CN105078937A - Application of dicyclogermacrane type sesquiterpenoids in preparing anticomplementary drugs - Google Patents
Application of dicyclogermacrane type sesquiterpenoids in preparing anticomplementary drugs Download PDFInfo
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- CN105078937A CN105078937A CN201410201402.8A CN201410201402A CN105078937A CN 105078937 A CN105078937 A CN 105078937A CN 201410201402 A CN201410201402 A CN 201410201402A CN 105078937 A CN105078937 A CN 105078937A
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Abstract
The invention belongs to the pharmaceutical field of traditional Chinese medicine, and relates to dicyclogermacrane type sesquiterpenoids as shown in formula I and novel application thereof in preparing anticomplementary drugs. Two dicyclogermacrane type sesquiterpenoids are separated from the petroleum ether extraction part of a dried whole-plant extract of viola yedoensis makino, and tests prove that the two dicyclogermacrane type sesquiterpenoids have different inhibitory effects on both the classical pathway and the alternative pathway of a complement system: CH50 and AP50 are respectively 0.14-0.22mg/ml and 0.33-0.44mg/ml. The sesquiterpenoids disclosed by the invention can be used for preparing anticomplementary drugs and for further preparing medicines for treating complement-related diseases.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to dicyclo germacrane sesquiterpenoids in Herba Violae and preparing the novelty teabag in anticomplement medicament.
Background technology
Report display, the multiple major diseases such as systemic lupus erythematosus (sle), rheumatoid arthritis, adult respiratory distress syndrome rise because the excessive activation of complement system caused.Anticomplement medicament research is one of focus and emphasis of world pharmaceutical research for many years always, but still lacks ideal medicine to this type of disease at present, is therefore badly in need of efficient, low toxicity, single-minded novel complement inhibitor clinically.Research and develop from natural product complement inhibitor be in recent years one be subject to more and more important research field paid close attention to, it has the features such as cost is low, toxicity is low.Chinese scholars has been separated from the multiple natural product comprising marine organisms etc. is had the inhibiting monomeric compound of complement system in a large number, and the research and development for anticomplement medicament provide wide prospect.
Chinese medicine Herba Violae is the dry herb of Violaceae Chinese violet (ViolayedoensisMakino).Its property bitter, acrid, cold; GUIXIN, Liver Channel; There is heat-clearing and toxic substances removing, effect of removing heat from blood detumescence; To swell and ache etc. for jaundice interior-heat, furuncle swelling toxin, sore throat the treatment of disease.Forefathers only concentrate on antiviral and the aspect such as antibacterial to the pharmacological research of Herba Violae, chemical constitution study has found some flavonoids, Coumarins, alkaloids and cyclic peptide compound, but up to now, there is not yet about dicyclo germacrane sesquiterpenoids has the report of Complement inhibition effect.
Summary of the invention
The object of this invention is to provide the new material with anticomplementary activity, be specifically related to dicyclo germacrane sesquiterpenoids in Herba Violae, especially dicyclo germacrane sesquiterpenoids isobicyclogermacrenal (1) and madolinY (2) in Herba Violae.
A further object of the present invention is to provide dicyclo germacrane sesquiterpenoids in above-mentioned Herba Violae and is preparing the purposes in anticomplement medicament.
The present invention's application modern pharmacology screening technique, carries out anticomplementary activity evaluation study to being separated the material obtained.Be separated from the petroleum ether extraction position of the dry herb ethanol extraction of Herba Violae (Violayedoensis) and obtain 2 sesquiterpenoids materials, and two compounds described in confirming all there is inhibitory action in various degree to complement system classical pathway and alternative pathway.
Active dicyclo germacrane sesquiterpenoids of the present invention has the chemical constitution of formula I:
The preferred isobicyclogermacrenal of dicyclo germacrane sesquiterpenoids of the present invention (1) and madolinY (2).Wherein, each substituent group and compound as shown in table 1.
Table 1
In the present invention, when double bond is Δ
1 (10), R
1for H, R
2during for H, compound is isobicyclogermacrenal (1); ; When double bond is Δ
10 (15), R
1for OH, R
2during for OH, compound is madolinY (2).
