AU2011252628A1

AU2011252628A1 - Snow lotus extract, preparing method and application thereof

Info

Publication number: AU2011252628A1
Application number: AU2011252628A
Authority: AU
Inventors: Mo Chen; Kang Cheng; Cheng Fang; Beibei Liu; Luo LV; Laiji Ma; Weiguo Xiong; Ya ZHAO; Le ZHU
Original assignee: Shanghai Jahwa United Co Ltd
Current assignee: Shanghai Jahwa United Co Ltd
Priority date: 2010-05-13
Filing date: 2011-03-10
Publication date: 2012-12-06
Also published as: WO2011140856A1; CN102240254A

Abstract

A method for preparing snow lotus extract comprises a step of solvent extraction. The solvent is selected from water, methanol, ethanol, isopropyl alcohol, 1-butanol, ethylene glycol, 1,2-propylene glycol, 1,3-propylene glycol, 1,3-butylene glycol, acetone, ethyl acetate, or the combination thereof. The obtained snow lotus extract has the effects of anti-oxidation and resistance to UVA and UVB radiations. The snow lotus extract is used as an active ingredient in the manufacture of cosmetics or medicaments for skin anti-aging, especially anti-photoaging.

Description

SNOW LOTUS FLOWER EXTRACTS, PREPARATION METHODS AND USE THEREOF FIELD OF THE INVENTION The present invention relates to the fields of natural medicinal chemistry and cosmetics, specifically to use of a Chinese herb medicine, snow lotus flower, in cosmetics or medicines for anti-aging application, preferably anti-light aging application. BACKGROUND OF THE INVENTION Studies have showed that both free radicals and ultraviolet rays are among the causes of skin aging, and light aging is principally caused by 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 aging, especially anti-light aging products have been released one by one; and more related researchs are being conducted. However, natural Chinese herb medicine additives for anti-light aging remained unseen. DESCRIPTION OF THE INVENTION The purpose of the invention is to provide a preparation method of snow lotus flower extracts, wherein said method comprises: (1) extraction of the snow lotus flower with a solvent, said solvent being selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof. Preferably, the extraction in step (1) is selected from soaking, decoction, refluxing, diacolation, or a combination thereof. Preferably, the preparation method further comprises: (2) separation of the extracted liquid from the residue to obtain the extracted liquid; 1 (3) optional recovery of the solvent; (4) optional dissolution or dilution. Preferably, the solvent used in step (4) is selected from water, ethanol, isopropanol, I-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof. Another purpose of the invention is to provide an alternative preparation method of snow lotus flower extracts, wherein said method comprises: (1) extraction of the snow lotus flower with a solvent, said solvent being selected from methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1,2-propanediol, 1,3 propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof; (2) separation of the extracted liquid from the residue to obtain the residue; (3) extraction of the residue obtained in step (2) with water. Preferably, the extraction in step (1) and the extraction in step (3) are independently selected from soaking, decoction, refluxing, diacolation, or a combination thereof. Preferably, the preparation method further comprises: (4) separation of the water-extracted liquid from the residue to obtain the water extracted liquid; (5) concentration, ethanol precipitation, and separation, to obtain the precipitates; (6) optionally, dissolution of the precipitates obtained in step (5), to form a solution. Preferably, the solvent used in step (6) is selected from ethanol, isopropanol, 1 butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof. Yet another purpose of the invention is to provide the snow lotus flower extracts obtained by the preparation method of the invention as described above. Preferably, the form of said snow lotus flower extracts is selected from solution, powder, ointment, film, or cream. The snow lotus flower extracts obtained according to the method of the invention can be directly used for the prevention of skin aging, preferably light aging, and also can be incorporated into cosmetics and pharmaceuticals as functional additives or active ingredients. 2 Yet another purpose of the invention is to provide the use of snow lotus flower extracts in the preparation of an anti-aging, preferably anti-light aging, cosmetic or pharmaceutical. Preferably, said snow lotus flower extracts are the above-mentioned snow lotus flower extracts prepared according to the method of the invention. Yet another purpose of the invention is to provide an anti-aging, preferably anti-light aging, cosmetic or pharmaceutical which comprises the snow lotus flower extracts. Preferably, said snow lotus flower extracts are the above-mentioned snow lotus flower extracts prepared according to the method of the invention. In the present invention, snow lotus flower 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 Kar.et Kir., Saussurea laniceps Hand-Mazz., S.gnaphaloides (Royle) Sch.-Bip., and Smedusa Maxim. Snow lotus flowers, 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. Snow lotus flowers 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 "snow lotus flowers, 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 "snow lotus flowers are 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." The snow lotus flower extracts prepared according to the method of the invention have the effects of scavenging free-radicals and the effect of anti-oxidation, and at the same time they can help the skin against the ultraviolet radiation of UVB and UVA, protect the skin, reduce cell damage, prevent the decrease in dermal collagen, and 3 efficiently prevent the skin from aging, especially light aging, without any irritating effect on the skin. EMBODIMENTS Example 1 One hundred grams of Saussurea laniceps Hand-Mazz were taken, to which 8-fold amount (fold amount refers to the ratio between the volume of the solvent (ml) and the weight of the snow lotus flower (g), hereinafter the same) of 75% ethanol (v/v) 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 1000 mg /ml, thus obtaining extracts A in the form of solution. Example 2 One hundred grams of S.gnaphaloides (Royle) Sch.