CN113388045A - Extraction method of dendrobium officinale with antibacterial effect and compound killing preparation - Google Patents
Extraction method of dendrobium officinale with antibacterial effect and compound killing preparation Download PDFInfo
- Publication number
- CN113388045A CN113388045A CN202010178027.5A CN202010178027A CN113388045A CN 113388045 A CN113388045 A CN 113388045A CN 202010178027 A CN202010178027 A CN 202010178027A CN 113388045 A CN113388045 A CN 113388045A
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- CN
- China
- Prior art keywords
- extract
- dendrobium officinale
- extraction
- precipitate
- bionic
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- 241001523681 Dendrobium Species 0.000 description 1
- DCXXMTOCNZCJGO-UHFFFAOYSA-N Glycerol trioctadecanoate Natural products CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N65/00—Biocides, pest repellants or attractants, or plant growth regulators containing material from algae, lichens, bryophyta, multi-cellular fungi or plants, or extracts thereof
- A01N65/40—Liliopsida [monocotyledons]
- A01N65/42—Aloeaceae [Aloe family] or Liliaceae [Lily family], e.g. aloe, veratrum, onion, garlic or chives
-
- 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
- A61K8/72—Cosmetics or similar toiletry preparations characterised by the composition containing organic macromolecular compounds
- A61K8/73—Polysaccharides
-
- 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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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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
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9794—Liliopsida [monocotyledons]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
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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
- A61Q19/10—Washing or bathing preparations
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
Abstract
The invention discloses a preparation method for extracting a dendrobium officinale extract and a compound killing preparation by combining a semi-bionic enzyme and an ultrasonic method, wherein the extraction method of the dendrobium officinale extract comprises the following steps: washing, drying, crushing, semi-bionic enzymolysis, semi-bionic ultrasonic treatment, decoloring treatment, concentration, washing and purification. The invention uses a semi-bionic enzyme auxiliary extraction method, firstly carries out enzymolysis, and secondly carries out bionic ultrasonic extraction, thereby optimizing the extraction conditions and extracting the dendrobium officinale extract. The herba Dendrobii extract prepared by this method has good antibacterial and antiviral effects, and can be made into disinfectant and disinfectant preparations such as spray, gel, ointment, hand sanitizer, wet towel, etc. by mixing with other components.
Description
Technical Field
The invention relates to a preparation method for extracting a dendrobium officinale extract and a compound killing preparation by combining a semi-bionic enzyme and an ultrasonic method, belonging to the technical field of preparation of killing products.
Background
Dendrobium officinale (Dendrobium officinale Kimura et Migo) is the stem of Dendrobium officinale of Orchidaceae, and has various chemical components, including phenanthrene, bibenzyl, fluorenone, sesquiterpene, coumarin, extract, steroid, volatile oil, etc. besides alkaloid and polysaccharide.
The herba Dendrobii extract refers to extract compound extracted from plants of Dendrobium. Modern researches show that the dendrobium officinale polysaccharide has the effects of enhancing immunity, resisting oxidation, resisting bacteria, reducing blood sugar, reducing blood pressure, resisting tumors and the like. The polysaccharide is a natural biological macromolecule formed by polymerizing monosaccharides, and has a complex structure, a large molecular weight and a large polarity. With the intensive research on the polysaccharide, more and more polysaccharides are separated, and the polysaccharide is found to have a plurality of biological activities and potential application values. However, polysaccharides are complex in structure and various in variety, and are often combined with lipids and proteins to form polysaccharide complexes, which brings great difficulty to the extraction of plant polysaccharides. The current extraction methods commonly used for the polysaccharide comprise a solvent extraction method, an ultrafiltration method, an enzymolysis method, an ultrasonic-assisted extraction method, a supercritical fluid extraction method, a microwave-assisted extraction method, a flash extraction method, a high-pressure cooking method and the like. However, the traditional hot water extraction method has higher temperature and long time, and the extracting solution is not suitable for long-time storage; the acid-base solvent of the acid-base extraction method can damage the polysaccharide structure, and the equipment can be corroded due to too high acid-base concentration; the novel extraction method can damage the structure of the polysaccharide and reduce the activity of the polysaccharide when the microwave extraction time is too long, and the microwave radiation can damage the human body; as a new technology, the supercritical fluid extraction has less research, strong dissolving capacity to oil-soluble components and low water solubility, high equipment cost and difficult equipment cleaning; the ultrasonic extraction is to utilize the mechanical effect, the cavitation effect and the thermal effect of ultrasonic waves to destroy plant cell walls, accelerate the transmission of components in cytoplasm, improve the dissolution speed of effective components in plant cells, eliminate the interference of other solvents, but cause the degradation of soluble polysaccharide when the ultrasonic time is too long; the enzymolysis method utilizes the specificity characteristic of enzyme to carry out enzymolysis on cell walls, accelerates the dissolution of effective components in plant cells, has high extraction rate and good specificity, reduces the environmental pollution caused by the use of a large amount of solvents, but because the activity of the enzyme is influenced by various factors, the extraction condition needs to be strictly controlled to keep the activity of the enzyme in the extraction process. The single-use extraction method has the problem of low yield. For example, the CN104926956A invention discloses a method for ultrasonic-optimized extraction of dendrobium officinale polysaccharide, which comprises the following steps: firstly, drying and crushing the dendrobium officinale in vacuum; then adding water into the dendrobium officinale powder according to the solid-to-liquid ratio of 1:30, stirring and mixing, and then carrying out ultrasonic water bath heating extraction, wherein the ultrasonic power is 300W-400W, the ultrasonic frequency is 45Hz, the extraction temperature is 50-65 ℃, the heat preservation extraction time is 1h-1.5h, the average yield of the dendrobium officinale polysaccharide in the ultrasonic optimized extraction process is 15.3%, which is 10.2% higher than that in the conventional water extraction process. In the extraction comparison of the water extraction method and the enzyme explanation method, the Yangye et al adopts an orthogonal test design to put 1g of powder into a round-bottomed flask, add different amounts of distilled water, calculate time and times at a specific temperature and mainly analyze the influence of water temperature, water amount, extraction time and times during extraction on polysaccharide extraction. Filtering with absorbent cotton after each extraction, placing the filtrate into a volumetric flask, and measuring the polysaccharide content to obtain the polysaccharide extract with the optimal yield of 12.07%. In addition, single polysaccharide is reported to be used for preparing disinfectants, but few reports are reported on the preparation of disinfectant by using compound polysaccharide for enhancing broader-spectrum antibiosis.
