CN110279758B

CN110279758B - Peony leaf dry powder, preparation method thereof and application of peony leaf dry powder as xanthine oxidase inhibitor

Info

Publication number: CN110279758B
Application number: CN201910613936.4A
Authority: CN
Inventors: 王四旺; 张妍; 林奋
Original assignee: Shaanxi Fengdan Zhengyuan Biotechnology Co ltd
Current assignee: Shaanxi Fengdan Zhengyuan Biotechnology Co ltd
Priority date: 2019-07-09
Filing date: 2019-07-09
Publication date: 2022-02-01
Anticipated expiration: 2039-07-09
Also published as: CN110279758A

Abstract

The invention relates to a peony leaf dry powder, a preparation method thereof and application of the peony leaf dry powder as a xanthine oxidase inhibitor, wherein the preparation method of the peony leaf dry powder comprises the following steps: (1) taking dried peony leaves, adding a proper amount of water, heating to reflux temperature, extracting for 1 hour, filtering, and collecting filtrate and filter residue; (2) adding a proper amount of water into the filter residue collected in the step (1), heating to the reflux temperature, extracting for 40 minutes, filtering, and collecting filtrate; (3) and (3) combining the filtrates collected in the steps (1) and (2), concentrating the filtrates into thick paste with the relative density of 1.10-1.15, spray-drying to obtain dry powder, and sieving with a 100-mesh sieve to obtain the peony leaf dry powder.

Description

Peony leaf dry powder, preparation method thereof and application of peony leaf dry powder as xanthine oxidase inhibitor

Technical Field

The invention belongs to the field of natural plant extraction, and particularly relates to peony leaf dry powder, a preparation method thereof and application of the peony leaf dry powder as a xanthine oxidase inhibitor.

Background

Peony is a perennial deciduous shrub of Paeonia of Paeoniaceae, and has bright color, reputation of "beautiful sky and fragrance" and "king of flower", and connotations of "richness and luck" and "prosperity and prosperity". Peony can be enjoyed and has high edible and medicinal value, for example, peony seeds can extract oil, root bark can be used as medicine, flowers can be used for making tea, and the like. However, no report about the peony leaf extract as a xanthine oxidase inhibitor exists at present. The invention provides peony leaf extract dry powder, a preparation method thereof and application of the peony leaf extract dry powder as a xanthine oxidase inhibitor.

Disclosure of Invention

The peony leaf dry powder is characterized in that the preparation method of the peony leaf dry powder comprises the following steps:

(1) taking dried peony leaves, adding a proper amount of water, heating to reflux temperature, extracting for 1 hour, filtering, and collecting filtrate and filter residue;

(2) adding a proper amount of water into the filter residue collected in the step (1), heating to the reflux temperature, extracting for 40 minutes, filtering, and collecting filtrate;

(3) and (3) combining the filtrates collected in the steps (1) and (2), concentrating the filtrates into thick paste with the relative density of 1.10-1.15, spray-drying to obtain dry powder, and sieving with a 100-mesh sieve to obtain the peony leaf dry powder.

The dried peony leaves in the step (1) are the peony leaves with the leaf axis and the leaf stalks removed and dried in the shade to constant weight, the proper amount of water is preferably 10 times of the weight of the dried peony leaves, and the water is preferably deionized water or distilled water;

the proper amount of water in the step (2) is preferably 8 times of the weight of the dried peony leaves, and the water is preferably deionized water or distilled water.

Another embodiment of the present invention provides a method for preparing the above-mentioned peony leaf dry powder, which is characterized by comprising the steps of:

Another embodiment of the present invention provides a pharmaceutical composition, wherein the pharmaceutical composition comprises the above-mentioned peony leaf dry powder as an active ingredient. The pharmaceutical composition also optionally comprises auxiliary materials with pharmaceutical and/or food approved addition amount, such as flavoring agent, excipient, anti-moisture agent, excipient, suspending agent, etc. The pharmaceutical composition can be made into tablet, capsule, granule, powder or table powder, meal replacement powder, meal preparation powder, oral liquid, injection (including lyophilized powder for injection), etc. The pharmaceutical composition also optionally includes other xanthine oxidase inhibitors.

