CN108740006A - Wild jujube antibacterial sensitizing activity ingredient and its preparation method and application - Google Patents

Wild jujube antibacterial sensitizing activity ingredient and its preparation method and application Download PDF

Info

Publication number
CN108740006A
CN108740006A CN201810435099.6A CN201810435099A CN108740006A CN 108740006 A CN108740006 A CN 108740006A CN 201810435099 A CN201810435099 A CN 201810435099A CN 108740006 A CN108740006 A CN 108740006A
Authority
CN
China
Prior art keywords
antibacterial
petroleum ether
wild jujube
silica gel
sample
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN201810435099.6A
Other languages
Chinese (zh)
Other versions
CN108740006B (en
Inventor
林金水
王贵锋
成娟丽
田野
张向前
王延峰
王晶瑶
杨雯
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zhiyong Biotechnology Tangshan Co ltd
Original Assignee
Yanan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Yanan University filed Critical Yanan University
Priority to CN201810435099.6A priority Critical patent/CN108740006B/en
Publication of CN108740006A publication Critical patent/CN108740006A/en
Application granted granted Critical
Publication of CN108740006B publication Critical patent/CN108740006B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C3/00—Preservation of milk or milk preparations
    • A23C3/08—Preservation of milk or milk preparations by addition of preservatives
    • 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)
    • A61K36/72—Rhamnaceae (Buckthorn family), e.g. buckthorn, chewstick or umbrella-tree
    • A61K36/725—Ziziphus, e.g. jujube
    • 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
    • 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
    • A61K2236/35—Extraction with lipophilic solvents, e.g. Hexane or petrol ether
    • 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
    • A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
    • 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
    • A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
    • 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

Landscapes

  • Health & Medical Sciences (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Engineering & Computer Science (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biotechnology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oncology (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Communicable Diseases (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Organic Chemistry (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Epidemiology (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention discloses a kind of wild jujube antibacterial sensitizing activity ingredients and its preparation method and application, the active constituent by 49.59% 1, 3- dichlorohydrins, the 1 of 5.49%, 1- dichlormethyl ethers, 0.96% carbon trichloride, the 1 of 7.81%, 1, 2, tetra- chloro-2-propenes of 3-, 1.33% lauric acid, 1.34% tetradecylic acid, 0.87% palmitoleic acid, 7.37% palmitic acid, 9.75% dibutyl phthalate, 2.02% anti-form-1 3- octadecenoic acids, 1.88% oleamide, 3.06% beta- amyrins, 0.93% α-calamus alcohol, 6.20% lupeol and 1.42% ursol aldehyde composition.The present invention has the antibacterial activity and antibacterial sensitizing activity of wide spectrum, can be used to prepare antibacterials and antibacterial sensitizer, can be used as potential antiseptics for natural food and is applied to food industry.