Dicyclo sesquiterpenoids of the present invention is prepared by following method:
Get dry Herba Violae herb 20kg, pulverize, (50L × 5 time) are extracted with 95% ethanol room temperature merceration, merge extractive liquid, is also concentrated into without alcohol taste, extractum is diluted with water to 2.5L, successively with equal-volume petroleum ether (60-90 DEG C), ethyl acetate, n-butanol extraction (each 2.5L × 3 time), merge petroleum ether extraction liquid and be concentrated into dry, obtaining petroleum ether extract 323g.(200g) is separated through silica gel column chromatography at petroleum ether extraction position, successively with petroleum ether-ethyl acetate (petroleum ether, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1) gradient elution, obtain 7 streams part (Fr.A-G), through silica gel column chromatography, (petroleum ether-ethyl acetate is eluant wherein to flow part Fr.E (22.2g), 30:1, 20:1, 15:1, 10:1, 5:1) with SephadexLH-20 (chloroform-methanol, 1:1) purification repeatedly, be separated finally by half preparative HPLC [methanol-water (65:35) is eluant] and obtain 2 sesquiterpenoidss, be respectively isobicyclogermacrenal (1, 19mg) with madolinY (2, 31mg).
In the present invention,
Compound 1 (isobicyclogermacrenal): colorless oil liquid, [α]
d 25=-29 ° (c0.10mg/ml, acetone),
1h-NMR (400MHz, acetone-d
6) δ: 9.30 (1H, s, H-15), 6.33 (1H, d, J=10.2Hz, H-5), 5.02 (1H, dd, J=9.4, 6.8Hz, H-1), 2.65 (H, m, H-3a), 2.44 (1H, m, H-2b), 2.24 (1H, ddd, J=12.6, 3.6, 3.1Hz, H-9b), 2.13 (H, m, H-3b), 1.99 (1H, ddd, J=12.9, 12.2, 3.2Hz, H-9a), 1.95 (1H, m, H-8a), 1.93 (1H, m, H-2a), 1.62 (3H, s, H-14), 1.21 (3H, s, H-12), 1.13 (1H, dd, J=9.6, 4.9Hz, H-6), 1.10 (3H, s, H-13), 1.00 (1H, m, H-8b), 0.55 (1H, m, H-7),
13c-NMR (100MHz, acetone-d6) δ: 193.1 (C-15), 156.3 (C-5), 143.7 (C-4), 134.3 (C-10), 124.3 (C-1), 40.0 (C-9), 38.5 (C-7), 29.7 (C-6), 28.5 (C-13), 27.5 (C-2), 23.6 (C-8), 23.4 (C-3), 21.3 (C-11), 17.4 (C-14), 16.4 (C-12), ESI-MS:m/z241 [M+Na]
+.
Compound 2 (madolinY): colourless oil liquid, [α]
d 25=+104 ° (c0.10mg/ml, acetone),
1h-NMR (400MHz, CDCl
3) δ: 9.25 (1H, s, CHO), 6.31 (1H, d, J=8.8Hz, H-5), 5.04 (1H, s, H-15a), 4.94 (1H, s, H-15b), 4.57 (1H, d, J=11.2Hz, H-1), 3.99 (1H, J=11.2Hz, H-12b), 3.87 (1H, d, J=11.2Hz, H-12a), 2.73 (1H, m, H-3b), 2.45 (1H, m, H-3a), 2.43 (1H, m, H-2b), 2.32 (1H, m, H-7), 2.27 (1H, m, H-9b), 2.16 (1H, m, H-2a), 2.14 (1H, m, H-9a), 2.05 (2H, m, H-8), 1.86 (1H, m, H-6), 1.26 (3H, s, H-13),
13c-NMR (100MHz, CDCl
3) δ: 194.0 (CHO), 151.8 (C-5), 146.1 (C-4), 145.6 (C-10), 117.6 (C-15), 88.5 (C-1), 73.0 (C-12), 36.1 (C-9), 33.2 (C-11), 28.0 (C-3), 27.3 (C-2), 25.4 (C-6), 21.9 (C-8), 21.0 (C-7), 11.4 (C-13).
Above-mentioned sesquiterpenoids is by classical pathway and the test determination of alternative pathway In Vitro Anti complement activity, result shows, above-mentioned sesquiterpenoids all has inhibitory action (as shown in table 2) to the classical pathway of complement system and alternative pathway, the inhibitory action (mean ± SD, n=3) of table 2. compound 1 and 2 pairs of complement system classical pathwaies and alternative pathway
Dicyclo germacrane sesquiterpenoids of the present invention can be used for preparing anticomplement medicament.
Sesquiterpenoids of the present invention can prepare the medicine for the treatment of and complement-associated disease further; Described comprises the diseases such as systemic lupus erythematosus (sle), rheumatoid arthritis, adult respiratory distress syndrome with complement-associated disease.
