rexresearch.com


Joshua HOOD, et al.

Melittin vs HIV / Cancer









http://news.wustl.edu/news/Pages/25061.aspx
March 7, 2013

Nanoparticles loaded with bee venom kill HIV

By

Julia Evangelou Strait



[ Nanoparticles (purple) carrying melittin (green) fuse with HIV (small circles with spiked outer ring), destroying the virus’s protective envelope. Molecular bumpers (small red ovals) prevent the nanoparticles from harming the body’s normal cells, which are much larger in size. ]

Nanoparticles carrying a toxin found in bee venom can destroy human immunodeficiency virus (HIV) while leaving surrounding cells unharmed, researchers at Washington University School of Medicine in St. Louis have shown. The finding is an important step toward developing a vaginal gel that may prevent the spread of HIV, the virus that causes AIDS.

“Our hope is that in places where HIV is running rampant, people could use this gel as a preventive measure to stop the initial infection,” says Joshua L. Hood, MD, PhD, a research instructor in medicine.

The study appears in the current issue of Antiviral Therapy.

Bee venom contains a potent toxin called melittin that can poke holes in the protective envelope that surrounds HIV, and other viruses. Large amounts of free melittin can cause a lot of damage. Indeed, in addition to anti-viral therapy, the paper’s senior author, Samuel A. Wickline, MD, the J. Russell Hornsby Professor of Biomedical Sciences, has shown melittin-loaded nanoparticles to be effective in killing tumor cells.

The new study shows that melittin loaded onto these nanoparticles does not harm normal cells. That’s because Hood added protective bumpers to the nanoparticle surface. When the nanoparticles come into contact with normal cells, which are much larger in size, the particles simply bounce off. HIV, on the other hand, is even smaller than the nanoparticle, so HIV fits between the bumpers and makes contact with the surface of the nanoparticle, where the bee toxin awaits.

“Melittin on the nanoparticles fuses with the viral envelope,” Hood says. “The melittin forms little pore-like attack complexes and ruptures the envelope, stripping it off the virus.”

According to Hood, an advantage of this approach is that the nanoparticle attacks an essential part of the virus’ structure. In contrast, most anti-HIV drugs inhibit the virus’s ability to replicate. But this anti-replication strategy does nothing to stop initial infection, and some strains of the virus have found ways around these drugs and reproduce anyway.

“We are attacking an inherent physical property of HIV,” Hood says. “Theoretically, there isn’t any way for the virus to adapt to that. The virus has to have a protective coat, a double-layered membrane that covers the virus.”

Beyond prevention in the form of a vaginal gel, Hood also sees potential for using nanoparticles with melittin as therapy for existing HIV infections, especially those that are drug-resistant. The nanoparticles could be injected intravenously and, in theory, would be able to clear HIV from the blood stream.

“The basic particle that we are using in these experiments was developed many years ago as an artificial blood product,” Hood says. “It didn’t work very well for delivering oxygen, but it circulates safely in the body and gives us a nice platform that we can adapt to fight different kinds of infections.”

Since melittin attacks double-layered membranes indiscriminately, this concept is not limited to HIV. Many viruses, including hepatitis B and C, rely on the same kind of protective envelope and would be vulnerable to melittin-loaded nanoparticles.

While this particular paper does not address contraception, Hood says the gel easily could be adapted to target sperm as well as HIV. But in some cases people may only want the HIV protection.

“We also are looking at this for couples where only one of the partners has HIV, and they want to have a baby,” Hood says. “These particles by themselves are actually very safe for sperm, for the same reason they are safe for vaginal cells.”

While this work was done in cells in a laboratory environment, Hood and his colleagues say the nanoparticles are easy to manufacture in large enough quantities to supply them for future clinical trials.

Hood JL, Jallouck AP, Campbell N, Ratner L, Wickline SA.
Cytolytic nanoparticles attenuate HIV-1 infectivity. Antiviral Therapy. Vol. 19: 95 - 103. 2013







http://www.dreamstime.com/royalty-free-stock-images-melittin-principal-component-bee-venom-image29150679



Melittin, the principal component of bee venom
ID 29150679 © Leonid Andronov | Dreamstime.com



US2012100186
NANOPARTICULATE-BASED CONTRACEPTIVE/ANTI-HIV COMPOSITION AND METHODS

Inventor: WICKLINE SAMUEL A / LANZA GREGORY [US]

Nanoparticulate compositions which employ membrane-integrating peptides to effect contraception and/or protection against infection by sexually transmitted virus are described.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority from U.S. provisional application 61/405,108 filed 20 Oct. 2010. The contents of this document are incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention is in the fields of protection against conception and against HIV infection. More particularly, the invention concerns vaginal preparations that specifically interact with sperm and/or HIV using nanoparticulate delivery systems.

BACKGROUND ART

[0003] There is a well recognized need for protection against HIV transmitted through sexual intercourse as well as an option for contraception, particularly in societies where women have little control over reproduction and sexual interaction. The present invention provides women with means to practice contraception and to protect themselves against HIV infection using a vaginal preparation which can be administered using a simple applicator and does not require cooperation or permission from sexual partners.

[0004] The basis for the compositions of the invention resides in perfluorocarbon-based nanoparticles (PFC-NP) that are targeted to sperm or to HIV and that carry a membrane-integrating peptide, i.e., a peptide which forms pores in or lyses cell membranes. U.S. Pat. No. 7,943,168 ('168 patent), incorporated herein by reference, describes such perfluorocarbon nanoparticles which are associated with membrane-integrating peptides. Briefly, the nanoparticles comprise perfluorocarbon cores coated with a lipid/surfactant layer as described, for example, in U.S. Pat. Nos. 7,255,875 and 7,186,399 (the “Lanza patents”), also incorporated herein by reference. The various membrane-integrating peptides that can be associated with the nanoparticles are also described in the above-cited '168 patent and include membrane-lytic peptides and cell-penetrating peptides as well as pore-forming peptides. In particular, melittin and its analogs are described.

[0005] As further noted in the above-referenced '168 patent, the nanoparticulates bearing the membrane-integrating peptides may be targeted. Targeting agents can include antibodies, aptamers, peptidomimetics and the like. A description of such targeting agents and means for attachment thereof is also found in the above-referenced Lanza patents as well as U.S. Pat. Nos. 7,255,875, 7,566,442 and 7,344,698, also incorporated herein by reference.

DISCLOSURE OF THE INVENTION

[0006] The invention is directed to compositions designed for application to the vaginal vault which compositions comprise nanoparticles targeted to sperm wherein said nanoparticles further contain membrane-integrating peptides or comprise nanoparticles targeted to sexually transmitted viruses, such as HIV, which nanoparticles further contain membrane-integrating peptides or wherein the composition comprises both. It is desirable, in preventing infection by sexually transmitted viruses for the nanosnares or nanoparticles to be targeted. However, untargeted nanosnares may also be used for this indication. The same nanoparticles may target both sperm and virus.

[0007] In another aspect, the invention concerns methods to prevent conception and/or protect a subject against virus infection in a subject which method comprises administering to the vagina of the subject the compositions of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1A, 1B, and 1C show the effect of free melittin as compared to melittin associated with PFC-NP on the viability of vaginal epithelium.

[0009] FIG. 2 shows the results of an in vitro experiment whereby HIV infection is prevented by melittin-containing nanoparticles of the invention.

[0010] FIGS. 3A and 3B show the effect of melittin coupled PFC-NP on virus infectivity of strains HIV-p120 and HIV-p134.

[0011] FIG. 4 is a graph demonstrating the effect of CD4 coupled PFC-NP on coupling of the particles to HIV.

[0012] FIGS. 5A-5D show the effect of free melittin or melittin-containing PFC-NP on sperm motility and viability.

[0013] FIG. 6 demonstrates that SPAM1 antibody can successfully target sperm.

MODES OF CARRYING OUT THE INVENTION

[0014] In general, “a” or “an” refer to one or more than one of the referent unless the opposite intention is clear from the context.

[0015] The compositions of the invention contain thousands of trillions of nanoparticles per intervaginal dose wherein these nanoparticles comprise one or more membrane-integrating peptides. In some embodiments, these nanoparticles, sometimes called herein “nanosnares”, are targeted specifically to sperm or to sexually transmitted viruses, such as HIV. These nanoparticles are typically perfluorocarbon nanoparticles (PFC-NP) and carry a potent toxin in the form of a membrane-integrating peptide that results in the formation of pores in the sperm or virus when these are fused to the nanoparticles. In the case of virus, specific targeting is not necessary since the nanoparticles are substantially larger than the virus particles. Nevertheless, efficiency may be improved by providing a targeting ligand. In the case of sperm, however, targeting is needed for efficient fusion because the fusion event establishes the proximity necessary for formation of a hemi-fusion stalk (<5 nm) in a process driven passively by the energy stored in the lipid membrane of the PFC-NP. Since cells and sperm are a great deal larger than the nanoparticles, non-targeted nanoparticles even comprising multiple copies of the membrane-integrating peptide may not be sufficient to affect the viability of the cells or motility of the sperm. Since only sperm, and not endothelial cells are targeted, nontargeted cells (but not virus) are spared and the nanoparticles in the composition are destructive only to the targeted sperm. As noted above, both targeted and non-targeted particles that comprise the membrane-integrating peptide are effective against virus infections that are sexually transmitted, such as herpes or papillomavirus, or HIV.

[0016] To target sperm, the nanoparticles may be associated with a targeting agent for the αvβ3 integrin, which is a well known docking site on the sperm cap. The targeting agent for this integrin may be an antibody specific for the integrin or an immunospecific portion thereof, an aptamer, or may be a peptidomimetic, such as those described in U.S. Pat. No. 7,566,442, incorporated herein by reference. Alternatively, other known sperm-associated receptors can be targeted. In addition to targeting the sperm per se, progesterone can be added to the composition since it is a chemoattractant for sperm that swim up a hormonal gradient sensed through their cap progesterone receptors. Progesterone mimics could also be included as the targeting agent on the nanoparticles.

[0017] Targeting agents for sperm also include antibodies or fragments thereof that are specifically immunoreactive with ligands on the surface of the sperm. (“Antibodies”, of course, include any immunoreactive portion of conventional antibodies, including recombinantly produced single chain antibodies, chimeric antibodies, polyclonal antibodies or monoclonal antibodies, antibody mimics, such as aptamers or peptidomimetics and the like.) A particularly useful antibody which might be used, or a fragment of which might be used, is the SPAM antibody marketed by Sigma-Aldrich that is specific for sperm.

[0018] For capture of HIV, the targeting ligands may be those that bind to gp41 and/or gp120 epitopes. Here, too, antibodies or aptamers could be employed. Alternatively or in addition CD4, CCR5 and CXCR4 peptides that imitate the viral membrane fusion process for T cells may be used. However, as noted above, effective defense against viral particles in general, including HIV, herpes and papillomavirus may be effected in the vaginal vault using nanosnares containing membrane-penetrating peptides that do not comprise targeting agents.

[0019] The composition may include nanoparticles targeted to sperm or nanoparticles targeted to virus or both types of nanoparticles. It is also possible to include targeting ligands to both virus and sperm on the same nanoparticle, or to employ non-targeted nanoparticles for virus protection.

[0020] For the targeted nanoparticles useful in the invention, the number of molecules of targeting ligand per nanosnare will vary depending on its nature. However, typically, the number of targeting ligands per nanoparticle is between 10 and 500, alternatively between 20 and 100 or between 20 and 30.

[0021] The targeted nanoparticles further comprise toxic membrane-integrating peptides, which are exemplified by melittin. Melittin forms pores in lipid membranes that are too large to be repaired by standard membrane repair mechanisms and thus result in discharge of DNA from sperm or RNA from HIV, rendering both ineffective. This effect is confined in the vaginal vault to the targeted sperm and/or to virus particles for the reasons set forth above, i.e., fusion to the target is needed to effect pore formation in the case of cells as opposed to viruses. In addition, the nanoparticles are too large (100-500 nm, typically 250 nm) to penetrate the vaginal mucosa and thus their action is confined to the vaginal vault and they remain in place until washed away.

[0022] As used herein, the word “peptide” is not intended to impose an upper limit on the number of amino acids contained. Any peptide/protein which is capable of effecting cell penetration can be used in the methods of the invention. The nature of the lipid/surfactant layer can be adjusted to provide a suitable environment for the peptides/proteins used in the invention depending on the specific characteristics thereof. Thus, the nature of the lipids and surfactants used in this layer are selected so as to accommodate cationic peptides, anionic peptides, neutral peptides, hydrophobic peptides, hydrophilic peptides, amphipathic peptides, etc.

[0023] Membrane-integrating peptides useful in the invention include lytic peptides such as melittin and the classic pore forming peptides magainin and alamethicin (Ludtke, S. J., et al., Biochemistry (1996) 35:13723-13728; He, K., et al., Biophys. J. (1996) 70:2659-2666). Pore forming peptides can also be derived from membrane active proteins, e.g., granulysin, prion proteins (Ramamoorthy, A., et al., Biochim Biophys Acta (2006) 1758:154-163; Andersson, A., et al., Eur. Biophys. J. (2007) DOI 10.1007/s00249-007-0131-9). Other peptides useful in the invention include naturally occurring membrane active peptides such as the defensins (Hughes, A. L., Cell Mol Life Sci (1999) 56:94-103), and synthetic membrane lytic peptides (Gokel, G W., et al., Bioorganic & Medicinal Chemistry (2004) 12:1291-1304). Included as generally synthetic peptides are the D-amino acid analogs of the conventional L forms, especially peptides that have all of the L-amino acids replaced by the D-enantiomers. Peptidomimetics that display cell penetrating properties may be used as well. Thus “cell penetrating peptides” include both natural and synthetic peptides and peptidomimetics.

[0024] One particular class of membrane-integrating peptides useful in the invention has the general characteristics of melittin in that it comprises a hydrophobic region of 10-20 amino acids adjacent to a cationic region of 3-6 amino acids. Melittin itself is formed from a longer precursor in bee venom and has the amino acid sequence

[0000]

(SEQ ID NO: 1)

GlyIleGlyAlaValLeuLysValLeuThrThrFlyLeuPro- 

AlaLeuIleSerTrpIleLysArgLysArgGlnGln-NH2.

[0025] Various analogs of melittin can be identified and tested as described in U.S. Pat. No. 5,645,996, for example. Other designs for peptides useful in the invention will be familiar to those in the art. In the melittin analogs, the hydrophobic region is preferably 15-20 amino acids long, more preferably 19-21 and the cationic sequence is preferably 3-5 or 4 amino acids long.

