Preparation method and application of platycladus orientalis alcohol extract for preventing alopecia

Abstract

The invention discloses a preparation method and application of a platycladus orientalis alcohol extract for preventing alopecia. The preparation method of the platycladus orientalis alcohol extract comprises the following steps: ultrasonically dissolving the platycladus orientalis alcohol extract with distilled water, and sequentially extracting with petroleum ether, ethyl acetate and n-butyl alcohol to obtain the platycladus orientalis alcohol extract. The platycladus orientalis alcohol extract can significantly improve the growth speed of androgenetic alopecia model mouse hair and accelerate the transition of the hair follicle period from the rest period to the growth period, and the treatment effect of the platycladus orientalis alcohol extract is superior to that of minoxidil (positive drug). When the platycladus orientalis alcohol extract is applied to preparation of anti-alopecia products, the in-vitro transdermal efficiency is high, an action target can be effectively reached, and the platycladus orientalis alcohol extract is non-irritant to skin, safe and reliable.

Description

Technical Field

[0001] The present invention relates to a preparation method and application of a Platycladus orientalis alcohol extract for preventing hair loss, belonging to the field of pharmaceutical technology. Background Art

[0002] Hair loss is divided into two major categories: physiological hair loss and pathological hair loss, and there are obvious differences in its pathogenic roots, mechanisms of action, and treatment methods. Androgenetic alopecia (AGA) is the most common type of pathological hair loss clinically. With the acceleration of people's living rhythm and the increase in work intensity, this type of hair loss shows a trend of getting younger, bringing huge mental pressure and psychological burden to patients. Currently, the commonly used western medicines for clinical treatment of androgenetic alopecia are minoxidil and finasteride, but both drugs are prone to recurrence after discontinuation, and can also cause side effects such as skin irritation, scalp itching, hirsutism on the face and hands, dizziness, anorexia, and tachycardia. In recent years, with the pursuit of healthy, natural, and green products by people, natural anti-hair loss products containing plant active substances have been increasingly favored by consumers.

[0003] Platycladus orientalis, as a traditional medicinal plant, has been widely used in the research and development of anti-hair loss products. CN101926973B discloses a traditional Chinese medicine semen for promoting hair growth, removing dandruff, relieving itching, and preventing hair loss by subcutaneous penetration and its preparation method, which is prepared from raw materials such as Polygonum multiflorum Thunb., Platycladus orientalis, Notopterygium incisum Ting ex H. T. Chang, Angelica sinensis (Oliv.) Diels, Eclipta prostrata L., Zanthoxylum bungeanum Maxim., Mentha haplocalyx Briq., and Zingiber officinale Rosc. The extraction method of its composition is water extraction after mixing various raw materials. The traditional water extraction method only extracts water-soluble components in the raw materials such as polysaccharides, flavonoids, or phenolic substances, and the water-insoluble active components cannot be extracted, and the transdermal absorption effect of the composition of this invention is not clear. Summary of the Invention

[0004] The purpose of the present invention is to provide a preparation method and application of a Platycladus orientalis alcohol extract for preventing hair loss in view of the deficiencies of the existing technology. The Platycladus orientalis alcohol extract can significantly increase the hair growth rate of androgenetic alopecia mice and accelerate the transition of the hair follicle cycle from the resting phase to the growth phase. This alcohol extract has high transdermal efficiency in vitro, can effectively reach the action target, and is mild and does not damage the scalp, and can be used to prepare a new type of natural anti-hair loss preparation.

[0005] The preparation method of the Platycladus orientalis alcohol extract of the present invention includes the following steps:

[0006] Step 1: Extract Platycladus orientalis with an ethanol solution, filter and collect the filtrate, combine, concentrate, and dry the obtained filtrate to obtain a Platycladus orientalis ethanol extract;

[0007] Step 2: Add distilled water to the Platycladus orientalis ethanol extract obtained in Step 1 and dissolve it by ultrasonic treatment. Then add petroleum ether, ethyl acetate, and n-butanol for fractional extraction. The upper organic phases are successively combined, and after drying, the Platycladus orientalis petroleum ether extract, ethyl acetate extract, and n-butanol extract (abbreviated as Platycladus orientalis alcohol extract) are respectively prepared.

[0008] Furthermore, in Step 1, crush Platycladus orientalis and sieve it through a 20-mesh sieve; mix the Platycladus orientalis powder with an ethanol solution having a volume concentration of 70 vt%, reflux and extract in a water bath at 80 °C for 1 h, repeat the extraction 3 times, and collect the extract after centrifugation; concentrate the obtained extract and then freeze-dry it to obtain the Platycladus orientalis ethanol extract.

