) meaning included.
Dragon's head is an annual plant native to Asia and Europe that blooms in tiers of blue flowers from early summer to summer. It is widely cultivated as an herb and for decorative purposes, and is easily available.
In this specification, the term "vitamin C" has the same meaning as that generally understood by those skilled in the art, and specifically includes ascorbic acid and its salts.
The ascorbic acid may be the L-form, which is known to be highly physiologically active, or may be a DL mixture containing it.
Examples of the salt of ascorbic acid include sodium salt, potassium salt, calcium salt, and the like.
Ascorbic acid and/or a salt thereof may be coated with an oil or fat, or may be incorporated into a liposome.
As for the oil-coated ascorbic acid and/or a salt thereof, preparation methods are disclosed in, for example, JP-A-2001-17093 and JP-A-2004-123636, and therefore those in a form obtained by these methods or methods similar to these methods can be used.
Alternatively, commercially available products such as "VC-80R" (manufactured by NOF Corp.), "Ascorbic Acid-95R" (manufactured by NOF Corp.), and "Vitacoat C-95" (manufactured by Yokohama Oil Industries Co., Ltd.) may be used.
As for liposomal ascorbic acid and/or a salt thereof, preparation methods are disclosed in, for example, JP-T-2004-529163A and JP-A-2012-140395A, and therefore those in a form obtained by these methods or methods similar thereto can be used.
Alternatively, commercially available products such as "Vitamin C Zeal" (manufactured by Aurea Biolabs) and "Lypo-C [Lipocapsule] Vitamin C" (manufactured by Spic) may be used.
In this specification, the term "vitamin C transporter" has the same meaning as that generally understood by those skilled in the art. Specifically, it is a protein that penetrates the lipid bilayer membrane of cells in biological tissues and transports extracellular vitamin C into the cells. Two subtypes, SVCT1 (gene name: SLC23Al) and SVCT2 (gene name: SLC23A2), are known in humans.
In general, SVCT1 is expressed mainly in the liver, lungs, kidneys, intestines, and skin, and has a higher maximum rate of vitamin C uptake than SVCT2, so it is thought to play a role in maintaining a constant vitamin C concentration in the body.
On the other hand, SVCT2 is mainly expressed in the brain, eyes, liver, etc., and has a higher affinity for vitamin C than SVCT1, so it is thought to play a role in efficiently absorbing low concentrations of vitamin C.
The dragon's head extract used in the present invention is not particularly limited as long as it contains the components of the dragon's head plant, and there are no particular limitations on the form of the extract.
In addition, there are no particular limitations on the type, cultivation region, cultivation period, etc. of the cultivated dragon head used as the raw material.
For convenient preparation, a primary extract obtained by adding a solvent to the raw material and treating it for a certain period of time can be used.
The obtained primary extract may be subjected to processing such as filtration, concentration, purification and separation using ion exchange resins or liquid chromatography, and freeze-drying, as necessary.
The parts of the plant used as the raw material include the leaves, stems, flowers, roots, or a mixture of two or more of these parts, the whole plant except the roots, or the whole plant including the roots.
Of these parts, it is preferable to use the entire plant except for the roots in terms of the content of dragon head ingredients and yield.
The extraction can be carried out by a conventionally known method.
For example, the raw plant material, one or more parts of dragon's head, or its juice, or a processed product obtained by concentrating, drying, powdering, etc., can be soaked or heated under reflux for a predetermined period of time in a suitable solvent at low or high temperature.
The solvent used for extraction is not particularly limited, and any solvent usually used for plant extraction can be selected and used.
Examples include water, alcohols, glycols, ketones, esters, ethers, and halogenated carbons.
Examples of the alcohols include ethanol, methanol, and propanol.
Examples of glycols include ethylene glycol, diethylene glycol, butylene glycol, propylene glycol, and glycerin.
Examples of the ketones include acetone and methyl ethyl ketone. Examples of the esters include ethyl acetate, propyl acetate, and ethyl formate. The ethers include diethyl ether and the like. Examples of halogenated carbons include chloroform and dichloromethane. The solvent used for extraction may be a mixture of two or more of these solvents, or the products obtained by performing extraction with two or more solvents may be mixed to obtain an extract, or extraction with two or more solvents may be performed sequentially.
The extract may be prepared in any form, such as a liquid, suspension, paste, gel, oil, or emulsion.
Alternatively, it may be dried to a solid state, or may be dried by freeze-drying, spray-drying, or the like to a powder state. In this case, a drying aid such as dextrin or an emulsifier such as sucrose fatty acid ester may be added. The extract may also be in the form of dragon head extract powder.
As for the dragon's head extract that can be prepared as described above, plant flavonoids can be mentioned as components for standardizing its quality.
For example, Reference 1 (Chao Wu et al., "Phytochemical composition profile and space-time accumulation of secondary metabolites for Dracocephalum moldavica Linn. via UPLC-Q/TOF-MS and HPLC-DAD method" Biomedical Chromatography, 2020, DOI: 10.1002 /bmc.4865.) and Reference 2 (Ozek G, et al., “Preparative Capillary GC for Characterization of Five Dracocephalum Essential Oils from Mongolia, and their Mosquito Larvicidal Activity. According to Nat Prod Commun, 2016, PMID: 30549618, dragon head extract contains (E)-nerolidol, (Z)-γ-curcumyl 2-methyl butyrate, 1,8-cineole, 1-octen-3-ol, 4,7-(endo)-dimethylbicyclo(3.2.1)-oct-3-en-6(exo)-ol, 8-hydroxy-salvigenin, acetate, acetate-7-O-β-D-glucuronide, agastachoside, apigenin, apigenin-7-O-β-D-glucuronide, carvacrol, carvone, caryophyllene. oxide, Chrysoeriol, Cirsimartin, cis-Dihydrocarveol, cis-p-Mentha-2,8-dien-1-ol, Diosmetin-7-O-β-D-glucuronide, Disometin, Esculetin, Eudesma-4(15),7-dien-1-β-ol, Gardenin B, Geranial, Geranic acid, Geraniol, Geranyl acetate, Geranyl formate, Hexahydrofarnesyl acetone, Homoplantaginin, Humulene epoxide-II, Isorhamnetin, Kaempferol, Limonene, Limonene glycol, Luteolin, Luteolin-7-O-β-D-glucuronide, Methyl eugenol, Neral, p-Menth-1-en-9-al(isomer 1), p-Mentha-1,8-dien-10-al(=Limonen-10-al), p-Mentha-1,8-dien-10-ol, p-Mentha-1,8-dien-10-yl acetate, Quercetin, Rosmarinic acid, Spatulenol, Takakin-8-O-β-D-glucoside, Terpinen-4-ol, Thymol, Tilianin, T Contains orilenol, trans-Carveol, trans-p-Mentha-2,8-dien-1-ol, α-Pinene, α-Terpineol, β-Caryophyllene, etc.
However, it goes without saying that plant extracts generally contain a complex mixture of a wide variety of components derived from the plant, and it is not necessary for the individual components of the dragon's head extract that can be prepared as described above to be identified in order to use it in the present invention.
The vitamin C absorption promoter of the present invention uses the dragon head extract, which can be prepared as described above, as an active ingredient, and is intended to promote the absorption of bioavailable vitamin C by administering this together with vitamin C to humans or non-human animals.
The method of administration for this purpose is not particularly limited, but for example, oral administration is preferred.
Oral administration can promote the absorption of vitamin C via the vitamin C transporter expressed in the intestinal epithelium. It can also be administered transdermally. Transdermal administration can promote absorption of vitamin C via vitamin C transporters expressed in the skin epithelium. When administering, it is not necessarily necessary to prepare and administer a composition containing dragon's head extract and vitamin C together. It is also possible to use dragon's head extract and vitamin C each contained in a separate form and administer them separately, but without any time gap.
The vitamin C absorption enhancer of the present invention can be used as an oral preparation by adding a pharma- ceutically acceptable base material or carrier as necessary and formulating it into the form of, for example, tablets, granules, powders, capsules, drinks, jellies, lozenges, or oral disintegrating agents using known formulation methods.
Similarly, the composition can be formulated by known formulation methods into, for example, creams, lotions, packs, ointments, gels, patches, tics, liniments, sprays, etc., and used as external preparations for the skin.
In the vitamin C absorption enhancer of the present invention, the content of the dragon head extract may be appropriately determined taking into consideration the relationship between the amount used and the effective dosage when it is in various forms.
Typically, the content of the dragon's head extract may be in the range of 0.01 to 10% by mass, or in the range of 0.01 to 5% by mass, or in the range of 0.01 to 2% by mass, calculated on a dry matter basis.
Meanwhile, in the present invention, a vitamin C supplement composition may be provided by utilizing the vitamin C absorption enhancer provided as above.
In other words, by combining the dragon head extract with vitamin C to form a composition, a vitamin C supplement composition that can be used in the form of food or drink can be obtained.
Vitamin C contained in the vitamin supplement composition of the present invention may be in any of the forms described above, i.e., (1) ascorbic acid and/or a salt thereof, (2) oil-coated ascorbic acid and/or a salt thereof, (3) liposomal ascorbic acid and/or a salt thereof, etc.
In any non-limiting embodiment of the present invention, all three forms of vitamin C listed above may be included.
In general, the behavior of vitamin C in the body after oral ingestion is described in the literature (Murata et al., "Plasma concentration and urinary excretion of vitamin C when 1000 mg and 2000 mg of vitamin C are orally administered" (1995) Vitamin Vol. 69, No. 3 (March), pp. 175-182.
