CN105982899A - Application of tryptantrin in preparation of anti-complement medicine - Google Patents

Abstract

The invention belongs to the field of traditional Chinese medicine pharmacy, and relates to the use of Qingdanone in the preparation of anti-complement drugs. The present invention separates the indole alkaloid compound indole alkaloid Qingdan ketone from the powder of the traditional Chinese medicine Qingdai, and adopts modern pharmacological research methods to confirm that it has a strong inhibitory effect on the activation of the classical pathway and the bypass pathway of the complement system, and its CH ₅₀ value is 0.024± 0.006mg/ml, AP ₅₀ value is 0.058±0.012mg/ml. The compounds are useful in the preparation of complement inhibitors.

Description

Qingdainone purposes in preparing anticomplement medicament

Technical field

The invention belongs to field of traditional Chinese medicine pharmacy, relate to qingdainone purposes in preparing anticomplement medicament.

Background technology

Research shows that the excessive activation of complement system can initiating system lupus erythematosus (SLE), rheumatoid pass The scorching multiple major disease such as (RA), adult respiratory distress syndrome (ARDS) of joint.Anticomplement medicament is studied for many years Carry out the focus and emphasis of always world pharmaceutical research.But at present this type of disease is still lacked ideal controlling Treat medicine, be badly in need of efficient, low toxicity, single-minded novel complement inhibitor the most clinically.Grind from natural product Study carefully exploitation complement inhibitor be in recent years one by more and more important research field paid close attention to, it has cost The features such as low, toxicity is low.Chinese scholars separates from the multiple natural product including marine organisms To the multiple monomeric compound with complement system inhibitory action, the research and development for anticomplement medicament provide Wide prospect.

Indigo Naturalis (Indigo Naturalis) is acanthaceous vegetable acanthaceous indigo Baphicacanthus cusia (Nees) Bremek., polygonaceae plant polygonum tinctorium ait. Polygonum tinctorium Ait. or cruciferae isatis Isatis The leaf of indigotica Fort. or the processed prepared dried powder of stem and leaf or agglomerate.Its property saline taste is trembled with fear, and returns Liver Channel. Can heat-clearing and toxic substances removing, blood cooling and ecchymoses removing, pathogenic fire purging arresting convulsion.For maculae caused by violent heat pathogen, heat in blood tell nosebleed, chest pain hemoptysis, aphtha, Mumps, sore throat, pediatric epilepsy scared.Existing pharmacological research shows, Indigo Naturalis has antibacterial, antiinflammatory, antitumor, anti- The effects such as oxidation, treatment psoriasis.Alkaloid therein is its main active, has antitumor, disease-resistant Poison, antiinflammatory and the biological activity such as antibacterial.

So far, there is not yet the report of the anticomplementary activity of qingdainone about deriving from Indigo Naturalis.The application sends out A person of good sense intends the new purposes providing the indole alkaloid qingdainone deriving from Indigo Naturalis in preparing anticomplement medicament.

Summary of the invention

It is an object of the invention to provide the qingdainone new purposes in preparing anticomplement medicament, be specifically related to source In the indole alkaloid qingdainone of the Indigo Naturalis purposes in preparing anticomplement medicament.

Present invention application modern pharmacology screening technique, studies anticomplementary activity material in Chinese medicine Indigo Naturalis, from The ethyl acetate extract isolated indole alkaloid compounds qingdainone of Indigo Naturalis ethanol extraction, and carry out The test of In Vitro Anti classical pathway of complement and In Vitro Anti alternative pathway of complement are tested, as a result, it was confirmed that described indole is raw Classical pathway and the alternative pathway of complement system are activated and all have stronger suppression to make by alkaloids compounds qingdainone With.

