CA2823422A1 - Anti-aging chinese herbal medicine composition, preparation method and use thereof - Google Patents

Abstract

Disclosed is a traditional Chinese medicinal herbal extract comprising extracts in water or solvent of 10 to 50 parts by weight of Saussurea involucrate, 10 to 50 parts by weight of Cistanche deserticola and 20 to 60 parts by weight of Astragalus membranaceus. The solvent is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate or a combination thereof. The extract and the traditional Chinese medicinal herbal composition containing the extract have the effects of anti-oxidation, promoting dermal fibroblast proliferation and anti-UV irradiation, and can be used as active components in the preparation of cosmetics for preventing skin aging, particularly photo-aging.

Description

Anti-aging Chinese herbal medicine composition, preparation method and use thereof Field of the invention The present invention relates to cosmetics field, particularly to an extract of Chinese herbal medicines, its preparation and use, as well as a Chinese herbal medicine composition containing the extract of Chinese herbal medicines having anti-aging, especially anti-photo-aging effect, its preparation and its use in cosmetics.
Background of the invention Studies have indicated that the skin photo-aging is mainly caused by both the medium-wavelength ultraviolet (UVB, 290-320 nm) together with the long-wavelength ultraviolet (UVA, 320-400 nm) in the sunlight. Ultraviolet rays significantly hasten the aging of human skin, with one-fold higher risk of skin aging and 10-year earlier aging occurrence in high-exposure population as compared with low-exposure population. The long-term exposure to the ultraviolet rays of sunlight results in the light aging of the skin and can lead to serious cosmetic issues, therefore arising professional attentions in recent years. Anti-photo aging products have been released one by one; and more related researches are being conducted. However, the main functional supportive additives in current anti-photo-aging cosmetics in domestic and foreign market are biochemical agent and synthesized compound, anti-photo-aging additives of Chinese herbal medicine having certain traditional Chinese medicine theory guidance remained unseen.
Description of the invention The technical problem to be solved by the present invention is to provide an extract of Chinese herbal medicines.
The extract of Chinese herbal medicines according to the present invention is an extract by water or solvent of 10-50 parts by weight of Saussurea involucrate, parts by weight of Cistanche deserticola and 20-60 parts by weight of Astragalus membranaceus, wherein the said solvent is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, =

=
1,3-butanediol, acetone, ethyl acetate or combinations thereof.
In the above formulations, Saussurea involucrate, Cistanche deserticola and Astragalus membranaceus all have specific anti-aging effects.
In the present invention, Saussurea involucrata refers to the flower, i.e., the medicinal part, of the snow lotus plant. Snow lotus is a herbaceous plant, belonging to subgenus Amphilaena, genus Saussurea, family Asteraceae, and mainly comprises the whole plant with flowers of Saussurea involucrata Karet Kir, Saussurea laniceps Hand.-Mazz., S.gnaphaloides (Boyle) Sch.-Bip., and S.medusa Maxim. Saussurea involucrata mainly produced in Sichuan, Yunnan, Tibet, Xinjiang, Gansu, Qinghai, etc. Upon blooming from June to July, the whole plants thereof are plucked, cleaned from soil, dried, cut into pieces and used in the form of crude materials.
Saussurea involucrata can expel the heat, nourish the blood, keep balance between Yin and Yang and has the tonifying effect, which is called "the celestial grass of thousand years" in Daozang more than one thousand years ago. Yijiuyoushihua recites "Saussurea involucrata, which are grown in the ancient non-thawing snow during one thousand years, look like a lotus flower and could be about 3.3 meters in height. Being used for brewing liquor, it will boast a feeling of spring scenery. It means that Yin reaches extreme so that Yang germinates." In Gangmushiyi, it is recited that "Saussurea involucrata is produced in the northwest of Yili as well as Jinchuan and other cold regions where snow persists from the spring through summer, and in snow there are grasses similar to lotus with a single stem, which stands from snow in a lovely posture."
Cistanche deserticola refers to the dried fleshy stem, i.e., the medical part of Cistanche deserticola or Cistanche tubulosa plant having scale leaf. Cistanche deserticola is the dried fleshy stem having scale leaf of Cistanche deserticola Y.C. Ma or Cistanche tubulosa (Schrenk) Wight. It is plucked mostly in spring when the young sprout not emerging or just emerging and removed the flower, cut into pieces and dried. Cistanche deserticola is mainly distributed in Inner Mongolia, Gansu, Xianjiang, Qinghai, etc.

