Method for obtaining a purified substance from bee venom and anti-ageing cosmetic product comprising the purified substance

Abstract

The present invention relates to a method for separating and purifying melittin (Mn), apamin (Ap), mast cell degranulation (MCD) peptide and other components present in bee venom (BV). The method allows the beneficial components of the venom to be retained in the purified BV substance and allergens to be eliminated, using ultrafiltration and silica gel chromatography. The combination of the two techniques eliminates allergenic and harmful components, such as the enzymes phospholipase A2 (PLA2) and hyaluronidase (HYA), and a purified substance (referred to as “PDA”) is obtained from the venom. The invention also relates to an anti-ageing cosmetic product comprising the purified substance, wherein the cosmetic product is selected from a cosmetic serum.

Claims

1. Method for preparing a purified bee venom comprising: a) separating by molecular weight, the components present in bee venom (BV), solubilizing it in deionized water, and then filtering it through a 0.45 and 0.22 membrane pm to remove impurities, including particulate matter; b) eliminate the phospholipase (PLA2) and hyaluronidase (HYA) components by ultrafiltration, and obtain a filtrate (FVB) containing MCD, Ap and Mn (<5% w/w), and free of PLA2 and HAY and a concentrate (CVB) comprising Mn (>90% of the total) and Ap (≥2% of the total) and traces or below the detection limit MCD and PLA2 and HYA; c) recovering Mn, Ap and traces or below the MCD detection limit of said CVB obtained from step b), by chromatography on Silica gel (G60); and d) resuspending said FVB obtained in step b) and said CVB resulting from the chromatography of step c), and storing at 4° C.

2. The method of claim 1 wherein said bee venom is selected from a bee venom powder preparation.

3. The method of claim 1 wherein said bee venom is obtained from a hive.

4. The method of claim 1 wherein in that step b) further comprising monitoring the elimination of PLA2 and HAY.

5. The method of claim 4 wherein in that said monitoring comprising performing enzymatic assays and reverse phase HPLC high performance chromatography.

6. The method of claim 1 wherein further comprising lyophilizing the purified bee venom obtained from step d).

7. The method of claim 1 in that step c) further comprising concentrating by rotary evaporation at 40° C.

8. Purified bee venom comprising apamin (Ap), melittin (Mn), MCD and trace elements including minimin, procamine A, B, secarpine, tertiapine, melittin F, cardiopep, protease inhibitors, norepinephrine, dopamine, histamine, carbohydrates including glucose and fructose, amino acids, gamma aminonobutyric acid, beta aminoisobutyric acid, minerals including phosphorus, calcium, magnesium.

9. Anti-aging serum formulation comprising a purified bee venom comprising Mn, Ap, MCD and selected trace elements of sugars, amino acids and minerals and a cosmetic vehicle composition comprising propolis and royal jelly, where the concentration of the purified bee venom is 0.006-0.012% w/w and where the concentration of the cosmetic vehicle composition is 10% w/w.

10. The formulation of claim 8 also comprising 2% w/v hyaluronic acid and vitamin E.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0011] FIG. 1. Sample of bee venom (BV) of Apis Mellifera obtained from the hives of using traps with electro-stimulation (Ketitlen, Colombia).

[0012] FIG. 2. HPLC chromatogram of BV. BV sample of Apis Mellifera bees Carnica type from Puerto Varas diluted in water. The major peaks represent: (1) Ap (Apamina); (2) MCD (Mast Cell Degranulating Peptide); (3) PLA2 (Phospholipase A2) and (4) Mn (Melittin).

[0013] FIG. 3. Tricine gel -SDS-PAGE 16% of samples separated using the Amicon® Ultra centrifugal Filter system of 10 Kda for 40 min. 1) Molecular weight marker; 2) sample Concentrated fraction; 3) Filtered fraction.

