CN105820201B - The method and application thereof of pinocembrin -7-O- β-D-Glucose glycosides is extracted from radix fici simplicissimae fruit - Google Patents
The method and application thereof of pinocembrin -7-O- β-D-Glucose glycosides is extracted from radix fici simplicissimae fruit Download PDFInfo
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- CN105820201B CN105820201B CN201610322974.0A CN201610322974A CN105820201B CN 105820201 B CN105820201 B CN 105820201B CN 201610322974 A CN201610322974 A CN 201610322974A CN 105820201 B CN105820201 B CN 105820201B
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- pinocembrin
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- fici simplicissimae
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H17/00—Compounds containing heterocyclic radicals directly attached to hetero atoms of saccharide radicals
- C07H17/04—Heterocyclic radicals containing only oxygen as ring hetero atoms
- C07H17/06—Benzopyran radicals
- C07H17/065—Benzo[b]pyrans
- C07H17/07—Benzo[b]pyran-4-ones
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B7/00—Preservation or chemical ripening of fruit or vegetables
- A23B7/14—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10
- A23B7/153—Preserving or ripening with chemicals not covered by groups A23B7/08 or A23B7/10 in the form of liquids or solids
- A23B7/154—Organic compounds; Microorganisms; Enzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
- C07H1/06—Separation; Purification
- C07H1/08—Separation; Purification from natural products
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
A kind of method and application thereof of 7 O β D glucosides of flavone compound-pinocembrin of extracting and developing that the invention discloses one kind from radix fici simplicissimae fruit, belongs to botanical pesticide technology field.Pass through macroporous resin column chromatography particular by by radix fici simplicissimae fruit crude extract, the isolated compound of method of 20 gel filtration chromatographies of Sephadex LH and recrystallization, the compound has preferable bacteriostatic activity to citrus postharvest pathogen-Italy's mould, there is preferable disease resistance to Penicillium italicum, can be used for developing into a kind of novel plant source citrus antisepsis antistaling agent.
Description
Technical field
The invention belongs to botanical pesticide technology fields, and in particular to one kind from radix fici simplicissimae fruit extracting and developing one
The method and its purposes in citrus post-harvest fresh-keeping of kind flavone compound-pinocembrin -7-O- β-D-Glucose glycosides.
Background technology
Radix fici simplicissimae fruit (Ficus simplicissima Lour.) is the fruit of Moraceae fig cleft palm banyan,
It records in over the years《Chinese Pharmacopoeia》In plurality of Chinese ancient book.Radix fici simplicissimae is the genuine traditional Chinese medicine material of In Guangdong Province, is had
Replenish qi to invigorate the spleen effect well, there is the good reputation of " panacis quinquefolii radix ".Radix fici simplicissimae is mild-natured, sweet in flavor, pungent, has strengthening the spleen and tonifying the lung, dampness removing to relax
The work(of muscle can be used for the diseases such as spleen deficiency edema, deficiency of food inability, pulmonary tuberculosis cough, night sweat, arthralgia pain due to rheumatism, postpartum agalorrhea, civil in Guangdong
Usage history is long.Modern pharmacology research shows that radix fici simplicissimae can protect stomach lining, mitigate hepatic injury, improve immunity of organism
Power, the damage for mitigating cerebral hypoxia ischemia.Animal acute toxicity experiment is research shows that 14d after mouse gavaging radix fici simplicissimae water extract
Interior, for animal without death, each internal organs are without exception.Show that radix fici simplicissimae toxicity is extremely low, biological safety is good.About radix fici simplicissimae
Antibacterial activity research, rarely seen radix fici simplicissimae water boiling and extraction liquid is to Escherichia coli, Bacillus subtillis, staphylococcus aureus
There is inhibiting effect.