[0026] The toxicity of such peptides is affected by a number of factors, including the charge status, bending modulus, compressibility, and other biophysical properties of the membranes as well as environmental factors such as temperature and pH. The presence or absence of certain moieties (other than the targeted epitope) on the cell surface may also effect toxicity.

[0027] Illustrated below is the membrane-integrating peptide melittin, which is a water-soluble, cationic, amphipathic 26 amino acid alpha-helical peptide. Suchanek, G., et al., PNAS (1978) 75:701-704. It constitutes 40% of the dry weight of the venom of the honey bee Apis mellifera. Although a candidate for cancer chemotherapy in the past, melittin has proved impractical because of its non-specific cellular lytic activity and the rapid degradation of the peptide in blood. Attempts have been made to stabilize melittin by using D-amino acid constituents (Papo, N., et al., Cancer Res. (2006) 66:5371-5378) and melittin has been demonstrated to enhance nuclear access of non-viral gene delivery vectors (Ogris, M., et al., J. Biol. Chem. (2001) 276:47550-47555 and Boeckle, S., et al., J. Control Release (2006) 112:240-248). The ultimate effect of melittin is to cause the formation of pores in a cell membrane, and membranes of internal cell organelles, so as to damage the cell and lead to cell death. As noted in the examples below, these proteins are also toxic to viruses.

[0028] In another embodiment a peptide from the Bcl-2-family proteins is employed based on activating or inhibitory activity, for example, BH3 domain peptides (Danial, N. N., et al., Cell (2004) 116:205-219). After penetrating to the cellular interior the peptides cause activation or inhibition of the endogenous Bcl-2-family or associated proteins in the cells (Walensky, L. D., et al., Mol Cell (2006) 24:199-210). Thus, the cellular machinery of apoptosis can be regulated to a variety of therapeutic goals.

[0029] In PFC-NP, the core is inert and nontoxic but facilitates fusion by mobilizing component lipids and relaxing lipid membrane structures.

[0030] A variety of means can be employed to couple the targeting agent and the membrane-integrating peptide to the nanoparticles but one advantageous method is through fusion with a peptide linker which is a truncated form of melittin that retains its membrane-binding potential but deletes its lytic capacity. This linking peptide is described in an article by Pan, H., et al., FASEB J. (2010) published online 24 Mar. 2010. This peptide and effective analogs are also described in WO2009/151788, incorporated herein by reference for the description of these peptides and methods for employing these peptides as linkers to couple any desired moiety to the PFC-NP. This linker can be inserted into the lipid layer of the PFC-NP using a 10-minute mixing procedure that drives the peptide to form a hydrophobic interaction with the lipid layer. Alternatively, a component of the lipid/surfactant layer may be used.

[0031] However, melittin may simply be passively loaded onto the PFC-NP. The hydrophobic portions of melittin are sufficiently compatible with the lipid/surfactant layer to effect coupling.

[0032] Targeted PFC-NP are prepared as described in the above-referenced patents. Targeting ligands to virus or other sperm cell marker that are peptides may be fused to the linker peptide described above to obtain up to 2,000-30,000 total copies of each associated with each nanoparticle. Thus, each of the nanoparticles may also contain about 10-1,000 targeting ligands. Gentle centrifugation removes any unbound ligands. Targeting ligands may be attached to a phospholipid anchor. This is coupled to a component of the lipid/surfactant layer and formulated into the particle itself.

[0033] Similarly, a multiplicity of toxin molecules may be associated with the nanoparticles. In the case of melittin, the hydrophobic α-helical portion of the protein serves as a linker whereby the lytic portion is associated with the nanoparticle. Alternative lytic or pore-forming membrane-integrating peptides may be fused to this linker and associated with the nanoparticles as well. The level of toxic pore-forming molecules associated with the nanoparticles can also be varied from just a few to more than 20,000. The pore-forming peptide or lytic peptide may be coupled to a component of the lipid/surfactant layer, as well, in order to associate the toxin with the nanoparticles.

[0034] The preparation of successfully derivatized nanoparticles can be verified by means known in the art. For example, flow cytometry may be used to identify and count nanoparticles successfully as associated with targeting ligands and toxins.

[0035] Efficacy as a contraceptive may be evaluated in vitro by demonstrating disrupted motility of sperm at selected concentrations of targeted nanoparticles by computer assisted semen analysis and viability of sperm may be tested by dye exclusion and apoptosis staining. Efficacy against virus, such as HIV, may be evaluated by calculating the viral load remaining in the supernatant of a mixture of virus and nanoparticles following 5-30 minute incubations with continuous mixing at 37° C. and low-speed centrifugation to pellet nanoparticle-virus complexes with visual confirmation of complexes by TEM. In addition, targeted nanoparticles incubated in viral cultures are assessed for efficacy of antiviral activity by incubating these cultures with cells that are candidates for viral infection, and observing infection rates.

[0036] The nanoparticles described above are formulated into suitable preparations for vaginal administration.

[0037] Vaginal Formulations

[0038] The nanosnares of the invention are specifically formulated in a composition suitable for vaginal administration. These formulations differ markedly from pharmaceutical compositions in general. Specifically, they are designed to provide a suitable residence time in the vagina and are adjusted for pH and release characteristics that are suitable for this environment. The formulations, when marketed, would be labeled appropriately to limit their use to vaginal administration.

[0039] Suitable vaginal preparations may be in the form of aerosols, foams, gels, creams, suppositories or tablets; typically these are in the forms of foams or gels or dissolvable waffles. The excipients in such compositions are typically polyethylene glycols, emulsifying agents, lanolin, starch, algins, polysorbates, xanthan gums, glycerol and the like. Preparation of vaginal compositions is well known in the art and is described, for example, in U.S. Pat. Nos. 5,725,870 and 6,706,276 incorporated herein by reference. Deodorants, colorants and other cosmetic materials may be added as well.

[0040] In addition to direct application to the vaginal vault, the vaginal formulations containing the nanosnares of the invention may be applied to condoms. Formulations designed to be retained at the surface of the condom until use are within the skill of the art. Typically, gels or creams can be used for this purpose. This embodiment is especially useful for nanosnares targeted to sperm, an analogy to contraceptive creams that are often applied to condom surfaces. However, the nanospheres designed to inhibit infectivity of sexually transmitted virus may be included as well. The surface may be either the inner or outer surface of the condom or both.

[0041] The formulations may contain a single type of nanosnare—i.e., nanosnares that comprise at least one membrane-integrating peptide and which either further comprise a targeting ligand for a sexually transmitted virus, or further comprise a targeting ligand for sperm or do not comprise a targeting ligand or that further comprise both a targeting ligand for sexually transmitted virus and a targeting ligand for sperm or combinations of the foregoing.

[0042] Usage

[0043] For use, the vaginal preparations of the invention are used in effective amounts. As prepared as a suppository or tablet, typically the suppository or tablet is in the range of 0.1-10 grams or 1-5 grams; as a cream or gel, similar quantities may be employed. The mode of application is dependent on the nature of the composition; for liquid or gel compositions, an applicator is generally employed. Use of coated condoms is also contemplated. The application should be carried out prior to the beginning of vaginal intercourse, generally 1 to 30 minutes, up to 12 hours prior to intercourse. Intermediate times such as 2 hours, 6 hours, etc., are also acceptable. The nature of carriers and excipients and their mode of application is understood in the art.

[0044] The following examples are intended to illustrate but not to limit the invention.

[0045] Preparation A

[0046] Preparation of Perfluorocarbon Nanoparticles

[0047] A. Perfluorocarbon nanoparticles were synthesized as described by Winter, P. M., et al., Arterioscler. Thromb. Vasc. Biol. (2006) 26:2103-2109. Briefly, a lipid surfactant co-mixture of egg lecithin (98 mol %) and dipalmitoyl-phosphatidylethanolamine (DPPE) 2 mol % (Avanti Polar Lipids, Piscataway, N.J.) was dissolved in chloroform, evaporated under reduced pressure, dried in a 50° C. vacuum oven and dispersed into water by sonication. The suspension was combined with either perfluoro-octylbromide (PFOB), or perfluoro-15-crown ether (CE) (Gateway Specialty Chemicals, St. Peters, Mo.), and distilled deionized water and continuously processed at 20,000 lbf/in<2 >for 4 min with an S110 Microfluidics emulsifier (Microfluidics, Newton, Mass.) to obtain an emulsion of perfluorocarbon nanoparticles (PFC-NP).

[0048] B. Alternatively, a lipid film containing 92.8 mol % lecithin (phosphatidyl choline), 5 mol % cholesterol, and 2.2 mol % MPB-PEG-DSPE was prepared using rotary evaporation. This lipid film representing the 2% surfactant portion was emulsified with sonication in the presence of 20% perfluorocarbon (perfluoro-octyl-bromide, PFOB), 1.85% glycerin and 76.15% water. The emulsion was then prepared into nanoparticles using microfluidization at 20,000 psi. Finished 2 mol % MPB-PEG-DSPE PFOB nanoparticles were sized (281 nm) using dynamic light scattering.

[0049] Preparation B

[0050] Coupling PFC-NP to Targeting Ligand

[0051] αvβ3-integrin targeted nanoparticles were made by incorporating 0.1 mole % peptidomimetic vitronectin antagonist conjugated to polyethylene glycol (PEG)2000-phosphatidylethanolamine (Avanti Polar Lipids, Inc.) replacing equimolar quantities of lecithin in the procedure of Preparation A.

[0052] The αvβ3-integrin targeting ligand linked to phosphatidyl ethanolamine has the formula:

[0000]
<img class="EMIRef" id="084535472-emi-c00001" />


[0053] Preparation C

[0054] Preparation of Melittin-Containing Nanoparticles

[0055] Perfluorocarbon nanoparticles were incubated in a 900 μM solution of melittin at 4° C. protected from light for 3 days. The nanoparticles were then centrifuged at 1000 rpm for 5 minutes and washed with PBS three times. Nanoparticles were stored under argon at 4° C. until use. For comparison, blank nanoparticles of the same concentration were obtained by incubating with PBS rather than melittin according to this protocol.

[0056] In more detail, 100 μL of 10 mM melittin or 100 μL of PBS was added to 1000 μL of blank nanoparticles and incubated at 4° C. protected from light with gentle shaking for 3 days.

[0057] The level of suspension in the vial used in the mixture is marked and then centrifuged at 1000 rpm for 5 minutes and washed 4× with PBS. The nanoparticles are resuspended to original volume in PBS and stored under argon at 4° C. until use.

[0058] In the alternative, melittin was dissolved in 100 mM KCl (pH 7, 10 mM HEPES) at 0.1 mM and 2-20 mL was added to 50 μl of nanoparticle suspension with mixing. After incubation at room temperature for 10 min, the nanoparticles were washed twice by centrifugation (100 g, 10 min) to remove the unbound melittin. The melittin in the supernatant was quantified by measuring the tryptophan fluorescence (described below). Depending on the amount of melittin added, the melittin-loaded nanoparticles yielded molar lipid/melittin ratios ranging from 1,000 to 40.

[0059] In still another alternative, the PFC-NP prepared in Preparation A, paragraph B or similar targeted nanosnares were incubated at a concentration of 0.91 mM melittin in water with rotation at 4° C. for 72 hours to load melittin. Nanosnares were isolated by low speed centrifugation for 20 min. at 1000 g to “softly” pellet the nanosnares. Nanosnare supernatants were analyzed for unbound melittin using an Eclipse™ plate reader at excitation 280 nm and emission 300-500 nm Single maximum emission peak sizes corresponding to the amount of melittin present were then compared to a standard 0.91 mM melittin emission peak and used to calculate supernatant and corresponding nanoparticle pellet concentrations of melittin for the nanosnares.

Example 1

Vaginal Epithelium Toxicity

[0060] In order to function satisfactorily, the compositions of the invention must not be toxic to the vaginal epithelium. This example demonstrates that although free melittin not coupled to nanoparticles is toxic, melittin coupled to untargeted nanoparticles is not toxic at concentrations useful in the invention.

[0061] Immortalized vaginal epithelial cells (VK2/E6E7) were obtained from ATCC (CRL-2616) and propagated by the suggested protocol. For toxicity studies, 7500 cells were added to each well of a 96-well plate and allowed to attach for 24 hours. Melittin or melittin coated PFC-NP were then added and incubated with the cells for 12 hours at 37° C. with shaking at 500 rpm. Cells were washed once with media and incubated with MTT reagent for 4 hours. The colored product was solubilized in DMSO and absorbance at 570 nm was measured using a plate reader.

[0062] In more detail, cells were detached from a T75 flask by rinsing with 2-3 mL of trypsin, followed by adding 5 mL trypsin and incubating at 37° C. for 15 minutes. Five mL DMEM-FBS is then added to stop the trypsin, and the mixture is centrifuged at 900 rpm for 5 minutes, supernatant is removed and the cells are resuspended in 5 mL of Keratinocyte-Serum Free Media (Invitrogen™). The cells are diluted to approximately 75,000 cells/mL, and 100 μL of the cell suspension is added to each well of a 96-well plate and incubated for 24 hours at 37° C. The media are replaced with 100 μL of media containing the melittin or melittin-loaded nanoparticles at various concentrations and incubated for 12 hours at 37° C. with shaking at 500 rpm. The test solutions are then removed and the cells washed once with media.

[0063] To each well containing 100 μL of media, 20 μL of MTT reagent (5 mg/mL MTT in PBS) were added, and incubated for 4 hours at 37° C. The media are removed and 75 μL of DMSO is added to each well and incubated for 10 minutes at 37° C. with shaking at 500 rpm. Absorbance is read at 570 nm using a plate reader.

[0064] Results are shown in FIGS. 1A-1C. As shown in FIG. 1A, concentrations of melittin as low as 1 μM dramatically decrease the viability of vaginal epithelium cells. However, as shown in FIG. 1B, concentrations of melittin up to 20 μM do not decrease epithelial cell viability. In fact, as shown in FIG. 1C, nanoparticles containing melittin at concentrations up to 20 μM appear to have a somewhat positive effect on cell viability. Controls with nanoparticles not containing melittin (blank NP), but comparable in concentration to the melittin-containing nanoparticles show substantially no effect.

Example 2

Contraception Cream

[0065] PFC-NP targeted to αvβ3 are prepared as described in U.S. Pat. No. 7,566,442 incorporated herein by reference. Melittin was associated with the particles by dissolving it at a concentration of 0.1 mM in 100 mM KCl, pH 7 and 0-20 ml is added to 50 μl of the nanoparticle suspension. After incubation at room temperature for 10 minutes, the nanoparticles are washed twice by centrifugation to remove unbound melittin. The melittin-loaded nanoparticles yield molar lipid/melittin ratios ranging from 1,000-40.

[0066] One gram of the particles thus prepared is added to a mixture of polyethylene glycol 400, polyethylene glycol 6,000 and hexantriol to form a water-soluble vaginal cream.

