CN102600290A - Application of lychee seed flavonoids compounds in terms of preparation of medicine for treating herpes simplex virus encephalitis - Google Patents
Application of lychee seed flavonoids compounds in terms of preparation of medicine for treating herpes simplex virus encephalitis Download PDFInfo
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- CN102600290A CN102600290A CN2012100708016A CN201210070801A CN102600290A CN 102600290 A CN102600290 A CN 102600290A CN 2012100708016 A CN2012100708016 A CN 2012100708016A CN 201210070801 A CN201210070801 A CN 201210070801A CN 102600290 A CN102600290 A CN 102600290A
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Abstract
The invention discloses application of lychee seed flavonoids compounds in terms of preparation of a medicine for treating virus encephalitis caused by infection of herpes simplex virus (HSV). The application specifically embodies that the lychee seed flavonoids compounds obviously suppress the HSV in mice during in-vivo test, the infection symptom of the mice is relieved along with increase of concentration of the medicine, mortality of the mice is reduced, mean survival time of the mice is prolonged, encephalitis symptom of the mice is obviously relieved, the mice are remarkably different from mice in a virus control group, and the curing effect is positively correlated with the concentration of the medicine.
Description
Technical field
The present invention relates to the purposes of Semen Litchi flavone compound on preparation treatment herpes simplex virus encephalitis medicine, belong to medical technical field.
Background technology
The infection of herpes simplex virus (HSV) in the crowd is very general; And comparatively serious be that HSV infects and can cause herpes simplex virus encephalitis (HSVE); Claim acute necrotizing encephalitis again, case fatality rate is up to 50%-70%, and survivor's disability rate is high; Disordered brain function is in various degree often arranged, and sequela is serious.Clinical treatment HSV infection at present is main with antiviral drugs such as iodouracil desoxyriboside, vidarabine, acycloguanosine, but these drug side effectes are bigger, and is obvious like symptom of digestive tract, the irritated and drug resistance phenomenon of normal appearance.Therefore, the new anti-herpes simplex virus medicament of research and development has important practical significance and social meaning.
Semen Litchi is the dry mature seed of sapindaceous plant Fructus Litchi (Litchi chinensis Sonn), has another name called Li Ren or litchi nuclear, sweet in the mouth, little hardship, Ancient Times in China pharmacopeia record Semen Litchi is warm in nature, return liver, kidney channel, can cold relieving pain relieving and promoting QI to circulate and dispersing the agglomeration of the pathogens; The chemical constituent of Semen Litchi has steroid class, saponin, volatile oil, tannin, fatty acid, polymerization anthocyanidin, saccharide, protein and inorganic salt.Flavone compound is the main component in the Semen Litchi extract, has characteristics such as toxicity is low, side effect is little.The Semen Litchi flavone compound is except that having blood sugar lowering, transferring the multiple efficacies such as blood fat and antioxidation; Also has antivirus action; It has several diseases toxic actions such as external anti-hepatitis B virus (HBV), influenza virus (IFV), respiratory syncytial virus (RSV), SARS virus existing bibliographical information; We also find it in the external effect that anti-herpes simplex virus is arranged early stage, but it does not appear in the newspapers to the therapeutical effect by the caused herpes simplex encephalitis of herpes simplex infections as yet.
Summary of the invention
Technical problem to be solved by this invention is to provide the application of Semen Litchi flavone compound in the medicine of the herpes simplex encephalitis that preparation treatment herpes simplex virus causes.
The present invention finds that the Semen Litchi flavone compound can improve the cause death survival rate of infection animal of herpes simplex virus in vivo, accelerates the removing of virus in the tissue, can be used for the herpes simplex encephalitis patient's that herpes simplex infections causes treatment clinically.
The method for distilling of the Semen Litchi flavone compound that the present invention adopts, its process is following: Semen Litchi is broken into powder crosses 40 mesh sieves, take by weighing the Semen Litchi powder; Add alcoholic solution, adopt microwave pretreatment, constant temperature extracts and intermittently concussion; Through after the extracted twice; With extracting liquid filtering, after the dehydrated alcohol that adds doubling dose after concentrating left standstill a period of time, fully post precipitation removed by filter macromole impurity such as protein.Decompression recycling ethanol changes water over to after concentrating, and is mixed with the Semen Litchi leachate that is used for resin absorption.In the ground conical flask of band plug, add the good adsorbent resin of pretreatment and the Semen Litchi leachate of above-mentioned preparation, under room temperature, vibrate, regulate about upper prop liquid pH=5.0, saturated under the room temperature with corresponding flow velocity absorption; Earlier use water wash during desorbing, the reuse alcohol desorption, the consumption of control desorbing agent flow velocity and desorbing agent is collected required composition then, concentrates and removes organic solvent, and it is higher to obtain purity at last, active better Semen Litchi flavone compound.
