CN104906118A - Medicine for treating H1N1 influenza virus infection - Google Patents
Medicine for treating H1N1 influenza virus infection Download PDFInfo
- Publication number
- CN104906118A CN104906118A CN201510332559.9A CN201510332559A CN104906118A CN 104906118 A CN104906118 A CN 104906118A CN 201510332559 A CN201510332559 A CN 201510332559A CN 104906118 A CN104906118 A CN 104906118A
- Authority
- CN
- China
- Prior art keywords
- medicine
- ilicis asprellae
- flos ilicis
- influenza virus
- saponin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Steroid Compounds (AREA)
Abstract
The invention relates to medical products containing organic effective components, and in particular relates to a monomer composition for treating H1N1 influenza virus infection. The effective component is prepared from 3,4,5-trimethoxyphenol-beta-D-5-O-caffeoyl-apiose-(1-6)-beta-D-heteroside and 3-O-beta-L-sulfating arabinose-19-O-sulfating hydroxyl-oleanolic acid-28-O-beta-D-glucose ester at the mass ratio of 1 to (0.5-1). The medicine is significant in treatment effect on the H1N1 influenza virus infection.
Description
Technical field
The present invention relates to the pharmaceutical products containing organic effective component, be specifically related to the medicine containing Triterpenoids sapogenins compounds and phenolic acid compound.
Background technology
Influenza is called for short influenza, is the respiratory tract disease caused by influenza virus, has that popular wide, infectiousness is strong, sickness rate high, and the mortality rate in child, old man and high-risk group is higher.Influenza virus is that one of human health threatens greatly.The major way of infected by influenza is answered to be vaccine and Drug therapy.To the Influenza epidemic situation of possibility large-scale outbreak, Drug therapy is the means of best control transmission of influenza virus.At present, anti-influenza virus medicament comprises at the arbidol HCl of Russia's listing and 4 anti-influenza virus medicaments of U.S. FDA approval, and the latter is divided into M2 ionophorous protein inhibitor and neuraminidase (NA) inhibitor according to the difference of mechanism of action.In recent years, greater advance is achieved to the research of anti-influenza virus medicament.M2 ionophorous protein inhibitor is the influenza clinical treatment medicine gone on the market the earliest, but there is neurotoxicity, and long-term taking easily produces drug resistance strain and to defects such as Type B influenza are invalid.Although current antiviral Western medicine kind is more, most selectivity is low and untoward reaction is comparatively large, and Chinese medicine has its particular advantages at anti-virus aspect, and untoward reaction is relatively low, not easily forms drug resistance.Chinese medicine can be divided into two large classes according to resisiting influenza virus approach: a class has direct antiviral effect, wherein great majority are antipyretic and antidotal type Chinese medicine, as Flos Lonicerae, and Herba Houttuyniae, Radix Isatidis, Folium Isatidis, Radix Scutellariae, Rhizoma Coptidis etc.; And another kind of by strengthening the indirect resisiting influenza virus effect of immunocyte ability orientation, as the Radix Astragali, Radix Salviae Miltiorrhizae etc. can inducement interferon and immunoglobulins.Study more clearly Anti-flu ingredient and mainly contain two classes: one is polyphenols, not only can suppress influenza virus protein matter and RNA synthesis, also can suppress the adsorption of influenza virus simultaneously; Another kind of is Flavonoid substances, can suppress the activity of influenza virus sialidase and suppress film fusion.Chinese medicine has good antivirus action, both directly can kill virus and not injure body tissue, can transfer again the enthusiasm of immune defense system, and the active immunity mechanism of excitating organism, enhancing immunity, plays antivirus action indirectly by Initiative Defense.Thus, Chinese medicine produces drug resistance at Western medicine, virus morphs, still there is potential therapeutical effect, there is adaptability and superiority widely; And Chinese herbal medicine enriches in china natural resources, cheap, presents wide research and development prospect.Therefore, develop determined curative effect, treat influenza infection safely and efficiently medicine very necessary.