Example 3

Fusogenic Anti-HIV “Nanosnares” Decrease Infectivity of HIV to TZM-b1 Cells

[0067] Vesicular stomatitis virus (VSV) pseudotyped HIV-1 is a strain of HIV that demonstrates several fold increases in the levels of transfection over amphotropic HIV by utilizing an endocytic entry mechanism. TZM-b1 cells are specially engineered HeLa cells designed to express CD4 and either CXCR4 or CCR5 which are required for HIV fusion. The cells also contain HIV-TAT inducible luciferase. When TZM-b1 cells are infected, firefly luciferase is produced. Cells to be assessed for infection are lysed and luciferin is added. Fluorescence is produced by infected cells, and the level of fluorescence, in relative fluorescence units, corresponds to the level of viral infection.

[0068] In this assay, PFC-NP (25 μl) providing 1 μM melittin in a nanoparticle/virus solution and virus (HIV or VSV pseudotyped HIV) (50 μl) were mixed, and then added to 4×10<4 >cultured cells in 50 well plates and incubated overnight at 37° C. The level of infection was measured as described above.

[0069] The results are shown in FIG. 2. As shown, when HIV alone or VSV pseudotyped HIV alone was added to the cells, high levels of infection occurred. However, the level of viral infectivity of either virus combined with the melittin-containing nanoparticles of the invention is dramatically mitigated.

[0070] The procedure set forth above was repeated comparing HIV-1p120 which is a CXCR4-dependent strain and p134 which is a CCR5 dependent strain. These results are shown in FIG. 3A and FIG. 3B. As shown, the infectivity of either p120 or p134 in the presence of nanoparticles alone (blank nanoparticles) is not effectively reduced; however, in the presence of melittin-containing nanoparticles with an effective concentration of 10 μM melittin, infectivity is dramatically decreased.

Example 4

Effect of Targeted Nanosnares

[0071] The nanosnares of this example were prepared as follows.

[0072] A lipid film containing 92.8 mol % lecithin (phosphatidyl choline), 5 mol % cholesterol, and 2.2 mol % MPB-PEG-DSPE was prepared using rotary evaporation. This lipid film representing the 2% surfactant portion was emulsified with sonication in the presence of 20% perfluorocarbon (perfluoro-octyl-bromide, PFOB), 1.85% glycerin and 76.15% water. The emulsion was then prepared into nanoparticles using microfluidization at 20,000 psi. Finished 2 mol % MPB-PEG-DSPE PFOB nanoparticles were sized (281 nm) using dynamic light scattering.

[0073] For preparation of the CD4 anti-HIV targeting motif, 300 μg CD4 (Pro Sci recombinant sCD4)/0.0111 μmoles was dissolved in 2.1 ml of 0.1 M PBS pH 8.0 then mixed with 5 μL of 7.3 nM 2-iminothiolane in 0.1 M PBS, 10 mmol EDTA buffer pH 8.0, flushed with argon gas and incubated at RT for 1 hour. For conjugation of CD4 to the nanoparticles, 15 ml of 2 mol % MPB-PEG-DSPE PFOB was added into 2.1 ml of modified CD4 solution and incubated at room temperature for 2 hours. Then 7 mg cysteine was added to quench MPB reactivity and incubated for another 2 hours. Finally, the nanoparticle suspension was then dialyzed in 2 L PBS buffer three times at intervals of 2, 3, and 2 hours to remove free unreacted sCD4 and excess 2-iminothiolane thus producing CD4 nanosnares.

[0074] To produce melittin loaded CD4 nanosnares, CD4 nanosnares were incubated at a concentration of 0.91 mM melittin in water with rotation at 4° C. for 72 hours to load melittin. Nanosnares were isolated by low speed centrifugation for 20 min. at 1000 g to “softly” pellet the nanosnares. Nanosnare supernatants were analyzed for unbound melittin using an Eclipse™ plate reader at excitation 280 nm and emission 300-500 nm. Single maximum emission peak sizes corresponding to the amount of melittin present were then compared to a standard 0.91 mM melittin emission peak and used to calculate supernatant and corresponding nanoparticle pellet concentrations of melittin for the nanosnares. Melittin only nanosnares were created using the same method in the absence of sCD4. “Blank” nanosnares were created using the same method without sCD4 or melittin.

[0075] In the resulting products PFC-NP comprising melittin contained about 20,000 melittin molecules per nanosnare and PFC-NP coupled to CD4 contained about 20-30 CD4 molecules per nanosnare.

[0076] HIV-1 producing 293T cell supernatants were harvested 48 h postlipofection, filtered, and assayed for p24 antigen content by enzyme-linked immunosorbent assay. Viruses were resuspended in culture media, aliquoted and stored at −80° C. Equal amounts of virus based on p24 content were used in each experiment.

[0077] HIV-1p134 viral strain was coincubated with blank nanospheres, melittin-bearing nanospheres, CD4-labeled nanospheres or CD4-melittin containing nanospheres at the same concentration levels of nanoparticles in each case. Ninety (90) μL of virus were added to 10 μL of the various nanoparticulate stocks. The final concentration with regard to nanosnares containing melittin is 0.06 mM melittin. To test whether the virus particles can be captured by the nanospheres, advantage is taken of the comparatively large size of the nanoparticles as compared to virus such that the nanoparticles are segregated by centrifugation at 1000×G. To detect capture of the virus, the level of the viral protein p24 in the supernatant and precipitate was compared. The initial p24 value was the same for every sample, confirming that the same level of virus particles was present in each sample.

[0078] As shown in FIG. 4, CD4-containing nanospheres show a larger percentage of p24 in the precipitate as compared to supernatant in contrast to nanospheres that do not contain CD4.

Example 5

Effect of Melittin-Containing Particles on Sperm Motility and Viability

[0079] Human semen samples were obtained from the Washington University in vitro fertilization laboratory and stored at 37° C. until use. Semen was diluted in EmbryoMax® Human Tubal Fluid (HTF) (Millipore) and free melittin or melittin-coupled nanoparticles in HTF was added to achieve a final sperm concentration of 10 million sperm per mL. (For nanoparticles, samples were pipetted up and down 3 times every 10 minutes to keep the nanoparticles suspended.) Samples were incubated in 96-well plates for 30 minutes at 37° C. with shaking at 150 rpm to prevent sperm from settling. Afterwards, Viadent® stain (Hoechst 33258, Hamilton Thorne) was added to achieve a final stain concentration of 10 μg/mL and samples were incubated for an additional 5 minutes. Sperm motility and viability were determined using an IVOS® Computer Assisted Sperm Analyzer (Hamilton Thorne).

[0080] The results are shown in FIGS. 5A-5D. As shown, both sperm viability and sperm motility are impeded in free melittin, but the presence of melittin on nanoparticles in comparable amounts shows no effect. The ability of nanoparticles to protect sperm (and endothelial cells) but not viruses from melittin poisoning is explained by the difference in size of the virus as compared to sperm or epithelial cells. These results indicate that sperm targeting is needed to effect inhibition of motility and viability of sperm. Means to provide sperm-targeting are illustrated in the next example.

Example 6

Successful Sperm Targeting

[0081] This example shows successful sperm targeting by the sperm-specific antibody SPAM1 (Sigma) as compared to control rabbit IgG (Thermo Scientific).

[0082] Sperm samples were centrifuged at 400 g for 5 minutes, the supernatant removed and the cells were fixed with 4% paraformaldehyde for 20 minutes at room temperature.

[0083] Sperm were again centrifuged at 400 g for 5 minutes, the supernatant removed and the pellet blocked in PBS, 2% BSA, 5% normal serum (serum of the animal in which secondary antibody was generated) for 60 minutes at room temperature. 0.5% Triton® X-100 was added if needed to permeabilize the sperm.

[0084] Sperm were again centrifuged at 400 g. The supernatant was removed and incubated overnight at 4° C., suspended in PBS, 2% BSA containing either the anti-SPAM1 antibody (Sigma-Aldrich, 1:100 dilution, 2 μg/mL) or control rabbit IgG (Thermo Scientific, 2 μg/mL).

[0085] After washing the pellet three times in PBS, 2% BSA at room temperature, the pellets were incubated in PBS, 2% BSA containing the appropriate secondary antibody (e.g., Alexa Fluor®-488 goat anti-rabbit IgG, Invitrogen, 1:250 dilution) or TO-PRO® dye (Invitrogen, 1:500 dilution, 2 μM) for 45 minutes at 4° C., and again washed three times in PBS at room temperature.

[0086] Five μL of the stained solutions were placed on a slide and smeared by passing another glass slide over the surface, and covered with a cover slip containing 15 μL of Vectashield® (Vector Labs), sealed with nail polish and visualized using confocal microscopy (60× objective).

[0087] The results are shown in FIG. 6 demonstrating that the SPAM antibody successfully targets sperm.



KR20150049865
METHOD FOR ISOLATING MELITTIN FROM BEE VENOM

   
Inventor: CHUNG BONG YOUL, et al.

[0001] The present invention relates to a method of separating melittin from bee venom, and more particularly to a method and a pure bee venom melittin from separation by using a cation-exchange resin yield.

[0002] The venom has been recognized medical treatment effect from the example. 4th century BC Hippocrates is called the bee venom as a "strange and even mysterious about", was intended for use in the treatment of inflammation such as arthritis. Also, our country was treating the disease as bongryo from Koguryo of progenitor Dongmyeong of Goguryeo time, when the Shilla sinmunwang time and Huh authored the Donguibogam ryeoteumyeo dozen diseases with needle in court, Emperor Gojong when using bee venom seen as a treatment of civilians needle It was our deep-rooted folk medicine.

[0003] Recent studies, bee venom has anticancer effects, rheumatoid arthritis, multiple sclerosis or arthritis pain in degenerative diseases such as relaxation, lower blood pressure in the blood lymph cells and the regeneration and growth of red blood cells, stimulate the adrenal adrenaline secretion, the local area It is known that the injection operation and an improvement in the bactericidal activity of blood reflux.

[0004] It should be noted is that bee venom treatment is 10 000 allergy sufferers because it does cause hives or difficulty breathing as one out of every people must have the prior allergic reaction test. There are also symptoms of itchy swollen after bee venom therapy, also it algi the body aches badly.

[0005] is a major component of bee venom, which is over 40 peptides, carbohydrates, enzymes, reported that consists of amino acids, of which 40-50% is melittin, a phospholipase A2, accounting for 10-12%.

[0006] melittin is hemolytic action, as well as antibacterial and antifungal action and this, in recent years anti-cancer activity, anti-inflammatory, anti-pain action, a number of effects as the compositions of antiviral agents has been reported. On the other hand, it phospholipase A2 (phospholipase A2: PLA2) is the blood pressure reduction, blood flow improving effect it is hemolytic action, to liberate the phospholipids of cell membranes makes cells stronger. In particular, the anti-inflammatory effects of bee venom is melittin, APA Min, mast cell granules reduced peptide (MCD peptide), ahdol Lapin, it is known that protease inhibitors such acts.

[0007] However, phospholipase A2, causes inflammation, which is known as the most powerful allergen of bee venom components. In addition, 0.5 ~ 1% of the histamine (histamin) may cause irritation and itching. APA is a civil and mast cell granules are reduced peptide neurotoxins block the potassium channels in neurons.

[0008] Accordingly, to remove the toxic component from the bee venom, and to develop a method that can separate only melittin has been urgently required.

[0009] In the Republic of Korea Patent No. 10-0744755 call, the bee venom into the buffer solution and then centrifuged to remove the surface lipids, the supernatant of pH 3.0 ~ pH 4.0 acetate buffer as the first gel filtration chromatography to Meli Separate the tin, and the separated melittin with hydrochloric acid solution the second gel filtration chromatography to obtain the desalted and then neutralized with NaOH, so that the cause of the enzyme phospholipase A2 in allergic generation-free method to remove the melittin It is disclosed.

[0010] However, the method has a low yield of pure melittin melittin and phospholipase this resolution of the lipase A2 is not good, go through desalination and the neutralization step because the process is complex, and the purification time is long, the purification efficiency is low Issues have.

[0011] Republic of Korea Patent Registration No. 10-0744755

[0012] The present invention has been made to solve the above problems, one object of the present invention to provide a method of separation in high yield from the pure bee venom melittin.

[0013] Another object of the invention is to provide a process for easily producing a high efficiency from the bee venom melittin to pure.

[0014] In one embodiment of the present invention relates to a method of separating from the bee venom melittin, separation method of the present invention

[0015] (i) of bee venom is dissolved in distilled water or a buffer solution and then centrifuged to remove the precipitate and to obtain a bee venom solution jijilcheung and purified by taking the middle aqueous layer.

[0016] (ii) solidifying the equilibrium phase with the distilled water or a buffer solution and injected into a cation exchange resin column purified bee venom solution;

[0017] (III) to the distilled water or a buffer solution, a salt solution containing from 0.2 to 5 M in a step of 100% to the slope (gradient) eluted in 0.

[0018] In one embodiment of the invention, the venom is a shock hazard in that it contains doknang bee, physical methods, chemical methods such as extraction by obtained by commercially available.

[0019] There is bee venom is added to the distilled water or a buffer solution in step (i) have to be limited to that amount, considering the convenience of working with distilled water or buffer solution, 100 parts by weight of 0.5 to 10 parts by weight is added to the It is preferred.

[0020] In one embodiment of the present invention, distilled water or a buffer solution may be a neutral or alkaline, melittin and pH 7 to 12 in the resolution aspect of phospholipase A preferably, pH 10 to 11.5 is more preferred .

[0021] The buffer solutions include phosphate buffer solution, Tris buffer solution, potassium chloride buffer solution and the like can be used, but is not limited thereto.

[0022] The distilled water is added to adjust the pH, such as sodium hydroxide.

[0023] The distilled water or a buffer solution, it is preferable to use the same solution in step (i) to (iii).

[0024] The purified bee venom solution obtained in step (i) can remove impurities that are insoluble passed through a 0.2 to 0.45 um filter.

[0025] In addition, the purified bee venom solution can be used in dialysis for one night at 2 to 25 ° C using a dialysis membrane.

[0026] As the step (ii) a polymer cation exchange resin is introduced the functional group with a cation exchange capacity in the, polymer matrix, for example, polystyrene (polystyrene), polyphenolic (polyphenolic) resin, cellulose (cellulose ), polyacrylamide (polyacrylamide), the test is, sepharose (Sepharose), and the like can be used however, and the like. As the functional group with a cation exchange capacity, for example, RCH2SO3 <-> H <+>, C6H5SO3 <-> H <+>, C6H5PO3 <2 -> (Na <+>) 2, RCOO <-> Na <+>, C6H5CH2N (CH2COO <-> H <+>) 2, carboxymethylcellulose (Carboxymethyl), phosphate (phosphate), sulfoethyl (sulfoethyl), sulfopropyl (sulfopropyl), diethyl (2-hydroxypropyl quaternary amino) (diethyl (2-hydroxylpropyl quaternary amino)), and the like can be used however, and the like. Preferably phosphate-Sepharose or Sepharose carboxymethylcellulose may be used.

