CN114634535A - Separation and purification method of anoectochilus formosanus glycoside and application of anoectochilus formosanus glycoside in soothing cosmetics - Google Patents

Separation and purification method of anoectochilus formosanus glycoside and application of anoectochilus formosanus glycoside in soothing cosmetics Download PDF

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CN114634535A
CN114634535A CN202111561937.2A CN202111561937A CN114634535A CN 114634535 A CN114634535 A CN 114634535A CN 202111561937 A CN202111561937 A CN 202111561937A CN 114634535 A CN114634535 A CN 114634535A
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glycoside
anoectochilus
extract
filtrate
anoectochilus formosanus
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王希丽
张亚芬
林晓锋
潘发伍
胡蓉青
萧自智
饶焕文
林建平
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Juwenlee Fujian Cosmetics Co ltd
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    • C07H1/00Processes for the preparation of sugar derivatives
    • C07H1/06Separation; Purification
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    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61QSPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00Preparations for care of the skin
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Abstract

A separation and purification method of Anoectochilus roxburghii glycoside and its application in soothing cosmetics are provided, wherein an Anoectochilus roxburghii extract capable of inhibiting NO release is screened from Anoectochilus roxburghii by adopting RAW264.7 cell model, the main component is Anoectochilus roxburghii glycoside, and the preparation method of the extract is designed and optimized; meanwhile, the extract can inhibit the activation of TRPV1 on human embryonic kidney cells caused by capsaicin stimulation, reduce the calcium ion inflow of cells caused by inflammatory mediators, avoid the initiation of corresponding physiological changes and the like, and further prove that the extract has the effect of relieving.

Description

Separation and purification method of anoectochilus formosanus glycoside and application of anoectochilus formosanus glycoside in soothing cosmetics
Technical Field
The invention belongs to the technical field of extraction, separation, transformation and application of natural plant active ingredients, and particularly relates to a preparation method and application of an anoectochilus formosanus extract with a relieving effect.
Background
Anoectochilus roxburghii is a common name of Anoectochilus roxburghii, is a plant of Orchidaceae (Orchidaceae) Kaliptae (Anoectochilus), and more than 17 plants of the genus are currently found in China. Anoectochilus roxburghii is produced in Fujian province and is a genuine herb in Fujian province.
According to records in Xinhua Bencao gang Yao, the whole herb of Anoectochilus roxburghii can be used as a medicine, has sweet taste and mild nature, and has the effects of cooling blood, calming liver, and clearing heat and detoxicating.
At present, anoectochilus roxburghii has been approved as food by the national food and drug administration, so that the anoectochilus roxburghii has a wide market in the fields of food and health food. However, the application and research of anoectochilus formosanus in the field of cosmetics are relatively few. Chinese patent CN2017106813818 adopts water extraction, alcohol extraction and other methods to obtain anoectochilus roxburghii extract, and the extract can inhibit melanin and can be used as a raw material for whitening cosmetics. CN2016107666883 proposes a separation and purification process of herba Anoectochili Roxburghii flavone, and indicates that the flavone has antioxidant activity and tyrosinase activity inhibiting effect.
Sensitive skin (sensitive skin) refers to a high-response state of skin under physiological or pathological conditions, which may show subjective symptoms such as burning, stabbing pain, itching and tightness, with or without objective signs such as erythema, telangiectasia and desquamation. In recent years, as people attach more and more importance to the appearance, the cleaning and care of the skin of the people are intentionally enhanced, but the excessive cleaning of the skin and the improper use of cosmetics easily induce the sensitive skin, and the incidence rate of the sensitive skin tends to increase. In addition, physiological stress such as overtime and mental stress can increase secretion of corticosterone, and the corticosterone is converted into cortisol (endogenous glucocorticoid) by enzymes in skin tissues, and the cortisone can reduce skin inflammation in a short period of time, but also reduce skin immunity and repair capacity, so that sensitive skin is developed. [i]
The domestic and foreign researches have considered that after the sensitive skin is generated and the skin barrier is damaged, the nerve endings are stimulated by the outside, the transient receptor potential vanillic acid subtype 1(TRPV1) distributed in the sensory nerve endings, keratinocytes and mast cells expresses some receptors capable of sensing temperature, pain and the like on the surfaces of the keratinocytes, such as TRPs. For example, TRPV1 and TRPV1 trigger nerve reactions in two ways, on one hand, electrical signals are transmitted to nerve centers to generate symptoms such as tingling and itching, and on the other hand, calcium ion influx triggered by TRPV1 activation can trigger neurogenic inflammation, and finally, symptoms such as skin redness and erythema are caused. [ ii ] therefore, TRPV1 can be used for evaluating a cosmetic raw material having soothing efficacy.