Generally, the traditional technology has lower extraction rate of the dendrobium officinale polysaccharide, and the emerging technology has the limitations of degrading the polysaccharide, damaging the polysaccharide structure, reducing the polysaccharide activity, lowering the equipment cost and the like due to the overlong extraction time of the single extraction technology. Therefore, the extraction method for improving the extraction yield of the dendrobium officinale polysaccharide with low cost and the preparation of the sterilizing agent for enhancing the broad-spectrum antibacterial performance in various scenes have good practical value.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides an extraction method of dendrobium officinale extract and a preparation method of a compound insecticide. The dendrobium officinale extract is extracted by a semi-bionic enzyme-ultrasonic method in a combined manner, and the extraction method has high extraction efficiency and better antibacterial and antiviral effects.
Semi-bionic extraction method (SBE) of Chinese medicine is from the perspective of biopharmacology, reflects the environment of simulating oral administration and gastrointestinal tract transportation and absorption by clinical medication comprehensive action of Chinese medicine, and adopts selected pH value to successively extract so as to obtain active substance with high component content. The extraction efficiency and the efficacy of the extract are different due to different extraction conditions in the extraction process. The semi-bionic, enzymatic degradation and ultrasonic methods are combined, and the three extraction methods are combined to exert respective advantages in the extraction of the dendrobium officinale polysaccharide, so that the extraction speed is accelerated, the extraction time is shortened, the polysaccharide structure is prevented from being damaged, the polysaccharide degradation is effectively prevented, the extraction yield is improved, and the polysaccharide activity is not damaged. The traditional Chinese medicine is not a monomer component but a collection of a plurality of active components, and the curative effect of the traditional Chinese medicine is not only related to the original components, but also depends on the interacting metabolites in the extraction process. According to the characteristics that most of effective components are unknown and basic finishing characteristics of traditional Chinese medicines, the semi-bionic method is used for guiding and separating the biological activity of the extract, the process of absorption and transportation of oral medicines through gastrointestinal tracts is simulated, the dendrobium officinale is sequentially extracted through acidic and alkaline solvents with fixed pH, and meanwhile, the combined extraction method is combined with an enzyme degradation method and an ultrasonic auxiliary method, the dendrobium officinale polysaccharide can be maximally extracted at the temperature close to the human body without high temperature, and the combined extraction method can promote the dissolution of the polysaccharide, improve the extraction rate of the polysaccharide, shorten the production period and reduce the cost on the basis of not changing the efficacy of the polysaccharide. The semi-bionic method can fully play the comprehensive action characteristics of the components of the mixture, is beneficial to controlling the quality of the dendrobium officinale polysaccharide by using the monomer components, and simultaneously overcomes the defect that the effective components are easy to damage by high-temperature decoction of the traditional semi-bionic extraction method by combining enzyme catalysis and ultrasound. The dendrobium officinale polysaccharide has high molecular weight and viscosity, and can regulate the whole immunologic function to inhibit virus replication or block virus infected cells by entering cells or being adsorbed on the surfaces of the cells, directly kill the virus and prevent the virus from causing cytopathic effect. The polysaccharide prepared by the extraction method has high activity and better adhesion effect on viruses.
The dendrobium officinale extract is extracted by a semi-bionic enzyme-ultrasonic method in a combined manner, and the extraction method has high extraction efficiency and better antibacterial and antiviral effects.
In order to solve the technical problems, the invention adopts the technical scheme that:
washing Dendrobium officinale with clear water, adding complex enzyme, adjusting pH to 2-3 and 7-8, reacting, centrifuging after reaction, mixing the precipitate with 70% ethanol, adjusting pH to 2-3 and 7-8 with phosphate buffer respectively, ultrasonic extracting for 2-3 times, mixing the supernatants, decolorizing, and concentrating.
