阅读说明：本技术 白及中2-异丁基苹果酸葡萄糖氧基苄酯类提取物的制备方法及用途 (Preparation method and application of 2-isobutyl malic acid glucoxybenzyl ester extract in rhizoma bletillae ) 是由 段营辉 陈惠玲 黄澜 朱樵苏 黄惠琼 李玲玲 于 2019-11-04 设计创作，主要内容包括：本发明公开了一种白及中2-异丁基苹果酸葡萄糖氧基苄酯类提取物的制备方法及用途,属于医药、日用化工领域。所述白及的提取物优选为块茎和/或须根。所述白及2-异丁基苹果酸葡萄糖氧基苄酯类提取物可作为酪氨酸酶抑制剂应用于制备黑色素生成抑制剂或美白剂的化妆品、医药品或皮肤外用剂,对紫外线照射后的皮肤晒黑及色斑、老年斑、雀斑等皮肤色素沉着具有良好的预防、改善及治疗效果。(The invention discloses a preparation method and application of 2-isobutyl malic acid glucoxybenzyl ester extract in rhizoma bletillae, and belongs to the fields of medicines and daily chemicals. The rhizoma bletillae extract is preferably tuber and/or fibrous root. The bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract can be used as a tyrosinase inhibitor to be applied to preparing cosmetics, medicaments or skin external preparations of melanin generation inhibitors or whitening agents, and has good effects of preventing, improving and treating skin pigmentation such as skin tanning, color spots, senile plaques, freckles and the like after ultraviolet irradiation.)

1. A bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract is characterized in that: the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract contains the following compounds: 1- (4-gluconoxybenzyl) -2-isobutylmalate-2-glucoside, 4- (gluconeoxy) -cis-glucyloxybenzyl cinnamate, 1- (4-gluconoxybenzyl) -2-isobutylmalic acid, 1, 4-bis (4-gluconoxybenzyl) -2-isobutylmalate-2-glucoside, 1, 4-bis (4-gluconoxybenzyl) -2-isobutylmalate-2- (6-acetyl) glucoside, 1, 4-bis (4-gluconoxybenzyl) -2-isobutylmalate-2- (4-cinnamoyl) glucoside and 1, 4-bis (4-glucosyloxybenzyl) -2-isobutylmalate-2- (4-cinnamoyl-6-acetyl) glucoside.

2. The extract of glucoxybenzyl 2-isobutylmalate according to claim 1, wherein: the mass percentage of the 1, 4-di (4-glucosyloxybenzyl) -2-isobutyl malate is not less than 20%.

3. The extract of glucoxybenzyl 2-isobutylmalate according to claim 1, wherein: in the bletilla striata 2-isobutyl malic acid glucosyloxybenzyl ester extract, the peak area corresponding to 1- (4-glucosyloxybenzyl) -2-isobutyl malate-2-glucoside accounts for 0.51-3.14%, the peak area corresponding to 4- (glucosyloxy) -cis-cinnamic acid glucosyloxybenzyl ester accounts for 2.94-5.01%, the peak area corresponding to 1- (4-glucosyloxybenzyl) -2-isobutyl malic acid accounts for 4.37-8.51%, the peak area corresponding to 1, 4-bis (4-glucosyloxybenzyl) -2-isobutyl malate-2-glucoside accounts for 4.02-9.26%, and the peak area corresponding to 1, 4-bis (4-glucosyloxybenzyl) -2-isobutyl malate accounts for 22.69-35.58%, the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (6-acetyl) glucoside accounts for 5.05-10.16%, the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (4-cinnamoyl) glucoside accounts for 1.60-3.74%, and the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (4-cinnamoyl-6-acetyl) glucoside accounts for 3.45-8.42%.