[0027] was observed melittin is shown to bind to the cation exchange resin at pH 12 or less, the higher the pH of binding was weaker. This is because the isoelectric point (pI) of melittin is pH 12.2.

[0028] On the other hand, phospholipase A2 was passed through the column without binding as the cation exchange resin is at pH 10 to 12. Therefore, according to the present invention can be completely separated from the phospholipase A2 from melittin.

[0029] step (iii) in step (ii) with distilled water or 0.2 to 5 100% gradient of solution at 0 was added a salt of M for buffer in the (gradient) elution ionic strength in accordance with the salt concentration (conductivity ) it can be separated and combined components to the cation exchange resin by. The higher the bonding strength, while increasing the salt concentration of the components can be separated.

[0030] The salts that can be used without restriction as the ionic strength is adjustable, sodium chloride (NaCl) is preferred.

[0031] The eluted fractions containing the melittin in the above step (iii) can be confirmed through electrophoresis, HPLC of the standard sample.

[0032] Thus, the collection of fractions containing only the melittin and melittin can be obtained a highly pure lyophilized. The resulting melittin is phospholipase A2 is toxic ingredients such as antimicrobial agents removed completely without side effects occur, such as allergic, anti-inflammatory drugs, anti-cancer drugs, anti-viral agents can be used effectively, cosmetics, quasi-drugs, and more.

[0033] According to the present invention, a highly purified melittin does not contain other components in addition to from bee venom melittin it can be isolated in a high yield. Separation method of the present invention can be simply prepared at a high efficiency from the bee venom melittin to pure using a cation exchange resin column.

[0034] Figure 1 is a diagram showing the purified bee venom in Example 1, the cation exchange resin column chromatography to give the result. 2 is a diagram showing the electrophoresis results of the purified bee venom fraction separated cation exchange resin column chromatography in Example 1. 3 is a view showing the purified bee venom in Example 2 cation exchange resin column chromatography to give the result. Figure 4 is a view showing the purified bee venom in Example 2 cation exchange resin column chromatography to obtain the electrophoresis results of the separated fractions. 5 is a view showing the purified bee venom in Example 3, the cation exchange resin column chromatography to give the result. 6 is a diagram showing the electrophoresis results of the fractions isolated by chromatography cation-exchange resin column of purified bee venom in Example 3. 7 is a view showing the purified bee venom in Example 4 cation-exchange resin column chromatography to give the result. 8 is a diagram showing the electrophoresis results of the purified fraction isolated by our bee venom in Example 4 was purified by cation-exchange resin column. 9 is a view showing the purified bee venom in Example 5, the cation exchange resin column chromatography to give the result. 10 is a diagram showing the electrophoresis results of the purified fraction isolated by our bee venom in Example 5 was purified by cation-exchange resin column. 11 is a view showing the effect of N- terminal sequence analysis of the melittin fraction obtained in Example 1-5. 12 is a view showing the results of HPLC analysis of the melittin fraction obtained in Example 1-5.

[0035] will hereinafter be described more specifically to the present invention by way of example. These examples are only for illustrating the present invention, the scope of the present invention is not limited to these examples it is apparent to those skilled in the art.

[0036] Example 1:

[0037] dissolving the venom 10 mg of distilled water (pH 7.0) in 1 ml and bee venom is making sure that the molten, separated 30 minutes centrifugation at 3,000 rpm, and using a syringe and were taken to the middle of the aqueous phase between the sludge and jijilcheung . Then, the dialysis membrane (1kDa dialysis tubing, Cellu Sep) dialysis was carried out overnight at 4 ° C using.

[0038] The injection of purified bee venom solution to cation exchange resin column (SP capto Impres, GE) was then equilibrated with distilled water (pH 7.0). After check the balance, distilled water and eluting solution a gradient with 0 to 100% 1 M NaCl in the (pH 7.0) (gradient) was separated melittin.

[0039] Measurement of each eluted fraction in absorbance 280 nm and to determine the composition of each fraction in electrophoresis and Coomassie blue staining (Coomassie blue staining), are shown the results in Fig. 1 and 2, respectively.

[0040] can be seen from the results, the binding at pH 7.0 to phospholipase A2 and both melittin a cation exchange resin, one when the elution gradient to M NaCl in sequence phospholipase A2 fraction and melittin fraction is obtained load there was.

[0041] Example 2:

[0042] distilled water, except that (pH 7.0) using 10 mM sodium phosphate (sodium phosphate) pH 10.6 in place, the separation of melittin in the same manner as in Example 1, and measured the absorbance of each of the eluted fractions Check the ingredients and the results are shown in FIGS.

[0043] From the above results, Phospholipase A2 from pH 10.6 is removed without binding to flowthrough the cation exchange resin, when 1 M NaCl elution gradient in the load was confirmed melittin fraction is obtained.

[0044] Example 3:

[0045] distilled water (pH 7.0) but using 10 mM sodium phosphate (sodium phosphate) pH 11.5 in place, the separation of melittin in the same manner as in Example 1, and measured the absorbance of each of the eluted fractions The result is to make the components shown in Fig. 5 and 6.

[0046] From the above results, Phospholipase A2 from pH 11.5 is removed without binding to flowthrough the cation exchange resin, when 1 M NaCl elution gradient in the load was confirmed melittin fraction is obtained.

[0047] Example 4:

[0048] Except for using the 10 mM KCl pH 12 instead of distilled water (pH 7.0) and are separated by melittin in the same manner as in Example 1, and that by measuring the absorbance of each of the eluted fractions were confirmed component The results are shown in Figure 7 and 8.

[0049] From the above results, Phospholipase A2 from the pH 12 is removed in the flowthrough without bonding the cation exchange resin, when 1 M NaCl elution gradient in the load was confirmed melittin fraction is obtained.

[0050] Example 5:

[0051] Except for using a distilled water (pH 12) (the solution was added sodium hydroxide to a final concentration of 5 mM in distilled water) instead of distilled water (pH 7.0), and then remove the melittin in the same manner as the Example 1, and , measuring the absorbance in each eluted fraction. The results confirm the components 9 and shown in Fig.
[0052]

[0052] From the above results, Phospholipase A2 from the pH 12 is removed in the flowthrough without bonding the cation exchange resin, when 1 M NaCl elution gradient in the load was confirmed melittin fraction is obtained.

[0053] Experimental Example 1:

[0054] the melittin fraction obtained in Example 1-5 was quantified using BCA Kit result, 2.2, 2, 3.5, 3.5, and it was confirmed Latin pure melamine is obtained a load of 3 mg.

[0055] Experimental Example 2:

[0056] Except for using bee venom 100 mg and is in Example 1 to the same manner as 5 to remove the melittin, and the obtained melittin fraction was quantified using a BCA kit results, respectively, 25, 28, 34 33, it was confirmed Latin pure melamine is obtained a load of 30 mg.

[0057] Example 3:

[0058] Except for using the readings from 1 g and is, in Example 1 to the same manner as 5 to remove the melittin, and the obtained melittin fraction was quantified using a BCA kit results, respectively, 243, 280, 340 , 348, 330 mg of Latin was pure melamine is obtained to check luggage.

[0059] Example 4:

[0060] Example 1 to the N- terminal sequence analysis obtained in the melittin fraction. 5, and the results are shown in Fig. Results confirmed the N- terminal sequence was as follows.

[0061] Gly-Ile-Gly-Ala-Val

[0062] is an amino acid sequence identified in the test was the same as that found in the literature (http://en.wikipedia.org/wiki/Melittin).

[0063] In addition, by analyzing the melittin fraction by HPLC, and the results are shown in Figure 12. Control group was tested using a commercially melittin (M2272, Sigma) and phospholipase A2 (P9279, Sigma).

[0064] melittin to sell melittin fraction and commercially obtained from 1-5 Example of RT (7.3 ~ 7.4 min) was found to have the same. In addition, phospholipase A2 was not detected at all.



WO2014196674
PREPARATION METHOD FOR ISOLATED, PURIFIED BEE VENOM HAVING ALLERGIC COMPONENTS ISOLATED

   
Inventor: SHEN CHANG ZHE / WON HA YOUNG

The present invention relates to a preparation method for isolated, purified bee venom having allergic components isolated, capable of preparing the isolated, purified bee venom fundamentally blocking allergy induction while maintaining a pharmacological effect by reducing or removing allergy-inducing components in bee venom, as an ultrafiltration method. The present invention comprises: a first step for arranging purified bee venom powder; a second step for preparing a bee venom solution by mixing the purified bee venom powder with distilled water; a third step for removing the allergic components included in the bee venom solution by filtering the bee venom solution through an ultrafiltration membrane having a cut-off size of 10kDa (kilo-Dalton); and a fourth step for harvesting the isolated, purified bee venom having the allergic components removed, wherein, in the second step, the distilled water being mixed with the purified bee venom powder corresponds to 1000ml to 1500ml per 1g of the purified bee venom powder, in the third step, the allergic components removed through the ultrafiltration membrane are A2 (phospholipase A2) and hyaluronidase, and, in the fourth step, the harvested isolated, purified bee venom comprises 4% by weight or more of apamin and 50% by weight or more of melittin.



KR101364506
PREPARATION OF BEE VENOM REMOVED ALLERGIC INGREDIENTS

The present invention relates to a method for producing an isolated and purified bee venom without allergenic ingredients to produce the isolated and purified bee venom having a medicinal effect while basically preventing allergy induction by reducing or removing the allergenic ingredients in the bee venom using ultrafiltration. The method comprises: a first step for preparing the purified bee venom powder; a second step for making a bee venom solution by mixing the purified bee venom powder and distilled water; a third step for removing the allergenic ingredients contained in the bee venom solution by filtering the bee venom solution through an ultrafiltration membrane with cut-off size of 10 kDa; and a fourth step for obtaining the isolated and purified bee venom without the allergenic ingredients. At the second step, 1000-1500 ml of the distilled water is mixed with 1 g of the purified bee venom powder. At the third step, the allergenic ingredients removed through the ultrafiltration membrane are phospholipase A2 and hyaluronidase. The isolated and purified bee venom obtained at the fourth step contains at least 4% of apamin and at least 50% of melittin. [Reference numerals] (AA) START; (BB) END; (S11) Preparing purified bee venom powder; (S12) Making a bee venom solution by mixing the purified bee venom powder and distilled water; (S13) Removing the allergenic ingredients contained in the bee venom solution by filtering the bee venom solution through an ultrafiltration membrane with cut-off size of 10 kDa; (S14) Obtaining the isolated and purified bee venom without the allergenic ingredients



WO2013135103
POLYPEPTIDE CARRYING MELITTIN, NANOPARTICLE CARRYING MELITTIN AND USE THEREOF
   
Inventor: LUO QINGMING, et al.

[0001] The present invention relates to the allergenic ingredient separated purified bee venom production method, and more particularly essentially blocked by a holding while causing allergic pharmacological effects to reduce or eliminate allergens component of bee venom by ultrafiltration method purified bee venom allergy components which can be produced is directed to a method for producing separated purified bee venom.

[0002] The venom and bee venom with Iran, relieve arthritis, arteriosclerosis relief, back pain relief, treatment of skin wounds, antibacterial and anti-inflammatory action to strengthen the immune system, as well as the purpose of beauty even skin It is known to participate in the whitening and anti-wrinkle effects.

[0003] The purified bee venom (Purified Bee Venom, PBV) is this venom collected from the bees using bee venom gathering unit process rough results such as natural drying, accept purification, sterile and lyophilized, purified bee venom and bee venom's bioactive It is known to be similar.

[0004] The composition has the physiological activity of purified bee venom is shown in the following Table 1.

[0007] The purified bee venom are peptides, proteins (eg. Enzymes) and including an active amine, such as a low molecule is composed of 40 or more materials, the main active ingredient as shown in Table 1, peptide (apamin that the molecular weight is comprised of the following 11kDa (kilo-Dalton), melittin, MCD ), the active amine (histamine, dopamin) and amino acids.

[0008] The protein in the composition is from about 13kDa or more bee venom phospholipase that has the molecular weight of A2 (Phospholipase A2, PLA2), it is a hyaluronic claim (Hyaluronidase), phosphatase (phosphatase), a-glucosidase (α-Glucosidase ) is composed of components such as, primarily blood cell membrane destruction, blood clotting, vasodilatation, and transmitted, promoting blood circulation, etc. The physiologically active role in promoting the hydrolysis of the protein,

[0009] In particular, to a phospholipase A2 and hyaluronidase venom is a composition for inducing a powerful allergic reaction, which is known to cause a serious safety problem, the user ought to be with a hypersensitivity to bee venom. (Stefan Bogdanov; Bee Venom: composition, Health, Medicine: A Review, Bee Product Science (2011))

[0010] and to limit the effective dose of the venom Because of this side effect, and the dosage of the physiologically active effects of bee venom is venom also limited to a problem can not be completely limited depending on the implementation.

[0011] Patent Publication No. 10-2012-0003178 discloses that disclosure relates to a composition for skin whitening and moisture containing bee venom extract, pre-treatment of bee venom or bee venom extract extracted from bee venom, and to do this as an effective component, skin whitening, and skin It discloses a composition or cosmetic composition which has a moisturizing effect.

[0012] However, in the prior art as described above do not include the process of removing it by phospholipase A2 and hyaluronidase for inducing a strong allergic reaction the composition of the readings from the extract prepared by conventional techniques with the active ingredient and a cosmetic composition, if the user uses with a hypersensitivity to the bee venom can cause a serious safety problem.

[0013] In addition, also the methods for the addition of inhibitors designed to inhibit the allergic reactions, allergic reactions to the conventional, but which do not address the fundamental allergies, side effects caused by inhibitors of allergic reactions, which can cause a problem.

[0014] Also, to a conventional bee venom by phospholipase A2 and hyaluronidase a way as to remove the HPLC (high performance liquid chromatography), or although the gel filtration (gel filtration) methods, separation or purification process according to the progress of the yield is dramatically low, it is difficult to mass production problem.