[0014] FIGS. 4A and 4B. HPLC chromatograms BV fractions. FIG. 4A: HPLC of the CBV sample obtained by Amicon®; shows the peaks for Mn (4), PLAA2 (3), MCD (2) and Ap (1). FIG. 4B: Chromatogram of FBV obtained by Amicon®, the peaks represent Mn (4), MCD (2) and Ap (1).

[0015] FIG. 5. Silica gel G60 chromatography. Elution profile of the fractions monitored by absorption at 280 nm.

[0016] FIG. 6. HPLC chromatogram of purified BV (PDA). The chromatographic profile of the PDA is shown, where Mn (4) is observed; MCD (2) and Ap (1).

[0017] FIGS. 7A-7D. Effect of PDA and BV on Staphylococcus aureus and Staphylococcus epidermis microorganisms. Viability of S. aureus and S. epidermis. FIG. 7A: % Viability versus time in hours when S. aureus is exposed to 8 pg/mL PDA and BV. FIG. 7B: A decrease in viability is observed when S. aureus is exposed to Kanamycin (positive control), BV and PDA. FIG. 7C: % Viability versus time in hours when S. epidermis is exposed to 8 pg/mL PDA and BV. FIG. 7D: A decrease in viability is observed when S. epidermis is exposed to Kanamycin (positive control), BV and PDA.

[0018] FIGS. 8A and 88. Effect of PDA and BV on the viability of HDFa cells. The cells were incubated in a range of 1 pg/mL to 100 pg/mL in incubated for 3 hours (FIG. 8A) or 24 hours (FIG. 8B).

[0019] FIG. 9. Wound closure test. The effect of PDA and BV Ha on HDFa cells over time is evaluated. ***=P-value<0.0001.

[0020] FIG. 10. Effect of PDA on wound closure in HDFa cells. Control cells and PDA cells were incubated and photographed at different times. The red line shows the wound caused by the tip.

[0021] FIGS. 11A and 11B. Percentage of release of the enzyme Lactate Dehydrogenase (LDH). FIG. 11A: HDFa cells are exposed to an MOI of 5 of S. aureus and different concentrations of MS extracts for 24 hours. FIG. 11B: HDFa cells when exposed to an MOI of 5 of S. epidermis and different concentrations of MS extracts for 24 hours.

[0022] FIG. 12. Percentage COX-2 inhibition by PDA and BV. Values expressed as mean±SD where, n−3, statistical significance performed by ANOVA followed by Tukey's multiple comparison test. (#P<0.0001 comparing between both concentrations, P<0.0001 comparing with the control at the same concentration).

[0023] FIGS. 13A-13C. Percentage of formation of reactive oxygen species (ROS). FIG. 13A. they are exposed for 3 hours to S. aureus and different concentrations of PDA. FIG. 13B. When treated for 3 hours with different concentrations of PDA, and later for 3 hours with S. aureus. FIG. 13C. When cells are treated with different concentrations of PDA and BV. “ ”=P-value<0.0001.

[0024] FIGS. 14A-14C. Percentage of formation of reactive oxygen species (ROS). FIG. 14A. They are exposed for 3 hours to S. epidermis and different concentrations of PDA and BV. FIG. 14B. When treated for 3 hours with different concentrations of PDA and BV, and later for 3 hours with S. epidermis. FIG. 14C. When cells are treated with different concentrations of PDA and BV. ****=P-value<0.0001.

DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention relates to a method for purifying bee venom comprising the following steps: [0026] a) separate the beneficial components present in complete bee venom (BV) from the allergenic components, solubilizing it in deionized water and then filtering it through a 0.45 and 0.22 pm membrane to remove particulate matter; [0027] b) characterize the protein composition of the filtrate resulting from step a) by high performance chromatography (HPLC), in reverse phase: [0028] c) separating the low molecular weight components of the filtrate characterized from step b) by ultrafiltration, including said low molecular weight components Ap. MCD, sugars, amino acids and Mn and evaluating the absence of PLA2 and HYA by SDS-PAGE electrophoresis, HPLC and enzyme assays. Enzymatic assays evaluate enzymatic activity (EnzChek® Phospholipase A2 Assay (Invitrogen) and Enzymatic Assay of Hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), and subsequent storage at 4° C.; [0029] d) determine the amount of Mn in the concentrate that is retained in the filter (CBV) of step c) by HPLC, SDS-PAGE, which is subsequently purified by Silica Gel G60 column chromatography, and the purity of Mn is determined, that is, free of PLA2 and HAY by HPLC, SDS-PAGE and measurement of enzymatic activity using EnzChek® Phospholipase A2 Assay (Invitrogen) and a turbidometric Enzymatic Assay of Hyaluronidase assay (Sigma-Aldrich, St. Louis, MO, USA), in the fractions resulting from silica gel chromatography, quantifying the content of peptides present and yield, [0030] e) joining the fraction obtained from silica gel free of PLA2 and HYA with the FBV, lyophilized and the final concentration is determined.

[0031] The purified obtained was called PDA “purified from BV”. This PDA is the active ingredient for the preparation and formulation of a Serum for cosmetic purposes.

[0032] Likewise, the present invention is related to a cosmetic product that includes the purified product described in the previous paragraph, and its preparation method.

[0033] In addition, according to the present invention, the method for preparing a purified BV, optionally comprises in step a), the preparation of bee venom powder that is subsequently diluted in water, to then carry out a filtration to remove impurities (0.45 and 0.22.m), and subsequently ultrafiltration and removal of harmful components PLA2 and HYA and recovery of Mn and peptide components (Ap and MCD) by Silica Gel chromatography. According to the method for preparing a purified BV in the present invention, PLA2 and HYA, which induce a strong allergic reaction, are removed by a combination of ultrafiltration with silica gel G60, the evaluation of allergen removal is monitored, by specific enzyme assays described throughout the process and HPLC.

[0034] Example 1: Characterization and purification of the BV components: In FIG. 1, the BV (2 g) collected from the hives is shown. To obtain the BV, electro-stimulation (Ketitlen, Colombia), then it was dissolved in sterile deionized water at a concentration of 4 mg/mL, it was filtered using a 0.45 and 0.22.m membrane to remove debris and impurities present. Using reverse phase HPLC chromatography (250×4.6 mm, LUNA C185 pm, 100 Å, Phenomenex), the crude extract of BV from Apis Mellifera was characterized in terms of its main protein components (FIG. 2). The total concentration of the protein content of BV in each stage of the process was determined by HPLC and by the bicinchoninic acid (BCA) method, which consists of a compound capable of forming an intense purple complex with Cu.sup.1+ ions in an alkaline medium. (Smith, P K, et al. 1985. Measurement of protein using bicinchoninic acid. Anal Biochem 150:76-85) and read at 562 nm, using a bovine serum albumin (BSA) standard curve.

TABLE-US-00001 TABLE 1 Concentration proteins presents in whole BV in mg/ml determined by HPLC Compound μg/ml % p/p Mn 38.1 ± 25.5 38.2 ± 2.6   Ap 19.95 ± 2    2 ± 0.2 MCD 9.95 ± 1   1 ± 0.1 PLA2 199.9 ± 6.5  12 ± 1.3  HYA ND ND ND, Not detected

