CN1644199A - Use of ampeloptin in preparation of antivirus medicines - Google Patents
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Abstract
A broad-spectrum composite antiviral medicine for HBV, influenza virus and/or coronavirus contains ampeloptin and pharmacologically acceptable excipient. Its preparing process and the application of ampeloptin in preparing it are also disclosed.
Description
The invention belongs to natural medicine field.
Document Walter Karrerr, Birkhauser Verlag, Bassel undstuttgart (1958), P.652, NO:1640 discloses the structural formula of ampelopsin,
But the activity of ampelopsin is not studied fully.
The applicant is surprisingly found out that ampelopsin has purposes widely at pharmaceutical field.
The applicant finds that ampelopsin has broad-spectrum disease resistance toxic action, particularly anti-hepatitis virus, influenza virus and/or coronavirus.The inventor finds that also ampelopsin has blood sugar lowering and effect for reducing blood fat, can be prepared into blood sugar lowering and hypolipidemic.Ampelopsin can directly be used as medicine, can also make pharmaceutical composition with pharmaceutical acceptable carrier or mixed with excipients.The ampelopsin that wherein preferably contains 2% to 98% weight ratio adds pharmaceutical acceptable carrier or excipient to 100% weight ratio.Can also be in prior art be mixed with pharmaceutical composition with the compatible active constituents of medicine of ampelopsin, wherein said compatible active constituents of medicine is the flavonoid medicine preferably, and described flavonoid medicine is ampelopsin preferably.Flavonoid medicament contg and ampelopsin content weight ratio can be 2-80: 20-98 in the compositions.The present composition can be prepared into tablet, granule, capsule, medicine for external use.The dosage that preferred active component is used the people is 0.01-5 gram/kg body weight/sky, but also can decide as the case may be, is not limited to this scope.
The regular convention formula of tablet can be: ampelopsin 10%-90% weight ratio, and lactose 90%-10% weight ratio randomly adds an amount of sodium carboxymethyl cellulose, magnesium stearate, 50% ethanol is an amount of.The regular convention formula of electuary can be: ampelopsin 5-100% weight ratio, and (sucrose+dextrin) 95-0% weight ratio, sucrose: dextrin=2: 1, randomly add an amount of other auxiliary agent, total amount is 100%.Capsular regular convention formula can be: ampelopsin 10-100% weight ratio, and starch 90-0% weight ratio randomly adds an amount of other auxiliary agent, and total amount is 100%.But those skilled in the art also can be mixed with other dosage form with technology well known in the art and carrier as required.
Ampelopsin can extract from ampelopsis, also can prepare with above-mentioned document disclosed method.Described ampelopsis comprises ampelopsis cantoniensis, a leaf beautiful Fructus Vitis viniferae, Ampelopsis grossedentata, Cayratia japonica (Thunb.) Gagnep., Radix Ampelopsis, Northeastern Caulis seu folium ampelopsis brevipedunculatae and Vitis Amurensis.Described extracting method can be: decoct vitaceae by all kinds of solvents (water and/or organic solvent), decoct one or many, decoction liquor is through concentrating cooling, standing over night, detect the active ingredient of precipitation and supernatant, get and have active precipitation, after chromatographic isolation, obtain ampelopsin of the present invention.Decoct used solvent preferred water, alcohols, esters, ketone, ethers and strong polar organic solvent, more preferably low-level chain triacontanol, low-grade fatty acid ester has ketone and ether and/or water, the most preferably methanol of 3-12 carbon atom, ethanol, propanol, isopropyl alcohol, n-butyl alcohol, isobutanol, sec-butyl alcohol, the tert-butyl alcohol, ethyl acetate, methyl acetate, Ethyl formate, acetone, methyl ethyl ketone, ether, methyl ethyl ether, methyl tertiary butyl ether(MTBE), dichloromethane, chloroform, carbon tetrachloride, dimethyl sulfoxine, N, dinethylformamide and/or water or the like.The chromatographic isolation filler can be a polyamide, polyacrylamide, macroporous adsorbent resin, cation exchange resin, anion exchange resin, preferred polyamide, polyacrylamide, macroporous adsorbent resin, more preferably polyamide.
Principle of the present invention is to follow the trail of the drug activity of ampelopsin by setting up the pharmacological screening model, finds its pharmaceutical usage.Extracorporeal bacteria inhibitor test and antivirus test show that ampelopsin has anti-inflammation and broad-spectrum disease resistance toxic action, particularly anti-hepatitis virus, influenza virus and/or coronavirus.Test proves that also ampelopsin has blood sugar lowering and effect for reducing blood fat, has the function of prevention and/or recovery alcoholic liver injury, has hepatoprotective, the function of enhance immunity systemic immunity power.
Ampelopsin can be mixed with pharmaceutical composition by conventional preparation technique of drug world and carrier.The present composition can be prepared into tablet, granule, capsule, medicine for external use.But those skilled in the art also can be mixed with other dosage form with technology well known in the art and carrier as required.
Brief description of drawings
Fig. 1 is the comparison of oral ampelopsin treatment group and the horizontal suppression ratio of the clear DHBV-DNA of viral infection matched group Sanguis Anas domestica in duck hepatitis B virus infection duck body.Be the inhibitory action (first experiment) of ampelopsin to DHBV infected duck serum DHBV-DNA.
