CN113004354B - Dammarane type tetracyclic triterpene compound and anti-gout application thereof - Google Patents
Dammarane type tetracyclic triterpene compound and anti-gout application thereof Download PDFInfo
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Abstract
The invention relates to an extract of cyclocarya paliurus, in particular to a cyclocarya paliurus dichloromethane active part and dammarane type tetracyclic triterpene compound, and an extraction method and application thereof. The structure of the cyclodammarane type tetracyclic triterpene compound is shown as a formula I or a formula II:
Description
Technical Field
The invention relates to an extract of cyclocarya paliurus, in particular to a cyclocarya paliurus dichloromethane active site and dammarane type tetracyclic triterpene compound (comprising two structural forms of 3, 4-split ring and non-split ring), and an extraction method and application thereof.
Background
Cyclocarya paliurus (Bata1) Iljinsk is a plant of cyclocarya paliurus of Juglandaceae (Juglauiaceae), is a unique single species plant in China, and is widely distributed in Anhui, Jiangsu, Zhejiang and other places. Since the 80 s in the 20 th century, experts and scholars at home and abroad mainly carry out a great deal of research on the aspects of resource cultivation, chemical components, biological activity, product development and research and the like of cyclocarya paliurus. The results show that the cyclocarya paliurus has various physiological activities and pharmacological functions beneficial to human bodies.
Triterpenes (triterpenoids) are important natural products with wide distribution and various structural types in nature, and have various biological activities of reducing blood sugar and blood pressure, resisting inflammation, reducing blood fat, resisting tumor and the like. Researches show that the triterpenoid has good anti-inflammatory activity, can inhibit NO generation under the condition of not influencing cell viability, has better effect than a Nitric Oxide Synthase (NOS) inhibitor, and has inhibitory activity on TNF-alpha and IL-1 beta induced by LPS. Researches show that the triterpenoid saponin can obviously reduce arterial blood pressure, reduce the contents of renin activity (PRA), angiotensin II (Ang II) and Aldosterone (ALD) in plasma of spontaneous hypertension rats, has certain inhibition effect on renal hypertension and myocardial hypertrophy of the spontaneous hypertension rats, can reverse the pathological state of the left ventricle reconstruction of the spontaneous hypertension rats, and the mechanism of the triterpenoid saponin is respectively related to the regulation of inflammatory reaction induced by an Ang II/p 38MAPK pathway and the inhibition of the expression of a myocardial tissue inflammatory factor TGF-beta 1.
Cyclocarya paliurus leaves contain various medicinal chemical components including carotene, protein, flavonoid, polysaccharide, triterpenes and other compounds, wherein dammarane type triterpenes and prenylflavonoids are widely concerned due to the diversity of structures and pharmacological activities. At present, the two components are mostly focused on the research of blood sugar reduction and inflammation, and the research on the activity and mechanism in other aspects is less, so that the specific elucidation of other application activities and action mechanisms of the dammarane type triterpene and the isopentenyl flavone has great significance for wide clinical application and innovative drug development.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide an extraction method and application of cyclocarya paliurus dichloromethane active site and cyclodammarane type tetracyclic triterpene compounds, and carry out systematic research on gout active ingredients and drug effect substance basis improvement.
In order to solve the technical problems, the technical scheme of the invention is as follows:
a dammarane-type tetracyclic triterpene compound (comprising two structural forms of 3, 4-split ring and non-split ring) has a structure shown in formula I or formula II:
wherein R is 1 Selected from hydrogen, C1-C6 alkyl; r 2 Selected from hydrogen, L-arabinose, D-cinchona sugar, D-glucose; r is 3 Is selected from
R 4 And R 5 Independently selected from L-arabinose, D-cinchona sugar, D-glucose, D-xylose and L-arabinose furan; r 6 Is selected from
Preferably, the dammarane type tetracyclic triterpene compound has the following structural formula:
the invention also provides a method for extracting the dammarane type tetracyclic triterpene compound from cyclocarya paliurus, which comprises the following steps:
s1, heating and refluxing the cyclocarya paliurus leaves by using ethanol/water, and concentrating to obtain an extract;
s2, dispersing the extract with water, extracting with dichloromethane, and concentrating the extract to obtain dichloromethane extraction concentrated solution;
and S3, analyzing, separating and purifying the dichloromethane extraction concentrated solution by adopting HPLC-DAD, TLC and UPLC-MS/MS methods.
