CN108210530A - A kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid and preparation method thereof - Google Patents
A kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid and preparation method thereof Download PDFInfo
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- CN108210530A CN108210530A CN201810287495.9A CN201810287495A CN108210530A CN 108210530 A CN108210530 A CN 108210530A CN 201810287495 A CN201810287495 A CN 201810287495A CN 108210530 A CN108210530 A CN 108210530A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/23—Apiaceae or Umbelliferae (Carrot family), e.g. dill, chervil, coriander or cumin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/55—Liquid-liquid separation; Phase separation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/40—Chemical, physico-chemical or functional or structural properties of particular ingredients
- A61K2800/52—Stabilizers
- A61K2800/524—Preservatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/70—Biological properties of the composition as a whole
- A61K2800/72—Hypo-allergenic
Abstract
The present invention is suitable for field of plant extraction, provides a kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid and preparation method thereof, this method, including：20~50 parts of centellas are taken, removal of impurities is broken to be placed in extraction vessel；40~100 parts of water, 10~25 parts of enzyme preparations, 1~5 part of alcohols material, 1~5 part of salts substances are added in into extraction vessel, form centella mixture, and the mixture is placed in immersion 15min~30min in 20 DEG C~45 DEG C of thermostat water bath；Ultrasound 1h~2h at 20 DEG C~45 DEG C is placed in, forms crude extract；Treat crude extract stratification, isolated supernatant；By supernatant by resin chromatography chromatographic column, and washing impurity-removing is carried out with water；It is eluted by the use of propylene glycol as eluant, eluent, collects eluent, eluent is centella extracting solution.The recovery rate of asiaticosid can be effectively improved using the present invention, obtained centella extracting solution not only good compatibility；And with natural anticorrosion performance, mildly non-stimulated to human skin, antiallergic, anti-oxidant and bacteriostasis are strong, safe.
Description
Technical field
The invention belongs to field of plant extraction more particularly to a kind of biological enzyme processing and corrosion-resistant centella plant extracts
Liquid and preparation method thereof.
Background technology
Centella is Umbelliferae Centella plant, and all herbal medicine is one of Chinese medicine that China applies earliest, traditional Chinese medicine
It takes the history for having more than 2,000 years with external application orally using centella, is recorded in earliest《Sheng Nong's herbal classic》.Version in 2015《Middle traditional Chinese medicines
Allusion quotation》Centella is recorded as parts of generic medicinal plants, recording it has effects that clearing heat and promoting diuresis, removing toxicity for detumescence, is mainly used for damp and hot Huang
The treatment of subcutaneous ulcer, heatstroke diarrhea, carbuncle sore tumefacting virus, traumatic injury etc..Main active of the asiaticosid for centella, modern pharmacology
Promote wound healing research shows that asiaticosid has, repair cicatrix of skin, allergy, anti-inflammatory, whitening, anti-oxidant and other effects.
Existing asiaticosid extracting method period length, the recovery rate of asiaticosid extract that is relatively low, and extracting
It is usually powdered, therefore the compliant applications in all kinds of medical product systems have limitation (for example, coordinating with other substances
It is mixed during use uneven etc.).In addition, it is organic molten to use methanol, ethyl alcohol etc. in existing asiaticosid extracting method
Agent promotes the preservative substances such as extraction and the different thiophene miaow quinoline ketone of methyl to improve the antiseptic property of Gotu Kola P.E, still, through adjusting
Research and development are existing, these above-mentioned ingredients there are certain allergic reaction (for example, red swelling of the skin, itch etc.), therefore pacify human skin
Full property is not high.
It can be seen that existing centella extracting method, there are asiaticosid recovery rate is not high, the compatibility of extract should
With with limitation, and remaining allergen easily causes the allergic reaction of human skin in extract, and safety is not high
The problem of.
Invention content
The embodiment of the present invention provides a kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid and preparation method thereof,
Existing centella extracting method is aimed to solve the problem that there are asiaticosid recovery rate is not high, the compatibility application of extract has limitation
Property, and the problem of remaining allergen easily causes the allergic reaction of human skin in extract, and safety is not high.
The embodiment of the present invention is achieved in that a kind of biological enzyme processing and the system of corrosion-resistant centella plant extraction liquid
Preparation Method, the preparation method include the following steps：
Step 1：Take 20~50 parts of centellas, removal of impurities cleans, it is broken be placed on it is spare in extraction vessel；
Step 2：40~100 parts of water, 10~25 parts of enzyme preparations, 1~5 part of alcohols material, 1~5 are added in into extraction vessel
Part salts substances, form centella mixture, and centella mixture is placed in 20 DEG C~45 DEG C of thermostat water bath and is impregnated
15min~30min；
Step 3：Centella mixture is placed in ultrasound 1h~2h at 20 DEG C~45 DEG C, forms crude extract；
Step 4：Treat crude extract stratification, isolated supernatant；
Step 5：Supernatant is made to pass through resin chromatography chromatographic column, and washing impurity-removing is carried out with water；
Step 6：It is eluted by the use of propylene glycol as eluant, eluent, collects eluent, eluent is centella plant extract
Liquid.
Preferably, the enzyme preparation for glucose oxidase, lysozyme and cellulase, pectase, Co-Q10 or
In zytase any one or at least two mixture.
More preferably, the enzyme preparation is cellulase, pectase, zytase, glucose oxidase and lysozyme
Mixture.
Preferably, the alcohols material for any one in 1,2- hexylene glycols, butanediol, propylene glycol or pungent glycol or
The mixture of person at least two.
More preferably, the alcohols material is propylene glycol.
Preferably, the salts substances be sodium chloride, potassium chloride, magnesium chloride either any one in sodium carbonate or extremely
Mixture two kinds few.
More preferably, the salts substances are sodium chloride.
Preferably, take 25 parts of centellas, removal of impurities cleans, it is broken be placed on it is spare in extraction vessel；
100 parts of water, 2.5 parts of cellulase, 2 parts of pectase, 2.5 parts of zytase, Portugal are added in into the extraction vessel
2 parts of carbohydrate oxidase, 3.5 parts of lysozyme, 5 parts of propylene glycol, 5 parts of sodium chloride form centella mixture, and the centella are mixed
It closes object and is placed in 25 DEG C of thermostat water bath and impregnate 30min；
The centella mixture is placed in ultrasound 1.5h at 25 DEG C, forms crude extract.
