CN118121639B - Extraction method of effective components of purslane and application of effective components in uric acid reduction - Google Patents

Abstract

The present invention provides a method for extracting effective ingredients of purslane and its application in reducing uric acid, belonging to the technical field of drug purification. The present invention adopts a two-stage extraction, which can effectively extract water-soluble compounds and fat-soluble compounds in purslane, and has the advantages of simple and easy process. Through research on it, it is found that the purslane extract prepared by the present invention acts on targets such as xanthine oxidase, adenosine triphosphate binding transporter G superfamily member 2, glucose transporter 9 and urate transporter 1, and plays a role in preventing and treating hyperuricemia by reducing the production of uric acid in the body and promoting the excretion of uric acid, and protecting the kidneys. It is in line with the advantages of traditional Chinese medicine with multiple components, multiple targets and overall regulation.

Description

A method for extracting effective components of purslane and its application in lowering uric acid

Technical Field

The invention relates to the technical field of drug purification, and in particular to a method for extracting effective components of purslane and application of the method in reducing uric acid.

Background technique

Purslane (Portulaca oleracea L., PO) is an annual herbaceous plant of Caryophyllales and Portulacaceae. It is produced in all parts of China and is one of the wild plants that can be used as both medicine and food. In ancient Chinese medicine, it has the functions of clearing away heat and dampness, detoxifying and reducing swelling, reducing inflammation, quenching thirst, and acting as a diuretic. Modern research has confirmed that Purslane has multiple functions such as lowering blood sugar, inhibiting bacteria, resisting oxidation, and enhancing immunity.

Hyperuricemia (HUA) is a chronic disease caused by increased serum uric acid levels due to impaired purine metabolism and/or reduced uric acid excretion. With the continuous improvement of living standards, dietary habits have gradually shown a trend of high purine content, causing the incidence of hyperuricemia to increase year by year and once became a metabolic disease that threatens human health.

Uric acid (UA), also known as 2,6,8-trihydroxypurine, is the end product of the synthesis and metabolism of purine substances (guanine, adenine, etc.) in the human body. It is a weak organic acid that is slightly soluble in water. About two-thirds of the uric acid in the human body is synthesized from endogenous purines, and one-third comes from exogenous high-purine diets. In addition to the purine synthesis and metabolism pathway, the excretion mechanism of uric acid in the kidneys also plays a key role in hyperuricemia. About two-thirds of the uric acid formed in the human blood is excreted through the kidneys, and one-third is excreted from the body through extrarenal pathways such as the intestines. At present, the research on hyperuricemia often takes the inhibition of purine/uric acid synthesis and the promotion of uric acid renal metabolism as research targets.

Chinese patent CN112043736A provides a method for preparing the active components of purslane, using water as a solvent, heating and extracting to obtain a water extract, and then filtering through an ultrafiltration membrane with a molecular weight cutoff of 5K-15K, and taking the filtrate to obtain the active components of purslane. The components extracted by this method have a large molecular weight, and the extracted contents are macromolecular substances such as polysaccharides, proteins and polypeptides.

"Research on the uric acid-lowering and kidney-protective effects of Portulaca oleracea" (Lv Youwei et al., Food Industry Science and Technology, 2022, 43(2):6), the alkaloid part was extracted using the acid-base extraction method.

The ingredients obtained by the above methods only reduce serum uric acid levels by inhibiting the activity of xanthine oxidase in the body.

Summary of the invention

In view of this, the present invention provides a method for extracting the effective ingredients of Portulaca oleracea. The process is simple and easy, the extract has comprehensive ingredients, and not only acts on xanthine oxidase, but also promotes ABCG2 and inhibits the expression of GLUT9 and URAT1 proteins, and has a multi-component, multi-target, and multi-pathway synergistic effect, thereby achieving the effect of reducing the uric acid level in the body.

The method for extracting the effective components of purslane of the present invention comprises the following steps:

(1) pre-treating purslane to obtain purslane powder;

(2) extracting the purslane powder with water, repeating the extraction operation 2 to 3 times to obtain a water extract and a water extraction residue;

(3) Extracting the water extraction residue with chloroform, repeating the extraction operation 2 to 3 times to obtain a chloroform extract;

(4) The water extract obtained in step (2) and the chloroform extract obtained in step (3) are mixed and concentrated to obtain a purslane extract.

Preferably, the pretreatment method in step (1) is: drying the fresh purslane in the sun, and then crushing it into powder that passes through a 40-80 mesh sieve.

