CN103893280A - Application of bistort rhizome extract in preparation of antiviral drug and extraction method for bistort rhizome extract - Google Patents
Application of bistort rhizome extract in preparation of antiviral drug and extraction method for bistort rhizome extract Download PDFInfo
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Abstract
The invention discloses a preparation method and novel application of a bistort rhizome extract. The bistort rhizome extract uses bistort rhizome as a raw material and is prepared through extraction of bistort rhizome by using an organic solvent. The novel application of the bistort rhizome extract is that the bistort rhizome extract can be used for preparing drugs or pharmaceutical compositions capable of resisting herpes viruses, human papilloma viruses and vesicular stomatitis viruses as the extract has a good antiviral effect.
Description
Technical field
The invention belongs to the field of Chinese medicines, be specifically related to Rhizoma Bistortae extract in preparation the application in antiviral drugs and the preparation method of this Rhizoma Bistortae extract.
Background technology
Rhizoma Bistortae, for the rhizome of polygonaceae plant Rhizoma Bistortae (Polygonum bistorta L.), be used for the treatment of clinically bacillary dysentery, enteritis, pulmonary tuberculosis, chronic tracheitis, and research is found, Rhizoma Bistortae has obvious analgesic activity, (the Chinese Clinical rehabilitation such as protective effect, DNA cleavage activity to coronary artery injury, 2005,9(6); Time precious 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 virus subfamily, be divided into 1 type and 2 types (HSV-1 and HSV-2) according to antigenic difference, can cause lip herpes, herpetic keratitis, herpetic dermatitis, Herpes genitalis, kaposi's disease etc., sometimes be also the cause of disease of meningitis, encephalitis, HSV-1 often causes that waist infects above, especially the infection at eye and position, oral cavity, HSV-1 also can cause genital herpes (10%).
Human papillomavirus HPV(Human papillomavirus) be that a kind of papillary tumor vacuolating virus A that belongs to papovaviridae belongs to, it is spherical DNA viruses, people is its unique host, HPV is relevant with various diseases, be divided into many hypotypes, to organizing vicious transformation degree difference, can be divided into high-risk-type, middle danger type and low risk three classes by it.HPV is different from other pathogen; its not causing inflammation property reaction; but inspire the propagation of host cell; be tumprigenicity growth, can cause the generation of malignant tumor, as skin carcinoma, carcinoma of tongue etc.; at the end of last century; the chronic infection of having confirmed in the world epithelium of cervix uteri HPV is the cause of disease of cervical cancer, and HPV often can cause the mankind's various warts in addition, as verruca vulgaris, verruca plana, plantar wart, periungual wart and condyloma acuminatum etc.Condyloma acuminatum is one of topmost sexually transmitted disease (STD) of China, has at least 10 types relevant with it, and wherein the most common with HPV6,11 types again, current therapeutic modality is difficult to directly start with from virus, therefore cures the symptoms, not the disease.
Vesicular stomatitis virus VSV(Vesicular stomatitis virus) be the member of Rhabdoviridae, vesiculovirus genus, be sub-thread minus-stranded rna virus, can cause the diseases such as herpetic stomatitis.Herpetic stomatitis is the acute height contagious disease of multiple mammiferous one.With animals such as horse, cattle, pigs, compared with susceptible, sheep and goat also can infect.People also occasionally has infection, causes influenza-like symptom, and severe patient can cause encephalitis.Primary disease is classified as category-A disease by International Office of Epizootics (OIE), is external zoosis at China's herpetic stomatitis, and country passes in and out in animal quarantine object and classifies VSV as two class epidemic diseases.
Up to now, not yet find that Rhizoma Bistortae extract has the document that suppresses HSV virus, HPV virus and VSV virus function.
Summary of the invention
Present inventor finds in the process of the medical usage of the Rhizoma Bistortae that studies for a long period of time, and Rhizoma Bistortae extract has significant inhibitory action to HSV-1, HPV6/11 and VSV virus.
One object of the present invention is to provide a kind of preparation method of preferred Rhizoma Bistortae extract, take organic solvent as extracting solvent, from Rhizoma Bistortae, extracts and obtains Rhizoma Bistortae extract.
Wherein, described organic solvent is selected from: methanol, ethanol, acetone, propanol, n-butyl alcohol, ethyl acetate, preferably, described organic solvent is that volume fraction is 50%～95% ethanol water.
