WO2020121017A1

WO2020121017A1 - Cream synthesis from platelet mediators with herbal penetration enhancers to increase the skin's collagen

Info

Publication number: WO2020121017A1
Application number: PCT/IB2018/059802
Authority: WO
Inventors: Hamidollah AFRASIABIAN
Original assignee: Afrasiabian Hamidollah
Priority date: 2018-12-09
Filing date: 2018-12-09
Publication date: 2020-06-18

Abstract

This invention using complementary medicine to the synthesis of anti-aging cream which is effective, non-invasive and based on scientific principles. So, we used platelet mediators which are approved by FDA and the safe herbal penetrators with many useful properties. For the first time, the musk has been used as an effective penetration enhancer in the formulation with unexpected positive effects. The randomized clinical trial showed the final product will help the skin rejuvenation.

Description

Cream Synthesis from platelet mediators with herbal penetration enhancers to increase the skin’s collagen

Description

Technical field

The technical field of this invention is related to skin’s collagen Cream.

Technical Problems

Skin is a natural barrier, so it is necessary to employ enhancement strategies to improve topical bioavailability. Therefore, many different approaches have been developed to overcome the impervious nature of the skin’s outer layer (stratum corneum) by various physical, chemical, biochemical enhancement strategies. But, we should note that the physical and chemical permeation enhancers have different side effects. While the natural products have got the potential to enhance the permeation of drug through the skin by reversibly reducing the skin barrier resistance. They are safe, non-toxic, pharmacologically inert, non-irritating and non- allergenic to use.

The goal of this work is using complementary medicine to the synthesis of anti-aging cream which is effective, non-invasive and based on scientific principles. So, we used platelet mediators which are approved by FDA and the safe herbal penetrators with many useful properties. For the first time, the musk has been used as an effective penetration enhancer in the formulation with unexpected positive effects. The randomized clinical trial showed the final product will help the skin rejuvenation.¹

Background Art

In recent years, modem medicine through the skin for therapeutic purposes has been proposed. The physical strategies involve phonophoresis, electrophoration, iontophoresis, magnetophoresis, micro fabricated needle and laser technologies. Recently, non-invasive laser is one of new methods that increase skin collagen, which is named as "non-invasive laser procedure".²

Currently, platelet-rich plasma (PRP) injection method into the skin is one of effective invasive methods.³ But, the patients have faced to symptoms such as swelling, allergic reaction, infection, cellulites, surgical complications. In another work, the keratin layer of the skin has been removed and nano-ingredients material has been employed.⁴

About a thousand years ago, Ibn Sina one of the Iranian scientists in field of traditional medicine said "drugs have two part as soft and thick segment, the soft may penetrate into the skin".⁵ In this study, we have used the combination of modem medicine and ideas of the ancient Iranian medicine simultaneously. Platelet mediator as the best biological tool with a variety of growing factors (FGFs) can be used to produce collagen (see supplementary section). It was blended with the herbal extracted involving hedera helix, licorice extract and musk in the new formulation. All mentioned compounds has been mixed with wheat germ and phytosterols. We will discuss why we select these ingredients according to the supplementary section and the following explanations.

Publication Number: 107648135, Title: Ginseng compounded cream for improving collagen structure of human skins, and preparation method thereof

The invention provides a ginseng compounded cream for improving the collagen structure of human skins. The ginseng compounded cream comprises, by weight, 3-10 parts of ginseng extract, 1-5 parts of collagen tripeptides, 3-10 parts of Folium Phyllostachys extract, 0.1-0.5 part of capsaicin, 3-10 parts of glycerin monostearate, 1-5 parts of tretinoin, 20-30 parts of glycerin and 10-20 parts of water. All above components have synergistic effects, so the collagen structure of human skins is remarkably improved, and the diameters and the spacing of fibrils are structure, thereby collagen fibers have good cohesiveness and stability, skin pores are shrunk, and ultraviolet radiation, blue light radiation and other damages are resisted.