The present invention has been made to solve the problem described above, Phospholipase inducing strong allergic response A2 is a (Phospholipase A2) and hyaluronic by removing the (Hyaluronidase) ultrafiltration to from purified bee venom, allergic reactions from basically the provision of purified bee venom production method capable of producing a secure purified bee venom in order. The present invention is separated and purified bee venom produced that can be produced basically safe purified bee venom from allergic reactions by reducing the delete is the in hyaluronic inducing a strong allergic reaction to purified bee venom ultrafiltration, and Phospholipase A2 to provide a method of the object. The present invention is to increase the yield by removing a component contained in the purified bee venom allergy ultrafiltration, and to provide an easy mass-production method for producing purified bee venom in order. Another object of the present invention is to provide cosmetics containing the purified bee venom comprises a venom of the present invention, separation and purification. However, an object of the present invention is not limited to the above-mentioned object, another object is not mentioned will be apparent to those skilled in the art can be understood from the following description.

Purified bee venom production process according to the present invention in order to achieve the object described above, the first step, the second step is mixed with distilled water, the purified bee venom powder for producing a bee venom solution, the bee venom solution to prepare purified bee venom powder a cut-off (cut-off) size 10kDa (kilo-Dalton) the ultrafiltration membrane (ultrafiltration membrane) to the filtration step 3 to remove the allergic (allergy) component included in the bee venom solution and the allergic component is canceled a fourth step of obtaining a purified bee venom, and wherein in the second step, the distilled water is mixed with the purified bee venom powder is 1000㎖ to 1500㎖ per 1 g of the powder purified bee venom, in the third step, the ultrafiltration an allergic component is removed through the membrane is a Phospholipase A2 (Phospholipase A2) and is a hyaluronic claim (Hyaluronidase), in the fourth step, the obtained said purified bee venom is pain Min (apamin) at least 4% by weight, mellitic The tin (melittin) and characterized in that it comprises at least 50% by weight. In addition, the purified bee venom production process according to the present invention are tablets comprising the steps of: preparing a bee venom powder, the purified second step the bee venom powder was mixed with distilled water to prepare a bee venom solution, the cut-off size of the bee venom solution 30kDa ultrafiltration a fourth step of obtaining the third phase, and the purified bee venom the allergic component is canceled, which was filtered in the filtration membrane to remove the allergenic components contained in the bee venom solution, wherein in the second step, the purified bee venom powder and The mixture is distilled to a 100㎖ 200㎖ per 1 g of the powder purified bee venom, in the third step, the allergenic component is removed through the ultrafiltration membrane is a hyaluronic claim, and in the fourth step, the separation and purification of the obtained Bee venom is pained Min 2.5% by weight, at least 45% by weight of the melittin and the phospholipase A2 characterized in that it comprises 1% by weight or less. In addition, cosmetics containing purified bee venom according to the invention is characterized in that it comprises the purified bee venom of the present invention as an active ingredient.

According to purified bee venom production method of the present invention, which induces a strong allergic reaction Phospholipase A2 is a (Phospholipase A2) and hyaluronic by removing the (Hyaluronidase) ultrafiltration to from purified bee venom, essentially separated safe from allergic reactions It has the advantage that it can produce a purified bee venom. Further, according to purified bee venom production method of the present invention, the purified bee venom ultrafiltration remove to the in hyaluronic inducing strong allergic reactions, Phospholipase A2 possible reduction in bee venom essentially safe separation and purification from the allergic response to There is the advantage that can be produced. Further, according to purified bee venom production method of the present invention, the components contained in the purified bee venom allergy increased the yield by removing by ultrafiltration, and mass production is easy advantage. Further, according to purified bee venom containing cosmetic of the present invention, purified bee venom Preparation of purified include bee venom, as an active ingredient the anti-wrinkle effect and represents the whitening effect and at the same time, allergic reactions contained in bee venom according to the method of There is benefit from the essentially secure.

[0027] Figure 1 is a flow diagram of a method for producing purified bee venom according to the exemplary embodiment of the present invention. Figure 2 is a flow diagram of a method for producing purified bee venom according to another embodiment of the present invention. 3 is an electrophoresis image showing the results of tests according to the experimental example of the present invention. Figure 4 is a HPLC (High-performance liquid chromatography) analysis graph of purified bee venom components in accordance with Experimental Example 2 of the present invention. 5 is a graph showing a test result of wrinkle-improving effect of purified bee venom according to Example 3 of the present invention. 6 is a graph showing the test results, the whitening effect of purified bee venom according to Example 4 of the present invention.

[0028] Hereinafter, a detailed description of the preferred embodiments of the invention will be described with reference to the accompanying drawings. In the following description, well-known functions or specific description of the configuration is determined to unnecessarily obscure the subject matter of the present invention, a detailed description thereof will be omitted.

[0029] The embodiment according to the concept of the present invention can be applied to a variety of changes can have a variety of forms, so will be described in detail herein and illustrated in the drawings certain embodiments or applications. However, this is not intended to limit the particular form disclosed embodiments of the concept of the present invention, it should be understood to include all modifications, equivalents and substitutes included in the spirit and scope of the present invention.

[0030] an element is "connected" to another element may or "connected" that, when referred to, but that is directly connected or may be connected to other components, other components are present in the medium it should be understood that there may be. On the other hand, when an element is "directly connected" to another element or "directly connected" that mentioned, it is to be understood that the other component is not present in between. Components other representation for explaining the relationship between, that is to be interpreted "between" and "directly between", etc. Similarly, "directly adjacent to" or "- the neighboring" and.

[0031] As used to describe particular embodiments only term used herein, it is not intended to be limiting of the present invention. Expression in the singular is not clearly different meaning in the context, include a plural meaning. In this specification, "comprise" or "gajida" and the term is intended to designate a staking features, numbers, steps, operations, elements, parts or combinations thereof is present, one or more other characteristics, numbers , steps, operations, elements, the presence or addition of parts or a combination of these ones but do not preclude.

[0032] Figure 1 is a flow diagram of a method for producing purified bee venom according to the exemplary embodiment of the present invention.

[0033] As purified bee venom production method according to one embodiment of the invention shown in FIG. 1, it is possible to prepare the purified bee venom powder (S 11). In this case, be prepared with purified bee venom Bee venom powder can be collected from seoyangjong bees (Apis Mellifera).

[0034] Here, there purified bee venom powder can be prepared by conventional various methods, specifically, the bee venom collected from bees by bee venom collection device to air-dry, acceptable tablet, purified bee venom powder through the process of sterilization and freeze-dried It can be prepared, the thus prepared powder was purified bee venom and bee venom may be similar to the physiological activity is collected from bees, but the present invention is not limited thereto.

[0035] Next, the mixed powder and distilled water to purified bee venom and bee venom solution is prepared (S12). At this time, the distilled mixture is preferably purified bee venom 1000㎖ to 1500㎖ per 1 g of powder.

[0036] Here, when the amount is less than 1000-ml of distilled water are mixed, the viscosity of the bee venom solution is increased, and thus the permeability of the bee venom solution to the ultrafiltration membrane will be described later filtration (ultrafiltration membrane) is lowered, to lower the yield after filtration There we have a problem. Further, if the amount exceeds 1500 ml of distilled water are mixed, amount of time required for the filtration and drying of the bee venom solution is uneconomical because the longer a problem.

[0037] In addition, the yield of purified bee venom in the case of distilled water to adjust the mixed powder 1000㎖ to purified bee venom 1500㎖ per 1g may be greater than or equal to 60%.

[0038] The bee venom solution and the cut-off (cut-off) was filtered with the size 10kDa (kilo-Dalton) the ultrafiltration membrane (ultrafiltration membrane) to remove the allergenic components contained in bee venom solution (S 13).

[0039] an approximate molecular weight of the tablet is the same as the major component of bee venom powder following Table 2.

[0040] [Table 2]

[0042] As shown in Table 2, which is known as a strong allergic component Phospholipase A2 (Phospholipase A2) and is in the molecular weight of the hyaluronidase (Hyaluronidase) is approximately 13kDa and 38kDa, respectively. The ribosome, the mitochondria, the case of a biomolecule, such as viruses, in general, to the molecular weight Dalton (Dalton) indicates the approximate molecular weight, and estimate the size (size) in the molecular weight.

[0043] Thus, purified bee venom production method according to one embodiment of the present invention, by setting the ultrafiltration membrane cut-off size of the 10kDa, in such a way as to not pass through the material having a higher size, allergic component of force included in the bee venom solution You can remove the fabric first with lipase A2 and hyaluronidase. In addition, filtration, by applying pressure to the bee venom solution was filtered quickly proceed, it is possible to reduce the filtration time.

[0044] to obtain the back, purified bee venom allergy component removed (S14). At this time, the resulting purified bee venom is pain Min (apamin) at least 4% by weight, melittin (melittin) and may include at least 50% by weight.

[0045] Figure 2 is a flow diagram of a method for producing purified bee venom according to another embodiment of the present invention.

[0046] purified bee venom production process according to another embodiment of the present invention 2, the first preparation of purified bee venom powder (S21). At this time, purified bee venom powder can be prepared in the same manner as the purified bee venom powder according to the exemplary embodiment of the present invention.

[0047] Next, the mixed powder and distilled water to purified bee venom and bee venom solution is prepared (S22). Here, the distilled mixture is preferably purified bee venom 100㎖ to 200㎖ per 1 g of powder.

[0048] and filtered bee venom solution with a cut-off size of 30kDa ultrafiltration membrane for removal of the allergenic component included in the bee venom solution (S23).

[0049] In particular, when the amount of less than 100 ml of distilled water are mixed, the viscosity of the solution increased and the bee venom thus ultrafiltration membrane permeability is lowered, there is a problem that can lower the yield after filtration. Further, if the amount of more than 200 mL of distilled water to be mixed, with reference to Table 2, the concentration of the allergenic component of the Phospholipase A2 and dilution, ultrafiltration membrane for phosphorylation strong allergic component is increased because the transmission of the lipase A2 to the phospholipase A2 has a problem that can not be reduced as much as possible.

[0050] Thus, purified bee venom production process according to another embodiment of the present invention in a distilled water, purified bee venom 100㎖ to 200㎖ per 1g powder to be mixed to produce the bee venom solution, hyaluronic by setting the ultrafiltration membrane cut-off size of the 30kDa removing Ronnie oxidase and a phospholipase A2 can be reduced as much as possible. In addition, filtration, by applying pressure to the bee venom solution was filtered quickly proceed, it is possible to reduce the filtration time.

[0051] to obtain the back, purified bee venom allergy component removed (S24). At this time, purified bee venom that is obtained may include pain Min (apamin) of 2.5% or more, melittin (melittin) at least 45% by weight, the phospholipase A2 is 1 wt% or less.

[0052] As described above, according to purified bee venom production method according to one embodiment of the invention, the force leading to a strong allergic reactions included lipase A2 (Phospholipase A2) and hyaluronidase is ultrafiltration from the purified bee venom claim (Hyaluronidase) in by removing by, there is an advantage to essentially produce a safe separation and purification from the venom allergies.

[0053] Further, according to purified bee venom production process according to another embodiment of the present invention, the purified bee venom ultrafiltration remove to the in hyaluronic inducing strong allergic reactions and possible reduction in allergic response of Phospholipase A2 from the advantage that you can basically prepare a safe purified bee venom.

[0054] Further, according to purified bee venom production method of the present invention, the components contained in the purified bee venom allergy increased the yield by removing by ultrafiltration, and mass production is easy advantage.

[0055] The cosmetics include purified bee venom containing purified bee venom according to another example prepared according to the method of producing a purified bee venom example and another example of the invention of the present invention as an active ingredient.

[0056] As described above, another example of purified bee venom Cosmetics according to the purified bee venom method wrinkles contained in bee venom, including purified bee venom produced by the active ingredient to improve the effect of the invention of the present invention There are basically safe point and represents the whitening effect and at the same time from the allergic response.

[0057] In the following based on the embodiments for the present invention will be described in detail. Presented embodiments are not intended to limit the scope of the present invention as illustrative.

[0059] 1) Preparation of purified bee venom powder

[0060] seoyangjong bees (Apis Mellifera) from bee venom (bee venom) to collect the electric shock method, dissolved the gathering by bee venom in distilled water, the mean filtration with a 3.0 ㎛ of filter paper pores are formed (filter paper) and soil , dust and pollen (花粉), etc. were removed.

[0061] 0.45 [micro] m to 0.2 [micro] m, and filtered through a membrane filter having pores of a (membrane filtration) to remove impurities and the balance was freeze-dried to prepare a powdered purified bee venom.

[0062] 2) separating allergic ingredients

[0063] The distilled water filtered through a membrane filter of 0.2 [micro] m prepared in advance purified bee venom powder and 1: 1000 (g / ml) was prepared by mixing the solution at a ratio of bee venom.

[0064] and using a Millipore series 8400 stirred cell, by a cut-off size of the ultrafiltration membrane filtered bee venom solution of 10kDa (Ultracel PL regenerated cellulose, 76 mm) to remove the allergenic components, to give the purified bee venom. At this time, a pressure of 0.4 MPa, the bee venom solution in the cell and filtered until it is concentrated to about 5 ml to 10 ml.

[0065] Purification of bee venom is obtained in the form of a powder and dried by freeze-drying.

[0067] 1) Preparation of purified bee venom powder

[0068] In Example 2 of the present invention to prepare a purified bee venom powder in the same manner as in the first embodiment of the present invention.

[0069] 2) separating allergic ingredients

[0070] The distilled water filtered through a membrane filter of 0.2 [micro] m prepared in advance purified bee venom powder and 1: 100 (g / ml) was prepared by mixing the solution at a ratio of bee venom.

[0071] and using a Millipore series 8400 stirred cell, by a cut-off size of the filtration membrane of 30 kDa bee venom solution to ultrafiltration (Ultracel, RC 76 mm) to remove the allergenic components, to give the purified bee venom. At this time, a pressure of 0.4 MPa, the bee venom solution in the cell and filtered until it is concentrated to about 5 ml to 10 ml.

[0072] Purification of bee venom is obtained in the form of a powder and dried by freeze-drying.

[0074] Electrophoresis Testing

[0075] 18% or 20% of the gel (gel) composition ((i) 18% of the gel composition: distilled water, 1.3 ml, 30% acrylamide mix (acrylamide Mix) 6.0 ml, 1.5 M Tris-HCl (Tris (hydroxymethyl) HCl-aminomethane) (pH8.8) 2.5 ml, 10% SDS (sodium dodecyl sulfate) 100 [mu] l, 10% ammonium persulfate (ammonium persulfate) 100 [mu] l, TEMED (tetramethylethylenediamine) 4 [mu] l; (II) gel composition of 20% : distilled water 700㎕, 30% acrylamide mix ml 6.6, 1.5 M Tris-HCl (pH8.8) 2.5 ml, 100 [mu] l 10% SDS, 10% ammonium persulfate (ammonium persulfate) 100 [mu] l, 4 [mu] l TEMED create) a , about 10 ml was dispensed into a glass plate degree.