In addition, sensitive skin is also an inflammatory reaction in nature, and NO plays a key regulatory role in inflammation and immune response. In the immune system, the synthesis of NO is an important mark for macrophage activation and is an important step for macrophage to generate pathological and immune effects, human body macrophages can generate NO under the condition of inflammatory diseases, inhibit or kill pathogenic microorganisms, and meanwhile, inflammatory immune reaction can be carried out under the participation of B lymphocytes and T lymphocytes, so that a sequence of physiological symptoms are triggered. Therefore, inhibition of NO release is commonly used to screen and evaluate anti-inflammatory and anti-allergic active materials.
The content of the anoectochilus formosanus glycoside in the anoectochilus formosanus is high (up to 25 percent), and the anoectochilus formosanus glycoside is easy to dissolve in water and is very convenient to extract. However, Anoectochilus roxburghii also contains flavones, pigments, inorganic salts, plant polysaccharides and the like, wherein the pigments and the inorganic salts influence the use of the Anoectochilus roxburghii in cosmetics. In addition, the roxburgh anoectochilus terminal bud glycoside has a fragile lactone ring structure and a fragile C-O glycoside structure, and can be easily hydrolyzed at high temperature or under the conditions of strong acid and strong base, so that the extraction and purification process parameters of the roxburgh anoectochilus terminal bud glycoside are limited to a certain extent.
Chinese patent 2015105110163 adopts petroleum ether extraction, methanol extraction, D101 crude separation, silica gel column subdivision, D101 purification and other processes to obtain the Anoectochilus roxburghii glycoside with purity of more than 95%, and the process is complex and uses methanol, chloroform and other toxic solvents.
Document [ iii ] discloses a method for separating off Anoectochilus roxburghii glycoside by using a combination of ODS and silica gel column, but this method is difficult to scale up and expensive in process cost.
Disclosure of Invention
Aiming at the problems, the invention provides a separation and purification method of anoectochilus formosanus glycoside and application of the anoectochilus formosanus glycoside in soothing cosmetics.
In order to realize the purpose of the invention, the invention adopts the following technical scheme:
a separation and purification method of anoectochilus formosanus glycoside comprises the following process steps:
(1) pulping: putting fresh anoectochilus formosanus and water into a pulping machine according to a proportion and pulping into slurry;
(2) coarse filtration: conveying the feed liquid to a bag filter for filtering, and collecting filtrate; washing the filter residue with water, and collecting the filtrate; mixing the two filtrates;
(3) fine filtering: fine filtering the filtrate with diatomite filter, and collecting fine filtrate;
(4) purifying: after the fine filtrate is pressurized by a pump, impurities in the feed liquid are removed through an activated carbon column and an ion exchange resin column in sequence, and the permeate is collected;
(5) concentration: concentrating the permeate at low temperature in vacuum to obtain extract;
(6) and (3) extraction: mixing the extract with an extractant, standing, and collecting supernatant;
(7) concentrating and drying: concentrating the supernatant under reduced pressure to obtain the anoectochilus formosanus glycoside.
Further, the ion exchange resin used is a cation exchange resin.
Further, the extracting agent is one or a mixture of ethyl acetate and absolute ethyl alcohol.
Further, the ion exchange resin used was LX-S1860 ion exchange resin.
Further, the extractant is a mixture of ethyl acetate and absolute ethyl alcohol, and the ratio of the absolute ethyl alcohol to the ethyl acetate is 2: 8.
The roxburgh anoectochilus terminal bud glycoside is added into the cosmetics with the relieving effect, and the addition amount is 0.01% -20%. The preferred addition amount is 2% to 5%.
By adopting the technical scheme, the invention has the following main innovation points and beneficial effects:
(1) by adopting the process, the extraction rate of the anoectochilus formosanus glycoside is more than 90%, the purity is more than 75%, and impurities such as pigment and the like are avoided due to the existing process. Anoectochilus formosanus glycoside is hydrophilic micromolecule, and has high solubility in water or absolute ethyl alcohol. According to the property, in the process, activated carbon is used for removing part of pigments and most of fat-soluble components, mineral substances and water-soluble pigments are removed through an ion exchange resin column, and finally, anhydrous ethanol is used for removing water-soluble saccharides and proteins.
(2) The process is simple, the equipment investment is low, and the method is suitable for batch production.