The Semen Litchi flavone compound has therapeutical effect to the mouse infection herpes simplex virus.Infect administration in back 24 hours, along with the increase of drug dose, mouse death rate reduces, and the mean survival time prolongs.
The Semen Litchi flavone compound is inhibited to herpes simplex virus in animal body.Infect administration in back 24 hours, can reduce body inner virus expression of nucleic acid level, alleviate tissue (brain) pathological change, reduce virus titer in the tissue (brain, lung, liver).
The present invention can obtain the flavone compound that content is higher, purity is higher from Semen Litchi, the flavone compound of extraction has the good curing effect to herpes simplex encephalitis due to the herpes simplex infections.
Description of drawings
Fig. 1 .PCR detects HSV-1 nucleic acid in the HSV-1 infecting mouse different tissues
The 1:100bp standard molecular weight; 2,8,10: normal mouse brain, lung, hepatic tissue; 3,4,5,6: the virus infected mice cerebral tissue; 7: the virus infected mice lung tissue; 9: the virus infected mice hepatic tissue; 11: the virus-positive contrast; 12: blank.
The detection of Fig. 2 .HSV-1 infecting mouse model
HSV-1 infecting mouse cerebral tissue HE dyeing mirror shows that down the mice forebrain has tangible lymphocytic infiltration (Fig. 2 B); And distinctive hemorrhagic necrosis (Fig. 2 C) arranged; And normal group does not have any pathological change (Fig. 2 A), this shows successfully to have set up the herpes simplex infections mouse model.
Fig. 3. Semen Litchi flavone compound for treating HSV-1 infected group mouse brain tissue pathological slice
A. dose groups (83.13mg/kg/d) C. low dose group (41.56mg/kg/d) among high dose group (166.25mg/kg/d) B..
The specific embodiment
Embodiment 1: the extraction and purification of Semen Litchi flavone compound
Semen Litchi is broken into powder crosses 40 mesh sieves, take by weighing a certain amount of Semen Litchi powder, the concentration that adds respective volume according to solid-liquid ratio at 1: 12 is the 60vol% alcoholic solution; Behind effect 150S under the 700W frequency microwave; Constant temperature extracts and intermittently concussion, through after the extracted twice, with extracting liquid filtering; The dehydrated alcohol that adds doubling dose after concentrating leaves standstill a period of time, and fully post precipitation removes by filter macromole impurity such as protein.Decompression recycling ethanol changes water over to after concentrating, and is mixed with the Semen Litchi leachate that is used for resin absorption.In the ground conical flask of band plug, add the good X-5 adsorbent resin of 600mg pretreatment and the Semen Litchi leachate of the above-mentioned preparation of 450ml; And adjustment sample solution mass concentration is 0.5mg/ml; In 25 ℃ of following vibration (180r/min) 24h of room temperature; Regulate upper prop liquid pH=5.0, saturated under the room temperature with flow velocity 2ml/min absorption; Earlier use water wash during desorbing, reuse 85vol% alcohol desorption, control desorbing agent flow velocity is 0.5ml/min, the consumption of desorbing agent is that 50ml/ criticizes, and collects required composition then, concentrates to remove organic solvent.
Embodiment 2: the qualitative evaluation and the assay of flavone in the Semen Litchi extract
1) at first get Semen Litchi extract, be dissolved in 60vol% ethanol, with HCl-Mg, AlCl
3, Pb (Ac)
3React all positive, show and contain flavone compound in the Semen Litchi extract.