Preferably, in S1, the cyclocarya paliurus leaves are dried and pulverized.
Preferably, in S1, the concentration of ethanol is 70%, the ratio of the mass of the cyclocarya paliurus leaves to the ethanol is 1:10, the heating temperature is 120 ℃, the heating time is 2h, the concentration temperature is 60 ℃, and the concentration time is 2-3 days.
Preferably, in S2, the extraction is carried out for three times by using dichloromethane, the ratio of the mass of the cyclocarya paliurus leaves to the volume of the dichloromethane used each time is 1:10, the concentration temperature is 60 ℃, and the concentration time is 24-48 hours.
Preferably, in S3, the dichloromethane part is eluted by silica gel column chromatography with a gradient of CH2Cl2: CH3OH (100:1-0:100), TLC spots are developed by 10% EtOH-H2SO4, samples are analyzed by HPLC-DAD and observed under ultraviolet lamps of different wave bands, and 9 parts (A-I) are obtained by concentration and combination;
performing TLC thin-layer analysis on the A-I part, heating and developing by using 10% concentrated sulfuric acid/ethanol, wherein a Fr.I thin-layer plate shows a purplish red strip, HPLC-DAD analysis mainly comprises tail end absorption, and the I part is preliminarily determined to be a triterpene enrichment part;
firstly, FrI is subjected to polyamide column chromatography to remove interference components such as pigments, tannins and the like, then silica gel column chromatography, small-pore resin column chromatography and reverse ODS column chromatography are adopted, the triterpene is tracked by an UPLC-MS/MS method in the whole separation process, TLC spots are subjected to color development by 10% EtOH-H2SO4, a sample is analyzed by HPLC-DAD, and the dammarane type tetracyclic triterpene compound is effectively separated; and finally, purifying the target compound by adopting a semi-preparative high performance liquid phase.
The active site is the dichloromethane extraction concentrated solution of cyclocarya paliurus, and the main component of the active site is dammarane type tetracyclic triterpene compounds.
The application of the active site in preparing the medicine for improving gout and reducing uric acid.
Further, the separation and purification process of the dammarane type tetracyclic triterpene compound comprises the following steps:
the invention adopts HPLC-DAD, TLC and UPLC-MS/MS methods to track, analyze, separate and purify the dichloromethane extraction concentrated solution. 400g of a dichloromethane fraction was subjected to silica gel column chromatography and gradient elution with a CH2Cl2: CH3OH system (100:1-0:100), the obtained fractions were subjected to TLC spot color development with 10% EtOH-H2SO4 developer, the samples were analyzed by HPLC-DAD under ultraviolet light observation of different wavelength bands, and finally concentrated and combined to obtain 10 fractions (I-X). TLC thin layer analysis and HPLC analysis of the 10 fractions were performed sequentially, and it was found that the thin layer plates at the IX and X sites showed purple red bands, whereas HPLC-DAD analysis was mainly terminal absorption, and the two sites were preliminarily determined to be triterpene-rich sites, so that the two fractions were mainly separated during the course of the subject. The IX sites were eluted through a polyamide column (ethanol/water, 0-95%) to give five fractions (A-E). IXA (33.2g) was further purified by silica gel column chromatography (CH2Cl2-MeOH,50:1-0:100) followed by C18 reverse phase column chromatography with methanol-water system gradient elution (20% -100%) to give compound 1(2.3mg) and compound 14(4.5 mg). IXB and IXC were combined and separated by a reverse phase column C18 using a MeOH/H2O gradient system to yield five fractions (IXBC-1-IXBC-5). IXBC-2(3.5g) is subjected to