CN103070967B - Water extract of houttuynia cordata thunb and preparation method and application of water extract - Google Patents
Water extract of houttuynia cordata thunb and preparation method and application of water extract Download PDFInfo
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Abstract
The invention belongs to the technical field of biological pharmacy, and particularly relates to a water extract of houttuynia cordata thunb and a preparation method and an application of the water extract. Total polysaccharide obtained from the water extract of the houttuynia cordata thunb has activity to inactivate or inhibit replication of hand-foot-mouth disease viruses, namely an enterovirus-71 (EV-71) and coxsackie A-16 (CVA16); polysaccharide with the antiviral activity is further found to be polysaccharide with molecular weight greater than 100kDa; therefore, the polysaccharide can be applied in preparation of a medicine for preventing the hand-foot-and-mouth disease viruses. As an edible aquatic herbaceous plant, the houttuynia cordata thunb has the characteristics of extensive source, small toxic and side effects, low price, simple extraction process and the like, and has important development and application prospects.
Description
Technical field
The invention belongs to biological pharmacy technical field, be specifically related to a kind of Herba Houttuyniae water extraction liquid and its preparation method and application.
Background technology
Herba Houttuyniae (Houttuynia cordata Thunb) is the perennial herbaceous plant of Saururaceae Houttuynia.Be distributed widely in Central China, the southeast and southwestern each provinces and regions.Herba Houttuyniae nutritive value is very high, is one of conventional wild vegetable.There is effect of heat-clearing and toxic substances removing, eliminating carbuncle evacuation of pus, inducing diuresis for treating stranguria syndrome simultaneously, belong to Ministry of Public Health determine be the plant resources of medicine and food.
Herba Houttuyniae chemical composition mainly comprises the compositions such as volatile oil, alkaloid, flavonoid, organic acid, polysaccharide.Wherein volatile oil main component is decanoylacetaldehyde (houttuynine sodium bisulfite), lauryl aldehyde etc.Alkaloidal composition mainly contains pyridine alkaloid etc.Flavonoid substances comprises the compositions such as Quercetin, Quercitroside, isoquercitrin, hyperin, rutin.In addition, water soluble polysaccharide is made up of glucose, fructose, arabinose etc.Both at home and abroad the activity research of Herba Houttuyniae is mainly concentrated on volatile oil and flavone compound at present, and for Herba Houttuyniae water soluble ingredient, particularly the bioactivity research of water soluble polysaccharide composition is less, has no report both at home and abroad for the research of polysaccharide composition anti-hand-foot-and-mouth-disease virus activity in Herba Houttuyniae.
Hand-foot-mouth disease (Hand, foot and mouth disease, HFMD) is a kind of infectious disease of the serious threat children's health that broke out in recent years of the Asian-Pacific area, main infection 0-5 year infant.Cardinal symptom comprises clinically, and blister measles ulcer appear in the mucosas such as hands, foot, lip; Patient with severe symptoms is even dead with meningitis, encephalitis, pulmonary edema, pneumorrhagia etc.Hand-foot-mouth disease is from 2008 since the outburst of Chinese Fuyang is listed in Class C infectious disease by Ministry of Public Health, and hand-foot-mouth disease M & M accounts for Class C infectious disease first place always.In addition, also not for effective vaccine and the medicine of hand-foot-mouth disease virus, hand-foot-mouth disease situation is extremely severe clinically, and it is extremely urgent that developmental research prevents and treats vaccine and the medicine of hand-foot-mouth disease virus.Enterovirns type 71 (Enterovirus71, EV71) and COxsackie A16(Coxsackievirus A16, CVA16) be two main pathogens that cause hand-foot-mouth disease, belong to nonencapsulated, little RNA enterovirus.The features such as plant is edible Chinese traditional herbs particularly, having source abundant, cheap, safety and low toxicity, therefore, develop plant resources, isolation identification antiviral activity composition, and the research of exploitation antiviral drugs has huge application prospect.
Summary of the invention
The problem that the present invention need to solve is to provide a kind of Herba Houttuyniae water extraction liquid and preparation method thereof and in the application preventing and treating in hand-foot-mouth disease virus drugs.