Penicilliosis and Penicillium digitatum (Penicillium caused by Italian mould (Penicillium italicum)
Digitatum green mould caused by), is the most important two kinds of diseases for causing citrus postharvest decay to occur, and rotting rate is generally
10%~30%, up to 40%~50% or more when serious, cause huge economic loss.For a long time, main useization
It is fresh-keeping to citrus progress as antistaling agent to learn synthesis medicament, these chemical bactericides can kill or inhibit the life of pathogenic microorganism
It is long, there is preferable corrosion-resistanting fresh-keeping effect, but can equally cause medicament residual, pollution environment, the drawbacks such as drug resistance.In addition, some
Chemical preservative have potential carcinogenic, mutagenesis, teratogenesis possibility.
In recent years, people start to turn to the citrus plants source antistaling agent that safety and environmental protection is found from natural plants.It is natural to plant
Material resource antistaling agent have many advantages, such as efficiently, it is low toxicity, low-residual, pollution-free, safe to use, upper easily get consumer reception applying.
In addition, medicine based on herbs is not likely to produce drug resistance and will not cause problem of environmental pollution.Therefore, it is necessary to be sought from natural plants
Look for, develop natural plant source antiseptic agent citrus is carried out it is fresh-keeping to reduce dependence to chemical agent.
Pinocembrin -7-O- β-D-Glucose glycosides is a kind of flavone compound, is separated to this compound from plant at present
Report it is less, be primarily present in Acacia pennata, pull root vegetables, Viscμm articulatum Burm f, Spondias axillaris, Hawk tea, rose, green peel
In the plants such as wood, semen litchi, glycyrrhiza glabra and blue or green money willow.There is not yet pinocembrin -7-O- β-D-Glucose glycosides in radix fici simplicissimae fruit
Report, the present invention is isolated pinocembrin -7-O- β-D-Glucose glycosides from radix fici simplicissimae fruit for the first time.
Invention content
A kind of the object of the present invention is to provide one kind from radix fici simplicissimae fruit isolated flavone compound-pinocembrin-
The method of 7-O- β-D-Glucose glycosides.
It is a further object to provide pinocembrin -7-O- β-D-Glucose glycosides answering in citrus post-harvest fresh-keeping agent
With.
The structure of chromocor compound provided by the invention is as follows：
The above-mentioned purpose of the present invention is achieved by following technical solution：
(1) radix fici simplicissimae fruit 40-50 DEG C of drying 2h in air dry oven, pulverizer smash it through 40 mesh sieve, take under sieve
Medicinal powder, the ethanol solution for being 90% using volume fraction is Extraction solvent, according to solid-liquid ratio (kg: L)=1：(10-40) ultrasound
The temperature of wave assisted extraction 30-90min, ultrasonic wave assisted extraction are 50-80 DEG C, repeat extraction 3 times, merge extracting solution and mistake
Filter, recycles ethyl alcohol, and filtrate is concentrated into no alcohol taste to get radix fici simplicissimae berry extract medicinal extract；
(2) the radix fici simplicissimae berry extract medicinal extract in step (1) is suspended in the distilled water of 10-20 times of medicinal extract weight,
Through D101 macroporous resin column chromatographies, efflux first is eluted to without color, again respectively successively with 5 times of 30% second of column volume with pure water
Alcohol, 5 times of 50% ethyl alcohol of column volume and 5 times of 95% ethanol gradient elutions of column volume collect 95% ethanol eluate, are concentrated into no alcohol
Taste is to get 95% ethanol elution concentrate；
(3) 95% ethanol elution concentrate in step (2) is dissolved in methanol, 95% ethanol elution concentrate and methanol
Mass volume ratio (g：ML) it is (1.0-5.0)：10, which is centrifuged into 3-10min in 5000-8000r/min on centrifuge,
It takes supernatant Sephadex LH-20 gel filtration chromatographies to detach, is eluted with methanol, collect 8-10 column volume eluent, thin layer
Chromatography merge target compound flow point, be concentrated under reduced pressure, into obtained concentrate be added methanol and be heated to concentrate can just
It all until dissolving, is put into 4 DEG C of refrigerators, it is target compound that light yellow precipitate, which is precipitated,.