Description

Wild jujube antibacterial sensitizing activity ingredient and its preparation method and application
Technical field
The present invention relates to field of plant extraction, and in particular to a kind of wild jujube antibacterial sensitizing activity ingredient and preparation method thereof and Using.
Background technology
Wild jujube, medical and edible dual purpose plant, nutritive value are high.Contain a large amount of vitamin C in fresh wild jujube, there is " vitamin C King " title.The researchs such as Duke confirm that sour jujube fruit flesh contains a variety of organic acids such as malic acid, oxalic acid, citric acid, calcium, iron, The several kinds of mineral elements such as magnesium, zinc and a large amount of soluble sugar, 17 kinds of amino acid (including 7 kinds of essential amino acids), cycli phosphate Adenosine (cAMP) and vitamin C, B1, B2, beta carotene.Can eat raw or make after fruit maturation it is dry, make it is dry after grinds, be made Wild jujube face, by northern Shensi etc., regional people like.Wild jujube is also equipped with the potential quality of deep processing, wherein with Wild Jujube Juice, Wild Jujube wine, acid The deep processed products such as jujube fruit vinegar, wild jujube fruit jam are most liked by masses.Fruit stone shell can be processed into high-grade fuel.It can also be from wild jujube Pigment, edible pectin etc. are extracted in fruit.
One of Chinese Famous medicine ancient books《Sheng Nong's herbal classic》Existing description, wild jujube " it is sour flat, trusted subordinate's fever and chills are cured mainly, Heresy knot gas is poly-, painful limbs, arthritis with fixed pain caused by dampness, long term usage five viscera settling, macrobiosis of making light of one's life by commiting suicide " there is prodigious medical value, have to wild jujube today Thousands of years medicinal histories.Wild jujube also has the effect of nourishing the liver, calming heart, tranquilizing the mind, arrest sweating, bowl spares benefit liver, hard muscles and bones to help cloudy gas, grows Mend and other effects.It is reported that fat content is abundant in spina date seed, there is tranquilizing soporific, improve learning and memory, cardiac stimulant and antitumor etc. Multiple biological activities；Spina date seed flavones has effects that calm hypnosis, anti-oxidant, tranquilizing the mind；Jujuboside is urged with calmness The multiple biological activities such as dormancy, decompression, antidepression, reducing blood lipid, protection cardiac muscle；Spina date seed polysaccharide has enhancing humoral immunity of organism The function immune with histocyte is improved, and can be very good to protect the mouse by radioactive damage, it is general to reduce its cell damage Rate；Spina date seed total alkaloid has apparent tranquilizing soporific and anticonvulsant action.Vahedi etc. also reports wild jujube with anti- Disease, face nursing and life prolonging, anti-aging, anticonvulsion, decompression and reducing blood lipid and other effects effect, often drinking Wild Jujube Juice can replenish qi to invigorate the spleen, and improve The colour of skin, is particularly suitable for old man and woman is edible.In China important wild fruit tree and resources of medicinal plant library, wild jujube is it Important component part has extremely important developing and utilizingpotentiality.
Bacterial drug resistance is increasingly enhanced, and new antibacterial strategy is studied there is an urgent need to us, finds new antibacterials.It grinds The strategy that drug-resistance bacteria medicine processed uses mainly has 3 kinds：1. screening has novel mechanism or new role target spot, new chemical The antimicrobial of structure；2. under the guidance of the researchs such as mechanism of action, resistance mechanism and structure-activity relationship, modify existing antibiotic with The chemical constitution of antimicrobial；3. the drug of existing antibacterials effect, i.e. antibacterial sensitizer can be enhanced by finding.Such drug The characteristics of be itself not have simultaneously or only there is weak antibacterial action, but can change or the phenotype of modified bacteria, carry Antibacterial activity that is high or restoring existing antibacterials.Since such drug not will produce bacterium directly selection pressure, The generation of the antibody-resistant bacterium for such drug will not be induced.Antibacterial sensitizer has reverse bacterial drug resistance, reduction combination anti- The advantages of raw element usage amount and antibody-resistant bacterium are not likely to produce, plant is the main source of such drug, it has been reported that antibacterial increase Quick dose has the chemical combination such as scutelloside, flavones, diterpene, triterpene, jamaicin, tellimagrandin I, corilagin, ECg, EGCg Object.Also there are the substances such as triterpene, flavones in wild jujube, whether they also have antibacterial sensitizing activity, up for further in the future Research.
Because polysaccharide can enhance the sensibility of bacterial antibiotic by inhibiting the formation of bacterial biof iotalm, and triterpenes Compound itself other than with antibacterial action also with very strong antibacterial sensitization, so in addition to triterpenic acid can in wild jujube Can have antibacterial sensitization outside, contained by a large amount of polysaccharide, unsaturated fatty acids compound, phenolic compound and life Object alkaloid compound all has potential antibacterial sensitization.Therefore, it using wild jujube fruit as research object, excavates and identifies its institute The antibacterial sensitizing activity ingredient contained will provide theoretical foundation and technical support for the deep processing of wild jujube, while expand wild jujube function Ingredient while being expected to lay the foundation for the exploitation of novel antibacterial sensitizer in the application in antiseptics for natural food field.
Invention content
To solve the above problems, the present invention provides a kind of wild jujube antibacterial sensitizing activity ingredient and preparation method thereof and answering With.
To achieve the above object, the technical solution that the present invention takes is：
Wild jujube antibacterial sensitizing activity ingredient, the ingredient are made of the raw material of following mass percent：49.59% 1,3- bis- Chloropropyl alcohol, 5.49% 1,1- dichlormethyl ethers, 0.96% carbon trichloride, 7.81% 1,1,2,3- tetra- chloro-2-propene, 1.33% lauric acid, 1.34% tetradecylic acid, 0.87% palmitoleic acid, 7.37% palmitic acid, 9.75% adjacent benzene two Formic acid dibutyl ester, 2.02% anti-form-1 3- octadecenoic acids, 1.88% oleamide, 3.06% beta- amyrins, 0.93% α-calamus alcohol, 6.20% lupeol and 1.42% ursol aldehyde.
The present invention also provides the preparation methods of above-mentioned wild jujube antibacterial sensitizing activity ingredient, include the following steps：
S1, ripe Wild jujube fruit is taken, selection has no mechanical damage, size color is uniform, no disease and pests harm fruit, washes away Surface contaminants are put into 45 DEG C of drying of baking oven, smash, cross 80 mesh sieve, obtain wild jujube powder, and total 10kg is put into refrigerator in valve bag and protects It deposits spare.
S2, after the wild jujube powder of gained is handled with petroleum ether degreasing and chloroform in material liquid volume ratio 1: 3 ratio mix after, It is placed in ultrasonic washing instrument, ultrasonic power 800W, 15-20 DEG C of ultrasonic temperature, ultrasonic 50min, filters, filter residue natural air drying, Filtrate is put into Rotary Evaporators, and 40 DEG C, rotating speed 120rpm of bath temperature, 4 DEG C of condensation temperature, pressure 0.09MPa waits for that solvent is waved To the greatest extent, chloroform extract is sucked out for hair；
S3, silica gel column chromatography is carried out to chloroform extract；
S31, sample treatment
Wild jujube chloroform extract 20g, methanol dissolving, mixes well, and centrifuges 5000rpm, 6min, takes supernatant；
S32, wet method dress post
170g 200-300 mesh silica gel is placed in beaker, eluent petroleum ether (polarity is minimum) submergence silica gel is added, fills Divide stirring, is added after the bubble in silica gel discharges in the chromatographic column that internal diameter is 9.5cm；One is added while settling While column outer wall is beaten with rubber pipette bulb, until cylinder is smooth；After silica gel adds, throwing away makes eluent stream drop a period of time, stands overnight；
S33, wet method loading
The sample handled well and 40g 100-200 mesh silica gel are mixed well, stirs in 65 DEG C of water-baths and is fully evaporated Methanol, the method for then using wet method dress post are packed into chromatographic column；Be added one layer of quartz sand, place into a little cotton, to prevent Cylinder is upset when eluant, eluent is added, to influence separating resulting；
S34, elution
After first-class sample, with petroleum ether, petroleum ether: chloroform 1: 1, petroleum ether: ethyl acetate 1: 1 elutes successively, collects respectively Each elution phase (filtered solution)；Petroleum ether elutes phase, is named as Fr.1；Petroleum ether: chloroform 1: 1 elutes phase, is named as Fr.2；Stone Oily ether: ethyl acetate 1: 1 elutes phase, is named as Fr.3.
Each elution phase is collected in S35, Rotary Evaporators vacuum distillation, recycles organic solvent, and 45 DEG C are dried in vacuo Fr.1, Fr.2, Fr.3, Cord blood；
S4, silica gel column chromatography separation is carried out to Fr.3；
S41, sample treatment
R.3, sample F is dissolved with ethyl acetate, if there is insoluble matter, a few drop chloroforms is added, fully dissolve spare；
S42, wet method dress post
The mode of wet method dress post is used to be packed into internal diameter in 60g 200-300 mesh silica gel in the chromatographic column of 4.7Cm, to stay overnight；
S43, wet method loading
By processed sample F r.3 with 12g 100-200 mesh silica gel mixings, stirs in 65 DEG C of water-baths and fully steam Then dry solvent is packed by the way of wet method loading in chromatographic column, one layer of quartz sand is added, places into a little cotton；
S44, elution
After first-class sample, petroleum ether and petroleum ether are used successively: ethyl acetate 10: 1 elutes, collect petroleum ether: ethyl acetate 10: 1 elution phase, is named as Fr.2a；