2 =
Desert cistanche is first recorded in "Sheng Nong's herbal classic". It is recorded that "Desert cistanche has sweet, salty taste, is warm in propertyand can tonify kidney, benefit essence and blood and moisten the intestines and free the stool.
It is mainly used to treat the diseases such as impotence, infertility, metrorrhagia, soreness and weakness of waist and knees, cold pains and weak muscle and joint."
Desert cistanche has always been used as an imperial delicacy worshiped by the western countries thus it is also called Cistanche deserticola. Cistanche deserticola can tonify kidney, benefit the blood and moisten the face, adjust the endocrine, promote metabolism and strengthen the body. It also has anti-oxidation effect.
It is said in "Medical Theory" that Cistanche deserticola can nourish marrow, moisten the face, prolong life and has great benefits" and "it can supplement the deficiency of essence, dark face and internal lesion caused by overexertion.
Astragalus membranaceus is also named as astragalus and is a general name of both the plants and the Chinese medicinal materials. Astragalus membranaceus refers to the dried root, i.e., the medical part of plant Astragalus membranaceus (Fisch.) Bge. var mongholicus (Bge.) Hsiao or Astragalus membranaceus (Fisch.) Bge.. Astragalus membranaceus is the dried root of legume plant Astragalus membranaceus (Fisch.) Bge. var mongholicus (Bge) Hsiao or Astragalus membranaceus (Fisch.) Bge.. It is plucked in spring and autumn and removed the fibrous root and root head and dried. Astragalus membranaceus is produced in North, East north, Inner Mongolia and west north in China and mainly produced in Shanxi, Heilongjiang, Liaoning and Hebei. There are also some distributions in Sichuan, former Soviet Union, North Korea and Mongolia. Astragalus membranaceus (Fisch.) Bge. is mainly distributed in East North, North, Gansu, Sichuan and Tibet in China. Astragalus membranaceus (Fisch.) Bge. var mongholicus (Bge) Hsiao is mainly produced in Inner Mongolia, Shanxi and Heilongjiang and now it is planted broadly.
The Chinese medicinal materials Astragalus membranaceus has effects on reinforcing qi and consolidating the exterior, promoting diuresis and detumescence, detoxification and apocenosis and promoting granulation. The medical applications of Astragalus membranaceus have a history of more than 2000 years. Current studies have indicated that Astragalus membranaceus contains saponins, sucrose,

3 =
polysaccharides, various amino acids, folic acid and various trace elements such as selenium, zinc, copper and the like, which have effects on enhancing immune functions, protecting livers, promoting diuresis, resisting aging, resisting stress, reducing blood pressure and wider antibacterial effect. Astragalus membranaceus has the effects on tonifying, retaining youthful looks and moisturizing face, and thus can enhance the body resistance, anti-oxidize and postpone aging.
In the current prescription, Astragalus membranaceus acts as the monarch drug by balancing and nourishing qi and blood and enhancing skin immune function, Cistanche deserticola and Saussurea involucrate act as the minister drug, wherein Cistanche deserticola can strengthen the body, supplement the essence and nourish the marrow and Saussurea involucrate can nourish the blood, resist photo-aging and oxidation. The root, stem and flower of the medical plant are taken together to resist the skin aging by selecting the amounts and controlling the compatibility, selecting the rational combination ratio of Astragalus membranaceus, Cistanche deserticola and Saussurea involucrate in combination with the studies of ancient cosmetics. It has comprehensive effects, wherein the composition is more suitable to be used in the cosmetics and has better synergy effects of Chinese medicines, compared with the a single Chinese medicine.
Preferably, the extract of Chinese herbal medicines according to the present invention is an extract by water or solvent of 30 parts by weight of Saussurea involucrate, 20 parts by weight of Cistanche deserticola and 50 parts by weight of Astragalus membranaceus, wherein the said solvent is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate or combinations thereof.
Preferably, the extract is carried out by a method selected from decoction, refluxing, soaking, diacolation or a combination thereof.
Preferably, the extract according to the present invention can be in a form selected from solution, powder, ointment, film or cream.
The present invention also provides a Chinese herbal medicine composition

