CN1739597A - Cajan seed extract with herpesvirus resisting activity - Google Patents

Abstract

The present invention provides one kind of ethanol extract of cajan seed with herpesvirus resisting activity, and the main component of the cajan seed extract is flavone compounds with relatively high pharmacological activity and molecular weight measured in LC/MS/MS method of 200-600. The cajan seed extract has obvious herpesvirus resisting effect and is one kind of virus inhibitor with wide development foreground.

Description

A kind of Semen Cajani extract with herpesvirus resisting activity

Technical field

The present invention relates to a kind of Semen Cajani ethanol extraction with antiviral activity.

Background technology

Semen Cajani (Cajanus cajan) originates in India, and whole world cultivated area is about 5,200,000 hectares at present, mainly is distributed in Asia, Africa and South America (Zhao Zhenzhong, 2003).China cultivation Semen Cajani mainly is distributed in the torrid zone, subtropical zone, i.e. provinces and regions (Gu Yong etc., 2000) such as Yunnan, Guangxi, Sichuan, Chongqing, Guangdong, Guizhou, Hainan, Fujian, Hunan, Jiangxi, Jiangsu.

The Semen Cajani whole body is precious, and economy, the ecological value are high, also have the medicinal and health-care effect of number of significant simultaneously.The medical value of Semen Cajani develops the earliest in China, record in " Luchuan book on Chinese herbal medicine ": Folium Cajani " flat, light, slightly poisonous ".Further point out in " Chinese medicine sea ": Folium Cajani cures mainly children's's chickenpox, and pain is swollen.In recent years, the report that extracts, separates relevant medicinal ingredient in China research Semen Cajani is less, mainly contain medicinal plants developmental research institute of the Chinese Academy of Medical Sciences (year) and once studied anti-antiinflammatory composition in the Folium Cajani, draw 11 kinds of crystallised component (Li Zhenghong etc., 2001), be accredited as apigenin-8-C-glucoside, salicylic acid, hentriacontane, cupreol, naringenin-4 ', 7-dimethyl ether and β-Amyrin etc.In addition, Lin Li (year) etc. instruct the extraction separation of effective ingredients in plant with bacteriostatic test, the ethyl acetate of producing the extraction of Folium Cajani extract from Hainan is partly separated and has been identified 6 chemical compounds, apigenin-8-C-glucoside, Saponaretin, apigenin, luteolin, naringenin-4 ', 7-dimethyl ether and cupreol, in these known compositions, the vegetable flavonoid with higher pharmacologically active accounts for the overwhelming majority.

Also there are some research reports pharmacologically active aspect to Semen Cajani in recent years, mainly is that the Chinese medicine Folium Cajani has magical effect to the treatment ischemic necrosis of femoral head.Hainan People's Hospital treats wound, burn infection and decubital ulcer etc. with Semen Cajani and has obtained curative effect (Liu Zhongqiu etc., 1998) preferably according to experience among the people.The pharmacological action of Sun Shaomei etc. (1995) research Folium Cajani effective ingredient cajanin shows: the cajanin preparation has permeability, analgesia, the antiinflammation of obvious suppression blood capillary, toxicity is little, animal has no adverse reaction in effective dosage ranges, and its antiinflammatory action is better than salicylic acid.Liu Zhongqiu (1998) etc. are on the basis of the antibacterial and anti-inflammation functions that confirms Semen Cajani, with pharmacodynamicss such as antiinflammatories is index, to give birth to arteries and veins become in the osteocomma principal agent---the different extracts of Folium Cajani screen, carry out the mouse corrosion disease studied on Pharmacology of Inhibiting with alcohol extract, water extract respectively, the result shows, the antiinflammatory action that Folium Cajani water is put forward part is the strongest, but has verified the effect of utilizing Folium Cajani water decoction removing toxic substances and promoting subsidence of swelling among the people.Though extraction and separating technology about the Semen Cajani effective ingredient remain further to be studied, certainly vegetable flavonoid has played crucial effects in its performance pharmacological effect active process.

Virus is minimum pathogenic microorganism, but the propagation of virus is the widest, is one of infectious disease that sickness rate is the highest in the world at present.More clinically disease is relevant with viral infection.According to the foreign statistic document announcement, nearly 3/4 infectious disease is caused that by virus different viral infection causes different illness, need be at this viral antiviral treatment.

Herpes is the infectious skin disease that is caused by herpesvirus, shows as pain or superficial sensory disturbance, is one of common clinical frequently-occurring disease, good being sent out season in spring and autumn.Though the clinical symptoms of herpes zoster is not very serious, can cause severe complications sometimes, and the herpes zoster patient is because of spinosity sample pain or burning pain, and occurs nervously, frightened, and mood agitation, anxiety usually influence work and sleep, and therefore harm is bigger.(Shang Xiaohong, 2005; Wu Weizhen, 2004)

Herpesvirus is divided into herpes simplex, herpes zoster, cytomegalovirus and epstein-barr virus, and this viroid can form latent infection in human body, causes the different tissues disease, outbreak repeatedly under certain environment.