Each elution phase is collected in S45, Rotary Evaporators vacuum distillation, recycles organic solvent, and 45 DEG C are dried in vacuo Fr.2a, i.e., , Cord blood.
Applicant by repetition test find, above-mentioned wild jujube antibacterial sensitizing activity ingredient have wide spectrum antibacterial activity and Antibacterial sensitizing activity can be used to prepare antibacterials and antibacterial sensitizer, can be effectively inhibited when mass fraction is 0.5% The breeding of microorganism in milk, extends the fresh keeping time of milk, can be used as potential antiseptics for natural food and is applied to food work Industry.
Description of the drawings
Fig. 1 is that wild jujube active material extracts flow chart in the embodiment of the present invention.
Fig. 2 is that wild jujube chloroform extract isolates and purifies flow chart.
Fig. 3 is influences of the Asia-MIC Fr.2a to strain growth situation；
Wherein；(A) influence (B) Asia-MIC Fr.2a dialogues of-MIC Fr.2a in Asia to P. aeruginosa growth situation Color reads influences of influence (C) Asia-MIC Fr.2a of coccus growing state to Acinetobacter bauamnnii growing state.
Fig. 4 is the influence that Asia-MIC Fr.2a form bacterial biof iotalm.
Fig. 5 is influences of the Asia-MIC Fr.2a to strain growth situation；
Wherein；(A) influence (B) Asia-MIC Fr.2a of Asia-MIC Fr.2a to Escherichia coli Growth situation are to typhoid fever bar Influences of influence (C) Asia-MIC Fr.2a of bacterium growing state to shigella flexneri growing state
Fig. 6 is influences of the 1/4 MIC Fr.2a to bacterial motility
Fig. 7, which is Fr.2a, must influence milk pH value.
Fig. 8 is influences of the Fr.2a to total plate count in milk.
Specific implementation mode
In order to make objects and advantages of the present invention be more clearly understood, the present invention is carried out with reference to embodiments further It is described in detail.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to limit this hair It is bright.
Embodiment
One, the extraction of wild jujube active material
Test method
Raw material disposal
Ripe Wild jujube fruit, selection has no mechanical damage, size color is uniform, no disease and pests harm fruit, washes away surface Dirt is put into 45 DEG C of drying of baking oven, smashes, crosses 80 mesh sieve, and total 10kg is put into refrigerator in valve bag and saves backup.
Extraction process (as shown in Figure 1)
Utilize petroleum ether, chloroform, ethyl acetate, 80% methanol, hot water opposed polarity solvent and auxiliary ultrasonic extraction side Method extracts active material from wild jujube powder successively.Petroleum ether degreasing 2 times, is put into ultrasonic wave according to material liquid volume than 1: 3 every time Ultrasound 50min in cleaning device, ultrasonic power 800W, 15-20 DEG C, filtering, filter residue natural air drying, filtrate is put into Rotary Evaporators, 40 DEG C, rotating speed 120rpm of bath temperature, 4 DEG C, pressure 0.09MPa of condensation temperature wait for solvent volatilization to the greatest extent, then will be in revolving bottle Ligroin extraction is sucked out.Wait for that the volatilization completely of the petroleum ether in filter residue to the greatest extent, then carries out chloroform extraction, is then acetic acid second successively Ester, the extraction of 80% methanol, operating method are identical as petroleum ether degreasing method.Obtained each section extract is poured out respectively or It is sucked out, is then drawn into the 2.0ml centrifuge tubes weighed, centrifuge tube is put into vacuum concentration instrument, temperature 45 C, until having Solvent volatilization to the greatest extent, claim extract weight.
The organic reagent volatilization completely for waiting for the filter residue extracted by 80% methanol to the greatest extent, carries out hot-water extraction, according to feed liquid body Product ratio 1: 3 is put into ultrasound 50min in ultrasonic washing instrument, and ultrasonic power 800W 50 DEG C, takes out 4200rpm, centrifuges 3min, Supernatant filters, and absolute ethyl alcohol is added in filtrate and the volume ratio of absolute ethyl alcohol 1: 3, mixes well, is stored at room temperature 3h, will stand The solidliquid mixture arrived 4 DEG C, is centrifuged 5min, stays precipitation, abandon supernatant with 8000rpm.1000ml ddH are added in precipitation2O is multiple Molten polysaccharide, is put on magnetic stirring apparatus and stirs 10-20min, so that polysaccharide fully dissolves.Into the polysaccharide system dissolved Trichloroacetic acid is added, makes final concentration of the 2% of trichloroacetic acid, is stored at room temperature 30min, mixed liquor 8000rpm, is centrifuged by 4 DEG C 5min stays supernatant, abandons precipitation (albumen).Absolute ethyl alcohol is added to the volume ratio of supernatant and absolute ethyl alcohol 1: 3, is stored at room temperature 30min, 8000rpm, 4 DEG C centrifuge 5min, stay precipitation, abandon supernatant, precipitation washed once with absolute ethyl alcohol again, then must precipitate i.e. Through Deproteinated hot water extract, then to dry packing, weighing.
As a result with analysis
By the extraction of the wild jujube active material to opposed polarity range, petroleum ether extraction has been obtained from the sample of 10kg Object 73.28g, chloroform extract 278.16g, ethyl acetate extract 7.79g, 80% methanolic extract 1717.04g, hot water Extract 356.83g, as shown in table 1.
The recovery rate and yield of 1 zizyphus jujube extract of table
This part experiment obtains the extract of opposed polarity range by ultrasonic wave assisted extraction method, and substance has been carried out just The separation of step obtains good experiment effect, obtains ligroin extraction, chloroform extract, ethyl acetate extraction respectively Object, 80% methanolic extract, hot water extract, their yield be respectively 73.28g, 278.16g, 7.79g, 1717.04g, 356.83 g；Recovery rate is respectively 0.733%, 2.782%, 0.078%, 17.170%, 3.568%.The method is easy to operate, It is low for equipment requirements, it is efficient, it is suitble to the extraction of big quantity of material, while also the extraction for the active material of other plants provides Certain reference value.Two, zizyphus jujube extract antibacterial activity in vitro research
Whether this part experiment has bioactivity for the extract for the opposed polarity range that the upper part Experiment of verification obtains, It is tested using filter paper enzyme, pseudomonas aeruginosa, Acinetobacter bauamnnii, staphylococcus aureus, bacillus subtilis are selected in experiment 1 kind of fungi of totally 8 kinds of bacteriums and Pichia pastoris is used as confession for bacterium, Escherichia coli, Candida albicans, typhoid bacillus, shigella flexneri Try bacterial strain.
Test method
The preparation of bacterium solution：By each bacterial strain from activation is taken out in -80 DEG C of ultra low temperature freezers to solid LB media, 37 DEG C are fallen Overnight incubation is set, single bacterium colony is marked, is inoculated in 5ml LB culture solutions from picking single bacterium colony on the culture dish of each bacterial strain, 37 DEG C, 220rpm shakes overnight incubation.
The preparation of culture medium
LB liquid medium：Water dissolution 2g tryptones are distilled with 150ml, 1g yeast extracts, 2g sodium chloride, stirring makes Dissolving, with sodium hydroxide solution adjust pH to 7.0-7.2, add distilled water to 200ml, be sub-packed in teat glass, often manage It is spare to be stored in room temperature after cooling by 5ml, 121 DEG C of high pressure steam sterilization 20min.
LB solid mediums：Water dissolution 10g tryptones, 5g yeast extracts, 10g sodium chloride, stirring are distilled with 900ml It is allowed to dissolve, adjusts pH to 7.0-7.2 with sodium hydroxide solution, add distilled water to 1L, be sub-packed in the 250ml of the agar powder containing 1.5g In triangular flask, it is spare to be stored in room temperature after cooling by every bottle of 100ml, 121 DEG C of high pressure steam sterilization 20min.The culture medium is used for The culture of bacterium.
YPD solid mediums：Solution 1：10g yeast extracts accurately are weighed, 20g peptones add water to mend to 900ml, such as 20g agar powders, 121 DEG C of high pressure steam sterilization 20min are added in solid medium processed.Solution 2：20g glucose accurately is weighed, is added Water is mended to 100ml, filtration sterilization or 115 DEG C of high pressure steam sterilization 15min.Solution 1 and solution 2 are mixed, protected after cooling It is spare to be stored in room temperature.The culture medium is used for culture yeasts bacterium.
The preparation of sample：
The ligroin extraction extracted from wild jujube, chloroform extract, ethyl acetate extract, 80% methanol are carried Object, hot water extract is taken to be sequentially added into petroleum ether, chloroform, ethyl acetate, methanol, water, it is 1000mg/ to be adjusted to concentration The solution for standby of ml.
The preparation of bacterial strain flat board
Spare LB solid mediums and YPD solid mediums are heated to melting completely in micro-wave oven, dried in the air to 50 DEG C Left and right, aseptically down in 90mm plates, each plate pours into 25ml or so, after its solidification, draw 100 μ l it Before in the bacterium solution to tablet shaken, then with sterile coating rod coating uniformly to dry, then 2 sterile filters are sticked on culture medium The scraps of paper (a diameter of 6mm), it is spare that each bacterial strain prepares 24 tablets.
The measurement of antibacterial circle diameter
It is drawn on 10 μ l sample solutions to filter paper with pipettor, does blank control with corresponding sample lysate, each Sample repeats three tablets, is put into constant incubator after marking, 37 DEG C of culture 12h of bacterium, 28 DEG C of culture 48h of fungi. Observation experiment with bacterial colony counting instrument as a result, measure antibacterial circle diameter size, and film recording after the test.
As a result with analysis
The bacteriostatic test result of 2 five kinds of extracts of table
Note：Antibacterial circle diameter diameter containing filter paper (Φ=6mm)