Flos Ilicis Asprellae is the dry root of holly plant Folium Mume Ilicis Purpureae Ilex Asprella (Hook.et Arn.) Champ.ex Benth., and main product is in the ground such as Guangdong, Guangxi, and its nature and flavor are bitter, micro-sweet, cool, there is heat-clearing and toxic substances removing, promoting the production of body fluid to quench thirst, relieving sore throat and diminishing swelling, effect of eliminating stasis to stop pain, is used for the treatment of cold, fever, cough due to lung-heat, calentura Tianjin wound is thirsty, laryngopharynx swelling and pain, traumatic injury stasis of blood pain etc. are also one of conventional raw materials of guangdong herbal tea.Modern pharmacology research shows that it has the effects such as antiinflammatory, antiviral, heart tonifying.Wang Ningsheng [ZHANG A L, QI Y, LI B G, et a1.Phenolic and Iriterpene glycosides from the stems of llex litseaefolia [J] .J Nat Prod, 2005,68:1531-1535.] etc. report, have the compositions such as phenolic acid, saponin, steroidal and alkaloid in Flos Ilicis Asprellae.Open also for the application for a patent for invention of CN 101879200A discloses a kind of preparation method of holly root anti-influenza effective part, a kind of holly root anti-influenza effective part that the method obtains is total saponin of Radix Ilicis Asprellae, and wherein characteristic saponin is Cheng oxysteroid 3-O-β-D-Fructus Vitis viniferae glycoside.But the effect of total saponin of Radix Ilicis Asprellae treatment H1N1 type influenza infection is still not ideal enough described in above-mentioned patent application.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of medicine for the treatment of H1N1 type influenza infection, and this medicine can significantly improve the effect for the treatment of H1N1 type influenza infection.
The technical scheme that the present invention solves the problem is:
A kind of medicine for the treatment of H1N1 type influenza infection, this medicine is made up of effective ingredient and the acceptable adjuvant of medical science, it is characterized in that, described effective ingredient is by 3, 4, 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides and 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester composition, the quality proportioning of the two is 3, 4, 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides ︰ 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester=1:0.5 ~ 1.
Of the present invention 3,4,5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides and 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester can adopt conventional method to extract from Chinese medicine Flos Ilicis Asprellae or other plant and obtain, also can by synthesize or additive method obtains.
Shown in the following formula I of chemical constitution of above-mentioned 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester, above-mentioned 3,4, shown in the following formula II of chemical constitution of 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides
The effective ingredient of medicine of the present invention is by separate from Flos Ilicis Asprellae 3,4,5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides and 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester two compound monomer composition, the Be very effective of its anti-H1N1 type influenza virus.
Accompanying drawing explanation
The IR figure of Fig. 1 compounds I of the present invention
Fig. 2 compounds I of the present invention
1h-NMR schemes
Fig. 3 compounds I of the present invention
13c-NMR schemes
The DEPT figure of Fig. 4 compounds I of the present invention
The HSQC figure of Fig. 5 compounds I of the present invention
The HMBC figure of Fig. 6 compounds I of the present invention
Fig. 7 compound ii of the present invention
1h-NMR schemes (500MHz)
II of Fig. 8 compound of the present invention
13c-NMR schemes (150MHz)
Detailed description of the invention
Following by compound 3 for convenience of description, 4,5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides is referred to as Flos Ilicis Asprellae 639 phenolic acid, and compound 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester is referred to as Flos Ilicis Asprellae 925 saponin.
Example 1 (preparation of Flos Ilicis Asprellae 925 saponin)
One, the preparation of compound
(1) Radix Ilicis Asprellae medical material 20kg, be ground into coarse powder, adding 8 times amount volumetric concentrations is the ethanol of 70%, and point 4 cold-macerations extract, each 12h, merge 4 extracting solution, decompression recycling ethanol, obtain dry extractum 638g, extractum adds 4L water makes suspension, divide 3 extractions by the ethyl acetate of 12L, leave standstill 1h at every turn, obtain Ethyl acetate fraction (185g).
(2) Ethyl acetate fraction dissolves, cross AB-8 macroporous resin, be eluted to distilled water and there is no color, use successively again 4 times of column volumes (Φ 15 × 61cm) volumetric concentration be 20%, 50%, 70%, 95% ethanol gradient elution, collect 50% alcohol elution decompression and solvent recovery, obtain total saponin of Radix Ilicis Asprellae extract 86g, total saponin content 89.73%.