MCI (micro-porous carbon dioxide) resin column chromatography and eluted by a methanol-water system (20% -100%) to obtain six sub-components (IXBC-2-a-IXBC-2-f). IXBC-2-b was isolated by semi-preparative liquid chromatography to give compound 3(2.0mg), compound 4(3.2 mg); IXBC-2-c was isolated by semi-preparative liquid chromatography to give compound 20(1.8mg), compound 22(1.5 mg). Chromatography of fraction IXD (78.2g) on silica gel column eluted with EtOAc-MeOH system afforded 8 subfractions (IXD-1-IXD-8). IXD-3(18.4g) was further purified by column chromatography on silica gel eluting with CH2Cl2-MeOH system to give four fractions (IXD-3-a-IXD-3-d). Subsequently, IXD-3-b separated compound 18(3.8mg) by semi-preparative liquid chromatography, and IXD-3-c separated compounds 23(3.8mg) and 15(4.8mg) by semi-preparative liquid chromatography. IXD-4(23.0g) was purified by MCI chromatography on a small pore resin column eluted with methanol-water (20% to 100%) to give six subfractions (IXD-4-a-IXD-4-e). Compounds 13(8.4mg) and 26(4.5mg) were isolated from IXD-4-c by semi-preparative HPLC, and Compound 9(5.6mg) and Compound 12(3.8mg) were isolated from IXD-4-d. Fraction X (60g) was also subjected to column chromatography on a polyamide column, gradient elution was carried out using a methanol-water system gradient (0-100%), and 5 fractions (Fr.A-Fr.E) were combined according to HPLC analysis. XB (13.2g) was further subjected to silica gel column chromatography (CH2Cl2/MeOH,10:0-0:10) and C18 reverse phase column chromatography (MeOH/H2O, 10% -100%) to give compound 19(4.2mg) and compound 24(4.5 mg). XC was eluted with a C18 reverse phase column, MeOH/H2O gradient, yielding five fractions (XC-1-XC-5). IXC-2 Compound 2(2.8mg), Compound 5(3.6mg) was isolated by semi-preparative liquid chromatography. IXC-3(4.5g) was purified by gel column MCI eluting with MeOH-H2O system (20% -100%) to give four subfractions (XC-3-a-XC-3-d). XC-3-c isolated Compound 6(1.8mg), Compound 21(1.5mg) therefrom by semi-preparative HPLC, XC-3-d isolated Compound 8(4.8mg), Compound 10(2.5mg) therefrom by semi-preparative HPLC. The XD fraction was further treated with silica gel and eluted with CH2Cl2-MeOH to give four fractions (XD-1-XD-4). Subsequently, compound 25(4.8mg), compound 7(5.3mg), compound 11(3.6mg), compound 16(3.5mg) and compound 17(5.8mg) were isolated from XD-3 by semipreparative liquid chromatography.
The dammarane type tetracyclic triterpene compound 1-26 is:
the invention also provides application of the dammarane type tetracyclic triterpene compounds 1-26 in preparation of medicines for improving gout and reducing uric acid.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention provides a brand-new method for extracting dammarane type tetracyclic triterpene compounds 1-26 from cyclocarya paliurus, which is simple and repeatable.
2. The invention provides a new application of cyclocarya paliurus dichloromethane active site and dammarane type tetracyclic triterpene compounds 1-26, researches the pharmaceutical mechanism of the cyclocarya paliurus dichloromethane active site and is expected to become a new medicine for reducing uric acid and improving gout.
Drawings
FIG. 1 is a graph of the effect of QQL on the synovial membrane of joints in a rat gouty arthritis model;
figure 2 is the DEPT 135 spectrum of compound 23;
figure 3 is the DEPT 135 spectrum of compound 24;
FIG. 4 is an HMBC spectrum of compound 24;
FIG. 5 is a DEPT 135 spectrum of Compound 5;
FIG. 6 is an HMBC spectrum of compound 5;
FIG. 7 is a NOESY spectrum of Compound 5;
FIG. 8 is a key correlation plot of H-H-COSY, HMBC, NOESY for Compound 5;
figure 9 is the DEPT 135 spectrum of compound 6;
figure 10 is the DEPT 135 spectrum of compound 7;
FIG. 11 is a NOESY spectrum of Compound 7;
figure 12 is the DEPT 135 spectrum of compound 25.
