CN109748981B

CN109748981B - Alkali extraction method of pachyman and application thereof

Info

Publication number: CN109748981B
Application number: CN201810309596.1A
Authority: CN
Inventors: 刘宏伟; 孙珊珊; 宝丽; 汪锴; 韩俊杰
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2017-11-02
Filing date: 2018-04-08
Publication date: 2021-02-02
Anticipated expiration: 2038-04-08
Also published as: CN109748981A

Abstract

The present invention belongs to the field of polysaccharide preparation and its application. The application specifically discloses an alkali extraction method of pachyman, which comprises the following steps: (1) pulverizing Poria sclerotium, adding 3-10 times of anhydrous alcohol, reflux extracting, and collecting alcohol extraction residue; (2) adding distilled water with the weight 3-10 times of the filter residue into the filter residue, performing reflux extraction, and collecting water extraction filter residue; (3) adding the water extraction filter residue into NaOH aqueous solution which is 10-60 times of the weight of the water extraction filter residue and is 0.75mol/L-2mol/L of the water extraction filter residue, leaching at room temperature, and collecting alkali extraction filtrate; (4) adding HCl into the alkali extraction filtrate to neutralize until the pH value is 6-7, and obtaining a precipitate. It can be used for treating and preventing metabolic syndrome, hyperglycemia, hyperlipemia, non-alcoholic fatty liver, weight loss, regulating intestinal flora, and health food.

Description

Alkali extraction method of pachyman and application thereof

Technical Field

The invention relates to the technical field of polysaccharide extraction, in particular to the technical field of alkali extraction of pachyman.

Background

Metabolic Syndrome (MS) is a clinical condition in which various metabolic abnormalities including obesity, insulin resistance, dyslipidemia, impaired glucose tolerance, hypertension, and the like occur in the same individual. The incidence of metabolic syndrome is rising year by year in recent years, and the international diabetes alliance estimates that 1/4 people are about to suffer from metabolic syndrome in the world in 2005, and recent survey results show that the incidence of metabolic syndrome patients in China is increased from 13.8% to 33.9% in 2000, and the number of the sick people reaches 4.54 hundred million, which is mainly related to the rapid development of urbanization, industrialization, population aging and economy. The metabolic syndrome is a high risk factor of chronic diseases such as type 2 diabetes mellitus, cardiovascular and cerebrovascular diseases and the like, and the chronic diseases seriously damage the life and health of human beings and cause great psychological and economic burden on patients and families thereof. Therefore, the high incidence of metabolic syndrome has attracted the attention of the world health organization, and its prevention and treatment are very slow.

Poria cocos is a dried sclerotium of Wolf (Schw.) Wolf of Poria cocos (a fungus of Polyporaceae), is a traditional Chinese medicinal material commonly used in China, is sweet, light and neutral in nature, enters spleen, stomach and kidney meridians, has the effects of promoting water and eliminating dampness, and benefiting spleen and calming heart. Can be used for treating qi deficiency, fatigue, edema, phlegm and fluid retention, emesis, diarrhea, heat stranguria, spermatorrhea, palpitation, and amnesia. The famous medical science Zhang Zhongjing of the Han Dynasty recorded in the book of choice of golden plaque & Youhao (national treatise on the study of Yokou province) created forty recipes such as "Poria cocos Sini Tang", "Wuling san", etc.; the book of famous medical prescription is compiled with more than two hundred of traditional Chinese medicine prescriptions since Han and Tang, wherein one fifth of the traditional Chinese medicine prescriptions contains Poria. Statistics show that: in 158 prescriptions of traditional Chinese medicine, 80% of tuckahoe exists. Pachyman and triterpene are the major active ingredients.

Disclosure of Invention

The invention aims to provide a pachyman alkali extraction method and application thereof in treatment and prevention of metabolic syndrome.

