CN103083292B - Shikonin compound and purpose thereof in preparing anticomplement medicines - Google Patents
Shikonin compound and purpose thereof in preparing anticomplement medicines Download PDFInfo
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- CN103083292B CN103083292B CN201110339029.9A CN201110339029A CN103083292B CN 103083292 B CN103083292 B CN 103083292B CN 201110339029 A CN201110339029 A CN 201110339029A CN 103083292 B CN103083292 B CN 103083292B
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Abstract
The invention belongs to the field of pharmacy, and relates to a purpose of a shikonin compound in preparing anticomplement medicines. According to the invention, the shikonin compound is extracted from an ethyl acetate extract of a boraginaceae plant arnebia euchroma (Royle) Johnst. With an in-vitro complement activity evaluation test, it is verified that the compound has high inhibition effect upon classical pathway and alternative pathway of a complement system. The CH50 of the inhibition effect of the compound upon the classical pathway of the complement system is 0.16+- 0.02 - 0.63+- 0.09mM, and AP50 of the inhibition effect of the compound upon the alternative pathway of the complement system is 0.24 +-0.04 - 0.41+-0.04mM. The compound can be used for preparing anti-complement medicines.
Description
Technical field
The invention belongs to field of traditional Chinese medicine pharmacy, relate to shikonin compounds and prepare the novelty teabag in anticomplement medicament, described shikonin compounds comprises compound shikonin (1), 2,3-dimethyl-2-amylenes acyl shikonin (2) and acetylshikonin (3).
Background technology
The various diseases such as excessive activation meeting initiating system lupus erythematosus, rheumatoid arthritis, adult respiratory distress syndrome of complement system.At present desirable medicine be there is no to this type of disease.Research about anticomplement medicament is the focus and emphasis of world pharmaceutical research for many years always.Research display, the cost directly researching and developing complement inhibitor from natural product is low, and most of active component can directly be digested and assimilated by body as a part for natural product, from natural origin, therefore find the new medicine with anticomplementary activity be in recent years subject to people and more and more pay close attention to.Scholar both domestic and external is separated and obtains a large amount of having the inhibiting monomeric compound of complement system from the natural product comprising marine organisms, and the research and development for anticomplement medicament provide wide prospect.
Lithospermum euchromum Royle (Arnebia euchroma (Royle) Johnst) is Boraginaceae (Boraginaceae) Lithospermum (Arnebia) plant, perennial herb.The dry root bitter in the mouth of lithospermum euchromum Royle, cold in nature.There is the function of removing heat from blood and promoting blood circulation, heat-clearing and toxic substances removing, laxation relieving constipation, can be used for prevention measles, calentura macule, jaundice, purpura, tell nosebleed hematuria, stranguria with blood, dysentery, carbuncle sore tumefacting virus, erysipelas, eczema, burn, constipation with heat retention.Research in recent years finds that it has anticancer, antiinflammatory, the effect such as antibacterial.Clinical can be used for, treats acute hepatitis, chronic hepatitis, liver cirrhosis (ascites); The dermatosis such as verruca plana, psoriasis; Topical application treatment burn and promotion wound healing; Eye drop is used for the treatment of herpes simplex keratitis, has certain curative effect to epitheliated type dendroid and shallow essential layer dendritic keratitis; Oil preparation is used for the treatment of infant dermatitis, eczema, vaginitis, cervicitis etc.Mainly concentrate on separation and Structural Identification to the research of the chemical composition of Radix Arnebiae (Radix Lithospermi) in recent years, be therefrom separated and obtain the compound such as some benzoquinone classes, monoterpene Radix Arnebiae (Radix Lithospermi) phenols, there is not yet the report to the inhibited compound of complement system up to now.Be badly in need of efficient, low toxicity, single-minded novel complement inhibitor in clinical practice to come out.
Summary of the invention
The object of this invention is to provide the new material with anticomplementary activity, be specifically related to shikonin compounds, in particular to compound shikonin (1) wherein, 2,3-dimethyl-2-amylenes acyl shikonin (2) and acetylshikonin (3).
A further object of the present invention is to provide above-mentioned shikonin compounds and is preparing the purposes in anticomplement medicament.
The present invention's application modern pharmacology screening technique, anticomplementary activity material in plant amedica is studied, be separated from the ethyl acetate extract of the alcohol extract Boraginaceae (Boraginaceae) Lithospermum (Arnebiae) plant lithospermum euchromum Royle (Arnebia euchroma (Royle) Johnst) dry root and obtain shikonin compounds, comprise compound shikonin (1), 2,3-dimethyl-2-amylene acyl shikonin (2) and acetylshikonin (3), and confirm that it all has inhibitory action to the classical pathway of complement system and alternative pathway.
Active Radix Arnebiae (Radix Lithospermi) chlorins compound of the present invention has the chemical constitution of following general structural formula:
Wherein, as R=CH=C (CH
3)
2time, compound is shikonin (1); Work as R=CH
2cCH
3=C (CH
3)
2time, compound is 2,3-dimethyl-2-amylene acyl shikonin (2); Work as R=CH
3time, compound is acetylshikonin (3).