4 having anti photo-aging effect, which comprises the above extract.
The extract of Chinese herbal medicines and the Chinese herbal medicine composition according to the present invention can be used directly to prevent the skin from aging, especially from photo-aging, or can be added to the cosmetics as functional additives. The extract of Chinese herbal medicines and the Chinese herbal medicine composition according to the present invention have anti-aging effect, especially anti-photo-aging effect, and can help the skin against the oxidation, facilitate the proliferation of dermal fibroblast, especially help the skin against the ultraviolet radiation of UVA and UVB, protect the skin, reduce the damage, prevent the skin from photo-agingõ without any irritating effect on the skin. Said cosmetics can be cosmetic skin cream, eye cream, facial mask, cleansing milk, lotion or essence.
The present invention also provides a method for preparing the above extractof Chinese herbal medicines, comprising:
Mixing 10 to 50 parts by weight of Saussurea involucrate, 10 to 50 parts by weight of Cistanche deserticola and 20 to 60 parts by weight of Astragalus membranaceus, soaking with water and/or solvent for 0.5-1 H and extracting, wherein said solvent is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate or combinations thereof;
Filtering, extracting the filtering residue again and combining the filtrates;
Concentrating the filtrate to about 2-fold amount of medicinal materials (V/W) (fold amount refers to the ratio between the volume of the solvent (m1) and the total weight of the medicinal materials (g));
Then carrying out alcohol precipitation;
Removing the solvent from the supernatant from the alcohol precipitation.
The product of alcohol precipitation can be formulated into various dosage forms, such as solution, powder, ointment, film or cream. Preferably, the alcohol used for the alcohol precipitation is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butylene glycol, acetone, ethyl acetate or combinations thereof.

The extracted solution according to the present invention can be formulated into a composition with a concentration of crude drug of 50-1000 mg/ml by adding water.
Embodiments The present invention will be further described in detail with reference to the following examples.
Example 1 Pure Chinese medicinal materials of Saussurea involucrate 10g, Astragalus membranaceus 60g and Cistanche deserticola 30g for a total weight of 100g were mixed thoroughly, to which 10-fold amount (fold amount refers to the ratio between the volume of the solvent (m1) and the total weight of the medicinal materials (g), hereinafter the same) of 75% (v/v) ethanol was added. The mixture was kept for cold soaking for 48 h and then filtered. Ethanol was recovered from filtrate; and the resultant extracts were dissolved with water to make a solution with a concentration of crude drug of 500 mg/ml, thus obtaining extract A in the form of solution.
Example 2 Pure Chinese medicinal materials of Saussurea involucrate 30g. Astragalus membranaceus 30g and Cistanche deserticola 40g for a total weight of 100g were mixed thoroughly, to which 10-fold amount of methanol was added. The mixture was subjected to refluxing extraction for 2 h, and filtered. Methanol was recovered from filtrate; and the resultant extracts were dissolved with water to make a solution with a concentration of crude drug of 50 mg /ml, thus obtaining extract B in the form of solution.
Example 3 Pure Chinese medicinal materials of Saussurea involucrate 40g, Astragalus membranaceus 50g and Cistanche deserticola lOg for a total weight of 100g were mixed thoroughly, and decocted with 10-fold amount of water for about 2 h, concentrated and filtered. The supernatant was made into a solution with a concentration of crude drug of 1000 mg /ml by adding water, thus obtaining extract C
in the form of solution.

Example 4 Pure Chinese medicinal materials of Saussurea involucrate 30g, Astragalus membranaceus 40g and Cistanche deserticola 30g for a total weight of 100g were mixed thoroughly, and decocted with 10-fold amount of water for about 2 h, concentrated, precipitated after 2-fold amount of ethanol was added, and then filtered.
Ethanol was recovered from filtrate; and the resultant extracts were dissolved with water and filtered to make a solution with a concentration of crude drug of 500 mg/ml, thus obtaining extract D in the form of solution.
Experiment 1- DPPH experiment Free radical is a class of chemicals with active reactivity and extremely high oxidbillity. The radiation of ultraviolet UVA/UVB can generate numerous free radicals, which exert damage to the body and accelerate aging. DPPH free radical scavenging assay was used herein to determine the in vitro anti-oxidation activity of the extracts of the examples.
The extracts of the examples were diluted with distilled water to two concentrations, 1% and 0.5% (v/v), respectively. Two milliliters of each of the diluted extracts of the Examples, 0.5 ml of DPPH solution in ethanol at a concentration of 500 mon, and 1.5ml water were then sequentially added into a same test tube and shaken thoroughly. After being left stand for 30 min; its optical density (OD) was measured at 517 nm by using a corresponding mixture liquid as the blank control. The scavenging rates for DPPH of the extracts of the examples were calculated according to the following formula:
The scavenging rate ( % ) = (1 T ¨ TO * 100 %
wherein:
T -- the optical density of DPPH + the extracts of the Examples, TO -- the optical density of the extracts of the examples + solvents, C -- the optical density of DPPH + solvents.
The results of evaluation on the anti-oxidation ability of the Chinese herbal medicine composition according to the present invention were shown in Table I.