Detailed description of the invention:

The characteristics of ingredients:

Licorice extract

Licorice extract have effectively treated skin disorders such as rosacea, eczema, psoriasis and dermatitis. Also, it has been known to fade dark spots and lighten skin. One of the ingredients is glabridin which acts to depigment. Pigmentation is caused by exposure to the sun's ultra violet rays on the other hand, potent antioxidants in licorice extract fight skin-damaging free radicals.^6,7 Licochalcone, another ingredient in licorice, helps to regulate the oil production. In addition, Glycyrrhiza glabra was reported to show improvement in the viscoelastic and hydration properties of the skin.⁸

Wheat germ

Wheat germ is widely recognized as a nutritious raw material for incorporation into food and cosmetic products. Wheat germ was found to be a rich source of carbohydrate (46.07 gr/lOOgr), protein (31.69 gr/lOOgr), and fatty acid (10.29 gr/lOOgr) especially linoleic, palmitic, oleic, linolenic acids. Also, it has antioxidant and antibacterial activity.^9,10 But it is a great source of vitamins such as E, Be, zinc, copper, phosphorus, manganese, and folic acid. So, it can act as an anti-aging agent for the skin. In addition, we used it as a humidifier.

Brief description of the figures:

Figure 1. The chemical structure of muscone

Figure 2. The chemical structure of phytosterols

Figure 3. The structure of skin layers

Figure 4. The chemical structure of ceramide

Figure 5. The disulfide linkage in keratin

Figure 6. The schematic pathways for skin permeation

Figure 7. The comparison between different configurations of fatty acids

Figure 8. The main chemical constituents of collagen

Figure 9. The schematic of collagen fibril formation

Figure 10. The schematic of platelet constitution

Detailed description of the figures:

Musk

Musk was a name originally given to a substance with a strong odor obtained from a gland of the male musk deer. Nowadays modern use of natural musk pods occurs in traditional Chinese medicine. According to the Chinese article, the chemical composition of musk has been identified.^12,13 All potential musk chemical compositions of Chinese forest musk deer were identified by GC/MS chromatograms of musk diethyl ether and ethyl alcohol extracts. One of the most important compound (Figure 1) was 3-Methylcyclopenta decanone (muscone). The percentage of muscone were about 30%. Also, the other important components were acetal, 3- ethyl-3-hydroxy-5a-androstan-17-one, and cholesterol. Regarding to the retention time of the main ingredients of musk we can deduce that these compounds specially muscone (R_t = 30 min) are useful for skin permeation because the polarity of it is moderate and it has both lipophilic and hydrophilic property, so it is excellent as a penetration enhancer. As reported in Chinese work the musk gland microbiota were investigated. They found many functional pathways related to ketone metabolism over represented in musk deer.

For example, aldosterone -regulated sodium reabsorption, metabolism of terpenoids and polyketides, flavone, isoflavonoid and flavonol biosynthesis pathways. So, the microbiome plays an important role in-human odor production and the number of certain microorganisms is strongly correlated with the intensity of the odor emitted. The skin's location at the interface with the outside world, therefore makes it the most subject to environmental influences that will affect the microbiota.¹⁴

Also, we can use the synthetic musk because of economic savings.

Phytosterols

Cholesterol is a vital component of human epidermal keratinocyte membranes and lipid micro environments on the cell surface known as lipid rafts.¹⁵ It was demonstrated that UVA radiation (320- 400 nm) reduces the content of cholesterol in plasma membranes and lipid rafts as a consequence of sphingomyelin hydrolysis and conversion to ceramide. Cholesterol plays a crucial role for stabilization of rafts which ideally need a cholesterol to ceramide ratio of > 1. Cholesterol depletion increased the susceptibility of keratinocytes for UVA-induced gene expression, whereas pre treatment with cholesterol completely abrogated the UVA stress response. Interestingly the cholesterol molecule could be mimicked by plant sterols like campesterol, b-sitosterol and stigmasterol.¹⁶ These major plant sterols (figure 2) can therefore reduce photosensitivity and protect from photo-aging when applied topically to the human skin. Another symptom for skin aging is the loss of dermal collagen fibres linked to wrinkle formation. The mechanism behind this, is widely elucidated and explained by proteolytic collagen degradation due to matrix metalloproteinase MMPs. These events are initiated by UVA and UVB radiation and other environmental stimuli. Cell based in-vitro assays proved that treatment of human keratinocytes with phytosterols was able to inhibit MMP expression.