Shikonin compounds of the present invention is prepared by following method:
Lithospermum euchromum Royle root coarse powder 20kg, with after 95% ethanol repeatedly merceration, seepage pressure effects several under room temperature, decompression and solvent recovery, obtain extractum 820g, extractum is suspended in distilled water, with petroleum ether, ethyl acetate and n-butanol extraction, obtains acetic acid ethyl ester extract 420g.Acetic acid ethyl ester extract 180g is through silica gel column chromatography, with petroleum ether (60 ~ 90 DEG C), petroleum ether (60 ~ 90 DEG C)-acetone, acetone gradient elution, gained flow point carries out silica gel column chromatography repeatedly with different eluant, SephadexLH-20 purification and preparative hplc, separation obtains compound shikonin (1), 2,3-dimethyl-2-amylene acyl shikonin (2) and acetylshikonin (3).
In the present invention, shikonin (1): dark red powder,
1h NMR (400MHz, CDCl
3, ppm): δ 12.51 (1H, d, J=6.2Hz), 12.35 (1H, d, J=6.2Hz), 8.19 (2H, s), 8.11 (1H, s), 5.36 (IH, t, J=7.2Hz), 5.19 (1H, s), 2.55 (2H, m), 2.08 (3H, t, J=0.9Hz), 1.86 (3H, t, J=0.9Hz), 1.61 (3H, s), 1.51 (3H, s).
In the present invention, 2,3-dimethyl-2-amylene acyl shikonin (2): red oil,
1h-NMR (400MHz, CDCl
3, ppm): δ 12.40 (1H, s, 8-OH), 12.24 (1H, s, 5-OH), 7.18 (2H, s, H-6,7), 7.01 (1H, s, H-3), 6.01 (1H, t, J=7.3, H-11), 5.12 (1H, t, J=6.5, H-13), 2.96,3.00 (2H, dd, H-12), 2.00 (3H, s, H-23), 1.68,1.53,1.30,1.25 (Me × 4, each s, H-15,16,21,22).
In the present invention, acetylshikonin (3): red oil,
1h-NMR (400MHz, CDCl
3, ppm): δ 12.59 (1H, s, 8-OH), 12.43 (1H, s, 5-OH), 7.19 (2H, s, H-6,7), 6.99 (1H, s, H-3), 6.03 (1H, t, J=7.3Hz, H-11), 5.15 (1H, t, J=6.5, H-13), 2.62,2.49 (2H, m, H-12), 2.14 (3H, s, H-18).
Above-mentioned shikonin compounds is through external classical pathway and the evaluation test of alternative pathway anticomplementary activity, and result confirms, described compound all has remarkable inhibitory action (as shown in table 1) to the classical pathway of complement system and alternative pathway.Wherein, 2,3-dimethyl-2-amylene acyl shikonin (2) classical pathway to complement system has the strongest inhibitory action, 2,3-dimethyl-2-amylene acyl shikonin (2) concentration (CH needed for 50% haemolysis
50) be 0.16 ± 0.02mM, suitable with positive control heparin action intensity; Shikonin (1) alternative pathway to complement system has the strongest inhibitory action, shikonin (1) concentration (AP needed for 50% haemolysis
50) be 0.24 ± 0.04mM, slightly weak compared with the effect of positive control heparin.
Shikonin compounds of the present invention can be used as active constituents of medicine, prepares anticomplement medicament further.
Table 1 is compound 1 ~ 3 couple of complement system classical pathway (CH
50) and alternative pathway inhibitory action (AP
50) data.
Table 1
aμ g/ml, NA non-activity.
Accompanying drawing illustrates:
Fig. 1 is lithospermum euchromum Royle ethyl acetate extract gained shikonin compounds 1-3 extraction and isolation flow chart.
Detailed description of the invention
Embodiment 1 prepares shikonin compounds
Lithospermum euchromum Royle dry root 20kg, coarse powder under room temperature with 95% ethanol repeatedly merceration, seepage pressure effects for several times after, decompression and solvent recovery, obtain extractum 820g, extractum is suspended in distilled water, with petroleum ether, ethyl acetate and n-butanol extraction, obtains acetic acid ethyl ester extract 420g.Get acetic acid ethyl ester extract 180g, through silica gel column chromatography, with petroleum ether (60 ~ 90 DEG C), petroleum ether (60 ~ 90 DEG C)-acetone, acetone gradient elution, gained flow point carries out silica gel column chromatography repeatedly with different eluant, SephadexLH-20 purification, is separated and obtains compound shikonin (1), 2,3-dimethyl-2-amylene acyl shikonin (2) and acetylshikonin (3), concrete steps are as follows:
1, petroleum ether-acetone (10: 1) eluting gained stream part, through column chromatography repeatedly, stream part is through SephadexLH-20 purification, and chloroform-methanol (1: 1) eluting, obtains compound (1) (7mg).Employing method of spectroscopy is analyzed, and its structure is defined as shikonin.