Preferably, in the water extraction process described in step (2), the mass ratio of purslane powder to water is 1:10-15; the extraction temperature is 60-70° C., and the extraction time is 2-3 h.

Preferably, in the chloroform extraction process described in step (3), the mass ratio of the water extraction residue to the chloroform solvent is 1:10-15, the extraction temperature is 60-70° C., and the extraction time is 2-3 h.

Preferably, the concentration in step (4) is evaporation concentration.

More preferably, the evaporation concentration is carried out using a vacuum rotary evaporator.

The purslane effective components extracted by the invention are used for preparing medicine for treating hyperuricemia.

Preferably, the drug is an oral preparation, and the oral preparation includes at least one of tablets, oral liquids, capsules, pills and granules.

More preferably, the drug contains pharmaceutically acceptable excipients.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a method for extracting effective ingredients of purslane, wherein the purslane extract of the method adopts a two-stage extraction, can effectively extract water-soluble compounds and fat-soluble compounds in purslane, and has the advantages of simple and easy process. Through research, it is found that the purslane extract prepared by the present invention acts on targets such as xanthine oxidase, adenosine triphosphate binding transporter G superfamily member 2, glucose transporter 9 and urate transporter 1, and plays a role in preventing and treating hyperuricemia and protecting the kidneys by reducing the production of uric acid in the body and promoting the excretion of uric acid. It is in line with the advantages of traditional Chinese medicine with multi-component, multi-target and overall regulation. In addition, the purslane used in the present invention is highly safe for the human body and has certain development potential.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the inhibitory effects of different extraction methods on xanthine oxidase, a key target of hyperuricemia;

FIG2 is a graph showing the effects of different drugs on the blood uric acid level in hyperuricemia mice;

FIG3 is a graph showing the effects of different drugs on serum xanthine oxidase activity in hyperuricemia mice;

FIG4 is a graph showing the effects of different drugs on creatinine levels in hyperuricemia mice;

FIG5 is a graph showing the effects of different drugs on urea nitrogen levels in hyperuricemia mice;

FIG6 is a graph showing the effect of different drugs on the expression level of antibodies against adenosine triphosphate binding transporter G superfamily member 2 in hyperuricemia;

FIG7 is a graph showing the effects of different drugs on the expression level of glucose transporter 9 antibody in hyperuricemia;

FIG8 is a graph showing the effects of different drugs on the expression level of urate transporter 1 antibody in hyperuricemia;

Figure 9 shows the kidney pathological morphology of mice in different groups (HE, ×400).

Detailed ways

The present invention will be further described below in conjunction with the embodiments.

Example 1

A method for extracting effective ingredients from purslane, comprising the following steps:

(1) Dry fresh purslane in the sun (Purslane was collected in Mianyang, Sichuan, and stored after drying, the same below), and then grind it into powder that passes through a 40-80 mesh sieve;

(2) Take the crushed purslane, add 10 times (the volume of water is 10 times the volume of the dried purslane) 60°C water to soak and extract for 3 hours, during which ultrasonic assisted extraction is used, and vacuum filtration is performed. The extraction operation is repeated 3 times, and the extracts are combined to obtain the purslane water extract and water extraction residue;

(3) extracting the water extraction residue with chloroform to obtain a chloroform extract; wherein the mass ratio of the water extraction residue to the chloroform solvent is 1:15, the extraction temperature is 60°C, and the extraction time is 3 hours;

(4) The water extract obtained in step (2) and the chloroform extract obtained in step (3) are mixed and concentrated using a vacuum rotary evaporator to obtain an extract, and the extract is vacuum dried to obtain a purslane extract.

Comparative Example 1

The extraction steps of purslane water extract are as follows:

(1) Dry fresh purslane in the sun, and then grind it into powder that can pass through a 40-80 mesh sieve;

(2) Take the crushed purslane, add 10 times (the volume of the liquid is 10 times the volume of the dried purslane) 60°C water and soak for extraction for 3 hours. During the extraction, ultrasonic-assisted extraction is used, and vacuum filtration is performed. The extraction operation is repeated 3 times. The extracts are combined to obtain the purslane water extract, and vacuum drying is performed to obtain the purslane water extract.

Comparative Example 2

The extraction steps of chloroform extract of purslane are as follows:

(1) Dry fresh purslane in the sun, and then grind it into powder that can pass through a 40-80 mesh sieve;

(2) Take the crushed purslane, add 10 times (the volume of the liquid is 10 times the volume of the dried purslane) 60°C chloroform to soak and extract for 3 hours, during which ultrasonic-assisted extraction is used, and vacuum filtration is performed. The extraction operation is repeated 3 times, and the extracts are combined to obtain the purslane chloroform extract, which is then vacuum dried to obtain the purslane chloroform extract.