[0009] Furthermore, in Step 2, weigh the Platycladus orientalis ethanol extract, add distilled water for ultrasonic dissolution according to the mass-to-volume ratio of 1 g: 10 mL, add an equal volume of petroleum ether, shake vigorously, and then let it stand for layering. Remove the lower-layer solution, repeat 3 times, and combine the upper organic phases. After drying, the petroleum ether phase extract is prepared; add an equal volume of ethyl acetate to the combined lower-layer phase after petroleum ether extraction, shake vigorously, and then let it stand for layering. Remove the lower-layer solution, repeat 3 times, and combine the upper organic phases. After drying, the ethyl acetate phase extract is prepared; add an equal volume of n-butanol to the combined lower-layer phase after ethyl acetate extraction, shake vigorously, and then let it stand for layering. Remove the lower-layer solution, repeat 3 times, and combine the upper organic phases. After drying, the Platycladus orientalis alcohol extract is prepared.

[0010] The present application adopts the method of fractional extraction. Fractional extraction separates components in order from low polarity to high polarity, and the active components of the extract are relatively more concentrated. Petroleum ether first extracts components with relatively low polarity such as oils, waxes, and steroids; the remaining phase is then extracted with ethyl acetate to obtain components with medium polarity such as flavonoids and terpenoids; finally, n-butanol extraction obtains components with relatively high polarity such as phenols and organic acids. When directly extracting with petroleum ether, ethyl acetate, and n-butanol, the distribution coefficients of different components in each solvent are different, and the active components have a certain solubility in each organic solvent, so they cannot be enriched, resulting in a low content of active components in the effective part, thus affecting the anti-hair loss effect.

[0011] Application of the Platycladus orientalis alcohol extract of the present invention in the preparation of anti-hair loss functional products.

[0012] Furthermore, the hair loss is androgenetic alopecia.

[0013] Zhang Yan from Shenyang Pharmaceutical University published a doctoral thesis titled "Study on the Active Parts and Active Ingredients CE (Volatile Oil) of Platycladus orientalis for Hair Growth and Their Preparations". This study conducted cell experiments on the Platycladus orientalis volatile oil and co-solvent extracts such as ethyl acetate and n-butanol to detect their effects on promoting hair growth. The results showed that the n-butanol, ethyl acetate extracts, and volatile oil of Platycladus orientalis all showed varying degrees of hair growth promotion effects. Among them, the Platycladus orientalis volatile oil obtained by the water distillation method has a better effect on preventing chemotherapy-induced alopecia and alopecia areata.

[0014] In the early stage, the research group of the applicant was oriented by activity (anti - hair loss), and through a large number of screening tests, it was found that the ethanol extract of Platycladus orientalis had a significant anti - hair loss effect among the water extracts and ethanol extracts of more than 20 traditional Chinese medicines such as Panax ginseng, Astragalus membranaceus, and Scutellaria baicalensis (CN119157795A). To further explore the effective anti - hair loss active components of the ethanol extract of Platycladus orientalis, the applicant carried out an anti - hair loss activity screening test on the fractionated extracts of the ethanol extract of Platycladus orientalis by constructing a mouse model of androgenetic alopecia, and further clarified the therapeutic effect of the n - butanol extract of Platycladus orientalis on pathological alopecia (androgenetic alopecia). The efficacy experiment showed ( Figure 2 ~6) that the anti - hair loss effect of the n - butanol group of Platycladus orientalis was better than that of the petroleum ether group and the ethyl acetate group, and even better than that of the positive group (the clinical drug minoxidil), approaching the effect of the blank control group; at the same time, it was proved that the anti - hair loss components in the n - butanol extract of Platycladus orientalis of the present invention were further enriched, and the content of active ingredients was greatly increased.

[0015] The beneficial effects of the present invention are reflected in:

[0016] 1. The preparation process of the ethanol extract of Platycladus orientalis of the present invention is simple. Using the extract of Platycladus orientalis as the raw material, the components are natural, green and low - toxic, mild to the skin, and will not cause irritation and damage to the scalp and hair, and are suitable for long - term use.

[0017] 2. The ethanol extract of Platycladus orientalis of the present invention further separates and purifies the components in the ethanol extract by using the difference in the solubility of active components in different solvents, so that the anti - hair loss active components are enriched. Compared with the positive drug group (minoxidil), its anti - androgenetic alopecia and transdermal absorption effects are more prominent. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a process flow chart for the preparation of the ethanol extract of Platycladus orientalis of the present invention.