phytosterols was reported to be used in 177 cosmetic products such as lipsticks, non-spray deodorant, eye make-up, face powders and... . The phytosterols are used in all of the FDA's cosmetic category groups except baby product. They are used at maximum concentration ranging from 0.000001%-8%. Most of them have been used as skin-conditioning agents or skin protectant or antioxidant.^{17 19}

Figure 2:

1- Cholesterol

2- Campsterol

3- B-Sitosterol

4- Stigmasterol

5- Brassicasterol

Materials and Methods

This study is a randomized clinical trial; 106 patients referred to the clinic with positive cases from skin wrinkles along with 20 MHz frequency ultrasound images of their skin faces. All individual enrolled in the study were randomly divided into two groups.¹ For group A, the platelet cream was prepared including emulsion of Lecithin and Eucerin as basic cream (this cream purchased from Farabi Pharmaceutical Company of Iran). 10 cc of blood was taken from each patient, then placed in a centrifuge machine at a speed of 1200 rpm for 15 minutes (the centrifuge tube contains an anticoagulant). Finally, after completion of the plasma centrifuge, plasma plate was added to the cream base. At first, the amount of plasma solution is 5 cc. Then 5 cc of herbal extracts from Hedera helix, Musk and Licorice extract were added. After that, the mixture combined with Wheat germ and Phy to sterols (extracted from cotton oil). Finally, the total creams of group A were 70 cc.

For group B, 60cc of basic cream group A along with lOcc glycerin was added as placebo. Consequently, 70 cc cream including placebo submitted to group B people. The cream was delivered to everyone in the two groups for one month's use. All people were advised to wash their face 2 hours before bed, rub it with a relatively rough texture, then dry the face. Then rub 2.3 g of the cream into the face skin slowly, also for each person, certain dishes with a capacity of 2.3 g of cream were prepared. In this study, the numerical comparison of the difference in skin tissue collagenization in two groups was very important. For the measurement of collagen before and after using the cream, ultrasound images of 20 MHz frequency were used. Therefore, the B-mode scanner and the DUB- USB-Luneburg Germany devices were used in current project. Sonographic images of both groups were obtained before and after the use of platelet and placebo creams. The reflection of the frequency transmitted to the skin tissue appears as an increase in the amount of brightness in the skin. Whatever collagen content per unit volume, collagen fibers are seen in bright or gray stripes. But the basis for comparing the tissue repair of the skin should be expressed in numerical units. In this image, the reflection energy of ultrasound radiation was evaluated based on the base ROI, to transform into a meaningful and numerical physical quantity; the radius energy square was expressed as the numerical quantity as shown in equation. Finally, a comparison was made by converting the numerical values of these values.

[RE=(ROI)²]

Results

The results in the various aspects include; there is no significant correlation between demographic conditions of the participants and the amount of collagen in the skin through numerical evaluation of RE . The demographic conditions in this research include sex, occupation, economic status and literacy level had no effect on the response to treatment but the age demographic index had a significant effect on response to treatment. Moreover, the effective factor in responding to treatment is the amount of skin collagen compression; the better collagen production is in the skin with a higher collagen density. Based on the calculations performed in this study, the minimum and maximum of the RE value or the reflectance energy square was 10 and 25 respectively. But the amount of change in either the production or increase of collagen in the worst and best case was obtained 1 and 7 respectively. Noticeably, only one person in group A had the highest increase in collagen content. In group A, 2 patients left the study due to an allergic reaction, and 3 individuals did not continue the study. As in group B, 5 patients did not go on the study.

In group A (intervention group), 13 patients did not respond to the use of cream. In other words, we had 27.03 percent negative or no response, while 35 patients responded to the intervention, with a positive therapeutic response of 72.91%.