Herpes simplex virus (HSV) is divided into HSV-1 type and HSV-2, finds HSV-6 again after the further investigation of carrying out in recent years, 7,8 types.It is a kind of systemic disease that HSV infects, virus enters human body through respiratory tract, oral cavity, genitals mucosa or damaged skin, submersible in human body normal mucosa, blood, saliva and local sensory ganglion and most organ, all separable in nearly all internal organs and the mucodermis to HSV.Primary infection is mainly seen in immunologic hypofunction infant or severe malnutrition or the child of other infection is arranged.After primary infection was disappeared, sustainable the diving of virus occupied in the body.These viral carriers that are more than half are arranged among the normal person approximately, but through port, nasal discharge and become the source of infection, because HSV does not produce permanent immunity in human body, so when body resistance against diseases goes down, as suffer from certain infectious fever, gastrointestinal dysfunction, menstruation, gestation, focal infection, overtired, when emotional environment changes, the HSV that hides in the body promptly is excited and falls ill.Thereby seeking new anti-herpesvirus medicament becomes the task of top priority.

Antiviral class medicine is the big class medicine that development in recent years is got up, now become attract people's attention on the domestic and international medical market enliven one of medicine.Found vidarabine from 1975, acyclovir (ACV in 1977 particularly, acycloguanosine) after the appearance, antiviral drugs is really starting, however, kind surplus such medicine of listing has only 20 at present only is about 1/14th of an anti-infectives, and there are certain toxic and side effects or antiviral effect not obvious mostly, are difficult to be approved by patient.

List of references:

Zhao Zhenzhong. a kind of tropical and subtropical zone plant---Semen Cajani that development potentiality is arranged very much. Fujian hotwork science and technology, 2003,28 (3): 31～32 paddy are brave, Zhou Rong, Zou Hengfang etc. Semen Cajani cultivation technique and comprehensive utilization. the Xi'nan College of Forestry journal, 2000,20 (4): 213～217 Lee are at prime, the letter Zhou Dynasty, Gu Yong, Zhang Jianyun. Chinese Semen Cajani present Research and DEVELOPMENT PROSPECT. forestry scientific research, 2001,14 (6): 647～681 Liu mid-autumns, Zhou Hua, woods is encouraged, Liao Huifang, Zhou Liling. give birth to arteries and veins and become Folium Cajani Study on extraction in the osteocomma. Chinese patent medicine .1998,20 (3): 7～9 Sun Shao U.S.s, Song Yumei, Liu Jian etc. cajanin preparation pharmacological research. Chinese herbal medicine, 1995,26 (3): 147～148 Liu mid-autumns, Zhou Hua, woods is encouraged, Liao Huifang, Zhou Liling. give birth to arteries and veins and become Folium Cajani Study on extraction in the osteocomma. Chinese patent medicine .1998,20 (3): 7～9 is still little red. and acupuncture and moxibustion therapy herpes zoster overview .2005,25 (2): 82～84 5 is big auspicious. the cost-effectiveness analysis of different antiviral treatment herpes zoster. international medical and health Leader, 2004,10 (16): 181～182

Summary of the invention

The present invention is an experiment material with the ethanol extraction of Semen Cajani stem and leaf, and it has significant herpesvirus resisting activity through experiment confirm first, thereby has established the chemistry and biology basis for developing this new new antiviral drug, and can be directly used in the exploitation new antiviral drug.

Description of drawings

Fig. 1 is the process chart of Semen Cajani extract preparation.The LC/MS/MS analysis result shows that the molecular weight of this extract main constituent is 200-600, contrasts with standard substance, determines that Main Ingredients and Appearance is: apigenin-8-C-glucoside, Saponaretin, luteolin, apigenin, kaempferol, isorhamnetin, see accompanying drawing 2-7.Fig. 2 is Semen Cajani sample total ion current figure; Fig. 3 is apigenin-8-C-glucoside mass spectrum (wherein 431.0 molecular ion peaks for it); Fig. 4 is Saponaretin mass spectrum (wherein 431.5 molecular ion peaks for it); Fig. 5 is the mass spectrum (wherein 284.7 molecular ion peaks for it) of luteolin; Fig. 6 is the mass spectrum (wherein 269.2 molecular ion peaks for it) of apigenin; Fig. 7 is the mass spectrum (wherein 286.3 molecular ion peaks for it) of kaempferol; Fig. 8 is the mass spectrum (wherein 315.2 molecular ion peaks for it) of isorhamnetin; Fig. 9 is active effect of Semen Cajani extract extracorporeal antivirus effect and pattern analysis figure.

The specific embodiment

Embodiment 1: the preparation of Semen Cajani extract

Semen Cajani extract obtains (seeing flow chart 1) through the following steps:

The Semen Cajani stem and leaf of gathering is dried in the shade, pulverize 65～80% ethanol that the back adds 10 times of amounts, reflux 2～5h, extract is by filter paper filtering, and supernatant suitably dilutes by requirement of experiment through 0.22 μ m membrane filtration degerming.