2. petroleum ether-acetone (10: 1) eluting gained stream part, with petroleum ether-ethyl acetate (5: 1) for developing solvent, preparative hplc, obtains (2) (4mg).Employing method of spectroscopy is analyzed, and its structure is defined as 2,3-dimethyl-2-amylene acyl shikonin.
3. petroleum ether-acetone (10: 1) eluting gained stream part, silica gel column chromatography, with petroleum ether-ethyl acetate (3: 1) for eluant, gained stream part is through preparative hplc, with petroleum ether-acetone (5: 1) for developing solvent, compound (3) (21mg).Employing method of spectroscopy is analyzed, and its structure is defined as acetylshikonin.
Embodiment 2 In Vitro Anti classical pathway of complement is tested
Get complement (guinea pig serum) 0.1ml, add BBS and be mixed with 1: 5 solution, become 1: 10,1: 20,1: 40,1: 80,1: 160,1: 320 and 1: 640 solution with BBS two-fold dilution.Getting 1: 1000 hemolysin, each concentration complement and each 0.1ml of 2%SRBC is dissolved in 0.3ml BBS, 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 absorbance at 405nm.Experiment arranges full haemolysis group (0.1ml 2%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.Get complement and the compound shikonin (1) of critical concentration respectively, 2,3-dimethyl-2-amylene acyl shikonin (2) and acetylshikonin (3) mixing, 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 compound control group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated by after the compounds of this invention absorbance deduction respective compound matched group absorbance.Using the compounds of this invention concentration as X-axis, haemolysis suppression ratio is mapped as Y-axis.Calculate CH
50value.Result shows, and the classical pathway of described compound to complement system has remarkable inhibitory action (as shown in table 1).Wherein, 2,3-dimethyl-2-amylene acyl shikonin (2) classical pathway to complement system has the strongest inhibitory action, 2,3-dimethyl-2-amylene acyl shikonin (2) concentration (CH needed for 50% haemolysis
50) be 0.16 ± 0.02mM, suitable with positive control heparin action intensity.
Embodiment 3 In Vitro Anti alternative pathway of complement is tested
Get complement (human serum) 0.2ml, add AP diluent preparing and become 1: 5 dilute solution, and two-fold dilution becomes 1: 10,1: 20,1: 40,1: 80,1: 160,1: 320 and 1: 640 solution.Get each concentration complement 0.15ml, AP diluent 0.15ml and 0.5%RE 0.20ml, mixing, is placed 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 absorbance at 405nm.Experiment arranges full haemolysis group (0.20ml 0.5%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.Get complement and the compound shikonin (1) of the critical concentration determined respectively, 2,3-dimethyl-2-amylene acyl shikonin (2) and acetylshikonin (3) mixing, after 37 DEG C of pre-water-bath 10min, add 0.2ml 0.5%RE.Be placed into low-temperature and high-speed centrifuge by after every pipe 37 DEG C of water-bath 30min, 5000rpm, 4 DEG C, get often pipe supernatant 0.2ml after centrifugal 10min respectively and, in 96 orifice plates, under 405nm, measure its absorbance.Experiment arranges compound control group, complement group and full haemolysis group simultaneously.Hemolysis rate is calculated by after the compounds of this invention absorbance deduction respective compound matched group absorbance.Using the compounds of this invention concentration as X-axis, haemolysis suppression ratio is mapped as Y-axis, calculates AP
50value.Result shows, and described compound all has remarkable inhibitory action (as shown in table 1) to the alternative pathway of complement system.Wherein, shikonin (1) alternative pathway to complement system has the strongest inhibitory action, shikonin (1) concentration (AP needed for 50% haemolysis
50) be 0.24 ± 0.04mM, slightly weak compared with the effect of positive control heparin.
Claims (2)
1. the shikonin compounds with following general structural formula is preparing the purposes in anticomplement medicament,
Work as R=CH
2cCH
3=C (CH
3)
2time, compound is 2,3-dimethyl-2-amylene acyl shikonin (2);
Work as R=CH
3time, compound is acetylshikonin (3).
2. by purposes according to claim 1, it is characterized in that, described 2,3-dimethyl-2-amylene acyl shikonin (2) classical pathwaies to complement system have inhibitory action, 2,3-dimethyl-2-amylene acyl shikonin (2) concentration (CH needed for 50% haemolysis
50) be 0.16 ± 0.02mM.
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Non-Patent Citations (3)
| Title |
|---|
| Shikonin,a Component of Chinese Herbal Medicine,Inhibits Chemokine Receptor Function and Suppresses Human Immunodeficiency Virus Type1;Xin Chen,et al.;《Antimicrobial Agents and Chemotherapy》;20030930;第47卷(第9期);第2810-2816页 * |
| 新疆紫草细胞培养物的化学成分研究;向桂琼等;《植物学报》;19921231;第34卷(第6期);第470-474页 * |
| 长白山产紫草化学成分研究;刘浩宇等;《北京中医药大学学报》;20091130;第32卷(第11期);第773-775页 * |
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