WO2020097766A1

WO2020097766A1 - Application of cannabis sativa extract or cannabidiol in scar treatment

Info

Publication number: WO2020097766A1
Application number: PCT/CN2018/115076
Authority: WO
Inventors: 于朝晖; 张可; 谭昕; 吴亚南; 李萌
Original assignee: 汉义生物科技（北京）有限公司
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2020-05-22

Abstract

Disclosed is an application of a Cannabis sativa extract or cannabidiol in scar treatment. Also disclosed are an application of a Cannabis sativa extract or cannabidiol in preparing a drug or cosmetic for scar treatment and a use of a Cannabis sativa extract or cannabidiol in preparing a drug or cosmetic for inhibiting tyrosinase activity or promoting fibroblast proliferation.

Description

Application of cannabis extract or cannabidiol in removing skin scars

Technical field

The invention relates to the field of medicines and cosmetics, in particular to the application of cannabis extracts or cannabidiol in removing skin scars.

Background technique

Cannabis (scientific name: Cannabis sativa L.) is a plant of the cannabis family and cannabis genus, also known as hemp, hemp, hemp, hemp seed, jute, and jute, and has important agricultural and medical value. Hemp plants have good application value in different parts. Commonly used parts include hemp roots, hemp seeds, hemp stems, hemp flowers and hemp leaves.

Hemp stalks have similar physical and mechanical properties as hardwood. The hemp stalk core is the remaining part of the hemp stalk after removing the bast fibers. The shape is a hollow structure, and the cavity is linear or elliptical, often different from the shape, and there are many fiber cross sections. Fine pores, this structure makes the hemp stalk core rich in oxygen, making anaerobic bacteria unable to survive. The high output of hemp stalks and stalk cores is conducive to industrial application. Cannabis flower, also known as "Mabo", male plants loose flowers, complex raceme, with pedicel, pollinated by wind, can spread about 12km in open area; female plant inflorescence is compact, spike-shaped, female flower is small, no Flower stalks and petals, each flower has only one pistil, surrounded by a green bract leaf. Cannabis leaves have simple and compound leaves, and are mostly palm-shaped compound leaves. The order of the leaves is opposite and alternate. The leaves are green with short hairs and are easy to fall off. The leaf is supported by the petiole, the length of the petiole is generally between 3-15cm, when the growth is strong, the quality of the leaf accounts for 24% -25% of the mass of the entire plant; in the late growth period, due to the factors such as the shedding of the leaf, the quality of the leaf only accounts for the entire plant 8% -14% of mass.

Cannabinoids are unique terpene secondary metabolites in cannabis and have complex biological activities. At present, more than 70 cannabinoids have been isolated, and the main active ingredients are tetrahydrocannabinol (THC) and cannabidiol (CBD). Both THC and CBD have neuroprotective effects. Because of its strong hallucinogenic properties, THC has limited application, CBD has no addiction, and has anti-spasmodic, anti-rheumatic arthritis, and anti-anxiety effects.

Acne is a common skin inflammatory disease, which is related to factors such as excessive sebaceous gland secretion, excessive bacterial growth, clogged pores, endocrine, mood, genetics, dietary habits, environment, and use of inappropriate cosmetics. A large number of sebum secretion and discharge obstacles are prone to secondary bacterial infections. There are a variety of microorganisms in the hair follicles such as Propionibacterium acnes, Staphylococcus albicans and Malassezia furfur, among which Propionibacterium acnes infection is the most important.

The general symptoms of acne are mild, but improper treatment often leaves varying degrees of depression or hypertrophic scars. Traditional Chinese medicine recognizes scars as evidence of blood stasis, so treatment drugs often take products that promote blood circulation and remove blood stasis; modern medicine believes that the specific mechanism of scar formation is not yet clear. It is generally believed that there is excessive proliferation of fibroblasts and inhibition of apoptosis during scar formation.

The effector cells of scar formation are fibroblasts. For keloids and hypertrophic scars, fibroblasts have extensive heterogeneity. Increased collagen synthesis and decreased degradation of fibroblasts lead to excessive accumulation of collagen in pathological scars. The abnormal proliferation of fibroblasts is the main reason for the hyperplasia and persistence of pathological scars. Fibroblasts have obvious abnormal response to factors such as cytokines. The contraction of hypertrophic scars is directly related to the heterogeneity of fibroblasts.

The important factor that affects scar formation and regression is collagen renewal. The renewal process mainly depends on the action of collagenase, which is mainly produced by fibroblasts. When connective tissue is formed, new collagen is continuously synthesized. At this time, collagenase not only continues to decompose the remaining collagen, but also the newly synthesized collagen. The deposition and decomposition of collagen fibers form a dynamic balance, which stabilizes collagen.

