CN112851738A - South Taiwan African leaf steroid compound and application thereof in preparation of anti-gout drugs - Google Patents

Abstract

South Taiwan African leaf steroid compound and application thereof in preparing anti-gout drugs belong to the field of medicines. Provides a south African leaf steroid compound vernonacums B of Taiwan and application thereof in preparing anti-gout medicaments. Extracting south African leaves with 60% ethanol under reflux, concentrating to obtain 60% ethanol extract of south African leaves, extracting partial extract with dichloromethane, recovering solvent to obtain extract, separating with chromatography methods such as normal phase silica gel column chromatography, reverse phase silica gel column chromatography, and purifying with preparative high performance liquid chromatography to obtain steroid compounds. The activity of reducing uric acid of the mice is verified, and the liver and kidney tissues of the mice are subjected to tissue slice observation, so that no damage is found, and the application of the Taiwan south African leaf steroid compound in preparing anti-gout drugs is proved to have no obvious liver and kidney toxicity. Has potential value for further development as a uric acid lowering drug and for treating gout.

Description

South Taiwan African leaf steroid compound and application thereof in preparation of anti-gout drugs

Technical Field

The invention belongs to the field of medicines, and particularly relates to a south Taiwan African leaf steroid compound and application thereof in preparation of anti-gout drugs.

Background

Gout is caused by that the concentration of blood uric acid is increased to form urate crystal due to purine metabolic disorder in vivo, and the urate crystal is accumulated at joints, kidneys and other parts to cause diseases such as gouty nephropathy, gouty arthritis and the like. With the provision of living standard of people, the intake of exogenous purine is greatly increased, and unhealthy living style is easy to cause purine metabolism abnormality in vivo, so that the number of gout patients in China is increased year by year. At present, the main strategy for treating gout is mainly to reduce the concentration of blood uric acid in a body, and a clinically common medicament is allopurinol. However, allopurinol as purine analogues has negative effects on the normal metabolism of the human body, and serious side reactions such as anaphylactic reaction, gastrointestinal reaction and liver and kidney injury exist, which bring great limitation to the clinical application of allopurinol.

Vernonia amygdalina Delile, a name of south Africa, is native to Africa and has been introduced in Taiwan, southeast Asia, etc. In taiwan, south african leaves are used to treat diseases such as hypertension, hyperlipidemia, cancer and gout. The applicant discloses the pharmaceutical use of the south African leaf alcohol extract in Chinese patent application CN201710260528.6, in particular the use of the south African leaf alcohol extract in the preparation of drugs for preventing and/or treating obesity and hyperlipidemia, pharmacological experiments show that the south African leaf alcohol extract can reduce the weight of obese people, improve visceral fat accumulation and show the prevention and treatment effects of obesity; and lipid content in serum and liver is reduced, so that hyperlipidemia and fat accumulation in liver tissue caused by high-fat diet are improved, and the effects of preventing hyperlipidemia, inhibiting the progress of hyperlipidemia conditions or improving the morbid state are achieved.

At present, chemical components separated from south African leaves comprise steroid compounds, flavonoid compounds, sesquiterpene lactone compounds, lignans, fatty acids and the like, reported activities mainly comprise cancer resistance, malaria resistance and the like, however, at present, the south African leaves have few researches and reports on gout resistance, and relevant research documents do not exist, so that the south African leaves are researched on gout resistance.

Disclosure of Invention

The invention aims to provide a south Taiwan african leaf steroid compound and application thereof in preparing anti-gout drugs.

The south African leaf steroid compound in Taiwan is vernonacums B, and the structure of the compound is analyzed by utilizing the technologies of nuclear magnetic resonance, mass spectrum and the like, and the structure is as follows:

the south Taiwan African leaf steroid compound is prepared by the following method:

1) taking 400g of dried south Africa leaf blades, cutting the dried south Africa leaf blades into pieces by using scissors, putting the cut south Africa leaf blades into a 10-L round-bottom flask, adding 12 times of 60% ethanol solution, heating, refluxing and extracting for 2.5 hours, pouring out the first extracting solution, adding the same amount of fresh 60% ethanol, extracting for one time again, filtering, combining the two extracting solutions, concentrating by using a rotary evaporator to remove ethanol, adding a proper amount of pure water into the residual water solution, and freeze-drying the residual water solution into powder by using a freeze dryer to obtain 60g of dried powder.