Detailed Description
The present invention will be described in detail with reference to examples. It should be noted that the embodiments and features of the embodiments of the present invention may be combined with each other without conflict.
Example 1
1.1 processing cyclocarya paliurus medicinal materials: dried leaves (10Kg) of cyclocarya paliurus, after being crushed, are heated and refluxed at 120 ℃ by 70 percent ethanol for extraction (100L; 2 x 2h), and are concentrated at 60 ℃ to obtain extractum, and the concentration time is 2 days. Dispersing the extract with water, sequentially extracting with dichloromethane, ethyl acetate, and n-butanol 10L each time for 3 times, concentrating the extractive solution at 60 deg.C for 24 hr to obtain concentrated extractive solutions of different polar parts. The dichloromethane part is mainly dammarane type tetracyclic triterpene compounds marked as QQL-ST, and the n-butanol part is mainly flavonoid compounds marked as QQL-HT.
1.2 the biological activity is guided to screen the cyclocarya paliurus active site: an acute gout model is established by inducing rats with sodium urate to track and screen the active site of the cyclocarya paliurus for improving gout:
1.3 active site ingredient Studies
1.3.1 triterpene extraction, separation and purification: the active site is subjected to systematic chemical component research by modern chromatographic separation technologies such as macroporous resin, polyamide, silica gel, HW-40C gel, Sephadex LH-20 gel, ODS, PrepHPLC, PrepTLC and the like. Tracking, analyzing, separating and purifying by HPLC-DAD, TLC and UPLC-MS/MS methods according to the characteristic UV absorption and TLC color reaction of dammarane type tetracyclic triterpenoids.
In the experiment, the dichloromethane extraction concentrated solution is tracked, analyzed, separated and purified by HPLC-DAD, TLC and UPLC-MS/MS methods. 400g of a dichloromethane fraction was subjected to silica gel column chromatography and gradient elution with a CH2Cl2: CH3OH system (100:1-0:100), the obtained fractions were subjected to TLC spot color development with 10% EtOH-H2SO4 developer, the samples were analyzed by HPLC-DAD under ultraviolet light observation of different wavelength bands, and finally concentrated and combined to obtain 10 fractions (I-X). TLC thin layer analysis and HPLC analysis of the 10 fractions were performed sequentially, and it was found that the thin layer plates at the IX and X sites showed purple red bands, whereas HPLC-DAD analysis was mainly terminal absorption, and the two sites were preliminarily determined to be triterpene-rich sites, so that the two fractions were mainly separated during the course of the subject. The IX sites were eluted through a polyamide column (ethanol/water, 0-95%) to give five fractions (A-E). IXA (33.2g) was further purified by silica gel column chromatography (CH2Cl2-MeOH,50:1-0:100) followed by C18 reverse phase column chromatography with methanol-water system gradient elution (20% -100%) to give compound 1(2.3mg) and compound 14(4.5 mg). IXB and IXC were combined and separated by a reverse phase column C18 using a MeOH/H2O gradient system to yield five fractions (IXBC-1-IXBC-5). IXBC-2(3.5g) is subjected to MCI (micro-porous carbon dioxide) resin column chromatography and eluted by a methanol-water system (20% -100%) to obtain six sub-components (IXBC-2-a-IXBC-2-f). IXBC-2-b was isolated by semi-preparative liquid chromatography to give compound 3(2.0mg), compound 4(3.2 mg); IXBC-2-c was isolated by semi-preparative liquid chromatography to give compound 20(1.8mg, compound 22(1.5 mg). IXD (78.2g) was subjected to silica gel column chromatography and eluted with EtOAc-MeOH system to give 8 subfractions (IXD-1-IXD-8). IXD-3(18.4g) was further purified by silica gel column chromatography