CN105985402B - A kind of sterol lactam compound Lasiosphaera fenzlii lactams and its purposes in anticomplement medicament is prepared - Google Patents
A kind of sterol lactam compound Lasiosphaera fenzlii lactams and its purposes in anticomplement medicament is prepared Download PDFInfo
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- CN105985402B CN105985402B CN201510052295.1A CN201510052295A CN105985402B CN 105985402 B CN105985402 B CN 105985402B CN 201510052295 A CN201510052295 A CN 201510052295A CN 105985402 B CN105985402 B CN 105985402B
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- lasiosphaera fenzlii
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Abstract
The invention belongs to field of traditional Chinese medicine pharmacy, is related to a kind of sterol lactam compound and its purposes in anticomplement medicament is prepared.Present invention sterol lactam compound Lasiosphaera fenzlii lactams isolated from the fructification of Lasiosphaera fenzlii, the compound is the ergosterol of B cyclic lactams, through anti-complement activity evaluation test, as a result confirming described sterol lactam compound Lasiosphaera fenzlii lactams, it has stronger inhibitory action to complement system classical pathway and alternative pathway activation, its CH50It is worth for 0.032 ± 0.007mg/ml, AP50It is worth for 0.067 ± 0.009mg/ml.The compound can be used for preparing complement inhibitor.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, is related to a kind of sterol lactams noval chemical compound Lasiosphaera fenzlii lactams and its resists preparing
Purposes in complement medicine.
Background technology
Prior art discloses the excessive activation of complement system can trigger systemic loupus erythematosus (SLE), rheumatoid to close
A variety of major diseases such as section scorching (RA), ARDS (ARDS).Anticomplement medicament research is always generation for many years
The focus and emphasis of boundary's study of pharmacy.But still lack ideal medicine to such disease at present, therefore clinically
It is badly in need of efficient, less toxic, single-minded new complement inhibitor.It is one in recent years that complement inhibitor is researched and developed from natural products
By the important research field of more and more concerns, it has the characteristics that cost is low, toxicity is low.Domestic and foreign scholars are from including sea
Isolated a variety of monomeric compounds with complement system inhibitory action in a variety of natural products including foreign biology, for anti-benefit
The research and development of body medicine provide wide prospect.
Lasiosphaera fenzlii also known as horse Hui Bao, grey steamed stuffed bun etc., it is a kind of common macro fungi based on medicinal, there is detumescence, stop
Blood, clearing lung-heat, relieving sore-throat, removing toxic substances etc. act on.Version in 2010《Chinese Pharmacopoeia》Regulation, Lasiosphaera fenzlii is Hui Bao sections fungi lasiosphaera fenzlii Reics
(Lasiosphaerafenzlii Reich.), calvatia gigantea (Calvatiagigantea (Batsch ex Pers.) Lloyd) or
The drying fructification of purple Lasiosphaera fenzlii (Calvatia lilacina (Mont.et Berk.) Lloyd).Research shows existing
Main active is sterol in chemical composition, additionally containing terpene, fitter acids and its ester, amino acid, polysaccharide etc., is had anti-
The pharmacological activity such as bacterium, anti-inflammatory, cough-relieving, antitumor, suppression free radical.
Present invention is intended providing a kind of sterol lactams noval chemical compound isolated from Lasiosphaera fenzlii, is named as in Lasiosphaera fenzlii
Acid amides, the compound structure novel and unique, for the ergosterol structure of B cyclic lactams, its structure and bioactivity are showed no report
Road.
The content of the invention
It is an object of the invention to provide a kind of sterol lactam compound Lasiosphaera fenzlii lactams and its preparing anticomplement medicament
In purposes.
The present invention applies modern pharmacological research method, from the ethanol extract Ethyl acetate fraction of Lasiosphaera fenzlii fructification
A kind of isolated sterol lactams noval chemical compound Lasiosphaera fenzlii lactams, and anticomplement work is carried out to isolated monomeric compound
Property evaluation, as a result confirm its to complement system classical pathway and alternative pathway activation have stronger inhibitory action.
Lasiosphaera fenzlii lactams of the present invention has following chemical constitution：
Compound of the present invention is prepared by following methods：
Take Lasiosphaera fenzlii raw material crush, alcohol reflux extraction, merge extract solution simultaneously be concentrated to give medicinal extract, add water to be suspended, respectively with etc.
Petroleum ether, ethyl acetate and extracting n-butyl alcohol, combining extraction liquid are simultaneously concentrated to dryness, and Ethyl acetate fraction is through silica gel column chromatography
The purifying of the means such as separation, Sephadex LH-20 column chromatographys and reversed-phase HPLC, isolated compound Lasiosphaera fenzlii lactams.