In group B (placebo group) no significant change was observed, where in 48 people, only 6 individuals responded, which was also very minor [RE = 1-3]. The success rate of placebo was only 12.5%, with a little collagenization, but compared with group A was significantly lower.

Conclusion One of the most important current medical science questions, and in particular the topics of the World Health Organization (WHO), is access to precise methods for evaluating and investigating allegations of traditional and complementary medicine. So, traditional and alternative medicine seems to be a great source of experiences that will provide the basis for future research ideas in field of modern medical science.

In this study 106 patients as the population of study were participated to reconstruction of skin collagen and increasing of skin tissue. Based on ultrasound technology, images of 20 MHz frequency were obtained from all individuals before the start of the study, and were reported numerically based on the reflectance energy square (RE²). The participants randomly divided into two groups as the intervention group (A) and the control or placebo group (B). At the end of this study, the response rate was 72.91% in group A.

Supplementary section

Structure of Skin

Structure of the skin can be divided into epidermis, dermis and hypodermis (figure 3). The epidermis can be subdivided into different layer. The outer layer is stratum corneum. It is typically 10-20 pm thick and compose of corneocytes which are non-living cells without nuclei and cytoplasmic organelles but are filled with keratin and are interspersed in a lipid-enriched extracellular matrix. Since diffusion across the stratum corneum is considered as the major pathway of skin permeation, the structural properties of this layer have been extensively studied to elucidate it’s barrier function.^20,21

Ceramides, cholesterol and saturated free fatty acids are the main constituents of the extracellular matrix in approximately equimolar proportions.²² Each ceramide molecule consists of a sphingoid moiety, containing a polar head group and a hydrocarbon chain which -NH2 functional group of it can reacted with a fatty acid as shown in figure 4.²³

Figure 4:

6- Polar head group

7- Sphingoid moiety

8- Fatty acid moiety

According to the recent investigations, ceramides are fully extended in the stratum corneum with the two hydrocarbon tails in opposite directions.²⁴ The rest of epidermis is composed of keratinocytes (95%). Keratin is a fibrous structural protein from alanine, leucine, arginine and cysteine. It is two polypeptide chain with disulfide cross-linked (Figure 5). Consequently, we found the first barrier for penetration of a drug has both lipophilic and hydrophilic structures.

- The dermis is composed mainly of collagen and elastin.

- The hypodermis is composed mainly of subcutaneous fat.

Skin permeation enhancers

Permeation enhancers are defined as substances that are capable of promoting penetration of drugs into skin. There are two pathways for permeation (Figure 6). The first is trans epidermal (diffusing across the skin layers containing Intracellular or Intercellular routes), and the second is appendageal pathway (through hair follicles or sweat ducts).

Figure 6: 9- Epidermis

10- Dermis

11- Subcutaneous Layer

12- Transappendageal Route

13 - Intracellular Route

14- Intracellular Lipid Matrix

15- Corneocytes

16- Sebaceous Gland

17- Hair Follicle

18- Sweat Duct

The trans epidermal pathway is usually the predominant route and it is comfortable, convenient and non-invasive way to administer drugs.²⁵ But, the greatest obstacle in the transdermal drug delivery is stratum comeum because it provides a rate-limiting step for the delivery of drugs. So, many different approaches have been developed to overcome the impervious nature of stratum corneum by various physical, chemical, biochemical enhancement strategies. The physical strategies involve phonophoresis, electrophoration, iontophoresis, magnetophoresis, micro fabricated needle and laser technologies.