2) Taking 50g of dried south African leaf 60% ethanol extract, adding a proper amount of distilled water to disperse into a suspension with a proper concentration, extracting with dichloromethane saturated by equal volume of water for 3 times, combining the extraction solutions for 3 times, concentrating into a dried extract by using a rotary evaporator to obtain 8.17g of south African leaf dichloromethane layer extract, further separating by using chromatographic methods of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and gel column chromatography, and purifying by combining preparative high performance liquid chromatography to obtain the steroid compound.

The south Taiwan African leaf steroid compound can be applied to the preparation of anti-gout drugs.

The main in vivo gout model of the invention is mouse hyperuricemia caused by intraperitoneal injection of hypoxanthine and subcutaneous injection of Potassium Oxonate, and the uric acid reducing effect of the south African leaf extract is evaluated by intragastric administration of the south African leaf extract, so that the anti-gout activity of the south African leaf extract is indirectly evaluated.

The results of an analysis experiment on the uric acid reducing activity of mice on the methylene dichloride layer extract of south Africa leaves show that the methylene dichloride extract layer has an obvious uric acid reducing effect on a mouse hyperuricemia model caused by oteracil potassium and hypoxanthine, the uric acid reducing activity is good, and the liver and kidney tissues of the methylene dichloride layer are subjected to tissue slice observation, so that no damage is found, and the methylene dichloride layer extract of south Africa leaves has no obvious hepatotoxicity or hepatotoxicity. The chemical components of the dichloromethane layer are further subjected to systematic separation and identification, a large number of steroid compounds are obtained through separation, and the compounds are subjected to in vitro anti-gout model xanthine oxidase inhibition experiment screening, and the result shows that the compound 21(vernonacums B) can obviously inhibit the activity of xanthine oxidase, so that the application prospect of the steroid compounds in south Africa leaves in treating gout is shown, and the steroid compounds have potential value in further being used as uric acid lowering drugs and treating gout.

Drawings

FIG. 1 is a graph showing the uric acid lowering effect of different dosage groups of the leaf extract of south Africa according to the present invention.

FIG. 2 is a comparative graph showing the uric acid lowering effect of the leaf extract of south Africa and its different solvent extracts according to the present invention.

FIG. 3 is a graph showing the comparison of the inhibitory activities of the steroid xanthine oxidase in the leaves of south Africa according to the present invention.

Detailed Description

The following examples will further illustrate the present invention with reference to the accompanying drawings.

In this example, ethanol extract of south africa leaves in taiwan, petroleum ether, dichloromethane, ethyl acetate and n-butanol extract thereof were subjected to activity tracking separation, and steroid compounds were obtained from the dichloromethane layer extract of south africa leaves after separation as drugs for treating gout.

The main experimental method and results of the invention are as follows:

1 materials and instruments

(1) Material

Vernonia amygdalina Delile, a leaf of south Africa, belonging to the genus Vernonia of the family Compositae, was purchased from Taiwan, China. 64 Kunming mice (18-22 g) were purchased from Beijing Wittingle laboratory animal technology, Inc

(2) Main reagent information is shown in Table 1

TABLE 1

(3) The experimental apparatus is shown in Table 2.