The macroreticular resin that wherein step (2) uses can also in addition to D101 types other low poles and low polar macroporous tree
Fat.
Target compound pinocembrin -7-O- β-D-Glucose glycosides produced by the present invention can add customary adjuvant and prevention mandarin orange is made
The pharmaceutical composition of tangerine postharvest pathogen is adopted rear anti-corrosive fresh-keeping for citrus, is prevented by Italian mould (Penicillium
Italicum green mould caused by penicilliosis caused by) and Penicillium digitatum (Penicillium digitatum).
King waits quietly extracting Extractive by system solvent, uses petroleum ether, dichloromethane, ethyl acetate, water successively
It is saturated butanol solvent partition, then by macroreticular resin post separation extracting n-butyl alcohol position, with ethanol-water system gradient elution,
It is divided into 10%, 30%, 50%, 95%4 part, 95% part is detached repeatedly with silicagel column, and combination ODS,
Sephadex LH-20 and preparative HPLC isolate and purify out pinocembrin -7-O- β-D-Glucose glycosides (Wang Jing, Lu Weili, Zhang Yi
Dragon, Tang Minfang, Tang Wenjian, chemical constitution study [J] the Medical University Of Anhui journal of Li Jun Hawk teas, 2014,49 (4):
479-483.).It pays bright wait and Herba Lysimachiae Clethroids extract is extracted by system solvent, use petroleum ether, ethyl acetate, n-butanol extraction successively
It takes, Ethyl acetate extract is repeatedly through silica gel column chromatography, Sephadex LH-20 column chromatographys and thin-layer chromatography etc., finally from acetic acid second
Isolated pinocembrin -7-O- β-D-Glucose the glycosides of ester moiety (pays bright, Wei Lin, Yu Juan, pulls the chemical composition of root vegetables towards newly-risen sun recklessly
Study [J] Chinese Pharmaceutical Journals, 2013,48 (22):1911-1914).Liu Hongyan extracts rose extraction by system solvent
Object, successively use petroleum ether, chloroform, ethyl acetate, extracting n-butyl alcohol, ethyl acetate extract repeatedly through silica gel column chromatography,
Sephadex LH-20 column chromatographys are finally partially separated to obtain pinocembrin -7-O- β-D-Glucose glycosides (Liu Hong from ethyl acetate
Chemical constitution study [J] research and development of natural products of swallow roses, 2013,25 (1):47-49.).The above method all makes
With a large amount of organic solvent, silica gel column chromatography, even preparative high-performance liquid chromatographic technology, what is needed is of high cost, is unfavorable for
The preparation of large sample amount.
The advantage of the invention is that organic solvent usage amount is few, operating procedure is simple, is suitble to large-scale production and preparation Himalayan pine
Element -7-O- β-D-Glucose glycosides.
Description of the drawings
Fig. 1 is the structural formula of pinocembrin -7-O- β-D-Glucose glycosides made from embodiment 1；
Fig. 2 is the nuclear magnetic resonance spectroscopy of pinocembrin -7-O- β-D-Glucose glycosides made from embodiment 1；
Fig. 3 is the carbon-13 nmr spectra of pinocembrin -7-O- β-D-Glucose glycosides made from embodiment 1；
Fig. 4 is inhibition figure of the pinocembrin -7-O- β-D-Glucose glycosides to penicillium italicum；
Fig. 5 is influence diagram of the pinocembrin -7-O- β-D-Glucose glycosides to penicillium italicum silk microscopic morphology；
Fig. 6 is influence diagram of the pinocembrin -7-O- β-D-Glucose glycosides to penicillium italicum silk increment；
Fig. 7 is influence diagram of the pinocembrin -7-O- β-D-Glucose glycosides to penicillium italicum silk relative conductivity；
Fig. 8 is influence diagram of the pinocembrin -7-O- β-D-Glucose glycosides to penicillium italicum chitinase activity；
Fig. 9 is pinocembrin -7-O- β-D-Glucose glycosides on the active influence of penicillium italicum beta-1,3-glucanase.