Preferably, it is described to treat the crude extract stratification, isolated supernatant, including：Treat the crude extract
Stratification is centrifugally separating to obtain supernatant, wherein, centrifugal speed 8000r/min, centrifugation time 15min.
The embodiment of the present invention additionally provides a kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid, the accumulated snow
Careless plant extraction liquid extracts to obtain using the preparation method of above-mentioned centella plant extraction liquid.
Preferably, Guangdong region, Hubei Regional, Hainan region or Jiangsu area can be selected in the above-mentioned centella for extraction
One kind in the centella of domain or at least two.
More preferably, the centella in Guangdong region is selected.
Below to raw material involved in the extraction of above-mentioned centella the effect of and effect are described in detail：
Cellulase and pectase：It can make the mass degradations such as cellulose, the pectin in cell wall or cytoplasm, destroy
The compact texture of cell wall causes cell wall and cytoplasm structure to generate the variations such as loose part, expansion, collapse, reduces thin
The resistance to mass tranfer that the mass transfers such as cell wall, cytoplasm barrier spreads active ingredient from intracellular to Extraction medium promotees from mass transfer angle
Improve effective component extraction rate.
Zytase：Zytase can crush plant cell wall, and the xylan degrading for improving nutrient is relatively low for the degree of polymerization
Small molecule segment, destroy the structure of cell wall, the nutriment that cell wall fetters released.Wherein, xylan is to plant
The important component of object hemicellulose, xylan is complicated, the degradable participation for needing a variety of enzymes, and zytase is to participate in
Most important one kind in degradation enzyme system.Zytase refers to can be by xylan degrading into the total of one group of enzyme of oligosaccharide and xylose
Claim.
Glucose oxidase：Effectively fast decoupled glucan, dissolving bacterial plaque bacterial plaque can be inhibited to be formed, bacterium can be effectively removed
Planting group.
Lysozyme：It is a kind of alkaline enzyme that can be hydrolyzed and polysaccharide is sticked in pathogenic bacteria.Mainly by destroying the N- second in cell wall
β-Isosorbide-5-Nitrae glycosidic bond between acyl muramic acid and n-acetylglucosamine makes the insoluble glutinous polysaccharide of cell wall resolve into soluble sugar
Peptide causes cell wall rupture content to escape and make bacterolysis.Lysozyme can also directly be tied with negatively charged virus protein
It closes, forms double salt with DNA, RNA, apoprotein, make virally inactivated.Therefore, which has the works such as antibacterial, anti-inflammatory, antiviral
With.Lysozyme is safe as preservative.Lysozyme is a kind of native protein, and property is very stable.
Co-Q10：It is a kind of fat-soluble antioxidant, the nutrition of energy human activin cell and cellular energy has and improves
Body immunity enhances the functions such as anti-oxidant, anti-aging and enhancing human activity.It can go deep into cell simultaneously, it is new to strengthen cell
Into metabolic function, on the other hand consolidation binding ability between Polygonum Multiflorum Thunb Extract forms the reticular structure of elasticity in epidermis, repairs really
The wrinkle caused by fluid loss characteristics, and and then reach real moisture-keeping efficacy.
1,2- hexylene glycol, butanediol, propylene glycol, pungent glycol can dissolve certain plant ingredient, while safety
It is good, there is respective good characteristic.Such as 1,2- hexylene glycols, can efficiently permanent moisturizing, also have the effect of antisepsis and sterilization；Fourth two
Alcohol sense of touch is smooth, and property is stablized, and can obtain more soft skin sense；The viscosity and good hygroscopicity of propylene glycol, it is nontoxic；Pungent glycol tool
There are certain moisturizing and antibacterial activity.
Sodium chloride：The viscosity of system can be increased；Osmolyte regulator, salting-out agents can be done, there is certain shadow to the activity of enzyme
It rings, influences the dissolution that enzyme cracks substance in the speed and plant cell of cell wall.Sodium chloride also has the function of bactericidal antiphlogistic.
Potassium chloride：It is one kind of hydrochloride, white crystals or crystalline powder, soluble easily in water and glycerine is insoluble in alcohol, no
It is dissolved in ether and acetone.In food service industry, it can be used as salt substitute applied to agricultural production, aquatic products, livestock products, fermentation, seasoning, tank
Head, instant food etc. make low Na prod, to reduce the excessively high harmful effect to body of sodium content；It is also used for strengthening potassium (for people
Body electrolyte use), prepare sports drink etc..
Magnesium chloride：Inorganic raw material is important in chemical industry, for producing magnesium carbonate, magnesium hydroxide, magnesia etc.
Magnesium products also serve as raw material of antifreezing agent etc..Food additives, protein coagulation agent, deicing salt, freezing can be done in other fields
Agent, dust-proofing agent, refractory material etc..
Sodium carbonate：It is a kind of white powder soluble easily in water, solution alkaline.It is widely used in medicine, papermaking, metallurgy, glass
The industry such as glass, weaving, dyestuff, as food industry leavening.
The preparation method of centella plant extraction liquid provided in an embodiment of the present invention, utilizes enzyme preparation and the association of salts substances
Same-action can crack the cell wall of centella and the compact texture of cytoplasm, make cytoplasm and active ingredient (accumulated snow comprehensively
Careless glycosides) it dissolves out, the extraction yield of asiaticosid can be effectively improved；Obtained centella extracting solution is soluble liquid, therefore
Its compatibility in various product system can be improved well, that is, overcomes traditional settled powder grass extract in product
The limitation of compliant applications in system.In addition, compounding of this law by the salts substances of natural enzyme preparation and food-grade, structure
Natural protective system, favorable anti-corrosion effect not only extends the shelf-life of centella extracting solution, and cause is not contained in extracting solution
Make human skin that anaphylactoid ingredient (the different thiophene miaow quinoline ketone of such as methyl) occur, it is safe；Furthermore the centella extracting solution
Mildly non-stimulated to human skin, antiallergic and oxidation resistance are strong, but also have whitening, moisturizing, can alleviate skin silt blueness
The effect of.
Specific embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with specific embodiment, to this
Invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, not
For limiting the present invention.
Biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid provided in an embodiment of the present invention, utilize
The synergistic effect of enzyme preparation and salts substances can crack the cell wall of centella and the compact texture of cytoplasm, make comprehensively
Cytoplasm and active ingredient (asiaticosid) dissolution can effectively improve the extraction yield of asiaticosid；Obtained centella carries
Liquid is taken as soluble liquid, the good compatibility in various product system.In addition, pass through natural enzyme preparation and food-grade
The compounding of salts substances builds natural protective system, and antiseptic property is good, can extend the shelf-life of centella extracting solution；Together
When do not contain and cause human skin that anaphylactoid ingredient (the different thiophene miaow quinoline ketone of such as methyl) occurs, it is safe；Furthermore the product
The careless extracting solution of snow is mildly non-stimulated to human skin, and antiallergic and oxidation resistance are strong, also have effects that whitening, moisturizing.
Technical scheme of the present invention and technique effect are described further below by way of specific embodiment.
Embodiment 1：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 20 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 40 parts of water, 10 parts of enzyme preparations (including：2 parts of cellulase, 2 parts of pectase, wood are poly-
1 part of carbohydrase, 2 parts of glucose oxidase, 3 parts of lysozyme), 1 part of alcohols material (1 part of propylene glycol), 1 part of salts substances (sodium chloride 1
Part), centella mixture is formed, and the centella mixture is placed in 20 DEG C of thermostat water bath and impregnates 15min.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 20 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 2：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 25 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 50 parts of water, 12.5 parts of enzyme preparations (including：3.5 parts of cellulase, 2 parts of pectase,
1.5 parts of zytase, 3 parts of glucose oxidase, 2.5 parts of lysozyme), 2.5 parts of alcohols materials (2.5 parts of propylene glycol), 2.5 parts of salts
Substance (2.5 parts of sodium chloride) forms centella mixture, and the centella mixture is placed in 20 DEG C of thermostat water bath and is soaked
Steep 15min.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 20 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 3：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 25 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 50 parts of water, 12.5 parts of enzyme preparations (including：2 parts of cellulase, 3.5 parts of pectase,
1 part of zytase, 3.5 parts of glucose oxidase, 2.5 parts of lysozyme), 1 part of alcohols material (1 part of propylene glycol), 2.5 parts of salts substances
(1 part of sodium chloride, 1.5 parts of magnesium chloride) forms centella mixture, and the centella mixture is placed in 25 DEG C of water bath with thermostatic control
15min is impregnated in pot.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 25 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 4：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 25 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 50 parts of water, 14 parts of enzyme preparations (including：4 parts of cellulase, 3 parts of pectase, wood are poly-
1.5 parts of carbohydrase, 2 parts of glucose oxidase, 3.5 parts of lysozyme), 1 part of alcohols material (1 part of propylene glycol), 2.5 parts of salts substances (chlorine
Change 1 part of sodium, 1.5 parts of sodium carbonate), centella mixture is formed, and the centella mixture is placed in 25 DEG C of thermostat water bath
Interior immersion 30min.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 25 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 5：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 25 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 50 parts of water, 12.5 parts of enzyme preparations (including：3 parts of cellulase, 2.5 parts of pectase,
2.5 parts of zytase, 1.5 parts of glucose oxidase, 3 parts of lysozyme), 2.5 parts of alcohols materials (2.5 parts of propylene glycol), 2.5 parts of salts
Substance (2.5 parts of sodium chloride) forms centella mixture, and the centella mixture is placed in 25 DEG C of thermostat water bath and is soaked
Steep 30min.
(3) continue ultrasound 1.5h at a temperature of above-mentioned centella mixture being placed on 25 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 6：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 25 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 100 parts of water, 12.5 parts of enzyme preparations (including：2.5 parts of cellulase, 2 parts of pectase,
2.5 parts of zytase, 2 parts of glucose oxidase, 3.5 parts of lysozyme), 5 parts of alcohols materials (5 parts of propylene glycol), 5 parts of salts substances
(5 parts of sodium chloride) forms centella mixture, and the centella mixture is placed in 25 DEG C of thermostat water bath and is impregnated
30min。
(3) continue ultrasound 1.5h at a temperature of above-mentioned centella mixture being placed on 25 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 7：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 50 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 100 parts of water, 25 parts of enzyme preparations (including：6 parts of cellulase, 7 parts of pectase, bacteriolyze
6 parts of enzyme, 5 parts of Co-Q10,1 part of glucose oxidase), 5 parts of alcohols materials (2.5 parts of 1,2- hexylene glycols, 2.5 parts of propylene glycol), 5 parts
Salts substances (2.5 parts of sodium chloride, 2.5 parts of potassium chloride) form centella mixture, and the centella mixture are placed in 45 DEG C
Thermostat water bath in impregnate 30min.
(3) continue ultrasound 2h at a temperature of above-mentioned centella mixture being placed on 45 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 8：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 50 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 100 parts of water, 25 parts of enzyme preparations (including：6.5 parts of cellulase, glucose oxidase
6.5 parts, 6 parts of lysozyme, 6 parts of Co-Q10), 4.5 parts of alcohols materials (1.5 parts of propylene glycol, 3 parts of pungent glycol), 5 parts of salts substances
(5 parts of sodium chloride) forms centella mixture, and the centella mixture is placed in 40 DEG C of thermostat water bath and is impregnated
30min。
(3) continue ultrasound 2h at a temperature of above-mentioned centella mixture being placed on 40 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 9：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 30 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 60 parts of water, 15 parts of enzyme preparations (including：4 parts of cellulase, 2 parts of pectase, glucose
5 parts of oxidizing ferment, 2 parts of Co-Q10,2 parts of lysozyme), 4 parts of alcohols materials (2.5 parts of 1,2- hexylene glycols, 1.5 parts of pungent glycol), 3 parts
Salts substances (3 parts of sodium chloride) form centella mixture, and the centella mixture are placed in 40 DEG C of thermostat water bath
Impregnate 25min.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 40 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 10：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 30 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 60 parts of water, 15 parts of enzyme preparations (including：It is 2.5 parts of pectase, 3 parts of zytase, molten
4 parts of bacterium enzyme, 4 parts of Co-Q10,1.5 parts of glucose oxidase), 3 parts of alcohols materials (1.5 parts of butanediol, 1.5 parts of propylene glycol), 3 parts
Salts substances (1.5 parts of sodium chloride, 1.5 parts of potassium chloride) form centella mixture, and the centella mixture are placed in 25 DEG C
Thermostat water bath in impregnate 25min.
(3) continue ultrasound 1h at a temperature of above-mentioned centella mixture being placed on 25 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 11：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 45 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) added in into extraction vessel 80 parts of water, 13 parts of enzyme preparations (including：6 parts of lysozyme, 5 parts of glucose oxidase, fibre
Plain 2 parts of the enzyme of dimension), 3.5 parts of alcohols materials (1 part of 1,2- hexylene glycols, 1.5 parts of propylene glycol, 1 part of pungent glycol), 3 parts of salts substances (chlorine
Change 1.5 parts of sodium, 1 part of potassium chloride, 0.5 part of magnesium chloride), centella mixture is formed, and the centella mixture is placed in 30 DEG C
Thermostat water bath in impregnate 30min.
(3) continue ultrasound 1.5h at a temperature of above-mentioned centella mixture being placed on 30 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
Embodiment 12：
A kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, include the following steps：
(1) 35 parts of centellas is taken to remove impurity and stalk, cleans up, is placed on using crusher machine of squeezing the juice standby in extraction vessel
With；It is wherein preferred, the centella in Guangdong region can be used.
(2) 75 parts of water, 18 parts of enzyme preparations (8 parts of lysozyme, 5 parts of glucose oxidase, zytases are added in into extraction vessel
5 parts), 4 parts of alcohols materials (2 parts of 1,2- hexylene glycols, 2 parts of propylene glycol), 2.5 parts of salts substances (2 parts of sodium chloride, magnesium chlorides 0.5
Part), centella mixture is formed, and the centella mixture is placed in 35 DEG C of thermostat water bath and impregnates 25min.
(3) continue ultrasound 1.5h at a temperature of above-mentioned centella mixture being placed on 35 DEG C, form crude extract.
(4) above-mentioned crude extract stratification, isolated supernatant are treated；Preferably, centrifugal separation separation can be used
Supernatant is obtained, the condition of centrifugation is 8000r/min, 15min.
(5) by above-mentioned supernatant by resin chromatography chromatographic column, for example, can be by above-mentioned supernatant by long 457mm, directly
The resin chromatography chromatographic column of diameter 40mm, and carry out washing impurity-removing with water.
(6) it is eluted by the use of propylene glycol as eluant, eluent, collects eluent, which is centella plant extract
Liquid.
In embodiments of the present invention, centella plant extraction liquid is prepared using above-mentioned preparation process.
It should be noted that in an embodiment of the present invention, enzyme preparation is glucose oxidase, lysozyme and cellulose
Enzyme, pectase, Co-Q10 either any one in zytase or at least two mixture.For convenience of description, on
It states embodiment and illustrates only several preferred combinations therein, in practical applications, can arbitrarily choose glucose oxidase, bacteriolyze
Either any one in zytase or at least two enzymes are compounded for enzyme and cellulase, pectase, Co-Q10,
Stable natural anticorrosion system is built, the ratio that need to only keep the enzyme preparation shared in centella plant extraction liquid is 10~25
Part, just do not enumerate all combinations herein.
Based on it is above-mentioned identical the reasons why, the alcohols material that arrives involved in the embodiment of the present invention is 1,2- hexylene glycols, butanediol,
Propylene glycol either any one in pungent glycol or at least two mixture；Salts substances are sodium chloride, potassium chloride, chlorination
Magnesium either any one in sodium carbonate or at least two mixture.The various combinations of these above-mentioned components also differ
One enumerates herein.
The advantageous effect of embodiment in order to further illustrate the present invention is further done specifically below in conjunction with specific test example
It is bright：
Experiment one：HPLC measures asiaticosid content
1st, experimental principle and method：Containing abundant asiaticosid in centella, it is made by measuring in embodiment 1~12
Gotu Kola P.E naphthoquinones class content of material, so as to find out asiaticosid recovery rate.
Chromatographic condition：Zorbax sb C18 columns (4.6mm × 150mm)；Mobile phase A：Water, B：Acetonitrile；Gradient elution：0~
3min, A are by 80%~71%；3~16min, A keep 71%, B to keep 29%, rear run time 2min；Flow velocity：1mL/min；
Sample size：20μL；Temperature：30℃；Detection wavelength：205nm.
The drafting of standard curve：Accurate weighing 45.5mg asiaticosid samples, 25mL is settled to 50% methanol aqueous solution,
Standard sample solution is made.Prepare to pipette from standard sample solution, asiaticosid concentration is configured to 50% methanol aqueous solution
The respectively standard series working solution of 0.1mg/mL, 0.5mg/mL, 0.8mg/mL, 1.0mg/mL, 1.5mg/mL crosses 0.45 μm of filter
Film measures it under above-mentioned chromatographic condition and absorbs peak area, using external standard method, draws asiaticosid concentration with absorbing peak area
Regression curve.
The measure of asiaticosid in extracting solution：The centella plant extract prepare liquid of 1mL is drawn, with 50% methanol aqueous solution
25mL is settled to, with 0.45 μm of membrane filtration, sample detection.
2nd, experimental result：Test standard curve regression equation is：Y=2053X-23.516, related coefficient R=
0.9995；Wherein X is asiaticosid standard concentration (mg/mL), and Y is peak area.
Wherein, centella weight used in content/extraction of asiaticosid in asiaticosid recovery rate=100% × extracting solution
Amount.Detailed results see the table below 1.
Table 1
| Embodiment | Y | X(mg/mL) | Content/mg of asiaticosid in extracting solution | Recovery rate/% |
| Embodiment 1 | 1781 | 0.879 | 21.97 | 87.90 |
| Embodiment 2 | 1797 | 0.887 | 22.17 | 88.68 |
| Embodiment 3 | 1895 | 0.934 | 23.36 | 93.45 |
| Embodiment 4 | 1800 | 0.888 | 22.21 | 88.82 |
| Embodiment 5 | 1841 | 0.908 | 22.70 | 90.82 |
| Embodiment 6 | 1921 | 0.947 | 23.68 | 94.72 |
| Embodiment 7 | 1905 | 0.939 | 23.48 | 93.94 |
| Embodiment 8 | 1869 | 0.922 | 23.05 | 92.18 |
| Embodiment 9 | 1804 | 0.890 | 22.25 | 89.02 |
| Embodiment 10 | 1834 | 0.905 | 22.62 | 90.48 |
| Embodiment 11 | 1803 | 0.911 | 22.24 | 88.96 |
| Embodiment 12 | 1821 | 0.898 | 22.45 | 89.80 |
The active ingredient of Chinese medicine generally requires to abolish the obstruction of cell wall during by separation, but cell wall with
And hemicellulose, pectic substance, cellulose in cytoplasm etc. can form comparatively dense layer, under normal circumstances, active ingredient
It is difficult quickly dissolution, thus causes extracting cycle long, extraction yield is low.
However, the embodiment of the present invention uses enzymatic isolation method, can accelerate to give birth under enzyme preparation and the synergistic effect of salts substances
The speed of object reaction so that plant compact tissue is destroyed comprehensively under the action of enzymolysis, and the active ingredient of cell interior obtains
With dissolution, thus the extraction yield of asiaticosid can be effectively improved.
As the embodiment of the present invention, centella cellulase, zytase and pectase catalytic action and salt from
Under the synergistic effect of son, cell wall is quickly cracked, the substance dissolution in plant cell, so as to make active ingredient (centella
Glycosides) dissolution, and under the dissolving of polyalcohol, greatly improve the recovery rate of asiaticosid.It can be seen by the experimental data of upper table 1
Go out, the asiaticosid recovery rate of the embodiment of the present invention 1~12 is more than 85%.
Experiment two：Preservative efficacy testing
1st, test specimen：The centella plant extraction liquid sample that the embodiment of the present invention 1~12 provides；Experimental bacteria is large intestine bar
Bacterium ATCC 25922, staphylococcus aureus ATCC 6538, pseudomonas aeruginosa ATCC 9027, Candida albicans ATCC
10231。
2nd, test method：Microbiology challenge test method.
3rd, result evaluation：Using《European Pharmacopoeia》Result evaluation standard this result of the test is evaluated, have
Body refers to the following table 2.
Table 2
From the test result of upper table 2 it is found that 4 kinds of experimental bacterias (1 week) total plate count when the 7th day declines 100%, until the
It is tested at 28 days without growth, judgement by challenge, anti-corrosion effect is good, i.e., centella plant provided in an embodiment of the present invention carries
Take liquid that there is good antiseptic property.And the embodiment of the present invention uses natural the preventing of lysozyme and glucose oxidase compounding structure
Rotten system, antiseptic property it is further preferred that, can effectively extend shelf-life of extracting solution.
Experiment three：Ocular irritation/erosion test
1st, test specimen：The centella plant extraction liquid that the embodiment of the present invention 1~12 provides.
2nd, test method：SN/T 2329-2009 cosmetics eye irritation/corrosive chick chorioallantoic membrane experiment.
3rd, evaluation of test result standard：Stimulation standards of grading are divided into level below：IS ＜ 1 are nonirritant；1≤IS ＜ 5
For subexcite；5≤IS ＜ 9 are moderate irritation；IS >=10 are strong and stimulating/corrosivity.
4th, result of the test：The stimulation scoring of the centella plant extraction liquid provided through overtesting, 1-12 of the embodiment of the present invention
It is IS ＜ 1, i.e., centella plant extraction liquid provided in an embodiment of the present invention is mildly nonirritant to eye, safe.
Experiment four：Skin irritation test
(1) skin closure type patch test
1st, test specimen：The centella plant extraction liquid that the embodiment of the present invention 1~12 provides.
2nd, test method：From a certain 39 volunteers of regional choice, the age of volunteer, body was good between 20~45 years old
Health, in the recent period non-drug administration, nonbreast-fed women or gravid woman, non-constitution sensitive subjects.Volunteer is divided into 13 groups at random,
Every group of three people.Wherein, group 1 is blank control group, and the small interior of spot examination device does not place any substance, spot is tried the low sensitization glue of device
Band is pasted on the back of volunteer or forearm bends side, is gently pressed with palm and is allowed to equably stick on the skin；Group 2~groups 13 with
The processing method of group 1 is identical, unlike：The centella plant that will be provided respectively added with the 0.025mL embodiment of the present invention 1~12
Extracting solution is put into the small interior of spot examination device；I.e. group 2 it is corresponding be the embodiment of the present invention 1 provide centella plant extraction liquid, organize 3 pairs
Answer be the embodiment of the present invention 2 provide centella plant extraction liquid, group 4 it is corresponding be the embodiment of the present invention 3 provide accumulated snow
Careless plant extraction liquid ..., it is centella plant extraction liquid that the embodiment of the present invention 12 provides that group 13 is corresponding.Respectively
Group test duration is after 24 hours, 48 hours, and 30min after removal spot examination device, after impression disappearance, observation sticks the reaction of place's skin
Situation simultaneously records.
Wherein, skin closure type patch test dermoreaction grade scale see the table below 3.
Table 3
During entire experiment, the subject's of group 1- groups 13 is reacted negative by gesture area skin.Thus
As can be seen that centella plant extraction liquid provided in an embodiment of the present invention will not cause the allergy and stimulate the reaction of skin, safety
Property is good.
(2) repeatability is opening smears experiment
1st, test specimen：The centella plant extraction liquid that 1-12 of the embodiment of the present invention is provided.
2nd, test method：From a certain region select 39 volunteers, the age of volunteer between 20-45 Sui, body
Health, in the recent period non-drug administration, nonbreast-fed women or gravid woman, non-constitution sensitive subjects.Volunteer is divided into 13 at random
Group, every group of three people.Wherein, group 1 is blank control group, is bent using forearm as recipient site, 3 × 3cm of area2, recipient site
Drying is kept, avoids contact with before tested and during by gesture other external preparations, with ultra-pure water 0.05 ± 0.005g/ times, equably
Smear recipient site；Group 2- groups 13 are respectively test group, unlike group 1：The product provided using 1-12 of the embodiment of the present invention
The careless plant extraction liquid of snow equably smears recipient site 0.05 ± 0.005g/ times；Wherein, the 2 correspondence embodiment of the present invention 1 of group provides
Centella plant extraction liquid, group 3 corresponds to the centella plant extraction liquids that the embodiment of the present invention 2 provide, and it is real that group 4 corresponds to the present invention
The centella plant extraction liquid ... of the offer of example 3 is applied, group 13 corresponds to the centellas that the embodiment of the present invention 12 provide and plants
Object extracting solution.Each group carries out 2 times and equably smears recipient site daily, and long run test is completed experiment, observed daily tested after 7 days
The dermoreaction situation of the recipient site of person simultaneously records.During experiment, if there is such as 3 points or more in the following table 4
During dermoreaction situation, it can stop continuing to test and record result.
Wherein, the following table 4 tests dermoreaction judgment criteria table for opening smear of skin repeatability.
Table 4
During entire experiment, the dermoreaction by gesture position of the subject of group 1- groups 13 is negative.By
This, which can be seen that centella plant extraction liquid provided in an embodiment of the present invention, will not cause the allergy and stimulate the reaction of skin, peace
Good perfection.
Experiment five：Biocidal property is tested
1st, test specimen：Choose commercially available competing product 1 --- (supplier has centella extracting solution for Jiangxi Jin Yuan natural perfume materials
Limit company)；(supplier is competing product 2 --- centella extracting solution：Guangzhou Yu Tai Chemical Industry Science Co., Ltd)；Competing product 3 --- product
(supplier is the careless extracting solution of snow：Guangzhou Zi Hui cosmetics Co., Ltd), competing product 4 --- (supplier is centella extracting solution：Extensively
Zhou Jia fine jades bio tech ltd) and the present invention
The centella plant extraction liquid that embodiment 1-12 is provided is tested.
2nd, test method：With reference to the Ministry of Public Health《Disinfection technology standard》2008 editions.Test strain：It is staphylococcus aureus, big
Enterobacteria, Pseudomonas aeruginosa, Candida albicans.
It, will be in 1-12 of the embodiment of the present invention centella plant extraction liquid samples provided and above-mentioned 4 during experiment
Competing product are configured to a concentration of 1% sample respectively, and are positioned in the volumetric flask or conical flask of number 1-16, spare.And it prepares
Experiment bacteria suspension is averagely dispensed into the conical flask of number 1a-16a, then, each test liquid of equivalent is taken to be added to respectively
In the conical flask of 1a-16a, it will treat that test liquid is sufficiently mixed uniformly and is interacted with the test strain in bacteria suspension with experiment
After 20min, start to test and record the killing rate of the test strain in each conical flask.
3rd, result of the test：Result of the test refers to the following table 5, wherein, killing rate=(the strain quantity survived after experiment/initial
The quantity of strain) × 100%.
Table 5
As can be seen that the centella plant extract that the embodiment 1~12 in the present invention provides from the result of the test of upper table 5
Liquid for staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Candida albicans killing rate more than 90%, hence it is evident that it is excellent
In the sterilizing rate of competing product 1~4.Especially under the compound action of lysozyme and glucose oxidase, bacterial plaque can be inhibited to be formed, Neng Gouyou
Effect removal bacterium colony population.In the embodiment of the present invention, the centella plant extraction liquid containing lysozyme and oenoxydase is antibacterial
Effect is more preferable.
Experiment six：Inoxidizability is tested
1st, experimental method：Liquor potassic permanganate titration：Prepare the liquor potassic permanganate of 5mmol/L.
During the experiment, the centella plant extraction liquid and competing product that the 2mL embodiment of the present invention 1~12 provides are taken respectively
(competing product 1 --- centella extracting solution (supplier is Jiangxi Jin Yuan natural perfume materials Co., Ltd)；Competing product 2 --- centella is extracted
(supplier is liquid：Guangzhou Yu Tai Chemical Industry Science Co., Ltd)；(supplier is competing product 3 --- centella extracting solution：Guangzhou appearance is converged
Cosmetics Co., Ltd), competing product 4 --- (supplier is centella extracting solution：Guangzhou Jia Qi bio tech ltd)) in dry
In net 1-16 conical flasks, and the distilled water of 50mL is separately added into, then with the liquor potassic permanganate of 5mmol/L into rower
It is fixed, when the solution in conical flask from it is shallow it is dark brown become lavender, and colour-fast in 30s, as titration end-point, record consumption Gao Meng
The volume V (mL) of sour potassium.
2nd, experimental result：Refer to the following table 6.
Table 6
With liquor potassic permanganate redox reaction can occur for centella extracting solution, and the volume of the potassium permanganate of consumption is got over
It is more, illustrate that, containing more reducing substances in extracting solution, the inoxidizability that represent the extracting solution is stronger.As shown in Table 6, originally
The inoxidizability ability of centella plant extraction liquid is significantly stronger than commercially available four kinds of competing product in the embodiment 1~12 of invention.
Experiment seven：Anaphylaxis tests (hyaluronidase body outer suppressioning test)
1st, laboratory sample：Choosing commercially available competing product, ((supplier is natural for Jiangxi Jin Yuan for competing product 1 --- centella extracting solution
Fragrance Co., Ltd)；(supplier is competing product 2 --- centella extracting solution：Guangzhou Yu Tai Chemical Industry Science Co., Ltd)；Competing product
3 --- (supplier is centella extracting solution：Guangzhou Zi Hui cosmetics Co., Ltd), competing product 4 --- centella extracting solution (supply
Shang Wei：Guangzhou Jia Qi bio tech ltd)) and 1-12 of the embodiment of the present invention provide centella plant extraction liquid carry out
Test.
2nd, experimental method：Take 0.1mL 0.25mmol/L CaCl2Solution and 0.5mL hyaluronic acids enzyme solution are in 37 DEG C of constant temperature
Culture 20min in water-bath；Then add in sample solution 0.5mL, constant temperature incubation 20min；Add 0.5mL Sodium Hyaluronate liquid
37 DEG C of constant temperature 30min.After being subsequently placed at room temperature 5min, 0.1mL 0.4mol/L NaOH solutions and 0.5mL levulinics are added in
Ketone solution is put after heating 15min in boiling water bath, cools down 5min with ice water immediately；Ehrlich's reagent 1.0mL is eventually adding to be used in combination
3.0mL absolute ethyl alcohols are diluted, and 20min colour developings are placed, with its absorbance (A) of spectrophotometric determination.