4. The preparation method of the bletilla striata and 2-isobutyl glucosyloxybenzyl malate extract as claimed in any one of claims 1 to 3, comprising the following steps: pulverizing tuber or fibrous root of bletilla striata of Orchidaceae, soaking in extraction solvent for extraction or heating and reflux extracting in the extraction solvent, wherein the extraction solvent is at least one of water, methanol, ethanol, acetone and ethyl acetate; filtering or centrifuging to remove insoluble substances, concentrating the obtained extractive solution under reduced pressure, and drying to obtain dry extract to obtain rhizoma Bletillae extract; extracting and separating the bletilla striata extract by using a solvent or separating by using macroporous resin to obtain a bletilla striata 2-isobutyl malic acid glucosyloxybenzyl ester extract;

the solvent extraction separation comprises dispersing rhizoma bletilla extract in water, sequentially extracting with ethyl acetate and n-butanol for 3 times at equal volume, vacuum drying respectively, and extracting the n-butanol to obtain rhizoma bletilla 2-isobutyl malic acid glucosyloxybenzyl ester extract;

dissolving the bletilla striata extract in water, filtering, taking filtrate, performing open column chromatography by macroporous resin, and performing gradient elution by sequentially adopting water, 50% by volume of ethanol-water solution and 95% by volume of ethanol-water solution, wherein the elution extract of the 50% by volume of ethanol-water solution is the bletilla striata 2-isobutyl malic acid glucosyloxybenzyl ester extract.

5. The method of claim 4, wherein: the extraction temperature is 20-100 ℃, and the extraction time is 1-48 hours.

6. The method of claim 4, wherein: the macroporous resin comprises at least one of D101, HP-20, AB-8, X-5 and D1300.

7. Use of bletilla striata and 2-isobutyl glucosyloxybenzyl malate extract according to any one of claims 1 to 3 for preparing tyrosinase inhibitors.

8. Use of bletilla striata 2-isobutyl glucosyloxybenzyl malate extract as defined in any one of claims 1 to 3 as tyrosinase inhibitor in preparation of cosmetics, pharmaceuticals or skin external preparations for melanin production inhibitor or whitening agent.

9. Use according to claim 8, characterized in that: the mass content of the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract in cosmetics or medicines is 0.1-20% or 0.1-50% in terms of dry products.

Technical Field

The invention belongs to the fields of pharmaceuticals and daily chemicals, and particularly relates to a 2-isobutyl malic acid glucosyloxybenzyl ester extract from rhizoma bletillae, a preparation method thereof, and application of the extract as a tyrosinase inhibitor in the fields of medicines and cosmetics.

Background

The rhizoma Bletillae is dry tuber of Bletilla striata (Thunb) of Orchidaceae, also known as rhizoma Nardostachyos, radix Paeoniae alba, herba Glechomae, rhizoma Bletillae, etc. Li Shizhenshuo has its name: its root is white and even in conjunction with it, so it is recorded as Bai Ji and its use as a medicine is recorded in Shen nong Ben Cao Jing. Bletilla plants are widely distributed in China, and the Yangtze river basin is an ecological area with most abundant types of bletilla and most suitable climatic conditions and mainly produced in Guizhou province, Sichuan province, Yunnan province, Hunan province, Hubei province, Anhui province and the like. It is bitter, sweet, astringent and cool in nature, and enters lung, liver and stomach meridians. Has astringent, hemostatic, repercussive, and granulation promoting effects, and can be used for treating hemoptysis, hematemesis, traumatic hemorrhage, pyocutaneous disease, toxic swelling, chapped skin, scald due to hot water and fire, etc. The main chemical components in the rhizoma bletillae comprise polysaccharides, bibenzyls, phenanthrenes and derivatives thereof, flavonoids, 2-isobutyl malic acid glucose oxybenzyl ester compounds and the like (Phytochemistry,1933,33: 1481-. Modern pharmacological studies show that rhizoma bletillae has the effects of hemostasis, antibiosis, tumor resistance and the like (journal of pharmaceutical practice, 2011,29: 206-. In addition, the common bletilla pseudobulb is a high-grade cigarette tobacco strip adhesive, a wild ginseng broken hair repairing agent, a gastroscopy protective agent, a beauty mask and the like. Bletilla striata polysaccharide (also called bletilla striata gum) is also an important raw material in the pharmaceutical industry, and is used for treating tumors as processed plasma substitutes and vascular embolization agents.