[0035] Example 2: Purification of the bloactive components of apitoxin: For the purification, 4.0 mL of the filtered extract were used at a concentration of 4 mg/mL, and were passed using the 10 KDa Amicon® Ultra centrifugal Filter ultrafiltration device. Device that has a membrane that allows the separation of proteins or peptides with a molecular weight above 10 KDa, and allows proteins below that molecular weight to pass through. It was then centrifuged for 40 min at 40,000 g (4° C.). After centrifugation, a concentrated fraction (CBV: upper part) was obtained that contains proteins and molecules of size over 10 KDa and a filtered fraction called FBV (what passes through the filter), where low molecular weight proteins are found. The FBV and CBV obtained from Amicon® were lyophilized and analyzed by RP-HPLC and 16% SDS-PAGE gel electrophoresis to determine the presence of the protein in both fractions obtained. FIG. 3 shows that high PM proteins remained in the CBV and Mn (>90% of the total) and Ap (>2% of the total) and MCD (traces: below the detection limit) were dragged along, which was confirmed by HPLC (FIG. 4A) and in the FBV it can be verified that the Mn was not retained; however, PLA2 was completely removed (FIG. 4B). In addition, PLA2 and HYA activity was evaluated in the fractions (FBV and CBV) obtained using the EnzChek® Phospholipase A2 Assay kit from Invitrogen™ for the determination of PLA2. Briefly, a calibration curve was performed in a range of 0-10 units/mL of PLA2, which was incubated together with liposomes composed of DOPG and DOPC, which contain the fluorescent substrate BODIPYOPC-A2. The purified sample and VB are interpolated on the calibration curve and the percentage of enzyme remaining was calculated using Equation 1.

PLA2%=PLA2 in purified/PLA2 in raw poison×100  (equation 1)

[0036] To evaluate HAY activity, an Enzymatic Assay of Hyaluronidase turbidometric assay (Sigma-Aldrich, St. Louis, MO, USA) was performed. Briefly, 250 μl of the samples to be tested were mixed with 250 μl of enzyme diluent (20 mM sodium phosphate with 77 mM NaCl and 0.01% (w/v) BSA, pH 7.0 at 37° C.). The sample was allowed to stand for 10 min at 37° C. Then hyaluronic acid solution (500 μl) was added and mixed by shaking, followed by incubation at 37° C. for 45 minutes. After 45 min, 34 μl of mixture was added to 170 μl of acidic albumin solution and transferred to a 96-well plate. The solution was allowed to stand for 10 minutes at room temperature, and the measurement was performed at 600 nm. The percentage of HYA enzyme remaining was calculated using Equation 2.

HYA%=HYA in purified % HYA in raw poison×100  (equation 2)

[0037] In both cases, in the complete BV and in the CBV fraction, 100% activity was found for both enzymes, respectively, and 0% activity for both enzymes in FBV.

[0038] Example 3: Separation of melittin and peptide residues by silica gel. The CBV fraction contained Mn and traces of MCD and Ap, which were purified using a Silica gel G60 column. Silica gel is a polar adsorbent, slightly acidic, it has a powerful ability to absorb basic contents such as proteins that may be in the material to be purified. The 20 mL column was equilibrated with a mobile phase Butanol: Acetic acid: H.sub.2O (4:3:1), the sample was loaded in the same mobile phase (2 mL) and then washed with 3 volumes of the same, previous mobile phase, then with 3 volumes of mobile phase in a ratio of 5:3:1, and finally 3 volumes of 100% methanol. (FIG. 5). As shown in FIG. 5, the fractions eluted from the silica Gel G60 column were monitored by spectrophotometric measurement in the UV region. From fraction 1 to fraction 17 approximately corresponds to the elution with the mobile phase 4:3:1, where the elution of PLA2 and HYA is observed, these are initially eluted given their large size and low acidity, therefore which does not interact with silica gel. From fraction 1 1 to approximately fraction 33 corresponds to the elution with the 5:3:1 mobile phase, where a decrease in the acidity of the medium occurs and the release of Mn and the other peptides begins (FIG. 5).