Accompanying drawing explanation
Fig. 1.: the extraction and isolation flow chart of Herba Violae ethanol extract petroleum ether extraction position dicyclo germacrane sesquiterpenoids dicyclo germacrane sesquiterpenoids 1 and 2.
Detailed description of the invention
The preparation of embodiment 1. dicyclo germacrane sesquiterpenoids
Get dry Herba Violae herb 20kg, pulverize, (50L × 5 time) are extracted with 95% ethanol room temperature merceration, merge extractive liquid, is also concentrated into without alcohol taste, extractum is diluted with water to 2.5L, successively with equal-volume petroleum ether (60-90 DEG C), ethyl acetate, n-butanol extraction (each 2.5L × 3 time), merge petroleum ether extraction liquid and be concentrated into dry, obtaining petroleum ether extract 323g.(200g) is separated through silica gel column chromatography at petroleum ether extraction position, successively with petroleum ether-ethyl acetate (petroleum ether, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1) gradient elution, obtain 7 streams part (Fr.A-G), through silica gel column chromatography, (petroleum ether-ethyl acetate is eluant wherein to flow part Fr.E (22.2g), 30:1, 20:1, 15:1, 10:1, 5:1) with SephadexLH-20 (chloroform-methanol, 1:1) purification repeatedly, be separated finally by half preparative HPLC [methanol-water (65:35) is eluant] and obtain 2 compounds, be respectively isobicyclogermacrenal (1, 19mg) with madolinY (2, 31mg).
Embodiment 2. In Vitro Anti classical pathway of complement is tested
Get complement (guinea pig serum) 0.1ml, add the solution that barbitol buffer solution (BBS) is mixed with 1:5, become the solution of 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640 with BBS two-fold dilution.Getting 1:1000 hemolysin, each concentration complement and 2% sheep red blood cell (SRBC) each 0.1ml is dissolved in 0.3mlBBS, and mixing, puts into low-temperature and high-speed centrifuge after 37 DEG C of water-bath 30min, centrifugal 10min under 5000rpm, 4 DEG C of conditions.Get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure its absorbance at 405nm.Experiment arranges full haemolysis group (0.1ml2%SRBC is dissolved in 0.5ml tri-distilled water) simultaneously.Using the absorbance of tri-distilled water haemolysis pipe as full haemolysis standard, calculate hemolysis rate.With complement dilution factor for X-axis, the percentage of hemolysis that each diluted concentration complement causes is Y-axis mapping.Select the minimum complement concentration reaching similar high hemolysis rate as the critical complement concentration guaranteed needed for the normal haemolysis of system energy.Complement and the test sample of getting critical concentration mix, and after 37 DEG C of pre-water-bath 10min, add appropriate BBS, hemolysin and 2%SRBC.Put into low-temperature and high-speed centrifuge by after every pipe 37 DEG C of water-bath 30min, get often pipe supernatant 0.2ml under 5000rpm, 4 DEG C of conditions after centrifugal 10min respectively and, in 96 orifice plates, under 405nm, measure absorbance.Experiment arranges test sample matched group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated after test sample absorbance being deducted corresponding test sample matched group absorbance.Using test sample concentration as X-axis, haemolysis suppression ratio is mapped as Y-axis, calculates the concentration (CH that 50% suppresses test sample needed for haemolysis
50); Result shows (as shown in table 2), and compound 1 and 2 pairs of complement system classical pathwaies have inhibitory action (mean ± SD, n=3).