Melanin (Melanin) is a biopolymer of a variety of polyphenolic substances secreted by melanocytes in the basal layer of the epidermis, ranging in color from yellow (brown Melanin) to black (eumelanin). It can absorb solar ultraviolet ray, eliminate Reactive Oxygen Species (ROS), and prevent skin from being damaged by ultraviolet ray. The disorder of melanin production can cause sparrow disease, senile plaque, vitiligo and albinism. Ultraviolet rays in the sunlight activate melanin-producing enzymes in melanocytes in the epidermis to produce pigments. The pigmentation, which causes freckles and skin darkening, has become a major annoyance for young and middle-aged people, especially young women. In the process of a series of melanin generation, tyrosinase converts tyrosine into dopa and dopaquinone, and melanin is produced through polymerization through non-enzymatic oxidation. Tyrosinase is the first two steps of reactions that catalyze the production of melanin, and is the key rate-limiting enzyme. Therefore, the production of melanin can be delayed and the pigmentation of the skin can be improved by inhibiting the activity of tyrosinase.

Various tyrosinase inhibitors are currently used for improving or treating skin stains and are commercially available, such as L-ascorbic acid, kojic acid, and hydroquinone, but cannot meet the expanding consumer market demand due to the problems of poor stability, poor efficacy, cytotoxicity, and the like. The plant extracts from natural sources have the advantages of good safety, rich resources, low cost and the like and are widely concerned. For example, arbutin, a hydroquinone glycoside derivative derived from a plant ingredient, has no cytotoxicity at an active dose, is developed as a commercial product, and has been added to various whitening products.

Disclosure of Invention

The invention aims to provide a bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract.

The invention also aims to provide a preparation method of the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract.

The invention further aims to provide application of the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract as a tyrosinase inhibitor in the fields of medicines and cosmetics.

The purpose of the invention is realized by the following technical scheme: the bletilla striata 2-isobutyl malic acid glucose oxygen benzyl ester extract contains the following compounds: 1- (4-Glucosyloxybenzyl) -2-isobutylmalate-2-glucoside (dactylorhin E), 4- (gluconyloxy) -cis-glucyloxybenzyl cinnamate (bledormin J), 1- (4-Gluconoxybenzyl) -2-isobutylmalate (gymnoside I), 1, 4-bis (4-Gluconoxybenzyl) -2-isobutylmalate-2-glucoside (dactylorhin A), 1, 4-bis (4-Gluconoxybenzyl) -2-isobutylmalate (mileanine), 1, 4-bis (4-Gluconoxybenzyl) -2-isobutylmalate-2- (6-acetyl) glucoside (gymnoside III), 1, 4-bis (4-gluconoxybenzyl) -2-isobutylmalate-2- (4-cinnamoyl) glucoside (gymsoside V) and 1, 4-bis (4-gluconoxybenzyl) -2-isobutylmalate-2- (4-cinnamoyl-6-acetyl) glucoside (gymsoside IX).

The mass percentage of the 1, 4-di (4-glucosyloxybenzyl) -2-isobutyl malate is not less than 20%.

In the bletilla striata 2-isobutyl malic acid glucosyloxybenzyl ester extract, the peak area corresponding to 1- (4-glucosyloxybenzyl) -2-isobutyl malate-2-glucoside accounts for 0.51-3.14%, the peak area corresponding to 4- (glucosyloxy) -cis-cinnamic acid glucosyloxybenzyl ester accounts for 2.94-5.01%, the peak area corresponding to 1- (4-glucosyloxybenzyl) -2-isobutyl malic acid accounts for 4.37-8.51%, the peak area corresponding to 1, 4-bis (4-glucosyloxybenzyl) -2-isobutyl malate-2-glucoside accounts for 4.02-9.26%, and the peak area corresponding to 1, 4-bis (4-glucosyloxybenzyl) -2-isobutyl malate accounts for 22.69-35.58%, the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (6-acetyl) glucoside accounts for 5.05-10.16%, the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (4-cinnamoyl) glucoside accounts for 1.60-3.74%, and the peak area corresponding to 1, 4-bis (4-gluconoxybenzyl) -2-isobutyl malate-2- (4-cinnamoyl-6-acetyl) glucoside accounts for 3.45-8.42%.