-Bip. were taken, to which 8-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 500 mg /ml, thus obtaining extracts B in the form of solution. Example 3 One hundred grams of S.medusa Maxim. were 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 extracts C in the form of solution. Example 4 One hundred grams of Saussurea involucrata Kar.et Kir. were taken, to which 6 fold amount of ethanol was added for refluxing extraction. The mixture was filtered, and the residue was decocted with 10-fold amount of water for about 2 h, concentrated, precipitated after 2-fold amount of ethanol was added, and filtered; then the precipitate 4 was dissolved with water to make a solution with a concentration of crude drug of 500 mg /ml, thus obtaining extracts D in the form of solution. Example 5 DPPH experiments 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 pmol/L, and l.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 (%) = - T * 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 the experiments were shown in Table 1. Table 1. Evaluation on the Anti-oxidation Ability of Snow Lotus Flower Extracts Concentration Free radical Concentration Free radical scavenging rate scavenging rate Extracts A of Example 1 1% 39.82% 0.5% 12.80% Extracts B of Example 2 1% 42.26% 0.5% 13.96% Extracts D of Example 4 1% 47.86% 0.5% 17.44% The results of the experiments showed that, the extracts A of Example 1, the extracts B of Example 2 and the extracts D of Example 4 have superior antioxidant ability for free 5 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 10% and all the free radical scavenging rates at the concentration of 1% are greater than 30%, and the effect from the extracts D of Example 4 is the best. Example 6 The protective effect on the fibroblasts 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 Chinese herb medicine extracts 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/cm 2 ) / UVB (20-80J/cm 2 ) after being cultured in normal DMEM solution were used as the model group, and Fbs co-cultured using the extracts of the Examples at 1/1000 concentration along with DMEM solution and then irradiated with UVA/UVB were used as the drug-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 2 and Table 3. Table 2. UVA Radiation Cell proliferation rate P value Extracts A of Example 1 (90.13±4.68)% <0.01 Extracts B of Example 2 (92.25±4.00)% <0.0 1 6 Extracts D of Example 4 (94.88±4.98)% <0.01 Control group (100±4.32)% Model group (74.25±3.78)% <0.01 Table 3. UVB Radiation Cell proliferation rate P value Extracts A of Example 1 (93.27±3.51)% <0.01 Extracts B of Example 2 (95.86±2.85)% <0.01 Extracts D of Example 4 (97.54±3.53)% <0.0 1 Control group (100±5.68)% Model group (83.63±5.15)% <0.0 1 The results of the experiments showed that, all of the extracts A of Example 1, extracts B of Example 2 and extracts 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 extracts D of Example 4 is the best. Example 7 In vitro assay on the effect of UV radiation on dermal collagen 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/cm 2 ) / UVB (20-80J/cm 2 ) after being cultured in normal DMEM solution were used as the model group, and Fbs co-cultured using the extracts of the Examples at 1/1000 concentration along with DMEM solution and then irradiated with UVA/UVB were used as the extracts-treated group. After ultraviolet treatment for 24 h, the cell culture supernatant was collected and the content of the hydroxyproline in the culture supernatant was determined according to the method described in the instructions of hydroxyproline assay kit (Nanjing Jiancheng). The results of the experiments were shown in Table 4 and Table 5. 7 Table 4. UVA Radiation Collagen content (pg/ml) Recovery rate of the change in collagen (relative to model group) Extracts A of Example 1 4.3 34.38% Extracts B of Example 2 4.9 53.13% Extracts D of Example 4 5.1 59.38% Control group 8.2 Model group 3.2 Table 5. UVB Radiation Collagen content (pg/ml) Recovery rate of the change in collagen (relative to model group) Extracts A of Example 1 4.8 26.32% Extracts B of Example 2 5.4 42.11% Extracts D of Example 4 6.6 73.68% Control group 8.2 Model group 3.8 The results of experiments showed that, all of the extracts A of Example 1, the extracts B of Example 2 and the extracts D of Example 4 can reduce the effect of UV radiation on collagen content of dermal fibroblasts with a recovery rate of above 25% relative to collagen of model damage group, wherein the effect from the extracts D of Example 4 is the best, whose recover rate is 59.38% for UVA and 73.68% for UVB. 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 the decrease in dermal collagen, leading to the damage to the skin. The above experiment results have demonstrated that for the cultured skin fibroblasts irradiated with UVA/UVB in a certain intensity, incubation with the snow lotus flower extracts can effectively reduce the effect of UV radiation on the proliferation of the fibroblasts while effectively reducing the change in the dermal fibroblasts collagen to protect the skin, which indicates 8 that the snow lotus flower extracts can protect skin fibroblasts against the damage of ultraviolet radiation and are capable of resisting light aging. 9