(3) get 400gODS-C18 reverse phase silica gel, methanol is fully swelling in right amount, dress post, dissolve by proper amount of methanol, cross silicagel column, be respectively 20% by the volumetric concentration of 3 times of column volumes successively, 30%, 40%, 50%, the methanol elution gradient of 60%, 70%, eluent concentrates, volumetric concentration be 10% sulphuric acid ethanol TLC develop the color, get 50% eluting position, repeatedly cross silicagel column, purification obtains compound Flos Ilicis Asprellae 925-saponin (47.2mg), and crystallization is dissolved repeatedly, and recrystallization obtains sterling compound.
Two, the qualification of compound
The compound that this example obtains, is colorless needle crystals (methanol).UV λ max (MeoH) nm:202,10% ethanol solution of sulfuric acid displaing amaranth (TLC), Liebermann-Burehard and Molish reaction is the positive, infers that this compound is triterpene saponin componds.IR(KBr):νmax3438,2942,2886,1728,1637,1460,1388,1229,1072,1017,913,825,589cm
-1。HR-ESI-MS: quasi-molecular ion peak be m/z925 [M-H]-, daughter ion m/z 845 be [M – H – 80] – combine and infrared 1229,1072,1017cm-1 strong absworption peaks, infer contain SO
3, daughter ion m/z 763 is for [M – H – 162] – infers in structure sugary, and determination of elemental analysis result shows, and this compound sulfur-bearing is 6.610%, infers that the structural formula of this compound is C
41h
66o
19s
2.IR figure is shown in Fig. 1.
In 1H-NMR (Fig. 2) spectrum of the compound that this example obtains, seven methyl proton signals demonstrated on aglycon are δ H 1.21,0.96,0.85,1.13,1.62,1.15,0.98 (each 3H, s, 7 × CH3).41 C signals are provided in 13CNMR (Fig. 3), 8 quaternary carbon signals, δ C177.2 is ester carbonyl group C-28, δ C123.6, 144.2 be olefinic carbon signal, compose (Fig. 4) in conjunction with DEPT simultaneously and can find out that 123.6 for CH signal, so be attributed to C-12, δ C144.2 is attributed to C-13, the signal δ H 4.68 of two saccharide residue protons is demonstrated in 1H-NMR spectrum, d (7.2), 6.37, d (7.9), in conjunction with δ C106.4 in 13CNMR and DEPT spectrum, 73.9, 82.4, 74.7, 63.9 (secondary carbon) pentose is similar in conjunction with documentation & info all the other and β-D-R except δ C82.4, δ C88.7 is that 3 upper glycosidations connect oxygen carbon, δ C95.8, 74.1, 78.8, 71.0, 79.2, 62.1 is that 28 connected sugar of carbonyl and β-D-Glucose data message are similar.Comprehensive above information, infers that this compound is the oleanane-type triterpene saponin compounds also having two glycosyls.
It is C-12 that the hsqc spectrum (see Fig. 5) of this compound releases δ C123.6 according to 1D NMR, there are δ H5.50 and δ C123.6 to have to intersect peak in conjunction with HSQC, ownership δ H5.50 is H-12, and δ H5.50 (H-12) is relevant to δ H1.95, then δ H1.95 can be attributed to H-11, then be C-11 by HSQC ownership δ C24.1.δ H1.95 (H-11) is relevant to δ H1.76, and can belong to δ H1.76 is H-9, and in conjunction with HSQC, ownership δ C48.2 is C-9.δ H4.50 (H-3) is relevant to δ H1.76, then belonging to δ H1.76 is H-2, and in conjunction with HSQC, have δ H1.76 and δ C26.4 to have to intersect peak, then ownership δ C26.4 is C-2.