2) be solvent with 60vol% ethanol, with NaNO
2-Al (NO
3)
3-NaOH makes the absorption curve of rutin standard substance and Semen Litchi extract respectively as developer, and the two all has the last one absworption peak at 508nm, so select 508nm for measuring wavelength.Accurately take by weighing rutin standard substance 5.00mg,, be mixed with rutin standard solution (100 μ g/ml) with being settled in the 50ml volumetric flask behind the 50vol% dissolve with ethanol.Accurately measure rutin standard solution 0.00,1.00,2.00,3.00,4.00,5.0,6.0,7.0ml places the 25ml volumetric flask respectively, each adds 5wt% sodium nitrite solution 1ml and shakes up, and places 6min; Add 10wt% aluminum nitrate solution 1ml, shake up and place 6min, add 4wt% sodium hydroxide solution 10ml; Use the 60vol% ethanol dilution to scale then; Shake up and place 15min, reagent is that blank is made reference, measures absorbance; The chromogenic assay absorbance A, the use method of least square obtains regression equation and is: A=0.0049C (μ g/ml)+0.0027.Get Semen Litchi extract 1ml and place the 25ml volumetric flask, add 5wt% sodium nitrite solution 1ml successively, shake up, place 6min; Add 10% aluminum nitrate solution 1ml, shake up, place 6min, add 4% sodium hydroxide solution 10ml; Use 60vol% ethanol standardize solution then, shake up, place 15min; Mentioned reagent not contain Semen Litchi extract is made reference liquid, measures absorbance at the 508nm place, and the concentration that obtains flavone compound is 13.33333mgmL
-1Thereby, know that the flavone compound mass content is 1.98% in the Semen Litchi by inference.
Embodiment 3: the Semen Litchi flavone compound is to the toxic action of mice
Get 40 of kunming mices (male), 4～5 ages in week, 16 ± 1g.The completely random method is divided into 5 groups, 8 every group, wherein comprises a normal control group.With the Semen Litchi flavone compound with the distilled water gradient dilution become 3436.25,344.00,34.40,4 groups of 3.44mg/kg, mice after 4 hours, is irritated stomach by every 0.2ml on an empty stomach; Every day 1 time; Continuous 14 days, every day is supplement feed, moisture regularly, changes bedding and padding; Indoor temperature is 22 ± 2 ℃, and the record the weight of animals changes and death condition.Observe after 14 days; Find that the animal in 344.00mg/kg, 34.40mg/kg, three variable concentrations groups of 3.44mg/kg does not have poisoning and death condition, the result shows: the Semen Litchi flavone compound is safe administration concentration to kunming mice below 344.00mg/kg concentration with the gastric infusion approach.
Embodiment 4: anti-herpes simplex virus in the Semen Litchi flavone compound body
1, the cause a disease foundation of model: animal housing should keep that air is fresh, relative humidity 60%, 22 ± 2 ℃ of temperature.72 of 4 week～5 male Kunming mouses in age in week behind etherization and the infection site routine disinfection, carry out 10 times of serial dilutions (10 with virus
-1～10
-8), 8 mices of each concentration inoculation, viral total amount 0.02ml/, the brain inoculation, the skull portion of median line is departed from slightly in the position in the middle of mouse right ear and right eye leading edge line, and it is dark that entry needle is vertically thrust 2～3mm, slowly liquid medicine injection.The normal control group gives virus-free diluent.Observed for 2 evenings, record mouse invasion, death condition are pressed the Reed-Muench method and are calculated median lethal dose(LD 50) (Median lethal dose, LD
50).
The result for after infecting through after of short duration incubation period, hair, the back of a bow, tic, weight loss appear trembling, alarm.In brain, lung, hepatic tissue, all detect viral nucleic acid and exist, the result sees Fig. 1.The tissue pathological slice mirror shows the pathological change that obvious neuromechanism is arranged in the mice forebrain down, and with a large amount of lymphocytic infiltrations and hemorrhagic necrosis kitchen range, the result sees Fig. 2.This viral half causes dead mouse dosage (LD
50) be 10
-5.3/ 0.2ml.
2, interior resisting virus experiment: experiment mice is divided into 1. normal control group at random, irritates the clothes normal saline; 2. the viral infection matched group is irritated the clothes normal saline; 3. ACV treats matched group (100.00mg/kg/d), and existing bibliographical information ACV is best to the therapeutic effect of mouse infection herpesvirus when 100mg/kg/d; 4. Semen Litchi flavone compound low dose group (41.56mg/kg/d); 5. dose groups (81.13mg/kg/d) in the Semen Litchi flavone compound; 6. Semen Litchi flavone compound high dose group (166.25mg/kg/d); 12 of every group of mices, viral infection dosage adopts fatal dose (100LD
50) 0.02ml/ only, through the brain injection inoculation.Begin gastric infusion after 24 hours, 5 days courses of treatment.Observe the sings and symptoms of animal, write down the death toll and survival natural law of each treated animal, and the dead protective rate relatively respectively organized and The average survival time natural law (Mean time to death, MTA).Each is treated treated animal and respectively put to death 4 mices in the 5th day and the 10th day in treatment end back; Pluck eyeball and get blood and separation of serum; The aseptic internal organs such as brain, lung, liver of getting are used for histopathological examination, infectious virus separation and viral nucleic acid and detect simultaneously, and each treated animal is all observed after 14 days and put to death.