[0039] From fraction 34 to fraction 45, elution is performed with 100% MetOH, where a proton exchange with Silica occurs, generating a change in the interaction with Mn and Ap, which are released from the column. Subsequently, these fractions were concentrated in a rotary steamer at 40° C. The product obtained was resuspended in the FBV fraction (previously stored at 4° C.) thus obtaining the PDA, which was lyophilized at −80° C. (SpeedVac, Thermo Scientific®, model SC250EXP P2). In this way, a PDA is obtained that contains not only the peptides, but also the other constituents of BV are retained. To verify that the methodology used allowed us to separate the PLA2 and HYA enzymes from the other protein components, the already lyophilized PDA was dissolved in water to be evaluated for purity by HPLC (FIG. 6). In addition, to verify that the PDA was free of PLA2 and HYA, an enzymatic assay was performed to measure enzymatic activity using EnzChek® Phospholipase A2 Assay (Invitrogen™) and Enzymatic Assay of Hyaluronidase (Sigma-Aldrich, St. Louis, MO, USA), respectively. After the enzyme activity assay for both proteins, it was negative, as can be seen in FIG. 6 and Table 2, which summarizes all the fractions analyzed during the process. Table 3 shows the content of proteins present in the PDA.

TABLE-US-00002 TABLE 2 Phospholipase A2 (PLA2) and hyaluronidase (HYA) enzymatic Activity Sample PLA2% Activity HYA % Activity BV 100 100 CBC 99.4 ND FVB 0 0 PDA 0 0

TABLE-US-00003 TABLE 3 Protein concentration presents in PDA in μg/g determined by HPLC Compound μg/g % p/p Mn   663.4 ± 13.4 66.34 ± 0.7 Ap 204.1 ± 3 20.41 ± 0.3 MCD 132.5 ± 8 13.25 ± 0.8 PLA2 0 0 HYA ND ND ND: Not Detected

Example 4: Effect of PDA on Epithelial Microorganisms Microorganisms

[0040] A) Cytotoxic effect of PDA on S. aureus and S. epidermis: In order to evaluate the inhibition of the growth of these bacteria by PDA. The concentrations considered for the formulation of the Serum are from 0.006 to 0.012%. Therefore, its use on the skin considering 0.2 mL is approximately 12 to 24 g of PDA, within this range a decrease in the viability of both microorganisms is observed as shown in FIG. 7. Although, BV has a greater effect on S. aureus than PDA. This increased effect may occur due to the presence of other components in BV: on the other hand, a greater effect of PDA on the inhibition of the growth of S. epidermis is observed. Therefore, PDA is effective in decreasing the growth of opportunistic epithelial pathogens.

Example 5: Effects of PDA in a Skin Fibroblast Cell Model

[0041] Example 5A: Cytotoxic Effect of PDA on HFDa Cells: It is very important to assess whether concentrations that affect the viability of bacteria have a damaging effect on human epithelial cells. For this, the optimal concentrations that do not cause damage to the cells were determined. Therefore, the tests were performed using a range of concentrations between 0 and 100/mL.Math.BV was shown to be highly cytotoxic on cells in culture vs. PDA at concentrations of 25|g/mL, therefore, the complete BV is not suitable for direct use on the skin (FIG. 9).

[0042] Example 5B: Effect of PDA on the migration of HDFa cells. The ability of the PDA compound to induce cell migration was evaluated through a wound closure assay (Becker et al. 2014. Coagulase-negative staphylococci. Clinical Microbiology Reviews, 27:870-926). The cells were incubated with 10 and 25 ug/mL of PDA and BV. In addition, vehicle (ethanol) was used as a positive control. Cellular migration towards the wound was recorded by photographs under a microscope and taken at 0, 3, 6, 9, 24 and 48 hrs, from the start of treatment with the compounds (time 0 hrs). Images were analyzed with ImageJ 1.42q imaging software (National Institutes for Health. US). The percentage of cell migration was calculated with Equation 3:

% Migration=(Do−Dn)/Do×100  (equation 3)

where D.sub.o is the average distance between cells produced by the wound and D.sub.n is the average distance between cells at different times, from time 0. In Table 4 shows the migratory percentages of the HDFa cell line when exposed to different concentrations of PDA and BV between the initial time (0) and 48 hours.

