CN104771456B

CN104771456B - Application of bistort rhizome extract in inhibiting tyrosinase activity

Info

Publication number: CN104771456B
Application number: CN201410017256.3A
Authority: CN
Inventors: 王滔; 王永兵; 肖功胜; 宗伟英; 雷辉; 沈雯; 席晓荣
Original assignee: Huzhou R&D Center for Nutrition and Health of SIBS
Current assignee: Huzhou Zhongke nutrition and health innovation center; Shanghai Institute of Nutrition and Health of CAS
Priority date: 2014-01-15
Filing date: 2014-01-15
Publication date: 2020-01-17
Anticipated expiration: 2034-01-15
Also published as: CN104771456A

Abstract

The invention discloses a new application of a bistort rhizome extract, which is prepared by extracting bistort rhizome serving as a raw material with ethanol and purifying the bistort rhizome extract by macroporous adsorption resin. The bistort rhizome extract has a good tyrosinase activity inhibition effect, and can be used for preparing medicines or cosmetics for preventing and treating pigmentation diseases.

Description

Application of bistort rhizome extract in inhibiting tyrosinase activity

Technical Field

The invention belongs to the field of traditional Chinese medicines, and particularly relates to an application of bistort rhizome extract in inhibiting tyrosinase activity.

Background

The pathogenesis of most skin diseases with dyschromatosis such as freckle, chloasma and melanism is not completely clear, wherein the chloasma is the most difficult to treat, and cheeks, forehead, upper lip, nose and chin of the face are good hair areas, which are mainly caused by genetic factors, ultraviolet irradiation, pregnancy, oral contraceptives or antiepileptic drugs. The oral contraceptive is mainly seen in middle-aged and young women in east China, the incidence of the female in south east China accounts for about 0.25-4% of the number of patients diagnosed in dermatology, the incidence of the female in south east China is 50-70%, the incidence of the female in south east China is 8-29%, color spots can continuously exist after delivery or withdrawal of a part of patients, and the patients are greatly psychologically burdened by chromatodermatosis such as chloasma and the like due to the influence on the appearance of the patients. However, the causes of the diseases are not completely clear so far, so that most methods and medicaments for treating the skin pigmentation disorder have unsatisfactory treatment effects, and the defects of long treatment period, high toxic and side effects, poor patient compliance, unstable effect, different curative effects and the like generally exist. Therefore, the search for a decolorant with small toxic and side effects and definite curative effect becomes the direction of efforts of researchers.

Tyrosinase (Tyrosinase) is a key enzyme for the synthesis of melanin in organisms, has double functions of oxygenase and oxidase, and can catalyze monophenol into diphenol and oxidize the diphenol into quinone; quinones form the final reaction product melanin under non-enzymatic conditions. Tyrosinase inhibitors inhibit melanin production by inhibiting tyrosinase activity. Abnormal overexpression of tyrosinase can lead to pigmentation disorders in humans. Therefore, the tyrosinase inhibitor can achieve the purpose of reducing melanin in the skin by destroying the activity of tyrosinase, is further used for treating the current common pigmentation diseases such as freckles, chloasma, senile plaques, melanoma and the like, and can be widely applied to the industries of medicines and cosmetics.

Because of the potential application prospect of tyrosinase in the fields of medicine and daily chemical industry, the research on the activity of tyrosinase becomes the focus of research. At present, many tyrosinase inhibitors derived from chemically synthesized and natural products have been used as medicines, cosmetics (whitening agents), food additives and biopesticides. Hydroquinone, kojic acid and derivatives thereof, azelaic acid, glycolic acid, vitamin C and derivatives thereof, nicotinamide and other chemical substances can inhibit tyrosinase, and are also widely used in whitening cosmetics. Kojic acid is used as a tyrosinase inhibitor, and has toxic and side effects of carcinogenesis, anaphylaxis and the like due to instability; hydroquinone compounds have strong tyrosinase inhibitory activity, but their use has been banned in many countries because of their ability to induce skin disorders and their cytotoxicity, and some tyrosinase inhibitors work by using mercury (i.e., mercury) as a catalyst. Therefore, the existing tyrosinase inhibitor has defects of different degrees in various aspects such as inhibition effect, stability or safety, and the like, so that the continuous search for the tyrosinase inhibitor with good inhibition effect, stable property, safety and mildness still has very important significance.

With the continuous development and application of tyrosinase inhibitors and the trend of returning 'pure natural products' to the mainstream, the development and research of the tyrosinase inhibitors as natural products are more and more emphasized. Therefore, the search for safe and effective tyrosinase inhibitors from natural plants has become a current trend in research in the pharmaceutical and cosmetic industries.