Specifically, the extraction method of the dendrobium officinale extract provided by the invention comprises the following steps:
a. washing fresh Dendrobium officinale with clear water, removing silt, drying at 45 deg.C, pulverizing the dried sample with a pulverizer, and sieving with 60-100 mesh sieve.
b. Semi-bionic enzymolysis: adding a complex enzyme aqueous solution into the dried sample according to the material-liquid ratio of 1:20-1:40 by weight, wherein the complex enzyme comprises cellulase and protease, and the weight ratio of the cellulase to the protease is 1: 3-3: 1, preferably 1:2 to 2: 1. The reaction is carried out for 4-8h at 40-55 ℃ by using phosphate buffer solution to adjust pH2-3 and 7-8.
c. Semi-bionic ultrasound: after the enzymolysis reaction is finished, centrifuging to obtain a precipitate; mixing the precipitate with 6-12 times volume of 70% ethanol, adjusting pH to 2-3 and 7-8 with phosphate buffer solution, respectively, placing in ultrasonic extractor, setting temperature at 50-80 deg.C, ultrasonic frequency at 40kHz and ultrasonic power at 80-300w, performing ultrasonic water bath heating extraction for 1-2h, centrifuging, collecting supernatant, extracting the precipitate for 2 times, and mixing the supernatants for 3 times.
d. And (3) decoloring treatment: collecting supernatant, adding 0.1% active carbon for decolorization, filtering, and collecting filtrate.
e. Concentration: concentrating the filtrate at 50 deg.C under reduced pressure, distilling to 1/4 volume, adjusting pH to 2-3 and 7-8 with buffer solution, adding 3-6 times volume of 70% ethanol under stirring, standing at 20-30 deg.C for 4 hr, centrifuging, and collecting precipitate.
f. Washing: adjusting pH to 2-3 and 7-8, respectively, and washing the precipitate with 70% ethanol to obtain crude extract of herba Dendrobii.
g. And (3) purification: adding 3-6 times volume of 70% ethanol and phosphate buffer solution to adjust pH to 2-3 and 7-8, centrifuging, and collecting precipitate for 3 times to obtain pure extract of herba Dendrobii.
The yield of the dendrobium officinale extract prepared by the following method is more than 30%.
In the method, in the complex enzyme, the weight ratio of the cellulase to the protease is 1: 3-3: when the weight ratio of the sample to the complex enzyme is 1:30-1:40 at 1 hour, the yield of the prepared dendrobium officinale extract is more than 30%.
Furthermore, the invention provides a composition, namely the dendrobium officinale extract and other antibacterial agents or auxiliary materials are prepared into a killing composition, the weight percentage content of the dendrobium officinale extract is within the range of 0.5-30%, and the weight percentage content of the other antibacterial agents is within the range of 0.5-25%.
The other antibacterial agent is one or more of anoectochilus formosanus extract, rhizoma polygonati extract, dendrobium officinale flower extract or anoectochilus formosanus extract, preferably, the weight percentage content of the anoectochilus formosanus extract and the rhizoma polygonati extract is 0.5-22%, and the weight percentage content of the dendrobium officinale flower extract and the anoectochilus formosanus extract is 0.5-3%.
Further, the invention provides a series of killing preparations, wherein the killing preparations are prepared by taking the dendrobium officinale extract prepared by the invention as an active ingredient, adding common auxiliary materials of the killing preparations and adopting a conventional method in the field.
The disinfectant is spray, gel, hand sanitizer, cream and the like.
The technical scheme of the invention also comprises a process method for preparing the compound disinfectant, which comprises the following steps: step one, mixing 0.5-30% of dendrobium officinale polysaccharide by mass ratio with 0.5-25% of other antibacterial agents by mass ratio; step two, adding the purified water with the residual mass, and uniformly stirring; adding the formula amount of essence; and step four, quantitative filling, sealing, weight checking, leakage detection, capping and packaging. The preparation method can adjust the process conditions of individual links according to the actual compatibility of the components, for example, the step two is changed into the step two in which 1 percent of the stearin, 0.1 to 2 percent of the lanolin derivative, 1 percent of the olive oil, 0.5 percent of the glycerol and 0.5 percent of the emulsifier are added according to the mass ratio, and the purified water and the emulsification are added. On the basis of the various preparation methods, 3 parts of surfactant can be added in the step two instead, and the rest purified water is added and stirred uniformly.
The dendrobium officinale extract, the composition containing the dendrobium officinale extract and the sterilizing preparation taking the dendrobium officinale extract as a main active ingredient have obvious bacteriostatic effects on escherichia coli, staphylococcus aureus, bacillus subtilis and candida albicans and have obvious killing effects on viruses.
The invention uses a semi-bionic enzyme-assisted ultrasonic extraction method, firstly carries out semi-bionic enzymolysis, and then carries out semi-bionic ultrasonic extraction, thereby optimizing the extraction conditions, preparing the dendrobium officinale extract, having no pollutant emission in the whole process, being suitable for large-scale production, having higher ultrasonic extraction yield after the semi-bionic enzymolysis than the conventional extraction method, and being beneficial to the development and utilization of dendrobium officinale resources. Simple steps, low cost, environmental protection, less loss of effective components, short production period, strong operability, high yield and easy industrial production.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention introduces a semi-bionic method, simulates the environment of oral administration and transgastrointestinal absorption, and adopts the selected pH value to sequentially and continuously extract under the conditions of pH2-3 and pH7-8 respectively to obtain the high-content dendrobium officinale active extract.
2. The invention adopts a method of firstly performing semi-bionic enzymolysis and then performing semi-bionic ultrasonic extraction, and increases the enzymolysis process of the complex enzyme, and the complex enzyme consists of cellulase and protease. Hydrolysis of the 1, 4-glycosidic bond cleaves one cellobiose molecule at a time. Promote the damage of intercellular and intracellular structures of the dendrobium officinale, and improve the extraction efficiency of the extract.
3. The invention optimizes the extraction process: the enzymolysis conditions (the proportion of the complex enzyme, the enzymolysis temperature and the enzymolysis time) of the complex enzyme are optimized, and the dosage of the ethanol is optimized.