Embodiment 2: the Semen Cajani extract cell toxicant is analyzed

1. cell culture

RC-37 (African green monkey kidney cell) grows containing 5% hyclone and be in the DMEM monolayer culture base of 100mg/ml penicillin and streptomycin.Go down to posterity during cell fusion.Cell is put and is carried out cell toxicant and antiviral analysis on 24 holes and 6 orifice plates respectively.

2. viral

The HSV-1 Strain is provided by Heidelberg, Germany university pharmacy and molecular engineering institute (IPMB).Virus inoculation after the RC-37 cell grows up to monolayer adds virus in the culture bottle by titre dilution back, puts into CO ₂Cultivate in the incubator,, put into-20 degree refrigerators when cytopathy reaches 90% when above.Merge behind the multigelation 3 times.

3. cell toxicant analysis

(1) cell is put on 24 orifice plates, density is every hole 5*10 ⁴, cultivate 24h for 37 ℃.

(2) remove culture medium, add the fresh suitable dilution Semen Cajani ethanol extraction that contains, 10 repetitions of every concentration.The 1ml culture medium that contains 1% ethanol and do not have a medicine in contrast.

(3) after 4d cultivates, remove culture medium, the dimethyl diaminophenazine chloride methods analyst.Culture medium replaces with the DMEM of the dimethyl diaminophenazine chloride that contains 40mg/ml, cultivates 3h for 37 ℃.

(4) remove culture medium, with the 1%CaCl that contains 0.5% formaldehyde ₂In wash 1min.

(5) cell dissolves in containing 50% ethanol of 1% acetic acid afterwards, cultivates 5min in blender, goes out with the bonded dimethyl diaminophenazine chloride elution of cell like this.

(6) the 540nm place surveys OD, measures the dimethyl diaminophenazine chloride that absorbs; Matched group is 100%.

Experimental result shows that the maximal non-toxic concentration of Semen Cajani ethanol extraction is 48 μ g/ml.

Embodiment 3: the active effect of Semen Cajani extract extracorporeal antivirus effect

1. directly plaque analysis

(1) 2*10 ³The Semen Cajani ethanol extract of PFU and variable concentrations is at room temperature cultivated 1h.

(2) 37 of the virus of treated serial dilution ℃ of following adherent cell 1h.

(3) discard remaining inoculum, infection cell covers with the culture medium that contains 0.5% methylcellulose, and 6 repetitions are cultivated 4d for 37 ℃.

(4) cell monolayer 10% formalin fixed.

(5) remove fixative, use 1% violet staining, after a few minutes, remove crystal violet, washing once.Look into the plaque number, untreated cell is contrast.

2. antiviral pattern

In order to determine antiviral pattern, be divided into following four groups:

(1) pretreatment virus: with Semen Cajani extract pretreatment virus, hatch 1h under the two room temperature, join on 6 orifice plates, behind 37 ℃ of cultivation 1h, add the covering culture medium, in incubator, cultivate 4d.

(2) viral adsorption: Semen Cajani extract and virus are hatched jointly before infection, and join immediately on 6 orifice plates, behind 37 ℃ of cultivation 1h, add the covering culture medium, cultivate 4d in incubator.

(3) virus replication: virus directly is added on 6 orifice plates, behind 37 ℃ of cultivation 1h, adds covering culture medium and extract simultaneously, in incubator, cultivate 4d.

(4) pretreatment cell: before viral infection, use the Semen Cajani extract pretreatment cell, behind 37 ℃ of cultivation 1h, add virus, behind 37 ℃ of cultivation 1h, add the covering culture medium, in incubator, cultivate 4d.

Experimental result shows that with Semen Cajani extract pretreatment HSV-1 virus, suppression ratio can reach 100%, is higher than reported in literature; Be respectively 43%, 9% and 8% for absorption phase, duplicate stage with the suppression ratio of medicine pretreatment cell.So Semen Cajani extract has the obvious suppression effect to HSV-1 virus, be expected to become a kind of new drug for the treatment of herpes zoster.

Compositions of the present invention can with pharmaceutically acceptable carrier or solvent administration, can be by oral, whole body or topical.The form of compositions can be liquid preparation, solid preparation form administration; For topical, said composition can administration on solid support such as gauze or sponge, also can be prepared into ointment, suppository or spray and come local the use.Said composition can also be taken with the form of beverage, food or additive.

Claims (6)

1, a kind of Semen Cajani ethanol extraction with antiviral activity.This extract is made by 65%～80% alcohol heating reflux, 2～5h.

2, the described ethanol extraction of claim 1, its main component are apigenin-8-C-glucoside, Saponaretin, luteolin, apigenin, kaempferol, the isorhamnetin between the molecular weight 200-600.

3, the described ethanol extraction of claim 2 can form compositions with pharmaceutically acceptable carrier as activating agent.

4, acceptable carrier on the described materia medica of claim 3 comprises conventional solvent, the various excipient that uses in this area, separates and collapse agent etc.

5, the described compositions of claim 3 is forms such as capsule, tablet, pill, injection, oral liquid, electuary, syrup, powder, granule, suppository, ointment, spray, beverage, food or additive.

6, the compositions of using the claim 3 that suppresses viral effective dose can be treated the viral infection that is caused by herpesvirus.

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