In another embodiment of the invention, the application of the peony leaf dry powder or the pharmaceutical composition in preventing and/or treating diseases caused by abnormal activity of uric acid, creatinine and/or Xanthine Oxidase (XOD) is provided.

In another embodiment of the invention, the application of the peony leaf dry powder or the pharmaceutical composition as a Xanthine Oxidase (XOD) inhibitor is provided.

The reference substance of relative density of the invention is water; the peony leaves of the invention are preferably paeonia ostii leaves.

Drawings

Figure 1 is a graph of total water consumption in groups of mice given 2 weeks (note: compare to blank,^##P<0.01; in comparison with the set of models,^**P<0.01)。

FIG. 2 is a graph of total food intake in groups of mice given 2 weeks.

Figure 3 is a graph of serum uric acid levels in groups of mice dosed for 2 weeks (note: compared to blank,^##P<0.01; in comparison with the set of models,^*P<0.05，^**P<0.01)。

figure 4 is a graph of serum creatinine levels in groups of mice administered for 2 weeks (note: compared to the blank,^##P<0.01; in comparison with the set of models,^*P<0.05，^**P<0.01)。

FIG. 5 is a graph of serum urea nitrogen levels in groups of mice administered for 2 weeks.

FIG. 6 is a graph showing the serum Xanthine Oxidase (XOD) activity of mice in each group administered for 2 weeks (note: compared with blank group,^#P<0.05; in comparison with the set of models,^*P<0.05，^**P<0.01)。

FIG. 7 is a graph showing the serum total cholesterol levels of groups of mice administered for 2 weeks.

FIG. 8 is a graph showing the serum triglyceride content of each group of mice administered for 2 weeks.

FIG. 9 is a graph showing the high-density lipoprotein content in each group of mice administered for 2 weeks.

FIG. 10 is a graph showing the low density lipoprotein content in each group of mice administered for 2 weeks.

FIG. 11 is a UHPLC-QTOF-MS analysis chart of the peony leaf dry powder of the present invention; a: NEG negative ion pattern, B: POS positive ion mode diagram.

Detailed Description

Example 1

(1) Adding deionized water (5.0L) into dried peony leaves (500g), heating to reflux temperature, extracting for 1 hr, filtering, and collecting filtrate and residue;

(2) adding deionized water (4.0L) into the filter residue collected in the step (1), heating to reflux temperature, extracting for 40 minutes, filtering, and collecting filtrate;

(3) and (3) mixing the filtrates collected in the steps (1) and (2), concentrating the filtrates into thick paste with the relative density of 1.10-1.15, spray-drying to obtain dry powder, and sieving with a 100-mesh sieve to obtain the peony leaf dry powder (8.53 g).

The dried peony leaves in the example 1 of the present invention are paeonia ostii leaves with the leaf axis and the leaf stalk removed and dried in the shade to constant weight.

Example 2 chemical composition analysis of the inventive (example 1 prepared) peony leaf dry powder