However, the massive proliferation and inhibition of apoptosis of repair cells (mainly fibroblasts), the imbalance of collagen synthesis and decomposition in extracellular matrix, the massive production of some growth factors, and the close relationship between the three constitute the biological basis of pathological scar formation .

In the treatment of acne, the traditional Chinese medicines on the market are mainly taken orally, with few external use, and the main effects are clearing away heat and detoxifying, promoting blood circulation and removing blood stasis. Western medicines are mostly used for external use, such as tamotifene, tretinoin, meat Toxicine, bleomycin, TNF-α, etc. mostly play a role in scar treatment from the perspective of inhibiting fibroblasts, but due to unclear effects, poor efficacy, high recurrence rate, difficult to cure and other issues, affecting the promotion and application.

Patent US20160214920A1 mainly discloses a method for extracting cannabinoids from industrial cannabis, making full use of industrial waste materials to extract high-purity CBD, and mentions that CBD can be used to treat acne. Attila Oláh et al. (J Clin Invest, 2014 Sep 2, 124 (9): 3713–3724) studies have shown that CBD can inhibit adipogenesis and inhibit blood cell proliferation channels, while also playing an anti-inflammatory role, thus indicating that CBD has potential as a treatment for acne vulgaris Promising therapeutic agent. Patent CN106232130A discloses a method of extracting cannabis plant material using aloe, and using the extract to treat acne and other skin disorders. However, none of the above-mentioned prior art mentions the use of cannabis extract or cannabidiol in removing skin scars, especially acne marks left by acne, nor does it apply cannabis extract or cannabidiol to medicines or cosmetics. Remove skin scars or acne marks.

Summary of the invention

The inventor of the present application explored the use of cannabis extracts or cannabidiol in medicines and cosmetics to treat scars through processes. More surprisingly, the inventors found that the actives in cannabis extracts, such as CBD, are harmful to bacteria, especially acne Propionibacterium has the effect of killing and inhibiting, and can prevent the formation of acne; at the same time, the inventors have proved through experiments that cannabis extract or cannabidiol can promote the proliferation of epidermal cells and inhibit the activity of tyrosinase, promote the rapid removal of acne marks, and eliminate The current status of pigmentation in the scars, so as to reduce the effect of acne marks.

In order to achieve the above objectives, the present invention adopts the following technical solutions:

The invention provides an application of cannabis extract or cannabidiol in removing skin scars.

Cannabis extracts or cannabidiol have a good repair effect on newborn acne pits. Various traumas will cause different degrees of cell degeneration, necrosis and tissue defects, and must be repaired through cell proliferation and formation of intercellular matrix . In this repair process, fibroblasts play a very important role. Cannabis extract stimulates the proliferation and growth of fibroblasts, which can help the skin of acne pits to repair quickly in the early stage; in the later stage of wound repair, fibroblasts secrete collagenase to participate in the reconstruction of the tissue after repair and increase collagen synthesis. Accelerate the uniform healing of skin wounds and increase the local tensile strength and other biological effects.

At the same time, the repair mechanism of cannabis extract or cannabidiol on the scab part is: first, excessive accumulation of fibroblasts occurs in the scab part, and an appropriate exfoliating agent (such as organic acids) is needed to help eliminate excess surface cells ; Second, hemp extract can promote the proliferation of fibroblasts and release collagenase. At this time, collagenase not only continues to decompose the remaining collagen, but also the newly synthesized collagen. The deposition and decomposition of collagen fibers form a dynamic balance-making the place Skin tends to be consistent with other normal skin. In addition, fibroblast proliferation can accelerate cell exfoliation and accelerate the removal of melanin.

Preferably, the application of the cannabis extract or cannabidiol of the present invention in removing skin scars refers to removing skin scars for non-therapeutic purposes.

The cannabidiol (CBD) according to the present invention can be a chemical synthesis product, a biosynthesis product, a plant extract, or can be prepared by other methods. Preferably, the cannabidiol according to the present invention is a plant extract, and the plant extracting part may be the stalk core, flower, leaf, root and / or seed shell of cannabis Cannabis sativa L. Preferably the extract is hemp leaf Thing.

In an embodiment of the present invention, the hemp extract contains 50% -99% actives by mass percentage; preferably, the hemp extract contains 60% -99 actives by mass percentage %; Preferably, the cannabis extract contains 95% -99% actives by mass percentage. The active substance described in the present invention is a cannabinoid substance which removes tetrahydrocannabinol, and the active substance is selected from cannabidiol, cannabidiol, cannabidiol terpene, cannabicyclic terpene phenol, hypocannabidiol, tetrahydro One or two or more of hypocannabinol and cannabidiol; preferably, the active substance is cannabidiol.