Fig. 6 is shown in by HMBC analysis of spectrum collection of illustrative plates, and δ H5.50 (H-12) is long-range relevant to δ C42.0,48.2 (C-9), and in conjunction with DEPT spectrum, δ C24.1 is attributed to C-11, and δ C42.0 is attributed to C-14.δ H3.52 (H-18), 3.58 (H-19) are long-range relevant to δ C46.4 (C-17), and in conjunction with DEPT spectrum, δ C44.5 is attributed to C-18, and δ C, 80.9 are attributed to C-19.By in HSQC, have δ H0.96 and δ C16.7 (C-24) to intersect peak, can belong to δ H0.96 is H-24; δ H1.21 and δ C28.0 (C-23) intersect peak, and can belong to δ H1.21 is H-23.HMBC spectrum display δ H1.21 methyl hydrogen (H-23) and δ C88.7,39.4,55.8,16.7 long-range relevant, compose in conjunction with DEPT, δ C88.7 is attributed to C-3, and δ C39.4 is attributed to C-4.Also can see that δ 0.96 methyl hydrogen (H-24) and C-3, C-4, C-5, C-23 exist long-range relevant simultaneously.δ H0.85 methyl hydrogen (H-25) and δ C33.1,40.1,48.2,24.1 relevant, in conjunction with DEPT spectrum, δ C33.1,24.1 are attributed to C-7, and C-11, δ C40.1,40.1 can be attributed to C-8, C-9, and δ C17.5 is attributed to C-26.δ H1.62 methyl hydrogen and δ C40.1,144.2 (C-13), 42.0,29.0 long-range relevant, δ H1.15 is methyl hydrogen (H-29), in conjunction with HMBC spectrum, with δ C80.9,35.5,28.9,28.7 exist long-range relevant, then δ C80.9 is attributed to C-19 and moves to low field containing sulfonic group, composes δ C35.5 in conjunction with DEPT, 28.9 are attributed to C-20, C-21.Comprehensive above structural information draws Flos Ilicis Asprellae 925 saponin of the structure of this compound as shown in formula I.
Example 2 (preparation of Flos Ilicis Asprellae 639 phenolic acid)
One, the preparation of compound
(1) Radix Ilicis Asprellae coarse powder 20kg, according to gained Optimized Extraction Process, adding 10 times amount volumetric concentrations is the ethanol of 54%, gradation reflux, extract, and each 88min, gets filtrate, concentrates and reclaim ethanol to obtain extractum (385g), and is dissolved in water;
(2) getting appropriate pretreated X-5 macroporous adsorbent resin, take volumetric concentration as the ethanol of 40% is eluant, and elution flow rate is 2mL/min, obtains 40% macroporous resin eluting position Flos Ilicis Asprellae phenolic acid extract (86g).
(3) utilize ODS column chromatography to carry out crude separation, be respectively 30% with volumetric concentration, 40%, 50%, the methanol-eluted fractions of 60%, obtain 24 flow points.TLC and liquid phase carry out detecting, merging, obtain 9 flow points, flow point 4 crosses gel Sephadex LH-20 post, with methanol-0.1% formic acid (1:1v/v) eluting, efficient half preparation liquid phase purification is finally adopted to obtain Flos Ilicis Asprellae 639-compound of phenolic acid (29mg), crystallization is dissolved repeatedly, and recrystallization obtains sterling compound.Wherein, described methanol-0.1% formic acid to be volumetric concentration be 0.1% the mixed solution that mixes with methanol geometric ratio of formic acid.
Two, the qualification of compound
The compound separation that this example obtains is buff powder, HR-ESI-MS m/z 639.1925 [M-H]
-. determine that its molecular formula is C
29h
36o
16, degree of unsaturation is 12.After testing, its
1h-NMR,
13c-NMR data are as follows:
1H-NMR(500MHz，DMSO)δ/ppm:7.50(1H,d,J＝15.9Hz),6.27(1H,d,J＝15.9Hz)，7.06(1H,d,J＝2.2Hz),7.01(1H,d,J＝8.1Hz),6.76(1H,dd,J＝2.2,8.1Hz)，3.73(6H,s)and 3.57(3H,s),4.86(1H,d,J＝2.9Hz)and 4.80(1H,d,J＝7.6Hz),