(3) The extract (Anoectochilus roxburghii glycoside) has soothing effect by activity screening and testing. NO plays a key regulatory role in inflammation and immune response, and the extract can inhibit NO release from macrophages stimulated by LPS, thereby inhibiting the progress of inflammatory reaction. Meanwhile, the extract can inhibit the activation of TRPV1 on human embryonic kidney cells caused by capsaicin stimulation, reduce the calcium ion influx of cells caused by inflammatory mediators, avoid the triggering of corresponding physiological changes (such as pain) and the like, and reduce the sensitivity of skin itch or pain sense.
Description of the drawings:
FIG. 1 shows a control sample of Anoectochilus roxburghii (leaf source organism, purity is more than or equal to 95%);
FIG. 2 is Anoectochilus roxburghii extract (example 1, purity 78%);
FIG. 3 is a screen of anti-inflammatory activity in Anoectochilus roxburghii (1. mu.g/mL);
FIG. 4 shows that Anoectochilus roxburghii extract (Anoectochilus roxburghii glycoside) can inhibit NO release;
FIG. 5 shows that Anoectochilus roxburghii extract (Anoectochilus roxburghii glycoside) can inhibit the influx of calcium ions into cells caused by capsaicin.
Detailed Description
The present invention is further illustrated by the accompanying fig. 1-5 and the detailed description, which describe only some embodiments of the invention and are not intended to limit the scope of the invention. Other embodiments, which can be made by persons skilled in the art without any inventive contribution, fall within the scope of protection of the present invention.
A separation and purification method of anoectochilus formosanus glycoside comprises the following process steps:
(1) pulping: putting fresh anoectochilus formosanus and water into a pulping machine according to a proportion and pulping into slurry;
(2) coarse filtration: conveying the feed liquid to a bag filter for filtering, and collecting filtrate; washing the filter residue with water, and collecting the filtrate; mixing the two filtrates;
(3) fine filtering: fine filtering the filtrate with diatomite filter, and collecting fine filtrate;
(4) purifying: after the fine filtrate is pressurized by a pump, impurities in the feed liquid are removed through an activated carbon column and an ion exchange resin column in sequence, and the permeate is collected;
(5) concentration: concentrating the permeate at low temperature in vacuum to obtain extract;
(6) and (3) extraction: mixing the extract with an extractant, standing, and collecting supernatant;
(7) concentrating and drying: concentrating the supernatant under reduced pressure to obtain the anoectochilus formosanus glycoside.
The following is further detailed in several groups of specific examples:
example 1:
(1) taking 1kg of fresh Anoectochilus roxburghii (Anoectochilus roxburghii glycoside content is 3.02%) and 3kg of water according to a certain proportion, and pulping into serous fluid in a pulping machine.
(2) Pouring the slurry into a bag filter, and collecting a crude filtrate; washing filter residues with 1-2 kg of pure water, and collecting a crude filtrate; the two crude filtrates were combined.
(3) Fine filtering the filtrate with diatomite filter, and collecting the fine filtrate.
(4) After the fine filtrate is pressurized by a pump, the fine filtrate sequentially passes through an activated carbon column and an LX-S1860 ion exchange resin column, and the permeate is collected.
(5) Concentrating the permeate at 50 deg.C and-0.095 MPa to obtain 0.104kg extract.
(6) And (3) fully mixing the extract with 500mL of absolute ethyl alcohol, and pouring out supernatant liquid, namely the extract liquid.
(7) Concentrating the extractive solution at 50 deg.C under-0.095 MPa to obtain light yellow solid 35.4g, which has a content of 78.4% and a total yield of 91.7% by HPLC.
Example 2:
(1) taking 10kg of fresh Anoectochilus roxburghii (Anoectochilus roxburghii glycoside content is 3.37%) and 25kg of water according to a certain proportion, and pulping into pulp in a pulping machine.
(2) Pouring the slurry into a bag filter, and collecting a crude filtrate; washing filter residues with 3-5 kg of pure water, and collecting a crude filtrate; the two crude filtrates were combined.
(3) Fine filtering the filtrate with diatomite filter, and collecting the fine filtrate.
(4) After the fine filtration liquid is pressurized by a pump, the fine filtration liquid sequentially passes through an activated carbon column and an LX-S1860 ion exchange resin column, and the permeation liquid is collected.
(5) The permeate was concentrated at 50 deg.C and-0.095 MPa to obtain 1.22kg extract.