Fig. 2 is the clear DHBV-DNA dot blot hybridization of a Sanguis Anas domestica radioautogram 1 (first experiment).
Fig. 3 is the comparison of oral ampelopsin treatment group and the horizontal suppression ratio of the clear DHBV-DNA of viral infection matched group Sanguis Anas domestica in duck hepatitis B virus infection duck body.Be the inhibitory action (second batch experiment) of ampelopsin to DHBV infected duck serum DHBV-DNA.
Fig. 4 is the clear DHBV-DNA dot blot hybridization of Sanguis Anas domestica radioautogram 2 (second batch of experiment).
Fig. 5 is the comparison of oral ampelopsin treatment group and the horizontal suppression ratio of the clear DHBV-DNA of viral infection matched group Sanguis Anas domestica in duck hepatitis B virus infection duck body.Be the inhibitory action (three batch experiment) of ampelopsin to DHBV infected duck serum DHBV-DNA.
Fig. 6 is the clear DHBV-DNA dot blot hybridization of Sanguis Anas domestica radioautogram 3 (the 3rd batch of experiment).
Describe the present invention in detail by the following examples.But should be appreciated that these embodiment just illustrate the present invention, rather than in office where face limits the scope of the invention.
Embodiment 1: tablet:
Ampelopsin 25g,
Lactose 53g,
Sodium carboxymethyl cellulose 1.5g,
Magnesium stearate 0.5g,
50% ethanol 5ml
Said components is mixed the back compacting in flakes in mixer.
Embodiment 2: syrup
Ampelopsin 100g,
Sucrose 20g,
Dextrin 10g,
Ethanol 50ml
Mix mentioned component and obtain syrup.
Embodiment 3: electuary
Ampelopsin 55g,
Icing Sugar 345g,
Dextrin 145g,
Ethanol 5ml
Said components is mixed after drying, obtains electuary.
Embodiment 4: capsule
Ampelopsin 200g,
Starch 20g,
Behind the said components mix homogeneously, incapsulate.
Embodiment 5: tablet:
Ampelopsin 5g,
Ampelopsin 30g
Lactose 53g,
Sodium carboxymethyl cellulose 1.5g,
Magnesium stearate 0.5g,
50% ethanol 5ml
Said components is mixed the back compacting in flakes in mixer.
Embodiment 6: electuary
Ampelopsin 520g,
Icing Sugar 350g,
Dextrin 150g,
Ethanol 10ml
Said components is mixed after drying, obtains electuary.
Embodiment 7: the test of pesticide effectiveness of the external anti-hepatitis virus of ampelopsin
Select 2215 cell strains of hepatitis B virus (HBV) DNA transfection, with the titre of HBsAg and HBeAg in its culture supernatant as the index of estimating anti-HBV effect, the anti-HBV effect of research ampelopsin.The toxicity of tetramethyl azo azoles salt (MTT) colorimetric analysis detection of drugs pair cell is also adopted in this test, selects for use acyclovir as positive control.
1. materials and methods
1.1. tried thing: the crude drug (wherein the content of ampelopsin is 50%) that contains ampelopsin.
1.1.2 sweet Thailand of positive drug or injection acyclovir (Acyclovir for Injection), lot number: 20,000,409 2, Hubei Qianjiang Pharmaceutical Co., Ltd. produces, and the accurate word (1991) of medicine is defended No. 000354 in Hubei Province.
1.2 cell and reagent
2215 cells go out this laboratory to be preserved, and culture fluid is DMEM (a Gibco company), contains 10% hyclone in the culture fluid, 38ug/mlG418 (Geneticin, Gibco company), 0.03%L-glutamine (magnificent import packing), various each 1000iu/ml of penicillin and streptomycin.
MTT (Switzerland Fluka company), dimethyl sulfoxide (DMSO) (Serva company)
HBsAg, HBeAg test kit are provided by Shanghai industry Hua Sheng Science and Technology Ltd..Lot number: 08-2000-03,06-2000-02
1.3 cell culture and experiment grouping
2215 cells are according to a conventional method with 10
3-10
4The concentration in individual/hole is inoculated 96 well culture plates, every hole 0.1ml, 5%CO
2, 37 ℃ remove culture fluid after cultivating 24h, proportionately contain the culture fluid of medicine, wherein ampelopsin is established 6 concentration: 95ug/ml, 195ug/ml, 385ug/ml, 750ug/ml, 1500ug/ml, 3100ug/ml, acyclovir is established 6 concentration, i.e. 62.5ug/ml, 125ug/ml, 250ug/ml, 500ug/ml, 1mg/ml, 2mg/ml, and each concentration is established 5 parallel holes, establish simultaneously that not add the medicine hole be matched group, cultivate and gather in the crops supernatant after 5 days.
1.4 the cytotoxic assay of medicine
After the medication 5 days, each hole supernatant exhausts, culture hole adds the culture fluid that contains MTT again, and making the MTT ultimate density is 0.5mg/ml, 37 ℃ of cultivation 4h, discards culture fluid, every hole adds 100ulDMSO, vibration 10min, 490nm measures the OD value on the microplate reader, with absorbance (survivaling cell the is 100%) comparison of corresponding control wells, calculating survivaling cell percentage ratio is determined the maximal non-toxic dose concentration of medicine pair cell.