) describes that the time to reach the maximum concentration (Tmax) after oral administration of 1000 mg of ascorbic acid is about 3.0 hours. Also, see the literature (M. Levine et al., "Vitamin C pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance" Proc Natl Acad Sci USA (1996) Apr 16;93(8):p3704-9. ) states that the blood concentration of orally ingested ascorbic acid becomes saturated when high doses of ascorbic acid are administered. It is believed that such dose-dependent saturation of blood ascorbic acid concentration can be avoided by using vitamin C, which has a different absorption behavior. Specifically, for example, liposomal ascorbic acid has the effect of promoting the absorption of ascorbic acid, but the Tmax is slightly slower than that of normal ascorbic acid. Oil-coated ascorbic acid has an even slower Tmax because it takes longer for the ascorbic acid to be released from the coating. Therefore, by using these forms of vitamin C with different absorption behaviors in combination, the bioavailability of vitamin C can be improved.
The vitamin C supplement composition of the present invention can be used as an oral composition by adding a pharma- ceutically acceptable base material or carrier as necessary and formulating it into, for example, tablets, granules, powders, capsules, drinks, jellies, lozenges, or oral disintegrating agents using known formulation methods.
Similarly, the composition can be formulated by known formulation methods into, for example, creams, lotions, packs, ointments, gels, patches, tics, liniments, sprays, etc., and used as a composition for external use on the skin.
In the vitamin C supplementation composition of the present invention, the content of the dragon head extract in various forms may be appropriately determined taking into consideration the relationship between the amount used and the effective dosage.
Typically, the content of the dragon's head extract may be in the range of 0.01 to 10% by mass, or in the range of 0.01 to 5% by mass, or in the range of 0.01 to 2% by mass, calculated on a dry matter basis. Furthermore, the content of the vitamin C may be in the range of 1 to 99.9% by mass, 10 to 80% by mass, or 20 to 60% by mass, calculated as the content of ascorbic acid.
In a non-limiting optional aspect of the present invention, it is preferable that the vitamin C supplementation composition has a content of the above-mentioned vitamin C in an amount of 5 to 2,000 parts by mass, calculated as the amount of ascorbic acid, per 1 part by mass of the content of the above-mentioned dragon's head extract in terms of dry matter.
The ratio of vitamin C content to 1 part by mass of the dry matter content of dragon head extract may be in the range of 20 to 2,000 parts by mass, or may be in the range of 50 to 2,000 parts by mass, in terms of the amount of ascorbic acid.
In a non-limiting embodiment of the present invention, the dosage per serving is preferably 100 to 2000 mg of the above-mentioned vitamin C in terms of ascorbic acid, and 1 to 20 mg of the above-mentioned dragon's head extract in terms of dry matter.
The dosage for one intake may be 300-2000 mg of the above vitamin C in terms of ascorbic acid and 1-15 mg of the above dragon head extract in terms of dry matter, or 500-2000 mg of the above vitamin C in terms of ascorbic acid and 1-10 mg of the above dragon head extract in terms of dry matter.
In any non-limiting embodiment of the present invention, when the vitamin C contained in the vitamin supplement composition is in all three forms, namely (1) ascorbic acid and/or a salt thereof, (2) oil-coated ascorbic acid and/or a salt thereof, and (3) liposomal ascorbic acid and/or a salt thereof, it is preferable that the amount of vitamin C derived from each form is in the range of 1-10:1-10:0.01-1 (ratio of amount of form (1) above: amount of form (2) above: amount of form (3) above) in terms of the amount of ascorbic acid.
The ratio of vitamin C derived from each of the three forms may be in the range of 1-10:1-10:0.01-0.5 (the ratio of the amount of the above form (1):the amount of the above form (2):the amount of the above form (3)) in terms of the amount of ascorbic acid, or may be in the range of 1-10:1-10:0.01-0.1.
In a non-limiting optional embodiment of the present invention, the vitamin C supplement composition may be combined with other ingredients for food and beverages as necessary to form a food or beverage.
Examples of food and beverage ingredients that can be combined include dressings, sauces, furikake, syrups, powdered fruit and vegetable juices, sports drinks, and cereal foods.
As ingredients for food and beverages, food materials containing vitamin C such as kiwi, persimmon, strawberry, orange, lemon, mango, papaya, acerola, banana, yuzu, sudachi, grapefruit, paprika, cabbage, rape blossoms, bitter melon, bell pepper, broccoli, molokheiya, turnip, cauliflower, pea sprouts, kale, pumpkin, parsley, potato, sweet potato, seaweed, wakame seaweed, and kelp may be used.
In this case, the food material is a composition containing a predetermined amount of vitamin C as the above-mentioned vitamin C source, and the vitamin C in the material is contained mainly in the form of the above-mentioned (1) ascorbic acid and/or its salt. In this case, the above-mentioned preferred range of vitamin C content in the composition and the preferred range of the ratio to the dragon's head extract can be directly applied in terms of the amount of ascorbic acid.
As described above, according to the present invention, there are provided a vitamin C absorption enhancer containing dragon's head extract as an active ingredient, and a vitamin C supplement composition containing dragon's head extract and vitamin C.
However, the technical idea of the present invention is not limited to the composition of the agent or composition, and in another aspect, the present invention also provides the use of a dragon's head extract for the manufacture of a vitamin C absorption enhancer. In another aspect, the present invention provides use of an extract of dragon's head and vitamin C for the manufacture of a vitamin C supplement composition. In yet another aspect, the present invention provides a method for promoting vitamin C absorption by administering an extract of dragon's head together with vitamin C.
The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples.
Test Example 1 The expression level of SVCT2, a vitamin C transporter, was examined using Caco-2 cells, a human small intestine-like cell line.
Specifically, the expression level of SVCT2 mRNA when each test sample was applied was examined as shown below.
〔１．Cell Culture] A 6-well plate-type culture vessel containing 2 mL of liquid medium E-MEM (containing 20% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin) was prepared, and 2 x 10 Caco-2 cells were seeded into the vessel and cultured statically at 37°C and 5% CO. After the cells had settled on the bottom of the vessel, the test sample was applied.
〔２． Test Sample] Ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) (hereinafter, sometimes referred to as "VC"): dissolved in advance in liquid medium E-MEM at a predetermined concentration, and added to the medium at the time of application to Caco-2 cells so that the concentration at the time of application was 8.8 μg/mL or 88 μg/mL ascorbic acid.
Dragon head extract powder (product name "Dracobel Nu", manufactured by H. Holstein): dissolved in liquid medium E-MEM at a specified concentration in advance, so that the concentration at the time of application is the dragon head extract (hereinafter sometimes referred to as "DH"). ) was added to the medium at the time of application to Caco-2 cells so as to give a concentration of 1.8 μg/mL.
〔３． Application] Before the addition of the test sample, the medium was replaced with liquid medium E-MEM and cultured for 1 hour for acclimatization, and then the test sample was applied, and the cells were harvested 30 or 60 minutes later.
〔４．PCR] RNA was collected from the cells, and the expression level of SVCT2 mRNA was measured by real-time PCR. Specifically, an RNA extraction reagent (trade name "RNAiso Plus", manufactured by Takara Bio) was applied to the collected cells to extract total RNA, and a reverse transcription reagent (trade name "PrimeScript RT reagent Kit", manufactured by Takara Bio) was applied to the total RNA to synthesize cDNA. Using the obtained cDNA as a template and primers specific to SVCT2, the mRNA expression level was measured by real-time PCR using a real-time PCR reagent (product name "TB GreenPremix Ex Taq", manufactured by Takara Bio).
The results were normalized by dividing by the β-actin value obtained when real-time PCR was performed using the same cDNA as a template and primers specific to β-actin, and the expression level of SVCT2 mRNA was calculated by setting the relative value to 1 for the control group to which the test sample was not applied.
As a result, as shown in FIG. 1, the following became clear.
(1) As shown in FIG. 1, when ascorbic acid was applied at a concentration of 8.8 μg/mL for 60 minutes, the relative value of the SVCT2 mRNA expression level in Caco-2 cells was 0.89.
In contrast, when dragon head extract powder was further applied in combination as dragon head extract at a concentration of 1.8 μg/mL, the relative value increased to 1.14.
(2) As shown in Figure 1, Study 2, when ascorbic acid was applied at a concentration of 88 μg/mL for 30 minutes, the relative expression level of SVCT2 mRNA in Caco-2 cells was 0.66.
In contrast, when dragon head extract powder was further applied in combination as dragon head extract at a concentration of 1.8 μg/mL, the relative value increased to 0.81. (3) As shown in Figure 1, in Study 3, when ascorbic acid was applied at a concentration of 88 μg/mL for 60 minutes, the relative expression level of SVCT2 mRNA in Caco-2 cells was 0.67. Furthermore, when dragon head extract powder was used in combination as a dragon head extract at a concentration of 1.8 μg/mL, the relative value was 0.70.
From the above, it was revealed that application of ascorbic acid to Caco-2 cells relatively reduces the expression level of vitamin C transporter.
This was thought to be due to the negative regulation of ascorbic acid. On the other hand, application of dragon's head extract together with ascorbic acid to Caco-2 cells alleviated the above negative regulation. This is thought to be because the dragon head extract has the effect of increasing the expression level of vitamin C transporters.