Table 1 Concentration Free radical Concentration Radical Scavenging Rate Scavenging Rate 35.04% 0.5% 15.25%
Extract A of Example 1 1%
1% 19.92% 0.5% 8.9%
Extract B of Example 2 1% 42.89% 0.5% 19.64%
Extract C of Example 3 1% 54.54% 0.5% 20.31%
Extract D of Example 4 The results of the experiments showed that, the extract A of Example 1, the extract B of Example 2, the extract C of Example 3 and the extract D of Example 4 have superior antioxidant ability for free radical scavenging in vitro at the concentrations of both 1% and 0.5%, wherein all the free radical scavenging rates at the concentration of 0.5% are greater than 15% and all the free radical scavenging rates at the concentration of 1% are greater than 35%, and the effect from the extract D of Example 4 is the best.
Experiment 2- proliferation experiment of fibroblast The fibroblast in the skin is the main cells for collagen synthesis. The large amounts of proliferation of fibroblast, or the increased ability of fibroblast for synthesizing and secreting collagen can all increase the total amounts of collagen, thus postponing the skin aging. Hence, studying the effect of promoting proliferation of fibroblast is one of the important methods to evaluate the anti-aging activity of the sample. The present experiment uses the method of culturing human fibroblast in vitro and preliminarily evaluates the anti-aging effect of the Chinese herbal medicine composition according to the present invention based on the above skin aging mechanism.
The prepared cell suspension (formulated into a concentration of 5*1031m1) was seeded into a 96-well plate. Except the blank control, 20 ul extract of Chinese herbal medicines of Examples with corresponding concentrations was added to each well after the cell attachment. 4 concentration groups were set with 6 wells per group. The plate was placed into an incubation chamber (37 C and 5% CO2) to incubate for 241-1, then 20 ul MTT solution was added. The plate was continued to incubate in the incubation chamber (37 C and 5% CO2) for 4H. The enzyme panel was taken out, the culture solution in each well was aborbed carefully, and the DMSO was added and the wells was mix evenly. The optical density (570 nm) is measured by enzyme-linked immunometric meter and the relative proliferation rate is obtained by comparison with the control.
The results of proliferation activity of the anti-aging Chinese herbal medicine composition according to the present invention to the fibroblast were shown in Table 2.
Table 2 The concentrations of the Chinese herbal 1% 0.1%
0.01% 0.001%
medicine composition according to the present invention The proliferation Extract A of Example 1 20.22% 12.44% 8.98% 5.9%
rate for promoting Extract B of Example 2 17.81% 11.50% 7.43% 4.79%
dermal fibroblast Extract C of Example 3 35.62% 27.68% 23% 10.4%
Extract D of Example 4 25.04% 20.44% 13.22% 6.54%
The results of the experiments indicated that extract A of Example 1, extract B of Example 2, extract C of Example 3 and extract D of Example 4 all can promote the proliferation of the fibroblast and the effect from the extract C of Example 3 is the best, wherein the proliferation rate of fibroblast is promoted to be 10.4% at a concentration of 0.001%.
Experiment 3- Protective effect on the fibroblast against the damage of UV
radiation Light aging of the skin is principally caused by the medium-wavelength ultraviolet rays (UVB, 290-320 nm) together with the long-wavelength ultraviolet rays (UVA, 320-400 nm) in the sunlight, and the ultraviolet rays significantly hasten the aging of human skin. UVA/UVB radiation will result in the damage and death of fibroblasts and hurt the skin, leading to light aging. The anti-light aging effect of the extract of Chinese herb medicines of the Examples in protecting the cells against the damage from UV radiation was investigated herein by UV radiation assay.