In another embodiment of the present invention, the cannabis extract contains 1% -99% cannabidiol in mass percentage. Preferably, the cannabis extract of the present invention contains 50% -99% cannabidiol in mass percentage. More preferably, the cannabis extract contains 60% -99% cannabidiol in mass percentage. Particularly preferably, the cannabis extract of the present invention contains 95% -99% cannabidiol in mass percentage.

In an embodiment of the present invention, the medicine or cosmetics contain hemp extract or cannabidiol at a mass percentage of 0.01% -6%; preferably, the medicine or cosmetics contain hemp extract at a mass percentage Drug or cannabidiol is 0.01% -3%; further preferably, the medicine or cosmetic contains 0.1% -1% of cannabis extract or cannabidiol by mass percentage; further more preferably, the medicine Or the cosmetics containing hemp extract or cannabidiol by mass percentage is 0.4-0.6%; particularly preferably, the medicines or cosmetics containing cannabis extract or cannabidiol by mass percentage is 0.6%.

The scars of the present invention are selected from hypertrophic scars, superficial scars, atrophic scars, keloids, contracture scars or concave scars; preferably, the scars of the present invention are acne scars.

The invention also provides the application of cannabis extract or cannabidiol in the preparation of medicines or cosmetics for removing skin scars; preferably, the scars are acne scars.

The present invention also provides the use of cannabis extract or cannabidiol in the preparation of drugs or cosmetics that inhibit tyrosinase activity and promote the proliferation of fibroblasts; preferably, the scars are acne scars.

Preferably, the medicine or cosmetics of the present invention may be in any dosage form acceptable in the pharmaceutical or cosmetic field, including solid, liquid or semi-solid; the cosmetics include aerosol sprays, creams, emulsions, facial cleanser, Dispersions, foams, gels, skin toners, toners, lotions, mousses, ointments, powders, patches, hair waxes, solutions, hand pump sprays, product forms composed of sticks, and wet wipes Product form; the medicines are preferably ointments, emulsions, sprays, gels, patches; the medicines or cosmetics of the present invention may also contain a variety of optional components, such as abrasives, buffers, ingredients Films, humectants, sunscreens, fragrances, pigments, essential oils, emollients, skin soothing agents, pH adjusters, plasticizers, preservatives, additional skin conditioners, skin penetration enhancers, skin protectants, Suspending agents, emulsifiers, thickeners, solubilizers, sunscreens, ultraviolet absorbers or scattering agents, sunless tanning agents, antioxidants and / or free radical scavengers, vitamins and their derivatives, and other natural extracts Wait.

The cannabis extract of the present invention is obtained from a cannabis plant. The extraction site of the cannabis plant includes the outer shell of stalk core, flower, leaf, root and / or seed; the cannabis extract may be Extracts or combinations of extracts formed by extracting different plant parts of plants can also be extracts or combinations of extracts obtained by simultaneously extracting different plant parts or combinations thereof of cannabis plants. Preferably, the cannabis extract of the present invention is hemp leaf extract.

The cannabis extract of the present invention is obtained by conventional extraction methods in the pharmaceutical or cosmetic field, preferably by solvent extraction. The extraction solvent is selected from water, low molecular alcohol or its aqueous solution, acetate, ketone, ether or low boiling point hydrocarbon; the low molecular alcohol includes methanol, ethanol, butanol or propanol; the ethyl alcohol The acid ester includes methyl acetate or ethyl acetate; the ketone includes acetone; the ether includes methyl ether or ether; the low-boiling hydrocarbon includes aliphatic hydrocarbon, aromatic hydrocarbon or chlorinated hydrocarbon. Preferably, the extraction solvent is selected from water or ethanol solution.

In a specific embodiment of the present invention, the cannabis extract can be obtained by the following method:

1) Use extraction solvent or its mixture to heat and reflux the raw materials;

2) Filter to remove residues;

3) Extraction;

4) Adjust the pH value to 2-4;

5) Extract with extraction solvent, and then remove the solvent;

6) Perform chromatographic separation to obtain hemp extract.

Preferably, the step 1) uses 3-10 times the amount of extraction solvent or a mixture thereof to heat and reflux the cannabis extraction site for at least 1 hour; the extraction solvent mentioned in step 3) in the method for extracting cannabis extract is An aqueous solution of sodium hydroxide containing 20% by weight of ethanol; the pH adjusting agent in the step 4) is a mixed liquid of the extract and the 5% sulfuric acid solution; the chromatographic separation described in the step 6) in the extraction method of the cannabis extract The mobile phase mixture used consists of methanol / water and acetic acid or ethanol / water and acetic acid.