TABLE 2

Name of the instrument	Production home
		SHB-III model circulating water type multipurpose vacuum pump	ZHENGZHOU GREATWALL SCIENTIFIC INDUSTRIAL AND TRADING Co.,Ltd.
Milli-QB ultrapure water system	Millipore Inc. USA
		DZF-6050 type vacuum drying oven	Shanghai Jinghong experiment equipment Co Ltd
CA-1111 TYPE EYELA COOLING WATER CIRCULATING DEVICE	Shanghai Ailang instruments Ltd
		LC-8A preparative high performance liquid chromatograph	Shimadzu Japan Ltd
LC-20A preparative high performance liquid chromatograph	Shimadzu Japan Ltd
		Bruker ASCENDTM 600MHz NMR spectrometer	Bruker, Switzerland
Thermo Scientific Q active mass spectrometer	Thermo Fisher corporation of America
		PL602-L type electronic balance	Mettler-Torloduoshanghai Co Ltd
KQ-500E type ultrasonic cleaner	KUNSHAN ULTRASONIC INSTRUMENTS Co.,Ltd.
		High-speed refrigerated centrifuge	BeckMan Coulter, USA
Micro-pipette	Eppendorf Co of USA
		Electric heating constant temperature water bath	Shanghai Jinghong experiment equipment Co Ltd
DZTW type temperature-adjusting electric heating jacket	Yongguang medical instruments Co Ltd, Beijing
		Leica CM1900 freezing microtome	German card
Full-wavelength multifunctional microplate reader Varioskan Flash	Thermo Scientific
		Freeze dryer	Beijing Bo exert oneself Shi Instrument Co Ltd

Example 1: preparation of south African leaf extract

1. Preparation of 60% ethanol extract of leaves of south Africa

Taking 400g of dried south Africa leaf blades, cutting the dried south Africa leaf blades into pieces by using scissors, putting the cut south Africa leaf blades into a 10-L round-bottom flask, adding 12 times of 60% ethanol solution, heating, refluxing and extracting for 2.5 hours, pouring out the first extracting solution, adding the same amount of fresh 60% ethanol, extracting for one time again, filtering, combining the two extracting solutions, concentrating by using a rotary evaporator to remove ethanol, adding a proper amount of pure water into the residual water solution, and freeze-drying the residual water solution into powder by using a freeze dryer to obtain 60g of dried powder.

2. Preparation of extract of polar layer of south Africa leaves

Taking 50g of dried south Africa leaf 60% ethanol extract, adding a proper amount of distilled water to disperse into a suspension with a proper concentration, sequentially extracting with petroleum ether, dichloromethane, ethyl acetate and n-butanol which are saturated by equal volume of water for three times, combining three extraction solutions, and concentrating by using a rotary evaporator to obtain a dried extract, wherein the weight of each part is respectively 2.00g of petroleum ether layer (VA-A), 8.17g of dichloromethane layer (VA-B), 2.24g of ethyl acetate layer (VA-C) and 6.67g of n-butanol layer (VA-D).

Example 2: preparation of steroid compound vernonacums B in south Africa leaves

1. Separation, purification and identification of vernonacums B

Performing forward silica gel column chromatography on the dichloromethane layer to obtain 11 fractions, performing next reverse silica gel column chromatography on B8, repeatedly purifying by preparative high performance liquid chromatography to obtain a compound vernonacums B, and analyzing the structure of the compound by using the techniques such as nuclear magnetic resonance, mass spectrometry and the like, wherein the structure is as follows:

example 3: establishment of mouse hyperuricemia model

1. Preparation of experimental drugs

CMC-Na solution: dissolving 1g CMC-Na powder in 200mL distilled water to prepare 0.5% CMC-Na solution

Allopurinol: 0.05g of allopurinol solid is dissolved in 25mL of 0.5% CMC-Na to prepare a solution of 0.002 g/mL.

South african leaf 60% ethanol extract (VA): 10g of dried 60% ethanol extract powder of leaves of south Africa was dissolved in 25mL of 0.5% CMC-Na to prepare a 0.4g/mL solution.

South Africa leaf petroleum ether layer extract: 2g of petroleum ether layer extract was dissolved in 25mL of 0.5% CMC-Na to prepare a 0.08g/mL solution.

South african leaf dichloromethane layer extract: 5g of petroleum ether layer extract was dissolved in 25mL of 0.5% CMC-Na to prepare a 0.2g/mL solution.

Ethyl acetate layer extract of south africa: 2.24g of petroleum ether layer extract was dissolved in 25mL of 0.5% CMC-Na to prepare a 0.09g/mL solution.