[0019] Figure 2 It is the hair growth effect diagram of the mouse experiment of the present invention.

[0020] Figure 3 It is the hair coverage rate of the mice of the present invention from the 9th day to the 21st day of drug administration.

[0021] Figure 4 It is the average hair length of the mice of the present invention from the 9th day to the 21st day of drug administration.

[0022] Figure 5 It is the H&E - stained section of the skin of the mice of the present invention.

[0023] Figure 6 It is the result of counting the proportion of vellus hair in the skin sections of mice.

[0024] Figure 7 It is the measurement result of the cumulative transdermal rate of the ethanol extract of Platycladus orientalis on the skin of mice.

[0025] Note: The values are expressed as mean ± standard deviation (n = 4). Values with different letters indicate significant differences (P < 0.05 or P < 0.01). Compared with the model group, # P < 0.05 and ## P < 0.01. Specific implementation manners

[0026] The present invention will be described in detail below through examples. It must be pointed out that the following examples are used to illustrate the present invention and should not be construed as limiting the present invention.

[0027] Example 1:

[0028] The preparation method of the Platycladus orientalis alcohol extract in this example includes the following steps

[0029] 1. Crush Platycladus orientalis and pass through a 20-mesh sieve; mix the Platycladus orientalis powder with an ethanol solution, reflux and extract in a water bath at 80 °C for 1 h, repeat the extraction 3 times, collect the extract after centrifugation; combine and concentrate the obtained extract and then freeze-dry to obtain the Platycladus orientalis ethanol extract.

[0030] 2. Weigh 10 g of the Platycladus orientalis ethanol extract, add 100 mL of distilled water and dissolve it by ultrasonic treatment, pour it into a separating funnel, add 100 mL of petroleum ether, shake vigorously and then let it stand for 20 min, release the lower-layer solution, repeat the extraction 3 times, combine the upper-layer organic phase, and dry it to obtain the petroleum ether phase extract;

[0031] 3. Add 100 mL of ethyl acetate to the remaining phase, shake vigorously and then let it stand for 20 min, release the lower-layer solution, repeat the extraction 3 times, combine and dry the upper-layer ethyl acetate phase to obtain the ethyl acetate phase extract;

[0032] 4. Add 100 mL of n-butanol to the remaining phase, shake vigorously and then let it stand for 20 min, release the lower-layer solution, repeat the extraction 3 times, combine and dry the upper-layer n-butanol phase to obtain the Platycladus orientalis alcohol extract.

[0033] Example 2: Detection of anti-hair loss effect

[0034] In this example, the effect of the Platycladus orientalis alcohol extract on hair growth in androgenetic alopecia mice was investigated.

[0035] Test subjects: C57 / BL6 mice, male, with a body weight between 20 - 25 g, randomly divided into 9 groups, 4 mice in each group.

[0036] Model establishment of androgenetic alopecia mice: Each mouse was injected with testosterone propionate for 5 weeks at a dose of 5 mg / kg·d.

[0037] The groups were set as: blank group, model group, positive group, Zhangguang 101 group, Platycladus orientalis ethanol extract group, Platycladus orientalis petroleum ether phase extract group, Platycladus orientalis ethyl acetate phase extract group, Platycladus orientalis n-butanol phase extract group (alcohol extract), and Platycladus orientalis residue after alcohol extraction group.

[0038] Normal saline was prepared for the blank group and the model group, and a 5% minoxidil solution was prepared for the positive group. The product control group was the Zhangguang 101 group, and solutions with a concentration of 100 mg / ml were prepared for the Platycladus orientalis ethanol extract group and each fractionated extraction phase group.

[0039] Test method: Each group of mice was administered once a day, and 100 μL was taken and evenly applied to the depilated area. The medication was continued continuously until the skin entered the telogen phase. The transition time of each mouse from the telogen phase to the anagen phase was recorded. The hair growth of the back area of the mice was photographed every 3 days, and 40 newly grown hairs from the back depilated area of each group of mice were randomly plucked. The average hair length was measured with a vernier caliper, and the changes in hair coverage rate and average hair length of each test mouse over time were statistically analyzed. After 21 days, the mice were sacrificed by cervical dislocation after blood collection from the orbital cavity, and the skin of the back alopecia area was taken for H&E staining to observe the follicle morphology of the back skin of the mice and to count the proportion of vellus hairs.