(6) Mixing the extract with 3L of mixed solution (ethanol: ethyl acetate is 8:2), and pouring out supernatant to obtain extract; extracting the bottom extract once again; the two extracts were combined.
(7) Concentrating the extractive solution at 50 deg.C under-0.095 MPa to obtain white flake solid 372g with Anoectochilus formosanus glycoside content of 81.5% and Anoectochilus formosanus glycoside total yield of 92.7%.
Example 3:
a preparation method of essence with a relieving effect,
(1) the ingredients are as follows: water, glycerol, 1, 3-butanediol, anoectochilus formosanus glycoside, sodium hyaluronate, p-hydroxyacetophenone and caprylyl hydroximic acid. (except for the Anoectochilus formosanus glycoside, other raw materials can be purchased from the market.)
(2) The mixture ratio is as follows:
ingredients Proportioning Remarks for note
Pure water Balance of Solvent(s)
Glycerol 10.0% Moisture-retaining agent
1, 3-butanediol 5.0% Moisture-retaining agent
Anoectochilus formosanus glycoside 2.5% Functional component
Hyaluronic acid sodium salt 0.5% Thickening agent
P-hydroxyacetophenone 0.3% Preservative
Octanoyl hydroximic acid 0.1% Preservative agent
(3) The preparation method comprises the following steps: mixing the above materials, and stirring to dissolve.
To demonstrate the effectiveness of the invention, supplementary explanations were made:
1. determination of Anoectochilus formosanus glycoside content
The method comprises the following steps:
crushing a gold thread lotus sample by using a meat grinder, putting 1-2 g of the sample into a beaker, adding 50% ethanol, leaching at 60 ℃, and filtering; leaching the filter residue for 2 times; mixing the filtrates, placing in a 250mL volumetric flask, diluting to constant volume with 50% ethanol, shaking, collecting a little solution, filtering with microporous membrane, placing the filtrate in a sample bottle, and analyzing the sample with an instrument.
The equipment configuration: chromatography column (Inertsil ODS-3, 5 μ M, 4.6X 250mm), detector (SPD-M20A), binary gradient pump (LC-16), column incubator (Essentia CTO-16).
Chromatographic conditions are as follows: the mobile phase A is acetonitrile, and the mobile phase B is 0.2% phosphoric acid water solution; the flow rate is 1.0mL/min, and the elution method is gradient elution (0-5min: 0% A; 5-10min: 0% A → 10% A); the column temperature is 30 ℃, the detection wavelength is 200nm, and the sample injection amount is 5 mu L.
As a result: FIGS. 1 to 2
2. Soothing effect
The method comprises the following steps: inoculating RAW264.7 cells into a 48-well plate according to the density of 2 x 105 cells/well, culturing overnight for 14 +/-2 h at 37 ℃ in a 5% CO2 incubator, discarding the culture solution, adding 190 mu L of DMEM culture solution (without serum), 5 mu L of LPS working solution and 5 mu L of sample to be detected (or PBS buffer solution) into each well, incubating and culturing for 24h at 37 ℃ and 5% CO2, sucking the supernatant, and determining the NO content according to the instruction of a nitric oxide detection kit.
As a result: FIGS. 3 to 4
3. Relieving efficacy
The method comprises the following steps: HEK293 cells are inoculated in a 48-well plate according to the density of 5 multiplied by 104 cells/well, cultured overnight for 14 +/-2 h at 37 ℃ in a 5% CO2 incubator, the culture solution is discarded, 180 mu L of physiological saline (containing 2mM CaCl2) +10 mu L of sample to be detected (or PBS buffer solution) is added into each well, incubation culture is carried out for 1h, 10 mu L of capsaicin solution is added, incubation culture is carried out for 30min, the supernatant is discarded, the cells are washed for 3 times by the PBS buffer solution, the operation is carried out according to the instruction of the tissue calcium ion concentration fluorescence quantitative detection kit, and the fluorescence intensity is measured in a chemiluminescence gel imaging system.
As a result: FIG. 5
The invention screens anoectochilus formosanus extract (mainly containing anoectochilus formosanus glycoside) capable of inhibiting NO release from anoectochilus formosanus by adopting a RAW264.7 cell model, and designs and optimizes the preparation method of the extract. Meanwhile, the extract can inhibit the activation of TRPV1 on human embryonic kidney cells caused by capsaicin stimulation, reduce the calcium ion influx of cells caused by inflammatory mediators, avoid causing corresponding physiological changes (such as pain) and the like, and further prove that the extract has the effect of relieving.