13C-NMR(125MHz，DMSO)δ/ppm:125.6(C-1),116.0(C-2),145.9(C-3),148.8(C-4),113.8(C-5),121.6(C-6),145.7(C-7),115.1(C-8),166.7(C-9)，154.1(C-1),94.6(C-2,6),153.3(C-3,5),132.8(C-4),60.3(4-OCH
3),56.0(3,5-OCH
3)，101.0(C-1),75.6(C-2),76.9(C-3),70.2(C-4),76.7(C-5),67.9(C-6)，108.8(C-1),73.3(C-2),77.2(C-3),73.5(C-4),66.0(C-5)
See accompanying drawing 7,8,
1a pair trans olefinic proton 7.50 (1H, d, J=15.9Hz) is shown in H-NMR, 6.27 (1H, d, J=15.9Hz), 3 phenyl ring protons 7.06 (1H, d, J=2.2Hz), 7.01 (1H, d, J=8.1Hz), 6.76 (1H, dd, J=2.2,8.1Hz), 3 methyl protons 3.73 (6H, s), 3.57 (3H, s), 2 sugared anomeric proton 4.86 (1H, d, J=2.9Hz), 4.80 (1H, d, J=7.6Hz).By
13containing coffee acyl 125.6 (C-1) in C-NMR; 116.0 (C-2); 145.9 (C-3); 148.8 (C-4), 113.8 (C-5), 121.6 (C-6); 145.7 (C-7); 115.1 (C-8), 166.7 (C-9), 3; 4; 5-trimethoxyphenoxy 154.1 (C-1), 94.6 (C-2,6); 153.3 (C-3; 5), 132.8 (C-4), 60.3 (4-OCH
3), 56.0 (3,5-OCH
3), 2 glycosidation glycosyls comprise D-Glucose base 101.0 (C-1), 75.6 (C-2), 76.9 (C-3), 70.2 (C-4), 76.7 (C-5), 67.9 (C-6) and D-celery glycosyl 108.8 (C-1), 73.3 (C-2), 77.2 (C-3), 73.5 (C-4), 66.0 (C-5) are phenolic acid compound by above-mentioned this structure of feature initial guess.According to document [Nobutoshi Tanaka, Three New Hemiterpene Glycosides from Ilex macropoda, Chem, Pharm, 45 (9) 1533-1535 (1997)], identify that the compound that this example obtains is Flos Ilicis Asprellae 639 phenolic acid shown in structure formula II formula, its name is called 3, 4, 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides (3, 4, 5-trimethoxyhpenol β-D-5-O-Caffeoyl-apiofuranosyl-(1 → 6)-β-D-glucopyranoside).
Example 3:(granule)
The Flos Ilicis Asprellae 925 saponin 8g that Example 1 the is obtained and Flos Ilicis Asprellae 639 phenolic acid 12g that embodiment 2 obtains, adds 300g starch, 200g sucrose, mix homogeneously, soft material processed, granulate with 18 mesh sieves, 1h after 60 DEG C of dryings, 20 order granulate, the granule 130 bags of confessions being distributed into 4g every bag orally use.
Example 4:(capsule)
The Flos Ilicis Asprellae 925 saponin 155mg that Example 1 the is obtained and Flos Ilicis Asprellae 639 phenolic acid 155mg that embodiment 2 obtains, add the adjuvants such as 6000mg microcrystalline Cellulose, 5000mg carboxymethyl starch sodium, 4000mg sodium lauryl sulphate fully to mix, roll-in method is adopted to carry out dry granulation, mix with appropriate magnesium stearate again, being packed into 3# Capsules, making the capsule of 100 for orally using.
Example 5:(tablet)
The Flos Ilicis Asprellae 925 saponin 167mg that Example 1 the is obtained and Flos Ilicis Asprellae 639 phenolic acid 334mg that embodiment 2 obtains, add 4000mg starch, 2500mg cross-linked pvp, 3000mg carboxymethyl starch sodium mix homogeneously, with 75% alcoholic solution of 5%PVP as binding agent, soft material processed, granulates with 18 mesh sieves, 1h after 60 DEG C of dryings, appropriate Pulvis Talci is added after 20 order granulate, mixing, tabletting, making specification is that tablet 100 confessions of 100mg/ sheet orally use.
Example 6 (pharmacology, effect experiment)
Experiment Flos Ilicis Asprellae 639 phenolic acid associating Flos Ilicis Asprellae 925 saponin suppresses the effect of H1N1 type influenza virus
Experiment purpose: inquire into phenolic acid and Madin-Darby canine kidney(cell line) (MDCK) (MDCK) form of saponin on H1N1 type influenza infection and the impact of growth that Flos Ilicis Asprellae extracts, and compare from the drug effect that different Flos Ilicis Asprellae phenolic acid and saponin ratio produce.