Test Example 2 A test for evaluating the absorbability of ascorbic acid was carried out using Caco-2 cells, a human small intestine-like cell line.
Specifically, as described below, a monolayer of Caco-2 cells was formed on the inside (apical side) of a transwell, and the amount of ascorbic acid permeating to the outside (basal side) of the transwell when each test sample was applied was examined.
１ Cell culture] 0.5 mL of liquid medium E-MEM (containing 20% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin) was placed on the apical side of the transwell, and 1.5 mL was placed on the basal side. 6.6 x 10 Caco-2 cells were seeded into this and statically cultured under conditions of 37°C and 5% CO2. After the cells had settled on the bottom of the container, they were further cultured for 21 days or more to form a Caco-2 cell layer, which was then used for the application of the test sample.
〔２．Test sample] Ascorbic acid (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.): dissolved in advance in Hank's Balanced Salt Solution (HBSS) at a predetermined concentration, and added to HBSS at the time of application to the Caco-2 cell layer so that the concentration at the time of application was 880 μg/mL ascorbic acid. Liposomal ascorbic acid (product name "Vitamin C Zeal", manufactured by Aurea Biolabs): dissolved in advance in Hank's Balanced Salt Solution (HBSS) at a predetermined concentration, and added to HBSS at the time of application to the Caco-2 cell layer so that the concentration at the time of application was 880 μg/mL ascorbic acid. Dragon head extract powder (product name "Dracobel Nu", manufactured by H. Holstein Co., Ltd.): This was dissolved in advance in Hank's Balanced Salt Solution (HBSS) at a specified concentration, and added to HBSS at the time of application so that the concentration at the time of application was 18 μg/mL as dragon head extract.
〔３．Application] After formation of the transwell, the medium on the inside (apical side) and outside (basal side) was replaced with Hank's Balanced Salt Solution (HBSS), and the test sample was added to the inside (apical side) of the transwell. The cells were then allowed to stand for 2 hours at 37°C and 5% CO<sub>２</sub>, after which the permeate was separated. Before and after the permeation test, the electrical resistance of the cell layer was measured using a Millicell ERS-2 resistance measurement system (manufactured by MERCK) and confirmed to be 250 Ω·cm<sup>２</sup> or higher.
〔４．Quantitative Analysis] The ascorbic acid content in the recovered permeate was measured using a Vitamin C quantitative analysis kit (manufactured by Cosmo Bio).
As a result, as shown in FIG. 2, the following became clear.
(1) As shown in FIG. 2, when ascorbic acid was applied at a concentration of 880 μg/mL for 2 hours, the amount of ascorbic acid that permeated the Caco-2 cell layer was 1.07 μg.
In contrast, when dragon head extract powder was further applied in combination as dragon head extract at a concentration of 18 μg/mL, the permeation amount increased to 1.53 μg.
(2) As shown in FIG. 2, when liposomal ascorbic acid was applied at a concentration of 880 μg/mL for 2 hours, the amount of ascorbic acid that permeated the Caco-2 cell layer was 2.22 μg. In contrast, when dragon head extract powder was further applied in combination as dragon head extract at a concentration of 18 μg/mL, the permeation amount increased to 2.59 μg.
From the above, it was revealed that the dragon head extract has the effect of increasing the amount of ascorbic acid permeation in an absorbency evaluation system using Caco-2 cell layers.
In addition, the absorption of ascorbic acid was improved by incorporating it into liposomes, but the amount of ascorbic acid permeated was further increased by the combined use of dragon's head extract. Therefore, it is believed that the increased absorption of dragon's head extract occurs through a mechanism different from that of liposomal formulation, possibly due to the regulation of vitamin C transporter expression levels.
<Test Example 3> The effect of dragon head extract on the absorbability of vitamin C was examined.
Specifically, as shown below, we investigated the changes in serum ascorbic acid concentration when adult male volunteers ingested granules containing vitamin C and dragon head extract.
〔１． Study method: After review and approval by an external ethical committee, this study was a placebo-controlled, randomized, parallel-group, single-blind comparative study. Specifically, 11 healthy Japanese male volunteers aged 20 to 55 years who were not regularly using vitamin preparations, foods high in vitamins, or luxury items and who the principal investigator judged to be eligible to participate in this study were randomly assigned to two groups (test food A group, 5 subjects; test food B group, 6 subjects) based on age, and asked to take one packet (2.3 g) of each test food shown in Table 1 once a day in the morning, without water.
〔２．Test Items] The amount of vitamin C in the blood was measured by collecting one 9 mL sample of serum at eight points: before ingestion of the test meal and 1, 2, 3, 4, 6, 8, and 10 hours after ingestion, and measuring the serum ascorbic acid concentration using HPLC according to standard methods.
The blood samples were taken after fasting for 8 hours or more before ingestion of the test meal.
〔３．Results] FIG. 3 shows the results of measuring and averaging the time course of serum ascorbic acid concentration for each of the subject groups that ingested test food A or test food B before the start of the study and after 4 weeks had elapsed, and plotting the results as a graph. FIG. 4 shows the concentration-time curve area (AUC<sub>０－１０</sub>(μg·h/ml)) calculated from the graph in FIG.
As shown in Figures 3 and 4, before the start of the test, the absorption of vitamin C into the blood was increased when test food A containing dragon's head extract was ingested compared to when test food B without dragon's head extract was ingested.
Furthermore, after continuing to take the test meals for four weeks, there was a tendency for blood vitamin C concentrations to increase in both subject groups taking test meals A and B. This tendency for increase was more pronounced in the group of subjects who ingested test meal A containing dragon's head extract than in the group of subjects who ingested test meal B not containing dragon's head extract.
From the above, it is clear that dragon head extract has the effect of promoting vitamin C absorption in humans as well.
[Production Example 1] Of the raw materials shown in Table 2, each raw material except for the flavoring and sweetener was placed in a fluidized bed granulator (manufactured by Okawara Manufacturing Co., Ltd.) and granulated while uniformly spraying purified water under conditions of an inlet air temperature of 60°C and an exhaust air temperature of 35°C. Further, the flavoring and sweetener were added and granulated to prepare granules, thereby obtaining a vitamin C supplement composition according to the present invention.
[Preparation Example 2] Of the raw materials shown in Table 3, each of the raw materials except for the flavor, sweetener, and calcium stearate was placed in a fluidized bed granulator (manufactured by Okawara Manufacturing Co., Ltd.) and granulated while uniformly spraying purified water under conditions of an intake air temperature of 70°C and an exhaust air temperature of 35°C. The flavor, sweetener, and calcium stearate were added and mixed, and then the mixture was compressed using a tablet press (manufactured by Kikusui Co., Ltd.) at a tableting pressure of 1000 kg/cm<sup>２</sup> to prepare tablets with a diameter of 10 mm and a weight of 1500 mg per tablet, thereby obtaining a vitamin C supplementation composition according to the present invention.
[Production Example 3] The ingredients shown in Table 4 were mixed, and purified water was added to make up a total volume of 1 L. The mixture was then subjected to UHT sterilization to prepare a beverage, thereby obtaining a vitamin C supplement composition of the present invention.
PL428783
Method of obtaining a preparation containing liposomal vitamin C and a preparation containing liposomal vitamin C
Description of the invention
The subject of the invention is a method for obtaining a preparation containing liposomal vitamin C and a preparation containing liposomal vitamin C.
Due to the progressive impoverishment of the diet in valuable nutrients such as vitamins and minerals, actions are taken to reduce such deficiencies, among others, through supplementation.
However, there remains the problem of bioavailability of the preparations taken, understood as the extent to which they are absorbed and the extent to which the body excretes them without use.
For these reasons, to increase absorption and reduce side effects of oral administration, vitamin C is encapsulated in liposomes for oral administration.
From the description of the invention to international application WO 2012/094033 A1, a method is known for obtaining a preparation with a retained agent, including:
- solubilization of at least about 18 wt%. vesicle-forming lipids in approximately 1-12 wt%. an aqueous solvent at room temperature to form a lipid solution;
- separately, solubilizing a certain amount of this agent in 0.005-0.01% by weight.
EDTA, at room temperature to form a solution containing the agent;
- filtering the solution containing this agent;
- injecting a stream of lipid solution into the solution containing the agent while stirring;
- leaving the resulting lipid and the solution containing the agent to hydrate for at least an hour with frequent stirring.
After the hydration step, more lipid or thickener is added to form a gel.
Ascorbic acid or its salt is used as the agent, and alcohol is used as the aqueous solvent.
The lipids that form the vesicles contain at least about 45-50% phosphatidylcholine, with the phosphatidylcholine coming from egg or soy.
From the description of this application, a liposome composition is also known, consisting of:
- at least about 18 wt%. vesicle-forming lipids; 0.005-0.1% by weight EDTA;
- 10-12% by weight alcohol;
- liposomes with an average particle size of 200-500 nm and at least about 22 wt. ascorbic acid or its salt, enclosed in liposomes.
The lipids that form the vesicles constitute at least about 45-50% of the phosphatidylcholine.
The phosphatidylcholine lipids are soybean phosphatidylcholine, and the composition also contains 0-0.6% by weight. xanthan gum.
Ascorbic acid or a salt thereof is sodium ascorbate.
A method for producing a liposome preparation of an anticancer substance with a high degree of containment of the active substance is known from the Polish patent description number PL 197 938 B1.