In order to achieve the purpose, the invention adopts the following technical scheme:

(1) pulverizing Poria sclerotium, adding 3-10 times of anhydrous ethanol, reflux extracting for 1-4 times (each time for 1-4 hr), and collecting residue;

(2) drying the filter residue after ethanol extraction, adding distilled water according to 3-10 times of the weight of the filter residue, refluxing and extracting for 1-4 times, wherein each time is 1-4 hours, and collecting the filter residue.

(3) Drying the filter residue after water extraction, adding 0.75-2 mol/L NaOH aqueous solution according to 10-60 times of the weight of the filter residue, leaching for 1-4 hours at room temperature, and collecting the filtrate.

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, adding 3-5 times of distilled water into the precipitate, washing for more than 3 times to remove water-soluble impurities, and freeze-drying the obtained precipitate.

(5) Detecting the obtained pachyman with water2659-2424 HPLC (detector: evaporative light scattering, column: TSK G5000 PW)_XL)。

(6) The pachyman obtained by the method can be used for preparing medicines or foods for treating or preventing metabolic syndrome, especially medicines or foods for reducing blood sugar and losing weight.

From the results of the current animal experiments, the pachyman can effectively reduce the content of blood sugar and glycosylated hemoglobin in blood serum, so the pachyman can improve hyperglycemia and has good long-term control effect on the blood sugar. Simultaneously, can remarkably reduce the levels of total cholesterol, triglyceride and low-density lipoprotein in blood and liver, and has regulating effect on lipid metabolism. In addition, the pachyman can remarkably reduce the content of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in blood, and has liver protecting effect. The results of the test on the inflammatory factor TNF-alpha in serum revealed that this polysaccharide can reduce the level of inflammation. Thus, the pachyman can improve various symptoms of metabolic syndrome, including lowering blood sugar, improving lipid metabolism and liver function, reducing inflammation level, etc.

Drawings

FIG. 1 is a HPLC result chart for pachyman purity detection.

FIG. 2 is a chart showing HPLC results of hydrolysis and derivatization of pachyman.

FIG. 3 shows the ultraviolet spectrum of pachyman.

FIG. 4 is an infrared spectrum of pachyman.

FIG. 5 pachyman¹³And C, a spectrogram.

FIG. 6 is a view showing the results of slicing adipose tissues.

FIG. 7 is a diagram showing the results of liver tissue section.

FIG. 8 is a graph of intestinal flora after administration of pachyman.

FIG. 9 Enterobacter and abundance reduction plots.

FIG. 10 Enterobacter and abundance reduction plots.

Detailed Description

The method and its application are further illustrated by reference to specific examples. It will be appreciated by those skilled in the art that these descriptions are provided for a better understanding of the present invention and that the scope of the present invention is not limited thereto.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1

Extracting pachyman:

(1) weighing 500g of poria cocos sclerotia, crushing, adding 3.5L of absolute ethyl alcohol, performing reflux extraction for 3 times, each time for 2 hours, performing suction filtration, and collecting filter residues;

(2) drying the filter residue after ethanol extraction, adding 1L distilled water into 200g of the filter residue, refluxing and extracting for 2 times, wherein each time is 2 hours, performing suction filtration, and collecting the filter residue.

(3) Drying the filter residue after water extraction, adding 5g of the filter residue into 100mL of 0.75mol/L NaOH aqueous solution for leaching at room temperature for 3 hours, filtering, and collecting the filtrate.

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, washing the precipitate with 3-5 times of distilled water for more than 3 times to remove water-soluble impurities, and freeze-drying the obtained precipitate to obtain pachyman 3.45 g.

Example 2

And (3) detecting the purity and identifying the structure of pachyman:

and (3) purity detection: GPC measurement of pachyman obtained in example 1 was carried out by means of water2659-2424 high performance liquid chromatography (detector: evaporative light scattering detector, column: TSK G5000PW_XL7.8mm × 30cm,10 μm, mobile phase: water, flow rate: 0.4mL/min, column temperature: 30 ℃, drift tube temperature: 80 ℃, nebulizer mode: heating (80% power, 48 ℃), carrier gas: n is a radical of₂And sample introduction volume: 10 μ L). The results are shown in FIG. 1 as a single, symmetrical peak, demonstrating that the polysaccharide is a homogeneous component.