1. experiment material
1.1 cell strain Madin-Darby canine kidney(cell line) (MDCK) (MDCK), draw from wide Yi Huyansuo Viral Laboratory, T11 generation.The frozen conservation in this room.
The test medicine of 1.2 experimental grouies and correspondence thereof
Flos Ilicis Asprellae saponin and phenolic acid mix in 1:1,1:2,2:3 ratio respectively.
1.3 reagent and instrument newborn calf serum and DMEM culture medium (Gibco company); DMSO (Guangzhou Kang Yang Chemical Co., Ltd.); DIFCO company of the trypsin U.S.; Shanghai Sheng Gong bio-engineering corporation is acted on behalf of).Olympus PM-6 inverted microscope (Japanese OLYMPUS company); ; E-52AA Rotary Evaporators (Shanghai Yarong Biochemical Instrument Plant); BP221S electronic analytical balance (Shanghai precision instrument table company limited).
2. method
The mensuration of 2.1 Flos Ilicis Asprellae monomer compositions toxic concentration on mdck cell
After conventional recovery cell, treating the enough number of experiments Trypsin conventional digestion of Growth of Cells, is 1.0 × 10 with culture medium adjustment cell concentration
5individual/mL, is inoculated in 96 porocyte culture plates with 0.1mL/ hole.After Growth of Cells to tight monolayers, Flos Ilicis Asprellae monomer composition is divided into 3 groups by Flos Ilicis Asprellae phenolic acid and Flos Ilicis Asprellae saponin different proportion, and compositions 1 ratio is Flos Ilicis Asprellae saponin: Flos Ilicis Asprellae phenolic acid=1:1; Compositions 2 ratio is Flos Ilicis Asprellae saponin: Flos Ilicis Asprellae phenolic acid=1:2; Compositions 3 ratio is Flos Ilicis Asprellae saponin: each compositions of Flos Ilicis Asprellae phenolic acid=2:3 inoculates 4 multiple holes, and arrange phenolic acid group, saponin group, arrange Normal group and ribavirin group simultaneously, cell is placed in 35 DEG C of incubators and continues cultivation 4 days.Stop experiment after 4 days, judge the toxic action of testing sample to cell with microscopic examination medicine cytopathogenic effect method (CPE method).
To the inhibiting mensuration of H1N1 type influenza virus (CPE method) on 2.2 Flos Ilicis Asprellae monomer composition MDCK
After conventional recovery cell, treating the enough number of experiments Trypsin conventional digestion of Growth of Cells, is 1.0 × 10 with culture medium adjustment cell concentration
5individual/mL, is inoculated in 96 porocyte culture plates with 0.1mL/ hole.After Growth of Cells to tight monolayers, abandon supernatant and add each acute drug 0.1mL diluted by the DMEM culture medium containing 2% calf serum.Grouping, as 2.1, attacks 100TCID simultaneously
50viral suspension 0.1mL, after 35 DEG C of absorption 2h, abandons supernatant.The pastille culture medium rejoining respective concentration continues cultivation 4 days.After stopping experiment, judge the inhibitory action of testing sample to virus with CPE observational method.Establish the contrast of positive drug ribavirin, virus control and normal cell controls in experimentation simultaneously.
To the inhibiting mensuration of H1N1 type influenza virus (chicken red blood cell agglutination) on 2.3 Flos Ilicis Asprellae monomer composition MDCK
After conventional recovery cell, treating the enough number of experiments Trypsin conventional digestion of Growth of Cells, is 1.0 × 10 with culture medium adjustment cell concentration
5individual/mL, is inoculated in 96 porocyte culture plates with 0.1mL/ hole.After Growth of Cells to tight monolayers, abandon supernatant and add each acute drug 0.1mL diluted by the DMEM culture medium containing 2% calf serum.Grouping, as 2.1, attacks 100TCID simultaneously
50viral suspension 0.1mL, after 35 DEG C of absorption 2h, abandons supernatant.The pastille culture medium rejoining respective concentration continues cultivation 4 days.After stopping experiment, judge the inhibitory action of testing sample to virus with chicken erythrocyte agglutination method.Establish the contrast of positive drug ribavirin, virus control and normal cell controls in experimentation simultaneously.