According to this way:
a) a solution of the active substance in a solvent, preferably an alkanol, is combined with a solution of lipid components in an organic solvent, wherein the lipid components are a mixture of at least one phospholipid derivative and at least one alkylphenol derivative represented by the general formula 1, in which:
m - is an integer from 0 to 4;
R1 - means C=O or O;
X - represents the NR2R3 group;
Y - is -H, -O-, -N-, -OR4 -NR4 or a monosaccharide group,
wherein
(i) when R1 is C=O, then X is an NR2R3 group, wherein R2 is a hydrogen atom or a C1-C3 alkyl group, R3 - is a C1-C6 alkyl group substituted with at least one hydroxyl group, and Y is -H, -N- or -NR4, where R4 is hydrogen or a C1-C2 alkyl group; or also
Y is -N- and R1 and R2 together with Y form a saturated heterocyclic ring;
and
(ii) when R1 is O, then X is a monosaccharide group and Y is a hydrogen atom or a monosaccharide group connected to X through an oxygen atom,
b) solvents are stripped away,
c) the lipid film obtained in step b) is spread in an organic solvent, preferably alkanol, and freeze-dried,
d) the lyophilisate from step c) is hydrated with water to obtain the original preparation of multilayer liposomes,
e) the primary preparation is subjected to additional physical processes, obtaining a suspension of calibrated bilayer liposomes with a size of 50-200 nm, preferably 100 nm, and then
f) the liposome suspension with the possible addition of auxiliary substances, such as cryoprotective substances, is lyophilized.
The suspension in step c) is frozen by immersing the flask with the suspension, optionally with the addition of cryoprotective substances, in liquid nitrogen and then thawing at 40°C. These steps are repeated 7 to 10 times.
Studies using a Jeol 100C transmission electron microscope at magnifications of 5000-100,000× show that repeated freezing and thawing of the liposome suspension causes two changes that are important from the point of view of pharmaceutical applications: reducing the layering of liposomes and standardizing their sizes.
A similar task is performed by exposing a liposome suspension to short-term ultrasound action, i.e. sonication.
The actual liposome preparation of the active substance is obtained by hydrating the primary preparation, which involves shaking, mechanical mixing, or subjecting the preparation to ultrasound (sonication) in the presence of water, physiological saline solution, buffer and, optionally, a solution of other pharmaceutically acceptable substances.
The obtained hydrated liposome preparation consists of multi- and single-layer liposomes with an average size of 80-1500 nm.
Bilayer liposomes with a preferred size of 100-200 nm can be obtained by subjecting the liposome suspension to appropriate physical processes, such as freezing, extrusion, high-pressure homogenization or sonication.
An appropriate method for standardizing the size of liposomes is to extrude the suspension several times through a polycarbonate membrane with a pore diameter of 50-200 nm, preferably with a diameter of 100 nm.
Extrusion carried out several times allows three processes of producing a liposome preparation to be carried out in one unit operation: loading the bubbles with the active substance, calibrating the liposomes and sterilizing the preparation.
Hydration of the lyophilisate of an anticancer substance with the disclosed composition, even under the simplest conditions, by shaking the lipid components with an aqueous solution of the active substance, ensures obtaining a liposomal preparation with a high degree of encapsulation, exceeding 95%.
From this patent description, a pharmaceutical composition for parenteral administration is also known, characterized by the fact that it contains a liposome preparation of the active substance enclosed in liposome vesicles constituting a composition of lipid components in the proportion of 1 part by weight of the active substance per 10 to 30 parts by weight of lipid components, preferably 1 part by weight active substance into 10 to 20 parts by weight of lipid components, wherein the composition of lipid components includes at least one phospholipid derivative and at least one alkylphenol derivative represented by the general formula 1, in which:
m - is an integer from 0 to 4;
R1 - means C=O or O;
X - represents the NR2R3 group;
Y - is -H, -O-, -N-, -OR4, -NR4 or a monosaccharide group,
wherein
(iii) when R1 is C=O, then X is an NR2R3 group, wherein R2 is a hydrogen atom or a C1-C3 alkyl group, R3 is a C1-C6 alkyl group substituted with at least one hydroxyl group, and
Y is -H, -N- or -NR4, where R4 is hydrogen or a C1-C2 alkyl group;
or Y is -N- and R1 and R2 together with Y form a saturated heterocyclic ring; and
(iv) when R1 is O, X is a monosaccharide group and Y is a hydrogen atom or a monosaccharide group connected to X through an oxygen atom; and pharmaceutically acceptable carriers and/or excipients.
A pharmaceutically acceptable carrier is any substance or mixture of carrier substances without its own pharmacological effect, diluent or excipient known from pharmaceutical practice, intended for the administration of biologically active substances.
In connection with the invention, preferred carriers suitable for intravenous administration of the composition include, for example, sterile aqueous solutions, such as saline solution, solutions of carbohydrates, e.g., glucose, mannitol, dextrose, lactose, and aqueous solutions of buffers, e.g., phosphate buffer.
In addition, the composition may contain other excipients traditionally used to ensure iso-osmoticity, antioxidants, preservatives and others that are not incompatible with the active substance and the components of the lipid bilayer.
In a preferred embodiment of the invention, glucose is added to the pharmaceutical composition in an amount of approximately 5:1 (w/w) in relation to the lipid components.
Glucose serves as a filling and cryoprotective substance in the composition.
The amount of glucose used ensures appropriate iso-osmoticity and isohydricity of the composition, so that the preparation before use can be reconstituted only with water for injection, obtaining a solution with an appropriate glucose concentration (5%), without the need to use additional diluents of excipients.
The pharmaceutical composition may be in the form of a suspension of a liposomal preparation of an anticancer substance ready for administration, in the form of a lyophilisate for ex tempore reconstitution, or in the form of a concentrate for intravenous infusion.
The lyophilisate is available in a unit dosage form, preferably in an ampoule, containing a therapeutically effective amount of the active substance.
The composition in unit dosage forms is obtained by distributing the sterile suspension into ampoules under sterile conditions, freeze-drying and sealing tightly.
The lyophilisate is reconstituted with water for injection or other pharmaceutically acceptable diluent immediately before use.
The composition of the lipid layer according to the invention makes it possible to obtain a liposomal preparation of anticancer active substances with a favorable lipid/active substance ratio, characterized by high efficiency of encapsulating the active substance and appropriate durability.
The liposome preparation is used to produce lyophilized compositions for parenteral administration containing a therapeutically effective amount of the active substance.
Lyophilized compositions according to the invention are characterized by high stability when stored at temperatures from 4°C to 30°C.
Most known products, including pharmaceutical compositions, contain liposomal vitamin C in liquid form (gel).
This is a significant technological problem because it requires the use of special sachets or bottles filled with nitrogen, which after opening must be stored in the refrigerator and the contents consumed within 3 months.
An alternative solution is to use a capsule with liquid, liposomal vitamin C and filled with nitrogen, but this is a complicated and expensive technology.
The purpose of the invention is a method for obtaining a product in the form of liposomal vitamin C in powder form, which can be enclosed in any type of hard capsule (gelatin, cellulose, pullulan) and a product in the form of liposomal vitamin C in powder form, which can be enclosed in any type of hard capsule ( gelatin, cellulose, pullulan).
The essence of the method for obtaining a preparation containing liposomal vitamin C, in which, according to the invention, lecithin is dissolved in purified water in the temperature range of 5°C +/-5°C to 90°C +/-5°C, optimally at 70°C, in addition, vitamin C is dissolved in purified water in the temperature range of 5°C +/-5°C to 90°C +/-5°C, optimally at 50°C, and then the resulting solutions are combined with intensive stirring, characterized by that at a temperature from 15°C +/-5°C to 80°C +/-5°C, and preferably at a temperature of 50°C +/-5°C, inulin is dissolved in purified water, and then the resulting mixture is added to the homogenized emulsion created by combining an aqueous solution of lecithin and an aqueous solution of vitamin C, mixing them intensively at a temperature from 15°C +/-5°C to 80°C +/-5°C, and preferably at a temperature of 50°C +/-5°C, while the resulting mixture maintains a lecithin to inulin content ratio ranging from 1:0.1 to 1:5, and preferably a 1:0.88 lecithin to inulin ratio, then the resulting the suspension is dried in a spray dryer at a temperature ranging from 100°C +/-5°C to 200°C +/-5°C, and preferably at a temperature of 135°C +/-5°C. According to another feature of the invention, L-ascorbic acid and its esters are used as vitamin C.
According to another feature of the invention, approved salts of L-ascorbic acid, such as sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, magnesium L-ascorbate, zinc L-ascorbate, and L-ascorbyl 6-palmitate, are used as vitamin C.
According to a further feature of the invention, sunflower lecithin is used as lecithin.
According to another feature of the invention, soy lecithin is used as lecithin.
According to another feature of the invention, rapeseed lecithin is used as lecithin.
According to a further feature of the invention, egg yolk lecithin is used as lecithin.
According to another feature of the invention, the ratio of vitamin C to lecithin content is maintained in the range of 1:50, preferably 1:1.
According to another feature of the invention, the ingredients for preparing the liposome suspension are selected maintaining their percentages, in which vitamin C is 9.6%, lecithin is 9.6%, inulin is 8.5%, and water is 72.3%.
According to another feature of the invention, drying is carried out in a spray dryer at a temperature ranging from 100°C +/-5°C to 200°C +/-5°C, and preferably at a temperature of 135°C +/-5° C According to another feature of the invention, drying is carried out in a plate freeze dryer, in the temperature range from -200.8°C +/-5°C to -20°C +/-5°C, preferably at -50°C +/-5 °C.