Specific implementation mode
Embodiment 1：The preparation method of compound
Radix fici simplicissimae fruit 45 DEG C of drying 2h in air dry oven, pulverizer smash it through 40 mesh sieve, take the five fingers under sieve
Wild peach fruit powder end 5.0Kg, the alcohol solution dipping for being 90% with 50L volume fractions, ultrasonic wave assisted extraction 60min, extraction temperature
Degree is 60 DEG C, in triplicate until extracting solution color is thin out, merges extracting solution and filters, filtrate is concentrated under the conditions of 45 DEG C
Without alcohol taste, medicinal extract 350g there are.Medicinal extract is suspended in 4L distilled water, through D101 macroporous resin column chromatographies, is first eluted with pure water
To efflux without color, again respectively successively with 5 times of 30% ethyl alcohol of column volume, 5 times of 50% ethyl alcohol of column volume and 5 times of column volumes 95%
Ethanol gradient elution, water elution component are abandoned, and the concentrate drying of other elution fractions gets dry extract, spare.95% ethanol elution object passes through
It is dried to obtain 7.5g powder.95% alcohol elution dried powders of 7.5g are dissolved in 20mL methanol, 5000-8000r/
Min centrifuges 6min, takes supernatant through Sephadex LH-20 gel filtration chromatographies, and 8-10 column volume elution is collected in methanol elution
Liquid, the identical flow point of thin-layer chromatography combining data detection obtain A~I totally 8 flow points.Flow point E is target compound flow point after testing, is taken
250mg flow points E, which is dissolved in 3.0-5.0mL methanol and heats, makes it all dissolve, and is then placed in 4 DEG C of refrigerators, through methanol weight
Light yellow precipitate 21.5mg is precipitated in crystallization.
The structural identification of compound：1H-NMR (600MHz, DMSO-d6) shows a 1- substituted benzene ring signals δH 7.55
(2H, d, J=7.6Hz, H-2', 6'), 7.44 (3H, m, H-3', 4', 5'), and a pair of of meta position couple proton signal 6.21
(1H, d, J=1.5Hz, H-8), 6.16 (1H, d, J=1.5Hz, H-6), there are one 12.05 (5- of phenolic hydroxyl group proton signal
OH), 5.66 (1H, d, J=12.9, H-2), 4.99 (1H, d, J=7.4Hz, H-1 ") are the anomeric proton of sugar, determine that it is β-
Configuration,13C-NMR (150MHz, DMSO-d6) data show that the compound has 21 C, are composed in conjunction with H and determine that compound is a Huang
Ketose glycosides compound, ESI-MS provide quasi-molecular ion peak m/z 417.00 [M-H]-, other H signals are as follows：3.66 (1H,
A), 3.15~3.45 (6H, H-3a, 2 ", 3 ", 4 ", 5 ", 6 " b), 2.85 (1H, d, J=16.7Hz, H- by d, J=9.4Hz, H-6 "
3b)。13C-NMR (150MHz, DMSO-d6), 79.1 (C-2), 42.6 (C-3), 197.3 (C-4), 163.4 (C-5), 97.1 (C-
6),165.8(C-7),96.0(C-8),163.0(C-9),103.7(C-10),138.9(C-1'),127.2(C-2',6'),
129.1(C-3',4',5'),100.0(C-1”),73.5(C-2”),76.8(C-3”),69.9(C-4”),77.6(C-5”),
61.0(C-6”).Identify that the yellow mercury oxide is pinocembrin -7-O- β-D-Glucose glycosides.
Embodiment 2：Compound inhibits citrus to adopt rear penicillium italicum activity
1 material Italy mould (Penicillium italicum, ACCC 30399) is purchased from Chinese agriculture microbial bacteria
Kind collection.Culture medium：PDA and PDB (PDA for being free of agar powder) culture medium；Cell wall degrading enzyme inducing culture (KNO3
2.0g, K2HPO41.0g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO40.01g, vitamin B10.1g, L- aspartoyl
Amine 0.5g, chitin or glucan, CMC 10.0g, H2O 1000mL), pH is adjusted to 5.0,121 DEG C of sterilizing 20min after packing.