Whether the zizyphus jujube extract of obtained opposed polarity range is subjected to bacteriostatic test with Detection and Extraction object with antibacterial Effect, as a result as shown in table 2 and Fig. 3-Fig. 5, chloroform extract is to pseudomonas aeruginosa, Acinetobacter bauamnnii, golden yellow grape Coccus, bacillus subtilis, Escherichia coli, Candida albicans, typhoid bacillus, shigella flexneri, Pichia pastoris have preferably Fungistatic effect, the inhibition zone of generation is respectively：19.272 ± 0.451mm of pseudomonas aeruginosa, Acinetobacter bauamnnii 23.044 ± 0.602 mm, 12.155 ± 0.102mm of staphylococcus aureus, 17.768 ± 0.356mm of bacillus subtilis, Escherichia coli 23.578 ± 0.467mm, 22.778 ± 0.369mm of Candida albicans, 24.954 ± 0.544mm of typhoid bacillus, Fu Shi dysentery bars 26.554 ± 0.208mm of bacterium, 21.632 ± 0.603mm of Pichia pastoris；And other extracts then press down these bacterial strains without generating Bacterium circle, it is clear that faint without fungistatic effect or fungistatic effect.
Three, wild jujube antibacterial sensitizing activity extract
Experiment above section experiment in this part is extract obtained to be used in combination in vitro with ampicillin sodium salt (Amp), is surveyed Its fixed fungistatic effect to pseudomonas aeruginosa, and being compared with antibiotic is used alone, with for lower part experiment provide according to According to.
Test method
(1) preparation of bacterium solution：It is inoculated in 5mL from picking single bacterium colony on the pseudomonas aeruginosa strains culture dish newly activated In LB culture solutions, 37 DEG C, 220rpm shaking overnight incubations.Wavelength be 620nm when test bacterium solution OD, take OD620 be 1.0 when Bacterium solution, do blank control with LB culture solutions, and by its 10 doubling dilution to 10-8, by 10-6、10-7、10-8It is coated on LB tablets On, 37 DEG C, it is inverted overnight incubation, is counted when being OD=1.0, the concentration of pseudomonas aeruginosa, then it is diluted to 1 × 106cfu/ml。
(2) each extract-treated：Each extract is after precise, ligroin extraction, chloroform extract, ethyl acetate Extract, 80% methanolic extract are diluted the method for each extract with DMSO using 2 multiple proportions, make final concentration 1000mg/ Ml, 500mg/ml, 250mg/ml, 125mg/ml, it is 1000mg/ml that hot water extract is configured to final concentration also with distilled water, The solution of 500mg/ml, 250mg/ml, 125mg/ml.
(3) preparation of antibiotic：Ampicillin sodium salt accurately is weighed, is configured to make final concentration of 4096 μ with distilled water The solution of g/ml.
(4) it is loaded：For independent drug sensitive experiment, 100 μ l of LB culture solutions are added per hole, 100 μ l are added in first hole The antibiotic of a concentration of 4096 μ g/ml, then be therefrom sucked out in 100 μ l to second holes, and so on, make drug final concentration according to Secondary 2 times of dilution, then 100 μ l a concentration of 1 × 10 are added in every hole6The pseudomonas aeruginosa bacterium solution of cfu/ml, then bacterium solution is dense eventually Degree is 5 × 105The final concentration of cfu/ml, antibiotic are followed successively by 1024 μ g/ml, 512 μ g/ml, 256 μ g/ml, 128 μ g/ml, 64 μg/ml,32μg/ml,16μg/ml,8μg/ml,4μg/ml,2μg/ml,1μg/ml.For combining drug sensitive experiment, in independent medicine The 4 μ l of extract handled well are added on the basis of quick experiment in every hole again.It is 200 μ l bacterium solutions to set up positive control, negative Control is 200 μ l LB culture solutions
(5) reading of MIC：It sets and just sets culture in 37 DEG C of incubators for 24 hours, read as a result, negative hole shows limpid, use "-" It indicates；Positive hole display is muddy, is indicated with "+".MIC be can inhibit bacterial growth, breeding lowest concentration of drug, that is, use It visually observes, no bacterial growth hole is drug minimum concentration hole.
(6) calculating of Combination index (FIC)：FIC refer to it is used at the same time in 2 kinds or a variety of drugs, respectively Minimal inhibitory concentration (MIC) when the drug combination of kind drug and the sum of the ratio of MIC when independent medication.When FIC≤0.5 Think that there is synergistic effect between drug；FIC is then thought between drug between 0.5~4.0 without dependent interaction；FIC > 4.0 Shi Ze thinks there is antagonism between drug.
As a result with analysis
Researches show that Amp to be more than 1024 μ g/ml to the MIC value of pseudomonas aeruginosa, is in height drug resistance.In order to probe into wild jujube Whether the extract of each polarity range can enhance sensibility of the pseudomonas aeruginosa to Amp, this research is carried out between the two Combination is tested, as a result as shown in table 3-8.
MIC of the 3 five kinds of extracts of table to Elastolyticenzyme of pseudomonas aeruginosa
Influence of 4 ligroin extraction of table to Amp antibacterial activities
Negative hole shows limpid, is indicated with (-), the display of positive hole is muddy, is indicated with (+).
Influence of 5 chloroform extract of table to Amp antibacterial activities
Negative hole shows limpid, is indicated with (-), the display of positive hole is muddy, is indicated with (+).
Influence of 6 ethyl acetate extract of table to Amp antibacterial activities
Negative hole shows limpid, is indicated with (-), the display of positive hole is muddy, is indicated with (+).
Influence of 7 80% methanolic extract of table to Amp antibacterial activities
Negative hole shows limpid, is indicated with (-), the display of positive hole is muddy, is indicated with (+).
Influence of 8 hot water extract of table to ampicillin antibacterial activity
Negative hole shows limpid, is indicated with (-), the display of positive hole is muddy, is indicated with (+).
By ligroin extraction known to upper table 3-8, ethyl acetate extract, 80% methanolic extract, hot water extract In Vitro Bacteriostasis the results show that ligroin extraction, ethyl acetate extract, 80% methanolic extract, hot water extract to verdigris Pseudomonad does not have direct antibacterial activity, and ligroin extraction, ethyl acetate extract, 80% methanolic extract, heat Water extract is in a concentration of 20mg/ml still without increasing pseudomonas aeruginosa to the sensibility of Amp, therefore this research thinks it Do not have antibacterial sensitization.And chloroform extract can make Amp decrease beyond 1000 times to the MIC of pseudomonas aeruginosa, FIC < 0.5 (5/15+1/1024) thereby determine that chloroform extract has antibacterial sensitizing activity.
Therefore, wild jujube ligroin extraction, ethyl acetate extract, 80% methanolic extract, hot water extract and Amp pairs Pseudomonas aeruginosa antibacterial activity is without dependent interaction；Chloroform extract is in cooperate with work to pseudomonas aeruginosa antibacterial activity with Amp With, so, chloroform extract is made further research.
In the extract that the extracted obtained opposed polarity of wild jujube is determined by experiment, chloroform extract increases with antibacterial Quick activity, although still being contained a large amount of impurities in extract using the solvent segment processing of opposed polarity range when extraction, The antibacterial sensitizing activity substance of further investigation and exploitation wild jujube, it is necessary to it be isolated and purified and its constituent is carried out Analysis.
Test method
Refined and refined substance the component analysis (as shown in Figure 2) of wild jujube antibacterial sensitizing activity substance
(1) silica gel column chromatography is carried out to chloroform extract：
1. sample treatment wild jujube chloroform extract 20g, methanol dissolving, mix well, 5000rpm, 6min are centrifuged, respectively Supernatant, precipitation is taken to do bacteriostatic test, as a result supernatant has fungistatic effect, precipitates without fungistatic effect, so supernatant is taken to purify.
2. the 200-300 mesh silica gel of 170g is placed in beaker by wet method dress post, eluent petroleum ether is added (polarity is minimum) Submerge silica gel, be sufficiently stirred, after in silica gel without bubble after by silica gel be added column in (internal diameter 95mm), add while settling Add and beat column outer wall with rubber pipette bulb on one side, until cylinder is smooth.After silica gel adds, still makes eluent stream drop a period of time, stood Night.
3. wet method loading mixes well the 100-200 mesh silica gel of the sample handled well and 40g, filled in 65 DEG C of water-baths Stirring is divided to be evaporated methanol, the method for then using wet method dress post is packed into column.One layer of quartz sand is added, places into a little cotton, To prevent upsetting cylinder when eluant, eluent is added, to influence separating resulting.
4. after eluting first-class sample, with petroleum ether, petroleum ether: chloroform 1: 1, petroleum ether: ethyl acetate 1: 1 elutes successively divide Each elution phase (filtered solution) is not collected.Petroleum ether elutes phase (being named as Fr.1), petroleum ether: chloroform 1: 1 elutes phase and (is named as Fr.2), petroleum ether: ethyl acetate 1: 1 (is named as Fr.3).
5. each elution phase is collected in Rotary Evaporators vacuum distillation, organic solvent is recycled, 45 DEG C are dried in vacuo Fr.1, Fr.2, Fr.3, Cord blood.
6. bacteriostatic test Fr.1, Fr.2, Fr.3 do bacteriostatic test respectively.
(2) silica gel column chromatography separation is carried out to Fr.3
1. r.3 sample treatment sample F is dissolved with ethyl acetate, if there is insoluble matter, a few drop chloroforms is added, fully dissolve standby With.
2. the 200-300 mesh silica gel of 60g is used the layer that the mode of wet method dress post is packed into internal diameter as 4.7Cm by wet method dress post It analyses in column, overnight.
3. the processed sample F of wet method loading r.3 with the 100-200 mesh silica gel mixings of 12g, in 65 DEG C of water-baths It is sufficiently stirred solvent evaporated, is then packed into column by the way of wet method loading.One layer of quartz sand is added, places into a little cotton Flower.
4. after eluting first-class sample, with petroleum ether, petroleum ether: ethyl acetate 10: 1 elutes successively collects each elution phase respectively (filtered solution).Petroleum ether elutes phase (being named as Fr.1a), petroleum ether: ethyl acetate 10: 1 elutes phase (being named as Fr.2a).
5. each elution phase is collected in Rotary Evaporators vacuum distillation, organic solvent is recycled, 45 DEG C are dried in vacuo Fr.1a, Fr.2a, Cord blood.