TABLE-US-00004 TABLE 4 Effect of PDA and BV on HDFa cell migration. PDA (pg/mL) BV (pg/mL) Control Time 10 25 10 25 3 20.36 ± 0.14  4.54 ± 0.69 31.05 ± 0.85  3.01 ± 1.23 4.50 ± 1.16 6 26.60 ± 0.65 16.38 ± 0.92 48.19 ± 0.35 10.04 ± 0.86 4.10 ± 0.77 9 38.85 ± 1.22 37.34 ± 1.78 70.85 ± 2.14 11.53 ± 0.02 4.56 ± 0.95 24 55.03 ± 0.19 57.94 ± 0.70 91.73 ± 1.99 14.31 ± 0.93 4.05 ± 0.06 48 98.06 ± 2.84 99.30 ± 0.37 99.19 ± 0.23 26.86 ± 1.41 4.72 ± 0.72 *Values represent % migration over time in hours.

[0043] As seen in Table 4 and FIG. 10, the migration percentage is represented with respect to time and the effect of PDA and BV on this event. It is possible to observe how the PDA exerts a positive effect on the migratory capacity of the cells at concentrations of 25 pg/mL, at 9 hours of incubation there was a 70% closure of the wound compared to the control and at 24 hours a 92%. As an example of these tests, FIG. 10 shows images taken by microscopy, where it can be seen how the cells after the wound caused begin to migrate and this migration is increased in the presence of PDA: however, BV was harmful to cells, generating cell death, as shown in Table 4, where there was practically no cell migration. It has been reported that cell migration can be reduced during infection or cell aging (Kondo & Yonezawa, (1992). Changes in the migratory ability of human lung and skin fibroblasts during in vitro aging and in vivo cellular senescence. Mechanisms of ageing and development, 63: 223-233.). This process is increased when there is production of collagen and other extracellular matrix proteins.

[0044] Example 5C: Reduction of bacterial Infection by measuring the release of the enzyme Lactate Dehydrogenase (LDH): The HDFa line was infected with S. aureus and S. epidermis using an MOI of 5, adding PDA at concentrations of 10 and 25 pg/mL, the release of the enzyme LDH was evaluated. LDH is a damage marker which is released when the cell membrane is damaged, using medium as negative control and positive control of LDH release using Triton X-100 detergent. It is observed that a lower release of LDH is obtained over time when the infection is carried out with S. epidermis and PDA FIG. 11B), which is related to the greater susceptibility of this bacterium to PDA; also, a decrease in LDH is observed when infected with S. aureus (FIG. 11A). Therefore, PDA provides a protective effect at the cellular level.

[0045] Example 5D: Anti-inflammatory activity. Tests were carried out to evaluate its anti-inflammatory potential, by inhibition of the enzyme COX-2. The results are shown in FIG. 12. A 60% inhibition of the activity of the COX-2 enzyme is obtained when using PDA and 20% inhibition when using 3 pg/mL of BV. Celecoxib (a drug that inhibits the pro-inflammatory action of COX-2) was used as a control. Consequently, PDA has very positive pro-inflammatory effects, which is important against epithelial damage, and BV has no effect.

[0046] Example 5E: The effect of PDA on HFDa cells exposed to Infection induced by epithelial pathogens—S. epidermis and S. aureus—(FIGS. 13 and 14). These pathogens when they attack cells, generating an increase in free radicals at the intracellular level, triggering damage to macromolecules such as DNA, proteins and lipids and generating collagen destruction. PDA is capable of protecting fibroblasts from the harmful effect generated by pathogens that generate an increase in ROS and increase inflammation. This test could be extrapolated to other noxa such as UV rays. With which it shows that PDA has a powerful protective effect at the level of: stimulating migration, anti-inflammatory and reducing ROS (against bacterial noxa).