3. result
3.1 Flos Ilicis Asprellae monomer compositions are to the mensuration of MDCK Madin-Darby canine kidney(cell line) (MDCK) toxic action
The mensuration of table 1 different Flos Ilicis Asprellae monomer composition in vitro toxicity concentration
Note :-for Growth of Cells is normal, occur without pathological changes;
± be less than 10% of whole cell monolayer for cytopathy;
+ account for 25% of whole cell monolayer for cytopathy;
++ for cytopathy accounts for 50% of whole cell monolayer;
+++ for cytopathy accounts for 75% of whole cell monolayer;
++++for cytopathy accounts for more than 75% of whole cell monolayer.
3.2 Flos Ilicis Asprellae monomer compositions are to the mensuration of MDCK Madin-Darby canine kidney(cell line) (MDCK) antivirus action
Table 2 different Flos Ilicis Asprellae monomer composition antiviral effect in vitro (CPE observational method)
Note :-for Growth of Cells is normal, occur without pathological changes;
± be less than 10% of whole cell monolayer for cytopathy;
+ account for 25% of whole cell monolayer for cytopathy;
++ for cytopathy accounts for 50% of whole cell monolayer;
+++ for cytopathy accounts for 75% of whole cell monolayer;
++++for cytopathy accounts for more than 75% of whole cell monolayer.
3.3 Flos Ilicis Asprellae monomer compositions are to the inhibitory action of H1N1 type influenza virus
The inhibitory action (chicken red blood cell agglutination) of table 3 different Flos Ilicis Asprellae monomer composition anti-H1N1 type influenza virus
Note: #: display chicken erythrocyte condensation degree reaches more than 75%, represents drug on viral unrestraint effect.
+++: represent 50-75% chicken erythrocyte generation coagulation;
++: the chicken red blood cell generation coagulation representing 25%-50%;
+: represent the cell generation pathological changes being less than 25%;
-: there is not coagulation in display chicken erythrocyte, represents that drug on viral has inhibitory action.
4. conclusion
Result shows that Flos Ilicis Asprellae saponin monomer and phenolic acid monomer are 5mg/mL and following concentration no cytotoxicity at drug level, Flos Ilicis Asprellae monomer composition 1,2,3 drug level be 10mg/mL and following concentration time no cytotoxicity, after showing drug regimen, toxicity does not increase.
Can find out that Flos Ilicis Asprellae saponin monomer and the antiviral minimal effective concentration of phenolic acid monomer are obviously higher than the antiviral minimal effective concentration of Flos Ilicis Asprellae monomer composition 1,2,3 by result 3.2, after showing drug regimen, antivirus action has potentiation.Wherein the effect of compositions 2 is best.After red cell agglutination experiment simultaneously also demonstrate that drug regimen, curative effect strengthens.
Result shows that Flos Ilicis Asprellae monomer composition 1, the 2 and 3 i.e. ratio of Flos Ilicis Asprellae phenolic acid and Flos Ilicis Asprellae saponin has good inhibitory action to H1N1 type influenza virus in 1:0.5 ~ 1 under non-toxic concn.
Claims (3)
1. treat the medicine of H1N1 type influenza infection for one kind, this medicine is made up of effective ingredient and the acceptable adjuvant of medical science, it is characterized in that, described effective ingredient is by 3, 4, 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides and 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester composition, the quality proportioning of the two is 3, 4, 5-trimethoxy phenol-β-D-5-O-coffee acyloxy-celery sugar-(1 → 6)-β-D-Glucose glycosides ︰ 3-O-β-L-sulphation arabinose-19-O-sulfated hydroxyl-oleanolic acid-28-O-β-D-Glucose ester=1:0.5 ~ 1.
2. a kind of medicine for the treatment of H1N1 type influenza infection according to claim 1, is characterized in that, the weight percentage of described effective ingredient in medicine is 2% ~ 5%.