It should be noted that the description of "3-10 times the amount" in this application refers to the volume of the extraction solvent used is 3-10 times the mass of the extraction site, for example, the cannabis extraction site is 1g, and the amount of extraction solvent is 3mL-10mL.

1) Crush the raw materials to 10-40 mesh to obtain powder;

2) The resulting powder is cold-dipped with an extraction solvent to obtain an extraction solution;

3) Decolorize the obtained extract;

4) Concentrate under reduced pressure to obtain cannabis extract extract.

Preferably, in the step 2), 4-8 times the amount of 30% -60% ethanol is used for cold soak extraction 1-3 times, 0.5-2 hours each time; in the step 3), the decolorization should be applied 0.1-1wt % Activated carbon adsorption decolorization; the reduced pressure concentration at 70 ℃. Concentrated to a relative density of 1.01-1.03.

The cannabis extract or cannabidiol of the present invention can promote the proliferation of epidermal cells, and the extremely low toxicity of the cannabis extract or cannabidiol helps to protect the new cells after using the product and make the product more durable; cannabis extraction The substance or cannabidiol can also accelerate the proliferation of fibroblasts, which can help accelerate the transfer of keratinocytes to the stratum corneum, accelerate cell regeneration, and thus promote the shedding of parts containing acne marks to reduce the effect of acne marks; inhibit tyrosinase activity in vitro Experiments have shown that cannabis extract or cannabidiol has a certain whitening effect, which can help eliminate the current status of pigmentation at the scar, and help to restore the skin color at the scar to the rest of the skin; and cannabis extract or cannabidiol against bacteria It has inhibitory and killing effects and is suitable for the treatment and prevention of acne.

BRIEF DESCRIPTION

Hereinafter, the embodiments of the present invention will be described in detail with reference to the drawings, in which:

Figure 1: The effect of cannabis leaf extract (99% active substance content, 95% CBD) on the tyrosinase inhibition rate. As the concentration increases, the inhibition rate increases;

Figure 2: The effect of cannabis leaf extract (50% active ingredient, 49.8% CBD) on the tyrosinase inhibition rate. As the concentration increases, the inhibition rate increases;

Figure 3: The effect of different concentrations of cannabis leaf extract (99% active content, 95% CBD) on the relative survival rate of B16 cells;

Figure 4: After different concentrations of cannabis leaf extract (99% active content, 95% CBD) act on B16 cells, the intracellular tyrosinase activity changes, with arbutin as a positive control and cell blank as a negative control;

Figure 5: After different concentrations of cannabis leaf extract (99% active content, 95% CBD) act on B16 cells, the cells produce melanin changes, of which arbutin is a positive control and cell blank is a negative control;

Figure 6: The effect of different concentrations of cannabis leaf extract (A: 50% active content, 49.8% CBD; B: 99% active content, 95% CBD) on the proliferation of fibroblasts, of which the blank control is not Culture medium containing the drug to be tested.

detailed description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are only some, but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative efforts fall within the protection scope of the present invention.

Example 1 Preparation of cannabis extract

(1) Wash and dry the raw materials (marijuana leaf, hemp stalk core, hemp flower);

(2) Crush the raw materials after air-drying, and then pass through a 40 mesh sieve;

(3) The obtained powder is cold-extracted twice with 6 times the amount of 45% ethanol for 1.5 hours each time;

(4) Combine extracts and decolorize with 0.5wt% activated carbon;

(5) Under the temperature condition of 70 ℃, concentrate under reduced pressure to a relative density of 1.02 to obtain the extract of cannabis extract.

According to the above preparation method,

When the raw material is hemp leaf, hemp extract 1A is obtained;

When the raw material is hemp flower, hemp extract 1B is obtained;

When the raw material is hemp stalk core, hemp extract 1C is obtained;

When the raw materials are hemp leaves and hemp flowers, hemp extract 1D is obtained;

Among them, the cannabis extract 1A contains 50% of the active substance, and 90% of the cannabidiol in the active substance.

Example 2 Preparation of cannabis extract

(1) Use 9 times the amount of ethanol to heat and reflux the raw materials (cannabis leaf, hemp stalk core, hemp flower) for 1 hour;

(2) Remove the residue by filtration and remove the solvent under vacuum;

(3) Heat the obtained extract at a temperature of about 125 ° C for about 40 minutes;

(4) Subsequent chromatographic separation, in which the mobile phase mixture is composed of ethanol / water and acetic acid, that is, cannabis extract.

According to the above preparation method,

When the raw material is hemp leaf, hemp extract 2A is obtained;

When the raw material is hemp flower, hemp extract 2B is obtained;

When the raw material is hemp stalk core, hemp extract 2C is obtained;

When the raw materials are hemp leaf and hemp flower, hemp extract 2D is obtained;

Among them, the cannabis extract 2A contains 80% of the active substance and 92% of the cannabidiol in the active substance.

