CN115919934B

CN115919934B - Peony active substance with effect of inhibiting prostatic hyperplasia, extraction method and application

Info

Publication number: CN115919934B
Application number: CN202211595252.4A
Authority: CN
Inventors: 陶福占; 庞志勇; 孙振亚; 房盟盟; 唐悦
Original assignee: Heze Peony Industry Technology Research Institute
Current assignee: Heze Peony Industry Technology Research Institute
Priority date: 2022-12-12
Filing date: 2022-12-12
Publication date: 2023-11-17
Anticipated expiration: 2042-12-12
Also published as: CN115919934A

Abstract

The invention belongs to the technical field of traditional Chinese medicines; in particular to a peony active substance with the effect of inhibiting prostatic hyperplasia, an extraction method and application. The peony active substance comprises the following substances: a, oxidizing paeoniflorin; B-limonin-3-O-beta-D-glucoside; c-coumaroyl spermidine (Tricoumaroyl spermidine); d: paeoniflorin; the effective contents are 30-70 mug/mL, 1-5 mug/mL, 150-350 mug/mL, 80-130 mug/mL respectively; the extraction method of the active substances comprises the following steps: cleaning, draining, adding water, refluxing in constant-temperature water bath, and leaching for 3-6h; filtering, concentrating under reduced pressure, drying to obtain material B, and mixing the residue with Streptococcus thermophilus, bacillus cereus and Bacillus subtilis according to a ratio of 1-3:7-13: fermenting at a ratio of 0.5-2, extracting the fermentation liquor, distilling under reduced pressure to obtain crude extract, and drying to obtain a material A; mixing the material A and the material B, and carrying out ultrasonic grinding. The active substance prepared by the invention can effectively inhibit the SRD5A-II from resisting BPH, and the inhibition rate can reach more than 70 percent; the volume of the prostate is significantly reduced.

Description

Peony active substance with effect of inhibiting prostatic hyperplasia, extraction method and application

Technical Field

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a peony active substance with a prostate hyperplasia inhibiting effect and application thereof.

Background

Prostatic hyperplasia (hyperplasia of prostate) is one of the common diseases of middle-aged and elderly men. The prostatic hyperplasia is a symptom caused by the gradual enlargement of the prostate and the compression effect on the urethra and the outlet of the bladder, and the most common harm to the body is the initiation of various infections, especially the bacterial or germ infection of each organ of the urinary system, the possible complications of urinary tract infection, cystitis and other inflammatory diseases, thus the occurrence of urgent urination, frequent urination, painful urination, difficult urination and other bad symptoms, and even the formation of urinary retention; benign prostatic hyperplasia is quite common, and all men have more or less prostatic hyperplasia, but the ages and the conditions are different. In addition, prostatic hyperplasia can also affect sperm survival and quality, and fertility; causing inflammation of the urethra, damaging the kidneys, etc.;

the current methods for treating prostatic hyperplasia are: surgical treatment, but also surgical excision, but related scholars and researchers believe that excision affects human endocrine. Alternatively, the drug attempts to: (1) Alpha adrenergic receptor blockers (e.g., terazosin, doxazosin, tamsulosin, alfuzosin, or silodosin) can relax certain prostate and bladder neck muscles while potentially improving urine flow; however, alpha-blockers do not reduce the incidence of acute urinary retention, nor do they reduce the volume of the prostate, which may also grow with age; and also has the side effects of postural hypotension, dizziness, headache and the like. (2) an M receptor antagonist that alleviates bladder detrusor spasticity; however, the medicine has relatively more contraindications and relatively smaller application range. Although the traditional Chinese medicine takes effect relatively slowly, the traditional Chinese medicine has the characteristics of multiple components and multiple targets, and can treat complex diseases of human body more comprehensively and comprehensively.

Modern pharmacological researches show that peony pollen has good anti-inflammatory and antioxidant activities, and flavonoid compounds such as quercetin, apigenin and the like have anti-inflammatory and antioxidant pharmacological effects; monoterpene glycoside compounds have remarkable effects on anti-tumor, antioxidation, antidepressant, immunoregulation, blood replenishing and other aspects, and have small toxic and side effects, so that the medicinal value of the monoterpene glycoside compounds is increasingly focused; the peony contains various active substances, and the prior art for treating the prostate by the peony has the problems that the preparation of the composition from the peony pistil or pollen is applied to the treatment of the prostate disease, the specific active substances achieving the treatment effect are not specifically explored, and the pertinence is not strong; the present invention further explores the active substances in peony against benign prostatic hyperplasia.