6. bacteriostatic test Fr.1a, Fr.2a do bacteriostatic test respectively.
7. Fr.2a is detected by GC-MS.
(3) the GC-MS analyses of Fr.2a：Research and development technology Spot detection is composed by the Fr.2a sections of sending
Pre-treating method：It takes a small amount of sample to be dissolved with chloroform, shakes up, cross 0.45 μm of filter membrane, upper machine.Instrument model： Agilent 7000C gas chromatography-mass spectrometries.Chromatographic column：DB-5 capillary columns, 60m × 0.25mm × 0.25 μm；Sample introduction Temperature：300 DEG C, sampling volume：1 μ l, carrier gas：Helium, flow：1.2ml/min；Column temperature program：Initial temperature is 50 DEG C, is protected 5min is held, is heated to 300 DEG C with the rate of 10 DEG C/min, constant temperature 30min；Scanning range：30-500amu；Solvent delay： 7min。
The activity rating method of each component (includes the broad-spectrum antiseptic sensitizing activity of refined substance in chloroform extract separation process Evaluation method)：
(1) preparation of bacterium solution：It is inoculated in 5ml LB culture solutions from picking single bacterium colony on each strain culturing ware newly activated In, 37 DEG C, 220rpm shaking overnight incubations.When the OD620 for testing bacterium solution is 1.0, blank control is done with LB culture solutions, by it It is diluted to 1 × 106cfu/ml.
(2) preparation of each separation component：After each group lease making precise, respectively carried using DMSO dilutions using 2 multiple proportions The method for taking object, make final concentration of 1000mg/ml, 750mg/ml, 625mg/ml, 500mg/ml, 375mg/ml, 250mg/ml, 125mg/ml, 62.5mg/ml, 31.25mg/ml, 15.625mg/ml, it is spare.
(3) preparation of antibiotic：It is accurate weigh gentamicin (Gm), kanamycins (Km), tobramycin (Tob), Amp, Tetracycline (Tc), carbenicillin sodium salt (Cb), streptomysin (St), chloramphenicol (Cm), wherein Cm absolute ethyl alcohols and it is other anti- Raw element is configured to the solution of final concentration of 4096 μ g/ml with distilled water.
(4) it is loaded：For independent drug sensitive experiment, 100 μ l of LB culture solutions are added per hole, 100 μ l are added in first hole The antibiotic of a concentration of 4096 μ g/ml, then be therefrom sucked out in 100 μ l to second holes, and so on, make drug final concentration according to Secondary 2 times of dilution, then a concentration of 1 × 106cfu/ml bacterium solutions are added in every hole, then final concentration of 5 × 105 cfu/ml of bacterium solution, resists The final concentration of raw element is followed successively by 1024 μ g/ml, 512 μ g/ml, 256 μ g/ml, 128 μ g/ml, 64 μ g/ml, 32 μ g/ml, 16 μ g/ ml、8μg/ml、4μg/ml、2μg/ml、1μg/ml、0.5μg/ml、0.25μg/ml、0.125 μg/ml、0.063μg/ml、 0.031μg/ml.For combining drug sensitive experiment, the group handled well is added in every hole again on the basis of independent drug sensitive experiment Divide 4 μ l.It is 200 μ l bacterium solutions to set up positive control, and negative control is 200 μ l LB culture solutions.
(5) reading of MIC：96 orifice plates are put in 37 DEG C of incubators and just set culture 24 hours, read what 96 orifice plates were shown As a result, negative hole shows limpid, indicated with "-"；Positive hole display is muddy, is indicated with "+".MIC is that bacterium can be inhibited to give birth to Long, breeding lowest concentration of drug, that is, detect by an unaided eye, and no bacterial growth hole is drug minimum concentration hole.
(6) calculating of FIC：FIC refers to used at the same time in 2 kinds or a variety of drugs, and the joint of various drugs is used Minimal inhibitory concentration (MIC) when medicine and the sum of the ratio of MIC when independent medication.Think have between drug when FIC≤0.5 There is synergistic effect；FIC is then thought between drug between 0.5~4.0 without dependent interaction；Then think when FIC > 4.0 drug it Between have antagonism.
As a result with analysis
Take antibacterial sensitizing activity as isolating and purifying for the wild jujube chloroform extract being oriented to
Since chloroform extract can significantly increase sensibility of the pseudomonas aeruginosa to Amp, to chloroform extract into Row silica gel column chromatogram separating purification has obtained tri- components of Fr.1, Fr.2, Fr.3, and in-vitro antibacterial sensitizing activity is carried out to them Evaluation finds that Fr.1, Fr.2 do not have antibacterial activity to pseudomonas aeruginosa, and cannot reduce the MIC of Amp do not have antibacterial and increase Quick activity；Fr.3 is 15mg/ml to the MIC of pseudomonas aeruginosa, and the Fr.3 of 5mg/ml can be such that the MIC of Amp decrease beyond 1000 times, FIC < 0.5 (1/1024+5/15) embody antibacterial sensitizing activity.
It takes Fr.3 through silica gel column chromatography post separation, respectively obtains component Fr.1a, Fr.2a, body is carried out to Fr.1a, Fr.2a Outer antibacterial sensitizing activity evaluation, the results showed that：Fr.2a components have antibacterial activity, MIC 12.5mg/ml.5mg/ml's Fr.2a can make Amp drop to 1 μ g/ml, FIC <, 0.5 (1/1024+5/ by 1024 μ g/ml to the MIC of pseudomonas aeruginosa 12.5), illustrate that Fr.2a has preferable antibacterial sensitizing activity.It can not continue column chromatography to Fr.2a because of loss of activity Separation, therefore determine that Fr.2a groups are divided into antibacterial sensitizing activity refined substance.
Fr.2a components are subjected to GC-MS analyses, are analyzed by GC-MS it is found that containing 49.59% 1,3- dichloros in sample Propyl alcohol, 5.49% 1,1- dichlormethyl ethers, 0.96% carbon trichloride, 7.81% 1,1,2,3- tetra- chloro-2-propene, 1.33% lauric acid, 1.34% tetradecylic acid, 0.87% palmitoleic acid, 7.37% palmitic acid, 9.75% adjacent benzene two Formic acid dibutyl ester, 2.02% anti-form-1 3- octadecenoic acids, 1.88% oleamide, 3.06% beta- amyrins, The ingredients such as 0.93% α-calamus alcohol, 6.20% lupeol and 1.42% ursol aldehyde.
The broad-spectrum antiseptic sensitizing activity of Fr.2a is studied
(1) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to pseudomonas aeruginosa
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of pseudomonas aeruginosa, as a result table It is bright：MICs of the Fr.2a to pseudomonas aeruginosa：The Fr.2a of 12.5mg/ml, 5mg/ml can be such that the MIC of Gm is dropped to by 4 μ g/ml The MIC of 0.063 μ g/ml, FIC=0.416 < 0.5 (5/12.5+0.063/4), Km drop to 0.5 μ g/ml by 128 μ g/ml, The MIC of FIC=0.404 < 0.5 (5/12.5+0.5/128), Tob drop to 0.063 μ g/ml, FIC=0.432 by 2 μ g/ml The MIC of < 0.5 (5/12.5+0.063/2), Amp drop to 1 μ g/ml, FIC=0.401 < 0.5 (5/12.5 by 1024 μ g/ml + 1/1024), the MIC of Tc drops to 4 μ g/ml, FIC=0.463 < 0.5 (5/12.5+4/64) by 64 μ g/ml, the MIC of Cb by 128 μ g/ml drop to 0.5 μ g/ml, FIC=0.404 < 0.5 (5/12.5+0.5/128), and the MIC of St is declined by 32 μ g/ml To 2 μ g/ml, FIC=0.463 < 0.5 (5/12.5+2/32), show Fr.2a and these antibiotic it is strong cooperate with anti-verdigris Pseudomonad acts on；And when Fr.2a and Cm combinations, nothing shows antibacterial sensitizing activity, 0.5 (5/12.5+ of FIC=0.65 > 32/128)。
(2) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to Acinetobacter bauamnnii
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of Acinetobacter bauamnnii, as a result table It is bright：MICs of the Fr.2a to Acinetobacter bauamnnii：When 5mg/ml, the Fr.2a of each concentration and 8 kinds of antibiotic are combined, the equal > of FIC 0.5, nothing shows antibacterial sensitizing activity.
(3) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to staphylococcus aureus
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of staphylococcus aureus, as a result table It is bright：MICs of the Fr.2a to staphylococcus aureus：When 5mg/ml, Fr.2a are combined with gentamicin, to staphylococcus aureus With synergetic antibacterial effect, FIC=0.313 (1.25/5+0.031/1) or 0.251 (0.63/5+0.125/1)；With Km, Tob When combination, it is 0.313 also to have synergetic antibacterial effect, FIC to staphylococcus aureus；And the Fr.2a and Amp of each concentration, When Tc, Cb, St, Cm are combined, the equal > 0.5 of FIC, nothing show antibacterial sensitizing activity.
4) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to bacillus subtilis
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of bacillus subtilis, as a result table It is bright：MICs of the Fr.2a to bacillus subtilis：When 15mg/ml, Fr.2a and Gm, Km, Tob, Tc combination, to bacillus subtilis With coordinate repression, FIC < 0.5；And when the Fr.2a of each concentration and Amp, Cb, St, Cm combination, the equal > 0.5 of FIC, Nothing shows antibacterial sensitizing activity.
5) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to Escherichia coli
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of Escherichia coli, the results showed that： MICs of the Fr.2a to Escherichia coli：When 25mg/ml, Fr.2a and Gm, Km, Tob, Amp, Tc, Cb, St, Cm combination, to large intestine Bacillus has coordinate repression, the equal < of FIC 0.5.
6) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to Candida albicans
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of Candida albicans, the results showed that： MICs of the Fr.2a to Candida albicans：When 2.5mg/ml, Fr.2a and Gm, Km, Tob, Amp, Tc, Cb, St, Cm combination, dialogue Color, which reads coccus, has coordinate repression, FIC < 0.5.
7) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to typhoid bacillus
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of typhoid bacillus, the results showed that： MICs of the Fr.2a to typhoid bacillus：When 2.5mg/ml, Fr.2a and Gm, Km, Tob, Amp, Tc, Cb, St, Cm combination, to typhoid fever Bacillus has coordinate repression, FIC < 0.5.
8) Fr.2a and 8 kinds of Antibiotic combinations use the inhibiting effect to shigella flexneri
Fr.2a and 8 kinds of Antibiotic combinations, which use, evaluates the in-vitro antibacterial sensitizing activity of shigella flexneri, as a result table It is bright：MICs of the Fr.2a to shigella flexneri：When 2.5mg/ml, Fr.2a and Gm, Km, Tob, Amp, Tc, Cb, St, Cm combination, There is coordinate repression, FIC < 0.5 to shigella flexneri.
The influence that Fr.2a forms bacterial biof iotalm
Test method