Test Example 1

Effects of Portulaca oleracea extracts extracted by different methods on xanthine oxidase, a key target of hyperuricemia:

Xanthine oxidase can decompose xanthine to generate uric acid, so the activity of xanthine oxidase can be judged based on the generation of uric acid. At present, the research method for the inhibition test of xanthine oxidase is mainly spectrophotometry, and the test basis is that uric acid has a characteristic absorption peak at 295nm.

The specific method is:

(1) Prepare reagents

Xanthine (XA, purity: 98%), allopurinol (allopurinol, purity: 98%), and xanthine oxidase (XOD, 50u/mg) were purchased from Yuanye Biotechnology Co., Ltd.; yeast extract was purchased from Beijing Aoboxing Biotechnology Co., Ltd., and sodium carboxymethyl cellulose (CMC-Na) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

(2) Preparation of assay reagents

Preparation of xanthine solution: Dissolve 0.057 g XA in 1 mL of 1 mol/L NaOH solution, and then dilute to 250 mL with PBS (0.1 mmol/L; pH: 7.2~7.4) solution.

Preparation of xanthine oxidase (XOD) solution: Accurately weigh 3 mg of XOD and dissolve it in 30 mL of PBS, then dilute it 100 times before use. At this time, the enzyme concentration is about 8 U/mL.

(3) Preparation of test agents: 10 mg each of allopurinol, purslane extract (Example 1), purslane water extract (Comparative Example 1), and purslane chloroform extract (Comparative Example 2) were weighed and dissolved in dimethyl sulfoxide (DMSO) to prepare sample solutions of 10 mg/mL, 5 mg/mL, 4 mg/mL, 2.5 mg/mL, 2 mg/mL, and 1 mg/mL, respectively; the final concentrations of the test solutions in the system were 1000 μg/mL, 500 μg/mL, 400 μg/mL, 250 μg/mL, 200 μg/mL, and 100 μg/mL, respectively.

(4) Prepare mixtures A, B, and C: A is 0.5 mL XOD, 3.5 mL PBS, and 1 mL XA; B is 4 mL PBS and 1 mL XA; C is 0.5 mL XOD, 3 mL PBS, 1 mL XA, and 0.5 mL sample solution; D is 3.5 mL PBS, 1 mL XA, and 0.5 mL sample solution. Incubate each mixture at 37°C for 30 minutes. Then, terminate the reaction by adding 200 μL of 0.5 M hydrochloric acid, and measure the absorbance at 295 nm. Since the total reaction system is 5 mL and the sample volume is 5 mL, the final concentration is one tenth of the sample concentration. Calculate the inhibition percentage using formula (1):

(1)

A curve was drawn with the final concentration of the test solution in the system as the horizontal axis and the xanthine oxidase inhibition rate as the vertical axis to analyze the inhibitory ability of allopurinol and purslane extracts extracted by different extraction methods on xanthine oxidase. The results are shown in Figure 1.

As can be seen from Figure 1, the inhibitory ability of the purslane extract of the present invention on xanthine oxidase is better than that of the purslane water extract and the purslane chloroform extract extracted by a single method. The extracted components are more comprehensive and effective, and the IC50 value of the inhibitory effect on xanthine oxidase is 139.38 μg/mL.

Test Example 2:

Effects of Purslane Extract on Hyperuricemia Diseases:

Uric acid was determined using a uric acid determination kit; creatinine was determined using a creatinine determination kit; urea nitrogen was determined using a urea nitrogen determination kit; the above kits were all purchased from Nanjing Jiancheng Bioengineering Institute, and the specific operating steps were carried out according to the kit instructions.

This test example uses an animal model of hyperuricemia to evaluate the efficacy of the purslane extract extracted by the present invention, and to study the mechanism of the purslane extract in preventing and treating hyperuricemia. The specific experimental method is as follows:

(1) Prepare reagents

Preparation of 0.5wt% CMC-Na solution: weigh 5.0g CMC-Na and dissolve it in 1000mL normal saline, mix well to obtain 0.5wt% CMC-Na solution;

Preparation of allopurinol solution: weigh 0.1 g of allopurinol, dissolve it in 100 mL of 0.5 wt % CMC-Na solution, and mix well to obtain 1 mg/mL allopurinol solution; in the experiment, the oral administration dose was 0.15 mL, and the dosage for mice was converted to 7.5 mg/kg according to the human equivalent dose coefficient.