Example 3 Preparation of cannabis extract

(1) Use 7 times the amount of ethanol to heat and reflux the raw materials (cannabis leaf, hemp stalk core, hemp flower) for 1.5 hours;

(2) Remove residues by filtration;

(3) Extract at least twice with a 5% aqueous sodium hydroxide solution, where the aqueous sodium hydroxide solution contains 20 wt% ethanol;

(4) Mix the extract with 5% sulfuric acid solution to make the pH value about 3;

(5) Then use aliphatic hydrocarbons to extract twice, and remove the solvent under low temperature vacuum;

(6) Subsequent chromatographic separation, in which the mobile phase mixture is composed of ethanol / water and acetic acid, that is, cannabis extract.

According to the above preparation method,

When the raw material is hemp leaf, hemp extract 3A is obtained;

When the raw material is hemp flower, hemp extract 3B is obtained;

When the raw material is hemp stalk core, hemp extract 3C is obtained;

When the raw materials are hemp leaves and hemp flowers, hemp extract 3D is obtained;

Among them, hemp extract 3A contains 99% of the active substance, and 95% of the cannabidiol in the active substance.

Example 4 Preparation of facial cleanser for removing skin scars

Formula (mass percentage):

Phase A: Cocoamide MEA 2%, coconut oil amphoteric sodium acetate 7%, ethylene glycol distearate 1.5%, vitamin E acetate 0.1%, cannabis leaf extract (1A prepared in Example 1) 6 %, Coconut oil amphoteric acetic acid 1.4%, deionized water 81.74%;

Phase B: essence 0.05%, methyl isothiazolinone 0.01%;

Phase C: citric acid 0.2%.

Heat phase A to 78 ° C, stir until it is fully dissolved, then cool to 40 ° C, then add phase B to stir and mix evenly, and finally add phase C to adjust the pH to 5.5, lower the temperature to 30 ° C, and filter to get the facial cleanser.

Example 5 Preparation of liquid foundation for removing skin scars

Formula (mass percentage):

Phase A: sorbitan olive oleate 0.5%, lauryl PEG / PPG-18 / 18 polymethylsiloxane 3%, cetyl PEG / PPG-10 / 1 polydimethylsiloxane 3%, Dimethicone 4%, mineral oil 8%, isopropyl myristate 5%, magnesium stearate 1%, aluminum stearate 1%, propyl paraben 0.15%, titanium dioxide 8%, Pigment 1.5%;

Phase B: glycerin 6%, methyl paraben 0.15%, sodium chloride 1.5%, hemp leaf extract (2A prepared in Example 2) 0.01%, deionized water 57.19%.

Heat phases A and B to 80 ° C under good agitation. After fully dissolving, slowly add phase B to phase A, homogenize and mix the two phases uniformly, and cool to 30 ° C to obtain liquid foundation.

Example 6 Preparation of spray for removing skin scars

Formula (mass percentage):

Phase A: 1,2-propanediol 6%, ethylhexyl glycerol 0.1%, deionized water 90.4%;

Phase B: Cannabis leaf extract (3A prepared in Example 3) 3%;

Phase C: DMDM Hydantoin 0.5%.

Stir phase A and heat it to 85 ° C. After fully dissolved, stir and cool to 40 ° C. Add phase B and phase C materials. After stirring evenly, continue to cool to 30 ° C and filter to obtain spray.

Example 7 tyrosinase activity inhibition experiment

1. Experimental principle

Pigmentation often occurs at the location of the scar, and in order to restore the color of the skin at the scar to the normal skin color, a study of melanin synthesis is required. Melanin is a high-molecular biological pigment synthesized in melanocytes. It is synthesized in the cell, transported from melanocytes to keratinocytes, and dispersed to various layers of the epidermis as the keratinocytes mature, and finally disappears as the epidermis falls off. Aiming at its formation path, this experiment studied the whitening efficacy of industrial cannabis leaf extract through the methods of cell biology and molecular biology.

At the cellular level, the effects of industrial cannabis leaf extracts on the activity of tyrosinase and melanin content in melanocytes are mainly measured. At the molecular level, the effects on the expression of relevant and important genes during melanin synthesis are mainly studied. B16 cells were selected in this experiment because the basic structure of the B16 cell line, especially the melanin synthesis process is basically the same as that of normal human melanocytes, and human primary melanocytes are not easy to culture, so the evaluation of whitening efficacy In the process, B16 cells were selected as a suitable model.