While the foregoing description shows and describes the preferred embodiments of the present invention, it is to be understood that the invention is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the inventive concept as described herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. And that modifications and variations may be effected by those skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Reference documents:
[1]effect of Anoectochilus roxburghii extract on xanthine oxidase activity and hyperuricemia mice]Strait pharmaceuticals, 2017,29(07):12-14.
[1]Study of ethanol extracts of Zhenzzhi Yun, Chenqianwei, Jiantao, Chenwanqiu, Houjie, Tangchunling and Anoectochilus formosanus on anti-aging effect of naturally aging mice [ J]The Chinese medicinal materials 2020,43(05):1200-1204.
[1]Research progress of liver protection and action mechanism of Anoectochilus roxburghii (J)]Modern Chinese traditional medicine 1-13.
[1]Chen Y,Lyga J.Brain-Skin Connection:Stress,Inflammation and Skin Aging[J].Inflammation&Allergy- Drug Targets(Formerly Current Drug Targets-Inflammation&Allergy),2014,13(3).
[1]Inhibition of TRPV1 for the treatment of sensitive skin[J].Experimental Dermatology,2010,19(11):980-986.
[1]Du X M,Sun N Y,Tamura T,et al.Higher yielding isolation of kinsenoside in Anoectochilus and its antihyperliposis effect.[J].Biological&Pharmaceutical Bulletin,2001,24(1):65-69。

Claims (7)

1. A separation and purification method of anoectochilus formosanus glycoside is characterized in that: comprises the following process steps:
(1) pulping: putting fresh anoectochilus formosanus and water into a pulping machine according to a proportion and pulping into slurry;
(2) coarse filtration: conveying the feed liquid to a bag filter for filtering, and collecting filtrate; washing the filter residue with water, and collecting the filtrate; mixing the two filtrates;
(3) fine filtering: fine filtering the filtrate with diatomite filter, and collecting fine filtrate;
(4) purifying: after the fine filtrate is pressurized by a pump, impurities in the feed liquid are removed through an activated carbon column and an ion exchange resin column in sequence, and the permeate liquid is collected;
(5) concentration: concentrating the permeate at low temperature in vacuum to obtain extract;
(6) and (3) extraction: mixing the extract with an extractant, standing, and collecting supernatant;
(7) concentrating and drying: concentrating the supernatant under reduced pressure to obtain the anoectochilus formosanus glycoside.
2. The method for separating and purifying acteoside as claimed in claim 1, wherein the method comprises the steps of: the ion exchange resin used is a cation exchange resin.
3. The method for separating and purifying acteoside as claimed in claim 1, wherein the method comprises the steps of: the extractant is one or a mixture of ethyl acetate and absolute ethyl alcohol.
4. The method for separating and purifying acteoside as claimed in claim 1, wherein the method comprises the steps of: the ion exchange resin used was LX-S1860 ion exchange resin.
5. The method for separating and purifying acteoside as claimed in claim 1, wherein the method comprises the following steps: the extractant is a mixture of ethyl acetate and absolute ethyl alcohol, and the ratio of the absolute ethyl alcohol to the ethyl acetate is 2: 8.
6. Use of the roxburgh anoectochilus terminal bud glycoside in a soothing cosmetic, characterized in that the roxburgh anoectochilus terminal bud glycoside extracted according to claim 1, 2, 3, 4 or 5 is added to a soothing cosmetic in an amount of 0.01% to 20%.
7. The use of an anoectochilus formosanus glycoside in a soothing cosmetic as claimed in claim 6, wherein: the roxburgh anoectochilus terminal bud glycoside is added into the cosmetics with the relieving effect, and the addition amount is 2% -5%.
CN202111561937.2A 2021-12-20 2021-12-20 Separation and purification method of anoectochilus formosanus glycoside and application of anoectochilus formosanus glycoside in soothing cosmetics Pending CN114634535A (en)

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US5994533A (en) * 1997-08-11 1999-11-30 Council Of Scientific & Industrial Research Process for the recovery of tartaric acid and other products from tamarind pulp
US20090306199A1 (en) * 2008-03-19 2009-12-10 Wen-Chuan Lin Method for inhibiting activation of macrophages, inhibiting formation of osteoclasts, inhibiting function of osteoclasts, and/or activating osteoblasts
KR20100102439A (en) * 2009-03-11 2010-09-24 주식회사 한국화장품제조 A cosmetic composition comprising an extract of tissue cultured anoectochilus formosanus and a preparation method of the extract of tissue cultured anoectochilus formosanus
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