Preparation of benzbromarone (purity: 98%) solution: weigh 0.1g of benzbromarone, dissolve it in 100mL of 0.5wt% CMC-Na solution, and mix well to obtain 1mg/mL benzbromarone solution; in the experiment, the oral administration dose was 0.15mL, and the dosage for mice was converted to 7.5mg/kg according to the human equivalent dose coefficient.

(2) Experimental animals

SPF grade C57BL/6J mice, male, 18-22g, 6-8 weeks old, purchased from Liaoning Changsheng Biotechnology Co., Ltd., license number SCXK (Liao) 2020-0001, ethics review number: 2023YNPZSY0307. The experimental animals were housed in separate cages and adaptively fed for 7 days before the experiment. They had free access to water and food during the experiment. The temperature of the breeding environment was 22-25℃, the relative humidity was 60%, and there was a 12-hour light and 12-hour dark cycle per day.

(3) Animal grouping

Before the experiment, SPF male C57BL/6J mice were weighed and randomly divided into a normal group (Control), a model group (Model), an allopurinol group (Allopurinol), a benzbromarone group (Benzbromarone), a low-dose purslane extract group (LPO), a medium-dose purslane extract group (MPO), and a high-dose purslane extract group (HPO) according to their body weight, with 8 mice in each group. After grouping, the mice were numbered, and the body weight of each mouse was weighed, and the corresponding administration volume was recorded and calculated.

(4) Model establishment: The mice in the normal group were gavaged with 0.5wt% CMC-Na solution every morning, and the mice in the other groups except the normal group were gavaged with 25 g/kg yeast extract every morning for 14 consecutive days. Blood was collected from the mice in each group before gavage on the 15th day to measure the uric acid level in the serum of each group of mice. The results of the test using the kit showed that the blood uric acid level of the mice in each treatment group was significantly higher than that in the normal group, indicating that the model was successfully established and the next step of the experiment was carried out.

(5) Administration: Yeast extract was then continuously gavaged every morning to maintain clinical signs of hyperuricemia. The mice were administered medication in the afternoon for 7 consecutive days. The dosages for mice were calculated according to the human equivalent dose coefficient as follows: allopurinol group (7.5 mg/kg), benzbromarone group (7.5 mg/kg), low-dose purslane extract group (0.5 g/kg), medium-dose purslane extract group (1.0 g/kg), and high-dose purslane extract group (2.0 g/kg).

In the following experiments, each data is expressed as mean ± standard deviation (mean ± SD), and Graphpad Prism 8 software was used for drawing. SPSS17 software was used for statistical analysis. One-way ANOVA was used to compare the differences between the groups. When the variances were equal, the LsD method was used for multiple comparisons between the groups; when the variances were unequal, the Dunnett's T3 method was used for multiple comparisons between the groups. P < 0.05 was used as the standard to indicate a significant difference. The independent sample T test (Student's t test) was used to compare the normal group with the model group. Compared with the normal group Compared with the model group .

(6) Effects of Purslane extract on serum uric acid levels, xanthine oxidase activity, creatinine and urea nitrogen in hyperuricemia mice:

Two hours after oral administration on the last day, each mouse was euthanized, blood was collected, and samples were obtained. The removed kidneys were washed three times in pre-cooled PBS buffer. After fully removing blood and other impurities, the left kidney was fixed in paraformaldehyde and the right kidney was frozen in a -80°C refrigerator. The blood samples were centrifuged and serum was separated for the determination of uric acid, xanthine oxidase activity, creatinine, and urea nitrogen levels. The results are shown in Figures 2 to 5, respectively.

Figure 2 shows the effects of different drugs on the blood uric acid levels of hyperuricemia mice; Figure 3 shows the effects of different drugs on the serum xanthine oxidase activity levels of hyperuricemia mice; Figure 4 shows the effects of different drugs on the creatinine levels of hyperuricemia mice; Figure 5 shows the effects of different drugs on the urea nitrogen levels of hyperuricemia mice.

As can be seen from Figures 2 and 5, hyperuricemia can cause a significant increase in the uric acid level, xanthine oxidase activity, creatinine and urea nitrogen levels in the serum of mice in the hyperuricemia model group; allopurinol, benzbromarone and purslane extract can reduce the uric acid level in the serum of mice and inhibit the activity of xanthine oxidase. In addition, the purslane extract group can significantly improve the creatinine and urea nitrogen indicators of mice, indicating that purslane extract has the effect of preventing and treating the increase of uric acid caused by hyperuricemia, and can improve the kidney damage caused by uric acid.