PBS (4.5 ml) was added to 0.5 ml of each of the extracts of the Examples, to formulate a dilution with a concentration of 1/10 (v/v), which was subjected to suction filtration. Then 0.1 ml filtrate was added into 9.9 ml sterile PBS, finally obtaining a dilution in a concentration of 1/1000 (v/v). Skin fibroblasts (Fbs) cultured with normal DMEM solution were used as the control group, Fbs irradiated with UVA
(5-20J/cm2) / UVB (20-80J/cm2) after being cultured in normal DMEM solution were used as the model group, and Fbs co-cultured using the extracts of the Examples (0.15mg/m1) along with DMEM solution and then irradiated with UVA/UVB were used as the extracts-treated group. After ultraviolet treatment for 24 h, the optical density (OD value) of each well was measured using a plate reader with CCK-8.
The cell proliferation rate was calculated as the percentage of the OD value of each treated group relative to the OD value of negative control group; each test was run in triplicate.
The results of the experiments were shown in Table 3 and Table 4.
Table 3 UVA Radiation Cell proliferation rate P value Extract A of Example 1 (86.96 4.68)% <0.01 Extract B of Example 2 (79.80 3.80)% <0.01 Extract C of Example 3 (89.25 4.00)% <0.01 Extract D of Example 4 (95.8 9.9)% <0.01 Control group (100 7.6)%
Model group (73.4 2.3)% <0.01 Table 4 UVB Radiation Cell proliferation rate P value Extract A of Example 1 (88.27 3.51)% <0.01 Extract B of Example 2 (82.58 4.20)% <0.01 Extract C of Example 3 (90.86 2.85)% <0.01 Extract D of Example 4 (94 .1 6 .8)% <0.01 Control group (100 5.4)%
Model group (76.9 3.5)% <0.01 The results of the experiments showed that, all of the extract A of Example 1, extract B of Example 2, extract C of Example 3 and extract D of Example 4 can reduce the damage of UV radiation to fibroblasts and allow the cell proliferation rate to be recovered above 90%, wherein the effect from the extract D of Example 4 is the best.

Claims (13)

Claims:

1. An extract of Chinese herbal medicines, which is an extract by water or solvent of 10 to 50 parts by weight of Saussurea involucrate, 10 to 50 parts by weight of Cistanche deserticola and 20 to 60 parts by weight of Astragalus membranaceus, wherein said solvent is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate or combinations thereof.

2. The extract of Chinese herbal medicines according to claim 1, wherein the extraction is selected from soaking, decoction, refluxing, diacolation or combinations thereof.

3. The extract of Chinese herbal medicines according to claim 1 or 2, wherein it is in a form selected from solution, powder, ointment, film or cream.

4. A Chinese herbal medicine composition having anti-aging effect, comprising an extract of Chinese herbal medicines according to any one of claims 1-3.

5. Use of a Chinese herbal medicine composition of claim 4 in cosmetics.

6. A method for preparing an extract of Chinese herbal medicines, comprising steps of:
extracting 10 to 50 parts by weight of Saussurea involucrate, 10 to 50 parts by weight of Cistanche deserticola and 20 to 60 parts by weight of Astragalus membranaceus with water or solvent, wherein said solvent is selected from water, methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate or combinations thereof.

7. The method of claim 6 further comprising:
filtering, extracting the filtering residue again and combining the filtrates;
concentrating the filtrate to about 2-fold amount of medicinal materials (V/W), wherein the fold amount refers to the ratio between the volume of the solvent (m1) and the total weight of the medicinal materials (g);
carrying out alcohol precipitation;
removing the solvent from the supernatant from the alcohol precipitation.

8. The method of claim 6 or 7, wherein the alcohol used in alcohol precipitation is selected from methanol, ethanol, isopropanol, n-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butylene glycol, acetone, ethyl acetate or combinations thereof.

9. A Chinese herbal medicine extract prepared according to the method of any one of claims 6-8.

10. The Chinese herbal medicine extract of claim 9, wherein the Chinese herbal medicine extract is in a form selected from solution, powder, ointment, film or cream.

11. A Chinese herbal medicine composition having anti-aging effect, comprising a Chinese herbal medicine extract of claim 9 or 10.

12. Use of a Chinese herbal medicine extract of claim 11 in cosmetics.

13. The use of claim 5 or 12, wherein the cosmetics is cosmetic skin cream, eye cream, facial mask, cleansing milk, lotion or essence.