and eluted with CH2Cl2-MeOH system to give four fractions (IXD-3-a-IXD-3-d). IXD-3-b was then isolated by semi-preparative liquid chromatography to give compound 18(3.8mg), IXD-3-c was isolated by semi-preparative liquid chromatography to give compound 23(3.8mg) and 15(4.8 mg). IXD-4(23.0g) was subjected to MCI small pore resin column chromatography, methanol-water system (20% -100%) was eluted, six subfractions (IXD-4-a-IXD-4-e) were obtained. Compounds 13(8.4mg) and 26(4.5mg) were isolated from IXD-4-c by semi-preparative HPLC, and Compound 9(5.6mg) and Compound 12(3.8mg) were isolated from IXD-4-d. Fraction X (60g) was also subjected to column chromatography on a polyamide column, gradient elution was carried out using a methanol-water system gradient (0-100%), and 5 fractions (Fr.A-Fr.E) were combined according to HPLC analysis. XB (13.2g) was further subjected to silica gel column chromatography (CH2Cl2/MeOH,10:0-0:10) and C18 reverse phase column chromatography (MeOH/H2O, 10% -100%) to give compound 19(4.2mg) and compound 24(4.5 mg). XC was eluted with a C18 reverse phase column, MeOH/H2O gradient, yielding five fractions (XC-1-XC-5). IXC-2 Compound 2(2.8mg), Compound 5(3.6mg) was isolated by semi-preparative liquid chromatography. IXC-3(4.5g) was purified by MCI gel column eluting with MeOH-H2O system (20% -100%) to give four subfractions (XC-3-a-XC-3-d). Compound 6(1.8mg), compound 21(1.5mg) and XC-3-c were isolated therefrom by semi-preparative HPLC, compound 8(4.8mg), compound 10(2.5mg) and XC-3-d were isolated therefrom by semi-preparative HPLC. The XD fraction was further treated with silica gel and eluted with CH2Cl2-MeOH to give four fractions (XD-1-XD-4). Subsequently, compound 25(4.8mg), compound 7(5.3mg), compound 11(3.6mg), compound 16(3.5mg) and compound 17(5.8mg) were isolated from XD-3 by semi-preparative liquid chromatography.
1.3.3 structural validation: modern spectral techniques such as UV, IR, NMR, MS, CD, ORD, ECD, single crystal X-rays and the like are used for confirming the planar structure and the spatial configuration of each compound, and the system attribution of the spectral characterization and the spectral data is carried out.
The type of absorption of the compound and its presence or absence of the conjugated segment was first confirmed by uv absorption. The planar structure of the compound was then determined by 1D/2D NMR and its molecular weight was confirmed by mass spectrometry. The absolute configuration of the compound was confirmed by NOESY, ROESY, CD, ECD, etc.
1.4 chemical Structure
26 dammarane-type tetracyclic triterpenoids (1-26) are isolated, and are shown in Table 1.
TABLE 1 molecular information for Compounds 1-26
The new structure characterization results are as follows:
compound 23: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (ACN-H 2 O)λ max ：230nm。HRESIMS，m/z：754.5123[M+NH 4 ] + 。
compound 24: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (ACN-H 2 O)λ max ：230nm。HRESIMS，m/z：788.5159[M+NH 4 ] + 。
the structural formula is as follows:
TABLE 2 characterization data for Compounds 23-24
Compound 5: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (ACN-H 2 O)λ max :203nm。HRESIMS，m/z:637.3953[M-H] - 。
compound 6: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (CH 3 CN-H 2 O)λ max :203nm。HRESIMS，m/z:695.4378[M+CH 3 COO] - 。
the structure is as follows:
TABLE 3 characterization data for Compounds 5-6
Compound 7: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (CH 3 CN-H 2 O)λ max :203nm。HRESIMS，675.4082[M+Na] + 。
compound 25: white amorphous powder, which is easily soluble in methanol and insoluble in water;
HPLC-UV (ACN-H 2 O)λ max ：230nm。HRESIMS，m/z：799.4904[M+HCOO] - 。
the structure is as follows:
TABLE 4 characterization data for Compounds 7 and 25
The characterization profiles are shown in FIGS. 2-12.