The essence of the preparation containing liposomal vitamin C, which according to the invention also contains lecithin, is characterized by the fact that it is in the form of a powder containing inulin, with the ratio of vitamin C to lecithin ranging from 1:50, preferably 1:1, while the ratio of lecithin to inulin ranges from 1:0.1 to 1:5, preferably 1:0.88.
According to another feature of the invention, the vitamin C is L-ascorbic acid and/or its esters.
According to another feature of the invention, vitamin C contains salts of L-ascorbic acid approved for consumption, such as sodium L-ascorbate, potassium L-ascorbate, calcium L-ascorbate, magnesium L-ascorbate, zinc L-ascorbate, and L-ascorbyl 6-palmitate.
According to a further feature of the invention, the lecithin is sunflower lecithin.
According to another feature of the invention, the lecithin is soy lecithin.
According to another feature of the invention, the lecithin is rapeseed lecithin.
According to a further feature of the invention, the lecithin is egg yolk lecithin.
A beneficial effect of the invention is the solution of a method for obtaining a product in the form of liposomal vitamin C in powder form, which can be enclosed in any type of hard capsule (gelatin, cellulose, pullulan) and a product in the form of liposomal vitamin C in powder form, which can be enclosed in any type of hard capsule. type of hard capsule (gelatin, cellulose, pullulan).
The beneficial effects were confirmed by the test results illustrated in the drawing, where Fig. 1a shows the results obtained when testing the size of particles of the invention in an aqueous solution in the form of three independent measurements superimposed on each other, Fig. 1b shows the average result obtained from three independent measurements, while Fig. 1c shows the device's report of the average measurement, created by superimposing and averaging (Averange) the three previous graphs, and Fig. 2a shows the results obtained when testing the particle size of the invention after a two-hour incubation in 0.1 M HCl at a temperature of 37° C in the form of three independent measurements superimposed on each other, Fig. 2b shows the average result obtained from three independent measurements, while Fig. 2c shows the device's report of the average measurement, created by superimposing and averaging (Averange) the three previous graphs.
The analysis of the test results allows us to conclude that the tested invention is stable during a two-hour incubation in a 0.1 M HCl solution, as evidenced by marginal changes in distribution and, therefore, negligible aggregation of liposomes. This is also confirmed by photographic analysis, the image is clear.
In the case of competitive preparations available on the market, significant changes in the sizes of liposomes have been observed - completely new sizes appear, very different from the initial ones.
In two competitive products, the presence of liposomes with a size smaller than 100 nm can be observed, which indicates a nanomaterial that is not allowed in foodstuffs, and therefore in dietary supplements. Also, photographic analysis of market products indicates less stability, images are characterized by less clarity and a significant aggregation of structures.
Analyzing scientific publications and literature, it can be concluded that the small size of liposomes may have a beneficial effect on bioavailability.
Inulin as a carrier protects liposomes against aggregation in the raw material. This is evidenced by the fact that the liposomes remain very similar in size.
The method according to the invention and the preparation according to the invention will be explained in more detail by means of exemplary embodiments thereof.
Example 1
In the method of obtaining a preparation containing liposomal vitamin C, according to an exemplary implementation of the invention, 100.0 kg of sunflower lecithin SF100 is dissolved in 256.75 kg of purified water - the treatment is carried out at a temperature of 70°C +/-5°C. Regardless of this, 100.0 kg of vitamin C, in the form of L-ascorbic acid, is dissolved in 220.05 kg of purified water, and the treatment is carried out at a temperature of 50°C +/-5°C, while the water is used to dissolve the vitamin C served at 62°C +/-5°C. During this treatment, after adding vitamin C, the solution temperature drops to 50°C +/-5°C. With intensive mixing, the sunflower lecithin solution with water and the vitamin C solution with water are combined at a temperature of 25°C +/-5°C, and the resulting emulsion is homogenized in portions of 20 L. The homogenization time for each portion is appear as 5 minutes.
Moreover, at a temperature of 50°C +/-5°C, 88.46 kg of inulin dissolves in 298.5 kg of purified water.
At a temperature of 25°C +/-5°C, the inulin solution with water is added to the emulsion created by combining the sunflower lecithin solution with water and the vitamin C solution with water, stirring intensively.
Inulin is treated as a carrier that protects liposomes against aggregation in the raw material. After completing the process of obtaining the liposome suspension, it is dried in a spray dryer at a temperature of approximately 135°C +/-5°C.
Example 2
In the method of obtaining a preparation containing liposomal vitamin C, according to an exemplary implementation of the invention, 100.0 kg of vitamin C, in the form of magnesium L-ascorbate, is dissolved in 220.05 kg of purified water, and the treatment is carried out at a temperature of 80°C +/- 5°C, while water to dissolve vitamin C is administered at a temperature of 90°C +/-5°C; during this treatment, after adding vitamin C, the solution temperature drops to 80°C +/-5°C. Regardless of this, 200.0 kg of soy lecithin is dissolved in 256.75 kg of purified water - the treatment is carried out at a temperature of 90°C +/-5°C. After dissolving, cool the solution to 80°C +/-5°C. Vitamin C solution at temp.
80°C +/-5°C is introduced into the lecithin solution at temp.
80°C +/-5°C, stirring intensively for several minutes using a mechanical mixer. The mixture is homogenized in portions of 20 L. The homogenization time for each portion is assumed to be 5 minutes. Moreover, at a temperature of 50°C +/-5°C, 180 kg of inulin dissolves in 298.5 kg of purified water. At a temperature of 80°C +/-5°C, the inulin solution with water is added to the emulsion created by combining the soy lecithin solution with water and the vitamin C solution with water, stirring intensively. After completing the process of obtaining the liposome suspension, it is dried in a spray dryer at a temperature of approximately 190°C +/-5°C.
Example 3
The preparation containing liposomal vitamin C in the form of L-ascorbic acid, which also contains sunflower lecithin, according to an exemplary embodiment of the invention is in the form of a powder containing inulin, where the ratio of vitamin C to lecithin is 1:1, and the ratio of lecithin to inulin is 1 :0.88.
Example 4
The preparation containing liposomal vitamin C in the form of magnesium L-ascobate, which also contains soy lecithin, according to an exemplary embodiment of the invention, is in the form of a powder containing inulin, the ratio of vitamin C to lecithin is 1:5, and the ratio of lecithin to inulin is 1 : :2
The results of testing the stability of the product according to an exemplary process of the invention, obtained with the technology of the present invention, are presented in Table 1 and Table 2.
Parameters tested: appearance, pH, smell
Test temperature: 25°C
The test results indicate full stability in terms of appearance, pH and smell.
The results of testing the stability of the product according to the exemplary process of the invention, obtained by the method of the present invention, are presented in Table 1 and Table 2.
Parameters tested: appearance, pH, smell
Test temperature: 30°C
Table 1
Formulation
Table 2
+ - the product is fully stable
+/- - at least one key parameter outside the specification
- - complete loss of stability
The test results indicate full stability in terms of appearance, pH and smell.
Report on stability tests of liposomal dietary supplements containing vitamin C in 0.1 M HCl solution at 37°C
Methods:
Particle size analysis by PCS
Incubation of preparations in 0.1 M HCl solution
Invention research
Stability tests of liposomal dietary supplements containing vitamin C in 0.1 M HCl solution at 37°C
Particle size was measured using the NanoSizer ZS device (Malvern Instruments, Great Britain) in the "volume" function.
In the case of undiluted preparations, 10 μl of liposome samples (after thoroughly mixing the preparations) were diluted with deionized water to a volume of 1000 μl and placed in the instrument's cuvette, and then at least three independent size distribution measurements were made.
In the case of suspensions obtained after incubation of diluted preparations 1:133 with a 0.1 M HCl solution, 1 ml of such a suspension was placed in a cuvette.
Measurements of the behavior of the tested preparations were made as follows.
Appropriate amounts of preparations (376 μl) were mixed with 49.6 ml of 0.1 M HCl solution at 37°C contained in plastic Falcon centrifuge tubes with a volume of 50 ml to obtain a homogeneous suspension.
Then the test tubes were placed in a water bath at the same temperature and incubated for 2 hours
US2016367480
Vitamin C delivery system and liposomal composition thereof
The present invention relates to a vitamin C delivery system, and to a liposome composition thereof. In the present invention, sunflower lecithin is used as a liposome composition, thereby increasing stability and bioavailability of vitamin C. Also, the liposome composition does not use soybean recithin, thereby having an excellent effect of removing various side effects.
BACKGROUND ART
Vitamin C (ascorbic acid) improves immunity within the human body and when it is applied to the skin, it promotes formation of collagen which is a component of cartilage, capillary and muscle and therefore prevents skin damage caused by UV light and the production of wrinkles. It also prevents histamine production which induces allergic reactions and inhibits formation of melanin which causes skin aging. However, vitamin C is especially sensitive to the external environments such as oxygen, heat and light and therefore more prone to oxidation decomposing. These oxidation include two electron transfer processes and electrolysis of the hydrogen ion. As a result, Dehydro ascorbate radicals are formed as an oxidation intermediate and cause decomposition. Accordingly, it is not stable and used as a small dose of active ingredients in medicine, food and cosmetic field.