Adopting UHPLC-QTOF-MS detection technology, collecting peony leaf dry powder 100mg, adding 500 μ L of extract containing internal standard (methanol-water ratio is 4: 1, internal standard concentration is 5 μm L/mL), sampling, collecting 200 μ L, loading on machine for detection in 2mL sampling bottle, introducing mass spectrum by Progenesis QI software, treating by retention time correction, peak identification, peak extraction, peak integration, peak alignment, etc., identifying peak containing MSMS data by using secondary mass spectrum database and corresponding cracking rule matching method (fig. 11A and B), and confirming that peony leaf dry powder mainly contains L-proline, kahweol, alpha-linolenic acid, hydroxy paeoniflorin (oxypaeoniflorin), methyl gallate/3, 4, 5-trihydroxymethyl benzoate, brevifolic acid, orixine, paeoniflorin, quercetin, verbascoside/primisula japonica glycoside/uplii glycoside, gossypin, and/gossypin, luteolin-7-O-glucopyranoside/luteolin/cinnamyl glycoside, kaempferol, sugar alcohol-7-O-glucoside, apigenin, isorhamnetin, rhoifolin, cosmosiin/apigenin-7-glucoside, methoxyflavone-4-methoxyflavone/diosmin, 3 ', 5, 7-trihydroxy-4' -methoxyflavone/methoxyflavone, diglucoside, luteolin, apigenin/apigenin, benzoylpaeoniflorin, sucrose/beta-D-fructofuranosyl-alpha-D-glucopyranoside, raffinose, pyrogallol/pyrogallic acid, gallic acid, alizarin, emodin, Alisol A, 3, 4-dihydroxybenzoic acid, More than 40 chemical components such as salicylic acid, p-coumaric acid, L-tryptophan, ellagic acid, hyperin, 2-O-galloyl hyperin, 1,2,3,4, 6-pentan-O-gallic acid-beta-D-glucose, 4-hydroxybenzoic acid, sophoricoside and the like. The high-content chemical components are as follows: the peak area size in parentheses is estimated as follows: 1,2,3,4, 6-penta-O-gallic acid-beta-D-glucose (272626) > paeoniflorin (197622) > gallic acid (189958) > luteolin-7-O-glucopyranoside/luteolin/cinnolin (80209) > cosmosin/apigenin-7-glucoside (65799) > benzoylpaeoniflorin (64335) > sucrose/beta-D-fructofuranosyl-alpha-D-glucopyranoside (37964) > diglucoside of glutamic acid (27387) > rhoifolin (24949) > methoxyflavone-4-methoxyflavone/diosmin (15811) > sophoricoside (12881) > hydroxypaeoniflorin/oxypaeoniflorin (11164) > pyrogallol/pyrogallic acid (11079) > luteolin (04).

Example 3 animal experiment of the inventive (example 1-prepared) peony leaf dry powder

1 Material

1.1 Experimental animals

SPF grade male kunming mice, 30 ± 2g, were provided by the experimental animal research center of the university of military and medical air force [ certification No.: SCXK- (shan) 2015-007 ]. The feed is fed adaptively for 1 week, and the water is drunk in normal diet, the room temperature is kept at 22-26 ℃, and the humidity is 50% -70%. The treatment of the animals in the experimental process conforms to the guidance opinions about the animals which are good at being tested issued by the Ministry of science and technology.

1.2 drugs and reagents

Yeast powder (Oxoid, UK, lot # 2104085-02), adenine (Beijing Solebao science and technology, Inc.), uric acid assay kit (Shanghai Rongsheng biological medicine, Inc., lot # 20182400622), urea nitrogen assay kit (Shanghai Rongsheng biological medicine, Inc., lot # 20180704119), cholesterol assay kit (Changchun Hui Living Biotechnology, Inc., lot # 2019002), triglyceride assay kit (Changchun Hui Liangsheng Lisheng, Inc., lot # 2018008), high density lipoprotein cholesterol assay kit (Changchun Hui Liangsheng Liang Biotechnology, Inc., lot # 2019003), and low density lipoprotein cholesterol assay kit (Changchun Hui Liang Liangsheng Liheng Biotechnology, Inc., lot # 2019004).

1.3 instruments

An RT-9600 type semi-automatic biochemical analyzer, a Meigu molecular SpectraMax190 type enzyme-labeling instrument (American MD), an XK96-A type rapid mixer (Xinkang medical instruments Co., Ltd., Jiangyan), and a constant-temperature water bath.