The chemical strategies are highly dependent on the structure of skin and the physicochemical properties of the permeant such as molecular mass and hydrophilicity. Molecules that permeate the skin most readily have a molecular mass of <500Da and are moderately hydrophilic. Small hydrophilic molecules generally favor the Intracellular route over the intercellular route and vice versa for lipophilic molecules and the appendageal pathway maybe more important for highly hydrophilic molecules.²⁶ In general, skin permeation enhancers may exert their effects through one or a combination of the following mechanisms.^{27 29}

1. By solubilizing the skin-tissue components.

2. Interaction with intercellular lipids leading to disruption of the highly ordered lamellar structure.

3. Interaction with intracellular protein to promote permeation of drugs through the corneocyte layer.

4. Improved partition of the drugs, co-enhancers or co-solvents into the stratum corneum.

The latter three mechanism have been described as the lipid-protein-partition (LPP) theory.^30,31 In conclusion, the structure- activity relationship (SAR) studies can be used to correlate the enhancing potency of chemical enhancer with it’s structure or physiochemical parameters such as molecular shape, size, molecular geometry, solubility parameter, electronic effect, hydrophilicity and lipophilicity.³² For example, in the case of the intracellular region, since the comeocytes are composed of keratin, penetration enhancers such as surfactants and aprotic solvents may interact with polar head group of the keratin. These interaction result in the reduction of the binding forces between protein molecules, thereby changing the conformations of protein helices. But, we should note that, the physical and chemical permeation enhancers have different side effects.^33,34 Therefore, the natural products have got potential to enhance the permeation of drug through skin by reversibly reducing the skin barrier resistance. They are safe, non-toxic, pharmacologically inert, non-irritating and non- allergenic to use. The most important of them are

1. Water

The water content of human stratum corneum is typically around 15-20% of tissue dry weight and it can approach 400%. In general, increased tissue hydration appears to increase transdermal delivery of permeant. The water within this membrane is found in two states: The water present in stratum comeum can be assessed as bound i.e. is associated with some structural functional groups within the tissue (hydrogen bonding or covalent bond), and the free water which is available to act as a solvent within the membrane for polar parts of permeants.³⁵

2. Fatty acids

According to the investigations, saturated alky chain lengths of around C10-C12 attached to a polar head group yields a potent enhancer and unsaturated alky chains up to Cis (e.g. oleic, linoleic, linolenic arachidonic acids) can optimum the penetration especially cis configuration (figure 7) is expected to disturb intercellular lipid packing as pools more than the trans arrangemet.^{36 38} The formation of such pools would provide permeability defects within the bilayer lipids to facilitate permeation. Also, the fatty acids in transdermal formulations appear to reduce the skin irritation and sensitization which is most common problem associated with some drugs.

Figure 7:

19- Saturated Fatty Acid - Stearic Acid

20- Unsaturated Fatty Acid - Cis Oleic Acid

21- Trans Oleic Acid

3. Surfactants

Surfactants are added to formulations in order to solubilize lipophilic ingredients within the stratum comeum. They are divided to four groups non-ionic, anionic, cationic and zwitter ionic. Anionic and cationic surfactant have potential to damage human skin³⁹ and non-ionic surfactants tend to be widely regarded as safe.⁴⁰ But natural surfactants such as saponins have great potential for use as permeation enhancers.^41,42

4. Natural product

The natural products containing essential oils, terpenes, and herbal extract (e.g. flavonoids, alkaloids) permeation enhancers exhibit low toxicity while maintaining their enhancing activity.⁴³ For example, terpenes could form hydrogen bonds with intercellular lipids and the polar extracts of plant can be more effective for hydrophilic drugs penetration.⁴⁴ So, we should consider all of the points mentioned above to provide a targeted formulation.

Collagen and the reasons of aging

Collagen is protein molecules made up of amino acids. There are various types of collagen (30) that have been discovered but, the most common are type 1 through IV and specially type 1. The differences lie in the make-up of the alpha peptides. The primary amino acid sequence of collagen is glycine -proline-X or glycine-X-hydroxyproline (Figure 8). X can be any of the other 17 amino acids. Collagen is composed of 3 chains which are wound together to form a triple helix.

Figure 8:

22- Hydroxyproline

23- Glycine

24- Proline

The interaction of a-chains is stabilized via interchain hydrogen bonding making the molecule fairy resistant to attack by other molecules. Each a-chain is surrounded by a hydration sphere (hyaluronic acid) which allows a hydrogen bonding network to be present between the water molecules and the peptide acceptor groups.⁴⁵ The synthesis of fibrillary collagen is discussed in the following. 1. Transcription of mRNA.