The present invention to the total polysaccharides in Herba Houttuyniae water extraction liquid and water extraction liquid extract, separation and anti-hand-foot-and-mouth-disease virus activity identify respectively, determines that antiviral polysaccharide is mainly the polysaccharide that molecular weight is greater than 100kDa.Therefore, can develop and be prepared into one and prevent and treat hand-foot-mouth disease virus drugs.The preparation method of Herba Houttuyniae water extraction liquid of the present invention and total polysaccharides is specially, Herba Houttuyniae cured leaf is cleaned with deionized water, dry, according to plant quality and liquid volume than being 1:10(g/ml) add deionized water, 60 ° C to 90 ° C water bath heat preservation 2-5 hour, the centrifugal 10min of 1000g/min obtains filtrate, and filtering residue is pressed same method lixiviate 2 times, merging filtrate.Filtrate is through the centrifugal 10min of 5000g/min, and 0.45 μ m membrane filtration, removes impurity, lyophilization.In water extraction liquid, the extraction of total polysaccharides adopts the method for precipitate with ethanol to precipitate.Be specially, in water extraction liquid, add the dehydrated alcohol precipitation polysaccharide of 4 times of volumes, spend the night in 4 ° of C refrigerator precipitations, the centrifugal 20min of 5000g/min for flocculent deposit, retain precipitation, precipitation is used after dehydrated alcohol, washing with acetone 3 times successively, and vacuum drying obtains dry Herba Houttuyniae total polysaccharides.Adopt phenol sulfuric acid method to detect polyoses content.Obtained water extraction liquid and polysaccharide are carried out to the active detection of extracorporeal antivirus effect, find, water extraction liquid and total polysaccharides have the activity that suppresses significantly hand-foot-mouth disease virus EV71 and CVA16 infection, further experiment shows, active polysaccharide is deactivation EV71 virion directly, causes virally inactivated cannot further infection; Thereby suppress it and further infect and active polysaccharide can suppress CVA16 virus copying in host cell.Centrifugal by ultrafilter membrane ultrafiltration, the polysaccharide component that molecular weight is greater than 100kDa is concentrated and antiviral activity significantly improves.On the contrary, the polysaccharide component antiviral activity that molecular weight is less than 100kDa significantly declines.Illustrate that in Herba Houttuyniae polysaccharide, antiviral activity composition is the polysaccharide that molecular weight is greater than 100kDa.
The invention has the advantages that:
1. Herba Houttuyniae water extraction liquid and water soluble polysaccharide wherein have direct deactivation EV71 and suppress the feature that CVA16 copies, and can directly block the source of infection by external means, and inactivation of viruses infects, protection Susceptible population.
2. Herba Houttuyniae water extraction liquid and polysaccharide extracting process are simple, convenient.In Herba Houttuyniae, antiviral polysaccharide component is further identified, for molecular weight is greater than the active polysaccharide of 100kDa.
Herba Houttuyniae water extraction liquid or total polysaccharides can be separately or with other drug component compatibility, be prepared as water preparation, granule, capsule etc. as the medicine of preventing and treating hand-foot-mouth disease.
4. Herba Houttuyniae wide material sources are edible Chinese traditional herbs, safety and low toxicity.
Accompanying drawing explanation
Fig. 1. Herba Houttuyniae polysaccharide suppresses viral infection time graph
Fig. 2. Herba Houttuyniae polysaccharide is directly hatched the impact of virus on viral infection particle release
■ represents untreated viral group
Fig. 3. the response rate of the centrifugal gained each several part of ultrafiltration polysaccharide
The specific embodiment
The test method using in following embodiment if no special instructions, is conventional method.
The material, the reagent etc. that in following embodiment, use, if no special instructions, all can obtain from commercial channels.
African green monkey kidney cell Vero cell is purchased from Shanghai Chinese Academy of Sciences cell bank.COxsackie A16 (CVA16) and enterovirns type 71 (EV71) type strain BrCr strain are purchased from Wuhan University's Chinese Typical Representative culture collection center, and diagnosing disease control center, Zhu Cong Jiangsu, enterovirns type 71 Fuyang obtains type strain.