This vitamin C is taken as forms of tablet, capsule, powder and liquid, but it is not absorbed to the body effectively.
To improve the stability of vitamin C, U.S. Pat. No. 4,938,969 and EUP 533,667 B1 disclosed methods of adding antioxidants, two methods which are stabilizing vitamin C with multi-lamellar emulsion, and stabilizing vitamin C using oil-in-water emulsion in terms of formulation. In addition, to improve the stability of vitamin C itself, a method of modifying vitamin C structure with SA (Sodium ascorbylphosphate), MAP (Magnesium ascorbylphosphate), CAP (Calcium ascorbylphosphate), AAP (ascorbic acid polypeptide), EAE (Ethyl ascorbic ether) and AD (Ascorbyl Dipalmate), AEP (Ascoly ethylsilanol pectinate) have been suggested.
In addition to the above-mentioned methods, to improve the stability of vitamin C itself, methods to prevent Vitamin C decomposition caused by the external environments such as water, oxygen, heat and light using physiochemical binding of the vitamin C to high molecular chains and encapsulating into nano-type micro foam, namely the liposome, has also been recently suggested.
As an example of the materials for stabilizing vitamin C, cationic polymers and anionic polymers are well known. For example, anionic polymers are preferred which can enfold vitamin C in three dimensions isolating it from water and air and enabling it to absorb UV light. The anionic polymers may include monosaccharides, polysaccharides, cellulose, gelatin, hyaluronic acid, alginic acid, starch, and carboxymethyl cellulose (CMC). Cationic polymers should be able to be formed into vitamin C with an acid-base binder. For example, it may be a polymethylmethacrylate copolymer or a styrene copolymer comprising chitosan, polyethyleneimine, polyvinylpyrrolidone, amino acids or 4 kinds of ammonium (KR 10-0588464).
A liposome consists of vesicular structure based on lipid bilayer enfolding liquid composition comprised of phosphatide or sterol. Therefore, liposomes show variable physicochemical properties according to their sizes and surface charge. Recently, attention was drawn to liposomes as pharmaceutically acceptable carriers for diagnosis or treatment for diseases. Especially, research on a liposomes used as drug delivery systems have been dramatically developed, and in this regard, a Korean Publication No. 10-2011-71017 discloses a pharmaceutical composition and a formula for gene therapies of various diseases in contact with an active solution with a solution comprising amino acid compound or lipid in organic solvent to improve the efficacy of the delivery of carrier and producing collision stream. However, a liposome may induce agglutination of blood by interacting with various plasma proteins and can be captured by reticuloendothelial systems (RES). For example, the Kupfer cell in the liver or the fixed macrophage in the spleen may capture liposomes before they reach the target and this capture by RES inhibits selective transportation of liposome to target tissues or cells. In addition, liposomes are unstable as they are easy to make electrostatic reactions, hydrophobic reactions and van der waals reactions with plasma protein which may lead to a rapid removal from the ER before reaching the target during blood circulation. Moreover, further to the interaction of liposome with cells or proteins, the drug itself can interact with a lipid of the liposome which makes the capsulation difficult. Therefore, a liposome is unstable during its manufacturing process and the drug may leak before arriving at a tumor site and can be decomposed by cytotoxicity. Consequently, the researchers tried to explore long time-circulating liposomes which are able to be released within blood vessel only. The effort to transport a drug to a target region by extending circulation time of liposomes is disclosed in U.S. Pat. No. 4,501, 725. Furthermore, U.S. Pat. No. 4,920,016 discloses a method of hardening liposomal membrane using neutral phosphatides, and U.S. Pat. No. 6,083,530 discloses a method of formulating liposomes which enables the increase of the ratio of lipid to drugs up to 3-80 folds, and U.S. Pat. No. 6,132,763 discloses a method of inducing phosphatides with polyethylene glycol (PEG) and pegylated liposome.
In U.S. Pat. No. 6,132, 763, a surface of a liposome is coated with hydrophilic polymer such as polyethylene glycol (PEG) and therefore it prevent adhesions of various plasma proteins. The inventors named it stealth liposome. However, a formula comprising the pegylated phosphatides shows toxicity of the hand-foot syndrome causing a skin rash or ulcer (Kenneth B. Gordon, Cancer, Vol 75(8), 1995, 2169-2193).
Meanwhile, liposome compositions with high bioaffinity may cause problems such as bring changes of color and scent which results from extraction of natural materials.
The cell penetration ratio, of a liposome formula using phospholipids as a surfactant is formed to be increased (Biochem, Biophys. Acta, 1237, (1995)), but it has disadvantage of having an unstable structure since it consists of phospholipids only. Moreover, liposome formulas prepared using soybean lecithin or egg lecithin have high cell penetration ratio by penetrating into gaps between skin keratinocytes, however, the hardness of the structure is weak and therefor causes oxidation of double binding regions of unsaturated lecithin by oxygen, metallic ions and more. Therefore, the structure of the liposome is destroyed which causes changes in color and scent (Biophsics J. vol. 79 No. 1(2000): 328-339).
According to the inventor's research, the liposome formula made of sunflower lecithin has appropriate hardness for a stable liposome structure to prevent changes in color and scent, and also appropriate rigidity to enhance cell penetration ratio and affinity. Moreover, compared to soybean lecithin, sunflower lecithin does not have a risk of having characteristics of GMO (Genetically Modified Organism) and side effects such as thyroid related diseases, interruption of mineral uptake or soybean caused allergies. Especially, because as it is extracted by cold pressing and not by extraction with organic solvents, it does not have side effects caused by solvent extraction.
So far, there is no report regarding a vitamin C liposome composition using the liposome consisting of sunflower lecithin so as to uptake vitamin C stably into body.
TECHNICAL PROBLEM
Accordingly, the object of the present invention is to provide a vitamin C liposome composition using the liposome consisting of sunflower lecithin. Another object of the present invention is to provide vitamin C delivery system improving bioavailability by administrating the liposome formula orally to be absorbed into blood circulation system.
TECHNICAL SOLUTION
The object of the present invention is achieved by providing a vitamin C liposome composition comprising 500 mg of vitamin C, 100 mg of sunflower lecithin, 2 mg of candelilla wax, 288 mg of medium chain triglyceride (MCT), 100 mg of Glycerin and 10 mg of distilled water prepared by the following steps:
preparing a water phase compound forming liposomes by mixing vitamin C, glycerin and sunflower lecithin with distilled water followed by stirring;
the 1st emulsifying step that medium chain triglyceride (MCT) and candelilla wax is added to the water phase compound and stirred for 1 hr;
preparing a vitamin liposome composition through the 2<nd >emulsifying step wherein the emulsion is homogenized with a homogenizer at 5000 rpm for 2 hrs;
preparing soft or hard capsules as a product by filling them with the vitamin C liposome composition; and
determining the stability and bioavailability of the composition by measuring titer and bioavailability of the vitamin C.
ADVANTAGEOUS EFFECT
The present invention is advantageous in that it provides a liposome composition improving stability and bioavailability of vitamin C. In addition, the composition does not use soybean lecithin thereby resolving side effect thereof.
DESCRIPTION OF DRAWINGS
FIG. 1 demonstrates the vitamin C liposome structure manufactured according to the Example of the present invention.
FIG. 2a-2d represents the test results showing bioavailability of vitamin C after uptake of the vitamin C liposome composition of the present invention
FIG. 3 is a graph which shows results of comparative experiments measuring bioavailability of the vitamin C after uptake of the vitamin C liposome of the present invention with a vitamin C liposome consisting of conventional vitamin C powder and soybean lecithin.
X: Dr. JEUNG, JONG; administered 0.5 g of the inventive products (66.43 μM/L)
V: Dr. SHAH, HITENDRA; administered 1 g of the inventive products (103.9 μM/L)
602: BARRERA, JUANITA: administered 2 g of the inventive products (135.9 μM/L)
Dr. JEUNG, JONG; administered 3 g of the inventive products (181.5 μM/L)
MODE FOR INVENTION
Hereafter, the present invention would be described in detail through the examples and experimental examples. However, the description certainly does not limit the scope of this invention.
The vitamin C liposome composition of the present invention is produced by the steps comprising as follows:
(a) preparing a water phase compound forming liposomes by mixing vitamin C, glycerin and sunflower lecithin with distilled water followed by stirring;
(b) the 1st emulsifying step that medium chain triglyceride (MCT) and candelilla wax is added to the water phase compound and stirred for 1 hr;
(c) preparing a vitamin liposome composition through the 2<nd >emulsifying step where the emulsion is homogenized with a homogenizer at 5000 rpm for 2 hrs;
The significant point of the present invention is to prepare vitamin C filled liposomes using sunflower lecithin. Although methods of preparing liposomes are well known in the art, huge differences may occur in stability and biological characters according to liposome formable materials, solvents and the materials to be filled within liposomes, and its order when adding, the ratio of its composition and conditions of stirring.
According to the present invention, the oil ingredient from the oil phase of the step (b) can be selected from the group consisting of paraffin oil, α-bisabolol, stearyl glycyrrhetinate, salicylic acid, tocopheryl acetate, panthenol, glyceryl stearate, cetyl octanolate, isopropyl myristate, 2-ethylene isopelagonate, di-c12-13 alkyl malate, ceteatyl octanoate, butylene glycol dicaptylate, butylene glycol dicaprate, isononyl isostearate, isostearyl isostearate, triglycerides, beeswax, canauba wax, suctose distearate, PEG-8 beeswax, candelilla (euphorbia cerifera) wax, mineral oil, squalene, squalane, monoglyceride, diglyceride, triglyceride, middle chain glyceride, myglyol and cremophor. Also, medium chain triglyceride (MCT) is preferred for using as a subadditive and candelilla wax is preferred for using as an emulsifier.