[0040] Figure 2 This is the back skin state of each group of mice after administration. On the 9th day, the back skin of each group of mice gradually turned black, and hair grew on the back. On the 12th day, hair had grown on each group of mice, but the hair coverage areas were different, and the hair growth rates were different. Figure 3 This is the statistical situation of the hair coverage rate of each group of mice after administration. As time increased, the hair coverage rate of the mice in the Platycladus orientalis alcohol extract group of the present invention was always higher than that of the mice in other fractionated extraction phase groups during the same period, reaching 96.07±1.85% on the 21st day, an increase of 2.89% compared with the mice in the positive group. Figure 4 This is the statistical situation of the average hair length of each group of mice after administration. On the 21st day, the average hair length of the mice in the Platycladus orientalis alcohol extract group reached 7.44±0.37 mm, which was 1.11 times that of the mice in the positive group. The Platycladus orientalis alcohol extract of the present invention can significantly increase the hair growth rate of androgenetic alopecia mice and accelerate the transition of the hair follicle cycle from the telogen phase to the anagen phase.

[0041] The mouse hair follicle morphology sections are as Figure 5 shown. In the blank group, more than 80% of the hair follicles were in the anagen phase in the natural state, with a round and plump appearance and regular morphology (terminal hairs, as Figure 5 indicated by the red arrow in); while a small number of hair follicles were in the telogen phase in the natural state, atrophied, degenerated or even disappeared (vellus hairs, as Figure 5 indicated by the green arrow in). The proportion of vellus hairs in the mouse skin sections was counted as Figure 6As shown, the vellus hair ratio of the model group reached 57.62%, which was 1.91 times that of the positive group. Among the fractionated extracts, the vellus hair ratio of the Platycladus orientalis ethanol extract group was the lowest, at 27.24%, which was 2.9% lower than that of the positive group. The Platycladus orientalis ethanol extract group of the present invention has a therapeutic effect equivalent to that of the positive drug group in relieving hair loss.

[0042] Example 3: Detection of Cumulative Skin Permeation Rate of Mice

[0043] This example investigated the effect of Platycladus orientalis ethanol extract on transdermal absorption of the back skin of mice.

[0044] Preparation of samples, experimental membranes and receiving solutions: Prepare a 20 mg / mL Platycladus orientalis ethanol extract with 0.9% NaCl solution and store it at 4°C for later use; Cut 6 pieces of fresh mouse skin measuring 1.5 cm × 1.5 cm, remove subcutaneous fat, rinse 3 times with physiological saline and store at -20°C for later use; Prepare 1 L of 0.9% NaCl solution as the receiving solution.

[0045] Setting the program: Place the skin sample - test sample - silica gel pad - anti-evaporation glass sheet in the center of the upper part of the diffusion cell assembly in sequence. Set the volume of the receiving solution added to the receptor cell to 15 mL per tube, and set the sampling time points to 0, 2, 4, 6, and 8 h. Take out 5 mL of the receiving solution from the receptor cell at each time point and replenish an equal volume of the receiving solution.

[0046] Data processing: Use an ultraviolet spectrophotometer to measure and record the drug concentration in the receiving solution. According to the measured drug concentration and sampling volume, calculate the cumulative drug permeation rate at each time point using the following formula (1). Plot the transdermal absorption curve with time on the horizontal axis and cumulative permeation rate on the vertical axis.

[0047]

[0048] V: Volume of the receiving solution (mL).

[0049] C: Drug concentration in the receiving solution (g / L).

[0050] D: Dosage (mg).

[0051] Figure 7 is the transdermal absorption curve of Platycladus orientalis ethanol extract. The cumulative transdermal absorption rate of the drug increased continuously from 0 to 8 h and reached the absorption equilibrium at 8 h, with the highest permeation rate reaching 68.80%. The Platycladus orientalis ethanol extract of the present invention has strong transdermal absorption ability and can reach deep into the skin through the skin to promote the growth of skin hair follicles.

[0052] Example 4: Identification of Active Ingredients in Platycladus orientalis Ethanol Extract

[0053] This example is about the investigation of the anti-hair loss active ingredients of Platycladus orientalis ethanol extract.

[0054] GC-MS detection: Weigh the ethanol extract of Platycladus orientalis, dissolve it ultrasonically in n-butanol, and prepare a test solution with a concentration of 1 mg / mL. Perform gas chromatography using an HP-5MS chromatographic column, and use an EI source for mass spectrometry.