Bighead atractylodes rhizome is dry rhizome of Polygonum bistorta L. of Polygonaceae, named as Caoheche, Zishen, dried shrimp, Pandalus carinatus, etc., and is mainly distributed in Henan, Hebei, Jilin, Shandong, Liaoning, Sichuan, etc. Has sweet and astringent taste and cool property, has effects of clearing away heat and toxic materials, relieving diarrhea, relieving convulsion, calming endogenous wind, and stopping bleeding, and can be used for treating dysentery, diarrhea, cough due to lung heat, carbuncle, swelling, hemorrhoid, hemorrhage, etc. Modern researches show that bistort rhizome also has the effects of antioxidation, anti-inflammation, antivirus, analgesia, anti-aging, blood fat reduction and the like.

To date, no study and report that bistort rhizome and its extract have the effect of inhibiting tyrosinase activity has been found.

Disclosure of Invention

The inventor of the application finds that the bistort rhizome extract has a remarkable inhibitory effect on the activity of tyrosinase in the process of long-term research on the medical application of bistort rhizome.

The invention aims to provide application of bistort rhizome extract in inhibiting tyrosinase activity.

According to the present invention, the bistort rhizome extract can be used for preparing a medicine or a cosmetic for preventing and treating pigmentation diseases.

According to the invention, the bistort rhizome extract is prepared by extracting bistort rhizome from bistort rhizome by using ethanol as an extraction solvent.

According to the invention, the ethanol is ethanol with a volume fraction of 70%.

According to the invention, the extraction is heating reflux extraction, and is carried out for at least 1 time, and the extraction time is 1-4 h each time.

According to the preferred embodiment of the present invention, the extraction is heating reflux extraction 3 times, and each extraction time is 2 h.

According to the invention, the preparation of the bistort rhizome extract also comprises a step of macroporous adsorption resin purification.

According to the invention, the step of purifying the macroporous adsorption resin comprises the following steps: purifying the bistort rhizome alcohol extract by adopting macroporous adsorption resin, eluting with water, sequentially eluting with 30-90% ethanol by volume fraction, respectively collecting ethanol eluates, concentrating under reduced pressure, and freeze-drying under vacuum.

The invention has the beneficial effects that: in the research of the application, the bistort rhizome extract prepared by applying the extraction method disclosed by the invention has a strong inhibition effect on tyrosinase, has strong inhibition activity and good stability compared with the prior art-recognized whitening agent kojic acid, does not have the toxic and side effects of carcinogenesis, allergy and the like of kojic acid, can be used as a novel potential medicament or cosmetic for preventing and treating pigmentation diseases, has a certain effect of preventing, improving and treating skin pigmentation diseases such as freckles, chloasma, senile plaques and the like, and has a wide application prospect in the fields of medicaments and cosmetics.

Detailed Description

The present invention will be further described with reference to the following examples. It should be understood that the following examples are illustrative only and are not intended to limit the scope of the present invention.

Example 1 preparation of different extracts of bistort rhizome

1.1 preparation of ethanol extract of Bistormwoo rhizome

Taking 50g of bistort rhizome crude powder, adding 10 times of 70% ethanol, extracting under reflux for three times, each time for 2h, filtering, combining filtrates, concentrating the filtrate under reduced pressure, recovering ethanol to obtain an extract, and freeze-drying the extract in vacuum to obtain the bistort rhizome ethanol extract.

1.2 preparation of extracts from the elution sites of Bistor 30%, 60% and 90% ethanol

Extracting rhizoma Bistortae medicinal material coarse powder 50g with 500ml70% ethanol under reflux for 3 times, each time for 2 hr, filtering, collecting filtrate, concentrating under reduced pressure to recover ethanol, and concentrating to 100ml to obtain rhizoma Bistortae ethanol extract solution with concentration of 0.5g crude medicinal material coarse powder per 1 ml.

Purifying the bistort alcohol extract solution by using D101 type macroporous adsorption resin, wherein the concentration of the liquid medicine on the column is 0.5g crude drug per 1ml liquid medicine, the pH of the liquid medicine is 7, the ratio of the amount of the traditional Chinese medicine on the column to the amount of the resin is 1:6, and the adsorption rate is 3 Bv/h; in the elution process, 6Bv is eluted by water at the elution rate of 6 Bv/h; and then sequentially eluting with 30%, 60% and 90% ethanol with 6Bv concentration at 3Bv/h, respectively collecting 30%, 60% and 90% ethanol eluates, concentrating the collected 30%, 60% and 90% ethanol eluates under reduced pressure to recover ethanol to obtain extract, and freeze-drying the extract under vacuum to obtain the bistort rhizome 30%, 60% and 90% ethanol elution part extract.

Example 2 analysis of the tyrosinase inhibitory Activity of different extracts of bistort rhizome

Experimental materials: tyrosinase, kojic acid, L-DOPA were all purchased from Sigma.

The tested drugs are: dissolving rhizoma Bistortae different extracts (samples prepared in example 1) in DMSO to obtain 60mg/mL mother liquor; kojic acid was dissolved in DMSO to make a 15mg/mL stock solution.