1.5 detect
Release 4-5 dosage level forward according to the maximal non-toxic dose concentration of medicine pair cell, gather in the crops the detection that supernatant at different levels are used for HBsAg and HBeAg, detect and undertaken by operating instruction.
1.6 the evaluation methodology of the anti-HBV of medicine
With medicine the suppression ratio of HBsAg or HBeAg is estimated
2. result
2.1 the toxicity of medicine pair cell
Found that along with the raising with concentration, the toxicity of its pair cell increases, the maximal non-toxic concentration of pair cell (survivaling cell is at the drug level more than 90%) is 750ug/ml, and the positive drug acyclovir is 500ug/ml (table 1).
The toxicity of table 1 ampelopsin pair cell
Medicine name concentration (ug/ml) survivaling cell (%M ± S)
3100??????????????????47.50±1.29
Serpentis 1,500 82.70 ± 2.22
Portugal 750 91.00 ± 1.87
Grape 385 91.20 ± 5.72
Plain 195 93.20 ± 4.92
95????????????????????98.60±1.34
2000??????????????????76.64±4.96
Ah 1,000 87.40 ± 1.51
Former times 500 92.54 ± 1.76
Lip river 250 92.76 ± 8.84
Wei 125 92.06 ± 4.87
62.5??????????????????96.48±4.04
The external anti-HBV result of the test of table 2 medicine
Medicine name concentration (ug/ml) HbsAg suppression ratio HBeAg suppression ratio
(％M±S)?????????????(％M±S)
Serpentis 95 14.00 ± 3.703 23.61 ± 1.804
Portugal 195 21.42 ± 2.212 25.60 ± 8.022
Grape 385 31.61 ± 8.605 29.47 ± 4.924
Plain 750 50.23 ± 9.916 41.79 ± 8.905
Ah 62.5 19.48 ± 9.86 20.42 ± 1.52
Former times 125 25.88 ± 11.09 20.94 ± 10.55
Lip river 250 44.44 ± 5.94 21.62 ± 19.95
Wei 500 51.86 ± 7.14 28.44 ± 5.15
2.2 the anti-HBV effect of medicine
Ampelopsin and positive drug the results are shown in Table 2 to the inhibition of 2215 emiocytosis HBsAg, HBeAg, as can be seen from the table, under the used concentration of this test: ampelopsin to the suppression ratio of HBsAg, HBeAg respectively between 14.00 ± 3.703%-50.23 ± 9.916% and 23.61 ± 1.804%-41.79 ± 8.905%, along with its suppression ratio of increase of drug level increases gradually, when drug level reaches 750ug/ml, to the suppression ratio of HBsAg greater than 50%.
The positive drug acyclovir is respectively between 19.48 ± 9.86%-51.86 ± 7.14% and 20.42 ± 1.52%-28.44 ± 5.15% the secretion suppression ratio of HBsAg, HBeAg, when drug level is 500ug/ml, to the suppression ratio of HBsAg greater than 50%.
Above result shows that ampelopsin has certain anti-hepatitis B activity external.
Embodiment 8: ampelopsin in the duck body to the therapeutic effect of duck hepatitis B virus infection
In duck hepatitis B virus infection duck body, carry out therapeutic test, observe ampelopsin and whether suppress DHB.
An age in days Beijing duck intravenous injection DHB is adopted in experiment, begin after 7 days to 3 dosage groups 1.25 of the oral ampelopsin of duck, 2.5 and 5.0g/kg, 1 day 2 times, administration 10 days (Bid * 10), observe medicine to toxicity of duck and the influence of the clear DHB DNA of Sanguis Anas domestica (DHBV-DNA), and compare with lamivudine.
Experiment shows, 5.0g/kg is oral for the heavy dose of group of ampelopsin, 1 day 2 times 10 days, avirulence, first experiment, by the pairing statistics, the 5.0g/kg group, after the administration after the 5th, 10 day and the drug withdrawal 3 days clear DHBV-DNA of treatment group Sanguis Anas domestica have highly significant to descend (P＜0.01).By statistics in groups and matched group separately relatively, after the administration the 5th day, after the 10th day and the drug withdrawal 3 days, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant to descend (P＜0.01).2.5g/kg group, after the administration after the 5th, 10 day and the drug withdrawal 3 days clear DHBV-DNA of treatment group Sanguis Anas domestica (P＜0.05-0.01) is arranged significantly and highly significant decline.By statistics in groups and matched group separately relatively, after the administration after the 10th day and the drug withdrawal 3 days, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant to descend (P＜0.05).1.25g/kg group, after the administration the 5th day, by the pairing statistics, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant to descend (P＜0.01).By statistics in groups, after the administration the 5th day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had remarkable decline, (P＜0.05).Second batch of experiment 5.0g/kg group, by the pairing statistics, after the administration the 5th day, 3 days clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant and significantly descend after the 10th day and the drug withdrawal, (P＜0.01-0.05).By statistics in groups and matched group separately relatively, after the administration the 5th day, the 10th day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant and significantly descends, (P＜0.01-0.05).2.5g/kg group, by statistics in groups and matched group separately relatively, after the administration the 10th day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had remarkable decline, (P＜0.05).1.25g/kg group, the unrestraint effect.The 3rd crowd of experiment 5.0g/kg, pairing statistics, after the administration the 5th, 10 day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant and remarkable result.(P＜0.01-0.05)。By statistics in groups and matched group separately relatively, after the administration the 5th day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had highly significant to descend (P＜0.01).2.5g/kg group, by pairing statistics, after the administration after the 5th, 10 day and the drug withdrawal 3 days, the clear DHBV-DNA of treatment group Sanguis Anas domestica had significantly and highly significant decline, (P＜0.05,0.01,0.05).Statistics in groups, after the administration the 5th day, the 10th day, the clear DHBV-DNA of treatment group Sanguis Anas domestica had significantly and highly significant decline, (P＜0.05-0.01).1.25g/kg group, the unrestraint effect.The lamivudine matched group has the highly significant effect, and illustrative experiment is credible.Prompting: ampelopsin is effective to duck hepatitis B virus infection in the 3.4-10.0g/kg group.