Wherein, described in be extracted as heating and refluxing extraction, at least extract once, each extraction time is 1 ~ 4h.
Another object of the present invention is to provide application said extracted Rhizoma Bistortae extract prepared by method.
Another object of the present invention is to provide the application of application said extracted Rhizoma Bistortae extract prepared by method for the preparation of antiviral drugs.
Particularly, Rhizoma Bistortae extract is in the application of preparing in anti-herpesvirus, human papillomavirus, vesicular stomatitis virus medicine.Further, described herpesvirus is HSV-1, and described human papillomavirus is HPV6 and HPV11.
In addition, the present invention also aims to provide a kind of pharmaceutical composition, comprise described Rhizoma Bistortae extract and at least one pharmaceutically suitable carrier for the treatment of effective dose." treatment effective dose " refers to the amount of the extract that can reach therapeutic effect.Those skilled in the art can understand " treatment effective dose " and can and may share etc. with other treatment agent and different along with the use of the mode of administration, carrier.Preferably, the Rhizoma Bistortae extract of the present invention as active component that pharmaceutical composition of the present invention contains 0.1-99.9% percentage by weight." pharmaceutically suitable carrier " can not destroy the pharmaceutical active of Rhizoma Bistortae extract of the present invention, simultaneously its effective dose, and can bringing into play pharmaceutical carrier, to make the consumption of used time nontoxic to human body.Described pharmaceutically suitable carrier includes but not limited to: soft phospholipid, aluminium stearate, aluminium oxide, ion exchange material, self-emulsifying drug delivery system, tween or other surfactants, serum albumin, buffer substance are if phosphate, glycine, sorbic acid, water, salt, electrolyte are as sulfate protamine, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, magnesium silicate, satisfied fatty acid partial glycerol ester admixture etc.
Pharmaceutical composition of the present invention also can comprise the excipient substance that one or more are conventional, as binding agent (as microcrystalline Cellulose), filler (as starch, glucose, Lactis Anhydrous and lactose beadlet), disintegrating agent (as cross-linked pvp, crosslinked carboxymethyl fecula sodium, cross-linking sodium carboxymethyl cellulose, low-substituted hydroxypropyl cellulose), lubricant (as magnesium stearate) and absorption enhancer, absorption carrier, flavouring agent, sweeting agent, excipient, diluent, wetting agent etc.
Described pharmaceutical composition can and can pass through intestinal or non-intestinal or topical routes by the preparation of this area conventional method.Oral formulations comprises tablet, granule, suspension, capsule, solution etc., and non-intestinal drug delivery agent comprises injection.Local administration preparation comprises cream, patch, ointment, spray, gel etc.
The route of administration of described Rhizoma Bistortae extract and pharmaceutical composition can be oral, Sublingual, percutaneous, through muscle or subcutaneous, mucocutaneous, vein, urethra, vagina etc.
The consumption of described Rhizoma Bistortae extract and pharmaceutical composition can be according to the difference of route of administration, patient's age and body weight, disease serious degree and difference, and its daily dose can be 0.001～100mg/kg, can be in single or divided doses.
The present invention Rhizoma Bistortae extract is further proposed or take its compositions as primary activity composition in prevention with treat the purposes in the various diseases that caused by HSV-1 virus, HPV6/11 virus and VSV virus.
Beneficial effect of the present invention: find in the application's research, apply Rhizoma Bistortae extract prepared by extracting method of the present invention HSV-1, HPV6/11, VSV are all had to stronger inhibitory action, illustrate that Rhizoma Bistortae extract of the present invention has good antivirus action, can be used for preparing anti-herpesvirus, human papillomavirus, vesicular stomatitis virus medicine or pharmaceutical composition.
The specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.Should be understood that following examples are only for the present invention is described but not for limiting scope of the present invention.