[0047] Example 6: Formulation of an anti-aging serum containing PDA and Apitop®. The present invention provides a purified bee venom (PDA) and its use as a key ingredient to delay antiaging, it also comprises a suitable cosmetic vehicle composition, without perfumes, and its color is given only by natural components. The PDA provides Mn, Ap, MCD and other molecules present in smaller quantities in the BV, such as sugars, amino acids, minerals. The PDA is presented in a concentration of 0.006-0.012% p/p, which is mixed with Apitop® at 10% p/p (propolis, royal jelly, pollen, honey and vitamins C and D), in addition, it contains hyaluronic acid, at 2% w/v and vitamin E.

[0048] The Serum contains a suitable concentration of PDA (0.006 to 0.012% w/w) for topical application on the skin; In addition, it contains Mn, a penetrating peptide that can help all the other components of the Serum to pass through the dermis and the bio-active components achieve their regenerative action, protective against ROS. The formulation of the Serum is soft, without perfumes and has a unique smell and color, given by its natural components. The advantages of this Serum is that it has components such as PDA plus Royal Jelly and propolis, which have been tested in vitro, showing that it has anti-radical species activity (ANTIROS), regenerative properties for the skin (anti-aging) and anti-inflammatory. As indicated in the state of the art, previous patents have used the complete BV or solvent extractions, but without removing the allergenic agents (KR101394817B1). In this invention they refer to the viability of epithelial pathogens that cause acne and in our case both PDA and BV have an effect on bacteria. Nevertheless; BV has a strong cytotoxicity on human epithelial cells, generating their death at concentrations indicated in said invention. Patent EP 2460 526A formulates a cosmetic preparation focused on treating acne based only on BV and in this case they use test concentrations in HEK epithelial cells of 0.01-10 g/mL of BV. Finally, this formulation contains 0.001% BV, however, the possibility of allergic reactions that they may generate is not clear.

[0049] Example 7: Formulation of Serum: The raw materials 1, 2, 3, 4 (Table 5) are added in the order indicated in Table 5 at a temperature of 22° C. and homogenized until the formation of a gel which is leave for 24 hours for stabilization. Potassium sorbate (5, see Table 5) is then added, which is a natural preservative and surfactant that allows, at a certain temperature, to join different phases, such as vitamin E (fat-soluble) with the other components, lowering the surface tension of the emulsion, then components 7 and 8 are added (Table 5). After that, component 9 (Apitop®) is added and mixed with homogenization until the appropriate consistency is obtained, for which the pH is maintained at 6. Finally, the aqueous solution of active component 11 (PDA) prepared at 1% in water is added, and the mixture is carried out with a homogenizer at very low speed, in order to avoid denaturation of the protein components of the formulation. All the mixture is made in amber containers and protected from light.

TABLE-US-00005 TABLE 5 Formulation Serum 0.006-0.012 PDA Raw Material % weight 1 Carbopol 940 0.52 2 Distilled water 81.93  3 Microcare 1.26 4 Triethanolamine 0.26 5 Potassium sorbate  0.026 6 Oily vitamin E 1   7 Liponic EG-1 2   8 Hyaluronic acid 2   9 Apitop ® 10    Propolis extract 3*   Pollen 2.5* Honey 4.1* 10 Tween 80 1   11 PDA (purified from bee venom) 0.006-0.012 Apamine* 0.0029442* (traces) Melitine* 0.0079608* (traces) MCD* 0.0015900* (traces) Minimine Traces* Procamine A, B Traces* Secarpine Traces* Tertiapine Traces* Melitine F Traces* Cardiopep Traces* Protease inhibitors Traces* Noradrenaline Traces* Dopamine Traces* Histamine Traces* Hydrocarbons (glucose, fructose) Traces* Amino acids Traces* Gamma Aminobutiric acid Traces* Beta aminoisobutiric acid Traces* Minerals (P, Ca, Mg) Traces* Note: The * indicates its composition of superior active ingredient (Propolis extract and purified apitoxin). Traces*: below the detection limit.