Material and method
(1) medicine
Contain the crude drug (wherein the content of ampelopsin is 85%) of ampelopsin, prepare with normal saline.
The positive drug lamivudine is prepared with normal saline by Glaxo Wellcome drugmaker product.
(2) virus
DHB DHB DNA (DHBV-DNA) strong positive serum picks up from the Shanghai sheldrake ,-70 ℃ of preservations.
(3) animal
1. age in days Beijing duck is available from animal feeding field, progressive species duck field, Beijing.
(4) reagent
α-
32P-dCTP is available from the auspicious biotechnology of Beijing good fortune engineering company.The nick translation medicine box is available from Pu Luomaige company (Promega Co.); Sephadex G-50, Ficoll PVP is available from Sweden Pharmacia company; SDS West Germany Merck company product; Milt DNA, bovine serum albumin are Instite of Biophysics, Chinese Academy of Sciences's product; Nitrocellulose filter 0.45um Amersham company product.
(5) experimental technique
1. duck hepatitis B virus infection:
1 age in days Beijing duck, clear through the positive Sanguis Anas domestica of lower limb shin intravenous injection Shanghai sheldrake DHBV-DNA, every 0.2ml got blood in back 7 days in infection, separation of serum ,-70 ℃ of preservations are to be checked.
2. Drug therapy test:
DHBV infect duckling after 7 days random packet carry out the Drug therapy test, 6 every group, 3 dosage groups of administration component are respectively 1.25,2.5, the 5.0g/kg group is oral, 1 day 2 times, 10 days.If virus control group (DHBV) is with the physiologic saline for substitute medicine.The positive drug lamivudine, oral administration 50mg/kg, 1 day 2 times, 10 days.The 7th day is (T0) before the medication after infection, and medication the 5th day (T5) after medication the 10th day (T10) and the drug withdrawal the 3rd day (P3), is got blood from duck lower limb shin vein, separation of serum, and-70 ℃ of preservations are to be checked.
3. detection method:
It is clear to get above-mentioned Sanguis Anas domestica to be checked, and every batch with the time point film, measure Sanguis Anas domestica clear in the DHBV-DNA level dynamically, press nick translation test kit description method, usefulness
32P labelling DHBV-DNA probe, and make the clear dot blot hybridization of Sanguis Anas domestica, autoradiography diaphragm speckle is measured OD value (optical filter is 490nm) at enzyme mark detector, calculating serum DHBV-DNA density, with hybridization spot OD value as specimen DHBV-DNA level value.
(6) drug effect is calculated
1. calculate the meansigma methods (X ± SD) of every group of duck different time serum DNA OD value, and with (T0) OD value comparison before the 3rd day (P3) serum DHBV-DNA level after different time (T5, T10) and the drug withdrawal after every group of duck medication and the administration on the same group, adopt paired t-test, calculate t1, P1 value.Analyze the significance of difference, judge the inhibition effect of medicine viral infection.
2. calculate the inhibition % of different time (T5, T10) and the 3rd day (P3) serum of drug withdrawal DHBV-DNA after every group of duck medication, and mapping, respectively organize the clear DHBV-DNA suppression ratio of Sanguis Anas domestica dynamically.
3. with identical with the virus control group respectively time D HBV-DNA suppression ratio of drug treatment group different time DHBV-DNA suppression ratio relatively, adopt t check in groups, take statistics to learn and handle, calculate t2, P2 value, analyze the significance of difference, judge drug effect.
The result
After (one) 1 age in days Beijing duck infected DHBV, serum DHBV-DNA was dynamic
The DHBV-DNA results of dot sees Table 3 behind the DHBV-DNA infected duck oral normal saline.78 serum DHBV-DNA of three batches of experimental infections total positives.18 ducklings of virus control group infect the 7th day serum DHBV-DNA total positives in back, test in omnidistance 21 days steady substantially behind the serum DHBV-DNA level infection for three batches.
(2) the positive drug lamivudine is to the influence of DHBV infected duck serum DHBV-DNA
The oral positive drug lamivudine of DHBV infected duck 50mg/kg, 1 day 2 times, 10 days.The results are shown in Table 3, table 4.Pair analysis, before administration the 10th day (T10) and the administration OD value of (T0) relatively, decline has highly significant (P＜0.01), after the administration to serum DHBV-DNA suppression ratio and virus control group, become group analysis administration the 10th day (T10) significant differences to be arranged, (P＜0.01).3 days (P3) not statistically significants after the drug withdrawal.Rebound phenomenon is arranged.