(1) biofilm formation is tested：1. the single bacterium colony of each bacterial strain of picking is inoculated in 5ml LB culture mediums, 37 DEG C respectively, 220rpm overnight incubations；2. by respective OD620It is adjusted to 0.95, the fresh LB liquid medium for being diluted in 10ml with 1: 100 In, mixing is dispensed into 1.5ml centrifuge tubes with the amount of every pipe 1ml, the sample of respective concentration is added in each centrifuge tube Solution, blank control group are added the DMSO of corresponding amount, 100 μ l are respectively taken to be added in 96 hole PVC boards, and each processing sets eight repetitions, 37 DEG C just put culture for 24 hours, and the growing state that OD620 is used to assess bacterium is surveyed per 4h.3. culture bacterium solution, 96 orifice plate PBS are sucked out It washes twice, after air drying, dyeing 10min is carried out to the biomembrane of formation with 0.1% crystal violet, then two are washed with PBS It is secondary, observe result.4. eluting the crystal violet on 96 orifice plates in every hole on biomembrane with 95% ethyl alcohol, and measure per hole elutriant OD570。
(2) preparation of sample：Sample is weighed, DMSO is added and is dissolved to a concentration of 1000mg/ml, 500mg/ml, 250mg/ The sample solution of ml, 125mg/ml, 62.5mg/ml, 31.25mg/ml, 15.625mg/ml.
(3) phosphate buffered saline solution (PBS)：137mmol/L NaCl,2.7mmol/L KCl,10mmol/L Na2HPO4 2mmol/L KH2PO4.With 800ml distillation water dissolution 8g NaCl, 0.2g KCl, 1.44g Na2HPO4,0.24g KH2PO4. The pH value for adjusting solution with HCl adds water to 1L to 7.4.121 DEG C of high pressure sterilization 20min or filtration sterilization, are stored in after packing Room temperature is spare.
(4) 0.1% crystal violet dye liquors：0.1g crystal violets accurately are weighed, add distilled water to 100ml.
As a result with analysis
The use of antibacterials often promotes the formation of bacterial biof iotalm, and bacterial biof iotalm can enhance bacterium confrontation The drug resistance of bacterium drug makes drug effect be greatly reduced and even fails, and therefore, the influence that research Fr.2a forms bacterial biof iotalm is It is very necessary, but this experiment must be under the premise of Fr.2a does not influence bacterial growth situation.From the figure 3, it may be seen that working as When Fr.2a concentration≤1/2 MIC, have substantially no effect on pseudomonas aeruginosa, Acinetobacter bauamnnii growing state；When Fr.2a is dense When degree is 1/4 MIC, it is suppressed that partial white reads the growth of coccus, as Fr.2a concentration is when being increased to 1/2 MIC, can press down The growth of Candida albicans processed, the control group of the OD620 values of different time points than non-dosing are low.
Under the concentration for not influencing the growth of bacterium in Fr.2a, select to pseudomonas aeruginosa, Acinetobacter bauamnnii 1/32 The formation that the Fr.2a concentration of MIC to 1/2 MIC carries out biomembrane influences experiment；Candida albicans are arrived in selection in 1/32 MIC The formation that the Fr.2a concentration of 1/8 MIC carries out biomembrane influences experiment.Fig. 4 shows：Fr.2a concentration is in 1/32 MIC to 1/2 MIC is in the formation for dose-dependently promoting aeruginosa biofilm, wherein with most strong (the P < of 1/2 MIC facilitations 0.05).Fr.2a concentration is in the formation for dose-dependently promoting Candida albicans biomembrane in 1/32 MIC to 1/8 MIC, Wherein, most strong (P < 0.05) with 1/8 MIC facilitations, however Fr.2a concentration 1/32 MIC to 1/2 MIC to Bao Man not The formation but not influence of conspicuousness of lever bacterium biomembrane.
Influences of the Fr.2a to bacterial motility
Test method
(1) preparation of bacterial strain single bacterium colony：Bacterial strain is taken out from -80 DEG C of refrigerators, is activated onto LB tablets, is cultivated for 37 DEG C and wait growing It after going out lawn, is crossed with oese, 37 DEG C of cultures are to growing single bacterium colony.
(2) configuration of swimming campaign culture medium：1% tryptone, 0.5%Na Cl, 0.3% agar, 121 DEG C of high pressures are steamed Vapour sterilizing 20min.Specific method：It waits for that culture medium is cooled to 40-60 DEG C, 14ml culture mediums is taken to be added in empty sterile petri dish, it is empty 1ml physiological saline is added in white group thereto, and 1ml Fr.2a solution (the final concentration of 1/4MIC of Fr.2a) is added in Fr.2a groups, gently Mixing is vibrated, 5h is stood.Single bacterium colony is chosen with toothpick to be vertically stained with to swimming campaign media surface, 37 DEG C are just set constant temperature incubation For 24 hours, swimming motion conditions are observed and measure motion range diameter, every group is repeated 3 times.
(3) processing of sample solution：Accurately weigh Fr.2a, with distilled water be configured to concentration be respectively 93.75mg/ml, The white emulsion of 56.25mg/ml, 46.875mg/ml, 18.75mg/ml, 9.375mg/ml, fully shaking keep it equal as possible It is even.Make to be added to final concentration of 6.25mg/ml, 3.75mg/ml after culture medium, 3.125mg/ml, 1.25mg/ml, 0.625mg/ml。
As a result with analysis
Bacterial motility and bacterial biof iotalm are the pathogenic factor of bacterium, also affect the resistance to of bacterium to varying degrees Pharmacological property, since Fr.2a has antibacterial activity and influences the formation of bacterial biof iotalm, we have probed into Fr.2a and have been transported to bacterium The influence of dynamic property.By Fig. 3 (A) (B) and Fig. 5 it is found that 1/4 MIC Fr.2a are to pseudomonas aeruginosa, Acinetobacter bauamnnii, big Enterobacteria, typhoid bacillus, shigella flexneri upgrowth situation do not influence, so selection 1/4 MIC Fr.2a to they into Row motility analysis.
Since Fr.2a is insoluble in water, White Flocculus will be precipitated on swimming exercise stress when the concentration is too high.It is training Support 24 h after, measure motion range diameter, Fig. 6 the result shows that：The Fr.2a of 1/4 MIC is added in the medium, keeps verdigris false single The motion range of born of the same parents bacterium drops to 0.956mm by 16.089mm；Acinetobacter bauamnnii drops to 4.212mm by 8.485mm；Greatly Enterobacteria drops to 2.223mm by 10.278mm；Typhoid bacillus drops to 7.123mm by 47.076mm；Shigella flexneri by 53.173mm dropping to 6.850mm.These differences all have statistical significance (p < 0.05).Therefore, 1/4 MIC Fr.2a drops The low motility of bacterium.
Antisepsises of the Fr.2a in milk
Test method
(1) preparation of sample solution and contrast solution：Sterile water is added to be configured to solution Fr.2a, potassium sorbate, it is seen that Fr.2a forms the emulsion suspension liquid of white.
(2) Fr.2a, potassium sorbate solution are added in the plate to sterilize respectively at four, makes Fr.2a end mass fractions point Not Wei 0,0.3% and 0.5%, potassium sorbate end mass fraction be 0.5%.20ml fresh milks are drawn to the culture dish to sterilize In, add the staphylococcus aureus suspension mixing of 1ml.Plate is placed in 28 DEG C of incubators, is taken at interval of 12h Mixing liquid in culture dish measures its total plate count, METHOD FOR CONTINUOUS DETERMINATION 2 days.
(3) total plate count assay method：According to Microbiological detection of foods national standard GB/T47892-2008 total plate counts
1. being drawn in 1ml bacterium solutions to the centrifuge tube for filling 9ml sterile salines with pipettor, fully shaking mixing, system At the even liquid of 1: 10 sample.
2. it is drawn in 1ml to the centrifuge tube for filling 9ml sterile salines from 1: the 10 even liquid of sample with pipettor again, 1: the 100 even liquid of sample is made in fully shaking mixing.
3. according to 2 operating procedures, 10 times of even liquid of gradient dilution sample are prepared.
4. according to the estimation to sample pollution level, the even liquid of sample of 2-3 acceptable diluent degree is selected, draws 1ml to nothing In bacterium plate, each dilution do two it is parallel.Meanwhile it drawing in 1ml sterile salines to two sterile blank plates and doing Blank control.
5. 15-20ml to be cooled to 46 DEG C of LB culture mediums (can be placed in 46 DEG C ± 1 of thermostat water bath and keep the temperature) in time Pour plate, shaking plate makes it be uniformly mixed.
6. after agar solidification, it is put into 37 DEG C of 48 ± 2h of constant temperature incubation carton upside down culture.
Method for counting colonies：
(4) it can detect by an unaided eye, use bacterial colony counting instrument or magnifying glass when necessary, record extension rate and corresponding bacterium colony is total Number.Bacterium colony counting is indicated with Colony Forming Unit (CFU).Clump count is chosen to grow between 30-300CFU, without sprawling bacterium colony Plate count total plate count.The clump count of each dilution should use the average of two tablets.
As a result with analysis
The experiment proves that Fr.2a has the antibacterial of wide spectrum and Synergistic antimicrobial active, and many plant-source antibacterial active materials Good antisepsis is all had in food, therefore we probe into whether Fr.2a has antisepsis in food, with fresh Milk is research object, and the Fr.2a of various concentration is added into milk to observe anti-corrosion effect.Since Fr.2a is insoluble in water, When Fr.2a is added in milk, the emulsification of milk makes Fr.2a disperse more uniform, the dissolubility in milk It significantly increases.Fresh milk added with Fr.2a measures the sum of its pH value and bacterium colony, METHOD FOR CONTINUOUS DETERMINATION 2d, with physiology every 12h Brine is negative control, using 0.5% potassium sorbate as positive control.
With the extension of time, downward trend is presented in the pH value of each group, it is similar with 0.5% potassium sorbate of positive control, 0.5% Fr.2a can effectively inhibit the decline of milk pH value, this species diversity statistically significant (p < 0.01) (Fig. 7).
With the extension of time, downward trend after first rising is presented in the total plate count of each group, and all reach when for 24 hours The total plate count of peak, negative control is 2.99 × 109Cfu/ml, wherein compare negative control, 0.5%Fr.2a with 0.5% potassium sorbate is similar, all shows significant fungistatic effect, this species diversity has statistical significance (p < 0.05) (figure 8)。
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the principle of the present invention, it can also make several improvements and retouch, these improvements and modifications It should be regarded as protection scope of the present invention.