4. The whole process of the invention has the advantages of no pollutant discharge, less loss of effective components, low cost, short production period, strong operability and high yield, is easy for industrial production and is beneficial to the development and utilization of dendrobium officinale resources.
5. The dendrobium officinale extract obtained by the invention has strong adhesion, has better bactericidal and bacteriostatic effects on common pathogenic bacteria, and has better effect than the common dendrobium officinale extract.
6. The invention is innovative in that the natural plant components are adopted, no toxic or harmful chemical components are contained, no toxic or side effect is caused, no chemical residue is caused, and the long-term use can be realized.
7. The invention has natural antibacterial activity, wide antibacterial spectrum and obvious bacteriostatic effect on escherichia coli, staphylococcus aureus, bacillus subtilis and candida albicans, and has good killing effect on bacterial viruses.
The following examples are presented to further illustrate the present invention in conjunction with experimental data.
Detailed Description
Example 1 comparative experiment on separate enzymatic extraction method
References to the preparation of extracts of dendrobium officinale. Taking the dendrobium officinale, crushing and sieving with a 40-mesh sieve: adding 4 times of complex enzyme buffer solution (pectinase-cellulase-laccase (1-2:1-3:0.5-2.5), adding glacial acetic acid solution to adjust pH to 4.0, and controlling enzymolysis temperature at 50 deg.C for enzymolysis for 140 min; adding 5 times of drinking water, reflux extracting for 2 hr, filtering (120 mesh sieve), concentrating the filtrate to relative density of 1.30(60 deg.C), adding ethanol to make ethanol content of the solution reach 75%, stirring, standing for 12 hr, collecting precipitate, volatilizing ethanol, and vacuum drying at 70 deg.C to obtain the final product. The result is shown in table-1, and compared with the extract obtained by the extraction method, the yield of the method of firstly performing semi-bionic enzymatic hydrolysis and then performing ultrasonic extraction is improved by 9.4 percent, and the extraction method has higher extraction rate.
Example 2 comparative ultrasonic extraction method alone experiment
References to the preparation of extracts of dendrobium officinale. Taking dendrobium officinale, drying in vacuum at 50 ℃ for 2.5 hours, crushing and sieving with a 80-mesh sieve, and mixing the dendrobium officinale powder according to a solid-to-liquid ratio of 1:30 adding water, stirring, mixing, placing in a stainless steel cylinder, keeping in a sealed state, adding the container containing the feed liquid into an ultrasonic extractor, performing ultrasonic water bath heating extraction with ultrasonic power of 300W, ultrasonic frequency of 45Hz, extraction temperature of 50-65 ℃, keeping the temperature for 1-1.5 h, performing suction filtration after extraction is finished, and taking out dregs to obtain a suction filtration mixed solution; centrifuging the filtrate at 6000r/min under 5000-. The result is shown in table-1, and compared with the extract obtained by the extraction method, the yield of the method of firstly performing semi-bionic enzymatic hydrolysis and then performing ultrasonic extraction is improved by 9.8 percent, and the extraction method has higher extraction rate.
Example 3 comparative ultrasound-enzyme extraction method experiment
References to the preparation of extracts of dendrobium officinale. Drying herba Dendrobii at 65 deg.C, pulverizing 500g, refluxing with 15 times of 95% ethanol for 3 times, mixing the residues, oven drying, adding distilled water at solid-liquid weight ratio of 1/25, performing ultrasonic treatment at 400W for 25min, adjusting pH to 6.5, performing water bath at 45 deg.C, adding cellulase and pectinase 2.5% of the residues, performing enzymolysis for 2.5 hr, and heating to 55 deg.C. Adjusting pH to 6.8, adding papain with a mass of 1.5% of the residue, performing enzymolysis for 2.5 hr, heating to 92 deg.C for 25min, centrifuging, filtering, decolorizing, concentrating, dialyzing, adding 95% ethanol, standing at 25 deg.C for 18 hr, filtering, and volatilizing. The result is shown in table-1, and compared with the extract obtained by the extraction method, the yield of the method of firstly performing semi-bionic enzymatic hydrolysis and then performing ultrasonic extraction is improved by 4.2 percent, and the extraction method has higher extraction rate.
TABLE-1 comparison of extraction yields of polysaccharides from Dendrobium officinale Kimura et Migo by different extraction methods
Example 4 extraction method of Dendrobium officinale with antibacterial effect and compound killing preparation
The method comprises the following steps: preparation of dendrobium officinale extract
a. Washing fresh Dendrobium officinale with clear water, removing silt, drying at 45 deg.C, pulverizing the dried sample with a pulverizer, and sieving with 60 mesh sieve.
b. Semi-bionic enzymolysis: adding a complex enzyme aqueous solution into the dried sample according to the material-liquid ratio of 1:20 by weight, wherein the complex enzyme comprises cellulase and protease, and the weight ratio of the cellulase to the protease is 1: 3. the reaction was carried out at 40 ℃ for 8h with the pH of 2-3, 7-8 adjusted using phosphate buffer.
c. Semi-bionic ultrasound: after the enzymolysis reaction is finished, centrifuging to obtain a precipitate; sequentially mixing the precipitate with 6 times volume of 70% ethanol and phosphoric acid buffer solution to adjust pH to 2-3 and 7-8, placing in an ultrasonic extractor, setting temperature at 55 deg.C, ultrasonic frequency at 40kHz and ultrasonic power at 295w, performing ultrasonic water bath heating extraction for 1h, centrifuging, collecting supernatant, repeatedly extracting the precipitate for 2 times, and mixing the supernatants for 3 times.