The compound Lasiosphaera fenzlii lactams：White powder, molecular formula C28H41NO2,EI-MS m/z:423[M]+。1H-NMR
(400MHz,CDCl3)δH:7.45 (1H, s, NH), 5.76 (1H, s), 5.57 (1H, s), 5.25 (1H, dd, J=15.2Hz,
7.4Hz), 5.18 (1H, dd, J=15.2Hz, 8.3Hz), 1.30 (3H, s), 1.04 (3H, d, J=6.6Hz), 0.94 (3H, d, J
=6.8Hz), 0.85 (6H, d, J=6.6Hz), 0,66 (3H, s).13C-NMR(100MHz,CDCl3)δC:35.2 (C-1), 33.0
(C-2), 198.0 (C-3), 115.1 (C-4), 161.6 (C-5), 166.7 (C-6), 119.0 (C-7), 156.5 (C-8), 46.2
(C-9), 40.4 (C-10), 23.4 (C-11), 39.2 (C-12), 45.9 (C-13), 58.2 (C-14), 25.8 (C-15), 33.0
(C-16), 56.3 (C-17), 12.4 (C-18), 22.2 (C-19), 40.4 (C-20), 21.0 (C-21), 134.8 (C-22),
132.7 (C-23), 42.8 (C-24), 33.0 (C-25), 19.6 (C-26), 19.9 (C-27), 17.6 (C-28).
Lasiosphaera fenzlii lactams produced by the present invention has carried out external anticomplement classical pathway experiment and external anticomplement bypass way
Footpath is tested, and is tested and is determined through external anti-complement activity, the results showed that its activation of classical pathway and alternative pathway to complement system
There is stronger inhibitory action, its CH50And AP50Value is respectively 0.032 ± 0.007mg/ml, 0.067 ± 0.012mg/ml.Sun
Property control heparin CH50And AP50Value is respectively 0.026 ± 0.005mg/ml, 0.054 ± 0.016mg/ml.Acyl in described Lasiosphaera fenzlii
Amine can be used for preparing complement inhibitor.
Brief description of the drawings
Fig. 1 is the extraction separation process figure of Lasiosphaera fenzlii lactams in Lasiosphaera fenzlii.
Embodiment
Embodiment 1 prepares Lasiosphaera fenzlii lactams
Get it filled material Lasiosphaera fenzlii fructification 25kg, crushes, and extracts 3 times (50L × 3) with 95% alcohol reflux, each 2h, merging carries
Take liquid and be concentrated to give medicinal extract 0.85kg, add water (4L) to be suspended, respectively with isometric petroleum ether, ethyl acetate and extracting n-butyl alcohol
5 times, combining extraction liquid is simultaneously concentrated to dryness, and obtains acetic acid ethyl ester extract 160g；By Ethyl acetate fraction through silica gel (200-
300 mesh) pillar layer separation, successively with methylene chloride-methanol (50:1-0:1) gradient elution, 10 fractions (Fr.1-10) are obtained,
Wherein fraction Fr.6 (14g) again through silica gel column chromatography (methylene chloride-methanol, 30:1,20:1,10:1,5:1,3:1)、
Sephadex LH-20 column chromatographys (chloroform-methanol, 1:1) and HPLC (methanol-water, 20:80-80:20, gradient elution) etc. means
Purifying, isolated compound Lasiosphaera fenzlii lactams；
The compound Lasiosphaera fenzlii lactams：White powder, molecular formula C28H41NO2,EI-MS m/z:423[M]+。1H-NMR
(400MHz,CDCl3)δH:7.45 (1H, s, NH), 5.76 (1H, s), 5.57 (1H, s), 5.25 (1H, dd, J=15.2Hz,
7.4Hz), 5.18 (1H, dd, J=15.2Hz, 8.3Hz), 1.30 (3H, s), 1.04 (3H, d, J=6.6Hz), 0.94 (3H, d, J
=6.8Hz), 0.85 (6H, d, J=6.6Hz), 0,66 (3H, s).13C-NMR(100MHz,CDCl3)δC:35.2 (C-1), 33.0
(C-2), 198.0 (C-3), 115.1 (C-4), 161.6 (C-5), 166.7 (C-6), 119.0 (C-7), 156.5 (C-8), 46.2
(C-9), 40.4 (C-10), 23.4 (C-11), 39.2 (C-12), 45.9 (C-13), 58.2 (C-14), 25.8 (C-15), 33.0
(C-16), 56.3 (C-17), 12.4 (C-18), 22.2 (C-19), 40.4 (C-20), 21.0 (C-21), 134.8 (C-22),
132.7 (C-23), 42.8 (C-24), 33.0 (C-25), 19.6 (C-26), 19.9 (C-27), 17.6 (C-28).