N-butanol layer extract of south africa leaves: 4g of petroleum ether layer extract was dissolved in 25mL of 0.5% CMC-Na to prepare a 0.16g/mL solution.

Potassium oxonate: 250mg of dried Potassium Oxonate powder was dissolved in 25mL of 0.5% CMC-Na to prepare a 10mg/mL solution.

Hypoxanthine: 1.25g of dried hypoxanthine powder was dissolved in 25mL of 0.5% CMC-Na to prepare a 50mg/mL solution.

2. Group administration of mice

Dividing 64 Kunming mice into 8 groups, namely a control group, a model group, a positive drug group, a VA-A group, a VA-B group, a VA-C group and a VA-D group, wherein each group comprises 8 mice, and marking the head and the limbs by picric acid. Adaptive feeding for one week, during which normal drinking and food is given. Seven days later, dosing was started and mice were dosed at 100 μ L/10 g. The control group was given 0.5% CMC-Na, the model group was also given 0.5% CMC-Na, the positive group was given 0.002g/mL allopurinol, the VA group was given 0.4g/mL 60% ethanol extract of south African leaves, the VA-A group was given 0.08g/mL petroleum ether layer extract of south African leaves, the VA-B group was given 0.2g/mL methylene chloride layer extract of south African leaves, the VA-C group was given 0.09g/mL ethyl acetate layer extract of south African leaves, and the VA-D group was given 0.16g/mL n-butanol layer extract of south African leaves for seven days.

3. Mouse hyperuricemia model

In the sixth day, the night is fasted for 12h without water prohibition. 1h after administration on day seven mice were initially injected subcutaneously with 10mg/mL Potassium Oxonate (100. mu.L/10 g mouse body weight) and intraperitoneally with 50mg/mL hypoxanthine to create a hyperuricemia model. Mice were bled from the orbit after 1 h. The blood was allowed to stand at room temperature for 2h to allow the blood to stratify. The blood sample is centrifuged for 10min at 3000 rpm to obtain an upper serum sample.

4. Uric acid detection

According to the operation instruction of the uric acid detection kit, adding 10 mu L of distilled water into a white group in a 96-hole cell culture plate, adding 10 mu L of 400 mu M uric acid standard substance into a standard substance group, adding 10 mu L of serum samples into the other groups, then adding 250 mu L buffers into all the holes, placing the 96-hole plate in a 37-DEG constant temperature incubator, and measuring the absorbance value at the wavelength of 510nm after 10min, wherein the calculation formula is as follows:

the comparative picture of the uric acid reducing effect of different dosage groups of the south Africa leaf extract in the embodiment of the invention is shown in figure 1, and the comparative picture of the uric acid reducing effect of the south Africa leaf extract and different solvent extracts thereof is shown in figure 2. Referring to fig. 1 and 2, in the embodiment of the invention, 60% ethanol is used for carrying out reflux extraction on south African leaves, 60% ethanol extract is obtained after concentration, partial extract is sequentially extracted by petroleum ether, dichloromethane, ethyl acetate and n-butanol, and extract of each extraction layer is obtained after solvent is recovered. Experiments prove that the 60% ethanol extract, petroleum ether, dichloromethane, ethyl acetate and n-butanol extract have the function of reducing the blood uric acid level of the mouse with high uric acid. Screening the five extracts for reducing the uric acid activity of the mice, primarily screening a dichloromethane extraction layer with better uric acid reducing activity, and observing the liver and kidney tissues of the dichloromethane extraction layer by tissue sections, wherein no damage is found, which indicates that the extract and the extract thereof have no obvious hepatotoxicity or hepatotoxicity, and have potential value for further developing into a uric acid reducing medicine and treating gout.