2. Experimental methods

1. Determination of tyrosinase inhibition rate

1.1 Experimental method:

Collect cells in good logarithmic growth phase, digest with trypsin, complete the medium to stop digestion, count; adjust the cell suspension to 10 × 10 ⁴ cells / mL, inoculate in 96-well culture plate, add 100 μL of cell suspension to each well The edge wells were filled with sterile PBS and incubated overnight at 37 ° C in a 5% CO ₂ incubator. Pipette the medium, add 100 μL of sample per well for 72 h, add 50 μL of 1% TritionX-100 solution to lyse the cells, and quickly release Store in an ultra-low temperature refrigerator at -80 ℃ for 1h, and then thaw at room temperature to release the tyrosinase inside the cells. After pre-warming at 37 ℃, add 50μL of 1% levodopa solution, react at 37 ℃ for 2h, and measure at a wavelength of 490nm For the absorbance value of each well, set more than three auxiliary wells for each concentration, and take the average value.

1.2 Experimental samples:

Cannabis leaf extracts with concentrations of 0.1%, 0.5%, 1%, and 2% (99% active content, 95% CBD);

Cannabis leaf extracts with concentrations of 0.50%, 1%, 2%, and 3% (50% active content, 49.8% CBD).

1.3 Calculation formula of tyrosinase inhibition rate:

Tyrosinase inhibition rate = 1- (average absorbance value of each concentration-average absorbance value of control group) × 100%

2. B16-MTT experiment

2.1 Experimental method:

Collect the logarithmic phase cells, adjust the concentration of the cell suspension, add 100 μL per well (96-well plate), and plate to adjust the density of the cells to be tested to 500 cells / well (the edge wells are filled with sterile PBS). Incubate with 5% CO ₂ at 37 ° C until the cells grow to a certain density, add cannabis leaf extract (99% active content, 95% CBD) after changing the medium, so that cannabis leaf extract (99% active content, 95% CBD) ) Concentrations are 5.00%, 1.67%, 0.56%, 0.19% and 0.06%, respectively, and the culture fluid without cannabis leaf extract (99% active content, 95% CBD) is used as a control. Incubate with 5% CO ₂ at 37 ° C for 24 hours, add 20 μL of MTT solution (5 mg / mL, or 0.5% MTT) to each well, and continue culturing for 4 h. Centrifuge and discard the culture solution. After careful washing with PBS for 2-3 times, Then add the culture medium containing MTT. To stop the culture, carefully aspirate the culture medium from the well. Add 150 μL of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at an OD value of 490 nm of the enzyme-linked immunoassay detector.

2.2 Medium:

The medium used for B16 cell culture is: DMEM medium + 10% fetal bovine serum.

2.3 Formula for calculating the relative survival rate of cells:

Cell relative survival rate = (measured OD value-blank control OD value) / (cell control group OD value-blank control OD value) x 100%.

3. Determination of tyrosinase activity

3.1 Experimental method:

Collect cells in good logarithmic growth phase, digest with trypsin, complete the medium to stop digestion, count; adjust the cell suspension to 10 × 10 ⁴ cells / mL, inoculate in 96-well culture plate, add 100 μL of cell suspension to each well The edge wells were filled with sterile PBS and incubated overnight at 37 ° C in a 5% CO ₂ incubator. Pipette the medium, add 100 μL of sample to each well for 48 h, add 50 μL of 1% TritionX-100 solution to lyse the cells, and quickly release Store in an ultra-low temperature refrigerator at -80 ℃ for 1h, and then thaw at room temperature to release the tyrosinase inside the cells. After pre-warming at 37 ℃, add 50μL of 1% levodopa solution, react at 37 ℃ for 2h, and measure at a wavelength of 490nm For the absorbance value of each well, set more than three auxiliary wells for each concentration, and take the average value.

3.2 Experimental samples:

Cannabis leaf extracts with concentrations of 0.2%, 1%, and 5% (99% active content, 95% CBD).

Control: 33mM arbutin and cell blank.

3.3 Calculation formula of tyrosinase activity:

Tyrosinase activity = (average absorbance value of each concentration-average absorbance value of control group) × 100%

4. Determination of melanin content

Collect B16 cells in good logarithmic growth state, digest with trypsin, complete the medium to stop digestion, count; adjust the cell suspension to 5 × 10 ⁴ cells / mL, add to 6-well culture plate, add 2mL cells to each well The suspension was placed in an incubator at 37 ° C and incubated overnight in a 5% CO ₂ environment; aspirate the medium, add 2mL of cannabis extract of different concentrations to each well for 48h, aspirate the medium, digest with trypsin, and collect into a centrifuge tube During centrifugation, the cell pellet was obtained by centrifugation, washed once with PBS, lysed by adding 1M NaOH (10% DMSO) solution, and placed in an 80 ° C water bath for 30 min in the water bath; shake and mix, and absorb to a 96-well plate; and measure the absorbance value at 475nm .