2. Pre-pro-peptide formation.

3. Pro-collagen formation.

In this process, hydroxylation of lysines and prolines on polypeptide by the enzymes prolyl hydroxylase and lysyl hydroxylase (to produce hydroxyproline and hydroxylysine) occurs to aid cross-linking of the alpha peptides. The enzymatic step requires vitamin C as a cofactor.

Also, in this step, three of the hydroxylated and glycosylated propeptides (by adding either glucose or glactose onto the hydroxyl groups of lysines) twist into a triple helix forming procollagen which still has unwound ends.

4. Golgi apparatus modification.

In this step, oligosaccharides are added.

5. Formation of tropocollagen.

In this step, the enzymes known as collagen peptidases remove the loose ends of the procollagen.

6. Formation of the collagen fibril.

Fysyl oxidase, an extracellular copper-dependent enzyme, produces the collagen according the following process (figure 9).

Figure 9:

25- Tropokollagen-Strang

26- Fysin-Seitenketten

27- Fysyloxidase

28- Carbonylgruppen

29- Quervemetzug (Aldolkondensation)

In young skin, collagen is arranged in the pattern of a regular grid, while in aging skin collagen is arranged in tufts giving the impression of disorder. Elastic fibers, in turn, undergo calcification and fragmentation in the extracellular matrix, the amount of the metalloproteinase enzyme is increased leading to it’s degradation, as well as the accumulation of material containing malformed elastin in the papillary and reticular dermis. Over the years, when the amount of collagen decreases, leading to the dilution of the dermal-epidermal junction (DEJ) consequently, the formation of wrinkles. On the other hand, in young skin, hyaluronic acid is found on the periphery of collagen and elastin. Hyaluronic acid has water-binding properties which allows the skin to maintain a water-electrolyte imbalance. As a result of aging, the amount of hyaluronic acid in the skin is almost completely reduced. As it is known, we should notice to the existence of vitamin C and Cu element as cofactors in reconstruction of collagen.

Platelets

The most important role of platelets in their involvement in hemostasis by adhesion and aggregation on the wall of the damaged vessel. In addition, they take part in synthesis, storage and release of many different compounds (e.g. thrombin, adenine, nucleotides, serotonin, Ca²⁺ ions). At the same time, they are also a reservoir of many growth factors involved in blood coagulation, immune response and tissue healing.^46,47 The most important growth factors stored in thrombocytes are:

1. PDGF (Platelet Derived Growth Factor).

It is considered to be one of the most important and potent wound healing stimulators and also increase the mechanical strength of the skin fibroblasts that are activated by PDGF for proliferation and for the production of extracellular matrix components. 2. TGF-b (Transforming Growth Factor b).

3. EGF (Epidermal Growth Factor).

It’s action is to stimulate the migration and proliferation of epidermal cells. It accelerates skin metabolism and promotes the transport of active nutrients, resulting in increased collagen synthesis. It is also responsible for repairing damaged polysaccharides and glycoproteins that build cell membranes.

4. FGF (Fibroblast Growth Factor).

5. VEGF (Vascular Endothelial Growth Factor).

This cytokine acts via receptors mainly on the surface of endothelial cells triggering the signal that stimulates angiogenesis. This results in the construction of new vessels on the scaffold composed of collagen and other proteins of the extracellular matrix. VEGF was also shown to participate in the production of collagen fibers and stimulation of thrombocyte agglomeration and clot formation.

6. IGF (Insulin-like Growth Factor).

7. HGF (Hepatocyte Growth Factor).

On the other hand, structurally the platelet can be divided into four zones (Figure 10).

1. Peripheral zone.

It is rich in glycoproteins for adhesion, aggregation and so on.

2. Sol-gel zone.

It is rich in microtubules and microfilaments to correlate the inside of platelet with outside.

3. Membranous zone

The platelet plasma membrane is a standard bilayer composed of proteins and lipids.