3. a kind of medicine for the treatment of H1N1 type influenza infection according to claim 1 and 2, is characterized in that, described medicine is granule, oral tablet or capsule.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510332559.9A CN104906118B (en) | 2015-06-16 | 2015-06-16 | A kind of medicine for treating H1N1 type influenza infections |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510332559.9A CN104906118B (en) | 2015-06-16 | 2015-06-16 | A kind of medicine for treating H1N1 type influenza infections |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104906118A true CN104906118A (en) | 2015-09-16 |
| CN104906118B CN104906118B (en) | 2018-01-05 |
Family
ID=54075839
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510332559.9A Active CN104906118B (en) | 2015-06-16 | 2015-06-16 | A kind of medicine for treating H1N1 type influenza infections |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104906118B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1861102A (en) * | 2005-06-20 | 2006-11-15 | 张玉生 | Tablets for promoting prodn. of body-fluid and nourishing throat |
| CN101879200A (en) * | 2010-05-28 | 2010-11-10 | 东莞广州中医药大学中医药数理工程研究院 | Preparation of holly root anti-influenza effective part and determination method |
-
2015
- 2015-06-16 CN CN201510332559.9A patent/CN104906118B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1861102A (en) * | 2005-06-20 | 2006-11-15 | 张玉生 | Tablets for promoting prodn. of body-fluid and nourishing throat |
| CN101879200A (en) * | 2010-05-28 | 2010-11-10 | 东莞广州中医药大学中医药数理工程研究院 | Preparation of holly root anti-influenza effective part and determination method |
Non-Patent Citations (1)
| Title |
|---|
| 祁银德等: "岭南地区习用中药岗梅研究进展", 《亚太传统医药》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104906118B (en) | 2018-01-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104961791B (en) | A kind of new triterpenoid, preparation method and purposes in Radix potentillae anserinae | |
| CN102302685B (en) | Common lophatherum herb extract and preparation method and application thereof | |
| CN105943532A (en) | Application of diterpenoid compound to preparation of medicament for treating liver cancer | |
| CN103665082B (en) | Hemsleya cucurbitane tetracyclic triterpenoid compound, pharmaceutical compositions containing same and application of compound and pharmaceutical composition | |
| CN100584856C (en) | The purposes of a kind of Hederagenin, its preparation method and preparation antitumor drug thereof | |
| CN104945452B (en) | A kind of preparation method and applications of phillygenol glucuronic acid derivative | |
| CN104774232B (en) | Hemsleya amabilis cucurbitane tetracyclotriterpene compounds, pharmaceutical composition containing compounds and application thereof | |
| CN105524063A (en) | Novel terpene indole alkaloid compound, and preparation method and medical application thereof | |
| CN103626824A (en) | Hemsleya amabilis cucurbitane tetracyclic triterpene compound, pharmaceutical composition comprising compound and application of pharmaceutical composition and compound | |
| CN106008543A (en) | Novel diterpenoid compound and preparation method thereof | |
| CN106008485A (en) | Medicinal composition of glimepiride, and application thereof in biomedicines | |
| CN102988529B (en) | Preparation method and novel application of total phenolic acid in cherry stones | |
| CN105640971B (en) | Application of the total saposins in terms of preparing auxiliary hyperglycemic drug in prematurity Fructus Monordicae extract | |
| CN102267891B (en) | Novel triterpenoid compound and preparation method thereof | |
| CN101375841B (en) | Daucane type sesquiterpenes and preparation method and application thereof | |
| CN104906118B (en) | A kind of medicine for treating H1N1 type influenza infections | |
| CN102408464A (en) | Novel notriterpenoid saponin compound and preparation method and application thereof | |
| CN105998041B (en) | Rosmarinic acid -4-O- β-D-Glucose glycosides is preparing the application in controlling flu pharmaceutical in advance | |
| CN104873526B (en) | Application of the Flos Ilicis Asprellae oleanane glycoside compound in antithrombotic reagent is prepared | |
| CN105061550B (en) | A kind of steroid saponin compound extracting from RHIZOMA PARIDIS and purposes | |
| CN110922444B (en) | Panaxadiol type triterpenoid saponin with anti-inflammatory activity | |
| CN109091602B (en) | Effective component of semen allii tuberosi, extraction method and application thereof in preparing liver injury protection medicine | |
| CN103610682A (en) | Preparation method of 3(alpha)-hydroxyl-30-olive-12,20(29)-diene-28-acid and application in preparing anti-tumor drug | |
| CN103980198B (en) | Alkaloid Casuarinine H and the purposes in preparation treatment nerve degenerative diseases medicine thereof | |
| CN113637021B (en) | Active compound, extraction method and application thereof, pharmaceutical composition and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |