CN101695511B - Pomegranate rind extract and production method and application thereof - Google Patents

Pomegranate rind extract and production method and application thereof Download PDF

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CN101695511B
CN101695511B CN200910113503A CN200910113503A CN101695511B CN 101695511 B CN101695511 B CN 101695511B CN 200910113503 A CN200910113503 A CN 200910113503A CN 200910113503 A CN200910113503 A CN 200910113503A CN 101695511 B CN101695511 B CN 101695511B
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chromatographic column
ethanol
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pericarpium granati
column volumes
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CN101695511A (en
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顾政一
黄华
徐芳
赵军
姚华
刘砥威
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INST OF PHARMACOLOGY XINJIANG UYGUR AUTONOMOUS REGIONS
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INST OF PHARMACOLOGY XINJIANG UYGUR AUTONOMOUS REGIONS
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Abstract

The invention relates to the technical field of extracts extracted from pomegranate rind and production methods and applications thereof, in particular to a pomegranate rind extract and a production method and application thereof. The pomegranate rind extract contains a flavone component and a tannin component. The pomegranate rind extract has obvious function of resisting influenza viruses, hepatitis B viruses and HIV viruses and can be used for preparing a broad-spectrum antiviral medicament. The invention proposes the function of the pomegranate rind extract in aspects of resisting the influenza viruses, the hepatitis B viruses and the HIV viruses for the first time; a macroporous resin adsorption and separation technology is applied for the first time in the production method of the pomegranate rind extract, the flavone component in the pomegranate rind is enriched from a crude extract of the pomegranate rind, and the extract with the total flavone content of 30 to 70 percent by weight, which contains 10-30 percent by weight of tannin, is obtained. The method is simple and easy, has reliable process and is suitable for industrialized production.

Description

Pericarpium Granati extract and production method thereof and application
Technical field
The extract that the present invention relates to from the peel of plant Punica granatum L., to extract and the technical field of production method and application thereof are that a kind of Pericarpium Granati extract and production method thereof and application are a kind of Pericarpium Granati extract and production method and the application of low pole macroporous resin in the production method of Pericarpium Granati extract.
Background technology
Influenza is by first, second, the third three caused acute respiratory infectious disease of type influenza virus.Three type influenza virus are just glutinous coe virus, are tunicary sub-thread minus-stranded rna virus, and wherein, influenza A virus is maximum to the mankind's harm, can cause global flu outbreak; The B-mode local outburst that causes more; Third type is then mainly invaded infant.
After influenza virus is imported the crowd into, cause the distinguishing feature of influenza pandemic to be: outburst suddenly, rapid spread, influence wide, sickness rate height and with certain mortality rate.The source of infection mainly is the patient, and main route of transmission is that the air that has influenza virus flies the Zhu.Death normally secondary bacterial infection causes, and complication often betides infant, old people and chronic.
The harm of influenza is well-known, human still unable so far prevention worldwide flu outbreak once in per 10 to 50 years, bird flu, first type H in recent years 1N 1The popular of influenza not only damaged the healthy of people, and causes enormous economic loss and heavy social burden thus, therefore, prevents and treats the key problem that influenza has become countries in the world infectious disease control.But because the variability of influenza virus is high, the research and development of anti-influenza virus medicament is slow.In order to tackle being very popular of influenza more energetically, international the world of medicine is striving to find the active drug of preventing and treating influenza always.In China, from the Chinese medicine material, excavate the influenza active substance and more receive extensive concern.
Hepatitis B virus is a member in the hepadnavirus, gets into blood circulation behind the hepatitis B virus infection body, in leukocyte, breeds, and is positioned hepatic parenchymal cells at last, causes hepatitis B.The route of transmission of hepatitis B is that intestinal is propagated outward.After suffering from acute hepatitis B, 90% patient can clinical cure, obtains immunity after being ill; 10% patient transfers chronic hepatitis B to, and wherein some people will develop into liver cirrhosis and hepatocarcinoma.Hepatitis B is that pandemic infection is sick, and Southeast Asia, Africa and China belong to the district occurred frequently, and crowd's hepatitis B virus infection rate is up to 60%.Infection rate childhood period that the main epidemiological features of high incidence of hepatitis b being is very high, and they are as stable source of infection long-term existence socially, and the harm crowd will be again the victim of liver cirrhosis and hepatocarcinoma simultaneously.
Though hepatitis B virus gene engineering subunit vaccine may command viral infection is popular, because high incidence of hepatitis b is the economically less developed region, crew's planned immunization also can't realize.Because available clinically anti-hbv drug is very limited at present, and curative effect is all desirable not to the utmost, and a large amount of hepatitis B patients that exist in the society are carried out efficacious therapy, to improve its life quality, prolongs life cycle, is the difficult problem that international medical community need be captured.Developing hepatitis B virus resisting medicine safely and effectively, then is the direction that vast pharmacy worker makes great efforts.