The experimental method comprises the following steps: adding 25 mu L of 20 mu g/mL tyrosinase and 25 mu L of a sample to be detected into each hole of a 96-hole plate, uniformly mixing, incubating at 25 ℃ for 10min, adding 50 mu L of 6mM L-DOPA into each hole to start reaction, incubating at 25 ℃ for 5min in a dark place, and reading an absorbance value at 475nm (blank control is performed on each hole, and the influence of the color of an extract on the absorbance value is deducted by using buffer solution instead of tyrosinase).

The results of the experiment are shown in tables 1 and 2.

TABLE 1 inhibitory Effect of different bistort rhizome extracts on tyrosinase at a concentration of 30. mu.g/mL

TABLE 2 tyrosinase inhibitory IC50 of different bistort rhizome extracts

Sample (I)	IC50（μg/mL）
		Kojic acid	18.2
Ethanol extract of rhizoma Bistortae	29.8
		30% alcohol elution site	21.3
60% alcohol elution site	10.7
		90% alcohol elution site	21.0

As can be seen from the results in tables 1 and 2, the bistort rhizome extracts all show strong inhibition effect on tyrosinase, wherein the tyrosinase is inhibited best by the 60% alcohol elution part, and the IC50 is 10.7 mug/mL.

Example 3 inhibition of tyrosinase by different extracts of bistort rhizome with different incubation times

Experimental materials: tyrosinase, L-DOPA were purchased from Sigma.

The tested drugs are: different bistort rhizome extracts (samples prepared in example 1) were dissolved in DMSO to prepare a 60mg/mL stock solution.

The experimental method comprises the following steps: adding 25 mu L of 20 mu g/mL tyrosinase and 25 mu L of a sample to be detected into each hole of a 96-hole plate, uniformly mixing, incubating for 10min at 25 ℃, adding 50 mu L of 6mM L-DOPA into each hole to start reaction, incubating for 5min, 10min and 20min at 25 ℃ in a dark place, and reading an absorbance value at 475nm (blank control is performed on each hole, and the influence of the color of an extract on the absorbance value is deducted by using buffer solution instead of tyrosinase).

The results of the experiment are shown in table 3.

TABLE 3 inhibitory Effect of different extracts of Bistortae on tyrosinase

As can be seen from the results in Table 3, the tyrosinase inhibitory effect of the different bistort rhizome extracts decreases with the increase of the reaction time, wherein after incubation of 60% alcohol elution part for 5min, 10min and 20min, the tyrosinase inhibitory IC50 values are 10.7. mu.g/mL, 14.8. mu.g/mL and 28.1. mu.g/mL, respectively, and the inhibitory effect is the best.

Example 4 cytotoxicity assays of different extracts of bistort rhizome

Experimental materials: MTT was purchased from Sigma, DMEM, fetal bovine serum, and pancreatin were purchased from GIBCO.

Cell: human Umbilical Vein Endothelial Cells (HUVEC), human lung adenocarcinoma cells (A549), Vero were purchased from the cell bank of the Chinese academy of sciences.

The experimental method comprises the following steps: cells are inoculated in a 96-well plate with proper concentration, a sample to be detected is added in the next day for treatment for 48 hours, the culture medium is discarded, 100 mu L of 500 mu g/mL MTT solution is added for incubation for 4 hours, the solution is discarded, 150 mu L of DMSO is added, the micro-well plate is shaken for 15min, and the absorbance value is read at 490 nm.

The results of the experiment are shown in table 4.

TABLE 4 cytotoxicity test results of different bistort rhizome extracts on three cells

The results in table 4 show that the different bistort rhizome extracts have CC50 of more than 30 mug/mL on three cells, have no obvious cytotoxicity and can be widely applied to the fields of medicines, cosmetics and the like.

In conclusion, it is obvious to those skilled in the art that the bistort rhizome extract of the present invention has a good inhibitory effect on tyrosinase activity and can be used for preparing a medicine or cosmetic for preventing and treating pigmentation disorders.

Claims

1. The application of the bistort rhizome extract in inhibiting tyrosinase activity is characterized in that the bistort rhizome extract is prepared by extracting bistort rhizome with ethanol as an extraction solvent to obtain a bistort rhizome alcohol extract, wherein the bistort rhizome alcohol extract is obtained by extracting bistort rhizome with ethanol as an extraction solvent;

the ethanol is ethanol with the volume fraction of 70 percent; the extraction is heating reflux extraction, and is carried out for at least 1 time, and the extraction time is 1-4 h each time;

the bistort rhizome extract is also prepared by adopting macroporous adsorption resin for purification, and the macroporous adsorption resin comprises the following steps: purifying the bistort rhizome alcohol extract by adopting macroporous adsorption resin, eluting with water, sequentially eluting with 30-90% ethanol by volume fraction, collecting 30-60% ethanol elution parts, concentrating under reduced pressure, and freeze-drying under vacuum.

2. Use of bistort rhizome extract according to claim 1 for inhibiting tyrosinase activity for the preparation of a medicament or cosmetic for the prevention and treatment of pigmentation disorders.

3. The bistort rhizome extract according to claim 1, which is used for inhibiting tyrosinase activity, wherein the extraction is performed by heating and refluxing for 3 times, and each extraction time is 2 hours.