3. Experimental results

1. Inhibitory effect of cannabis extract on tyrosinase

It can be seen from Figures 1 and 2 that marijuana leaf extract (99% active content, 95% CBD) and hemp leaf extract (50% active content, 49.8% CBD) have inhibitory effects on tyrosinase. As the concentration of cannabis extract increases, the tyrosinase inhibition rate increases.

2. B16-MTT experiment results

As can be seen from Figure 3, within a certain range, the survival rate of B16 cells decreases with increasing concentration of cannabis leaf extract (99% active content, 95% CBD), with a certain concentration dependence. When the concentration of marijuana leaf extract (99% active content, 95% CBD) concentration was 5%, the relative survival rate of cells was the lowest at 90.88% for 48 hours, and the relative survival rate of cells at other concentrations was above 90.88%. There was no significant difference at different concentrations Sexual differences, showing no cytotoxicity.

3. Results of tyrosinase activity changes

The results of changes in intracellular tyrosinase activity of B16 cells with different concentrations (0.2%, 1%, 5%) of cannabis leaf extract (99% active content, 95% CBD) were investigated, as shown in Figure 4. In this experiment, 33mM arbutin was used as a positive control, and the cell blank was used as a negative control, and the experimental data was significantly analyzed (p <0.05).

The results showed that the extracts of cannabis leaf at concentrations of 0.2%, 1%, and 5% can significantly reduce the activity of intracellular tyrosinase. As the concentration of cannabis leaf extract increased, the activity of tyrosinase decreased, and the concentration was 0.2 %, 1%, and 5% of the cannabis leaf extract have similar inhibitory effects on tyrosinase activity as the positive control arbutin, while the 5% concentration of cannabis leaf extract has inhibitory effect on tyrosinase The best is to reduce the tyrosinase activity to 73.13%.

4. Result of melanin content change

After investigating different concentrations of cannabis leaf extract (99% active content, 95% CBD) on B16 cells, the amount of melanin produced by the cells was changed. The results are shown in Figure 5. In this experiment, 33mM arbutin was used as a positive control, and the cell blank was used as a negative control. A significant analysis of the experimental data showed that different levels of cannabis leaf extract (99% active content, 95% CBD) can be significant Reducing the amount of melanin synthesized in the cell can reduce the melanin content in B16 cells to 73.79%, and the positive control is 79.24%. With the increase in the concentration of cannabis leaf extract (99% active content, 95% CBD), The melanin content becomes low. There is no significant difference between the effects of different levels of cannabis leaf extract on melanin synthesis and the relative ratio of arbutin.

Example 8 Fibroblast proliferation experiment (MTT)

1. Experimental method

Collect the logarithmic phase cells, adjust the concentration of the cell suspension, add 100 μL per well (96-well plate), and plate to adjust the density of the cells to be tested to 5000 cells / well (the edge wells are filled with sterile PBS). Incubate with 5% CO ₂ at 37 ° C until the cells grow to a certain density. Change the test solution and add different concentration gradients of the drug to be tested. Take the culture solution without the drug to be tested as the control. Incubate 5% CO ₂ at 37 ° C for 24 hours, add 20 μLMTT solution (5 mg / mL, ie 0.5% MTT) to each well, and continue culturing for 4 h. To make the drug fully react with MTT, centrifuge and discard the culture solution. After carefully washing 2-3 times with PBS, add the culture solution containing MTT. To stop the culture, carefully aspirate the culture medium from the well. Add 150 μL of dimethyl sulfoxide to each well, and shake on a shaker at low speed for 10 min to fully dissolve the crystals. The absorbance of each well was measured at an OD value of 490 nm of the enzyme-linked immunoassay detector.

Cell viability = (measured well OD value-blank control OD value) / (cell control group OD value-blank control OD value) * 100%.

2. Experimental results

As shown in Fig. 6, cosmetics containing hemp leaf extract (99% active content, 95% CBD) and hemp leaf extract (50% active content, 49.8% CBD) at a concentration of 1% and below Non-toxic, and has the effect of promoting proliferation. Accelerating cell proliferation can help accelerate the transfer of keratinocytes to the stratum corneum, accelerate cell regeneration, and thus accelerate the shedding of parts containing acne marks to reduce the effect of acne marks.

Example 9 Human safety test

This test complies with the relevant provisions of the human body safety inspection method in the "Cosmetics Safety Technical Specifications".

The necessary toxicological test has been completed before this test, and the test result is qualified.

Human skin patch test-skin closed patch test

1. Test Purpose

Detect the potential of the test substance to cause adverse reactions in human skin.

2. Test materials

2.1 Test product

For the 3 drugs / cosmetics involved in Examples 4-6, the formulation of each product was selected for the human safety test), a total of 3 products.