2 method

2.1 preparation of high purine feed

Mixing yeast dry powder and adenine in the pulverized granulated feed, fully mixing uniformly, and re-pressing and molding. The content of the yeast dry powder in the feed is 10 percent, and the content of the adenine in the feed is 0.1 percent.

2.2 establishment, grouping and administration of mouse high xanthine oxidase Activity model

After the mice are adaptively raised for 1 week, the mice are randomly divided into a blank group, a model group and a peony leaf dry powder group (12.5, 25, 50, 100, 200 mg.kg)^-1) And allopurinol group (5mg kg)^-1) 10 each per group. The model group and each administration group were given high purine feed, the normal group was given ordinary feed, and the drinking water intake was recorded daily. The peony leaf dry powder is given with different dosages in each dosage group, allopurinol is given in allopurinol group, and physiological saline with the same amount is given in blank group and model group according to the proportion of 10 mL-kg^-1The administration was performed daily for 14 days.

2.3 specimen Collection and index Observation

On day 14 of high purine feed administration, 9: 00 fasting is not forbidden for 12h, each group of mice takes eyeballs and blood, stands for 2h at room temperature, centrifuges at 3500rpm for 15min, and separates serum. Rapidly separating mouse liver, making a part of the liver into 10% homogenate in ice bath, measuring XOD activity in the liver homogenate, fixing a part of the liver homogenate with 10% paraformaldehyde, and performing HE staining; rapidly separating bilateral kidneys, thymus and spleen, weighing, and calculating organ index; the heart and abdominal aorta were fixed in 10% paraformaldehyde and HE stained.

2.4 statistical analysis

SPSS 23.0 for data analysis, statistical processing, and data measurement

Showing that the comparison among the groups adopts a one-factor analysis of variance (LSD) method with P<A difference of 0.05 is statistically significant.

3 results

3.1 general case

The model group water intake was significantly increased (P) compared to the blank group<0.01); compared with the model group, the peony leaf dry powder (12.5mg kg)^-1) And allopurinol group (5mg kg)^-1) With significant difference (P)<0.01), the rest groups have no statistical difference; the blank group, model group and each administration group had no statistical difference in food intake for 2 weeks. The results are shown in FIGS. 1 and 2.

3.2 major visceral indices

The kidney body index, thymus index and spleen index of the blank group, the model group and each administration group are not statistically different.

3.3 serum uric acid, creatinine, Urea Nitrogen levels in mice

Compared with the model group, the dosages of the peony leaf dry powder (12.5, 25, 50, 100, 200 mg.kg-1) and the allopurinol group (5 mg.kg-1) can obviously reduce the serum uric acid level of mice (P is less than 0.05, and P is less than 0.01). The results are shown in FIG. 3.

The dosages of the peony leaf dry powder were 12.5, 25, 50, 100, 200 mg-kg, respectively, as compared with the model group^-1) And allopurinol group (5mg kg)^-1) All can obviously reduce the serum creatinine level (P) of the mouse<0.05，P<0.01). The results are shown in FIG. 4.

The blank group, the model group and each administration group have no significant difference in serum urea nitrogen content. The results are shown in FIG. 5.

3.4 xanthine oxidase Activity in serum and liver

Compared with the blank group, the serum Xanthine Oxidase (XOD) activity of the model group mice is obviously improved (P)<0.05); peony leaves compared with the model groupDosage of each dry powder (12.5, 25, 50, 100, 200 mg/kg)^-1) And allopurinol group (5mg kg)^-1) All can obviously reduce the activity (P) of the xanthine oxidase in serum of a model mouse<0.01，P<0.05). The results are shown in FIG. 6.

3.5 serum Total Cholesterol, triglyceride, high Density lipoprotein, Low Density lipoprotein levels

Although the serum total cholesterol and triglyceride contents of the blank group, the model group and each administration group have no statistical difference, the peony leaf dry powder has the tendency of reducing in each dosage group. The results are shown in FIGS. 7 and 8.