HIV belongs to the human retrovirus, for the RNA viruses of film is arranged.Behind the human infection HIV, Most patients needed just develop into AIDS and cause death in 7 to 10 years.The course of disease comes from that HIV destroys immune function of human body gradually and the final life-threatening all kinds of complication of secondary slowly.Yet the spread speed of AIDS is surprising, in short 20 years after coming to light; This disease has been diffused into each corner, the whole world; Though China belongs to the low district of sending out, the infected is also considerably beyond 1,000,000, and the propagation of AIDS has brought grave danger to human health.
Aspect the preventing and treating of AIDS, be the focus of studying in the world though infect with vaccine prevention always, because HIV very easily produces gene mutation and various reasons such as escape immunoprotection, vaccine development is the progress of achieving no breakthrough property so far.AIDS resisting poisons the exploitation of treating medicine and has obtained certain achievement, and a plurality of chemical medicine that gone on the market have shown certain effect to prevention mother-to-baby transmission and inadvertent contamination.Yet chemotherapeutics can not suppress virus replication fully, and defective has greatly limited its curative effect and clinical practice widely to rely on toxicity, the drug resistance of drug dose and cost an arm and a leg etc.Scientists is labouring for the substantial progress of AIDS resisting cytotoxic drug research and development.
The control of viral disease has become international medical community and has pressed for one of great difficult problem of solution; Worldwide pestilence such as influenza, hepatitis B and AIDS particularly; People's the life quality and even the economic development of society are had a far reaching influence; Therefore, the research of antiviral drugs is that field of medicaments presses for the scientific research task that makes a breakthrough.
In China, utilize Chinese medicine to prevent and treat viral disease one to enjoying attention because the advantage of Chinese medicine multicomponent, many target spots, multiple mechanism of action, from Chinese crude drug extraction separation to some antiviral effective ingredient demonstrate the excellent development application prospect.
Pericarpium Granati is the dry peel of Punicaceae plant Punica granatum L. (Punica granatum L), property acid, and puckery has the effect of astringing intestine to stop diarrhea, parasite killing, hemostasis.Punica granatum L. is one of fruit of giving priority to of China, its cultivated area forward large-scale development, but the Pericarpium Granati that accounts for pomegranate fruit nearly 1/3rd also do not fully utilize, and big portion goes out of use, and causes the serious wasting of resources.It is reported, contain the number of chemical composition in the Pericarpium Granati, mainly contain flavonoid, tannin class, 2-acetonylpiperidine and resinae and become to grade.
At present; From Pericarpium Granati, prepare extract; And the research that is used to develop antiviral drugs at home and abroad do not appear in the newspapers, and adopts the research of macroporous adsorption resin technology separating and purifying flavone constituents from Pericarpium Granati also not see formal report, and do not find relevant patent application or mandate.
Summary of the invention
The present invention provides a kind of Pericarpium Granati extract and production method and the application of low pole macroporous resin in the production method of Pericarpium Granati extract that is applied to prepare broad-spectrum antiviral medicament; Overcome the deficiency of prior art, this Pericarpium Granati extract contains the flavones ingredient of 30% to 70% (percentage by weight) and the tannin constituents of 10% to 30% (percentage by weight); Production method first Application low pole macroporous resin adsorption isolation technics flavones ingredient in the separation and purification Pericarpium Granati from the Pericarpium Granati crude extract of this Pericarpium Granati extract; This Pericarpium Granati extract can be used to prepare the broad-spectrum antiviral medicament of treatment influenza and/or diseases such as hepatitis B and/or AIDS especially.
One of technical scheme of the present invention obtains in the following manner: a kind of Pericarpium Granati extract that is used to prepare broad-spectrum antiviral medicament, it contains 30% to 70% flavones ingredient and 10% to 30% tannin constituents by weight percentage.
Be further optimization and/or selection below to one of technique scheme:
This Pericarpium Granati extract obtains by following step:
The first step, Pericarpium Granati coarse powder water and/or ethanol are slightly carried, wherein, water slightly carry for: the Pericarpium Granati coarse powder is added 8 times to 10 times water gagings, down extracts twice, each 2 hours to 3 hours, filter merging filtrate at 80 ℃ to 100 ℃; Ethanol slightly carry for: the Pericarpium Granati coarse powder is added 8 times to 10 times amount 30% to 70% ethanol, extracts twice, each 2 hours to 3 hours, filter merging filtrate at 60 ℃ to 80 ℃;
Second step was concentrated into 1/3rd of original volume with above-mentioned filtrating, added the dehydrated alcohol that is equivalent to 2 times of amounts of concentrated solution volume, placed deposition 24 hours, and filtering precipitate obtains alcoholic solution;
In the 3rd step, it is 3 to 7 that above-mentioned alcoholic solution is concentrated back adjusting pH value, last macroporous resin chromatographic column absorption; Wherein, macroporous resin adopts a kind of in HPD400, HPD600, D-101, D-201, the AB-8 type low pole macroporous resin;
The 4th step; To be equivalent to the above-mentioned chromatographic column of deionized water rinsing of 3 to 8 chromatographic column volumes, be that eluant carries out eluting with 30% to 70% ethanol again, the consumption of eluant is equivalent to 3 to 8 chromatographic column volumes; Flow velocity be 2 to 5 chromatographic column volumes/hour, collect ethanol elution and merge;
The 5th step concentrated the ethanol elution after the above-mentioned merging down at 60 ℃ to 90 ℃, reclaimed ethanol, again at 50 ℃ to 80 ℃ following drying under reduced pressure, promptly got required Pericarpium Granati extract.