The preparation method of the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract comprises the following operation steps: pulverizing tuber or fibrous root of bletilla striata of Orchidaceae, soaking in extraction solvent for extraction or heating and reflux-extracting in the extraction solvent, wherein the extraction solvent is one or more of water, methanol, ethanol and acetone, filtering or centrifuging to remove insoluble substances, concentrating the obtained extraction solution under reduced pressure, and drying to obtain rhizoma bletilla extract; extracting rhizoma Bletillae extract with solvent or separating with macroporous adsorbent resin to obtain 2-isobutyl malic acid glucosyloxybenzyl ester extract;

the solvent extraction separation comprises dispersing rhizoma bletilla extract in water, sequentially extracting with ethyl acetate and n-butanol for 3 times at equal volume, vacuum drying respectively, and extracting the n-butanol fraction to obtain rhizoma bletilla 2-isobutyl malic acid glucosyloxybenzyl ester extract.

The macroporous adsorption resin separation is to dissolve a bletilla striata extract in water, filter, take filtrate and pass macroporous resin D101 column chromatography, the macroporous resin includes but is not limited to D101, HP-20, AB-8, X-5, D1300 and other types, water, ethanol-water solution with volume percentage concentration of 50% and ethanol-water solution with volume percentage concentration of 95% are adopted in sequence for gradient elution, and the elution extract of the ethanol-water solution with volume percentage concentration of 50% is the bletilla striata 2-isobutyl malic acid glucose oxygen benzyl ester extract.

The extraction temperature is 20-100 ℃, and the extraction time is 1-48 hours; the separation is carried out by adopting a column chromatography separation method and/or an extraction method.

The bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract is used as a tyrosinase inhibitor to be applied to cosmetics and medicaments for inhibiting melanin generation or whitening.

The bletilla striata 2-isobutyl malic acid glucoxybenzyl ester extract provided by the invention can be used as medicines or cosmetics, and the use amount can be different according to different purposes.

When the medicine is prepared, the bletilla striata 2-isobutyl malic acid glucose oxygen benzyl ester extract is mixed with a conventional medicinal carrier to prepare external preparations such as powder, tablets or emulsion, paste and the like, and the dosage is proper to be 0.1-50%.

When the cosmetic is prepared, 0.1-20% of the extract can be added into a cosmetic matrix.

Compared with the prior art, the invention has the following advantages and beneficial effects: the invention provides a novel bletilla striata 2-isobutyl malic acid glucosyloxybenzyl ester extract with tyrosinase inhibitory effect, which can be used as a tyrosinase inhibitor, can be used for preparing cosmetics or medicaments for inhibiting melanin generation or whitening, and has good effects of preventing, improving and treating skin pigmentation such as skin tanning, color spots, senile plaques, freckles and the like after ultraviolet irradiation. In addition, the extract can be mixed with other compounds and extracts with related applications to prepare cosmetics or medicines for inhibiting melanin generation or whitening skin.

Drawings

The invention is further illustrated by the following figures and examples.

Fig. 1 is a liquid Total Ion Current (TIC) characteristic spectrum and an identification spectrum of main chromatographic peaks of a bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract, wherein a is the TIC characteristic spectrum of the bletilla striata 2-isobutyl malic acid glucose oxybenzyl ester extract, and b is the identification spectrum of the main chromatographic peaks.

FIG. 2 shows the inhibitory effect of arbutin and extracts of gluconyloxybenzyl 2-isobutylmalate on tyrosinase at different concentrations.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.