Example 4: establishment of in-vitro anti-gout model xanthine oxidase inhibition experimental model

Evaluation of anti-gout Activity of some monomeric compounds in vitro Xanthine oxidase inhibition experiments were used, and Xanthine oxidase (XAnthine oxidase, abbreviated as XOD, EC 1.17.3.2) is a complex oxidoreductase containing [2 Fe-2S ] cluster, molybdopterin and flavin prosthetic group. The xanthine oxidase can catalyze substrate xanthine (hypoxanthine) to generate uric acid and active oxygen, the uric acid has ultraviolet absorption under 295nm, and the method evaluates the xanthine oxidase inhibitory activity by detecting the uric acid generation amount under 295nm through a colorimetric method.

First, sample preparation

Approximately 1mg of each sample was placed in an EP tube, dissolved in DMSO, and prepared into a stock solution with a concentration of 100mM, which was diluted with time.

Second, reagent preparation

The concentrations and amounts of the reagents used are shown in Table 3.

TABLE 3

Sodium pyrophosphate buffer 2.659g of sodium pyrophosphate and 8.77mg of EDTA were dissolved in 100mL of ultrapure water to prepare a 0.1M sodium pyrophosphate buffer, and the pH was adjusted to 8.3.

Xanthine 1mg was dissolved in 13.15mL sodium pyrophosphate buffer to prepare a solution with a concentration of 500. mu.M, which was used as it was.

XO enzyme concentration is 10.7U/mg, 1mg XO is dissolved in 256. mu.L buffer solution to prepare 41.75U/mL solution, which is diluted for use.

Allopurinol 1mg allopurinol was dissolved in 14.69mL buffer to prepare a 500. mu.M solution, which was diluted with water

The experimental steps are as follows:

the final volume of the reaction was 200. mu.L, and all groups were repeated 3 times.

1. All reagents were allowed to come to room temperature (25 ℃) before use.

2. All samples, enzymes and substrates were diluted to the use concentration.

3. Add 80. mu.L of enzyme, sample, and positive drug to 96-well UV plate and incubate for 15min at room temperature.

4. The reaction was initiated by adding 80. mu.L of substrate to each well and allowed to react at room temperature for 30 min.

5. OD was measured at 295nm with a full wavelength reader.

6. Calculating an inhibition rate:

the reaction equation of the system is as follows:

third, the results of the Activity test

As shown in FIG. 3, the results of activity tests show that compounds 14-19,22,25,32-34 have weak inhibitory activity, and compound 21(vernonacums B) has strong inhibitory activity.

The experimental result shows that the compound 21(vernonacums B) can obviously inhibit the activity of xanthine oxidase, so that the steroid compound in the south Africa leaves has application prospects in the aspects of gout treatment medicines and the like, and has potential values of further serving as a uric acid reducing medicine and treating gout.

Claims (3)

1. The south Taiwan African leaf steroid compound is characterized in that the structural formula is as follows:

2. the method of preparing the steroids from south gulf african leaves according to claim 1, comprising the steps of:

1) taking 400g of dried south Africa leaf blades, shearing the dried south Africa leaf blades into pieces by using scissors, putting the cut south Africa leaf blades into a 10-L round-bottom flask, adding 12 times of 60% ethanol solution, heating, refluxing and extracting for 2.5h, pouring out the 1 st extracting solution, adding the same amount of fresh 60% ethanol, extracting for 1 time again, filtering, combining the extracting solutions, concentrating by using a rotary evaporator to remove ethanol, adding a proper amount of pure water into the residual water solution, and freeze-drying the residual water solution into powder by using a freeze dryer to obtain 60g of dried powder;

2) taking 50g of dried south African leaf 60% ethanol extract, adding a proper amount of distilled water to disperse into a suspension with a proper concentration, extracting with dichloromethane saturated by equal volume of water for 3 times, combining the extraction solutions for 3 times, concentrating into a dried extract by using a rotary evaporator to obtain 8.17g of south African leaf dichloromethane layer extract, further separating by using chromatographic methods of normal phase silica gel column chromatography, reverse phase silica gel column chromatography and gel column chromatography, and purifying by combining preparative high performance liquid chromatography to obtain the steroid compound.

3. Use of the south taiwan african leaf steroid compound according to claim 1 for the preparation of an anti-gout agent.

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