Two of technical scheme of the present invention obtains in the following manner: a kind of production method of Pericarpium Granati extract is undertaken by following step:
The first step, the Pericarpium Granati coarse powder is slightly carried with water and/or ethanol; In second step, add the impurity in the ethanol precipitation crude extract; In the 3rd step, alcoholic solution concentrates the back and regulates pH value, with the total flavones in the macroporous resin adsorption crude extract; In the 4th step, obtain eluent with ethanol eluting total flavones from the macroporous resin; The 5th step concentrated eluent and recovery ethanol, and drying under reduced pressure promptly gets Pericarpium Granati extract.Wherein, macroporous resin adopts a kind of in HPD400, HPD600, D-101, D-201, the AB-8 type low pole macroporous resin.
Be two further optimization and/or selection below to technique scheme:
The water that in the above-mentioned first step, the Pericarpium Granati coarse powder is added 8 times of amount to 10 times amounts extracts twice at 60 ℃ to 80 ℃, each 2 hours to 3 hours, filters merging filtrate.
In the above-mentioned first step, the Pericarpium Granati coarse powder added the ethanol of 8 times of amounts to 10 times amount 30% to 70%, extract twice, each 2 hours to 3 hours, filter merging filtrate at 60 ℃ to 80 ℃.
In above-mentioned second step, will filtrate adds the dehydrated alcohol that is equivalent to 2 times of amounts of concentrated solution volume after concentrating, and places and precipitates 24 hours, and filtering precipitate obtains alcoholic solution; In the 3rd step, alcoholic solution being concentrated back adjusting pH value is 3 to 7, last macroporous resin chromatographic column absorption; Wherein, the big pore resin is selected a kind of in HPD400, HPD600, D-101, D-201, the AB-8 type low pole macroporous resin for use; The 4th the step in to be equivalent to the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Be that eluant carries out eluting with 30% to 70% ethanol again; The consumption of eluant is equivalent to 3 to 8 chromatographic column volumes, flow velocity be 2 to 5 chromatographic column volumes/hour, collect ethanol elution and merge; In the 5th step, ethanol elution is concentrated down at 60 ℃ to 90 ℃, reclaim ethanol,, promptly obtain required Pericarpium Granati extract again at 50 ℃ to 80 ℃ following vacuum dryings.
Three of technical scheme of the present invention obtains in the following manner:
The application of low pole macroporous resin in the production method of above-mentioned Pericarpium Granati extract, its big pore resin are a kind of of HPD400, HPD600, D-101, D-201, AB-8 type low pole macroporous resin.
Four of technical scheme of the present invention obtains in the following manner: above-mentioned Pericarpium Granati extract is applied to prepare the broad-spectrum antiviral medicament to influenza virus and/or hepatitis B virus and/or HIV.
Pericarpium Granati extract of the present invention has tangible resisiting influenza virus, hepatitis B virus and HIV effect, can be used for preparing broad-spectrum antiviral medicament; The present invention proposes the effect of Pericarpium Granati extract aspect resisiting influenza virus, hepatitis B virus and HIV first; And in the production method of this Pericarpium Granati extract first Application macroporous resin adsorption isolation technics; Flavones ingredient from the Pericarpium Granati crude extract in the enrichment Pericarpium Granati; And obtain general flavone content and reach 30% to 70% (percentage by weight), contain the extract of 10% to 30% (percentage by weight) tannin class composition simultaneously.This method is simple, and technology is reliable, is fit to suitability for industrialized production.
The specific embodiment
The present invention does not receive the restriction of following embodiment, can confirm concrete embodiment according to the technical scheme and the practical situation of the invention described above.
Below in conjunction with embodiment the present invention is done further argumentation.
Through following embodiment Pericarpium Granati extract of the present invention is elaborated:
Embodiment 1: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 3 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 2: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 5 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 3: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils for 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 12 hours; It is 7 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out gradient elution with 70% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Every concentration ethanol consumption is 3 to 8 chromatographic column volumes, flow velocity be 2 chromatographic column volumes/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.Wherein, big pore adsorption resin is selected a kind of in the macroporous resin of models such as D-201, D-301, HPD400, HPD600, AB-8 for use.