[0076] hyeotgo to match the balance in good the separating gel, the back, staking gel composition of about 5 ml (STAKING gel composition: distilled water, 3.4 ml, 30% acrylamide, mix 830 [mu] l, 1.5 M Tris-HCl (pH 8.8) 630 [mu] l, [mu] l 50% SDS 10, 50 [mu] l 10% ammonium persulfate, TEMED and 5 [mu] l) and dispense, solidified into a comb before the gel hardens. Staking gel is solidified, and then, to remove the comb completed the SDS page gel.

[0077] The sample buffer and SDS page fraction to make a (2 x Laemmli Concentrate, Sigma, S3401) 1: 1 were mixed at a ratio, which was in the water bath of 90 ° C water bath for 5 minutes. And 5 [mu] l of a marker to comb of the gel, based on purified bee venom, separation according to Example 1 and 2 purified bee venom, the pain Min, phospholipase A2 and melittin loading each 30 [mu] l each, and by inserting the gel in SDS page tank It filled the running buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3).

[0078] SDS page power supply running sikyeotgo for about 30min from staking gel to 60V through, was running for about 60-90 min at 120V at separating gel.

[0079] components of the sample were separated by the molecular weight of the gel according to the current flowing in the power supply, coomassie brilliant bule R-250 and stained with the dye and destaining solution The results are shown in figure 3, and the color processing.

[0080] Figure 3 is an electrophoresis image showing the results of tests according to the experimental example of the present invention.

[0081] As shown in Figure 3, the reference tablet bee venom, an embodiment of the present invention 1 and 2 purified bee venom (10kDa, 30 kDa) of, pain Min (apamin 99.9%, STDA), phospholipase A2 (PLA2 90.7% , STDP) and melittin (melittin 96%, STDM) compared the band represents in each electrophoresis test.

[0082] Phospholipase A2 15-20kDa bands may appear on the molecular weight can be removed to check the readings from the separation and purification of Example 1, the purified bee venom in Example 2 it can be seen that reduction is almost non-existent. In addition, the band is of the hyaluronic to 50kDa can be confirmed by showing that removing both the purified bee venom in Example 1 and 2 of the present invention.

[0084] HPLC quantification

[0085] create a purified bee venom and bee venom in isolated and purified in Example 2 of Example 1, each at a concentration of mg 1.0 / ml, HPLC (High-Performance Liquid chromatography) equipment with only peptide analysis column (150 X 4.6 mm 4.0μm , 90Å, phenomenex174;) used, and water (0.2% TFA in water for HPLC) and acetonitrile (0.22% TFA in ACN) were each analyzed with a solvent.

[0086] Figure 4 is a HPLC (High-performance liquid chromatography) analysis graph of purified bee venom components in accordance with Experimental Example 2 of the present invention.

[0087] Remove the HPLC quantitative analysis result of purified bee venom each of Examples 1 and 2 it can be found as a component analysis, purified bee venom compared to the reference shown in Fig.

[0088] More specifically, according to HPLC quantitative analysis of the standard purified bee venom, a peak (peak) of the pain Min (apamin) and melittin (melittin) appeared in each of 10.4 min and 21.3 min, Phospholipase A2 ( phospholipase A2, PLA2) of the peak can be found to appear in 16.3 min.

[0089] The content available to the area of ​​this peak, specifically, is shown in the following [Equation 1].

[0090] [Equation 1]

[0092] Expression Purification Purification results for the readings from the value calculated by applying each one of the reading and the second embodiment of the first embodiment of the sample 1 is shown in the following Table 3.

[0093] [Table 3]

[0095] Therefore, the purified bee venom according to the first embodiment, as shown in Table 3, it can be seen that there is no PLA2. Furthermore, in the separation and purification of the bee venom PLA2 it can be seen that the second embodiment is reduced as much as possible to present in a 1% by weight or less.

[0097] Anti-wrinkle effect (Elastase inhibitory effect)

[0098] In Example 3 of the present invention the addition of substrate, 0.1 mM N-succinly- (Ala) 3-p-nitroanilide 200 ㎕ each purified bee venom and bee venom of the reference tablets of Examples 1 and 2 in 20㎕ and In addition, 0.1 mg / ml of Elastase (Porcine pancreas solution) was added to 10㎕ reacted for 10 minutes at 25 ° C, ELISA (Enzyme-linked immunosorbent assay) by measuring the absorbance at 410 nm as a reader for Elastase By calculating the inhibition rate (%), and confirms a wrinkle-improving effect.

[0099] Figure 5 is a graph showing a test result of wrinkle-improving effect of purified bee venom according to Example 3 of the present invention.

[0100] When confirming the anti-wrinkle effect on the purified bee venom of the reference tablet venom as in Example 1 and Example 2, as shown in Fig. 5, Example 1 and anti-wrinkle effects of purified bee venom in Example 2 It can be seen that 0.2 mg / ml, 0.5 mg / ml, 1 mg / ml, 2 mg / ml and 3 mg / ml indicating that the superior activity than the reference tablet bee venom both in the respective concentrations.

[0102] whitening effect (Tyrosinase inhibitory effect)

[0103] In Example 4 of the present invention to make by dissolving 21.2 mg tyrosine in the substrate solution 0.1 M PBS ml 46.8 (Phosphate buffered saline) (pH 6.8), also, based on the separation of purified bee venom as in Example 1 and 2 readings from each of the tablets were mixed with distilled water to prepare a test solution.
[0104]

[0104] Each of these test liquid 300 ㎕ the substrate solution 225 ㎕ mixed and, tyrosinase enzyme (tyrosinase enzyme is 210 U / ㎖ (diluted in PBS) was used) to 107 ㎕ added (PBS with 900 ㎕ to Fill) by After reaction for 15 minutes in the incubator (incubator) to 37 ° C, using a microplate reader and the absorbance is measured at 480 nm by ELISA, the whitening effect was confirmed.
[0105]

[0105] Figure 6 is a graph showing the test results of whitening purified bee venom according to Example 4 of the present invention.

[0106] When confirming the whitening effect of the purified bee venom of the reference tablet venom as in Example 1 and Example 2, 6, Example 1 and the whitening effect of purified bee venom of Example 2 0.1 mg / ml, it can be seen 0.5 mg / ml, 1 mg / ml, 3 mg / ml, 5 mg / ml, 8 mg / ml and the 10 mg / in each concentration of ml all shown excellent activity than that of the reference tablet venom .

[0108] disk diffusion (Disc diffusion method)

[0109] An experiment based on the purification of different concentrations on the disc (disc) culturing a strain of bee venom, Experimental Examples 1 and 2 was absorbed and purified bee venom of the strain was inoculated in the dispenser on a solid culture medium (dispenser). After 16 to 18 hours of incubation, the amount of bacteria observed eokjedae (inhibition zone) compared with the case of using the antimicrobial whether the control is based on purified bee venom.

[0110] In order to test the antimicrobial effect of the experimental examples 1 and 2 purified bee venom in accordance with Example 5 of the present invention, indicated by the Korea Culture Center of Microorganisms bacteria (gram-positive bacteria of Staphylococcus aureus, Streptococcus mutans and Gram-negative bacteria is Salmonella typhimurium ) it was used as received pre-sale three species.

[0111] In particular, after 24 hours of incubation for bacteria in the liquid-based medium, and activates the indicated bacteria. If the number of bacteria found at 660 nm indicated the OD value of 0.5 to 0.7, a triangular glass rod dispensed and a certain amount of sterilized and plated on solid medium to medium. Place the paper disk on the medium plated the instructions bacteria (paper disc), based on purified bee venom, experimental examples 1 and 2, then loading 40㎕ the concentration of purified bee venom by.

[0112]; for a Petri dish (petri dish) for 18 to 24 hours in the incubator, and the result of comparing the size of bacteria eokjedae (clear zone) are shown in Table 4.

[0113] [Table 4]

[0115] (In this case, S.aureus is Staphylococcus aureus, S.mutans is Streptococcus mutans, S.typhimurium is Salmonella typhimurium)

[0116] Referring to Table 4, the active ingredient of purified bee venom of Examples 1 and 2 of the present invention can determine the indicated antimicrobial effect on the same level compared to the standard tablet bee venom, purified bee venom according to the invention It can be confirmed exhibits a significant antimicrobial activity while essentially blocking to the allergens.
[0117]

[0118] The minimum inhibitory concentration test development - MIC (Minium Inhibitory Concentration)

[0119] Salmonella typhimurium and Staphylococcus aureus strains were cultured for 24 hours in LB (Luria-Bertani) broth, Streptococcus epidermidis strain BHI (Brain Heart Infusion) were used to culture for 24 hours in broth, standard tablet venom as in Example 1, and 2 Purification of readings from each half was diluted with distilled water to prepare the test solution at different concentrations.

[0120] Before inoculation the strains by measuring the absorbance at 650 nm measure the initial concentration of the strain, and added to the pure liquid medium and purified bee venom of the reference tablet venom as in Example 1 and 2 for each concentration of the tube after a certain amount It was inoculated with the strain.

[0121] After 24 hours incubation at 37 ° C, at an OD 650nm was measured to determine the concentration of the sample showing the same effect as the pure liquid medium and the minimum concentration for inhibition of bacteria MIC [1- (absorbance of sample treatment group / control group absorbance) and calculation of the formula X 100 The results are shown in Table 5.



CN102526116
Method for refining bee venom

   
Inventor: WENXIAN CHENG, et al.

A method for refining bee venom comprises the steps: preparing the bee venom, adding water of 5-10 times of the amount of the bee venom into the bee venom to perform dissolution and then perform high speed centrifugation, and water phase is obtained after the centrifugation; adding acetone with the 1-3 times of the volume of the water phase into the water phase to perform standing at the temperature of less than or equal to -8 DEG C until the precipitation finishes completely, adding the acetone in precipitate and smashing the mixture after separation, performing filtering separation and volatilizing the acetone in the precipitate to obtain a rough bee venom product;; using 0.08-0.12mol/L of acetum to dissolve the rough product, then using a sephadex G50 chromatographic column to perform the separation and purification, using acetum with the same concentration to perform elution and using an ultraviolet detector to perform on-line detection at 280nm of wavelength, collecting eluent of a third peak, adding activated carbon to perform slow stirring, and freezing and drying filtrate after the separation. In the refined bee venom, melittin content is greater than or equal to 76%, macromolecule substance content is less than 1.5%, and a lyophilized agent for clinical injection can be directly added.

A method for purifying bee venom, bee venom plus high speed centrifugation after taking 5-10 times the amount of water dissolved, the aqueous phase was separated; the aqueous phase was added 1-3 volumes of acetone at a temperature of ≤-8 ° C under stand to complete precipitation after adding acetone to the precipitate separated and mashed, separated by filtration, sediment shake off acetone to give crude venom; after crude dissolved 0.08-0.12mol / L acetic acid solution with dextran gel G50 isolated and purified by column chromatography, eluting with acetic acid solution of the same concentration, with UV detection at 280nm wavelength line detection, collecting a third eluate peak, activated carbon, stirred slowly, after separation of the filtrate was lyophilized. The purified bee venom, melittin content of ≥76%, the content of macromolecules <1.5%, can be directly applied for clinical injection lyophilized agent.

I. Technical Field

Separation and purification methods of the present invention relates to a venom, specifically refined method of bee venom.

II BACKGROUND ART

Bee venom is secreted by worker bees venom glands having an aromatic odor, stored in the poison sac, when the bees attack other When discharged from the tail sting species, it is a complex mixture, its chemical composition is complex, divided into polypeptides, macromolecules The amount of enzymes, small molecular weight amines and other substances, including peptides melittin (about 50% of bee venom dry weight), Apamin, MCD peptide, cardiac peptide, peptide safely, histamine peptides, enzymes including more than 55 kinds of enzymes substance, wherein the phospholipase A2 (Jane Said PLA2, an average molecular weight of 14,500) is the main substance produced by bees after a sting anaphylaxis, amines including histamine, children Catecholamines and other active amines, amine and produced by bee sting pain.

Bee venom for medical care in the private sector has a long history, modern studies have proven that bee venom has anti-inflammatory and analgesic effect, used to treat Treatment of rheumatoid arthritis, rheumatoid arthritis, ankylosing spondylitis rheumatic diseases, peripheral neuritis, neuralgia and other muscular diseases Disease has curative effect, but venom enzymes large molecular weight substances that can cause allergies, small molecular weight amines easy Causing pain, which greatly influenced the formulation to promote the use of bee venom in the clinic.

CN101088514A discloses a method for purifying a venom was dissolved in ethanol solution of the crude venom of bee venom removed Propolis, honey and other impurities, and then using a solvent extraction method to obtain purified bee venom. First crude venom was added 95% ethanol made from hanging Turbid solution was filtered to remove impurities and the filtrate was centrifuged to remove the liquid phase, the precipitate was dehydrated ethanol was added, the precipitate was then raised Water was added at 10 ± 2 ° C under extraction 12-15HR; extract was precipitated with ethanol, aqueous ethanol is removed, the precipitate was added to 0.15-0.2 Molar ammonium hydroxide solution and extracted with n-butanol, the precipitate was discarded, distilling the extract, the concentrate was freeze-dried to obtain To purified bee venom.

CN1416827A discloses a method for extracting venom. The method of the invention is described as follows: the living body hornet cut Yuchi placed inside a sealing cap, container, add more than 40% concentration of aqueous ethanol or above 40 ° C consumption of wine, to make a living Body hornet completely immersed in it, the vessel stamped seal and let stand at least three days, whichever is the lower part of the milky liquid in the container, filter.

CN101455287A discloses a method for the separation and purification of melittin, bee venom is rough water immersion, and the filtrate washed with ethanol The precipitate, the precipitate with ammonium hydroxide and n-butanol extract was precipitated with acetone, the precipitate was dissolved in urea acetate buffer A solution, the ion exchange column as an elution chromatography, collecting the strongest hemolytic activity under the absorption peak of V column chromatography fractions were concentrated, conc. Condensing fluid by G-10 sephadex column desalted, acetone precipitation, re-dissolving, precipitation desalting treatment, the precipitate was dissolved in acetate buffer Liquid B, in sephadex G-25 column in buffer B was used as a gradient of the absorption peak at the time of collection of concentrated fractions column chromatography Shrinkage, freeze-dried to obtain the electrophoretic desalting level melittin.

CN1088215A discloses a method for rapid separation of bee venom peptides from crude, the crude venom was dissolved in distilled water, Using ultrafiltration and dialysis bags of bee venom were isolated and purified.