Example 2
Research on effect of cyclocarya paliurus dichloromethane active site and dammarane type tetracyclic triterpene compound on improvement of gout
2.1 anti-gouty arthritis action
2.1.1 Experimental methods:
research on the effect of QQL-ST on gouty arthritis: selecting 40 quarantine certified SD rats, and randomly dividing males into 4 groups according to weight, wherein the 4 groups are respectively a normal control group, a model control group, a cyclocarya paliurus dichloromethane active part group and a gout capsule fixing group, and each group comprises 10 animals. The corresponding liquid medicine is given to each group by intragastric administration, and the equal volume of pure water is given to the normal control group and the model control group by intragastric administration. The administration is performed 1 time per day for 8 days. Except for a normal control group, the other groups are molded 30min after administration, SD rats are inhaled and anesthetized with isoflurane (inducing 1-4 percent and maintaining 0.25-2 percent), then the peripheries of knee joints of hind legs at two sides are shaved, the skin is disinfected by medical alcohol, the knee joints are slightly bent, the needles are inserted through the side faces of the joints, 0.5mL of sodium urate solution (25mg/mL) is injected into the knee joint cavities of the rats through supraclavicular ligaments by using a No. 6 syringe needle, and the acute gouty arthritis model is copied.
Anti-inflammatory action study of candidate compounds: taking rat macrophage-like cell line (RAW.264.7) cells at 1 × 10 6 The cells were inoculated in 96-well plates at a concentration of one mL and incubated at 37 ℃ for 12 hours. Then, the test sample (1, 3, 10, 30. mu.M) was added to the cell culture solution and cultured for 1 hour, LPS (1. mu.g/mL) was added thereto, and the cells were cultured together at 37 ℃ for 24 hours. 50. mu.L of the cell culture supernatant was placed in a new 96-well plate, and 50. mu.L of LGriess I solution and Griess II solution were added to each well in succession. After standing at room temperature for 10min, the absorbance (A) of the reaction product was measured at 540nm using a microplate reader. Meanwhile, the MTT method is used for measuring the cytotoxicity of each group of medicines.
2.1.2 Experimental results:
(1) influence on swelling degree of knee joint
Measuring the circumferences of the right knee joints of the rats in each group by adopting a line-laying method at 1h, 2h, 4h, 6h and 12h before and after the model building respectively, and calculating a swelling index, wherein the swelling index is (the circumference of the knee joint after the model building-the circumference of the knee joint before the model building)/the circumference of the knee joint before the model building is 100 percent.
As shown in table 5, the percent swelling of the rat joints in the model control group was significantly increased (P <0.01) compared to the normal control group, suggesting that the gouty arthritis model was successfully replicated; compared with a model control group, the joint swelling rate of rats in the QQQL-ST group after model building for 1, 2, 4, 6 and 12 hours is obviously reduced (P is less than 0.05 or P is less than 0.01), and the QQL-ST can obviously inhibit the swelling of a rat gouty arthritis model caused by sodium urate; the QQL-ST has a more lasting effect on inhibiting gouty arthritis swelling and has a better effect than gout relieving capsules.
TABLE 5 influence of QQL-ST on joint swelling in rat gouty arthritis model with sodium urate (S) ((S))
n＝10)
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05， ** P<0.01
(2) Effects on arthritic factors
As shown in Table 6, compared with the normal control group, the contents of inflammatory factors IL-1 beta and TNF-alpha in the joint cavity washing fluid of the rats in the model control group are obviously increased (P is less than 0.01); compared with a model control group, IL-1 beta and TNF-alpha in the joint cavity flushing fluid of the rat in the QQL-ST group are both remarkably reduced (P is less than 0.01), which indicates that the QQL-ST can remarkably inhibit the secretion of inflammatory factors of the rat gouty arthritis model caused by sodium urate, and meanwhile, the inhibition effect of the QQQL-ST on the secretion of the IL-1 beta and the TNF-alpha is remarkably stronger than that of the gout determining capsule.