Indole alkaloid compound qingdainone of the present invention has a following chemical constitution:

Described compound qingdainone: purple powder, EI-MS:m/z 363 [M]⁺, molecular formula is C₂₃H₁₃N₃O₂。¹H-NMR(DMSO-d₆,400MHz):δ_H11.85 (1H, s, NH), 9.24 (1H, d, J=8.0Hz), 8.63 (1H, D, J=8.2Hz), 8.34 (1H, d, J=8.1Hz), 8.33 (1H, s), 8.26 (1H, d, J=7.9Hz), 7.98 (1H, dd, J=7.0Hz, 8.3Hz), 7.76 (1H, d, J=7.6Hz), 7.70 (1H, dd, J=7.0Hz, 8.0Hz), 7.64 (1H, dd, J=7.1Hz, 7.7Hz), 7.59 (2H, m), 7.49 (1H, dd, J=7.1Hz, 8.4Hz), 7.13 (1H, dd, J=7.2 Hz,7.6Hz).。

Described compound qingdainone is prepared by following method:

Taking Chinese medicine Indigo Naturalis powder, 95% alcohol reflux 3 times, each 2h, united extraction liquid is also concentrated to give leaching Cream, add water suspendible, respectively with equal-volume petroleum ether, ethyl acetate and n-butanol extraction 5 times, combining extraction liquid And be concentrated to dryness, obtain acetic acid ethyl ester extract；By Ethyl acetate fraction through silica gel (200-300 mesh) post Chromatographic isolation, successively with methylene chloride-methanol gradient elution, obtains 8 streams part (Fr.1-8), wherein stream part Fr.5 Again through silica gel column chromatography (methylene chloride-methanol, 10:1,5:1,3:1,1:1), Sephadex LH-20 post color Spectrum (chloroform-methanol, 1:1) and reversed-phase HPLC (methanol-water, 20:80-80:20 gradient elution) purification, point From obtaining qingdainone.

Above-mentioned indole alkaloid compound qingdainone, through In Vitro Anti complement activity test determination, shows that it is to complement The classical pathway of system and alternative pathway activate all stronger Complement inhibition effect, its CH₅₀And AP₅₀Value point It is not 0.024 ± 0.006mg/ml, 0.058 ± 0.012mg/ml.Positive control heparin CH₅₀And AP₅₀Value point It is not 0.026 ± 0.005mg/ml, 0.054 ± 0.016mg/ml.

Accompanying drawing explanation

Fig. 1 is the extraction separation process figure of qingdainone in Indigo Naturalis.

Detailed description of the invention

Embodiment 1 prepares qingdainone

Taking Indigo Naturalis powder 5kg, 95% alcohol reflux 3 times (50L × 3), each 2h, united extraction liquid is also Being concentrated to give extractum 0.25kg, add water (4L) suspendible, respectively with equal-volume petroleum ether, ethyl acetate and positive fourth Alcohol extracts 5 times, and combining extraction liquid is also concentrated to dryness, and obtains acetic acid ethyl ester extract 50g.Ethyl acetate is extracted portion Position, through silica gel (200-300 mesh) pillar layer separation, is washed with methylene chloride-methanol (50:1-0:1) gradient successively De-, obtain 8 streams part (Fr.1-8), wherein stream part Fr.5 (4g) is again through silica gel column chromatography (dichloromethane-first Alcohol, 10:1,5:1,3:1,1:1), Sephadex LH-20 column chromatography (chloroform-methanol, 1:1) and reversed-phase HPLC Means purification such as (methanol-water, 20:80-80:20 gradient elutions), isolated qingdainone.