The external anticomplement classical pathway experiment of embodiment 2
Complement (guinea pig serum) 0.04ml is taken, barbitol buffer solution (BBS) is added and is configured to 1:10 solution, with BBS pairs
1 is diluted to again:20、1:40、1:80、1:160、1:320、1:640 and 1:1280 solution；Take 1:1000 hemolysins, 2% sheep are red
Each 0.1ml of cell (SRBC) and each concentration complement 0.2ml are dissolved in 0.2ml BBS, are mixed, and are put into after 37 DEG C of water-bath 30min low
Warm supercentrifuge, 5min is centrifuged under the conditions of 4000rpm, 4 DEG C.Every pipe supernatant 0.2ml is taken respectively in 96 orifice plates, in 405nm
Determine its absorbance；Experiment sets full haemolysis group (0.1ml 2%SRBC, 0.1ml hemolysins are dissolved in 0.4ml tri-distilled waters) simultaneously,
Using the absorbance of tri-distilled water haemolysis pipe as full haemolysis standard, hemolysis rate is calculated.Using complement dilution factor as X-axis, percentage of hemolysis
Mapped for Y-axis, the minimum complement concentration that selection reaches similar high hemolysis rate is critical needed for the normal haemolysis of system energy as ensuring
Complement concentration, the complement and test sample for taking critical concentration mix, and determine absorbance under 405nm as stated above, experiment is simultaneously
Test sample control group, complement group and full haemolysis group are set, test sample absorbance is deducted into corresponding test sample control group absorbance
Hemolysis rate is calculated after value, using test sample concentration as X-axis, haemolysis inhibiting rate is mapped as Y-axis, is calculated 50% and is suppressed needed for haemolysis
Concentration (the CH of test sample50), it is as a result 0.032 ± 0.007mg/ml, positive control heparin CH50It is worth for 0.026 ± 0.005mg/
ml。
The external anticomplement alternative pathway experiment of embodiment 3
Complement (human serum) 0.2ml is taken, adds AP dilutions (barbitol buffer solution, pH=7.4, Mg containing 5mM2+,8mM
EGTA) it is configured to 1:5 solution, and two-fold dilution is into 1:10、1:20、1:40、1:80、1:160、1:320 and 1:640 it is molten
Liquid；Each concentration complement 0.15ml, AP dilution 0.15ml and 0.5% rabbit erythrocyte (RE) 0.20ml is taken, is mixed, 37 DEG C of water-baths
Low-temperature and high-speed centrifuge is placed in after 30min, 5min is centrifuged under the conditions of 4000rpm, 4 DEG C.Every pipe supernatant 0.2ml is taken respectively in 96
Orifice plate, absorbance is determined in 405nm；Experiment sets full haemolysis group (0.20ml 0.5%RE are dissolved in 0.3ml tri-distilled waters) simultaneously, with
The absorbance of tri-distilled water haemolysis pipe calculates hemolysis rate, using complement dilution factor as X-axis, percentage of hemolysis Y as full haemolysis standard
Axle is mapped；Selection reaches the minimum complement concentration of similar high hemolysis rate as the critical complement ensured needed for the normal haemolysis of system energy
Concentration, take the complement of the critical concentration of determination to be mixed with test sample, determine its absorbance under 405nm as stated above, test
Test sample control group, complement group and full haemolysis group are set simultaneously, test sample absorbance is deducted into corresponding test sample control group and inhaled
Hemolysis rate is calculated after shading value, using test sample concentration as X-axis, haemolysis inhibiting rate is mapped as Y-axis, is calculated 50% and is suppressed haemolysis
Concentration (the AP of required test sample50), it is as a result 0.067 ± 0.009mg/ml, positive control heparin AP50Be worth for 0.054 ±
0.016mg/ml。
The reagent that use is tested in the present invention is techniques well known, commercially available.
Claims (4)
1. the sterol lactam compound Lasiosphaera fenzlii lactams of formula,
2. purposes of the sterol lactam compound Lasiosphaera fenzlii lactams in anticomplement medicament is prepared described in claim 1.
3. the purposes as described in claim 2, it is characterised in that described sterol lactam compound Lasiosphaera fenzlii lactams is to complement
The classical pathway of system suppresses.
4. the purposes as described in claim 2, it is characterised in that described sterol lactam compound Lasiosphaera fenzlii lactams is to complement
The alternative pathway activation of system suppresses.
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
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Patent Citations (1)
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 《Anti-complement constituents of Commelina communis and their targets》;Jiahong Jin et al.;《Journal of Chinese Pharmaceutical Sciences》;20121231;第21卷(第6期);全文 * |
| 《天然产物中的抗补体活性成分》;徐晗等;《中国天然药物》;20070930;第5卷(第5期);全文 * |
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