Determination of molecular weight: the molecular weight of the pachyman is measured by using a SEC-RI-DAWN EOS eighteen-angle laser scattering combined system.

The measurement conditions were as follows: a chromatographic column: SHODEX SB-807 HQ; column temperature: 25 ℃; flow rate: 0.75 mL/min; sample introduction volume: 10 mu L of the solution; concentration of sample solution: 4 mg/mL; the results are shown in the following table:

monosaccharide composition:

(1) 10mg of pachyman obtained in example 1 was put in an ampoule bottle, 2mL of 2N HCl was added, the mixture was sealed, hydrolyzed at 110 ℃ for 180min, cooled, washed repeatedly with methanol, and dissolved in 1mL of distilled water for further use.

(2) Detecting the pachyman hydrolysate and monosaccharide standard with water2659-2424 high performance liquid chromatograph (detector: evaporative light scattering detector, chromatographic column: Inertsil NH)₂4.6X 250mm 5 μm, mobile phase: water, flow rate: 0.6mL/min, column temperature: 30 ℃, drift tube temperature: 80 ℃, nebulizer mode: heating (80% power, 48 ℃), carrier gas: n is a radical of₂And sample introduction volume: 10 μ L). The detection results are shown in FIG. 2, wherein the hydrolysate is completely overlapped with the D-glucose standard, which indicates that the tuckahoe homogeneous polysaccharide is composed of D-glucose.

Ultraviolet, infrared and¹³the C spectrum information is shown in fig. 3-5.

Ultraviolet spectrum: the pachyman obtained in example 1 was prepared into a 2mg/ml solution with 0.1mol/L NaOH, and scanned at a wavelength of 190-400nm, as shown in FIG. 3, no absorption occurred at 260nm and 280nm, indicating that no impurities such as proteins, nucleic acids, etc. were contained.

Infrared spectrum: weighing 5mg of pachyman obtained in example 1, mixing with dried KBr powder, grinding in a mortar for 5 minutes, tabletting on a tabletting machine, and measuring by 4000-500 cm on an infrared spectrometer^-1IR of (c) is used. The results are shown in FIG. 4, 3395cm^-1: a hydroxyl group; 2914cm^-1: C-H stretching vibration; 890cm^-1: a beta-glycosidic bond.

¹³Spectrum C: 5mg of pachyman obtained in example 1 was dissolved in DMSO-d₆The C spectrum was measured, and as a result, as shown in FIG. 5, it was consistent with the C spectrum of monosaccharide, and it was presumed that the C spectrum was a homopolysaccharide containing one kind of monosaccharide. 103.1ppm is beta-heterohead C-1.

Example 3

Extracting pachyman:

(3) Drying the filter residue after water extraction, adding 5g of the filter residue into 100mL of 0.5mol/L NaOH aqueous solution for leaching at room temperature for 3 hours, filtering, and collecting the filtrate.

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, washing the precipitate with 3-5 times of distilled water for more than 3 times to remove water-soluble impurities, and freeze-drying the obtained precipitate to obtain pachymaran 0.65 g.

Example 4

Extracting pachyman:

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, washing the precipitate with 3-5 times of distilled water for more than 3 times to remove water-soluble impurities, and freeze-drying the obtained precipitate to obtain pachyman 3.33 g.

Example 5

Extracting pachyman:

(1) weighing 500g of poria cocos sclerotia, crushing, adding 1.5L of absolute ethyl alcohol, performing reflux extraction for 4 times, each time for 4 hours, performing suction filtration, and collecting filter residues;

(2) drying the filter residue after ethanol extraction, adding 2L distilled water into 200g of the filter residue, refluxing and extracting for 4 times, wherein each time is 4 hours, performing suction filtration, and collecting the filter residue.