(3) ampelopsin in DHBV infected duck body to the influence of the clear DHBV-DNA of Sanguis Anas domestica
Three batches of experimental results show:
1. 3 dosage groups are selected in first experiment for use, see Table 3, Fig. 1.Be respectively 1.25,2.5 and 5.0g/kg group, after (T0) and the administration after the 5th day (T5), 10 days (T10) and the drug withdrawal 3 days (P3), get Sanguis Anas domestica before administration, separation of serum detects DHBV-DNA OD value, does self comparison.The result shows: 5.0g/kg organizes after the 5th day (T5) of administration, the 10th day (T10) and drug withdrawal the 3rd day (P3), before clear DHBV-DNAOD value of Sanguis Anas domestica and the administration (T0) relatively, pair analysis has the inhibitory action of highly significant.(P＜0.01)。Become group analysis, administration the 5th day, after 10 days (T10) and the drug withdrawal the 3rd day (P3), administration group and matched group relatively have the inhibitory action of highly significant.(P＜0.01)。2.5g/kg group, after the 5th day (T5) of administration, the 10th day (T10) and drug withdrawal the 3rd day (P3), (T0) compares before Sanguis Anas domestica clear DHBV-DNA OD value and the administration, and pair analysis has significantly and the inhibitory action of highly significant.(P＜0.05-0.01)。Become group analysis, after administration the 10th day (T10) and the drug withdrawal the 3rd day (P3), administration group and matched group comparison have significant inhibitory effect.(P＜0.05)。1.25g/kg group, (T0) compares before the clear DHBV DNA of Sanguis Anas domestica OD value and the administration, and pair analysis in administration the 5th day (T5), has significant inhibitory effect.(P＜0.05)。Become group analysis, administration the 5th day (T5), administration group and matched group relatively have significant inhibitory effect.(P＜0.05)。
2. second batch of experimental result shows: see Table 3,4, Fig. 2.5.0g/kg group different time after administration, the clear DHBV-DNA of Sanguis Anas domestica, (T0) compares before OD value and the administration, the 5th day (T5), 10 days (T10) after the administration, with the 3rd day (P3) after the drug withdrawal, highly significant and remarkable inhibitory action are arranged, (P＜0.01-0.05), become group analysis, suppressing % with matched group analyzes, administration the 5th day (T5), the 10th day (T10) have highly significant and remarkable inhibitory action, (P＜0.01-0.05).2.5g/kg group, pair analysis, the 10th day (T10) has the highly significant inhibitory action after the administration, and (P＜0.01) becomes group analysis, suppresses % with matched group and analyzes, and administration the 10th day (T10) has remarkable inhibitory action, (P＜0.05).1.25g/kg group, inhibitory action is not obvious.
3. the 3rd crowd of repeated experiments result shows: see Table 3,4, Fig. 3.5.0g/kg after the group administration the 5th, 10 day, before DHBV-DNA and the administration relatively, pair analysis, highly significant and remarkable inhibitory action are arranged, and (P＜0.01-0.05), become group analysis suppresses % with matched group and analyzes, administration the 5th day (T5) has the highly significant inhibitory action, (P＜0.01).2.5g/kg group, pair analysis with comparison before the administration, after the 5th day (T5) of administration, the 10th day (T10) and the drug withdrawal the 3rd day (P3), has highly significant and remarkable inhibitory action, (P＜0.05,0.01,0.05).Become group analysis, suppress % with matched group and analyze, administration the 5th day (T5), the 10th day (T10) have significantly and the highly significant inhibitory action, (P＜0.05-0.01).1.25g/kg group, inhibitory action is not obvious.
Table 3 ampelopsin is clear in duck hepatitis B virus infection duck interior therapeutic group and viral infection matched group Sanguis Anas domestica
DHBV-DNA OD value relatively
| The experiment batch | Group | Dosage g/kg | Duck number (only) bid * 10 | The clear DHBV-DNA OD490 of Sanguis Anas domestica value (X ± SD) | ????P3 | ||
| ????T0 | ????T5 | ????T10 | |||||
| I | The normal saline ampelopsin | 1.25 2.5 5.0 | ????6 ????6 ????6 ????6 | ?0.958±0.06 ?0.928±0.08 ?0.907±0.07 ?1.275±0.23 | ?0.926±0.07 ?0.747±0.11** ?0.781±0.08* ?1.031±0.23** | ?0.930±0.07 ?0.851±0.11 ?0.686±0.09** ?0.927±0.14** | ?0.910±0.07 ?0.793±0.23 ?0.701±0.05** ?0.959±0.11** |
| II | The normal saline ampelopsin | 1.25 2.5 5.0 | ????6 ????6 ????6 ????6 | ?0.549±0.05 ?0.611±0.16 ?0.716±0.05 ?1.051±0.39 | ?0.528±0.04 ?0.534±0.08 ?0.667±0.07 ?0.680±0.32** | ?0.514±0.03 ?0.556±0.10 ?0.593±0.05** ?0.765±0.30** | ?0.515±0.03 ?0.555±0.10 ?0.672±0.07 ?0.826±0.27* |
| III | Normal saline ampelopsin 3TC | 1.25 2.5 5.0 0.05 | ????6 ????6 ????6 ????6 ????6 | ?0.802±0.05 ?0.966±0.06 ?1.372±0.23 ?1.374±0.09 ?1.554±0.17 | ?0.795±0.06 ?1.017±0.20 ?1.071±0.14* ?0.682±0.38** ?1.195±0.62 | ?0.755±0.02 ?0.970±0.23 ?0.993±0.11** ?0.923±0.43* ?0.657±0.20** | ?0.776±0.08 ?0.906±0.12 ?1.006±0.11* ?1.235±0.32 ?1.742±0.14 |
Statistical disposition: t1, P1: (T5, T10, P3) the clear DHBV-DNA OD of Sanguis Anas domestica value compares (paired t-test) with preceding (T0) OD value of infection to administration group different time.