Claims (4)

1. wild jujube antibacterial sensitizing activity ingredient, which is characterized in that by 49.59% 1,3- dichlorohydrins, 5.49% 1,1- bis- Chloromethyl ether, 0.96% carbon trichloride, the 1 of 7.81%, 1,2,3- tetra- chloro-2-propene, 1.33% lauric acid, the ten of 1.34% Tetracid, 0.87% palmitoleic acid, 7.37% palmitic acid, 9.75% dibutyl phthalate, 2.02% it is trans-- 13- octadecenoic acids, 1.88% oleamide, 3.06% beta- amyrins, 0.93% α-calamus alcohol, 6.20% Lupeol and 1.42% ursol aldehyde composition.
2. the preparation method of wild jujube antibacterial sensitizing activity ingredient, which is characterized in that include the following steps：
S1, ripe Wild jujube fruit is taken, selection has no mechanical damage, size color is uniform, no disease and pests harm fruit, washes away surface Dirt is put into 45 DEG C of drying of baking oven, smashes, crosses 80 mesh sieve, obtain wild jujube powder, spare；
S2, it after handling the wild jujube powder of gained with petroleum ether degreasing and after chloroform is in the ratio mixing of material liquid volume ratio 1: 3, is placed in In ultrasonic washing instrument, ultrasonic power 800W, 15-20 DEG C of ultrasonic temperature, ultrasonic 50min, filtering, filter residue natural air drying, filtrate Rotary Evaporators, 40 DEG C, rotating speed 120rpm of bath temperature are put into, 4 DEG C, pressure 0.09MPa of condensation temperature waits for solvent volatilization to the greatest extent, Chloroform extract is sucked out；
S3, silica gel column chromatography is carried out to chloroform extract；
S31, sample treatment
Wild jujube chloroform extract 20g, methanol dissolving, mixes well, and centrifuges 5000rpm, 6min, takes supernatant；
S32, wet method dress post
170g 200-300 mesh silica gel is placed in beaker, eluent petroleum ether is added and submerges silica gel, is sufficiently stirred, waits in silica gel Bubble discharge after add internal diameter be 9.5Cm chromatographic column in；On one side settling column is beaten with rubber pipette bulb while adding Outer wall, until cylinder is smooth；After silica gel adds, throwing away makes eluent stream drop a period of time, stands overnight；
S33, wet method loading
The sample handled well and 40g 100-200 mesh silica gel are mixed well, is stirred in 65 DEG C of water-baths and is fully evaporated methanol, Then the method for using wet method dress post is packed into chromatographic column；One layer of quartz sand is added, places into a little cotton；
S34, elution
After first-class sample, with petroleum ether, petroleum ether: chloroform 1: 1, petroleum ether: ethyl acetate 1: 1 elutes successively are collected and respectively washed respectively Out-of-phase；Petroleum ether elutes phase, is named as Fr.1；Petroleum ether: chloroform 1: 1 elutes phase, is named as Fr.2；Petroleum ether: ethyl acetate 1 : 1 elution phase is named as Fr.3；
Each elution phase is collected in S35, Rotary Evaporators vacuum distillation, recycles organic solvent, and 45 DEG C are dried in vacuo Fr.1, Fr.2, Fr.3, Cord blood；
S4, silica gel column chromatography separation is carried out to Fr.3；
S41, sample treatment
R.3, sample F is dissolved with ethyl acetate, if there is insoluble matter, a few drop chloroforms is added, fully dissolve spare；
S42, wet method dress post
The mode of wet method dress post is used to be packed into internal diameter in 60g 200-300 mesh silica gel in the chromatographic column of 4.7Cm, to stay overnight；
S43, wet method loading
By processed sample F r.3 with 12g 100-200 mesh silica gel mixings, stirred in 65 DEG C of water-baths be fully evaporated it is molten Then agent is packed by the way of wet method loading in chromatographic column, one layer of quartz sand is added, places into a little cotton；
S44, elution
After first-class sample, petroleum ether and petroleum ether are used successively: ethyl acetate 10: 1 elutes, collect petroleum ether: ethyl acetate 10: 1 is washed Out-of-phase is named as Fr.2a；
Each elution phase is collected in S45, Rotary Evaporators vacuum distillation, recycles organic solvent, 45 DEG C of vacuum drying Fr.2a are to get low Temperature preserves.
3. the application of wild jujube antibacterial sensitizing activity ingredient as described in claim 1, which is characterized in that can be used for preparing antimicrobial Object, antibacterial sensitizer and antiseptics for natural food.
4. application as claimed in claim 3, which is characterized in that antibacterial activity and antibacterial sensitizing activity with wide spectrum, in matter Amount score can effectively inhibit the breeding of microorganism in milk when being 0.5%, extend the fresh keeping time of milk.
CN201810435099.6A 2018-04-28 2018-04-28 Wild jujube antibacterial sensitization active component and preparation method and application thereof Active CN108740006B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810435099.6A CN108740006B (en) 2018-04-28 2018-04-28 Wild jujube antibacterial sensitization active component and preparation method and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810435099.6A CN108740006B (en) 2018-04-28 2018-04-28 Wild jujube antibacterial sensitization active component and preparation method and application thereof