2.2 Spot test materials

Qualified spot test equipment with an area not exceeding 50 mm ² and a depth of about 1 mm.

3. Subject selection

90 social volunteers that meet the requirements, aged 20-45 years old, 30 males and 60 females, are divided into 3 groups according to products, 30 in each group, and try the 3 types of products listed in Examples 4-6 In the formula, there are 10 men and 20 women in each group.

4. Test method

4.1 Take 0.020g-0.025g (solid or semi-solid) or 0.020mL-0.025mL (liquid) of the tested product respectively and put them into the chamber of the spot tester.

4.2 Control hole does not do any treatment.

4.3 Place the spot tester with the test substance on the back of the subject or the curved side of the forearm with hypoallergenic tape, and gently press it with your palm to apply it evenly on the skin for 24 hours.

4.4 Observe the skin reaction according to the standard in Table 3 at 30 min (after the indentation disappears), 24 h and 48 h after removing the spot of the test object.

Table 3 Classification criteria of skin reaction of closed skin patch test

5. Test results

The test results of all products showed negative reactions, proving that the safety of the cosmetics provided by the present invention is guaranteed, and there will be no adverse reactions such as skin irritation, sensitization (except for people who are allergic or allergic to this product);

The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical idea of the present invention, various simple modifications can be made to the technical solution of the present invention. These simple modifications All belong to the protection scope of the present invention.

In addition, it should be noted that the specific technical features described in the above specific embodiments can be combined in any suitable manner without contradictions. In order to avoid unnecessary repetition, the present invention The combination method will not be explained separately.

In addition, various combinations of various embodiments of the present invention can also be arbitrarily combined, as long as it does not violate the idea of the present invention, it should also be regarded as the content disclosed by the present invention.

Claims

The use of cannabis extract or cannabidiol in removing skin scars.
The use of cannabis extract or cannabidiol according to claim 1 in removing skin scars, characterized in that the scars are hypertrophic scars, superficial scars, atrophic scars, keloids, contracture scars Or concave scars; preferably, the scars are acne scars.
Application of cannabis extract or cannabidiol in preparing medicines or cosmetics for removing skin scars.
The use of cannabis extract or cannabidiol in the preparation of drugs or cosmetics that inhibit tyrosinase activity and promote the proliferation of fibroblasts.
The use according to claim 3 or 4, characterized in that the cannabis extract contains 50% -99% of active substance by mass percentage, and the active substance is cannabidiol to remove tetrahydrocannabinol Substance, preferably, the active substance is selected from one or two of cannabidiol, cannabidiol, cannabidiol terpene, cannabicyclic terpene phenol, hypocannabidiol, tetrahydrocannabinol, cannabidiol. the above.
The use according to claim 3 or 4, characterized in that the hemp extract contains cannabidiol at a mass percentage of 1% -99%; preferably, the hemp extract contains a mass percentage Cannabidiol is 50% -99%.
The use according to claim 3 or 4, characterized in that the medicine or cosmetic contains 0.01% -6% of cannabis extract or cannabidiol in mass percentage.
The use according to claim 3 or 4, characterized in that the medicine or cosmetic can be any dosage form acceptable in the field of pharmacy or cosmetics, and the medicine is preferably an ointment, emulsion, spray, gel, Patches; the cosmetics are selected from aerosol sprays, creams, lotions, facial cleansers, dispersions, foams, gels, toners, toners, lotions, mousses, ointments, powders, patches, hair wax , Solution, hand pump spray, product form composed of sticks, product form composed of wet tissues.
The use according to claim 3 or 4, characterized in that the cannabis extract is extracted from a cannabis plant, and the extraction site of the cannabis plant includes stalk core, flower, leaf, root and / or seed shell.
The use according to claim 9, characterized in that the extraction site of the hemp plant is hemp leaves.
The use according to claim 3 or 4, characterized in that the cannabis extract is obtained by solvent extraction, and the solvent is selected from water, low molecular alcohol or its aqueous solution, acetate, ketone, ether or low Boiling point hydrocarbons.
The use according to claim 3 or 4, characterized in that the hemp extract can be obtained by the following method:

1) Use extraction solvent or mixture to heat and reflux the extraction site of hemp plant;

2) Filter to remove residues;

3) Extraction;

4) Adjust the pH value to 2-4;

5) Extract with extraction solvent, and then remove the solvent;

6) Chromatographic separation to obtain cannabis extract; or,

1) Crush the extraction part of hemp plant to 10-40 mesh to obtain powder;

2) The resulting powder is cold-dipped with an extraction solvent to obtain an extraction solution;

3) Decolorize the obtained extract;

4) Concentrate under reduced pressure to obtain cannabis extract extract.