TABLE 6 QQL-ST on sodium urateInfluence of inflammatory factors in articular cavity of rat gouty arthritis model (ii)
n＝10)
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05
(3) Histopathological examination of the synovial membrane of the knee joint
24 hours after model building, the isoflurane (inducing 1-4 percent and maintaining 0.25-2 percent) of rats in each group is inhaled for anesthesia and then the abdominal aorta is exsanguinated and euthanased, joint synovial tissues are taken and placed in 10 percent neutral formalin solution for fixation, paraffin embedding and HE staining are carried out, the pathological examination of the joint synovial tissues is carried out, and the pathological changes of the knee joint are divided into 4 grades according to the pathological changes, wherein the 1 grade is not abnormal, and the 2-4 grades are respectively divided into light, medium and severe pathological changes according to the synovial tissue hyperplasia, interstitial edema and bleeding, and the infiltration degree of inflammatory cells and fibroblasts.
As shown in table 7 and fig. 1, compared with the normal control group, the level of joint lesions of the rats in the model control group is significantly increased (P <0.01), the types of lesions are mainly synovial tissue hyperplasia of 2-3 levels, and are accompanied by interstitial edema, vascular congestion, inflammatory cells and fibroblast infiltration, which indicates that the gouty arthritis model is successfully replicated; compared with a model control group, the joint pathological changes of rats in the QQQL-ST group are obviously reduced, the number of animals with pathological changes in various degrees is obviously reduced (P is less than 0.01), the QQL-ST is prompted to be capable of obviously improving the joint pathological changes of the rat gouty arthritis model caused by sodium urate, and the QQQL-ST is stronger than the goulidine capsules.
TABLE 7 influence of QQL-ST on pathological changes in joints of rat gouty arthritis model induced by sodium urate ((
n＝10)
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05。
(4) Triterpene compound anti-inflammatory activity evaluation
3.3.1 inhibition of LPS-induced NO production in RAW.264.7
NO is closely related to gouty arthritis, and the increase of the in vivo NO level can affect pathological processes from a plurality of ways, such as the initiation of nuclear factor NF-kB, the induction of proinflammatory cytokines TNF-alpha, IL-1 and the like, and finally the stimulation of synovial cell proliferation to cause irreversible damage to cartilage. The evaluation of anti-gouty arthritis activity can be carried out by taking mouse mononuclear macrophage (RAW.264.7) induced by LPS as a model and indometacin (Indo) as a positive control. In the experiment, a Griess reagent is adopted to detect NO in the culture supernatant of RAW264.7 cells; and (3) detecting the cell activity by using an MTT method, and inspecting the influence of the compound on NO generated by an LPS-induced RAW.264.7 cell model, wherein the NO content in the supernate is used as a detection index. The results are shown in tables 8 and 9.
TABLE 8 Effect on LPS-induced RAW.264.7 cell viability
Compared with the model group, # p <0.05, # p <0.01
TABLE 9 inhibition of NO production by LPS-induced RAW.264.7 cells
Compared with the model group, # p <0.05, # p <0.01
The experimental results show that: (1) compound 2 showed weak inhibitory effect on cell viability of raw.264.7 at high concentration; the remaining compounds had no significant effect on cell viability. (2) The detection result of the Griess reagent indicates that: most candidate compounds can inhibit NO secretion of RAW.264.7 cells induced by LPS to a certain degree, and the compound 5 has strong inhibition effect on NO secretion of RAW.264.7 cells induced by LPS, and has better effect than that of indomethacin serving as a positive medicament.
2.3 uric acid lowering action
2.3.1 methods of experiment:
selecting 40 male ICR mice qualified for quarantine, randomly dividing the mice into 4 groups according to weight, namely a normal control group, a model control group, a gout capsule group and a QQL-ST group, wherein each group comprises 10 animals. The animals of each group are administrated with liquid medicine with corresponding concentration through oral gavage according to 20mL/kg, and the normal control group and the model control group are administrated with pure water with equal volume through gavage for 1 time every day for 7 days continuously. A single 20mL/kg intraperitoneal injection of OAPS 300mg/kg replicates hyperuricemia model 1h before administration on day 7, and a normal control group and a blank administration group are given equal volume of 0.9% sodium chloride injection. The drug is administered 1h after the model building, and blood is collected for measuring blood uric acid 1h after the drug administration (fasting is not forbidden one day before blood collection).