Experimental result is: in the Semen Litchi flavone compound administration group, the high dose group animal is after observing 14 days, and survival rate is 67%, compares with the virus control group, shows notable difference (p＜0.01); But the survival rate of middle dose groups and low dose group is respectively 50% and 42%, compares with the virus control group, then equal no difference of science of statistics (p＞0.05).The mean survival time of Semen Litchi flavone compound high dose group and virus control group relatively have significant difference (p＜0.05); But the mean survival time of middle dose groups and low dose group and virus control group compare respectively, all no difference of science of statistics (p＞0.05).Detailed results is seen table 1.
Table 1. Semen Litchi flavone compound is to the effect of HSV-I induced mice viral encephalitis
Annotate: * *: compare p＜0.01 with the virus control group; * compare p＜0.05 with the virus control group.
This medicine is brought into play antivirus action through suppressing HSV-1 in the intravital propagation of Mus, shows that virus infection titer in infecting mouse brain, lung, the liver reduces with the increase of drug dose, and detailed results is seen table 2.
Virus titer in the table 2. Semen Litchi flavone compound for treating HSV-1 infecting mouse tissue
Annotate: *: compare p＜0.05 with the virus control group.
Mouse brain organizes HE dyeing back mirror to show down: the cranial nerve cell of the mouse brain tissue after the effect of Semen Litchi flavone compound and glial cell are downright bad, hemorrhage; Pathological change degree such as the lymphocyte of blood vessel, plasmocyte infiltrating are along with drug level increases gradually and weakens gradually.The result sees Fig. 3.
Claims (1)
1. the application of Semen Litchi flavone compound in the medicine of the viral encephalitis that preparation treatment herpes simplex virus causes.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113367327A (en) * | 2021-06-03 | 2021-09-10 | 暨南大学 | Application of ketogenic diet in preventing herpes simplex virus encephalitis |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4132782A (en) * | 1977-05-19 | 1979-01-02 | Samuel Bean | Method for suppressing herpes simplex virus |
| EP0548798A1 (en) * | 1991-12-18 | 1993-06-30 | Sanwa Kagaku Kenkyusho Co., Ltd. | Antiviral agent |
| CN1434052A (en) * | 2003-02-12 | 2003-08-06 | 复旦大学 | Cranesbill total flavonoid extract and use in preparation of anti-virus medicine |
| CN1872105A (en) * | 2006-04-08 | 2006-12-06 | 冯秀明 | Application of holly and extractive in preparing medication for anti virus |
-
2012
- 2012-03-16 CN CN2012100708016A patent/CN102600290A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4132782A (en) * | 1977-05-19 | 1979-01-02 | Samuel Bean | Method for suppressing herpes simplex virus |
| EP0548798A1 (en) * | 1991-12-18 | 1993-06-30 | Sanwa Kagaku Kenkyusho Co., Ltd. | Antiviral agent |
| CN1434052A (en) * | 2003-02-12 | 2003-08-06 | 复旦大学 | Cranesbill total flavonoid extract and use in preparation of anti-virus medicine |
| CN1872105A (en) * | 2006-04-08 | 2006-12-06 | 冯秀明 | Application of holly and extractive in preparing medication for anti virus |
Non-Patent Citations (4)
| Title |
|---|
| ANNELIESE VECKENSTEDT等: "Eifeet oi Treatment With Certain Flavonoids on Men qo Virus-Induced Encephalitis in Mice", 《ARCHIVES OF VIROLOGY》 * |
| I. MUCSI & B.M. PRFIGAI: "Inhibition of virus multiplication and alteration of cyclic AMP level in cell cultures by flavonoids", 《EXPERIENTIA》 * |
| 马亦林，李兰娟主编: "《传染病学》", 31 May 2011 * |
| 魏金亮等: "荔枝核黄酮体外抗单纯疱疹病毒的作用", 《武汉大学学报（医学版）》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113367327A (en) * | 2021-06-03 | 2021-09-10 | 暨南大学 | Application of ketogenic diet in preventing herpes simplex virus encephalitis |
| CN113367327B (en) * | 2021-06-03 | 2022-07-08 | 暨南大学 | Application of ketogenic diet in preventing herpes simplex virus encephalitis |
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Application publication date: 20120725 |