Allopurinol (5mg kg) compared to the model group^-1) Has a significantly reduced high density lipoprotein content (P)<0.05). Compared with the model group, the peony leaf dry powder (25mg kg)^-1) Has a significantly reduced low density lipoprotein content (P)<0.05). And (6) concluding.

The research result shows that the Xanthine Oxidase (XOD) activity in the serum of a mouse model induced by yeast and adenine is obviously improved, the Xanthine Oxidase (XOD) activity in the serum of each dosage group of the peony leaf dry powder is obviously reduced, and the result is superior to that of a positive drug allopurinol, which indicates that the peony leaf dry powder can be used as a Xanthine Oxidase (XOD) inhibitor.

EXAMPLE 4 acute toxicity test of the inventive (example 1-prepared) peony leaf dry powder

Preparing dry powder of folium Salviae Miltiorrhizae into 30.0% water suspension according to Maximum Tolerated Dose (MTD) determination method, and administering twice per day (total volume of 2 × 40ml/kg) to 40 mice (male and female half), with total dose of 24 g/kg; intragastrically administering 40.0% dry powder aqueous suspension of Danye to 40 rats (female and male half parts) twice a day (total volume of 2 × 20ml/kg), with a total dose of 16 g/kg; all observed for 2 weeks with no animal death. The experimental result shows that the toxicity of the medicine is very low, and the LD of the medicine cannot be measured when the medicine is used for intragastric administration of mice and rats₅₀. And (4) conclusion: the MTD of the peony leaf dry powder to mice and rats is 24g/kg and 16g/kg respectively; the dosage is 4000 and 2667 times the dosage (6.0mg/kg) which is planned to be recommended by clinical adults. Has far lower toxicity than the current clinically used drugs, is expected to be developed as xanthine oxidase inhibitor and is used for preventing and/or treating related diseases。

Example 5

(1) Adding deionized water (1.0L) into dried peony leaf shaft and petiole (100g), heating to reflux temperature, extracting for 1 hr, filtering, and collecting filtrate and residue;

(2) adding deionized water (0.8L) into the filter residue collected in the step (1), heating to reflux temperature, extracting for 40 minutes, filtering, and collecting filtrate;

(3) and (3) merging the filtrates collected in the steps (1) and (2), concentrating the filtrates into thick paste with the relative density of 1.10-1.15, performing spray drying to obtain dry powder, and sieving the dry powder with a 100-mesh sieve to obtain the dry powder (2.12g) of the leaf axis and the leaf stalk.

The dried peony leaf stalks and petioles in this example were obtained by drying the leaf stalks and petioles removed in example 1 in the shade to a constant weight.

The effects of the dry powders of the leaf axes and the leaf stalks on the Xanthine Oxidase (XOD) activity of the model mice were examined according to the method described in example 3, and as a result, it was found that the respective dosages of the dry powders of the leaf axes and the leaf stalks (12.5, 25, 50, 100, 200 mg. kg. multidot.^-1) Has no inhibitory effect on the activity of serum xanthine oxidase of a model mouse. Namely, the dry powder of the leaf axis and the leaf stalk does not have the function of the peony leaf dry powder for inhibiting the activity of Xanthine Oxidase (XOD).

Claims

1. The application of peony leaf dry powder in preparing a medicine for treating diseases related to hyperuricemia accompanied with abnormal rise of serum creatinine level comprises the following steps:

2. The use according to claim 1, wherein the dried peony leaf in step (1) is a peony leaf which is dried in the shade to a constant weight after removing the leaf axis and the leaf stalk, and the appropriate amount of water is 10 times the mass of the dried peony leaf.

3. The use according to claim 1, wherein the amount of water used in step (2) is 8 times the mass of the dried peony leaves.

4. Use according to any one of claims 2 to 3, characterized in that the water is selected from deionized or distilled water.