Related percentage ratio (%) in following examples, except special instruction, is mass percent.
embodiment 1, Rhizoma Bistortae extract preparation
Take Rhizoma Bistortae medicinal material coarse powder appropriate, add 10 times of amount 50% alcohol reflux 1 time, 1h/ time, filter, merging filtrate, filtrate decompression is concentrated into respect to containing crude drug amount 1g/ml, and concentrated solution is diluted with water to and is equivalent to, containing crude drug amount 0.25g/ml, stir, hold over night, the centrifugal supernatant that obtains, supernatant is evaporated to extractum, and extractum lyophilization obtains Rhizoma Bistortae extract (wherein content of tannin 35%).
embodiment 2, Rhizoma Bistortae extract preparation
Take Rhizoma Bistortae medicinal material coarse powder appropriate, add 10 times of amount 70% alcohol reflux 3 times, 2h/ time, filter, merging filtrate, filtrate decompression is concentrated into respect to containing crude drug amount 1g/ml, and concentrated solution is diluted with water to and is equivalent to, containing crude drug amount 0.25g/ml, stir, hold over night, the centrifugal supernatant that obtains, supernatant is evaporated to extractum, and extractum lyophilization obtains Rhizoma Bistortae extract (wherein content of tannin 40%).
embodiment 3, Rhizoma Bistortae extract preparation
Take Rhizoma Bistortae medicinal material coarse powder appropriate, add 10 times of amount 95% alcohol reflux 2 times, 4h/ time, filter, merging filtrate, filtrate decompression is concentrated into respect to containing crude drug amount 1g/ml, and concentrated solution is diluted with water to and is equivalent to, containing crude drug amount 0.25g/ml, stir, hold over night, the centrifugal supernatant that obtains, supernatant is evaporated to extractum, and extractum lyophilization obtains Rhizoma Bistortae extract (wherein content of tannin 43%).
embodiment 4, the inhibitory action of Rhizoma Bistortae extract to HSV-1 virus
Experiment material:
Cell: human lung adenocarcinoma cell (A549), purchased from Chinese Academy of Sciences's cell bank.
Virus: HSV-1-GFP.
Tested medicine: Rhizoma Bistortae extract (sample prepared by embodiment 1), be dissolved in DMSO, be prepared into 60mg/mL mother solution.
Experimental technique:
A549 cell is cultivated according to a conventional method, is inoculated in 96 orifice plates next day, after cell attachment, virus infected cell, after 1 hour, removes containing virus-culturing fluid, with certain density tested drug treating cell, fluorescence microscopy Microscopic observation GFP protein expression situation after 18 hours.Process cell as positive control using the acyclovir of 1 μ g/mL simultaneously.
Experimental result as shown in the following Table 1.
Table 1, the inhibition of Rhizoma Bistortae extract to HSV-1 virus
The experimental result of above table 1 shows, in human lung adenocarcinoma cell, Rhizoma Bistortae extract has obvious inhibitory action to HSV-1, and Rhizoma Bistortae extract is dose-dependence to the inhibition of HSV-1.
embodiment 5, the inhibitory action of Rhizoma Bistortae extract to HPV6/11 type virus
Experiment material:
HPV6/11 type viral source: HPV6,11 type viruses are taken from the wart body of untreated Condyloma acuminatum, grind to form tissue homogenate for subsequent use after normal saline cleans under aseptic condition.
Tested medicine: Rhizoma Bistortae extract (sample prepared by embodiment 2), be dissolved in DMSO, be prepared into 60mg/mL mother solution.
Key instrument: ABI7500 quantitative real time PCR Instrument.
HPV6/11 type FQ-PCR diagnostic kit: Da'an Gene Company, Zhongshan University.
Experimental technique:
Get Condyloma Acuminata homogenate 100 μ L, add tested medicine 10 μ L, control tube adds normal saline 10uL, feminine gender and positive control pipe are established in each experiment simultaneously, hatch for 37 ℃ and (in time gradient experiment, respectively hatch 5 minutes 10 minutes in 20 hours, 15 minutes, 30 minutes, 60 minutes).
Press kit method and extract DNA, get 2 μ L and add in reaction tube, put into ABI7500 quantitative real time PCR Instrument and react, quantitative result, by computer software automatic analysis, calculates its initial copy number.
Experimental result is as shown in following table 2 and table 3.
Table 2, the inhibitory action of Rhizoma Bistortae extract to HPV6/11 type virus
The experimental result of above table 2 shows, the Rhizoma Bistortae extract effect of 25 μ g/mL is after 20 hours, HPV6/11 virus is examined and is not measured, and blank pipe has the very HPV6/11 virus quantity of high concentration, illustrate that Rhizoma Bistortae extract has the effect of well killing HPV6/11 virus, and this action effect is dose-dependent effect.