Disclosure of Invention

In view of the above, the invention provides a peony active substance with the effect of inhibiting prostatic hyperplasia, an extraction method and application thereof. In the invention, peony is taken as a research object, active substances with the effect of resisting benign prostatic hyperplasia in the peony are researched through in vivo animal experiments, and the action mechanism of the active substances for resisting benign prostatic hyperplasia is primarily researched; provides scientific theoretical basis for further researching the prostatic hyperplasia resisting effect of peony pollen and developing safe and effective plant medicaments or health-care products.

The invention provides a peony active substance with a prostate hyperplasia inhibiting effect, which is characterized by comprising the following substances:

a, oxidizing paeoniflorin; B-limonin-3-O-beta-D-glucoside; c-coumaroyl spermidine (Tricoumaroyl spermidine); d: paeoniflorin;

preferably, the effective amounts of the 4 active substances are 30-70. Mu.g/mL, 1-5. Mu.g/mL, 150-350. Mu.g/mL, 80-130. Mu.g/mL, respectively.

Preferably, the effective amounts of the 4 active substances are 30-60. Mu.g/mL, 3-5. Mu.g/mL, 150-350. Mu.g/mL, 90-130. Mu.g/mL, respectively.

Preferably, the effective amounts of the 4 active substances are 35-60. Mu.g/mL, 3-5. Mu.g/mL, 200-350. Mu.g/mL, 100-130. Mu.g/mL, respectively.

Preferably, the effective amounts of the 4 active substances are 40-60. Mu.g/mL, 3-5. Mu.g/mL, 250-350. Mu.g/mL, 105-130. Mu.g/mL, respectively.

The invention also provides an extraction method of the 4 active substances, which comprises the following steps:

(1) Cleaning and draining fresh peony whole flowers, separating the whole flowers, adding water, and carrying out reflux extraction for 3-6h in a constant-temperature water bath at 70-90 ℃; filtering to obtain filtrate A and filter residue A; repeatedly leaching the filter residue A with water for 1-2 times under the same condition to obtain filter residue B and filtrate B, mixing the filtrate A, B, and collecting the filter residue B;

(2) Fermenting the filter residue B to obtain fermentation liquor, extracting the fermentation liquor by using macroporous resin to obtain extracting solution, performing reduced pressure distillation on the extracting solution to obtain crude extract, and drying to obtain a material A;

(3) Concentrating the combined filtrate A, B by reduced pressure distillation, and drying to obtain a material B;

(4) Mixing the material A and the material B, and performing ultrasonic grinding treatment for 30-60s to obtain the dry powder containing the active substances.

Preferably, in the step (1), when the petals are split, the petals are cut to a size of 1.5-2.5 cm, and the water adding condition is that: adding water until the peony material is completely immersed in water.

Preferably, in the step (2), the fermenting agent for fermentation is streptococcus thermophilus, bacillus cereus or bacillus subtilis; wherein, streptococcus thermophilus: bacillus cereus: the adding proportion of the bacillus subtilis is 1-3:7-13:0.5-2.

Preferably, in the step (2), the fermenting agent for fermentation is streptococcus thermophilus, bacillus cereus or bacillus subtilis; wherein, streptococcus thermophilus: bacillus cereus: the adding proportion of the bacillus subtilis is 2-3:7-11:1-2.

Preferably, in the step (2), the fermenting agent for fermentation is streptococcus thermophilus, bacillus cereus or bacillus subtilis; wherein, streptococcus thermophilus: bacillus cereus: the adding proportion of the bacillus subtilis is 2:7-9:1-2.

Preferably, in the step (3), the combined filtrate is concentrated to 1/3 to 1/2 of the original volume.

The peony active substance provided by the invention can be applied to health care products for inhibiting prostatic hyperplasia; further, it can be used in common foods with the effect of inhibiting prostatic hyperplasia. The common food includes, but is not limited to, pasta, dairy products, bean products, drinks and the like.

The peony active substance with the effect of inhibiting the prostatic hyperplasia, provided by the invention, is not added with any organic solvent in the extraction process, and has high safety index; the active substance has other beneficial effects, such as the tricosanthranide can induce cell autophagy, delay aging, and can also relieve alcohol, protect liver and protect liver; the average consumer can also use the product.

The beneficial effects of the invention are as follows:

1. according to the invention, several active substances with specific concentrations are obtained from peony through a specific extraction method, namely 30-60 mug/mL of paeoniflorin oxide, 3-5 mug/mL of limonin-3-O-beta-D-glucoside, 150-350 mug/mL of tricosaurus mongolicus spermidine and 220-390 mug/mL of paeoniflorin, when the effective contents of the 4 active substances are in the range, the inhibition effect on SRD5A-II is better, the SRD5A-II anti-BPH can be effectively inhibited, and the inhibition rate can reach more than 70%; so that the prostate index and the prostate volume are significantly reduced;

2. according to the detection of an experimental mouse, the active ingredient substance dry powder prepared by the fermentation strain can remarkably reduce the levels of SRD5A and E2 in rat serum, so that the generation of DHT is inhibited, and the symptom of prostatic hyperplasia is improved; this also indicates that the selection of the fermentation species is critical in the preparation of peony active;

3. in the preparation process, no organic solvent is added, so that the safety is high; the product has various effects, and the contained tricolorimide can induce autophagy, delay aging, and can also relieve alcohol, protect liver and protect liver; the average consumer can also use the product.

Drawings

FIG. 1 is a general morphology of prostate tissue from each group of rats;

fig. 2 is a histopathological analysis of the prostate of each group of rats.

Detailed Description

The present invention will now be further described in connection with specific embodiments in order to enable those skilled in the art to better understand the invention. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.

1 Experimental materials

1.1, material selection: the peony pollen is prepared from Paeonia ostii produced in the lotus city by weighing, breaking cell wall and pulverizing.

1.2 preparation method of peony active substance

Example 1

(1) Cleaning fresh peony whole flowers, draining, splitting the whole flowers, adding water until peony materials are completely immersed in water, and carrying out reflux extraction for 4 hours in a constant-temperature water bath at 85 ℃, wherein petals are cut to 1.5-2.5 cm in size when splitting; filtering to obtain filtrate A and filter residue A; repeatedly leaching the filter residue A with water for 2 times under the same condition to obtain filter residue B and filtrate B, mixing the filtrate A, B, and collecting the filter residue B;

(2) Filter residue B was treated with streptococcus thermophilus, bacillus cereus, bacillus subtilis at 2:9:1, fermenting to obtain fermentation liquor, extracting the fermentation liquor by using macroporous resin to obtain extract, performing reduced pressure distillation on the extract to obtain crude extract, and drying to obtain a material A;

(3) Concentrating the combined filtrate A, B by reduced pressure distillation to 1/3 of the original volume, and drying to obtain a material B;

(4) Mixing the material A and the material B, and performing ultrasonic grinding treatment for 40s to obtain the composite material.

Comparative example 1

Replacing the fermentation bacteria with lactobacillus plantarum, saccharomycetes and bacillus subtilis; the procedure is as in example 1.

Comparative example 2

Replacing the fermentation bacteria with bacillus subtilis, bacillus anthracis and lactobacillus delbrueckii; the procedure is as in example 1.

1.3 laboratory animals

Clean SD rats, males, weighing 180-220g, purchased from Shanghai national institute of family planning science laboratory animal manager, animal production license number: SCXK 2018-0006.

2 test method

Study of the pharmacodynamic action of active substances on benign prostatic hyperplasia rats

2.1 mold Forming period

Healthy clean-grade SD rats with the weight of 18-22 g are selected, the tail is numbered, the rats are adaptively fed for 4 days in a 12h/12h light-dark alternating environment, normal water and food are fed, the temperature of animal houses is strictly controlled within a range of 15-26 ℃, the relative humidity is kept within 50-90%, the rats are cleaned once every two days, and the mental states of the rats are closely monitored. After the adaptive feeding is finished, the experimental mice are divided into the following groups according to different weights by adopting a random number table method: blank control group (n=8), model group (n=40).

The testosterone propionate induction method is adopted to prepare a rat model of benign prostatic hyperplasia, and the rat model is administrated at the morning every day for 28 days, and the specific administration conditions are as follows:

(1) Blank control group: soybean oil was administered at 1 mL/kg.

(2) Model group: testosterone propionate (5 mg/kg) +soybean oil was administered and the mold was built by 1 ml/kg.

2.2 treatment period

Model group rats were divided into: model group (n=8), prostakang group (n=8), comparative example 1 (n=8), comparative example 2 (n=8), comparative example 3 (n=8). The administration dosage is strictly calculated according to a human and mouse dosage conversion formula, the normal control group is administrated by the gastric lavage of 0.5% CMC-Na with the same volume according to the weight of the normal control group, the administration is carried out by the gastric lavage in the morning every day, the administration is continued for 28 days, and the specific gastric lavage condition is as follows:

(1) Blank control group: the stomach was irrigated at 10mL/kg with 0.5% CMC-Na.

(2) Model group: the stomach was irrigated at 10mL/kg with 0.5% CMC-Na.

(3) Prostakang group: prostakang (1260 mg/kg) +0.5% CMC-Na was administered and the stomach was irrigated at 10 mL/kg.

(4) Comparative example 1: the active-containing dry powder (1260 mg/kg) +0.5% CMC-Na was administered and the stomach was irrigated at 10 mL/kg.

(5) Comparative example 2: the active-containing dry powder (1260 mg/kg) +0.5% CMC-Na was administered and the stomach was irrigated at 10 mL/kg.

(6) Example 1: the active-containing dry powder (1260 mg/kg) +0.5% CMC-Na was administered and the stomach was irrigated at 10 mL/kg.

Note that: CMC-Na is a common drug suspending agent, 1000mL of deionized water is firstly weighed and poured into a beaker when 0.5% CMC-Na solution is prepared, then 5g of CMC-Na powder is weighed and evenly spread on the water surface, the mixture is placed in a 60 ℃ oven for standing overnight (the beaker is covered with paper sheets to prevent dust from falling in the standing process), the mixture is fully swelled, the mixture is heated and stirred for the next day, the mixture is fully dissolved, and the mixture is filtered by clean gauze after being cooled for standby.

2.3 sample collection

The rats were anesthetized with 20% uratam intraperitoneal injection at a dose of 1mL/100g, fasted for 24 h. Taking blood from abdominal aorta, standing at room temperature for 30min to allow natural blood coagulation, centrifuging at 3000rpm for 5 min, separating serum, and storing in a refrigerator at-80deg.C for subsequent detection; after blood collection, the rats are killed by cervical dislocation, the prostate, liver, kidney and spleen of the rats are separated, physiological saline is used for washing, filter paper is used for sucking surface water, the volume of the prostate is measured, wet weights of all tissues are weighed, and the organ indexes of the prostate, liver, kidney and spleen are calculated. Rat left side prostate tissue was fixed with 10% paraformaldehyde and right side prostate tissue was stored in-80 ℃ freezer for use.

2.4 measurement of organ index

After the prostate, liver, kidney and spleen of the rat are separated and washed by normal saline, the surface water is sucked by filter paper, the volume of the prostate is measured, the wet weight of each tissue is weighed, and the index of each organ of the prostate, liver, kidney and spleen is calculated.

Organ index = wet tissue mass (g)/bulk mass (g) ×100%

2.5 HE staining of rat prostate tissue

After the experiment, the rat was isolated from the prostate, the excess water was removed, weighed and fixed with 4% paraformaldehyde for 24 hours, paraffin embedded after a conventional dehydration operation, cut into 4 μm sections and stained with hematoxylin-eosin (HE), and the pathological changes of the prostate tissue sections were observed under an optical microscope.

2.6 statistical analysis

Statistical analysis was performed by GraphPad Prism 9.0 software and expressed as mean±sd; one-way ANOVA (One-way ANOVA) was performed between groups, with statistical differences being considered as p < 0.05.

Initial detection of benign prostatic hyperplasia resisting mechanism by 3 peony active substance

3.1 detection of hormone content level in rat serum

After the experiment, rats were anesthetized, and the abdominal aorta was bled to collect serum samples. The amounts of AR, SRD5A and E2 in the rat serum were determined by ELISA.

4. Analysis of results

Table 1 rat body weight and prostate related index measurement results (n=6)

Group of	Body weight (g)	Prostate index (%)	Prostate volume (cm) ³ )
				K	346.625±17.50	0.170±0.049	0.613±0.155
M	324.25±38.526	0.250±0.039 ^##	1.167±0.207 ^##
				ZY	343.857±33.128	0.196±0.022 ^*	0.633±0.163 ^**
Comparative example 1	330.750±26.575	0.197±0.028 ^*	0.743±0.079 ^**
				Comparative example 2	347.833±38.732	0.183±0.034 ^**	0.683±0.075 ^**
Example 1	355.125±21.866	0.166±0.020 ^**	0.667±0.082 ^**

Note that: k: blank control group; m: a model group; ZY: a group of prostaglandins;

results are expressed as mean±sd; compared to blank (K), # denotes p < 0.01, # denotes p < 0.05; compared to model group (M), p < 0.01 and p < 0.05.

A general morphology of prostate tissue from each group of rats is shown in figure 1. As can be seen from FIG. 1, the prostate tissue of the blank group was of moderate size, smooth, shiny, pink in color and soft in texture. The prostate of the model group is obviously enlarged, the gland is dark red, the texture is hard slightly, and the surface of the gland is unsmooth. The prostate tissue of the Qianliekang group and example 1 was significantly reduced, pink, tough, and improved in surface smoothness compared to the model group. The volume of the prostate of comparative examples 1 and 2 is slightly reduced, the prostate is pink, and the surface smoothness is better than that of a model group.

Rat prostate histopathological analysis is shown in figure 2. The figure shows that the prostate epithelial cell monolayers of the blank group are closely arranged, the acinar epithelium has fewer folds and the epithelium is smooth; the prostate tissue of the rat in the model group shows obvious hyperplasia, the epithelial cells are arranged in a plurality of layers and protrude into the cavity in a nipple shape, and rough folds are increased; compared with the model group, in the comparative example 1, the gland epithelial cells still have partial increase and protrude into the gland cavity in a nipple shape, partial acinus still shrink, and the gland lumen is mostly restored to be normal; the comparative example 2 is obviously better than the model group, the glandular epithelial cells are arranged into a single layer, papilla is occasionally seen, and the glandular lumen is basically recovered to be normal; compared with the model group, the prostate health group and the example 1 have obviously reduced proliferation phenomenon, the gland epithelial cells are arranged into a single layer, the gland lumen is restored to be normal, and the fibrous tissue has no obvious proliferation.

As is clear from the above table, the experimental mice of example 1 showed a significant decrease in prostate index and prostate volume, while the body weight was maintained at the highest, which could reach 355g; this can indicate that example 1 plays a very significant role in treating the prostate, which is not achieved by comparative examples 1, 2. This also indicates that the selection of the fermentation species is critical in the preparation of peony actives.

Table 2 rat body weight and liver, kidney, spleen index measurement results (n=6)

Group of	Body weight (g)	Liver index (%)	Kidney index (%)	Spleen index (%)
					K	346.625±17.50	2.109±0.100	0.651±0.029	0.158±0.018
M	324.25±38.526	2.521±0.431**	0.753±0.054**	0.165±0.025
					ZY	343.857±33.128	2.091±0.079	0.678±0.035	0.176±0.009
Comparative example 1	330.750±26.575	2.228±0.098	0.703±0.017	0.172±0.026
					Comparative example 2	347.833±38.732	2.352±0.127	0.671±0.011	0.194±0.027*
Example 1	355.125±21.866	2.285±0.118	0.656±0.028	0.177±0.025

As is clear from the above table, the remaining active substances have no significant difference in liver and kidney index from the blank group, except that the comparative example 2 has a significant effect on spleen index.

The following table shows several active substances detected in the resulting dried fractions.

TABLE 3 active substance detection results

The present inventors have further studied the peony active ingredient of example 1 and comparative examples 1 and 2.

