CN103893280B - Application and its extracting method of the Rhizoma Bistortae extract in antiviral drugs is prepared - Google Patents
Application and its extracting method of the Rhizoma Bistortae extract in antiviral drugs is prepared Download PDFInfo
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Abstract
The invention discloses the preparation method and its new application of a kind of Rhizoma Bistortae extract, extract of the present invention is using adder-wort as raw material, is prepared with organic solvent extraction.The new application of the Rhizoma Bistortae extract of the present invention is that the extract has preferable antivirus action, available for preparing anti-herpesvirus, human papilloma virus, vesicular stomatitis virus drug or pharmaceutical composition.
Description
Technical field
The invention belongs to the field of Chinese medicines, and in particular to application and the fist of the Rhizoma Bistortae extract in antiviral drugs is prepared
The preparation method of conopsea extraction.
Background technology
Adder-wort is polygonaceae plant adder-wort（Polygonum bistorta L.）Rhizome, clinically for treat bacillary dysentery,
Enteritis, pulmonary tuberculosis, chronic bronchitis, and the study found that adder-wort has apparent analgesic activity, the protection to coronary artery injury
Effect, DNA lytic activities etc.（Chinese Clinical rehabilitation, 2005,9（6）；When treasure's traditional Chinese medical science traditional Chinese medicines, 2006,17（6）；Chinese herbal medicine,
2003,34（3））.
Herpes simplex virus HSV（Herpes simplex virus）Belong to herpetoviridae a Chordopoxvirinaes, according to antigen
The difference of property is divided into 1 type and 2 types（HSV-1 and HSV-2）, lip bleb, herpetic keratitis, herpetic skin can be caused
Inflammation, Herpes genitalis, kaposi's disease etc. are also sometimes meningitis, the cause of disease of encephalitis, and HSV-1, which often causes more than waist, to be infected, especially
Eye and the infection at oral cavity position, HSV-1 can also cause genital herpes（10%）.
Human papilloma virus HPV（Human papillomavirus）It is that a kind of papilloma for belonging to papovaviridae is empty
It steeps virus A to belong to, is spherical DNA virus, people is its unique host, and HPV is related with a variety of diseases, is divided into many hypotypes, by it
It is different to tissue vicious transformation degree, high-risk-type, middle danger type and low risk three classes can be divided into.HPV is different from other pathogens, it
Do not cause inflammatory reaction, but inspire the proliferation of host cell, grown in tumprigenicity, the generation of malignant tumour can be caused,
Such as cutaneum carcinoma, tongue cancer, the end of last century, the chronic infection for having confirmed that epithelium of cervix uteri HPV in the world are the causes of disease of cervical carcinoma, this
Outer HPV can often cause the various warts of the mankind, such as verruca vulgaris, flat wart, plantar wart, periungual wart and condyloma acuminatum.Condyloma acuminatum is
One of most important venereal disease in China, at least 10 types are associated, wherein again most common with HPV6,11 types, current controls
Treatment mode is difficult directly to start with from virus, therefore palliative.
Vesicular stomatitis virus VSV（Vesicular stomatitis virus）For Rhabdoviridae, vesiculovirus genus
Member, be sub-thread minus-stranded rna virus, the diseases such as vesicular stomatitis can be caused.Vesicular stomatitis is the one of a variety of mammals
The acute highly contagious disease of kind.More susceptible with animals such as horse, ox, pigs, sheep and goat can also infect.People's also even thoughts
Dye, causes influenza-like symptom, severe patient can cause encephalitis.This disease is by International Office of Epizootics（OIE）A class diseases are classified as, in China's water
Blister stomatitis is exotic animals disease, and country passes in and out and VSV is classified as two class epidemic diseases in animal quarantine object.
Up to now, not yet find that Rhizoma Bistortae extract has the text for inhibiting HSV viruses, HPV viruse and VSV virus functions
It offers.
Invention content
Present inventor has found during the medical usage for the adder-wort that studies for a long period of time, Rhizoma Bistortae extract to HSV-1,
HPV6/11 and VSV viruses have significant inhibiting effect.
It is an object of the present invention to provide a kind of preparation method of preferred Rhizoma Bistortae extract, using organic solvent to carry
Solvent is taken, is extracted from adder-wort and obtains Rhizoma Bistortae extract.
Wherein, the organic solvent is selected from：Methanol, ethyl alcohol, acetone, propyl alcohol, n-butanol, ethyl acetate, it is preferable that described
Organic solvent is the ethanol water that volume fraction is 50%~95%.
Wherein, described to be extracted as heating and refluxing extraction, at least extraction is primary, and each extraction time is 1 ~ 4h.
It is another object of the present invention to provide Rhizoma Bistortae extracts prepared by application said extracted method.
It is used to prepare it is another object of the present invention to provide Rhizoma Bistortae extract prepared by application said extracted method anti-
The application of virus drugs.
Particularly, Rhizoma Bistortae extract is in anti-herpesvirus, human papilloma virus, vesicular stomatitis virus drug is prepared
Using.Further, the herpesviral is HSV-1, and the human papilloma virus is HPV6 and HPV11.
In addition, the present invention also aims to provide a kind of pharmaceutical composition, the adder-wort including therapeutically effective amount carries
Take object and at least one pharmaceutical acceptable carrier." therapeutically effective amount " is the amount for the extract for referring to reach therapeutic effect.This field
Technical staff it will be appreciated that " therapeutically effective amount " can with the mode of administration, the use of carrier and may and other treatment
Agent shares etc. and different.Preferably, the pharmaceutical composition of the present invention contains the conduct activity of 0.1-99.9% weight percent
The Rhizoma Bistortae extract of the present invention of ingredient." pharmaceutical acceptable carrier " will not destroy the pharmaceutical active of the Rhizoma Bistortae extract of the present invention, together
When its effective dose, can play pharmaceutical carrier effect when dosage it is nontoxic to human body.Described pharmaceutical acceptable carrier includes but unlimited
In：Soft phosphatide, aluminum stearate, aluminium oxide, ion exchange material, self-emulsifying drug delivery system, tween or other surfaces activation
Agent, haemocyanin, buffer substance such as phosphate, amion acetic acid, sorbic acid, water, salt, electrolyte such as sulfate protamine, phosphoric acid
Disodium hydrogen, potassium hydrogen phosphate, sodium chloride, zinc salt, magnesium silicate, saturated fatty acid partial glyceride mixtures etc..
The pharmaceutical composition of the present invention may also include one or more common excipient substances, such as adhesive（Such as crystallite fibre
Dimension element）, filler（Such as starch, glucose, Lactis Anhydrous and lactose bead）, disintegrant（As cross-linked pvp, cross-linked carboxymethyl form sediment
Powder sodium, croscarmellose sodium, low-substituted hydroxypropyl cellulose）, lubricant（Such as magnesium stearate）And sorbefacient,
Absorption carrier, flavouring agent, sweetener, excipient, diluent, wetting agent etc..
Described pharmaceutical composition can be prepared by this field conventional method and can pass through enteron aisle or non-bowel or topic route
Administration.Oral preparation includes tablet, granule, suspension, capsule, solution etc., and non-intestinal drug delivery agent includes parenteral solution.Part
Drug-delivery preparation includes creme, patch, ointment, spray, gelling agent etc..
The administration route of the Rhizoma Bistortae extract and pharmaceutical composition can be oral, sublingual, percutaneous, through muscle or skin
Under, mucocutaneous, vein, urethra, vagina etc..
The dosage of the Rhizoma Bistortae extract and pharmaceutical composition can be according to administration route, the age of patient and weight, illness
The difference of severity and it is different, daily dose can be 0.001~100mg/kg, can be in single or divided doses.
Present invention further propose that Rhizoma Bistortae extract or being prevented and treated using its composition as primary activity ingredient
Purposes in the various diseases as caused by HSV-1 viruses, HPV6/11 viruses and VSV viruses.
Beneficial effects of the present invention：It is found in the research of the application, the adder-wort prepared using the extracting method of the present invention
Extract has HSV-1, HPV6/11, VSV stronger inhibiting effect, illustrates the Rhizoma Bistortae extract of the present invention and has preferably
Antivirus action, available for preparing anti-herpesvirus, human papilloma virus, vesicular stomatitis virus drug or pharmaceutical composition.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention is not for restriction the scope of the present invention.
Involved percentage in following embodiment（%）, it is mass percent unless otherwise indicated.
Embodiment 1, Rhizoma Bistortae extract preparation
It is appropriate to weigh adder-wort medicinal material coarse powder, 10 times of 50% alcohol refluxs of amount is added to extract 1 time, 1h/ times, are filtered, merging filtrate,
Filtrate decompression is concentrated into relative to 1g/ml containing crude drug amount, and concentrate, which is diluted with water to, is equivalent to 0.25g/ml containing crude drug amount, fully
It stirs evenly, stands overnight, centrifuge to obtain supernatant, supernatant is concentrated under reduced pressure into medicinal extract, and medicinal extract is freeze-dried to be extracted up to adder-wort
Object（Wherein content of tannin 35%）.
Embodiment 2, Rhizoma Bistortae extract preparation
It is appropriate to weigh adder-wort medicinal material coarse powder, 10 times of 70% alcohol refluxs of amount is added to extract 3 times, 2h/ times, are filtered, merging filtrate,
Filtrate decompression is concentrated into relative to 1g/ml containing crude drug amount, and concentrate, which is diluted with water to, is equivalent to 0.25g/ml containing crude drug amount, fully
It stirs evenly, stands overnight, centrifuge to obtain supernatant, supernatant is concentrated under reduced pressure into medicinal extract, and medicinal extract is freeze-dried to be extracted up to adder-wort
Object（Wherein content of tannin 40%）.
Embodiment 3, Rhizoma Bistortae extract preparation
It is appropriate to weigh adder-wort medicinal material coarse powder, 10 times of 95% alcohol refluxs of amount is added to extract 2 times, 4h/ times, are filtered, merging filtrate,
Filtrate decompression is concentrated into relative to 1g/ml containing crude drug amount, and concentrate, which is diluted with water to, is equivalent to 0.25g/ml containing crude drug amount, fully
It stirs evenly, stands overnight, centrifuge to obtain supernatant, supernatant is concentrated under reduced pressure into medicinal extract, and medicinal extract is freeze-dried to be extracted up to adder-wort
Object（Wherein content of tannin 43%）.
Embodiment 4, Rhizoma Bistortae extract is to the inhibiting effect of HSV-1 viruses
Experiment material：
Cell：Human lung adenocarcinoma cell（A549）, purchased from Chinese Academy of Sciences's cell bank.
Virus：HSV-1-GFP.
Test medicine：Rhizoma Bistortae extract（Sample prepared by embodiment 1）, it is dissolved in DMSO, is prepared into 60mg/mL mothers
Liquid.
Experimental method：
A549 cells are cultivated according to a conventional method, and next day is inoculated in 96 orifice plates, and after cell is adherent, virus infected cell, 1 is small
Shi Hou, removal handle cell, fluorescence microscopy Microscopic observation after 18 hours containing virus-culturing fluid with certain density test medicine
GFP protein expression situations.Cell is handled as positive control using the acyclovir of 1 μ g/mL simultaneously.
Experimental result is as shown in the following Table 1.
The inhibition of table 1, Rhizoma Bistortae extract to HSV-1 viruses
With upper table 1 the experimental results showed that, Rhizoma Bistortae extract has HSV-1 apparent inhibit in human lung adenocarcinoma cell
Effect, and Rhizoma Bistortae extract is in dose-dependence to the inhibition of HSV-1.
Embodiment 5, Rhizoma Bistortae extract is to the inhibiting effect of HPV6/11 types virus
Experiment material：
HPV6/11 type viral sources：HPV6,11 type viruses are derived from the wart of the Condyloma acuminatum of untreated, through life
It is spare that tissue homogenate is aseptically ground into after reason brine cleaning.
Test medicine：Rhizoma Bistortae extract（Sample prepared by embodiment 2）, it is dissolved in DMSO, is prepared into 60mg/mL mothers
Liquid.
Key instrument：ABI7500 fluorescence quantitative PCR instruments.
HPV6/11 type FQ-PCR diagnostic kits：Da'an Gene Company, Zhongshan University.
Experimental method：
100 μ L of Condyloma Acuminata homogenate are taken, add in 10 μ L of test medicine, control tube then adds in physiological saline 10uL,
Experiment sets negative and positive control pipe simultaneously every time, and 37 DEG C are incubated 20 hours（It is respectively incubated 5 minutes, 10 points in time gradient experiment
Clock, 15 minutes, 30 minutes, 60 minutes）.
DNA is extracted by kit method, 2 μ L is taken to add in reaction tube, ABI7500 fluorescence quantitative PCR instruments is put into and carries out instead
Should, quantitative result is automatically analyzed by computer software, calculates its initial copy number.
Experimental result is as shown in following table 2 and table 3.