Embodiment 3. In Vitro Anti alternative pathway of complement is tested
Get complement (human serum) 0.2ml, (barbitol buffer solution, pH=7.4, containing 5mMMg to add AP dilution
2+, 8mMEGTA) and liquid is mixed with the solution of 1:5, and two-fold dilution becomes the solution of 1:10,1:20,1:40,1:80,1:160,1:320 and 1:640.Get each concentration complement 0.15ml, AP diluent 0.15ml and 0.5% rabbit erythrocyte (RE) 0.20ml, mixing, 37 DEG C of water-bath 30min are placed on low-temperature and high-speed centrifuge, centrifugal 10min under 5000rpm, 4 DEG C of conditions.Get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure absorbance at 405nm.Experiment arranges full haemolysis group (0.20ml0.5%RE is dissolved in 0.3ml tri-distilled water) simultaneously.Using the absorbance of tri-distilled water haemolysis pipe as full haemolysis standard, calculate hemolysis rate.With complement dilution factor for X-axis, the percentage of hemolysis that each diluted concentration complement causes is Y-axis mapping.Select the minimum complement concentration reaching similar high hemolysis rate as the critical complement concentration guaranteed needed for the normal haemolysis of system energy.Complement and the test sample of getting the critical concentration determined mix, and after 37 DEG C of pre-water-bath 10min, add 0.2ml0.5%RE.Every pipe 37 DEG C of water-bath 30min are placed on low-temperature and high-speed centrifuge, under 5000rpm, 4 DEG C of conditions after centrifugal 10min, get often pipe supernatant 0.2ml respectively and, in 96 orifice plates, under 405nm, measure its absorbance.Experiment arranges test sample matched group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated after test sample absorbance being deducted corresponding test sample matched group absorbance.Using test sample concentration as X-axis, haemolysis suppression ratio is mapped as Y-axis, calculates the concentration (AP that 50% suppresses test sample needed for haemolysis
50); Result shows (as shown in table 2), and compound 1 and 2 pairs of complement system alternative pathways have inhibitory action (mean ± SD, n=3)
The reagent of testing employing in the present invention is techniques well known, commercially available.
The inhibitory action (mean ± SD, n=3) of table 2. compound 1 and 2 pairs of complement system classical pathwaies and alternative pathway
Claims (4)
1. the dicyclo germacrane sesquiterpenoids of formula Ι is preparing the purposes in anticomplement medicament:
Wherein, when double bond is Δ
1 (10), R
1for H, R
2during for H, compound is isobicyclogermacrenal (1); ; When double bond is Δ
10 (15), R
1for OH, R
2during for OH, compound is madolinY (2).
2., by purposes according to claim 1, it is characterized in that, described compound 1 and 2 pairs of complement system classical pathwaies.
3. by purposes according to claim 1, it is characterized in that, described compound 1 and 2 suppresses complement system alternative pathway.
4., by the preparation method of the dicyclo germacrane sesquiterpenoids of claim 1, it is characterized in that, it comprises:
Get dry Herba Violae herb, pulverize, 5 times are extracted with 95% ethanol room temperature merceration, merge extractive liquid, is also concentrated into without alcohol taste, extractum thin up, successively with equal-volume petroleum ether 60-90 DEG C, ethyl acetate, n-butanol extraction 3 times, merge petroleum ether extraction liquid and be also concentrated into dry, obtain petroleum ether extract, petroleum ether extraction position is separated through silica gel column chromatography, successively with petroleum ether-ethyl acetate (petroleum ether, 50:1, 30:1, 20:1, 10:1, 5:1, 1:1) gradient elution, obtain 7 streams part (Fr.A-G), through silica gel column chromatography, (petroleum ether-ethyl acetate is eluant wherein to flow part Fr.E, 30:1, 20:1, 15:1, 10:1, 5:1) with SephadexLH-20 (chloroform-methanol, 1:1) purification repeatedly, be separated finally by half preparative HPLC [methanol-water (65:35) is eluant] and obtain 2 sesquiterpenoidss, be respectively isobicyclogermacrenal (1) and madolinY (2).
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Citations (3)
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|---|---|---|---|---|
| CN102558278A (en) * | 2010-12-28 | 2012-07-11 | 复旦大学 | Triterpene compound and application thereof in preparation of anti-complementary medicament |
| CN103508922A (en) * | 2012-06-25 | 2014-01-15 | 复旦大学 | Dipeptide compound and use of the same in preparation of anti-complement drugs |
| CN103508919A (en) * | 2012-06-25 | 2014-01-15 | 复旦大学 | Alkaloid compound and use of the same in preparation of anti-complement drugs |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102558278A (en) * | 2010-12-28 | 2012-07-11 | 复旦大学 | Triterpene compound and application thereof in preparation of anti-complementary medicament |
| CN103508922A (en) * | 2012-06-25 | 2014-01-15 | 复旦大学 | Dipeptide compound and use of the same in preparation of anti-complement drugs |
| CN103508919A (en) * | 2012-06-25 | 2014-01-15 | 复旦大学 | Alkaloid compound and use of the same in preparation of anti-complement drugs |
Non-Patent Citations (1)
| Title |
|---|
| TIAN-SHUNG WU 等: "New Sesquiterpenes from the Roots and Stems of Aristolochia mollissima", 《JOURNAL OF THE CHINESE CHEMICAL SOCIETY》 * |
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