d. And (3) decoloring treatment: collecting supernatant, adding 0.1% active carbon for decolorization, filtering, and collecting filtrate.
e. Concentration: concentrating the filtrate at 50 deg.C under reduced pressure, distilling to 1/4 volume, adjusting pH to 2-3 and 7-8 with buffer solution, adding 3 times volume of 70% ethanol under stirring, standing at 20-30 deg.C for 4 hr, centrifuging, and collecting precipitate.
f. Washing: adjusting pH to 2-3 and 7-8, respectively, and washing the precipitate with 70% ethanol to obtain crude extract of herba Dendrobii.
g. And (3) purification: adding 3 times volume of 70% ethanol and phosphoric acid buffer solution into the crude extract, adjusting pH to 2-3 and 7-8, centrifuging, and collecting precipitate for 3 times to obtain pure extract of herba Dendrobii. The yield of the method of firstly performing semi-bionic enzymatic hydrolysis and then performing ultrasonic extraction is improved by 17.7 percent compared with the common extraction method, and the prepared dendrobium officinale polysaccharide is reserved;
step two: adding 25% of dendrobium officinale extract according to the mass ratio for later use;
step three: adding the residual mass of purified water, and uniformly stirring;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping, and packaging to obtain spray.
And (3) antibacterial testing: the insecticide is removed, the insecticide is dissolved into 5 gradients of 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL by using sterile water respectively, and filter paper sheets sterilized by dry heat at 120 ℃ are placed into the gradients for soaking for 0.5h for later use. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then adding 10mL of sterile physiological saline into the activated slant test tube, oscillating for several times, washing off the bacteria on the slant, and preparing into 106CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial rods are evenly coated, filter paper sheets soaked with polysaccharide are respectively carefully and flatly paved on the bacterial coating plate at a proper distance, the culture is carried out for 48h at the temperature of 30 ℃, the formation of an inhibition zone is observed, the diameter of a test group formed by the inhibition zone is measured, and the result is recorded after the diameter (6mm) of the filter paper sheets is subtracted. The experiment was repeated 3 times. The results were averaged. The results are shown in Table-2, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-2 bacteriostatic Activity of the disinfectant on different test bacteria
In vitro antiviral testing:
respectively growing Vero cells into single layers in 96-hole cell culture plates, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding influenza virus, parainfluenza virus, and respiratory syncytial virus solution 10 μ L/hole, 40 deg.C, and 5% CO2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-3, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-3 in vitro anti-influenza, parainfluenza, respiratory syncytial virus test results for the miticides
Example 5 extraction method of Dendrobium officinale with antibacterial effect and compound killing preparation
The method comprises the following steps: preparation of dendrobium officinale extract
a. Washing fresh Dendrobium officinale with clear water, removing silt, drying at 45 deg.C, pulverizing the dried sample with a pulverizer, and sieving with 100 mesh sieve.
b. Semi-bionic enzymolysis: adding a compound enzyme solution into the dried sample according to the material-liquid ratio of 1:40 by weight, wherein the compound enzyme comprises cellulase and protease, and the weight ratio of the cellulase to the protease is 3: 1. the reaction was carried out at 55 ℃ for 4h with pH2-3 adjusted using phosphate buffer, 7-8.
c. Semi-bionic ultrasound: after the enzymolysis reaction is finished, centrifuging to obtain a precipitate; sequentially mixing the precipitate with 12 times volume of 70% ethanol and phosphoric acid buffer solution, adjusting pH to 2-3 and 7-8, placing in an ultrasonic extractor, setting temperature at 65 deg.C, ultrasonic frequency at 40kHz and ultrasonic power at 135w, performing ultrasonic water bath heating extraction for 2h, centrifuging, collecting supernatant, repeatedly extracting the precipitate for 2 times, and mixing the supernatants for 3 times.
d. And (3) decoloring treatment: collecting supernatant, adding 0.1% active carbon for decolorization, filtering, and collecting filtrate.
e. Concentration: concentrating the filtrate at 50 deg.C under reduced pressure, distilling to 1/4 volume, adjusting pH to 2-3 and 7-8 with buffer solution, adding 6 times volume of 70% ethanol under stirring, standing at 20-30 deg.C for 4 hr, centrifuging, and collecting precipitate.
f. Washing: adjusting pH to 2-3 and 7-8, respectively, and washing the precipitate with 70% ethanol to obtain crude extract of herba Dendrobii.
g. And (3) purification: adding 6 times volume of 70% ethanol and phosphate buffer solution into the crude extract respectively, adjusting pH to 2-3 and 7-8, centrifuging, collecting precipitate for 3 times, and increasing yield by 19.6% compared with common extraction method. The prepared dendrobium officinale polysaccharide is reserved;
step two: adding 30% of dendrobium officinale extract, 5% of anoectochilus formosanus extract, 5% of rhizoma polygonati extract, 0.5% of dendrobium officinale flower extract and 0.5% of anoectochilus formosanus extract according to the mass ratio, and mixing for later use;
step three: adding 3 parts of surfactant, adding the rest purified water, and uniformly stirring;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping and packaging to obtain the hand sanitizer.
And (3) antibacterial testing: the insecticide is removed, the insecticide is dissolved into 5 gradients of 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL by using sterile water respectively, and filter paper sheets sterilized by dry heat at 120 ℃ are placed into the gradients for soaking for 0.5h for later use. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then adding 10mL of sterile physiological saline into the activated slant test tube, oscillating for several times, washing off the bacteria on the slant, and preparing into 106CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial rods are evenly coated, filter paper sheets soaked with polysaccharide are respectively carefully and flatly paved on the bacterial coating plate at a proper distance, the culture is carried out for 48h at the temperature of 30 ℃, the formation of an inhibition zone is observed, the diameter of a test group formed by the inhibition zone is measured, and the result is recorded after the diameter (6mm) of the filter paper sheets is subtracted. The experiment was repeated 3 times. The results were averaged. The results are shown in Table-4, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-4 bacteriostatic Activity of the disinfectant on different test bacteria
In vitro antiviral testing:
respectively growing Vero cells into single layers in 96-hole cell culture plates, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding influenza virus, parainfluenza virus, and respiratory tractSyncytial virus liquid 10. mu.L/well, 40 deg.C, 5% CO2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-5, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-5 in vitro anti-influenza, parainfluenza, respiratory syncytial virus test results for the miticides
Example 6 extraction method of Dendrobium officinale with antibacterial effect and compound killing preparation
The method comprises the following steps: preparation of dendrobium officinale extract
a. Washing fresh Dendrobium officinale with clear water, removing silt, drying at 45 deg.C, pulverizing the dried sample with a pulverizer, and sieving with 80 mesh sieve.
b. Semi-bionic enzymolysis: adding a complex enzyme aqueous solution into the dried sample according to the material-liquid ratio of 1:30 by weight, wherein the complex enzyme comprises cellulase and protease, and the weight ratio of the cellulase to the protease is 1: 1. the reaction was carried out at 50 ℃ for 6h with pH2-3 adjusted using phosphate buffer, 7-8.
c. Semi-bionic ultrasound: after the enzymolysis reaction is finished, centrifuging to obtain a precipitate; sequentially mixing the precipitate with 8 times volume of 70% ethanol and phosphoric acid buffer solution to adjust pH2-3 and 7-8, placing in an ultrasonic extractor, setting temperature at 65 deg.C, ultrasonic frequency at 40kHz and ultrasonic power at 190w, performing ultrasonic water bath heating extraction for 1.5h, centrifuging, collecting supernatant, repeatedly extracting the precipitate for 2 times, and mixing the supernatants for 3 times.
d. And (3) decoloring treatment: collecting supernatant, adding 0.1% active carbon for decolorization, filtering, and collecting filtrate.
e. Concentration: concentrating the filtrate at 50 deg.C under reduced pressure, distilling to 1/4 volume, adjusting pH to 2-3 and 7-8 with buffer solution, adding 5 times volume of 70% ethanol under stirring, standing at 20-30 deg.C for 4 hr, centrifuging, and collecting precipitate.
f. Washing: adjusting pH to 2-3 and 7-8, respectively, and washing the precipitate with 70% ethanol to obtain crude extract of herba Dendrobii.
g. And (3) purification: adding 5 times volume of 70% ethanol and phosphate buffer solution to adjust pH to 2-3 and 7-8, centrifuging, and collecting precipitate for 3 times to obtain pure extract of herba Dendrobii. Compared with the common extraction method, the method of firstly carrying out semi-bionic enzymatic hydrolysis and then carrying out ultrasonic extraction has the advantage that the yield is improved by 18.2 percent. The prepared dendrobium officinale polysaccharide is reserved;
step two: adding 20% of dendrobium officinale extract and 1% of other antibacterial agents according to the mass ratio, and mixing for later use;
step three: 1% of glyceryl stearate, 0.1-2% of lanolin derivative, 1% of olive oil, 0.5% of glycerol and 0.5% of emulsifier, adding purified water and emulsifying;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping, and packaging to obtain cream.
And (3) antibacterial testing: the insecticide is removed, the insecticide is dissolved into 5 gradients of 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL by using sterile water respectively, and filter paper sheets sterilized by dry heat at 120 ℃ are placed into the gradients for soaking for 0.5h for later use. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then 10mL of sterile physiological saline is added into the activated slant test tube, the tube is shaken for several times, and the slant is washed off10, to6CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial rods are evenly coated, filter paper sheets soaked with polysaccharide are respectively carefully and flatly paved on the bacterial coating plate at a proper distance, the culture is carried out for 48h at the temperature of 30 ℃, the formation of an inhibition zone is observed, the diameter of a test group formed by the inhibition zone is measured, and the result is recorded after the diameter (6mm) of the filter paper sheets is subtracted. The experiment was repeated 3 times. The results were averaged. The results are shown in Table-6, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-6 bacteriostatic Activity of the disinfectant against different test bacteria
In vitro antiviral testing:
taking Vero cells to grow into a monolayer in a 96-hole cell culture plate respectively, discarding a supernatant culture solution, washing the Vero cells with a serum-free culture medium solution for three times, adding 10 mu L/hole of influenza virus, parainfluenza virus and respiratory syncytial virus solution, and performing reaction at 40 ℃ under the condition of 5% CO 2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-7, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-7 in vitro anti-influenza, parainfluenza, respiratory syncytial virus test results for the miticides
Example 7 extraction method of Dendrobium officinale with antibacterial effect and compound killing preparation
The method comprises the following steps: preparing a proper amount of dendrobium officinale polysaccharide for later use;
step two: adding 10% of dendrobium officinale extract, 20% of anoectochilus roxburghii extract, 2.5% of rhizoma polygonati extract, 2% of dendrobium officinale flower extract and 0.5% of anoectochilus roxburghii extract according to the mass ratio, and mixing for later use;
step three: adding the residual mass of purified water, and uniformly stirring;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping, and packaging to obtain nanometer spray.
And (3) antibacterial testing: the insecticide is removed, the insecticide is dissolved into 5 gradients of 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL by using sterile water respectively, and filter paper sheets sterilized by dry heat at 120 ℃ are placed into the gradients for soaking for 0.5h for later use. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then adding 10mL of sterile physiological saline into the activated slant test tube, oscillating for several times, washing off the bacteria on the slant, and preparing into 106CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial stick is used for coating evenly, filter paper sheets soaked with polysaccharide are respectively carefully and flatly laid on the bacterial coating plate with moderate distance, the culture is carried out for 48h at 30 ℃, the formation of a bacteriostatic ring is observed, and the inhibition is carried outThe colony forming test group measured the diameter and the results were recorded after subtracting the diameter of the filter paper sheet (6 mm). The experiment was repeated 3 times. The results were averaged. The results are shown in Table-8, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-8 bacteriostatic Activity of the disinfectant on different test bacteria
In vitro antiviral testing:
respectively growing Vero cells into single layers in 96-hole cell culture plates, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding influenza virus, parainfluenza virus, and respiratory syncytial virus solution 10 μ L/hole, 40 deg.C, and 5% CO2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-9, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-9 in vitro anti-influenza, parainfluenza, respiratory syncytial virus test results for the miticides
Example 8 extraction method of Dendrobium officinale with antibacterial effect and compound killing preparation
The method comprises the following steps: preparing a proper amount of dendrobium officinale polysaccharide for later use;
step two: adding 0.5% of dendrobium officinale extract, 15% of anoectochilus formosanus extract, 5% of rhizoma polygonati extract, 3% of dendrobium officinale flower extract and 3% of anoectochilus formosanus extract according to the mass ratio, and mixing for later use;
step three: adding the residual mass of purified water, and uniformly stirring;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping, and packaging to obtain nanometer spray.
And (3) antibacterial testing: the insecticide is removed, the insecticide is dissolved into 5 gradients of 0.4, 0.6, 0.8, 1.0 and 1.2mg/mL by using sterile water respectively, and filter paper sheets sterilized by dry heat at 120 ℃ are placed into the gradients for soaking for 0.5h for later use. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then adding 10mL of sterile physiological saline into the activated slant test tube, oscillating for several times, washing off the bacteria on the slant, and preparing into 106CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial rods are evenly coated, filter paper sheets soaked with polysaccharide are respectively carefully and flatly paved on the bacterial coating plate at a proper distance, the culture is carried out for 48h at the temperature of 30 ℃, the formation of an inhibition zone is observed, the diameter of a test group formed by the inhibition zone is measured, and the result is recorded after the diameter (6mm) of the filter paper sheets is subtracted. The experiment was repeated 3 times. The results were averaged. The results are shown in Table-10, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-10 bacteriostatic Activity of the sterilizer against different test bacteria
In vitro antiviral testing:
respectively growing Vero cells into single layers in 96-hole cell culture plates, discarding supernatant culture solution, washing with serum-free culture medium solution for three times, adding influenza virus, parainfluenza virus, and respiratory syncytial virus solution 10 μ L/hole, 40 deg.C, and 5% CO2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-11, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-11 in vitro anti-influenza, parainfluenza, respiratory syncytial virus test results for the miticides
Example 9 extraction method of Dendrobium officinale with antibacterial effect and Compound preparation for killing bacteria
The method comprises the following steps: preparing a proper amount of dendrobium officinale polysaccharide for later use;
step two: adding 25% of dendrobium officinale extract and 15% of anoectochilus formosanus extract according to the mass ratio, and mixing for later use;
step three: adding 1% of glyceryl stearate, 0.1-2% of lanolin derivative, 1% of olive oil, 0.5% of glycerol and 0.5% of emulsifier, adding purified water, and emulsifying;
step four: adding a prescription amount of essence;
step five: quantitatively filling, sealing, checking weight, detecting leakage, capping, and packaging to obtain nanometer spray.
And (3) antibacterial testing: the killing agent is removed and dissolved into 0.4 and 0.6 respectively by using sterile water,5 gradients of 0.8, 1.0 and 1.2mg/mL, and a filter paper sheet sterilized by dry heat at 120 ℃ is put into the gradient and soaked for 0.5h for standby. Inoculating Escherichia coli, Staphylococcus aureus and Bacillus subtilis on beef extract peptone slant culture medium, and culturing at 30 deg.C for 24 hr for activation; the Candida albicans is inoculated on an improved Ma's agar culture medium and cultured for 2-3 days at 25 ℃ for activation. Then adding 10mL of sterile physiological saline into the activated slant test tube, oscillating for several times, washing off the bacteria on the slant, and preparing into 106CFU/mL bacterial suspension, then 100 μ L of the bacterial suspension is put on a corresponding solid agar plate, the bacterial rods are evenly coated, filter paper sheets soaked with polysaccharide are respectively carefully and flatly paved on the bacterial coating plate at a proper distance, the culture is carried out for 48h at the temperature of 30 ℃, the formation of an inhibition zone is observed, the diameter of a test group formed by the inhibition zone is measured, and the result is recorded after the diameter (6mm) of the filter paper sheets is subtracted. The experiment was repeated 3 times. The results were averaged. The results are shown in Table-12, which shows that the disinfectant has different degrees of inhibition effects on Escherichia coli, Staphylococcus aureus, Bacillus subtilis and Candida albicans, and the inhibition intensity is increased along with the increase of the concentration.