Hyaluronic acid enzyme inhibition rate=[(A-B)-(C-D)] × 100%/(A-B)；A (uses acetic acid for contrast solution absorbance
Buffer solution replaces sample solution), B (replaces sample solution and enzyme for control blank solution absorbance with hac buffer
Liquid), C is sample solution absorbance value, and D with hac buffer by sample blank solution absorbance (replacing enzyme solution)
Table 7
| Embodiment | Hyaluronic acid enzyme inhibition rate/% | Embodiment | Hyaluronic acid enzyme inhibition rate/% |
| Embodiment 1 | 94.32 | Embodiment 8 | 97.41 |
| Embodiment 2 | 93.41 | Embodiment 9 | 96.99 |
| Embodiment 3 | 95.12 | Embodiment 10 | 96.45 |
| Embodiment 4 | 94.27 | Embodiment 11 | 95.84 |
| Embodiment 5 | 96.99 | Embodiment 12 | 96.61 |
| Embodiment 6 | 98.17 | Competing product 1 | 50.66 |
| Embodiment 7 | 96.73 | Competing product 2 | 45.42 |
| Competing product 3 | 49.78 | Competing product 4 | 46.87 |
Hyaluronidase is allergic reaction participant, has strong correlation with allergy, many Claritins have high inhibition
The effect of hyaluronidase activity.This experiment provides the embodiment of the present invention 1~12 using hyaluronidase body outer suppressioning test
Centella plant extraction liquid and commercially available four kinds of competing product centella extracting solutions carry out anaphylaxis efficacy test, by upper table 7
Experimental result it is found that 1-12 of the embodiment of the present invention centella plant extraction liquid for hyaluronic acid enzyme inhibition rate 90% with
On, there is preferable antiallergic effect, antiallergic is apparently higher than commercially available competing product 1~4.
Experiment eight：White-skinned face function experiment (inhibits tyrosinase activity experiment)
1st, experimental method：It is A that l-tyrosine, sample liquid, PBS buffer solution are sequentially added number1、A2、B1、B2Each pipe of group
Middle mixing；Rack for test tube is put into 10~15min of water-bath in 37 DEG C of thermostat water baths；Tyrosinase is sequentially added into A1And B1Group is each
Guan Zhong, it shakes up, accurate timing, reaction 15min stops；The contact plate on 96 orifice plates successively measures its absorbance with microplate reader and counts
Calculate inhibitory activity against tyrosinase.
2nd, experimental result：The following table 8 is referred to, wherein, inhibitory activity against tyrosinase=[(A1-A2)-(B1-B2)]×
100%/(A1-A2), wherein, A1There is enzyme system absorbance for blank model；A2It is blank model without enzyme system absorbance；B1For sample
Product group has enzyme system absorbance；B2It is sample sets without enzyme system absorbance.
Table 8
Tyrosinase plays a crucial role during melanin biosynthesis, it controls melanocyte forming process, activity
Degree plays a major role to the quantity that pigment is formed and deposition.Whitening class substance is exactly by inhibiting tyrosinase activity or blocking
The generation that melanin is reduced from the oxidation reaction of tyrosine generation melanin achievees the effect that skin whitening.As shown in upper table 8, this
The centella plant extraction liquid that inventive embodiments 1~12 provide significantly inhibits tyrosinase, and inhibiting rate is up to
More than 85%, and be four kinds of competing product more than twice.That is, centella plant extraction liquid provided in an embodiment of the present invention is compared to compared with city
For selling competing product, there is good white-skinned face function.
Experiment nine：Moistening effect
1st, laboratory sample：The centella plant extraction liquid and four kinds of commercially available competing product that 1-12 of the embodiment of the present invention is provided are (competing
Product 1 --- centella extracting solution (supplier is Jiangxi Jin Yuan natural perfume materials Co., Ltd)；Competing product 2 --- centella extracting solution
(supplier is：Guangzhou Yu Tai Chemical Industry Science Co., Ltd)；(supplier is competing product 3 --- centella extracting solution：Guangzhou appearance remittanceization
Cosmetic Co., Ltd), competing product 4 --- (supplier is centella extracting solution：Guangzhou Jia Qi bio tech ltd)).
2nd, experimental method：Initial parameter index initial value (moisture content, oil content, the bullet of fixing arm are measured with skinanalysis apparatus
Property, collagenous fibres etc.), smear the centella extraction fluid samples of equivalent, smear uniform, wait for fully absorbed to skin within 1 minute, survey
It tries each parameter index value after smearing at fixing arm and records.
3rd, experimental result：Refer to the following table 9.
Table 9
| Test specimen | Moisture content initial value | After smearing 1min | After smearing 30min | After smearing 1h |
| Competing product 1 | 40% | 45% | 45% | 43% |
| Competing product 2 | 41% | 43% | 43% | 44% |
| Competing product 3 | 42% | 42% | 42% | 40% |
| Competing product 4 | 39% | 43% | 43% | 42% |
| Embodiment 1 | 41% | 60% | 59% | 59% |
| Embodiment 2 | 45% | 66% | 65% | 66% |
| Embodiment 3 | 42% | 67% | 55% | 56% |
| Embodiment 4 | 40% | 59% | 58% | 57% |
| Embodiment 5 | 41% | 60% | 60% | 59% |
| Embodiment 6 | 40% | 68% | 67% | 67% |
| Embodiment 7 | 39% | 61% | 60% | 60% |
| Embodiment 8 | 40% | 60% | 59% | 58% |
| Embodiment 9 | 38% | 58% | 58% | 57% |
| Embodiment 10 | 42% | 61% | 60% | 60% |
| Embodiment 11 | 41% | 60% | 60% | 58% |
| Embodiment 12 | 40% | 59% | 59% | 58% |
By upper table 9 it is found that compared to above-mentioned four kinds of competing product, the centella plant extraction liquid of 1-12 of embodiment of the present invention offers
The moisture of skin can significantly be increased, increase the water humidity of skin, the soft and smooth water for keeping skin is tender；It smears after absorbing 1h, skin
Moisture value added be kept essentially constant, being capable of long-acting moisturizing.
Experiment ten：Skin improves experience experiment
1st, test specimen：The centella plant extraction liquid and competing product (competing product 1 --- accumulated snow that 1-12 of the embodiment of the present invention is provided
Careless extracting solution (supplier is Jiangxi Jin Yuan natural perfume materials Co., Ltd)；(supplier is competing product 2 --- centella extracting solution：Extensively
Zhou Yutai Chemical Industry Science Co., Ltd)；(supplier is competing product 3 --- centella extracting solution：The Guangzhou appearance remittance limited public affairs of cosmetics
Department), competing product 4 --- (supplier is centella extracting solution：Guangzhou Jia Qi bio tech ltd)).