2.3.2 results of the experiment
As shown in table 10, compared with the normal control group, the serum UA of the mice in the model control group is significantly increased (P <0.01), indicating that the hyperuricemia model is successfully replicated; compared with a model control group, UA (P <0.01) of mice in the QQL-ST group is remarkably reduced after the mice are modeled, which indicates that QQL-ST can remarkably reduce the uric acid level of a hyperuricemia model caused by potassium thiocyanate, and the drug effect of QQL-ST is remarkably stronger than that of a gout determining capsule.
TABLE 10 influence of QQL-ST on the model of hyperuricemia in mice by Potassium Oxocyanate ((
n＝10)
Note: comparing with normal control group + P<0.01; comparison with model control group * P<0.05
2.4 inhibition of xanthine oxidase Activity
2.4.1 methods of experiment:
xanthine Oxidase (XOD), which is a key enzyme in purine catabolism in vivo, plays an important role in maintaining uric acid balance in blood in vivo, and is a target for uric acid lowering therapy. Xanthine oxidase produces superoxide ions and uric acid by catalyzing xanthine metabolism, and therefore experiments evaluated the inhibitory effect of compounds on xanthine oxidase by measuring the levels of superoxide ions and uric acid produced. Initial screening for uric acid lowering activity can be performed by using Allopurinol (Allopurinol) as a positive control. The effect of the drug on xanthine oxidase activity was evaluated by measuring the content of superoxide ion by NBT chromogenic reaction in which xanthine (50 μ M), xanthine oxidase (0.1U/ml), NBT (50 μ M) and candidate compounds at each concentration or positive control allopurinol (1 μ g/ml) were added to the reaction system, and phosphate buffer (50mM, pH 7.5) was added to the reaction system to a final volume of 100 μ L. The reaction was started by adding xanthine oxidase, the reaction was carried out at room temperature for 15min, and the absorbance value was measured at 560nm after the reaction was completed. Measurement of uric acid content the effects of the drug on xanthine oxidase activity were evaluated by adding xanthine (50. mu.M), xanthine oxidase (0.1U/ml) and candidate compound at each concentration or positive control allopurinol (1. mu.g/ml) to the reaction system, and adding phosphate buffer (50mM, pH 7.5) to the reaction system to a final volume of 100. mu.L. The reaction starts with the addition of xanthine oxidase, the reaction is carried out for 15min at room temperature, the absorbance value is measured at 295nm after the reaction is finished, and the inhibition rate of the xanthine oxidase is calculated.
TABLE 11 inhibition of xanthine oxidase activity in vitro
The data show that the active site and the compound have stronger effects of inhibiting gouty arthritis and reducing uric acid, the effect is better than that of a positive medicament, namely indometacin, and the active site and the compound can be used as a novel medicament. The monomeric compound has obviously better effects of inhibiting gouty arthritis and reducing uric acid than the activity of similar dammarane type tetracyclic triterpenoids disclosed in the prior art, and the excellent performance of the monomeric compound cannot be expected.
The foregoing examples are set forth to illustrate the present invention more clearly and are not to be construed as limiting the scope of the invention, which is defined in the appended claims to which the invention pertains, as modified in all equivalent forms, by those skilled in the art after reading the present invention.
Claims (3)
1. A dammarane type tetracyclic triterpene compound is characterized by having a structural formula as follows:
2. the application of an active site in preparing a medicament for improving gout is characterized in that the active site is dichloromethane extraction concentrated solution of cyclocarya paliurus, and the main component of the active site is dammarane type tetracyclic triterpene compound; the structural formula of the dammarane tetracyclic triterpene compound is as follows:
3. the application of the dammarane type tetracyclic triterpene compound in the preparation of medicines for improving gout is characterized in that the structural formula is as follows:
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