[0055] LC-MS detection: Weigh the ethanol extract of Platycladus orientalis, dissolve it ultrasonically in mass spectrometry-grade methanol, and prepare a test solution with a concentration of 1 mg / mL. Perform liquid chromatography using a Zorbox Eclipse Plus C18 chromatographic column. Mobile phase A is an aqueous solution of 0.05% formic acid (V / V), and mobile phase B is methanol. Use an HESI source for mass spectrometry.

[0056]

[0057]

[0058]

[0059] A total of 30 compounds were identified from the ethanol extract of Platycladus orientalis, including 10 flavonoids, 5 phenols, 9 organic acids, 1 lignan, 2 terpenoids, 2 glycosides, and 1 glyceride. Some of these compounds have been proven to have anti-hair loss effects in research. The total flavonoid component in Platycladus orientalis can activate hair matrix cells and promote blood circulation, enabling hair follicles with declining hair growth ability to revive and supplementing them with nutrients, thereby exerting the effects of hair nourishment and hair growth. It has been reported that phenolic compounds in Platycladus orientalis can reduce the level of dihydrotestosterone in animals and promote hair regeneration. Before ungraded extraction treatment, the volatile components of Platycladus orientalis leaves were mainly olefins, phenols, aldehydes and ketones, and esters. The main components analyzed by LC-MS were flavonoids, myricitrin, quercitrin, terpenoids, etc. The volatile components of the ethanol extract of Platycladus orientalis in the present invention are mainly phenolic, terpenoid and ester compounds, and the main components analyzed by LC-MS are organic acids, flavonoids and phenols. The fractional extraction treatment enriches some organic acids and sugars in the ethanol extract of Platycladus orientalis in the present invention, and removes some olefins, phenols and terpenoids. It is preliminarily speculated that the anti-hair loss effect of the ethanol extract of Platycladus orientalis in the present invention may be the result of the synergistic effect of compounds such as flavonoids, phenols and organic acids contained therein.

[0060] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. A method for preparing an alcohol extract of Platycladus orientalis for preventing hair loss, characterized in that The steps include: Step 1: extracting Platycladus orientalis with ethanol solution, filtering and collecting the filtrate, combining, concentrating and drying the obtained filtrate to obtain Platycladus orientalis ethanol extract; Step 2: Add distilled water to the Thuja orientalis ethanol extract obtained in step 1 for ultrasonic dissolution, add petroleum ether, ethyl acetate and n-butanol for graded extraction, combine the upper organic phases in sequence, and after drying, respectively obtain Thuja orientalis petroleum ether extract, ethyl acetate extract and n-butanol extract, referred to as Thuja orientalis ethanol extract.

2. The preparation method according to claim 1, characterized in that: In step 1, the Platycladus orientalis is crushed and passed through a 20-mesh sieve; the Platycladus orientalis powder is mixed with an ethanol solution with a volume concentration of 70 vt%, and extracted by reflux in a water bath at 80°C for 1 h, the extraction is repeated 3 times, and the extract is collected after centrifugation; the obtained extract is concentrated and freeze-dried to obtain the Platycladus orientalis ethanol extract.

3. The preparation method according to claim 1, characterized in that: In step 2, the ethanol extract of Platycladus orientalis is weighed, distilled water is added for ultrasonic dissolution, an equal volume of petroleum ether is added and shaken vigorously, the mixture is allowed to stand for stratification, the lower layer solution is removed, the process is repeated 2-3 times, the upper organic phase is combined, and the petroleum ether phase extract is obtained after drying; an equal volume of ethyl acetate is added to the lower combined phase after petroleum ether extraction and shaken vigorously, the mixture is allowed to stand for stratification, the lower layer solution is removed, the process is repeated 2-3 times, the upper organic phase is combined, and the ethyl acetate phase extract is obtained after drying; an equal volume of n-butanol is added to the lower combined phase after ethyl acetate extraction and shaken vigorously, the mixture is allowed to stand for stratification, the lower layer solution is removed, the process is repeated 3 times, the upper organic phase is combined, and the ethyl acetate phase extract is obtained after drying.

4. The preparation method according to claim 1, characterized in that: In step 2, the amount of distilled water added is 1 g:10 mL in terms of mass to volume ratio.

5. Use of the ethanol extract of Platycladus orientalis prepared by the preparation method according to any one of claims 1 to 4 in the preparation of a product with anti-hair loss function.

6. The use according to claim 5, characterized in that: The hair loss is androgenic alopecia.

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