Extraction and the detection of embodiment mono-Herba Houttuyniae water extraction liquid and polysaccharide
(1) extraction of Herba Houttuyniae water extraction liquid and polysaccharide
First dry Herba Houttuyniae plant is cleaned, oven drying at low temperature, to constant weight, is divided into two parts, a extraction for water extraction liquid, and another part is used for extracting Herba Houttuyniae polysaccharide.According to plant quality and liquid volume than 1:10(g/ml) add deionized water, in 60 ° C to 90 ° C water bath heat preservation 2-5 hour, the centrifugal 10min of 1000g/min obtains supernatant water extract, precipitation filtering residue is in the same way after lixiviate 2 times, merging filtrate, the centrifugal 10min of 5000g/min for supernatant, the filter membrane of 0.45 μ m filters.The direct freeze-drying of supernatant liquid obtains the water extraction liquid lyophilized powder of Herba Houttuyniae, or supernatant water extract adds the dehydrated alcohol of 4 times of volumes for precipitating polysaccharide, hold over night in 4 ° of C refrigerators, has a large amount of flocky precipitates to separate out, with the centrifugal 20min of 5000g/min, obtain precipitate.After precipitation is used dehydrated alcohol, washing with acetone 3 times successively, vacuum drying obtains dry Herba Houttuyniae total polysaccharides.
(2) Herba Houttuyniae polyoses content detects
Extract as stated above the dry Herba Houttuyniae polysaccharide of gained, weighing and calculating precipitate yield is 2.48%-2.75%, and computational methods are: yield %=(precipitate quality/former plant quality) × 100%.After precipitate with deionized water is redissolved, with glucose as a standard product, the content of measuring Herba Houttuyniae polysaccharide in alcohol precipitation by phenol sulfuric acid method is 75.3%-83.1%, polyoses content computational methods are: polyoses content %=(C × V × N/W) × 100%, wherein C is polysaccharide concentration, V is liquid volume, and N is extension rate, and W is precipitation quality.
Embodiment bis-Herba Houttuyniae water extraction liquid and polysaccharide anti-hand-foot-and-mouth-disease virus activity detect
(1) Herba Houttuyniae water extraction liquid and the toxotest of Herba Houttuyniae polysaccharide to cell
Cultivation of Vero in 96 orifice plates, after cell grows up to monolayer, after above-mentioned gained water extraction liquid lyophilized powder and precipitate with ethanol polysaccharide are redissolved with deionized water, be respectively the sample treatment Vero cell of 1 μ g/ml, 3 μ g/ml, 10 μ g/ml, 30 μ g/ml, 100 μ g/ml, 300 μ g/ml, 1000 μ g/ml with final concentration, three multiple holes are set.Normal group without drug treating is set simultaneously.In 37 ° of C, 5%CO
2in incubator, cultivate and observe the toxicity of sample to Vero cell, after cultivating 72 hours, the toxicity of the MTT method working sample that adopts classical detection cell viability to Vero cell.Specifically, adding final concentration is after the MTT cultured cell 4 hours of 0.5mg/ml, because normal living cells can be reduced into MTT water-insoluble bluish violet crystallization first a ceremonial jade-ladle, used in libation and be deposited in cell, so by removing after culture supernatant, add the DMSO of 100 μ l that first a ceremonial jade-ladle, used in libation is dissolved, measure light absorption value at 570nm place by microplate reader, the light absorption value of each group of cell is deducted after extinction background values, compared with the light absorption value of processed group cell and the light absorption value of Normal group cell, calculate the half toxic concentration CC of Herba Houttuyniae polysaccharide to Vero cell
50value, there is Cytotoxic concentration to 50% cell in the sample adding.CC
50be worth larger interpret sample less to Vero cytotoxicity.Result shows, the CC of Herba Houttuyniae water extraction liquid
50be greater than 1000 μ g/ml, and the CC of Herba Houttuyniae polysaccharide to Vero cell
50be 548 ± 11 μ g/ml.