*p1＜0.05，
**p1＜0.01。
Sum up: ampelopsin is tested at duck hepatitis B virus infection duck interior therapeutic, three batches of experimental results show: the duck hepatitis B virus infection duck is oral ampelopsin treatment in the 7th day after infection, 5.0g/kg the inhibition effect to infected duck serum DHBV-DNA level had the highly significant effect in one day 2 times 10 days, (P＜0.01), nontoxic renaturation reaction; 2.5g/kg group administration the 5th, 10 day in experiment has certain inhibitory action.Remarkable and highly significant effect are arranged, (P＜0.05,0.01).The 1.25g/kg group does not have obvious inhibitory action in the experiment.The oral inhibitory action to DHBV-DNA of positive drug lamivudine has the inhibitory action of highly significant.With repeatedly experimental result was consistent in the past.Above experimental result shows: ampelopsin treatment duck hepatitis B virus infection duck is effective, and effective dose is 2.5-5.0g/kg, one day 2 times 10 days.
Table 4. is oral ampelopsin treatment group and viral infection matched group in duck hepatitis B virus infection duck body
The comparison of the horizontal suppression ratio of the clear DHBV-DNA of Sanguis Anas domestica
Experiment drug dose duck is counted T5 suppression ratio (%) P3
T10
I virus control 6 3.32 2.91 4.90
1.25????6??????19.57*?????8.31????????14.80
Ampelopsin 2.5 6 13.52 23.68 22.03*
5.0?????6??????19.39**????26.76**?????23.96**
II virus control 6 3.51 5.86 5.62
1.25????6??????10.50??????6.68????????7.06
Ampelopsin 2.5 6 6.86 17.02* 6.16
5.0?????6??????36.35**????26.80*??????20.01
III virus control 6 0.88 5.65 2.99
1.25????6?????-5.34???????0.23????????5.94
Ampelopsin 2.5 6 19.93* 26.39** 23.56
5.0?????6??????50.82**????32.87???????9.53
3TC???????????????????50mg/kg?6??????23.88??????56.67**????-13.16
Statistical disposition: t2, P2: (T5, T10, P3) the clear DHBV-DNA level of Sanguis Anas domestica suppresses % relatively (t checks in groups) with infection preceding (T0) inhibition % and virus control group relatively to administration group different time.
*p2＜0.05，
**p2＜0.01，
***p2＜0.001。
Embodiment 9: the test of the function of ampelopsin enhancing immunity
1. ampelopsin is to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function
1.1 experiment purpose
Observe the influence of ampelopsin to the mice phagocytic function.
1.2 be subjected to the reagent thing
Title: ampelopsin
Solvent: hot distilled water
Consumption: 0.5ml/20g mice
1.3 control sample
Bifendate; Shanghai balance pharmaceutical factory, the accurate word (1995) of medicine is defended No. 3765-011 in Shanghai
1.4 animal
Mice, the Kunming kind, male, 19～21g.
Chicken red blood cell: 5% concentration
1.5 method
Kunming mouse, be divided into 5 groups at random, be matched group, ampelopsin 75,150,300mg/kg bifendate 150mg/kg, matched group gives normal saline, the equal oral administration of administration group, every day 1 time, totally 6 times, lumbar injection 0.5% lactoalbumin hydrolysate 1.5ml/ simultaneously of last administration, pneumoretroperitoneum was injected 5% chicken red blood cell 0.1ml in 24 hours, the blood-letting of breaking end after 30 minutes, use the normal saline flushing abdominal cavity, collect peritoneal macrophage and hatch half an hour for 37 ℃, centrifugal, the precipitate smear, dyeing, counting cells under the oil mirror calculates with following formula, and compares with matched group.
1.6 result
The results are shown in Table 5, the basic, normal, high dosage of ampelopsin can promote all Turnover of Mouse Peritoneal Macrophages to engulf chicken red blood cell and increase phagocytic index that bifendate 150mg/kg dosage group also can promote the Turnover of Mouse Peritoneal Macrophages phagocytic function.