2 experimental methods
1. pinocembrin -7-O- β-D-Glucose glycosides is to the inhibition of penicillium italicum
The inhibition poison that pinocembrin -7-O- β-D-Glucose glycosides grows penicillium italicum silk is measured using growth rate method
Power.The card punch that edge sterilizes is fallen from the penicillium italicum activated to be beatened to take bacteria cake (Ф=5mm), mycelia placed face down
In the pastille culture medium center solidified, 27 DEG C of cultures.Each 4 repetitions of processing, experiment are repeated 2 times.Cross after culture 7d
Interior extrapolation method measures colony diameter, compared with blank control, calculates what each acute drug processing grew mycelia using following formula
Inhibiting rate.According to biometrics niqueMin conversion table, percentage will be inhibited to be converted into niqueMin, use 7.05 softwares of DPS to try
A concentration of abscissa of middle setting is tested, inhibition niqueMin is ordinate, obtains the virulence regression equation and effective concentration EC of medicament50
And EC95。
2. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum filament shapes
Pinocembrin solution is added in the triangular flask of 50mL PDB culture mediums, its final concentration is made to reach 0.2528mgmL-1
(EC50With EC95The sum of half), pinocembrin -7-O- β-D-Glucose glycosides solution is not added with as a control group, to each triangular flask
200 μ L penicillium italicum suspensions of middle addition, are placed in 27 DEG C, after 180rpm shaken cultivations 24 and 48h, in 10x40 times of electronic display
Micro- microscopic observation.
3. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum spore germination
Under aseptic condition, Italian mould spore suspension is prepared, 9 are pressed with pipettor:1 ratio is dense with difference by bacteria suspension
Pinocembrin -7-O- the β of degree-D-Glucose glycosides solution instills in concave slide groove, makes pinocembrin -7-O- β-D-Glucose glycosides effect
Final concentration of 0.8,0.4,0.2,0.1,0.05,0.025mgmL-1, each handle and be repeated 3 times.Slide is placed in 27 DEG C of trainings
It supports, carries out microscopy afterwards for 24 hours, observe the sprouting situation of the Italian mould spore of each processing and control group, count under the microscope
The sprouting number of 100-150 spore calculates spore germination rate and inhibiting rate.
4. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum silk increment
Under sterile conditions, take 1.0mL bacteria suspensions that the 250mL triangular flasks equipped with 100mL PDB fluid nutrient mediums are added
In, pinocembrin -7-O- β-D-Glucose glycosides processing is added in 27 DEG C, 180rpm shaken cultivation 48h when mycelium pellet well-grown
Liquid makes final concentration of 0.2528mgmL-1, the sterile water of control addition equivalent.Respectively continue culture 0,3,6,12, for 24 hours
Sampling, with Buchner funnel collected by suction mycelia, after being rinsed 3 times to mycelia with distilled water, by mycelia in vacuum freeze drier
It is lyophilized to constant weight, as mycelial weight.Each processing is repeated 5 times, and experiment is repeated 2 times.
5. pinocembrin -7-O- β-D-Glucose glycosides influences penicillium italicum cell leakage
Electrical conductivity method measures：1.0mL bacteria suspensions are taken to be added in the 250mL triangular flasks equipped with 100mL PDB fluid nutrient mediums,
27 DEG C, 180rpm shaken cultivations 48h.The mycelia for weighing equivalent is respectively put into equipped with 100mL0.2528mgmL-1Pinocembrin -7-
In the 250mL triangular flasks of O- β-D-Glucose glycosides solution and 100mL sterile waters, continue to cultivate on 27 DEG C, the shaking table of 180rpm,
In processing 0,1,2,4,6h samplings, it is that 3000rpm centrifuges 10min in rotating speed, is measured and compareed using DDS-11A digital display conductivity meters
Solvent blank conductivity (the C of group and pinocembrin -7-O- β-D-Glucose glycosides processing0) and supernatant conductivity (Ct), finally will
Mycelia boiling water bath keeps the temperature 10min, after being cooled to room temperature, measures its conductivity (Cd).Each processing is in triplicate.With electricity relatively
Conductance (relative electric conductivity, REC) indicates cell leakage, the calculating of relative conductivity (%)
Formula is as follows：
Chitinase 6. (Chitinase, CHI) active measurement
The extraction of chitin enzyme solution：1.0g mycelia is taken, 5mL acetic acid-sodium acetate buffer solutions are added and (contains 0.1M, pH 5.2, contains
5mM beta -mercaptoethanols；1mM Macrogol 6000s；1mM EDTA；4%PVP and 0.05%Triton X-100), it grinds under ice bath
(4 DEG C, 12 000rpm, 20min) is centrifuged in after homogenate, it is spare to collect supernatant.