(2) Herba Houttuyniae water extraction liquid and the test of polysaccharide anti-hand-foot-and-mouth-disease virus activity
The CC to Vero cell according to the Herba Houttuyniae water extraction liquid recording and polysaccharide
50value, next we be respectively 0.1 μ g/ml with final concentration in Vero cell, 0.3 μ g/ml, 1 μ g/ml, 3 μ g/ml, 10 μ g/ml, 100 μ g/ml, 300 μ g/ml, the Herba Houttuyniae water extraction liquid of 500 μ g/ml and polysaccharide detect it and suppress hand-foot-mouth disease virus EV71 (BrCr strain and Fuyang strain) and CVA16 infection activity.Vero cell grows up to after monolayer in 96 orifice plates, with after the sample treatment Vero cell of above variable concentrations 2 hours, uses respectively the EV71 that infection multiplicity (MOI) is 0.3 (BrCr strain and Fuyang strain) and CVA16 vero cells infection.Each processed group arranges three multiple holes, and the Normal group that do not infect without drug treating and the infected group without drug treating are set simultaneously.The host cell form causing with microscope direct observing viral infection changes, cell rounding, the cytopathic effecies (CPE) such as fragmentation come off, and in conjunction with above-described MTT method, after infecting 72 hours, detect cell viability, according to formula (processed group light absorption value-untreated infected group light absorption value)/(Normal group light absorption value-untreated infected group light absorption value) × 100%, calculate inhibition percentage ratio and the half-inhibition concentration IC of medicine to viral infection
50value, IC
50be worth littlely, illustrate that medicine suppresses viral effect more remarkable.Result is as shown in table 1, Herba Houttuyniae water extraction liquid pair
Table 1
The IC that EV71 (BrCr strain and Fuyang strain) and CVA16 infect
50value is respectively 20.6 ± 2.5 μ g/ml, 8.9 ± 1.6 μ g/ml, 186.3 ± 20.1 μ g/ml, and the IC of polysaccharide to EV71 (BrCr strain and Fuyang strain) and CVA16 infection
50value is respectively 4.3 ± 0.3 μ g/ml, 2.8 ± 0.2 μ g/ml, 13.5 ± 1.3 μ g/ml.Result shows, Herba Houttuyniae water extraction liquid and Herba Houttuyniae polysaccharide can suppress the infection of EV71 and CVA16 virus significantly; And the activity of the polysaccharide precipitating in water extraction liquid is significantly higher than the antiviral activity of water extraction liquid, therefore, we can infer, the polysaccharide in Herba Houttuyniae water extraction liquid is antiviral main active.
(3) directly deactivation EV71 virion of Herba Houttuyniae polysaccharide, and by suppressing the infection of inhibition of DNA replication CVA16 in CVA16 born of the same parents
According to the IC of above mensuration
50and CC
50value, we choose the time graph that final concentration is the Herba Houttuyniae polysaccharide detection of drugs inhibition viral infection of 30 μ g/ml.Cultivation of Vero in 96 orifice plates, after cell grows up to monolayer, the Herba Houttuyniae polysaccharide that with final concentration is 30 μ g/ml joined in cell by the time shown in Fig. 1, at viral infection first 2 hours, add with virus simultaneously, and 2 hours, 4 hours, 8 hours, 10 hours, 12 hours, 24 hours (2,0,2,4,8,10,12,24hr) join in cell after viral infection, after treating viral infection 72 hours, suppress the suppression ratio of viral infection by above-mentioned MTT method detection of drugs.Result shows, the suppression ratio of the Herba Houttuyniae polysaccharide pretreatment cell of the 30 μ g/ml 2 hours infection to the strain of EV71 Fuyang and CVA16 is 100%.But, EV71 vero cells infection added medicine inhibition worse and worse after 2 hours, and contrary CVA16 infection cell adds medicine inhibition still remarkable after 8 hours, illustrate that Herba Houttuyniae polysaccharide can just can suppress the infection of EV71 before EV71 enters host cell, and its to suppress that CVA16 infects may be to play a role by suppressing the process such as copy of virus in host cell.Whether directly in order further to determine the inactivation of viruses of Herba Houttuyniae polysaccharide, we are Herba Houttuyniae polysaccharide and the 1x10 of 30 μ g/ml by final concentration
5tCID
50eV71 or 1x10
5the CVA16 of PFU mixes in the serum-free medium of 100 μ l, in 37 ° of C, hatches after 60min, gets mixed liquor 10 μ l and infect the Vero cell of 24 orifice plates, repeats 3 multiple holes, and the normal virus group of non-processor and viral group of deionized water processing are set simultaneously.After treating that virus absorption onto cell 2 as a child, culture medium is removed, with depletion of blood cleaning three times, to remove the virus of not absorption and residual medicine.After viral infection 48 hours, collect the cell and the supernatant that infect, with liquid nitrogen multigelation 3 times to discharge virion, by gradient dilution virus liquid superinfection Vero cell, counting CVA16 infects the plaque (PFU) causing and calculates viral titre, calculates EV71 virus titer by Reed-Muench method, after taking the logarithm, maps, as shown in Figure 2, the titre of the EV71 virus that Herba Houttuyniae polysaccharide is hatched is significantly lower than untreated fish group or deionized water processing viral group for result.Specifically, the infectious viral particle of the EV71 of drug treating group contrasts with untreated EV71 infected group, and medicine can suppress viral infection and reach 5 more than log, and 10
5doubly, directly deactivation EV71 virion of Herba Houttuyniae polysaccharide is described.On the contrary, the titre of the CVA16 virus of hatching with Herba Houttuyniae polysaccharide does not have marked difference with the virus group titre of not processed group or deionized water processing, illustrate that Herba Houttuyniae polysaccharide is not to bring into play antivirus action by direct deactivation CVA16 virion.To sum up experimental result shows, Herba Houttuyniae polysaccharide is mainly by direct deactivation EV71 virus, causes virally inactivated can not continuation to infect, and it suppresses viral infection effect by suppressing the process performances such as CVA16 copying in host cell.
Due to the composition complexity of polysaccharide, the change of molecular weight is huge, and its biological activity and molecular weight have very large relation.In order further to determine the molecular weight of antiviral activity polysaccharide in Herba Houttuyniae polysaccharide, we use the ultrafilter membrane ultrafiltration of 100kDa and 10kDa centrifugal after polysaccharide is redissolved with deionized water successively, gained trapped fluid is collected respectively, concentrated, lyophilizing, obtains the Herba Houttuyniae polysaccharide of different molecular weight ranges, according to formula (hold back rear polysaccharide quality/hold back the quality of front total polysaccharides) × 100%, calculate the response rate of each several part polysaccharide, and further detect anti-by MTT method
Table 2
Hand-foot-mouth disease virus activity, the IC of calculating each several part anti-hand-foot-and-mouth-disease virus
50value.As shown in Figure 3, the polysaccharide recovery that molecular weight is greater than 100kDa is 25.3% to result, and the polysaccharide recovery of molecular weight between 10-100kDa is 45.2%, and being less than 10kDa polysaccharide recovery is 28.2%.Wherein approximately there is 1.3% loss.Each several part antiviral IC
50be worth as shown in table 2, "-" represents not detect antiviral activity, the polysaccharide that is less than 10kDa by the visible molecular weight of table 2 does not have antiviral activity substantially, the relative total polysaccharides of the polysaccharide of molecular weight between 10-100kDa, antiviral activity is general, significantly increases with respect to its antiviral activity of total polysaccharides and molecular weight is greater than the polysaccharide of 100kDa.Can draw thus, the polysaccharide in Herba Houttuyniae water extraction liquid has significant anti-hand-foot-and-mouth-disease virus activity, and its there is antiviral activity be greater than the polysaccharide of 100kDa for molecular weight.
Claims (1)
1. the Herba Houttuyniae polysaccharide that molecular weight is greater than 100kDa is prevented and treated the application in the medicine of hand-foot-mouth disease viral infection as unique active component in preparation, and wherein said hand-foot-mouth disease virus is enterovirns type 71 and COxsackie A16 virus; The Herba Houttuyniae polyoses producing method that wherein said molecular weight is greater than 100kDa is: first dry Herba Houttuyniae plant is cleaned, oven drying at low temperature is to constant weight, add deionized water according to plant quality and liquid volume than 1:10g/ml, in 60 ℃ to 90 ℃ water bath heat preservation 2-5 hour, the centrifugal 10min of 1000g/min obtains supernatant water extract, supernatant water extract adds the dehydrated alcohol of 4 times of volumes for precipitating polysaccharide, hold over night in 4 ℃ of refrigerators, there is a large amount of flocky precipitates to separate out, with the centrifugal 20min of 5000g/min, obtain precipitate; After precipitation is used dehydrated alcohol, washing with acetone 3 times successively, vacuum drying obtains dry Herba Houttuyniae total polysaccharides; Herba Houttuyniae total polysaccharides uses the ultrafilter membrane ultrafiltration of 100kDa and 10kDa centrifugal after redissolving with deionized water successively, and gained trapped fluid is collected respectively, concentrated, and lyophilizing obtains the Herba Houttuyniae polysaccharide of corresponding molecular weight.
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