Table 5. ampelopsin to the influence of Turnover of Mouse Peritoneal Macrophages phagocytic function (± SD)
| Group | Dosage approach mg/kg | Number of animals (only) | Phagocytic percentage % | Phagocytic index |
| Ampelopsin | 75???po×6 | ????8 | ????31.2±3.4 ** | ????0.64±0.07 ** |
| 150??po×6 | ????8 | ????43.2±4.2 | ????0.85±0.02 ** | |
| 300??po×6 | ????8 | ????42.3±4.8 ** | ????0.81±0.06 ** | |
| Bifendate | 150??po×6 | ????8 | ????40.1±4.5 ** | ????0.88±0.03 ** |
| Matched group | Coordinative solvent | ????8 | ????23.0±5.3 | ????0.39±0.12 |
*Compare with model control group P＜0.01
2. ampelopsin is to the influence of mice spleen lymphocytes proliferation
2.1 experiment purpose
Observe the influence of ampelopsin to mice spleen lymphocytes proliferation.
2.2 be subjected to the reagent thing
Title: ampelopsin
Solvent: hot distilled water
Consumption: 0.5ml/20g mice
2.3 control sample
Bifendate; Shanghai balance pharmaceutical factory, the accurate word (1995) of medicine is defended No. 3765-011 in Shanghai
2.4 animal
Mice, C
57BL/6, ♂, 19～21g.
2.5 other materials
Canavaline (ConA): Sigma product, 50 μ g/ml concentration
3H-TdR: Shanghai nuclear research institute, radioactive concentration 1mci/ml
Culture medium: RPMI-1640 includes 15% calf serum, mercaptoethanol, Hepes etc.
2.6 method
C
57The BL/6 mice is divided into 5 groups at random, i.e. normal control group, ampelopsin 75,150,300mg/kg, bifendate 150mg/kg, every day, oral administration was 1 time, and continuous 7 days, after administration finishes, put to death under the animal aseptic condition and get spleen, the counting splenocyte, and the adjustment cell concentration is 1 * 10
7/ ml, every hole adds cell suspension 100 μ l on 96 porocyte culture plates, ConA 50 μ l, and culture fluid, each group is all established three multiple pipes, 37 ℃, 5%CO
2Cultivated 48 hours under the condition, add
3H-TdR 0.5 μ ci/ hole continues to cultivate 18 hours, with bull cell harvestor collecting cell, surveys the CPM value on liquid scintillation instrument, and compares with matched group, the results are shown in Table 6.
2.7 result
Table 6. ampelopsin is to the influence of mice spleen lymphocytes proliferation
| Group | Dosage approach mg/kg | Number of animals (only) | The CPM value (± SD) |
| Ampelopsin | ??75?????po×7 | ????4 | ????11763±1985 * |
| ??150????po×7 | ????4 | ????7547±1369 * | |
| ??300????po×7 | ????4 | ????7832±1298 * | |
| Bifendate | ??150????po×7 | ????4 | ????7188±1010 ** |
| Matched group | ??N.S. | ????4 | ????5448±917 |
*P＜0.05
*Compare with matched group P＜0.01
The result shows that Caulis seu folium ampelopsis brevipedunculatae (Caulis Ampelopsis Brevipedunculae) have the effect of tangible promotion mice spleen lymphocytes proliferation, and strong than other two groups with the low dosage effect.
Embodiment 10: acute toxicity test
After getting mice prerun, alternative is got 20 mices, and male and female half and half are given the mouse stomach administration with the Cmax and the maximum volume of ampelopsin, and 7d is observed in 24h administration 3 times continuously, situations such as record mice crawler behavior, defecation and diet.Result: observed 7 days, and do not see that animal had situations such as dystropy, death, body weight normal growth.Calculating its maximum filling stomach amount is 26.0gkg
-1
Embodiment 11: the experiment of ampelopsin resisiting influenza virus:
Separation of influenza virus and mirror (dripping) are fixed
Embryo Gallus domesticus at present commonly used separates and carries out influenza virus, and route of inoculation is the allantoic cavity of Embryo Gallus domesticus, occurs the characteristics of lesion of back chicken embryo death after 3 days and hemagglutinin occurs, and to this, we adopt the method to carry out evaluation to ampelopsin.We adopt the influenza virus of certain density extracting solution and 4 HAUs to mix at 1: 1, allow the two inject Embryo Gallus domesticus after 20 minutes again in the room temperature effect, and injection volume is every embryo 0.2ml.The Embryo Gallus domesticus of 9 to 11 ages in days is adopted in experiment, carry out the allantoic cavity inoculation, cultivate after 48 hours, placed liquid in 4 ℃, aseptic then its urine of getting, get supernatant through 1000rpm after centrifugal 10 minutes, packing is deposited for-20 ℃, the usefulness that is equipped with detection, the detection of urine divides blood clotting and blood to press down two kinds, and the erythrocyte of use comes prosperous cock, and method is a conventional method.