TABLE-12 bacteriostatic Activity of the miticides on different test bacteria
In vitro antiviral testing:
taking Vero cells to grow into a monolayer in a 96-hole cell culture plate respectively, discarding a supernatant culture solution, washing the Vero cells with a serum-free culture medium solution for three times, adding 10 mu L/hole of influenza virus, parainfluenza virus and respiratory syncytial virus solution, and performing reaction at 40 ℃ under the condition of 5% CO 2: adsorbing in an incubator for 2h, discarding the virus solution, and adding the disinfectant prepared in the example with the concentration of 5, 10, 20, 40 and 80mg/mL for culture. 3 duplicate wells were set, and virus control, normal cell control and blank zeroing wells were set simultaneously. Placing influenza virus, parainfluenza virus and respiratory syncytial virus group into a constant temperature box for rotary culture, adding 100 mu L MTT into each hole after culturing for 4h, adding 100 mu L dimethyl sulfoxide into each hole after 4h, standing for 10min, and measuring the OD value of each hole by using an enzyme-labeling instrument. The percent virus inhibition was calculated as (killer OD-virus OD)/(cell control OD-virus control OD) × 100. As shown in Table-13, the disinfectant prepared in this example had a significant effect of killing or inhibiting the proliferation of viruses.
TABLE-13 in vitro anti-influenza virus, parainfluenza virus, respiratory syncytial virus test results for the miticide
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention and do not limit the spirit and scope of the present invention. Various modifications and improvements of the technical solutions of the present invention may be made by those skilled in the art without departing from the design concept of the present invention, and the technical contents of the present invention are all described in the claims.
Claims (10)
1. The preparation method of the dendrobium officinale extract is characterized by comprising the following steps:
a. washing fresh Dendrobium officinale stems and leaves with clear water, drying, and pulverizing with a pulverizer;
b. semi-bionic enzymolysis: simulating the body fluid environment of the digestive tract of a human body, adjusting the pH value by using a phosphate buffer solution, and adding the dendrobium officinale dry powder into a compound enzyme solution for carrying out an enzymolysis reaction;
c. semi-bionic ultrasound: simulating the body fluid environment of the digestive tract of a human body, adjusting the pH value by using a phosphate buffer solution, and centrifuging after the enzymolysis reaction is finished to obtain a precipitate; adding ethanol into the precipitate, performing ultrasonic treatment, extracting in water bath, centrifuging, collecting supernatant, repeatedly extracting, and mixing the supernatants;
d. decoloring, concentrating and precipitating: after decoloring with active carbon, concentrating and distilling the obtained filtrate under reduced pressure, adjusting the pH, adding an ethanol solution, centrifuging and collecting precipitates; washing the precipitate with ethanol to obtain crude extract of herba Dendrobii.
2. The method of claim 1, wherein the size of the pulverized particles is between 60 and 100 mesh. Adjusting pH to 2-3, 7-8, and performing enzymolysis at 40-55 deg.C. The compound enzyme comprises cellulase and protease, and the weight ratio of the cellulase to the protease is 1: 3-3: 1. the weight ratio of the dendrobium officinale dry powder to the compound enzyme solution is 1:20-1: 40. The enzymolysis reaction is carried out for 4-8 hours.
3. The preparation method according to claim 1, adjusting pH to 2-3, 7-8, mixing with 6-12 times volume of 70% ethanol solution, extracting in water bath at 50-80 deg.C for 1-2 hr, centrifuging, collecting supernatant, repeatedly extracting the precipitate for 2 times, and combining the supernatants for 3 times; decolorizing with activated carbon, adding 3-6 times volume of 70% ethanol solution, adjusting pH to 2-3, 7-8, centrifuging, and collecting precipitate; washing the precipitate with 70% ethanol to obtain crude extract of herba Dendrobii.
4. The method of claim 1, wherein the purification step comprises: dissolving the obtained crude extract in 70% ethanol solution 3-6 times of the crude extract; centrifuging, and collecting precipitate to obtain high-purity herba Dendrobii extract.
5. The Dendrobium officinale extract prepared by the preparation method of claim 1 has certain antibacterial and antiviral effects.
6. The extract of iron sheet of claim 1, in combination with other antibacterial agents or adjuvants, is formulated into a disinfectant product. The content of the dendrobium officinale extract is in the range of 0.5-30%.
7. The other antibacterial agent according to claim 6 can be a composition of a disinfectant product of anoectochilus roxburghii extract, polygonatum extract, dendrobium officinale flower extract, anoectochilus roxburghii extract, etc.
8. A biocidal product composition according to claim 6 wherein the other antimicrobial agent is in the range of 0.5-25%.
9. The composition of claim 7, wherein the content of the extracts of Anoectochilus roxburghii and Polygonatum sibiricum is 0.5-22% of the extract of Dendrobium officinale flower and 0.5-3% of the extract of Anoectochilus roxburghii.
10. The dendrobium officinale extract and compound composition as claimed in any one of claims 5-7 can be prepared into a disinfectant product in the form of spray, gel, hand sanitizer, wet towel, paste, emulsion, etc.
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