III. SUMMARY

The present invention aims to provide clinical purified bee venom injection, the technical problem to be solved is to remove as much venom Enzymes and biogenic amines.
The purification method includes the preparation and purification of the crude venom venom crude, concrete steps are as follows:

1, dissolved: Take venom plus 5-10 times the weight of deionized water was dissolved after high speed centrifugation (3000rpm) 10-15 minutes, pour The supernatant, comprising the residue was brownish-gray residue was dissolved in deionized water together, separated by filtration after high-speed centrifugation, the combined aqueous phases, spare. The effect is to make high-speed centrifugation and the residue was separated grease-like impurity, and float on the surface of the transfer of the aqueous phase.

2, the precipitation: To the above aqueous phase was added 1-3 volumes of acetone at a temperature of ≤-8 ° C was allowed to stand under conditions to precipitate completely (solid Experience has shown that less than 10 hours), pour the supernatant was added to the precipitate and mashed acetone, isolated by filtration, sediment play Acetone will give the crude venom.

Experiments showed that in order to make the peptide melittin based precipitate completely (≥99%), the volume ratio of acetone and aqueous be ≥1, Take 1:1 better, this would save the amount of acetone.

3. Purification: acetic acid concentration 0.08-0.12mol L / dissolve the crude venom on sephadex G50 column to 0.08-0.12mol / L acetic acid solution was eluted with UV-3000 UV detector at 280nm wavelength online testing, when When to start collecting the third peak to peak down to 100mV third stop collecting. Activated carbon was added to the eluent, slowly stir Mixed with 0.5 to 1 hour, separated by filtration, the filtrate was lyophilized to give purified bee venom.

Experiments show that dextran gel G50 can effectively remove large molecular weight PLA2 and HAase (hyaluronidase molecular weight 45,000), and other enzymes, proteins, but also remove small molecular weight amines. The effect is not only activated carbon bleaching, while there Endotoxin removal efficiency, the activated carbon is added in an amount of 0.02-0.05% by mass eluent.

Bee venom Bee venom large molecular weight substances accounted for about 16% of the dry weight, by column chromatography, the content of high molecular weight substances < 1.5%, melittin content ≥76% (crude melittin content about 45%) of the purified bee venom can be directly processed for clinical injection With a lyophilized preparation.

IV. BRIEF DESCRIPTION

Figure 1 is a column chromatography on-line detection of UV spectrum.

V. DETAILED DESCRIPTION

Non-limiting examples are described below:

1.Preparation of (a) crude venom 1, was dissolved
Taking bee venom was added 8 times the weight of deionized water was dissolved, high-speed centrifugation (3000rpm) 12 minutes. The supernatant was decanted to Coupled with the residue deionized water (the same amount of water for the first time) the same method ,, high-speed centrifugal separation by filtration, the combined aqueous phases prepared use.

2, precipitation:
To the above aqueous phase was added 1 volume of acetone, allowed to stand at a temperature of -10 ° C for 12 hours, the supernatant was decanted, the precipitate Was further added acetone and mashed precipitate was filtered, the precipitate hypothermia shake off acetone to give the crude venom.

Refining (b) of crude 3, with 0.1mol L acetic acid solution / dissolution of the crude venom on sephadex G50 column [(2.0-10.0cm) × 100cm], In 0.1mol / L acetic acid solution, flow rate 5-30mL / min, and treated with UV-3000 UV detector at 280nm wavelength into Line online testing, when the third peak appears to start collecting, to the third peak down to 100mV stop collecting, was added to the eluent An appropriate amount of activated carbon, with slow stirring 1h, filtered and the filtrate was lyophilized to give purified bee venom. It was determined that melittin content of 79.56 percent, Macromolecules content of 1.29%.

Content melittin press "Chinese Pharmacopoeia" 2010 edition by HPLC VID prescribed an appendix.

Content of macromolecules using high performance liquid chromatography to measure the following conditions:

Reference: ribonuclease A (molecular weight 13,700). Provided by the Chinese Pharmaceutical and Biological Products.
Column: TSK gel G3000pw gel column (7.5mm × 300mm). Japan TOSOH.
Mobile phase: trifluoroacetic acid - acetonitrile - water mixture, mixing weight ratio of 0.025:30:70, number of theoretical plates ribonuclease A Peak of not less than 1,500.
Column temperature: 30 ° C.
Flow rate: 0.7mL / min.
Detection wavelength: 214nm.

(C) a lyophilized formulation
Mannitol 40g, water for injection to dissolve, add the right amount of activated carbon, heated to boiling for 15 minutes, filtered, and the filtrate cold To room temperature, purified bee venom 0.35g (to melittin dollars), stirred, sterile filtration, quantitative filling made 1,000 bottles (specifications 0.35mg / bottle) or 500 bottles (standard 0.7mg / vial) and freeze-dried to obtain.



US2012082656
COMPOSITION COMPRISING THE PURIFIED FRACTION ISOLATED FROM BEE VENOM FOR PREVENTING AND TREATING OF DEGENERATIVE BRAIN DISEASES

Inventor(s): YOON SEONG TAE, et al
 
PURPOSE: A composition containing purified bee venom is provided to suppress abnormal rotation motion and to protect nerve cells. CONSTITUTION: A pharmaceutical composition for preventing and treating neurodegenerative diseases contains isolated bee venom as an active ingredient. A crude purified bee venom is prepared by collecting honey bee, dissolving the dried bee in water, low alcohol of 1-4 carbon atoms or mixture solvent thereof, filtering, purifying impurities and freeze-drying. The bee venom contains 1-80% of phospholipase, 30-90% of melittin and 0.1-30% of apamin.

Solution to Problem

[0014] The term “purified extract” disclosed herein comprises the crude purified extract and the purified extract of bee venom such as honey bee, bumble bee, sweat bee etc, preferably, the purified extract of honey bee venom.

[0015] Specifically, the above-described crude purified extract of bee venom may comprise the extract prepared by the procedure comprising the steps of: dissolving dried bee venom collected from honey bee in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, subjecting the solution to filtration to remove impurities from the solution, and drying the filtrates with lyophilization to obtain the inventive crude purified extract of bee venom.

[0016] The above-described purified extract of bee venom may comprise the extract prepared by the procedure comprising the steps of; dissolving the dried crude purified extract of bee venom prepared in the above-step in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, subjecting the solution to at least one purification process selected from salting out method, solvent precipitation method, and dialysis membrane filtration in order to performing centrifugation or dialysis, and drying the filtrates with lyophilization to obtain the inventive purified extract of bee venom.

[0017] Preferably, the purified extract of bee venom may comprise the extract prepared by the procedure comprising the steps of; dissolving dried bee venom collected from honey bee in water, filtrating to remove impurities and drying with lyophilization to obtain the crude purified extract of bee venom at 1<st >step; dissolving the crude purified extract prepared in step 1 in the solvent such as distilled water to obtain water soluble extract of bee venom at 2<nd >step; subjecting the solution to dialysis membrane filtration using by membrane dialysis to collect the purified extract present in the membrane and drying the filtrates with lyophilization at 3<rd >step to obtain the inventive purified extract of bee venom having more potent pharmacological effect than the other extract of bee venom.

[0018] The inventive purified extract of bee venom having more potent pharmacological effect, may comprise melittin in an amount of ranging from about 30% to 90% (w/w %), preferably, about 35% to 80% (w/w %), more preferably, about 40% to 60% (w/w %), as an active ingredient.

[0023] The purified extract of bee venom may be prepared by the procedure comprising the steps of; dissolving dried bee venom collected from honey bee in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, filtrating to remove impurities and drying with lyophilization to obtain the crude purified extract of bee venom (designated as “HP-1” hereinafter) at 1<st >step; dissolving the crude purified extract prepared in step 1 in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, subjecting the solution to at least one purification process selected from salting out method, solvent precipitation method, and dialysis membrane filtration in order to performing centrifugation or dialysis, and drying the filtrates with lyophilization at 2<nd >step to obtain the inventive purified extract of bee venom.

[0024] In the 1<st >preferred embodiment of the present invention, the present invention also provides a method for preparing the purified extract of bee venom prepared by the procedure comprising the steps of; dissolving dried bee venom collected from honey bee in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, filtrating to remove impurities and drying with lyophilization to obtain the crude purified extract of bee venom comprising 10.2% phospholipase, 40.5% melittin, 3.8% apamine, 1.6% histamine, 1.1% dopamine and 0.3% adrenaline (designated as “HP-01” hereinafter).

[0025] In the 2nd preferred embodiment of the present invention, the present invention also provides a method for preparing the purified extract of bee venom comprising the steps of; dissolving dried bee venom collected from honey bee in water, lower alcohols such as methanol, ethanol, or butanol, preferably in water, filtrating to remove impurities and drying with lyophilization to obtain the crude purified extract of bee venom at 1<st >step; dissolving the crude purified extract prepared in step 1 in the solvent such as distilled water to obtain water soluble extract of bee venom at 2<nd >step; subjecting the solution to gel filtration chromatography and then protein dialysis membrane filtration using by membrane dialysis to perform salting out process at 3rd step; collecting the purified extract present in the membrane and drying the filtrates with lyophilization at 4th step to obtain the inventive purified extract of bee venom comprising 12.4% phospholipase, 48.4% melittin, 4.3% apamine, 0.9% histamine, 1.4% dopamine and 0.4% adrenaline (designated as “HP-01G” hereinafter).

Example 1

Preparation of the Crude Purified Extract of Bee Venom

[0061] 10.0 g of dried bee venom collected from honey bee was dissolved in waster and the impurities were removed by filtration using by syringe filter (Minisart RC 15, 0.20 microm, Sartorius Co. Germany). The filtrate was dried with lyophilizer (FDCF-12012, Operon Co. Korea) to obtain 9.76 g of dried crude purified extract of bee venom (designated as “HP-01” hereinafter). The dried powder was used in following experiments as a test sample.

Example 2

Preparation of Purified Extract of Bee Venom Using by Gel Filtration Chromatography

[0062] 100 mg of the dried crude purified extract prepared in Example 1 was dissolved in 1.0 ml of distilled water (HPLC grade) and subject to gel filtration chromatography according the condition disclosed in Table 1 in order to afford 20 fractions. Each fraction was added to protein dialysis membrane (Spectra/por 7, Spectrum Co. USA) and the membrane was dipped into a cylindrical glass flask containing 500 ml of distilled water (HPLC grade) to perform dialysis with stirring for 90 mins. After finishing desalting process with dialysis, the desalted solution present within the membrane was lyophilized for 3 days using by lyophilizer (FDCF-12012, Operon Co. Korea) and each purified fraction was collected to obtain 74 mg of purified extract of bee venom (yield: 74%) (designated as “HP-01G” hereinafter).

Example 3

Preparation of Purified Extract of Bee Venom Using by Salting-Out Method

[0065] 100 mg of the dried crude purified extract prepared in Example 1 was dissolved in 5.0 ml of distilled water (HPLC grade) to be adjusted to 20 mg/ml and the solution was subject to salting-in process by adding ammonium sulfate with stirring for 1 hour at room temperature to be 30% ammonium sulfate solution dropwisely. The solution was further stirred for 1 hour at room temperature and subjected to salting-out process by adding ammonium sulfate dropwisely to be 80% solution.

[0066] The solution was left alone for 2 hours at 0° C. to provide enough time to sufficient salting out process and centrifuged for 15 mins with the speed of 15,000 rpm by using ultra-speed centrifuges (Ultra 5.0, Hanil Science Medical Co. Ltd, Korea). The supernatant was collected and the precipitant was dissolved in 5 ml of distilled water (HPLC grade) in order that each one was subjected to be desalted and lyophilized to obtain 19 mg of purified extract of supernatant (designated as “HP-01AL” hereinafter) and 62 mg of purified extract of precipitant (designated as “HP-01AP” hereinafter) (total yield: 81%).

Example 4

Preparation of Purified Extract of Bee Venom Using by Solvent-Precipitation Method

[0069] 100 mg of the dried crude purified extract prepared in Example 1 was dissolved in 2.5 ml of distilled water (HPLC grade) and ethanol having kept at −20° C. was added thereto to be adjusted to 10 ml of 75% (v/v) ethanol.

[0070] The solution was left alone for 2 hours at 0° C. to provide enough time to sufficient precipitation process and centrifuged for 15 mins with the speed of 15,000 rpm by using ultra-speed centrifuges (Ultra 5.0, Hanil Science Medical Co. Ltd, Korea). The supernatant and the precipitant was collected and each one was subjected to be desalted and lyophilized to obtain 13 mg of purified extract of supernatant (designated as “HP-01SL” hereinafter) and 69 mg of purified extract of precipitant (designated as “HP-01SP” hereinafter) (total yield: 82%).

Example 5

Preparation of Purified Extract of Bee Venom Using by Ultra Speed Centrifuges

[0073] 100 mg of the dried crude purified extract prepared in Example 1 was dissolved in distilled water (HPLC grade) to be 10 ml and the sample was added to cartridge equipped with 50 kDa membrane filter (cartridge, Centrprep YM-50, Milipore Co. Ltd, USA). The sample was further centrifuged for 30 mins with the speed of 3,000 G by using ultra-speed centrifuges (Ultra 5.0, Hanil Science Medical Co. Ltd, Korea) to obtain two different fractions, i.e., high-molecular fraction having M. W. of more than 50 kDa (designated as “HP-02A50” hereinafter) and low-molecular fraction having M. W. of less than 50 kDa (designated as “HP-02B50” hereinafter).

[0074] The low-molecular fraction having M. W. of less than 50 kDa was added to cartridge equipped with 10 kDa membrane filter (cartridge, Centrprep YM-10, Milipore Co. Ltd, USA). The sample was further centrifuged for 30 mins with the speed of 3,000 G by using ultra-speed centrifuges (Ultra 5.0, Hanil Science Medical Co. Ltd, Korea) to obtain two different fractions, i.e., higher-molecular fraction having M. W. ranging from 10 kDa to 50 kDa (designated as “HP-03” hereinafter) and lower-molecular fraction having M. W. of less than 10 kDa (designated as “HP-04” hereinafter).

[0075] The collected fractions were lyophilized to obtain 10 mg of HP-02A50, 62 mg of HP-03, and 12 mg of HP-04, respectively (total yield: 84%).

[0076] The component of HP-02A50, HP-03 and HP-04 was analyzed using by HPLC according to the condition disclosed in Table 7 and the result was shown in Table 5.

[0077] As can be seen in Table 5, it has been confirmed that the amount of phospholipase, melittin, apamine, histamine, dopamine, and adrenaline is 76.2%, <0.1%, <0.1%, <0.1%, <0.1%, and <0.1% in HP-02A50; <0.1%, 43.2%, 6.2%, <0.1%, <0.1%, and <0.1%, in HP-03; and <0.1%, <0.1%, <0.1%, 12.8%, 8.1%, and 1.3% in HP-04, respectively whereas those are 10.2%, 40.5%, 3.8%, 1.6%, 1.1% and 0.3% in HP-01.