The antiviral experiment: the ampelopsin with variable concentrations carries out the antiviral experiment in Embryo Gallus domesticus
Table 7 hirst's hemagglutination inhibition test
| Sample concentration | Serum dilution | ||||||||
| Stock solution | 10× | 20× | 40× | 80× | 160× | 320× | 640× | 1280× | |
| Stock solution 1 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | ||
| Stock solution 2 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | ||
| Stock solution 3 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | |
| Stock solution 4 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | |
| 10 -5Stock solution 1 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ |
| 10 -5Stock solution 2 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ |
| 10 -5Stock solution 3 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | ++++ | |
| 10 -5Stock solution 4 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | |
| 10 -6Stock solution 1 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | ++++ | |
| 10 -6Stock solution 2 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | ++++ | |
| 10 -6Stock solution 3 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | |
| 10 -6Stock solution 4 | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++ | |
Remarks: 1, stock solution refers to the ampelopsin 4mg/ml of initial concentration
2,10
-5With 10
-6Finger carries out the dilution factor of medicine from ampelopsin stock solution
3, during result of determination by blood clotting intensity respectively with ++ ++, +++, ++ ,-expression coagulation appears with 50% erythrocyte ++ the blood clotting dilution factor be its blood clotting decline
Erythrocyte is the invalid main aperture of fine sand grain sample (pipe) end person +++, i.e. 100% coagulation; Erythrocyte evenly is laid on the end, hole (pipe), but the edge is whole to hole (pipe) end concentrator is not slightly +++, i.e. 75% coagulation; (pipe) end, form a ring-type to erythrocyte in the hole, has little coagulation piece person to be all around ++, i.e. 50% coagulation; (pipe) end, form wad to erythrocyte in the hole, but the edge is smooth inadequately, have slightly all around coagulation piece person for+, i.e. 25% coagulation; (pipe) end, form wad to erythrocyte in the hole, and the smooth neat person in edge is one, does not promptly have coagulation.
Above-mentioned experimental data proof ampelopsin has the resisiting influenza virus effect.
Claims (16)
1. a broad-spectrum antiviral and improve the pharmaceutical composition of immunity is characterised in that ampelopsin and pharmaceutical acceptable excipient that it contains the antiviral effective dose.
2. the pharmaceutical composition of claim 1, wherein said pharmaceutical composition institute antiviral is hepatitis B virus, influenza virus and/or coronavirus.
3. the pharmaceutical composition of claim 1 wherein contains the ampelopsin of 2% to 98% weight ratio.
4. each pharmaceutical composition of claim 1-3 wherein further contains the active constituents of medicine compatible with ampelopsin.
5. the pharmaceutical composition of claim 4, wherein said compatible active constituents of medicine is the flavonoid medicine, flavonoid medicament contg and ampelopsin content weight ratio can be 2-80: 20-98 in the compositions.
6. the pharmaceutical composition of claim 5, wherein said flavonoid medicine is an ampelopsin.
7. arbitrary pharmaceutical composition of claim 1-6 is in the purposes of preparation in the broad-spectrum antiviral medicament.
8. arbitrary pharmaceutical composition of claim 1-6 is in the purposes of preparation in the anti-hepatic-B virus medicine.
9. the purposes of arbitrary pharmaceutical composition of claim 1-6 in preparation anti-coronavirus medicine.
10. arbitrary pharmaceutical composition of claim 1-6 is in the purposes of preparation in the anti-influenza virus medicament.
11. the purposes of arbitrary pharmaceutical composition of claim 1-6 in preparation raising immunity medicine.
12. the purposes of ampelopsin in the preparation broad-spectrum antiviral medicament.
13. the purposes of ampelopsin in the preparation anti-hepatic-B virus medicine.
14. the purposes of ampelopsin in preparation anti-coronavirus medicine.
15. the purposes of ampelopsin in the preparation anti-influenza virus medicament.
16. the purposes of ampelopsin in preparation raising immunity medicine.
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| CN 03138055 CN1644199A (en) | 2003-05-30 | 2003-05-30 | Use of ampeloptin in preparation of antivirus medicines |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102772398A (en) * | 2011-05-13 | 2012-11-14 | 贵州省生物研究所 | Application of dihydromyricetin in preparation of drug preventing and treating influenza |
| CN110420274A (en) * | 2019-09-12 | 2019-11-08 | 江苏农牧科技职业学院 | A kind of pharmaceutical composition and its preparation method and application for treating canine coronavirus disease |
| WO2021175295A1 (en) * | 2020-03-06 | 2021-09-10 | 中国科学院上海药物研究所 | Application of myricetin compound in preparation of drugs for prevention and treatment of novel coronavirus pneumonia |
| WO2021243756A1 (en) * | 2020-06-03 | 2021-12-09 | 上海爱启医药技术有限公司 | Pharmaceutical use of myricetin in inhibition of novel coronavirus |
| CN115645393A (en) * | 2020-02-26 | 2023-01-31 | 上海科技大学 | Application of dihydromyricetin in resisting coronavirus |
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2003
- 2003-05-30 CN CN 03138055 patent/CN1644199A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102772398A (en) * | 2011-05-13 | 2012-11-14 | 贵州省生物研究所 | Application of dihydromyricetin in preparation of drug preventing and treating influenza |
| CN110420274A (en) * | 2019-09-12 | 2019-11-08 | 江苏农牧科技职业学院 | A kind of pharmaceutical composition and its preparation method and application for treating canine coronavirus disease |
| CN115645393A (en) * | 2020-02-26 | 2023-01-31 | 上海科技大学 | Application of dihydromyricetin in resisting coronavirus |
| WO2021175295A1 (en) * | 2020-03-06 | 2021-09-10 | 中国科学院上海药物研究所 | Application of myricetin compound in preparation of drugs for prevention and treatment of novel coronavirus pneumonia |
| WO2021243756A1 (en) * | 2020-06-03 | 2021-12-09 | 上海爱启医药技术有限公司 | Pharmaceutical use of myricetin in inhibition of novel coronavirus |
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