In addition, the vitamin C liposome compositions may be formed for oral administrations such as a tablet, a capsule and powder, but it is preferably formed, it may be formed into a soft or hard capsules with filling. The formula of the present invention may comprise conventional additives and excipients within the capsule formula. For example, it may be a humectant, a pH control agent, a metal chelating agent, a viscosity increasing agent and distilled water, but it is not limited thereto.
Hereafter, the present invention will be described in detail by examples. However, the description does not limit the scope of the invention.
EXAMPLE 1
Preparation of a Vitamin C Liposome Composition
The vitamin C liposome compositions of the present invention preferably have the component ratio described in Table 1.
TABLE 1
Component ratio of the vitamin C liposome compositions
Criteria Compound Amount
Vitamin C Water-phase Vitamin C Ascorbic acid 500 mg
liposome Compound Phosphatide Sunflower 100 mg
compositions (liposome) Preservative lecithin
Glycerin 100 mg
Distilled water Pure water 10 mg
Emulsifier Candelilla wax 2 mg
Sub-additive Medium chain 288 mg triglyceride (MCT)
Weight in total 1000 mg
The inventors of the present application prepared a vitamin C liposome composition according to the component ratio of Table 1. First, they added 100 mg of glycerin, 500 mg of vitamin C and 100 mg of sunflower lecithin in a flask with 10 mg of distilled water and stirred for 30 mins at 30 ° C. and therefore prepared a water phase compound filled with vitamin C in a liposome. In the above-mentioned flask, they added 288 mg of MCT as a sub-additive and 2 mg of candelilla wax as an emulsifier and stirred for 1 hr for the 1<st >emulsification. The product of the 1<st >emulsification were emulsified by homogenization for 2 hrs at 5,000 rpm so as to be appeared milky for the 2nd emulsification thereby preparing the vitamin C liposome composition of the present invention. The above-mentioned vitamin C liposome composition was prepared into a vitamin C liposome formula by filling it into a soft capsule. This formula was used for the material in the examples described below.
COMPARATIVE EXAMPLE 1-3
The inventors prepared capsules according to the example 1 with paraffin oil instead of candelilla wax (comparative example 1), ethanol instead of glycerin (comparative example 2) and soybean lecithin instead of sunflower lecithin (comparative example 3). The scheme of sunflower lecithin liposome structure of the vitamin C liposome structure according to the present invention is shown in FIG. 1.
EXPERIMENTAL EXAMPLE 1
Titration of Vitamin C
According to the example 1 and the comparative Example 1-3, vitamin C titer of the capsulated products were measured and compared. The results are shown in Table 2. The titers of vitamin C of each products were measured at 36.5 ° C. (body temperature) and 45° C. (Shelf temperature) after one month later from the production of each product. The results are calculated using the [Equation 1] below. The titers were measured by quantification of remaining vitamin C using HPLC (Waters Co. LTD.) To determine the amount of remaining vitamin C, the used wave length of detector was 254 nm, the column was Luna C18 column of phenomenex Co., Ltd. and the flow rate was 0.8 mL/min. The comparative amount of vitamin C was determined by drawing a standard measurement graph using the measured remaining amount and the peak of UV spectrophotometer at 266 nm. The spectrophotometer used was Helios β, a product of spectronic unicam Co. Ltd.
TABLE 2
Vitamin C Titers
Comparative Experiments
Condition Example 1 1 2 3
Room Temperature 95 93 93 95
36.5° C. 94 91 90 93
45° C. 93 87 85
A=titers of vitamin C of each product after 1 month post-production/primary titers of vitamin C of each product×100 (1)
As seen in Table 2, when the titers of the product of the Example 1 and the products of the comparative example 1-3 are compared, the inventive product shows improved effect on vitamin C stability at the temperature of 36.5 ° C. (body temperature) and 45° C. (Shelf temperature).
EXPERIMENTAL EXAMPLE 2
Bioavailability of the Inventive Vitamin C Liposome Product
The inventors performed clinical trials using with a vitamin C liposome product prepared through Example 1. The persons participating in the trial were Dr. JEONG, JONG (Male, age 47), BARRERA, JUANITA (Female, age 79) and Dr. SHAH, HITENDRA H (Male, age 68). In addition, Dr. JEONG, JUNG was administered with 3 g of the product orally on Jan. 28, 2016, whereas the dose used in the clinical trial was 0.5 g. The blood test was performed after 4 hrs and 15 mins post administration to confirm the stability of the product. The above clinical trials were requested to Quest Diagnostics and the results are shown in FIG. 2a-2d. In case of the Dr. JEONG, the measurement of vitamin C after 4 hrs and 15 mins of post- administration of 0.5 g of the product was 1.17 mg/dL (FIG. 2a) and therefore, 66.43 uM/L (1.17 mg/dL×55) of vitamin C was remaining. Likewise, in case of Ms. BARRERA, the measurement of vitamin C after 4 hrs and 15 mins of post-administration of 1 g of the product was 2.47 mg/dL (FIG. 2b) and therefore, 135.9 uM/L (2.47 mg/dL×55) of vitamin C was remaining. In the case of Dr. SHAH, the measurement of vitamin C after 4 hrs and 15 mins of post-administration of 1 g of the product was 1.89 mg/dL (FIG. 2c) and therefore, 103.9 uM/L (1.89 mg/dL×55) of vitamin C was remaining. Meanwhile, in case of Dr. JEONG, the measurement of vitamin C after 4 hrs and 15 mins of post-administration of 3 g of the product was 3.3 mg/dL (FIG. 2d) and therefore, 181.5 uM/L (3.3 mg/dL×55) of vitamin C was remaining. Lastly, there were no side effects such as vomiting, fever and dizziness when the testees were administered with the inventive product.
EXPERIMENTAL EXAMPLE 3
Comparison on the Bioavailability of the Vitamin C Liposome Product of the Present Invention with the Conventional Vitamin C Powder
The clinical data on bioavailability of the inventive vitamin C liposome product (Experimental Example 2) were compared with the conventional vitamin C liposome product which consists of vitamin C-powder and soybean lecithin (Empirical Labs Liposomal Vitamin C). As shown in FIG. 3, in case of Dr. JEONG, the bioavailability of vitamin C measured after administration of 0.5 g of the inventive product (x) was equivalent as that of 5 g of the conventional vitamin C powder. In the case of Dr. Shah, the bioavailability of vitamin C measured after administration of 1 g of the inventive product (v) was even higher than that of 5 g of the conventional vitamin C powder. In addition, in the case of Dr. JEONG, the bioavailability of vitamin C measured after administration of 3 g of the inventive product (□) was similar to that of 5 g of the product consisting of soybean lecithin. In conclusion, the inventors confirmed that the sunflower lecithin used to formulate vitamin C liposome product aids in showing improved bioavailability than using soybean lecithin.
INDUSTRIAL APPLICABILITY
The present invention uses sunflower lecithin as a component of the vitamin C liposome composition increasing the bioavailability, improvement of stability, and the titer measurement of vitamin C. In conclusion, the present invention is useful for the functional health food industry.
ES2105973
Liposomal composition for cellular regeneration of the skin.
Liposomal composition for cellular regeneration of the skin, consisting of a suspension of liposomes with a size of 75 to 300 mm which encapsulate each of the active principles glycolic acid, vitamin C and vitamin E. The composition comprises: Content of active principle Liposomal glycolic acid 5.0-30.0% 0.100-0.600% Liposomal vitamin C 5.0-30.0% 0.250-1.500% Liposomal vitamin E 0.0025-0.0100% Excipient made up to 100 ml
This invention relates to a composition that regenerates the intercellular tissue of the epidermis that stimulates the natural renewal capacity of the skin.
The outermost layer of the skin, called the stratum corneum, forms the permeable epidermal barrier that regulates the water content of the skin.
Morphologically, the stratum corneum is made up of two well-differentiated structures: corneocytes and the intercellular matrix.
Corneocytes are anucleated cells with a high keratin content.
They originate in the stratobasal layer of the epidermis through a differentiation process called cornification.
They are removed by detachment from the surface of the skin, through the natural process of peeling, and are replaced by new cells.
The peeling or removal of dead cells from the stratum corneum is the natural process of constant renewal of the epidermis.
With the passage of time or in prematurely aged skin, this process slows down; so that the cohesion of these dead cells increases and their elimination is slower, the regeneration capacity of young skin decreases, the thickness of the stratum corneum increases, and it is lost. elasticity and moisturizing and wrinkles appear.
It would therefore be convenient to have a means to compensate for this decrease in epidermal functioning.
Logically, such a medium should provide the natural structural constituents of the skin, non-allergens and contributors to increasing its flexibility, such as glycolic acid, procollagen vitamins and restorative principles.
Alpha-hydroxy acids (A.H.A.) or fruit acids are organic compounds of natural origin, characterized by having in their molecule a carboxyl function and a hydroxyl group in an adjacent position. Their properties are derived from the presence and position of these two functions.
They come from natural products, mainly fruit, sugar cane and milk.
Glycolic acid is the alpha-hydroxy acid with the smallest molecular size, containing two carbon atoms.