(7) Effect of Purslane Extract on Uric Acid Transporter in Hyperuricemia Mice:

Western blotting was used to measure the expression levels of adenosine triphosphate binding transporter G superfamily member 2 (ABCG2), glucose transporter 9 (GLUT9), and urate transporter 1 (URAT1) in the kidneys of each group of mice. The results are shown in Figures 6 to 8.

Figure 6 shows the effect of different drugs on the expression level of antibody of adenosine triphosphate binding transporter G superfamily member 2 in hyperuricemia. The results in Figure 6 show that hyperuricemia can cause a decrease in the expression level of adenosine triphosphate binding transporter G superfamily member 2 (ABCG2) in the mouse kidney, that is, a decrease in uric acid excretion level. Allopurinol, benzbromarone and purslane extract can increase its expression level and promote the excretion of uric acid in mice. The enhancement effect of purslane extract is dose-dependent, and the effect of the extract in the high-dose group is close to that of the positive drugs (allopurinol, benzbromarone), and there is no significant difference with the normal group.

Figure 7 shows the effects of different drugs on the expression level of glucose transporter 9 antibody in hyperuricemia. The results in Figure 7 show that hyperuricemia can increase the expression level of glucose transporter 9 (GLUT9) in the mouse kidney and promote the reabsorption of uric acid in the mouse kidney tissue. Compared with the model group, the allopurinol, benzbromarone and purslane extract groups can reduce their expression levels. The enhancement effect of purslane extract is dose-dependent, and the effects of the extracts in the medium and high dose groups are close to those of the positive drugs (allopurinol, benzbromarone), and there is no significant difference from the normal group.

Figure 8 is a graph showing the effects of different drugs on the expression level of urate transporter 1 antibody in hyperuricemia. The results in Figure 8 show that hyperuricemia can increase the expression level of urate transporter 1 (URAT1) in the mouse kidney and promote the reabsorption of uric acid by the mouse kidney tissue. Compared with the model group, the allopurinol, benzbromarone and purslane extract groups can reduce their expression levels. The enhancement effect of purslane extract is dose-dependent, and the effect of the high-dose group extract is close to that of the positive drugs (allopurinol, benzbromarone), and there is no significant difference from the normal group.

(8) Morphological observation of mouse kidney sections:

The results of kidney pathological morphology (HE, ×400) of mice in different groups are shown in Figure 9. Obvious eosinophilic changes and severe tubular vacuolation were observed in the model group. Eosinophilic changes and a certain degree of tubular vacuolation with loose structure were also observed in the allopurinol group, benzbromarone group and low-dose group. Compared with the model group, no obvious morphological abnormalities were observed in the tubular cells and tissue structure in the medium and high-dose groups.

The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims (6)

1. The application of the purslane effective component is characterized in that the purslane effective component is used for preparing a medicament for treating hyperuricemia;

The extraction method of the purslane active ingredient comprises the following steps:

(1) Pretreating herba Portulacae to obtain herba Portulacae powder;

(2) Extracting purslane powder with water for 2-3 times, and repeating the extraction operation to obtain water extract and water extraction filter residues;

(3) Extracting the water extraction filter residue by using chloroform, and repeating the extraction operation for 2-3 times to obtain chloroform extract;

(4) Uniformly mixing the water extract obtained in the step (2) and the chloroform extract obtained in the step (3), and concentrating to obtain a purslane extract;

In the water extraction process in the step (2), the mass ratio of the purslane powder to the water is 1:10-15; the extraction temperature is 60-70 ℃ and the extraction time is 2-3 h;

In the chloroform extraction process in the step (3), the mass ratio of water extraction filter residues to chloroform solvent is 1:10-15, the extraction temperature is 60-70 ℃, and the extraction time is 2-3 h.

2. The use of the purslane active ingredient according to claim 1, wherein the pretreatment method in step (1) is as follows: fresh purslane is dried in the sun and then crushed into powder which is sieved by a sieve with 40 to 80 meshes.

3. The use of the purslane active ingredient according to claim 1, wherein the concentration in the step (4) is evaporation concentration.

4. The use of the purslane active ingredient according to claim 3, wherein the evaporation concentration is performed by a vacuum rotary evaporator.

5. The use of the purslane active ingredient according to claim 1, wherein the drug is an oral preparation, and the oral preparation comprises at least one of a tablet, an oral liquid, a capsule, a dripping pill and a granule.

6. The use of the purslane active ingredient according to claim 5, wherein the medicament comprises pharmaceutically acceptable auxiliary materials.