2nd, alleviate the green effect experiment of skin silt：160 volunteers, the recent skin of 160 volunteers are selected from a certain region
There is green symptom of becoming silted up of colliding with, volunteer is divided into 16 groups at random, every group of 10 people.The above-mentioned laboratory sample of equivalent is taken to apply respectively
The skin smeared in volunteer becomes silted up at blueness, once in the morning and once at night, twice a day, observes the green alleviation situation of skin silt after two weeks and records.
3rd, experimental result：The skin remission effect pair for green part of before and after centella extracting solution, becoming silted up is used by volunteer
Than collecting related data using questionnaire form, effective evaluation is subject to the skin that goes recorded and becomes silted up green feedback effects data, in detail
Carefully it the results are shown in Table 10.
Wherein, go to become silted up green rate=100% × number (go become silted up green effect assessment good)/every group of total number of persons
Table 10
| Experimental example | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 | Embodiment 5 |
| Number (go become silted up green effect assessment good) | 8 | 7 | 8 | 9 | 9 |
| Go green rate (%) of becoming silted up | 80 | 70 | 80 | 90 | 90 |
| Experimental example | Embodiment 6 | Embodiment 7 | Embodiment 8 | Embodiment 9 | Embodiment 10 |
| Number (go become silted up green effect assessment good) | 10 | 9 | 8 | 10 | 9 |
| Go green rate (%) of becoming silted up | 100 | 90 | 80 | 100 | 90 |
| Experimental example | Embodiment 11 | Embodiment 12 | Competing product 1 | Competing product 2 | Competing product 3 |
| Number (go become silted up green effect assessment good) | 9 | 8 | 2 | 3 | 3 |
| Go green rate (%) of becoming silted up | 90 | 80 | 20 | 30 | 30 |
| Experimental example | Competing product 4 | / | / | / | / |
| Number (go become silted up green effect assessment good) | 4 | / | / | / | / |
| Go green rate (%) of becoming silted up | 40 | / | / | / | / |
Volunteer does not occur adverse reaction (such as facial redness, allergy etc.), and by upper table during two weeks experience
10 it is found that the centella plant extraction liquids that provide of 1-12 of the embodiment of the present invention for green effect of going to become silted up be substantially better than four kinds it is competing
Product, it is seen that this product has the anti-recovery capability of enhancing skin.
The preparation method of centella plant extraction liquid provided in an embodiment of the present invention, utilizes enzyme preparation and the association of salts substances
Same-action can crack the cell wall of centella and the compact texture of cytoplasm, make cytoplasm and active ingredient (accumulated snow comprehensively
Careless glycosides) it dissolves out, the extraction yield of asiaticosid can be effectively improved, recovery rate can reach more than 85%；Obtained centella is planted
Object extracting solution is soluble liquid, therefore can improve its compatibility in various product system well, that is, overcomes tradition
Settled powder grass extract compliant applications in product system limitation.In addition, this law by natural enzyme preparation and
The compounding of the salts substances of food-grade, builds natural protective system, and favorable anti-corrosion effect not only extends centella extracting solution
Shelf-life, and not contained in extracting solution causes human skin that anaphylactoid ingredient (the different thiophene miaow quinoline ketone of such as methyl) occurs,
It is safe；Furthermore the centella extracting solution is mildly non-stimulated to human skin, and antiallergic and oxidation resistance are strong, but also have
There are whitening, moisturizing, the effect of skin silt is green can be alleviated.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all essences in the present invention
All any modification, equivalent and improvement made within refreshing and principle etc., should all be included in the protection scope of the present invention.
Claims (10)
1. a kind of biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, which is characterized in that described
Preparation method includes the following steps：
Take 20~50 parts of centellas, removal of impurities cleans, it is broken be placed on it is spare in extraction vessel；
40~100 parts of water, 10~25 parts of enzyme preparations, 1~5 part of alcohols material, 1~5 part of salt are added in into the extraction vessel
Substance forms centella mixture, and the centella mixture is placed in 20 DEG C~45 DEG C of thermostat water bath and is impregnated
15min~30min；
The centella mixture is placed in ultrasound 1h~2h at 20 DEG C~45 DEG C, forms crude extract；
Treat the crude extract stratification, isolated supernatant；
The supernatant is made to pass through resin chromatography chromatographic column, and washing impurity-removing is carried out with water；
It is eluted by the use of propylene glycol as eluant, eluent, collects eluent, the eluent is centella plant extraction liquid.
2. as described in claim 1 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
Be, the enzyme preparation is glucose oxidase, lysozyme and cellulase, pectase, in Co-Q10 or zytase
Any one or at least two mixture.
3. as described in claim 1 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is, the alcohols material is 1,2- hexylene glycols, butanediol, propylene glycol either any one in pungent glycol or at least two
Mixture.
4. as described in claim 1 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
Be, the salts substances are sodium chloride, potassium chloride, magnesium chloride either any one in sodium carbonate or at least two it is mixed
Close object.
5. as claimed in claim 2 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is, the enzyme preparation is the mixture of cellulase, pectase, zytase, glucose oxidase and lysozyme.
6. as claimed in claim 3 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is, the alcohols material is propylene glycol.
7. as claimed in claim 4 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is, the salts substances are sodium chloride.
8. as described in claim 1 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is,
Take 25 parts of centellas, removal of impurities cleans, it is broken be placed on it is spare in extraction vessel；
100 parts of water, 2.5 parts of cellulase, 2 parts of pectase, 2.5 parts of zytase, glucose oxygen are added in into the extraction vessel
Change 2 parts of enzyme, 3.5 parts of lysozyme, 5 parts of propylene glycol, 5 parts of sodium chloride, form centella mixture, and by the centella mixture
It is placed in 25 DEG C of thermostat water bath and impregnates 30min；
The centella mixture is placed in ultrasound 1.5h at 25 DEG C, forms crude extract.
9. as described in claim 1 with biological enzyme processing and the preparation method of corrosion-resistant centella plant extraction liquid, feature
It is, described to treat the crude extract stratification, isolated supernatant, including：
It treats the crude extract stratification, is centrifugally separating to obtain supernatant, wherein, centrifugal speed 8000r/min, during centrifugation
Between be 15min.
10. a kind of biological enzyme processing and corrosion-resistant centella plant extraction liquid, which is characterized in that the centella plant extract
Liquid extracts to obtain using preparation method as claimed in any one of claims 1 to 9 wherein.
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| CN110604716A (en) * | 2019-10-15 | 2019-12-24 | 广州环亚化妆品科技有限公司 | Light protection plant composition and preparation method and application thereof |
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