Embodiment 4: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils in 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 5: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 6: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 7: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 8: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 9: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 5 to 10 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 10: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 11: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 12: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last AB-8 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 13: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 3 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 14: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 5 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 15: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils for 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 12 hours; It is 7 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out gradient elution with 70% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Every concentration ethanol consumption is 3 to 8 chromatographic column volumes, flow velocity be 2 chromatographic column volumes/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.Wherein, big pore adsorption resin is selected a kind of in the macroporous resin of models such as D-201, D-301, HPD400, HPD600, AB-8 for use.
Embodiment 16: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils in 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 17: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 18: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 19: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 20: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 21: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 5 to 10 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 22: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 23: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 24: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD400 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 25: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 3 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 26: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 5 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 27: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils for 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 12 hours; It is 7 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out gradient elution with 70% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Every concentration ethanol consumption is 3 to 8 chromatographic column volumes, flow velocity be 2 chromatographic column volumes/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.Wherein, big pore adsorption resin is selected a kind of in the macroporous resin of models such as D-201, D-301, HPD400, HPD600, AB-8 for use.
Embodiment 28: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils in 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 29: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 30: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 31: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 32: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 33: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 5 to 10 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 34: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 35: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 36: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last HPD600 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 37: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 3 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 38: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 5 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 39: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils for 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 12 hours; It is 7 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out gradient elution with 70% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Every concentration ethanol consumption is 3 to 8 chromatographic column volumes, flow velocity be 2 chromatographic column volumes/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.Wherein, big pore adsorption resin is selected a kind of in the macroporous resin of models such as D-201, D-301, HPD400, HPD600, AB-8 for use.
Embodiment 40: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils in 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 41: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 42: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 43: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 44: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 45: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 5 to 10 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 46: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 47: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 48: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-101 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 49: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 3 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 50: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils in 80 ℃ to 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; It is 5 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Amount of ethanol is 3 to 8 chromatographic column volumes, and flow velocity is 2 to 5 chromatographic column volumes per hour, collects ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 51: the Pericarpium Granati coarse powder adds 8 times of amount to 10 times water gagings and boils for 100 ℃ and carry twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 12 hours; It is 7 with dilute hydrochloric acid adjusting pH value that filtering precipitate, filtrating concentrate the back, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out gradient elution with 70% ethanol again with 3 to 8 chromatographic column volumes of deionized water flushing chromatographic columns; Every concentration ethanol consumption is 3 to 8 chromatographic column volumes, flow velocity be 2 chromatographic column volumes/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.Wherein, big pore adsorption resin is selected a kind of in the macroporous resin of models such as D-201, D-301, HPD400, HPD600, AB-8 for use.
Embodiment 52: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils in 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 53: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds the dehydrated alcohol that concentrates the long-pending 2 times of amounts of liquid and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 54: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 30% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 55: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 56: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 57: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 50% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 5 to 10 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 58: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 3 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 30% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 59: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 2 hours to 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 5 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 50% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 60: the ethanol that the Pericarpium Granati coarse powder adds 8 times of amounts to 10 times amount 70% boils for 60 ℃ to 80 ℃ carries twice, each 3 hours, filters; Merging filtrate is concentrated into 1/3rd of original volume, adds 2 times of amount dehydrated alcohol and places deposition 24 hours; Filtering precipitate, concentrating the back is 7 with dilute hydrochloric acid adjusting pH value, last D-201 type macroporous resin chromatographic column; Treat sample solution all after the absorption,, carry out eluting with 70% ethanol again with the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Amount of ethanol is 3 to 8 chromatographic column volumes, 2 to 5 chromatographic column volumes of flow velocity/hour, collect ethanol elution; Merge the back and concentrate in 60 ℃ to 90 ℃, concentrated solution in 50 ℃ to 80 ℃ drying under reduced pressure, promptly gets Pericarpium Granati extract again.
Embodiment 61: the mensuration of the foregoing description 1 to 60 gained Pericarpium Granati extract being carried out as follows general flavone content:
The preparation precision of reference substance solution takes by weighing 50 milligrams of control substance of Rutin that are dried to constant weight, puts in 25 milliliters of volumetric flasks, and it is an amount of to add methanol; Put that slight fever makes dissolving in the water-bath, put coldly, add methanol to scale; Shake up, precision is measured 10 milliliters, puts in 100 milliliters of volumetric flasks; Add water to scale, shake up, promptly get every milliliter of reference substance solution that contains 0.2 milligram of anhydrous rutin.
The preparation precision of standard curve is measured 1,2,3,4,5,6 milliliters of reference substance solution, puts respectively in 25 milliliters the volumetric flask, respectively adds water to 6 milliliters, adds 1 milliliter of 5% sodium nitrite solution; Mixing was placed 6 minutes, added 1 milliliter of 10% aluminum nitrate solution again, shook up; Placed 6 minutes, and added 10 milliliters of sodium hydroxide test solutions again, adding distil water shakes up to scale; Placing 15 minutes, is blank with the corresponding reagent, measures trap in 500 nanometers; With the trap is vertical coordinate, and concentration is abscissa, the drawing standard curve.
Sample determination precision respectively takes by weighing the foregoing description 1 to 60 gained Pericarpium Granati extract sample 1.0g, puts in 100 milliliters the volumetric flask, adds methanol to scale, shakes up, and precision is measured 10 milliliters; Put in 100 milliliters the volumetric flask, add water to scale, shake up, precision is measured 3 milliliters, puts in 25 milliliters of volumetric flasks; Add water to 6 milliliters, add 1 milliliter of 5% sodium nitrite solution, mixing was placed 6 minutes; Add 1 milliliter of 10% aluminum nitrate solution again, shake up, placed 6 minutes, add 10 milliliters of sodium hydroxide test solutions again; Adding distil water shakes up to scale, places 15 minutes, is blank with the corresponding reagent; Measure trap in 500 nanometers, read the amount that contains anhydrous rutin the sample solution, calculate the content of total flavones (in anhydrous rutin) in the Pericarpium Granati extract sample from above-mentioned standard curve, should be in 30% to 70% scope.
Embodiment 62: the mensuration of the foregoing description 1 to 60 gained Pericarpium Granati extract being carried out as follows content of tannin:
Sodium tungstate 100g, sodium molybdate 25g are got in the preparation of P-Mo-Wo acid test solution, add water 700ml and make dissolving, add hydrochloric acid 100ml, phosphoric acid 50ml; Reflux 10 hours is put coldly, adds lithium sulfate 150g, water 50ml and bromine 0.2ml again; Boil and remove residual bromine (about 15 minutes), cooling, thin up is to 1000ml; Filter, promptly get.It is yellow that test solution should be, and after placing, becomes green, can add bromine 0.2ml, boils to remove unnecessary bromine and get final product.
The preparation precision of reference substance solution takes by weighing gallic acid reference substance 50mg, puts in the brown measuring bottle of 100ml, is dissolved in water and is diluted to scale; Precision is measured 5ml, puts in the brown measuring bottle of 50ml, is diluted with water to scale; Shake up, promptly get (containing gallic acid 0.05mg among every 1ml).
The preparation precision of standard curve is measured reference substance solution 0.5,1.0,2.0,3.0,4.0,5.0ml; Put respectively in the brown measuring bottle of 25ml, each adds P-Mo-Wo acid test solution 1ml, adds water 11.5,11,10,9,8,7ml more respectively; Be diluted to scale with 29% sodium carbonate liquor, shake up.Placing 30 minutes, is blank with the reagent corresponding, the photograph ultraviolet visible spectrophotometry (" 2005 editions appendix VA of Chinese pharmacopoeia), measure absorbance in the wavelength of 760nm, be vertical coordinate with the absorbance, concentration is abscissa, the drawing standard curve.
It is an amount of that test sample is got in the preparation of sample solution, accurate claims surely, puts in the brown measuring bottle of 250ml, adds water 150ml, and placement is spent the night, and supersound process 10 minutes is put coldly, is diluted with water to scale, shakes up, and leaves standstill (making precipitation of solid material).Filter, discard filtrating 50ml just, precision is measured subsequent filtrate 20ml, puts in the brown measuring bottle of 100ml, is diluted with water to scale, shakes up, and promptly gets.
Measure the total phenols precision and measure sample solution 2ml, put in the brown measuring bottle of 25ml, the sighting target directrix curve prepares the method under the item; From " adding P-Mo-Wo acid test solution 1ml ", add water 10ml, measure absorbance in accordance with the law; From standard curve, read the amount (mg) of gallic acid in the sample solution, calculate, promptly get.
Measure the polyphenol precision that is not adsorbed and measure sample solution 25ml, add in the 100ml tool plug conical flask that fills casein 0.6g, close plug is put in 30 ℃ of water-baths and is incubated 1 hour; Jolting is constantly taken out, and puts coldly, shakes up; Filter, discard filtrating just, precision is measured subsequent filtrate 2ml, puts in the brown measuring bottle of 25ml; The sighting target directrix curve prepares the method under the item, from " adding P-Mo-Wo acid test solution 1ml ", adds water 10ml, measures absorbance in accordance with the law; From standard curve, read the amount (mg) of gallic acid in the sample solution, calculate, promptly get.
The calculating of content of tannin in the sample: the polyphenol amount of content of tannin=total phenols amount-be not adsorbed
Calculate the content of tannin in the Pericarpium Granati extract sample as stated above, should be in 10% to 30% scope.
In the present invention: propose the application of low pole macroporous resin in the Pericarpium Granati extract production method first.Used low pole macroporous resin is selected a kind of in D-101, D-201, HPD400, HPD600, the AB-8 type macroporous resin for use.
Describe through the broad-spectrum disease resistance toxic action of following experimental example Pericarpium Granati extract of the present invention:
Experimental example 1 Pericarpium Granati extract of the present invention is to the cytopathogenic inhibitory action of influenza virus.
1. material and method:
1.1 Strain: influenza first type virus (H 3N 272243 strains are prevented in hypotype Guangdong), influenza B virus (the anti-97-13 strain of Ji) is available from Virology Inst., Chinese Academy of Preventive Medical Science.