Publications (2)

Publication Number Publication Date
CN108740006A true CN108740006A (en) 2018-11-06
CN108740006B CN108740006B (en) 2022-03-25

Family

ID=64009797

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810435099.6A Active CN108740006B (en) 2018-04-28 2018-04-28 Wild jujube antibacterial sensitization active component and preparation method and application thereof

Country Status (1)

Country Link
CN (1) CN108740006B (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113499334A (en) * 2021-06-24 2021-10-15 延安大学 Wild jujube fruit antibacterial sensitization active refined substance and preparation method and application thereof

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ASMA HAMOOD AL-SAEEDI等: "Brine shrimp toxicity of various polarities leaves and fruits crude fractions of", 《SUSTAINABLE CHEMISTRY AND PHARMACY》 *
余明阳等: "《中国品牌报告2013》", 31 October 2012, 上海交通大学出版社 *
廖铁星: "《化学试剂危险物品安全储存养护手册》", 31 July 1991, 广西师范大学出版社 *
谢明勇: "《食品安全与卫生实用手册》", 30 June 2006, 江西科学技术出版社 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113499334A (en) * 2021-06-24 2021-10-15 延安大学 Wild jujube fruit antibacterial sensitization active refined substance and preparation method and application thereof

Also Published As

Publication number Publication date
CN108740006B (en) 2022-03-25

Similar Documents

Publication Publication Date Title
Bhattacharjee et al. Antibacterial activities of some plant extracts used in Indian traditional folk medicine
Azad et al. Phytochemical and microbiological evaluation of a local medicinal plant Bacopa monnieri (l.) Penn
Srivastav et al. Phytochemical extraction and characterization of roots of Withania Somnifera for its Anti-Bacterial, Anti-Oxidant, Anti-Inflammation and Analgesic activity
Alwhibi et al. Evaluating the antibacterial activity of fenugreek (Trigonella foenum-graecum) seed extract against a selection of different pathogenic bacteria
Hoekou et al. Therapeutic potentials of ethanolic extract of leaves of Holarrhena floribunda (G. Don) Dur. and schinz (apocynaceae)
CN109674778B (en) Lipid antibacterial composition, extraction method, detection method and application
CN108740006A (en) Wild jujube antibacterial sensitizing activity ingredient and its preparation method and application
Ekawati et al. Lime (Citrus aurantifolia) peel as natural antibacteria for wound skin infection caused by staphylococcus aureus
Ajaib et al. Firmiana Simplex: A Potential Source of Antimicrobials.
Khan et al. Phytochemical screening and antimicrobial activity of the Cichorium intybus (Familyasteraceae) and Medicago sativa (Familyfabaceae) Peshawar
Nurtjahyani et al. Antibacterial activity of garlic (Allium sativum) againts Gram-positive bacteria isolated from tiger shrimp (Penaeus monodon)
CN105145634B (en) It is a kind of to contain bactericide for killing excellent Lip river rhzomorph streptomycete fermentation product and its preparation method and application
CN102028190A (en) Method for producing refined pure hawthorn extract tablets
Mahmud et al. Antibacterial activity of alkaloid extracts from Ochrosia oppositifolia
Ulahannan et al. Antibacterial action of leaves of Aegle marmelos
br Karo et al. Antibacterial activity of flavonoid-rich fractions of Citrus maxima peel extract
Chaity et al. Screening of in vitro antibacterial activity of Rumex vesicarius (L.) leaves extract against twelve pathogenic bacterial
Rizki Identification of active compound and Antibacterial activity against gram-positive and gram-negative bacteria of Chromolaena odorata leaf extract
Airaodion et al. Investigation of antibacterial activity of Vernonia amygdalina leaf extracts against gram-positive and gram-negative bacteria
Aryanti et al. The effect of fraction and active compounds of Momordica balsamina L. on bacteria Salmonella typhi causing salmonellosis
CN105796579A (en) Anti-senile brain dementia drug
CN110251499A (en) Dandelion extract and its purposes in preparation prevention and treatment woman vagina disease medicament
Adetunde et al. Antimicrobial activities of the extract of shea tree (Vitellaria paradoxa Gaertn. F.) leaf and bark on some selected clinical pathogens
Coli Antimicrobial activity of Xylopia aethiopica (UDA) on Escherichia coli and Staphylococcus aureus isolates from gastroenteritic patients
Yusuf-Babatunde et al. Evaluation of Antimicrobial, Antioxidant and Gas Chromatography-Mass Spectrometry Profile of the Stem Bark Extract of bombax buonopozense p. Beauv.(Bombacaceae)

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant
TR01 Transfer of patent right

Effective date of registration: 20230803

Address after: No. 3 Chuangye Street, Industrial Cluster District, Leting County, Tangshan City, Hebei Province, 063000

Patentee after: Zhiyong Biotechnology (Tangshan) Co.,Ltd.

Address before: 716000 Yangjialing, Baota District, Yan'an City, Shaanxi Province

Patentee before: YAN'AN University

TR01 Transfer of patent right