TABLE 4 detection of peony active substance content (μg/mL) under different lactic acid bacteria

Project	Comparative example 1	Comparative example 2	Example 1
				Oxidized paeoniflorin	8-25	15-40	30-70
Lemon flavin-3-O-beta-D-glucoside	1-2	1-2	1-5
				Tri-coumaroyl spermidine	220-480	50-135	150-350
Paeoniflorin	25-40	30-55	80-130

TABLE 5 inhibition of SRD5A-II by peony active

Note that: results are expressed as mean±sd; compared to the blank group, p < 0.01 is indicated.

Experimental results show that the effective content of the 4 active substances is within a certain range, so that the SRD5A-II inhibition effect is good, and the SRD5A-II anti-BPH can be effectively and excessively inhibited.

The following table shows the results of the SRD5A, AR and E2 content measurements in rat serum:

TABLE 6 determination of SRD5A, AR and E2 content in serum

Group of	SRD5A(U/mL)	AR(ng/mL)	E2(pmol/L)
				K	93.214±4.594	7.624±0.615	104.343±8.128
M	107.009±4.985 ^##	11.173±0.674 ^##	123.217±3.597 ^##
				ZY	85.658±4.375**	9.565±0.867**	99.818±7.131**
Comparative example 1	81.055±3.892**	10.413±0.793	102.339±5.564**
				Comparative example 2	78.567±5.887**	10.141±1.270	97.299±5.583**
Example 1	75.444±3.711**	9.120±0.798**	95.669±8.923**

From the above table, the levels of SRD5A, AR and E2 were both significantly elevated (p < 0.01) in the model group compared to the blank group, indicating that testosterone propionate-induced benign prostatic hyperplasia models in rats increased SRD5A, AR and E2 levels in the serum of rats.

Comparative example 2 significantly reduced the AR level in the serum of rats (p < 0.05) compared to the model group, and both the prostakang group and example 1 significantly reduced the AR level in the serum of rats (p < 0.01), with little decrease in AR level but no significant difference (p > 0.05) compared to the model group in comparative example 1.

It can be shown that example 1 of the present invention is capable of very significantly reducing the levels of SRD5A and E2 (p < 0.01) in rat serum. Inhibiting DHT production by reducing SRD5A levels in rat serum, and improving prostatic hyperplasia symptoms by reducing AR and E2 levels in serum and regulating in vivo hormone balance.

Claims

1. The peony active substance with the effect of inhibiting the prostatic hyperplasia is characterized by comprising the following substances:

a, oxidizing paeoniflorin; B-limonin-3-O-beta-D-glucoside; c-coumaroyl spermidine (Tricoumaroyl spermidine); d paeoniflorin;

the effective contents of the paeoniflorin oxide, the limonin-3-O-beta-D-glucoside, the tricosamide and the paeoniflorin are respectively 30-70 mug/mL, 1-5 mug/mL, 150-350 mug/mL and 80-130 mug/mL.

2. The peony active substance according to claim 1, wherein the effective contents of paeoniflorin oxide, limonin-3-O-beta-D-glucoside, coumaroyl spermidine and paeoniflorin are 30-60 μg/mL, 3-5 μg/mL, 150-350 μg/mL and 90-130 μg/mL, respectively.

3. The peony active substance as defined in claim 1, wherein the active substance is extracted by the following method:

(2) Fermenting the filter residue B to obtain a fermentation liquor, extracting the fermentation liquor by using macroporous resin to obtain an extracting solution, carrying out reduced pressure distillation on the extracting solution to obtain a crude extract, and drying to obtain a material A, wherein the fermenting agent for fermentation is streptococcus thermophilus, bacillus cereus and bacillus subtilis, and the streptococcus thermophilus is: bacillus cereus: bacillus subtilis = 2:9:1, a step of;

(4) Mixing the material A and the material B, and carrying out ultrasonic grinding treatment for 30-60s.

4. A peony active substance according to claim 3, wherein in step (1), the petals are cut to a size of 1.5-2.5 cm when split, and water is added: adding water until the peony material is completely immersed in water.

5. A peony active substance according to claim 3, wherein in step (3), the combined filtrates are concentrated to 1/3-1/2 of the original volume.