Claims

1. A method for preparation of snow lotus flower extracts comprising: (1) extraction of the snow lotus flower with a solvent selected from water, methanol, ethanol, isopropanol, 1-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3 butanediol, acetone, ethyl acetate, or a combination thereof.

2. The method of Claim- 1, wherein the extraction of step (1) is selected from soaking, decoction, refluxing, diacolation, or a combination thereof.

3. The preparation method of Claim I or 2, further comprising: (2) separation of the extracted liquid from the residue to obtain the extracted liquid; (3) optional recovery of the solvent; (4) optional dissolution or dilution.

4. The method of Claim 3, wherein the solvent used in step (4) is selected from water, ethanol, isopropanol, 1-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof.

5. A method for preparation of snow lotus flower extracts comprising: (1) extraction with a solvent selected from methanol, ethanol, isopropanol, I butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3-butanediol, acetone, ethyl acetate, or a combination thereof; (2) separation of the extracted liquid from the residue to obtain the residue; (3) extraction of the residue obtained in step (2) with water.

6. The method of Claim 5, wherein the extraction in step (1) and the extraction in step (3) are independently selected from soaking, decoction, refluxing, diacolation, or a combination thereof.

7. The method of Claim 5 or 6, further comprising: (4) separation of the water-extracted liquid from the residue to obtain the water extracted liquid; (5) concentration, ethanol precipitation, and separation, to obtain the precipitates; (6) optionally, dissolution of the precipitates obtained in step (5) to form a solution. 10

8. The method of Claim 7, wherein the solvent used in step (6) is selected from ethanol, isopropanol, I-butanol, ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,3 butanediol, acetone, ethyl acetate, or a combination thereof.

9. Snow lotus flower extracts prepared according to the preparation method of any one of Claims 1-8.

10. The snow lotus flower extracts of Claim 9, wherein the form of said snow lotus flower extracts is selected from solution, powder, ointment, film, or cream.

11. Use of snow lotus flower extracts in the preparation of an anti-aging, preferably anti-light aging cosmetic or pharmaceutical.

12. The use of Claim 11, wherein said snow lotus flower extracts are the snow lotus flower extracts of Claim 9 or 10.

13. An anti-aging, preferably anti-light aging cosmetic or pharmaceutical comprising snow lotus flower extracts.

14. The cosmetic of Claim 13, wherein said snow lotus flower extracts are the snow lotus flower extracts of Claim 9 or 10. 11