1.2 laboratory sample: Pericarpium Granati extract sample SL1, SL2, SL3; Be light brown to dark-brown extract powder; General flavone content in the sample (percentage by weight) SL1 is 30%, SL2 is 50%, SL3 is 70%, and content of tannin (percentage by weight) SL1 is 30%, SL2 is 20%, SL3 is 10%.All be mixed with the original liquid of 10mg/ml before the experiment with high purity water, subsequent use after the filtration sterilization; The positive control drug ribavirin, specification 100mg/ sheet, clinical take 2/inferior, 3 times/day, be mixed with the original liquid of 10mg/ml with high purity water before the experiment, subsequent use after the filtration sterilization.
2. method and result
Sample is to the toxic mensuration of mdck cell:
Sample SL2, SL2, SL3 and positive control stock solution are all done 2 times of dilutions with culture fluid; Get 1: 5 to 1: 320 totally 7 dilution factors, each dilution medicinal liquid is added to has respectively inoculated the Hela cell and grown up in the culture plate of monolayer 100 microlitres/hole; 4 parallel holes of every concentration are established the cell contrast simultaneously.Culture plate is put 37 ℃, 5%CO 2Condition was cultivated four days, every day the observation of cell growing state, the minimum extension rate that does not occur obvious regression with cell is maximal non-toxic concentration (TC 0).And calculate 50% poisonous concentration (TC by the Reed-Muench method 50), the result sees table 1.
Sample is to the influence of pathological changes caused by virus:
Get the mdck cell that grows up to monolayer, inoculation influenza first type virus 10 -5(50% cell culture infective dose CCID 5015 times), influenza B virus 10 -2(CCID 5030 times), in 37 ℃ of absorption hypsokinesis in 2 hours venom of preventing or cure a disease, wash cell surface 2 times with the liquid of keeping that does not contain serum.Cell matched group, the different dilution administration groups with each sample of virus control group are established in experiment, every group of 4 parallel holes, and cell matched group, virus control group add isopyknic liquid of keeping, and administration group sample is got TC 0Following 5 doubling dilution degree administration respectively.37 ℃, 5%CO 2Condition is cultivated, every day observation of cell pathological changes situation, the cytopathy degree by six grade standards judge (: no pathological changes; ±: cytopathy accounts for below 10% of whole monolayer; 1: cytopathy accounts for 10% to 25% of whole monolayer; 2: cytopathy accounts for 25% to 50% of whole monolayer; 3: cytopathy accounts for 50% to 75% of whole monolayer; 4: cytopathy accounts for more than 75% of whole monolayer).
Record experimental result when virus control group cytopathy is 4.Press the Reed-Muench method and calculate medium effective concentration EC 50, and the selection index TI (TC of calculation sample 50/ EC 50), the result sees table 2.
According to the result of calculation of selection index TI, think that Pericarpium Granati extract SL1, SL2, SL3 all have inhibitory action to influenza A virus and Influenza B virus in cell in vitro is cultivated.Experimental result explanation Pericarpium Granati extract sample SL1, SL2, SL3 have tangible resisiting influenza virus effect.
Experimental example 4: Pericarpium Granati extract of the present invention is to the inhibitory action of human hepatitis B virus
1. experiment material
1.1 sample: Pericarpium Granati extract SL1, SL2, SL3, all be mixed with the 10mg/ml original liquid before the experiment with high purity water, in 4 ℃ of preservations, face the time spent to be diluted to the desired concn administration after the filtration sterilization with culture fluid; The positive control drug lamivudine is mixed with the stock solution of 1mg/ml with high purity water, is made into the desired concn administration with cell culture fluid before the experiment.
1.2 reagent: MEM culture medium, GiBco, Invitrogen Corporation; Pancreatin, Beijing three rich Radix Polygalae Bioisystech Co., Ltd; Calf serum, HEPES, Chinese medical courses in general institute biomedical engineering institute; Hepatitis B virus nucleic acid detection by quantitative reagent, cPCR-fluorescent probe, Da.
1.3 cell strain: 2.2.15 cell, hepatitis B virus (HBV) dna clone transfection human liver cancer cell (Hep G 2) 2.2.15 cell line, available from The People's Hospital of Peking University hepatopathy center, with the MEM culture fluid that contains glutamine in 37 ℃, 5%CO 2Cultivate under the condition, an about week goes down to posterity once.
2. method and result
Cytotoxic mensuration 2.2.15 cell inoculation 96 well culture plates when treating that cell grows up to monolayer, are inhaled and are abandoned supernatant, establish the administration group of cell matched group and SL1, SL2, each 6 concentration of SL3, every group 4 hole.Each sample stock solution is done 2 times of dilutions with culture fluid respectively, gets 1/5 to 1/320 totally 7 dilution factors, application of sample respectively, and the cell matched group adds culture fluid, and 200 μ l/ holes are in 37 ℃, 5%CO 2Condition is cultivated, and respectively organizes the medicinal liquid that concentration is answered in commutation on the 4th day, and the 8th day is index in the microscopically observation with cytopathy CPE, and degree is divided into Pyatyi, and destroying fully is 4; 75% is 3; 50% is 2; 25% is 1; Anosisly become 0.Calculate every concentration liquid average cell lesion degree and suppress percentage rate.Observe maximal non-toxic concentration TC 0, and press the Reed&Muench method and calculate the poisonous concentration TC of half 50, the result sees table 3.