Embodiment 2 In Vitro Anti classical pathway of complement is tested

Take complement (guinea pig serum) 0.04ml, add barbitol buffer solution (BBS) and be configured to the solution of 1:10, The solution of 1:20,1:40,1:80,1:160,1:320,1:640 and 1:1280 is become with BBS two-fold dilution. Take 1:1000 hemolysin, 2% sheep red blood cell (SRBC) each 0.1ml and each concentration complement 0.2ml and be dissolved in 0.2 In ml BBS, mixing, put into low-temperature and high-speed centrifuge after 37 DEG C of water-bath 30min, 4000rpm, 4 DEG C Under the conditions of centrifugal 5min.Take often pipe supernatant 0.2ml respectively and, in 96 orifice plates, measure its absorbance at 405nm. Experiment arranges full haemolysis group (0.1ml 2%SRBC, 0.1ml hemolysin is dissolved in 0.4ml tri-distilled water) simultaneously. Using the absorbance of tri-distilled water haemolysis pipe as full haemolysis standard, calculate hemolysis rate.With complement dilution factor as X-axis, Percentage of hemolysis is Y-axis mapping, selects to reach the minimum complement concentration of similar high hemolysis rate as guaranteeing system Critical complement concentration needed for the normal haemolysis of energy；The complement taking critical concentration mixes with test sample, as stated above Absorbance is measured under 405nm；Experiment arranges test sample matched group, complement group and full haemolysis group simultaneously.Will Test sample absorbance calculates hemolysis rate after deducting corresponding test sample matched group absorbance, makees with test sample concentration For X-axis, haemolysis suppression ratio is as the concentration (CH of test sample needed for Y-axis mapping, calculating 50% suppression haemolysis₅₀), Result is 0.024 ± 0.006mg/ml.Positive control heparin CH₅₀It is 0.026 ± 0.005mg/ml.

Embodiment 3 In Vitro Anti alternative pathway of complement is tested

Taking complement (human serum) 0.2ml, (barbitol buffer solution, pH=7.4, containing 5mM to add AP diluent Mg²⁺, 8mM EGTA) be configured to the solution of 1:5, and two-fold dilution become 1:10,1:20,1:40,1:80, The solution of 1:160,1:320 and 1:640.Take 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, 4000rpm, centrifugal 5min under the conditions of 4 DEG C.Take often that pipe supernatant 0.2ml is in 96 orifice plates, at 405nm respectively Measure absorbance.Experiment arranges full haemolysis group (0.20ml 0.5%RE is dissolved in 0.3ml tri-distilled water) simultaneously.With The absorbance of tri-distilled water haemolysis pipe, as full haemolysis standard, calculates hemolysis rate.With complement dilution factor as X-axis, Percentage of hemolysis is Y-axis mapping.Select to reach the minimum complement concentration of similar high hemolysis rate as guaranteeing system Critical complement concentration needed for the normal haemolysis of energy.The complement taking the critical concentration determined mixes with test sample, by upper Method of stating measures its absorbance under 405nm.Experiment arranges test sample matched group, complement group and the most molten simultaneously Blood group.Hemolysis rate is calculated after test sample absorbance is deducted corresponding test sample matched group absorbance.For examination Product concentration is mapped as Y-axis as X-axis, haemolysis suppression ratio, test sample needed for calculating 50% suppression haemolysis Concentration (AP₅₀), result is 0.058 ± 0.012mg/ml.Positive control heparin AP₅₀Value is 0.054 ± 0.016 mg/ml。

The reagent testing employing in the present invention is techniques well known, commercially available.

Claims (4)

1. the purposes of the indigo ketone of following formula structure in the preparation anti-complement medicine, 2. The use according to claim 1, characterized in that, said indigocin has a complement inhibitory effect on the classical pathway of the complement system. 3. The use according to claim 1, characterized in that, said indigocin has a complement inhibitory effect on the activation of the alternative pathway of the complement system. 4. according to the described purposes of claim 1, it is characterized in that, described compound indigo ketone is prepared by following method: Take the Chinese medicine Qingdai powder, 95% ethanol reflux extraction 3 times, 2 hours each time, combine the extracts and concentrate to obtain the extract, add water to suspend, extract 5 times with equal volumes of petroleum ether, ethyl acetate and n-butanol respectively, and combine the extracts and concentrated to dryness to obtain the ethyl acetate extract; the ethyl acetate extraction part was separated by silica gel, 200-300 mesh, column chromatography, and sequentially eluted with dichloromethane-methanol gradient to obtain 8 fractions, Fr.1 -8, where fraction Fr.5 is subjected to silica gel column chromatography, wherein, dichloromethane-methanol, 10:1, 5:1, 3:1, 1:1, Sephadex LH-20 column chromatography, wherein, chloroform-methanol, 1:1, and reversed-phase HPLC, methanol-water, 20:80-80:20, gradient elution, purification, and separation to obtain Qingdanone.