Table 2, Rhizoma Bistortae extract are to the inhibiting effect of HPV6/11 types virus
With upper table 2 the experimental results showed that, after the Rhizoma Bistortae extract of 25 μ g/mL acts on 20 hours, HPV6/11 viruses have been examined
It does not measure, and blank control pipe has the HPV6/11 virus quantities of very high concentration, illustrates that Rhizoma Bistortae extract has good killing
The effect of HPV6/11 viruses, and this function and effect are in dose-dependent effect.
Table 3, Rhizoma Bistortae extract are to the time-dependent effects of HPV6/11 HIV suppressions
With upper table 3 the experimental results showed that, Rhizoma Bistortae extract can be killed for 5 minutes completely under the concentration of 25 μ g/mL
HPV6/11 viruses.
Embodiment 6, Rhizoma Bistortae extract is to the inhibiting effect of VSV viruses
Experiment material：
Cell：Human lung adenocarcinoma cell（A549）, purchased from Chinese Academy of Sciences's cell bank.
Virus：VSV-GFP.
Test medicine：Rhizoma Bistortae extract（It is prepared by 3 method of embodiment）, it is dissolved in DMSO, is prepared into 60mg/mL mothers
Liquid.
Experimental method：
A549 cells are cultivated according to a conventional method, and next day is inoculated in 96 orifice plates, after cell is adherent, with VSV-GFP virus senses
Cell is contaminated, after 1 hour, removal handles cell, fluorescence is shown after 18 hours containing virus-culturing fluid with certain density Rhizoma Bistortae extract
Micro- Microscopic observation GFP protein expression situations.Simultaneously with the interferon-' alpha ' of 10IU/mL（IFNα）Cell is handled as positive control.
Experimental result is as shown in the following Table 4.
The inhibition of table 4, Rhizoma Bistortae extract to VSV viruses
With upper table 4 the experimental results showed that, Rhizoma Bistortae extract has VSV viruses apparent suppression in human lung adenocarcinoma cell
It makes and uses, and Rhizoma Bistortae extract is in dose-dependence to the inhibition of VSV viruses.
In conclusion Rhizoma Bistortae extract has good inhibiting effect to HSV-1, HPV6/11 and VSV virus, can be used for
It prepares the drug of anti-HSV-1, HPV6/11 and VSV virus or antiviral medicinal composition is further made, in addition to control
A effective amount of Rhizoma Bistortae extract is treated as outside active constituent, described pharmaceutical composition further includes one or more pharmaceutical acceptable carrier, institute
Conventional carrier as known in the art can be selected by stating pharmaceutical acceptable carrier；In addition, described pharmaceutical composition can also include one kind
Or a variety of common excipient substances, this will be apparent to the person skilled in the art.Without departing from essence of the invention
Under the premise of god, those skilled in the art can carry out it equivalent change and modification, and the change and modification equally fall into this Shen
Please appended claims coverage area.
Claims (3)
1. application of the Rhizoma Bistortae extract in antiviral drugs is prepared, which is characterized in that the virus is：Herpesviral, human milk
Head tumor virus, vesicular stomatitis virus, wherein,
The Rhizoma Bistortae extract is prepared via a method which to obtain：Using the ethanol water that volume fraction is 50%~95% to carry
Solvent is taken, is extracted from adder-wort and obtains Rhizoma Bistortae extract, described to be extracted as heating and refluxing extraction, at least extraction is primary, carries every time
It is 1~4h to take the time.
2. application of the Rhizoma Bistortae extract as described in claim 1 in antiviral drugs is prepared, which is characterized in that the bleb
Virus is HSV-1.
3. application of the Rhizoma Bistortae extract as described in claim 1 in antiviral drugs is prepared, which is characterized in that the human milk
Head tumor virus is HPV6 and HPV11.
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Address after: 313000 Hongfeng Road, Huzhou, Zhejiang 1366 Patentee after: Huzhou Zhongke nutrition and health innovation center Address before: 313000 Hongfeng Road, Huzhou, Zhejiang 1366 Patentee before: HUZHOU R & D CENTER FOR NUTRITION AND HEALTH, SHANGHAI INSTITUTES FOR BIOLOGICAL SCIENCES, CHINESE ACADEMY OF SCIENCE |
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Effective date of registration: 20211220 Address after: 313000 Hongfeng Road, Huzhou, Zhejiang 1366 Patentee after: Huzhou Zhongke nutrition and health innovation center Patentee after: Shanghai Institute of nutrition and health, Chinese Academy of Sciences Address before: 313000 Hongfeng Road, Huzhou, Zhejiang 1366 Patentee before: Huzhou Zhongke nutrition and health innovation center |