The inhibitory action 2.2.15 cell inoculation 96 porocyte culture plates (about 100,000/milliliter) that sample duplicates hepatitis B virus DNA, every hole 200 microlitres, 37 ℃, 5%CO 2Condition is cultivated, treat that cell grows up to monolayer after, establish cell matched group, lamivudine (1 mcg/ml) group, H1, H2, each four concentration of H3 and (all get TC 0And 3 following 2 times of dilution factors), every concentration 4 holes, application of sample, 200 microlitres/hole are in 37 ℃, 5%CO 2Condition is cultivated, and changes medicinal liquid one time on the 4th day, continues to cultivate, and in the 8th day collecting cell culture supernatant, measures hepatitis B virus DNA expression (copies/ml), the suppression ratio of calculation sample with fluorescence quantitative PCR method.Press the Reed&Muench method and calculate medium effective concentration IC 50, and calculating selection index SI, the result sees table 4.
Suppression ratio (%)=(matched group expression-administration group expression)/matched group expression * 100%
Selection index SI=TC 50/ IC 50
The selection index of three sample inhibition hepatitis B virus duplications shows that all greater than 2 Pericarpium Granati extract has tangible anti-HBV effect.
Test Example 4: Pericarpium Granati extract of the present invention is had a liking in the T cell line MT-4 cell that T lymphocyte virus I type infects the antigenic inhibitory action of HIV HIV-1 P24 the mankind
MT-4 cell suspension counting is used 100CCID 50HIV-1 IIIB infection cell, at 37 ℃, 5%CO 2Absorption is 1.5 hours in the incubator.With the RPMI-1640 flush away of serum-free viral adsorption not, the cell of infective virus is made into 2 * 10 with culture fluid 5Individual cells/ml is inoculated in the 96 porocyte culture plates, every hole 100 microlitres; Add the sample solution of 3 times of dilutions and positive control drug zidovudine (AZT) solution 100 microlitres of 5 times of dilutions more respectively.Each dilution factor repeats 3 holes, establishes cell matched group and virus control group simultaneously.Culture plate places 37 ℃, 5%CO 2Cultivate in the saturated humidity incubator.Sucking-off supernatant after 4 days ,-20 ℃ are frozen, and HIV-1P24 antigen titre to be measured, cell add the toxicity of MTT dyeing working sample pair cell.
Cytotoxicity is measured in MTT dyeing: the every hole of above-mentioned culture plate adds the MTT solution 10 microlitres dyeing of 5 mg/ml, 37 ℃, 5%CO 2Cultivate after 4 hours in the saturated humidity incubator, every hole adds 100 microlitre 50%DMF-17%Triton X-100 destaining solution, puts under 37 ℃ and spends the night, and on ELIASA, measuring wavelength is the trap value of 570 nanometers, the poisonous concentration (CC of the half of calculation sample 50).
Working sample suppresses the antigenic EC of HIV-1P24 in cell culture 50: dilute after the MT-4 supernatant of above-mentioned frozen infective virus melted, the result adjusts dilution ratio by preliminary experiment.Measure HIV-1 P24 antigen titre according to the explanation of HIV-1P24 antigen detecting agent box, application of sample group and virus control group compare, the EC of calculation sample 50And selection index TI (CC 50/ EC 50).The result sees the following form 5.
Pericarpium Granati extract SL1, SL2, SL3 show that to the inhibiting selection index SI of HIV-1 P24 antigen Pericarpium Granati extract of the present invention has tangible AIDS resisting toxic action in this experiment.
Can find out that from the foregoing description Pericarpium Granati extract of the present invention has the broad-spectrum disease resistance toxic action, can be used for preparing the broad-spectrum antiviral medicament of preventing and treating influenza, hepatitis B and AIDS.