The measurement of chitinase activity is with reference to the method for Boller and slightly modified, with every milligram of proteolysis glue chitin
Matter generates 1 × 10-9MoL N-Acetyl-D-glucosamines are a CHI active units (Umg-1protein)。
Beta-1,3-glucanase 7. (β -1,3-glucanase, GLU) active measurement
The extracting method of beta-1,3-glucanase liquid is identical as the extracting method of chitin enzyme solution.Beta-1,3-glucanase is lived
Property measure with reference to the method for Abeles and Forrence and slightly modified, generated with every milligram of protein breakdown laminarin in mycelia
1 μ g glucose is 1 GLU active units (Umg-1protein)。
8. data statistic analysis carries out statistical using SPSS 19.0 and 7.05 Software of Data Statistics of DPS to test data
Analysis carries out significant difference analysis with Duncan duncan's new multiple range methods, and significant difference is horizontal：Significantly (P<0.05)；Extremely significantly (P<
0.01)；Data processing and graphic making are carried out using 2003 softwares of Excel.
3 experimental results
1. pinocembrin -7-O- β-D-Glucose glycosides is to the inhibition of penicillium italicum
In test result such as Figure of description shown in Fig. 4, with gradually rising for compound concentration for the treatment of, to Italy
The fungistatic effect of mould also gradually increases；When concentration for the treatment of is 0.8mgmL-1When, the bacteriostasis rate of Italian mould is reached
100%, completely inhibit the growth of Italian mould.It is for statistical analysis using DPS v8.05 softwares, it obtains several with bacteriostasis rate
Rate value is Y, drug concentration (0.025~0.4mgmL-1) logarithm be X virulence regression equation：Y=7.8219+3.1150X,
Correlation coefficient r is 0.9836, illustrates that the concentration for the treatment of of pinocembrin -7-O- β-D-Glucose glycosides is linearly closed with bacteriostasis rate in significant
System；Also calculate EC simultaneously50And EC95Respectively 0.0846 and 0.4209mgmL-1。
2. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum filament shapes
In test result such as Figure of description shown in Fig. 5, the penicillium italicum silk cell wall of control group is smooth, thickness one
It causes, normal, the internal plasm of branch is evenly distributed (Fig. 5 A)；After pinocembrin -7-O- β-D-Glucose glycosides processing 12h, Italy
Mould mycelia ultimate swelling, mycelia winding kink, thickness are uneven (Fig. 5 B)；After processing for 24 hours mycelia is elongated, shrinkage, branch increase
It is more, or even there is content resolution, mycelia cavity and phenomenon of rupture (Fig. 5 C).
3. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum spore germination
As seen from Table 1, the spore of Italian mould is sprouted in the pinocembrin -7-O- β of various concentration-D-Glucose glycosides processing
Hair has significant inhibiting effect.With the increase of concentration for the treatment of, the spore germination rate of penicillium italicum gradually decreases, and inhibits
Effect enhances, and the extremely significantly (P of the difference between each processing and control group<0.01).At pinocembrin -7-O- β-D-Glucose glycosides
Manage a concentration of 0.4mgmL-1When, the spore germination rate of the processing is only 3.5%, and inhibiting rate is up to 96.3%.In conclusion tall
Song Su -7-O- β-D-Glucose glycosides has significant inhibition to the spore germination of Italian mould.
Inhibiting effect of 1 pinocembrin -7-O- β of the table-D-Glucose glycosides to Italian mould spore germination
Note：Data are the average value ± standard variance repeated three times in table, the same expression warp shown different size and write female
Deng Kenshi multiple checks are notable in 0.01 and 0.05 level difference respectively.
4. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum silk increment
By Fig. 6 in Figure of description it is found that during entire culture, the mycelium morphology factor of control group is in rising trend, until
When for 24 hours, increment reaches maximum value, and dry mycelial weight is 1.22 ± 0.032g100mL-1；And the Portugals pinocembrin -7-O- β-D- are added
Polyglycoside (0.2528mgmL-1) processing culture medium in mycelia growth obviously be suppressed, culture 6h in, the increment of mycelia
Drastically decline, then decline it is more gentle, until for 24 hours when mycelia increment reach minimum value, dry weight is 0.53 ± 0.011g
100mL-1, only the 43.4% of concurrent control group dry mycelial weight.It can be seen that pinocembrin grows tool to the mycelia of Italian mould
There is significant inhibition.
5. pinocembrin -7-O- β-influence of the D-Glucose glycosides to penicillium italicum cell leakage
Electrolyte can leak when cell membrane is by destroying, inside thalline is seeped into bacterium solution outside, to make the conductivity of bacterium solution
It increases, the size of mycelia relative conductivity can reflect the integrality of cell leakage indirectly.It can by Fig. 7 in Figure of description
Know, the relative conductivity of control group is in the variation tendency slowly risen；Pinocembrin -7-O- β-D-Glucose glycosides processing group is relatively electric
Conductance gradually rises with processing time, and ascensional range is significantly higher than control group (P<0.05), illustrate with the extension of time,
The cell leakage of Italian mould gradually increases, and a large amount of content leaks outward in cell so that bacterium solution conductivity is quick
Rise, destroys the integrality of cell leakage.
6. pinocembrin -7-O- β-D-Glucose glycosides is on the Italian active influence of mould cell wall degrading enzyme
Pinocembrin -7-O- β-D-Glucose glycosides is shown in specification to the active influence result of Italian mould cell wall degrading enzyme
Fig. 8 in attached drawing.From figure 8, it is seen that mycelia chitinase (CHI) activity of control group is in slow downward trend, entire
It is not significantly different during culture；And the mycelia CHI activity of pinocembrin -7-O- β-D-Glucose glycosides processing first significantly rises,
Reach maximum value when handling 2h, then slowly declines.CHI activity in processing 2,4,6h is respectively 55.6,50.7 and
52.2U·mg-1Protein is higher than the CHI activity of concurrent control group, it can be seen that pinocembrin significantly improves Italian mould
Cell degradation enzyme CHI activity.
Pinocembrin -7-O- β-D-Glucose glycosides equally can also increase another cell wall degradation phase in penicillium italicum
Enzyme β -1,3- dextranase (GLU) active variation is closed, it is a kind of lysozyme for decomposing fungal cell wall.By Figure of description
Middle Fig. 9 is it is found that mycelia β -1,3- dextranase (GLU) activity of control group is in the variation tendency slowly risen；And pinocembrin -7-
Mycelia β -1,3- dextranase (GLU) activity of O- β-D-Glucose glycosides processing is in steeply rise trend, and ascensional range is notable
More than control group.GLU activity in processing 2,4,6h is respectively 5.59,5.17 and 6.23Umg-1Protein compares the same period
The GLU activity of control group is high, it is seen that active increase of GLU can cause penicillium italicum cell wall that autolysis occurs.