The time-dependent effect that table 3, Rhizoma Bistortae extract suppress HPV6/11 virus
The experimental result of above table 3 shows, Rhizoma Bistortae extract, under the concentration of 25 μ g/mL, can be killed HPV6/11 virus in 5 minutes completely.
embodiment 6, the inhibitory action of Rhizoma Bistortae extract to VSV virus
Experiment material:
Cell: human lung adenocarcinoma cell (A549), purchased from Chinese Academy of Sciences's cell bank.
Virus: VSV-GFP.
Tested medicine: Rhizoma Bistortae extract (pressing embodiment 3 method preparations), be dissolved in DMSO, be prepared into 60mg/mL mother solution.
Experimental technique:
A549 cell is cultivated according to a conventional method, is inoculated in 96 orifice plates next day, after cell attachment, with VSV-GFP virus infected cell, after 1 hour, remove containing virus-culturing fluid, process cell, fluorescence microscopy Microscopic observation GFP protein expression situation after 18 hours with certain density Rhizoma Bistortae extract.Process cell as positive control using the interferon-ALPHA (IFN α) of 10IU/mL simultaneously.
Experimental result as shown in the following Table 4.
Table 4, the inhibition of Rhizoma Bistortae extract to VSV virus
The experimental result of above table 4 shows, in human lung adenocarcinoma cell, Rhizoma Bistortae extract has obvious inhibitory action to VSV virus, and Rhizoma Bistortae extract is dose-dependence to the inhibition of VSV virus.
In sum, Rhizoma Bistortae extract has good inhibitory action to HSV-1, HPV6/11 and VSV virus, can be used for preparing the medicine of anti-HSV-1, HPV6/11 and VSV virus, or further make antiviral medicinal composition, except the Rhizoma Bistortae extract take treatment effective dose is as active component, described pharmaceutical composition also comprises one or more pharmaceutically suitable carrier, and described pharmaceutically suitable carrier can be selected conventional carrier as known in the art; In addition, described pharmaceutical composition can also comprise the excipient substance that one or more are conventional, and this is apparent for a person skilled in the art.Before not departing from spirit of the present invention, put, those skilled in the art can be equal to and change and modify it, and described change and modification fall into the coverage of the application's claims equally.
Claims (10)
1. the preparation method of Rhizoma Bistortae extract, is characterized in that: take organic solvent as extracting solvent, from Rhizoma Bistortae, extract and obtain Rhizoma Bistortae extract.
2. the preparation method of Rhizoma Bistortae extract as claimed in claim 1, is characterized in that: described organic solvent is selected from: methanol, ethanol, acetone, propanol, n-butyl alcohol, ethyl acetate.
3. the preparation method of Rhizoma Bistortae extract as claimed in claim 2, is characterized in that: described organic solvent is that volume fraction is 50%～95% ethanol water.
4. the preparation method of Rhizoma Bistortae extract as claimed in claim 1, is characterized in that: described in be extracted as heating and refluxing extraction, at least extract once, each extraction time is 1 ~ 4h.
5. the Rhizoma Bistortae extract that prepared by the preparation method of the Rhizoma Bistortae extract as described in any one in claim 1～4.
6. the application of Rhizoma Bistortae extract as claimed in claim 5 in preparation antiviral drugs.
7. the application of Rhizoma Bistortae extract as claimed in claim 6 in preparation antiviral drugs, is characterized in that, described virus comprises: herpesvirus, human papillomavirus, vesicular stomatitis virus.
8. the application of Rhizoma Bistortae extract as claimed in claim 7 in preparation antiviral drugs, is characterized in that, described herpesvirus is HSV-1.
9. the application of Rhizoma Bistortae extract as claimed in claim 7 in preparation antiviral drugs, is characterized in that, described human papillomavirus is HPV6 and HPV11.
10. a pharmaceutical composition, is characterized in that, described pharmaceutical composition comprises the Rhizoma Bistortae extract as claimed in claim 5 for the treatment of effective dose, and at least one pharmaceutically suitable carrier.
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| CN1660373A (en) * | 2005-01-31 | 2005-08-31 | 北京正大绿洲医药科技有限公司 | Bistort drop pill and preparation method |
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| CN1660373A (en) * | 2005-01-31 | 2005-08-31 | 北京正大绿洲医药科技有限公司 | Bistort drop pill and preparation method |
Non-Patent Citations (2)
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| 梁波等: "中药拳参化学成分及药理活性研究进展", 《甘肃高师学报》 * |
| 附属医院儿科教研室: "中药"抗病毒注射液"及其治疗小儿肺炎疗效观察", 《山东医学院学报》 * |
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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 |