Example 6

Preparation of Purified Extract of Bee Venom Using by Dialysis Method

[0078] 500 mg of the dried crude purified extract prepared in Example 1 weighed by electronic balance (CP225D, Sartorius, Germany) was dissolved in 2 ml of distilled water (HPLC grade). The sample was added to protein dialysis membrane (Spectra/por 7, Spectrum Co. USA) and the membrane was dipped into a cylindrical glass flask containing 200 ml of distilled water (HPLC grade) to perform dialysis with stirring for 90 mins. After finishing desalting process with dialysis, the desalted solution present within the membrane was lyophilized for 3 days using by lyophilizer (FDCF-12012, Operon Co. Korea) to obtain 440 mg of purified extract of bee venom (yield: 88%) (designated as “HP-05” hereinafter).

[0079] The component of HP-05 was analyzed using by HPLC according to the condition disclosed in Table 7 and the result was shown in Table 5.

[0080] As can be seen in Table 6, it has been confirmed that the amount of phospholipase, melittin, apamine, histamine, dopamine, and adrenaline is 10.2%, 40.5%, 3.8%, 1.6%, 1.1%, and 0.3% in HP-05 whereas those are 10.2%, 40.5%, 3.8%, 1.6%, 1.1% and 0.3% in HP-01.



CN101455287
Melittin purification method


Inventor: WENLI ZHANG [CN]    

A separation and purification method of melittin includes: using water to soak the coarse bee venom, using ethanol to precipitate the filtrate, extracting the sediment using ammonium hydroxide and n-butanol, using acetone to precipitate the extract, dissolving the sediments in a carbamide acetate buffering liquid A, performing eluting chromatography on an ion exchange column, collecting the chromatography parts of an absorption honeybee V with the strongest hemolytic activity for concentrating, desalinizing the concentrated liquid through a dextran gelatin G-10, acetone precipitating, performing redissolving and desalinization precipitating process, dissolving the sediments in an acetate buffering liquid B, using the buffering liquid B to perform gradient elution on a dextran gelatin G-25, obtaining the electrophoresis level melittin through chromatography parts concentrating, desalinization and freeze dehydration when collecting an absorption honeybee II under the column. Compared with the prior method, the production period of the invention after process decreasing is shorten for 1.5 to 2 days, melittin yield has been improved for 7 to 8 percent, the purification cost is reduced about 30%, to achieve the industrial production of melittin.

A method of separation and purification of melittin, bee venom is rough water immersion, and the filtrate was precipitated with ethanol, the precipitate with ammonium hydroxide and n-butanol extract was precipitated with acetone, the precipitate was dissolved in acetic acid urea salt buffer A, the ion exchange column as an elution chromatography column hemolytic activity were collected under the strongest absorption bee V chromatography fractions concentrated. The concentrate was dried over sephadex G-10 desalting column, acetone precipitation , redissolved precipitation desalting treatment, the precipitate was dissolved in acetate buffer B, the sephadex G-25 column using a gradient of buffer B was used as eluting at the column chromatography to collect absorbed bee evaporated II parts concentrated, desalted freeze-dried to obtain electrophoresis grade melittin. After the process of the invention reduce production cycle time of 1.5 to 2 days, melittin yields increased by 7 to 8% higher than existing methods, purification costs by about 30%, to achieve the industrial production melittin.

TECHNICAL FIELD

The present invention relates to the separation and extraction process melittin, in particular separation and extraction side with crude venom melittin law.

Background technique

Among the many ingredients in bee venom to the content of melittin (melittin) the highest, the strongest biological activity, It has the most extensive clinical prospects. Although isolated reports at home and abroad purification process, but the process is complex, Harsh conditions, difficult to mass production; and a longer production cycle, generally at least need three days; severe loss of product, Income was only about 30 percent of melittin extracted high purification costs. It has been published patent number: 92114271.4 "From The method of "rapid separation bee venom peptide, is dissolved in water and crude venom, using ultrafiltration and dialysis Bags make bee peptide isolated ultrafiltration membrane cutoff molecular weight of 5,000 or more, a molecular weight cutoff dialysis bags 1000, which aims to separate the high molecular weight can cause allergic reactions in non-protein material, such as the use of the Method, melittin tetramer (molecular weight 11,360) was the early closure of the separation out, and melittin tetramer Body about 10 to 20% in bee venom, so make the extraction rate of melittin than the theoretical extraction rate of 20 to 30%; The method for the separation process, melittin electrophoresis purity of less than pure, so the application field is limited greatly system.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the drawbacks of the prior art, to improve existing chromatographic separation, Provide a process step is relatively small, less loss of melittin extraction and purification process, namely high yield, production Short cycle, the purified melittin purity level of melittin electrophoresis separation and purification methods.

The method of the present invention is a crude venom water immersion, solvent extraction, gel filtration column chromatography method, from Extracting venom melittin, its features are:

1) The crude venom water immersion, to remove impurities insoluble matter, the filtrate was precipitated with ethanol, removing ethanol aqueous solution, The precipitate was added an ammonium hydroxide solution and extracted with n-butanol, precipitate discarded, recovered by distillation of n-butanol, and concentrated The precipitate was added with acetone to obtain a precipitate 2;

2) The precipitate was dissolved in 2 urea acetate buffer A, the ion-exchange gel CM-Sephrose. FF column and eluted with Buffer A gradient elution chromatography;

3) collecting the hemolytic activity in the ion-exchange chromatography, gel column strongest absorption peak fractions were concentrated when V Shrink, concentrate through sephadex G-10 column desalted, desalted and concentrated to obtain a precipitate 3 was precipitated with acetone;

4) 3 precipitate was dissolved in guanidine hydrochloride, sulfite and p-chloro-mercury benzoate mixed solution, and then dissolved Was concentrated, then desalted by Sephadex, and precipitated with acetone precipitate 4;

5) The precipitate was dissolved in 4 acetate buffer B, the sephadex G-25 column and eluted with buffer Chong liquid B gradient elution chromatography, chromatography collect hemolytic activity when the strongest absorption peak II at column fractions were Concentrated, desalted on Sephadex G-10, the freeze-dried to give melittin.

Buffer A and Buffer B at the ion exchange column and sephadex gel column chromatography using The PH of 4.2 to 5.0, with a gradient elution gradient mixer. Guanidine hydrochloride, sodium sulfite and chlorine mercury Benzoic acid mixed solution of 0.15M ammonium hydroxide to adjust PH value of 7.0, guanidine hydrochloride, sodium sulfite and Concentration chloromercuribenzoate were 3M, 0.055M and 0.0023M. When crude venom immersion filtrate with 20 Fold ethanol precipitation; intermediate during desalting, precipitation with 3 volumes of acetone precipitation.

Adopting the method of production of melittin purity electrophoresis purity level to meet the more areas, especially Pro Bed application technology requirements; extraction rate of melittin were 7 to 8 percent higher than existing methods, reduction of process steps, Purification of the production cycle to reduce the 1.5 to 2 days than the original, purification costs about 30% lower than the original.

Below in conjunction with embodiments of the present invention will be described in detail.

Take crude venom 20g add 100ml of distilled water three times a dip, with a neutral filter paper, bee sting Impurities and other insolubles was discarded. The filtrate was precipitated with 20 ethanol, aqueous ethanol was removed, the precipitate was dissolved in 1 1200ml 0.15M ammonium hydroxide, and then added 800ml of n-butanol was shaken for 2hr. Separated by a separating funnel, The precipitate was discarded, recovered by distillation butanol extract, concentrate precipitated with acetone three times; precipitate 2 It was dissolved in 1000mL containing 4mol / L urea acetate buffer, PH 4.75, this is the buffer A, the ion exchange gel CM-Sephrose. FF column with Buffer A + 0.5mol / L sodium chloride gradient Elution. Eluent hemolysis test for biological activity tracking melittin collected at ion gel column Chromatography hemolytic activity of the strongest absorption peak fraction V when total 1000ml, concentrated by evaporation to 100ml. concentrated Reduction was precipitated with acetone three times by sephadex G-10 column desalination, desalination concentrate. 3 precipitate dissolved Solutions to 2000mL containing 3mol / L guanidine hydrochloride, 55mmol / L sodium sulfite and 2.3mmol / L for chlorine Mercury benzoic acid solution, the entire solution NH4OH adjusted pH 7.0, at 37 ± 2 ° C incubated 2hr, The solution was concentrated and evaporated to 200ml, then desalted by G-10 Sephadex precipitated with acetone three times. The precipitate was dissolved in 500mL 4 ammonium acetate buffer (0.05mo l / L, pH 4.75, this is the buffer B) In the sephadex G-25 column using a gradient elution buffer B (with a gradient mixer), eluted Hemolysis test was carried out with the active melittin track, collect hemolytic activity under the action column strongest absorption peak II When co-chromatography fractions 800ml, concentrated to 100ml, with sephadex G-10 desalting column, freeze-dried, Finally, lyophilized melittin was 9.4g. The product yield was 47% of melittin.



http://www.wikipedia.org

Melittin

PDB 2mlt EBI.jpg
Pfam PF01372
InterPro IPR002116
SCOP 2mlt
SUPERFAMILY 2mlt
TCDB 1.C.18
OPM superfamily 160
OPM protein 2mlt
CAS Number 20449-79-0 Yes
ChEBI     CHEBI:6736
ChEMBL     ChEMBL412927
ChemSpider 17290230
Jmol interactive 3D     Image
MeSH Melitten
PubChem 16133648
UNII     24VT8NVE75
Chemical formula C131H229N39O31
Molar mass  2846.46266
Except where otherwise noted, data are given for materials in their standard state (at 25 °C [77 °F], 100 kPa).   

Melittin is the principal active component of apitoxin (bee venom) and is a powerful stimulator of phospholipase A2. Melittin is a peptide consisting of 26 amino acids with the sequence GIGAVLKVLTTGLPALISWIKRKRQQ.

Biological effects

Melittin inhibits protein kinase C, Ca2+/calmodulin-dependent protein kinase II, myosin light chain kinase and Na+/K+-ATPase (synaptosomal membrane) and is a cell membrane lytic factor. Melittin is a small peptide with no disulphide bridge; the N-terminal part of the molecule is predominantly hydrophobic and the C-terminal part is hydrophilic and strongly basic.

Extensive work with melittin has shown that the venom has multiple effects, probably, as a result of its interaction with negatively charged phospholipids. It inhibits well known transport pumps such as the Na+-K+-ATPase and the H+-K+-ATPase. Melittin increases the permeability of cell membranes to ions, particularly Na+ and indirectly Ca2+, because of the Na+-Ca2+-exchange. This effect results in marked morphological and functional changes, particularly in excitable tissues such as cardiac myocytes. In some other tissues, e.g., cornea, not only Na+ but Cl− permeability is also increased by melittin. Similar effects to melittin on H+-K+-ATPase have been found with the synthetic amphipathic polypeptide Trp-3.[2]

Melittin also exhibits potent anti-microbial activity. For example, melittin has been shown to exert "profound inhibitory effects" on Borrelia burgdorferi, the bacteria that causes lyme disease.[3] Melittin has also been shown to kill the yeast Candida albicans[4] and to suppress Mycoplasma hominis and Chlamydia trachomatis infections.[5][6][7]

Potential therapeutic applications

At Washington University School of Medicine in St. Louis, very small nanobot "nanobee" devices are being developed to carefully deliver melittin, which is known to disrupt cellular walls and thus destroy cells, to tumor cells in animals.[8] In February 2013, it was reported that nanoparticles carrying melittin were effective in destroying HIV by eroding the double-layer viral envelope surrounding the virus. Possible applications include a vaginal gel that would target HIV intrusion prior to infection and as an intravenous treatment of extant HIV infections.[9]

It has been suggested that the regulation of S100B by melittin has potential for the treatment of epilepsy.[10]

References

Melitten - Compound Summary, PubChem.

Yang S, Carrasquer G (January 1997). "Effect of melittin on ion transport across cell membranes". Zhongguo Yao Li Xue Bao 18 (1): 3–5. PMID 10072885.

Lubke LL, Garon CF (July 1997). "The antimicrobial agent melittin exhibits powerful in vitro inhibitory effects on the Lyme disease spirochete". Clin. Infect. Dis. 25 (Suppl 1): S48–51. doi:10.1086/516165. PMID 9233664.

Klotz SA, Gaur NK, Rauceo J, Lake DF, Park Y, Hahm KS, Lipke PN (November 2004). "Inhibition of adherence and killing of Candida albicans with a 23-Mer peptide (Fn/23) with dual antifungal properties". Antimicrob. Agents Chemother. 48 (11): 4337–41. doi:10.1128/AAC.48.11.4337-4341.2004. PMC 525394. PMID 15504862.

Lazarev VN, Shkarupeta MM, Titova GA, Kostrjukova ES, Akopian TA, Govorun VM (December 2005). "Effect of induced expression of an antimicrobial peptide melittin on Chlamydia trachomatis and Mycoplasma hominis infections in vivo". Biochem. Biophys. Res. Commun. 338 (2): 946–50. doi:10.1016/j.bbrc.2005.10.028. PMID 16246304.

Lazarev VN, Stipkovits L, Biro J, Miklodi D, Shkarupeta MM, Titova GA, Akopian TA, Govorun VM (May 2004). "Induced expression of the antimicrobial peptide melittin inhibits experimental infection by Mycoplasma gallisepticum in chickens". Microbes Infect. 6 (6): 536–41. doi:10.1016/j.micinf.2004.02.006. PMID 15158186.

Lazarev VN, Parfenova TM, Gularyan SK, Misyurina OY, Akopian TA, Govorun VM (February 2002). "Induced expression of melittin, an antimicrobial peptide, inhibits infection by Chlamydia trachomatis and Mycoplasma hominis in a HeLa cell line". Int. J. Antimicrob. Agents 19 (2): 133–7. doi:10.1016/S0924-8579(01)00479-4. PMID 11850166.
"The Buzz: Targeting Cancer With Bee Venom". Wall Street Journal. September 28, 2009.

Hood JL, Jallouk AP, Campbell N, Ratner L, Wickline SA (2013). "Cytolytic nanoparticles attenuate HIV-1 infectivity". Antiviral therapy 18 (1): 95–103. doi:10.3851/IMP2346. PMID 22954649. Lay summary – Washington University in St. Louis (March 7, 2013).

Verma N, Karmakar M, Singh KP, Smita S (February 2013). "Structural and Dynamic Insights into S100B Protein Activity Inhibition by Melittin for the Treatment of Epilepsy". International journal of Computer Application. NSAAILS (1): 55–60. ISSN 0975-8887.



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