Claims (2)
1. a kind of method for extracting pinocembrin -7-O- β-D-Glucose glycosides in fruit from radix fici simplicissimae, it is characterised in that：Including following
Step：
(1) radix fici simplicissimae fruit 40-50 DEG C of drying 2h in air dry oven, pulverizer smash it through 40 mesh sieve, take the lower medicinal material of sieve
Powder, the ethanol solution for being 90% using volume fraction is Extraction solvent, according to solid-liquid ratio (kg: L)=1：(10-40) ultrasonic wave is auxiliary
Extraction 30-90min is helped, the temperature of ultrasonic wave assisted extraction is 50-80 DEG C, repeats extraction 3 times, merges extracting solution and filters, returns
Ethyl alcohol is received, filtrate is concentrated into no alcohol taste to get radix fici simplicissimae berry extract medicinal extract；
(2) the radix fici simplicissimae berry extract medicinal extract in step (1) is suspended in the distilled water of 10-20 times of medicinal extract weight, is passed through
D101 macroporous resin column chromatographies are first eluted to efflux without color, again respectively successively with 5 times of 30% ethyl alcohol of column volume, 5 with pure water
Times 50% ethyl alcohol of column volume and 5 times of 95% ethanol gradient elutions of column volume collect 95% ethanol eluate, are concentrated into no alcohol taste,
Up to 95% ethanol elution concentrate；
(3) 95% ethanol elution concentrate in step (2) is dissolved in methanol, the matter of 95% ethanol elution concentrate and methanol
Measure volume ratio (g：ML) it is (1.0-5.0)：10, which is centrifuged into 3-10min in 5000-8000r/min on centrifuge, is taken
Clear liquid is detached with Sephadex LH-20 gel filtration chromatographies, is eluted with methanol, and 8-10 column volume eluent, thin-layer chromatography are collected
Merge target compound flow point, be concentrated under reduced pressure, into obtained concentrate be added methanol and be heated to concentrate can just be whole
It until dissolving, is put into 4 DEG C of refrigerators, it is target compound that light yellow precipitate, which is precipitated,.
2. the method for extraction pinocembrin -7-O- β-D-Glucose glycosides in a kind of fruit from radix fici simplicissimae as described in claim 1,
It is characterized in that：Include the following steps：Radix fici simplicissimae fruit 45 DEG C of drying 2h, pulverizer in air dry oven smash it through 40 mesh
Sieve takes the radix fici simplicissimae fruit powder end 5.0Kg under sieve, is impregnated with 90% ethyl alcohol of 50L, ultrasonic wave assisted extraction 60min, extraction temperature
Degree is 60 DEG C, in triplicate until extracting solution color is thin out, merges extracting solution and filters, filtrate is concentrated under the conditions of 45 DEG C
Without alcohol taste, medicinal extract 350g there are；Medicinal extract is suspended in 4L distilled water, through D101 macroporous resin column chromatographies, is first eluted with pure water
To efflux without color, again respectively successively with 5 times of 30% ethyl alcohol of column volume, 5 times of 50% ethyl alcohol of column volume and 5 times of column volumes 95%
Ethanol gradient elution, water elution component are abandoned, and the concentrate drying of other elution fractions gets dry extract, spare；95% ethanol elution object passes through
It is dried to obtain 7.5g powder, 95% alcohol elution dried powders of 7.5g are dissolved in 20mL methanol, 5000-8000r/
Min centrifuges 6min, takes supernatant through Sephadex LH-20 gel filtration chromatographies, and 8-10 column volume elution is collected in methanol elution
Liquid, the identical flow point of thin-layer chromatography combining data detection obtain A~I totally 8 flow points；Flow point E is target compound flow point after testing, is taken
250mg flow points E, which is dissolved in 3.0-5.0mL methanol and heats, makes it all dissolve, and is then placed in 4 DEG C of refrigerators, through methanol weight
Light yellow precipitate 21.5mg is precipitated in crystallization.
Priority Applications (1)
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| CN107556353A (en) * | 2017-10-23 | 2018-01-09 | 梅州金柚康健康科技有限公司 | A kind of Pu Luning and its derivative preparation and its application in anti-inflammatory and suppressing panting calming medicine |
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