Be table 1 to table 5 below:
Table 1 sample is to the TC of MDCK cultured cell 0And TC 50
Sample Stock solution (mg/ml) TC 0(mcg/ml) TC 50(mcg/ml)
SL1 10 500 1349.6
SL2 10 250 682.5
SL3 10 125 387.2
Ribavirin 10 62.5 276.7
Table 2. Pericarpium Granati extract is to the inhibitory action of influenza virus
Sample Virus is planted EC 50Mcg/ml TI
SL1 The first type 174.3 7.7
B-mode 396.4 3.4
SL2 The first type 69.5 9.8
B-mode 135.8 5.0
SL3 The first type 45.2 8.5
B-mode 67.6 5.7
Ribavirin The first type 5.6 49.4
B-mode 23.0 12.0
The toxicity of table 3 sample in the 2.2.15 cell culture
Laboratory sample Original liquid concentration (mg/ml) TC 50(mcg/ml) TC 0(mcg/ml)
SL1 10 1357.6 500
SL2 10 705.2 250
SL3 10 394.8 125
Table 4 sample is the 8th day inhibitory action to HBV-DNA in the 2.2.15 cell culture
Table 5
Sample CC 50(mcg/ml) EC 50(mcg/ml) SI
SL1 1045.3 144.6 7.2
SL2 864.5 75.7 11.4
SL3 652.8 51.4 12.7
Zidovudine 14.0 0.0085 1647

Claims (3)

1. one kind is used to prepare the Pericarpium Granati extract to influenza virus and/or HIV, it is characterized in that this Pericarpium Granati extract contains 30% to 70% flavones ingredient and 10% to 30% tannin constituents by weight percentage; Wherein, this extract obtains by following step:
The first step, Pericarpium Granati coarse powder water and/or ethanol are slightly carried, wherein, water slightly carry for: the Pericarpium Granati coarse powder is added 8 times to 10 times water gagings, down extracts twice, each 2 hours to 3 hours, filter merging filtrate at 80 ℃ to 100 ℃; Ethanol slightly carry for: the Pericarpium Granati coarse powder is added 8 times to 10 times amount 30% to 70% ethanol, extracts twice, each 2 hours to 3 hours, filter merging filtrate at 60 ℃ to 80 ℃;
Second step was concentrated into 1/3rd of original volume with above-mentioned filtrating, added the dehydrated alcohol that is equivalent to 2 times of amounts of concentrated solution volume, placed deposition 24 hours, and filtering precipitate obtains alcoholic solution;
In the 3rd step, it is 3 to 7 that above-mentioned alcoholic solution is concentrated back adjusting pH value, last macroporous resin chromatographic column absorption; Wherein, macroporous resin adopts a kind of in HPD400, HPD600, D-101, D-201, the AB-8 type low pole macroporous resin;
The 4th step; To be equivalent to the above-mentioned chromatographic column of deionized water rinsing of 3 to 8 chromatographic column volumes, be that eluant carries out eluting with 30% to 70% ethanol again, the consumption of eluant is equivalent to 3 to 8 chromatographic column volumes; Flow velocity be 2 to 5 chromatographic column volumes/hour, collect ethanol elution and merge;
The 5th step concentrated the ethanol elution after the above-mentioned merging down at 60 ℃ to 90 ℃, reclaimed ethanol, again at 50 ℃ to 80 ℃ following drying under reduced pressure, promptly got required Pericarpium Granati extract.
2. one kind is used to prepare the production method to the Pericarpium Granati extract of influenza virus and/or HIV, it is characterized in that this method undertaken by following step:
The first step, the Pericarpium Granati coarse powder is slightly carried with water and/or ethanol; In second step, add the impurity in the ethanol precipitation crude extract; In the 3rd step, alcoholic solution concentrates the back and regulates pH value, with the total flavones in the macroporous resin adsorption crude extract; In the 4th step, obtain eluent with ethanol eluting total flavones from the macroporous resin; The 5th step concentrated eluent and recovery ethanol, and drying under reduced pressure promptly gets Pericarpium Granati extract; Wherein:
The water that in the first step, the Pericarpium Granati coarse powder is added 8 times of amount to 10 times amounts extracts twice at 60 ℃ to 80 ℃, each 2 hours to 3 hours, filters merging filtrate; In the first step, the Pericarpium Granati coarse powder added the ethanol of 8 times of amounts to 10 times amount 30% to 70%, extract twice, each 2 hours to 3 hours, filter merging filtrate at 60 ℃ to 80 ℃;
In second step, will filtrate adds the dehydrated alcohol that is equivalent to 2 times of amounts of concentrated solution volume after concentrating, and places and precipitates 24 hours, and filtering precipitate obtains alcoholic solution; In the 3rd step, alcoholic solution being concentrated back adjusting pH value is 3 to 7, last macroporous resin chromatographic column absorption; Wherein, the big pore resin is selected a kind of in HPD400, HPD600, D-101, D-201, the AB-8 type low pole macroporous resin for use; The 4th the step in to be equivalent to the deionized water rinsing chromatographic column of 3 to 8 chromatographic column volumes; Be that eluant carries out eluting with 30% to 70% ethanol again; The consumption of eluant is equivalent to 3 to 8 chromatographic column volumes, flow velocity be 2 to 5 chromatographic column volumes/hour, collect ethanol elution and merge; In the 5th step, ethanol elution is concentrated down at 60 ℃ to 90 ℃, reclaim ethanol,, promptly obtain required Pericarpium Granati extract again at 50 ℃ to 80 ℃ following vacuum dryings.
3. a Pericarpium Granati extract according to claim 1 is applied to prepare the antiviral drugs to influenza virus and/or HIV.
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