4. Organelle zone.

It is rich in platelet granules such as Alpha or Delta granules contain ADP, Ca and serotonin which are platelet-activating mediators

Figure 10:

30- Pheripheral Zone

31- Structural Zone

32- Membrance System

33- Organelle Zone

As can be seen, the platelet possesses a standard biological membrane composed of a phospholipid bilayer with polar head groups oriented toward the aqueous plasma and cytoplasm and nonpolar fatty acid tails the orient toward the center. The phospholipid layers contain the neutral and anionic ones. The most important one of them is phosphatidylinositol which support platelet activation by supplying arachidonic acid that becomes converted to the eicosanoids prostaglandin and thromboxane A2 during platelet activation. In this among, cholesterol stabilizes the membrane, maintains fluidity and helps control the trans membranous passage of materials. So, the platelet mediators are the best biological tool to promote the collagen production.

Advantages

1. Non-invasive and safe method which doesn’t hurt to the normal skin tissue.

2. This method isn’t painful and the skin of patients hasn’t been suffered as a result of swelling, blood disorder, or treatment of tissue damage such as infection. 3. This method is non-thermal, so it doesn’t cause burns in the skin such as laser techniques.

4. This cream uses from platelet of the own patient, so it is a natural and biological cream for the skin rejuvenation.

5. The herbal extracts with useful properties has been used as penetration enhancers which don’t have any side effects

6. For the first time, we used the musk ingredient to improve our formulation.

7. The production of an organic cream with herbal penetration enhancers is very important from the economic points of view in the cosmetics industry. Therefore, it can compete with similar expensive products.

8. The most important point in this project is the beginning of a pathway to assess the claims of complementary and traditional medicine along with modem medicine.

The Formula of Cream

Basic Cream + Herbal Penetration Enhancers + Platelet-rich Plasma + Wheat Germ

The First Step:

Preparation of the basic cream

Sweet Almond Oil (27% - 38%) + Beez wax (10% - 12%) + Lanolin (3% - 4%) (PEG 75) + Vegetable Glycerin (6% - 9%)

The Second Step:

Addition of herbal Penetration Enhancers

Crataegus Extract (2% - 4%) + Musk (4% - 6%) + Licoric Extract (3% - 5%)

The Third Step:

Addition of fibroblastic growing factor (FGF) from platelet plasma mediators by centrifugation of blood of the patient (lOcc)

(FGF: Hydroxy Triptamin + TGBF¹, TGBF² + Histamin + Albumidis + CA)

The Final Step:

Addition of Wheat germ extract as a humidifier + Phytosterols

Riboflavin _ Niasin + Ca/Se/Mg/ Vit E/ Vit B

Claims

Cream Synthesis from platelet mediators with herbal penetration enhancers to increase the skin’s collagen Claims

1. Cream Synthesis from platelet mediators to increase the skin’s collagen which is consisted of following components:

a. Basic Cream

b. Herbal Penetration Enhancers

c. Platelet-rich Plasma

d. Wheat Germ

2. According to claim 1, the Basic Cream which is consisted of following components:

a. Sweet Almond Oil (27% - 38%)

b. Beez wax (10% - 12%)

c. Lanolin (3% - 4%) (PEG 75)

d. Vegetable Glycerin (6% - 9%)

3. The herbal penetration enhancers of the Cream which is consisted of following components: a. Crataegus Extract (2% - 4%)

b. Musk (4% - 6%)

c. Licoric Extract (3% - 5%)

4. The Addition of fibroblastic growing factor from platelet plasma mediators by centrifugation of lOcc blood of the patient which is consisted of following components:

a. FGF: Hydroxy Triptamin

b. TGBF1, TGBF2

c. Histamin

d. Albumidis

e. CA

5. The addition of Wheat germ extract as a humidifier + Phytosterols which is consisted of

following components:

a. Riboflavin _ Niasin

b. Ca/Se/Mg/ Vit E/ Vit B

6. According to claim 3, this step takes 20-25 min to complete under 25-30c and constant

temperature.

7. According to claim 4, this step takes 5-8 min to complete under 25-30c and constant

temperature.

8. According to claim 5, this step takes 10-12 min to complete under 25-30c and constant

temperature.