Its small size facilitates skin absorption and improves effectiveness.
The A.H.A. Applied topically at low concentrations, they act in the innermost layers of the stratocornea, reducing the cohesion of the corneocytes by interfering with the ionic bonds and hydrogen bonds that hold them together. As a consequence, the external corneocytes detach in a way that is imperceptible to the naked eye.
They restore the skin's natural capacity for renewal.
The result of this reactivation is an epidermis of young cells and a decrease in the thickness of the stratum corneum.
The skin is much more elastic, fine, silky and velvety, the face is firmed and illuminated, small furrows and expression lines disappear, and wrinkles are attenuated.
This manifest regenerative effect is further enhanced by the rejuvenating action of A.H.A. since they stimulate the biosynthesis of glycosaminoglycans, collagen, elastin and reticulin, essential components of the dermis and responsible for eradicating small wrinkles.
By facilitating the progressive elimination of worm accumulations on the skin surface, A.H.A. also have a lightening and depigmenting effect on dark spots.
They are beneficial for any type of skin, even oily and seborrheic skin, since their exfoliating effect eliminates obstructions that prevent the correct drainage of the pilosebaceous follicle, the source of comedone formation.
It also reduces acne marks.
They vitalize the skin, they are not toxic, irritating, teratogenic, or photosensitizing.
Vitamin C is a water-soluble substance that, when applied to the skin, has the following effects:
Anti-radical: protects cell membranes from free radical attacks.
Anti-inflammatory and skin decongestant.
Procollagen effect: estimates the synthesis of collagen and glycosaminoglycans.
Regulates the acid-base balance of the skin.
Vitamin E is a fat-soluble vitamin essential for the human body. It is involved in numerous metabolic and functional processes as well as in the care and protection of the skin.
From a dermatological point of view, its activity is based on its extraordinary antioxidant properties, so that it protects cellular structures from the harmful effect of free radicals generated in countless physiological and biological processes and mainly responsible for cellular aging.
Vitamin E thus contributes to protecting and maintaining the integrity of cell membranes and the stability of epidermal structures, preventing aging and cellular deterioration.
Vitamin E strengthens the skin's defenses and makes it resistant to environmental aggressions, including ultraviolet rays.
Vitamin E also has an anti-inflammatory effect that provides rest and comfort to the skin.
It has been proven, however, that the simple application of the active ingredients indicated above would imply the use of large quantities of them and this would not translate into optimal effects either since a good part of the active ingredients would not be absorbed into the skin and, therefore , would not reach the lower layers of the skin, so its action would be partial and superficial, not exempt from toxic effects for the outermost layers of the skin.
It has now been discovered that the application of liposomal techniques for the transport of the aforementioned active ingredients results in a high moisturizing and restoring efficiency of the skin's natural capacity to maintain its optimal level of regeneration.
Liposomes are spherical vesicles whose interior is occupied by an aqueous cavity and whose envelope consists of a variable number of bimolecular layers of the phospholipid or sphingolipid type of natural origin.
Its size is variable, depending on the number of layers it contains, although the optimal size is 100-200 nm and the unilamellar structure, that is, a single bimolecular layer.
The liposome membrane has a composition very similar to that of cell membranes.
Because they are biologically compatible with the body's cells, they do not cause dermatological or tolerance problems, nor toxicity, they are totally safe.
The main property of liposomes is their ability to simultaneously encapsulate water-soluble substances and fat-soluble substances.
So that water-soluble substances are included, along with water, in the aqueous spaces inside the spheres, while any fat-soluble molecule, added to the solvent during the formation of the vesicle, is incorporated into the lipid bilayer.
They transport both types of substances in an aqueous medium.
When liposomes are applied to the surface of the skin, they quickly penetrate through the intercellular spaces to the innermost layers of the stratocornea and in some cases to the dermis.
Due to their chemical composition, analogous to the cell membrane, they come into contact with the cells and slowly release the encapsulated active ingredients, so that they progressively diffuse as the concentration of active ingredient in the external phase decreases.
This osmotic mechanism prolongs the action of the encapsulated substances and increases their effectiveness, while protecting the encapsulated molecules so that they reach the place of action intact.
Its main components, phospholipids and water, are natural constituents of the skin.
So, liposomes guarantee the supply of water to the epidermis and dermis, while preventing its loss due to the regenerative action of the epidermal barrier provided by phospholipids.
This results in increased hydration and flexibility of the skin.
These properties are added to those provided by the active ingredients carried in the liposome.
Without the targeted technology of liposomes, a much larger amount of active ingredient would be needed and its penetration into the skin would not be as effective since a good part, especially in hydrophilic molecules, would not be absorbed.
It is considered that the absorption of liposomed active ingredients in the skin is multiplied by a factor of 5 to 10 in the epidermis and 10 to 15 in the dermis, in relation to the same molecules without liposomes.
Considering the same effectiveness of action, the presence of liposomes allows the amount of active ingredient to be reduced, also reducing its possible side effects.
Consequently, and in accordance with the invention, a suspension composition is provided composed of liposomes that serve as vehicles for the transport of the active ingredients: glycolic acid, vitamin C and vitamin E, whose liposomes, when applied to the skin, penetrate through the interstices of the intercellular matrix, transporting glycolic acid, vitamin C and vitamin E throughout the stratum corneum and the epidermis, releasing them in contact with the cells.
Specifically, the invention provides a skin regenerating composition composed of a suspension of liposomes with a size of 75 to 300 nm that encapsulate each of the active ingredients: glycolic acid, vitamin C and vitamin E, characterized in that it comprises:
Content in active ingredient
Liposomed glycolic acid5.0-30.0%0.100-0.600%Liposomed vitamin C5.0-39.0%0.250-1.500%Liposomed vitamin E0.0025-0.0100%Excipient c.s.p. 100ml
The following example of preparation of the liposomal composition of the invention is offered below, whose example must be considered only illustrative and in no way limiting the scope of the invention.
Example
Pharmaceutical grade soy lecithin (phosphatidylcholine content: at least 92%) is dissolved in ethanol (96%, DAB 10), thus preparing solution I.
Next, and depending on the desired final concentration, the lipophilic agent (vitamin E) is dissolved in solution I.
Also depending on the desired final concentration, the hydrophilic agents (sodium salt of ascorbic acid and sodium salt of glycolic acid) are dissolved in water and, if necessary, an amphiphilic surfactant is added in a concentration of less than 1.4%, thus preparing solution II.
All these stages are carried out under aseptic conditions.
Finally, solutions I and II are combined, mixed thoroughly, diluted to the final concentration with sterile water, adjusted to the desired pH value and filtered under sterile conditions.
According to the skin absorption tests carried out with these liposomes, the skin absorption, in extent and depth, and therefore the effectiveness of an active ingredient is 10 to 15 times greater when administered in liposomal form than when applied without liposomes.
In this way, low doses of active ingredients administered in liposomes achieve high efficiency of action, while preventing toxicity and cellular "accustomization" due to excess product.
It is also important to highlight the beneficial effect of phospholipids, components of liposomes, in the hydration and regeneration of the skin, which potentializes the effectiveness of the active ingredients.
http://www.anti-agingresearchcenter.org/bio-technology/liposomal-encapsulated-vitamin-c.html
The Life, Health Implications of LET Vitamin C
Cardiologist, Thomas Levy, MD JD, a frequent Vitamin C lecturer and the author of two books on the subject theorizes in his book, Curing the Incurable, that it is likely the human body was not intended to get all its ascorbate (Vitamin C) from dietary sources. He presents eight evidences for this theory:
The need for ascorbate (Vitamin C) in the human body for basic maintenance of basic structure and function is essential and fluctuates greatly based on health status and environmental conditions.
Even in IV doses exceeding 200 grams per day, no toxicity for ascorbate has ever been documented.
Human livers have all the ingredients necessary to synthesize ascorbate except one — the enzyme L-Gulonolactone oxidase (GLO).
Humans have the gene required to produce GLO but it is defective in the vast majority of the population.
Some humans apparently synthesize ascorbate as not all individuals deprived of dietary ascorbate develop scurvy.
Most mammals, reptiles, and amphibians do synthesize ascorbate. Some of the larger mammals produce upwards of 100 grams daily.
Intravenous doses of ascorbate have shown powerful antioxidant, anti-toxin and anti-pathogenic properties in humans. (Dr. Levy cites many cases of this including
Fred Klenner’s use of ascorbate to cure 60 out of 60 cases of polio in the late 1940s.)
The uptake of ascorbate by the intestines is very inefficient.
Since Liposomal Encapsulation Technology can deliver virtually 100% of a nutrient directly to the bloodstream, it promises to eliminate the huge loss of bioavailability when dose sizes of actively transported nutrients are increased. This bioavailability chart was developed from a study done by J.L. Groff, S.S. Gropper, and S.M. Hunt which was published in the book Advanced Nutrition and Human Metabolism, West Publishing Co., 1995, pages 222-237.
Concerning the inefficiency of the body’s uptake of Vitamin C, studies show that the body has an increasing resistance to traditional forms of oral Vitamin C — tablets, powders, capsules — as dose size increases.
J.L. Groff (1995 - see chart at left) demonstrated that less than 2 grams of a 12 gram oral dose of Vitamin C actually gets to the bloodstream. Based on that study, 2 grams of liposomal encapsulated Vitamin C has the bio-availability equal to 24-500 mg tablets of the nutrient.