(3) Drying the filter residue after water extraction, adding 5g of the filter residue into 300mL of 0.8mol/L NaOH aqueous solution for leaching at room temperature for 4 hours, filtering, and collecting the filtrate.

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, washing the precipitate with 3-5 times of distilled water for more than 3 times to remove water-soluble impurities, and freeze drying the obtained precipitate to obtain pachyman 3.2 g.

Example 6

Extracting pachyman:

(1) weighing 500g of poria cocos sclerotia, crushing, adding 5L of absolute ethyl alcohol, performing reflux extraction for 3 times, each time for 3 hours, performing suction filtration, and collecting filter residues;

(2) drying the filter residue after ethanol extraction, adding 600mL of distilled water into 200g of the filter residue, refluxing and extracting for 3 times, wherein each time is 3 hours, performing suction filtration, and collecting the filter residue.

(3) Drying the filter residue after water extraction, adding 5g of the filter residue into 200mL of 2mol/L NaOH aqueous solution for leaching at room temperature for 2 hours, carrying out suction filtration, and collecting the filtrate.

(4) Adding HCl into the filtrate to neutralize to pH 6-7, vacuum filtering to obtain precipitate, washing the precipitate with 3-5 times of distilled water for more than 3 times to remove water-soluble impurities, and freeze drying the obtained precipitate to obtain pachyman 3.76 g.

Example 7

Pharmacological experiments are carried out on the pachyman extract prepared in the example 1, and the result analysis proves that the pachyman extract prepared by the method can be applied to preparing health-care products or medicines for preventing and treating metabolic syndrome, hyperglycemia and hyperlipidemia and losing weight.

(1) Example 1 Effect of pachyman on blood glucose and body weight in ob/ob mice:

ob/ob mice (7weeks, plus, SPF grade) were subjected to fasting blood glucose and body weight determination, and divided into 4 groups on the basis of average blood glucose and body weight, 10 mice per group were used as model group and administration group, respectively, and 10 ICR mice (7weeks, plus, SPF grade) were used as control group. The grouping situation is as follows: (1) blank control group: ICR mice (given the same dose of distilled water as the administration group); (2) model group: ob/ob mice (given the same dose of distilled water as the administration group); (3) pachyman high dose group: ob/ob mice (1 g/kg); (4) pachyman low dose group: ob/ob mice (500 mg/kg); (5) inulin (positive drug) group: ob/ob mice (5 g/kg). The administration was continued for 5 weeks.

After 35 days of administration, animals were fasted for 12 hours, sacrificed, blood was collected from the heart, and organs such as liver and kidney were collected on ice.

Measurement indexes are as follows:

1) effect of dosing on blood glucose and body weight: blood glucose values were measured from the tip of the tail on the day of administration (day 0), 7, 14, 21 and 28 days, and body weight and food intake were weighed (see tables 1-3).

2) Glucose Tolerance Test (OGTT): glucose tolerance was measured on day 30 of administration: animals were fasted for 12 hours and blood (0 hours) was taken, glucose (2.0g/kg) was orally administered, blood glucose values were measured by taking blood from the tail tips at 30, 60, and 120 minutes, respectively, and the curves of blood glucose with time were plotted and the area under the curve was calculated. (see Table 4).

3) Insulin Tolerance Test (ITT): on day 27 of dosing, animals were fasted and bled 2 hours after dosing (0), injected subcutaneously with insulin (0.4IU/kg b.w.,0.1ml/20g B.W.), blood glucose was measured at 40 and 90 minutes after insulin injection, respectively, and blood glucose was plotted against time and the area under the curve was calculated. (Table 5)

4) After the last dose, the drug was sacrificed under anesthesia, blood was taken, centrifuged at 3000rpm at 4 ℃ and the glycated hemoglobin content in serum and the insulin content in serum were measured (see Table 6).

5) After the last dose, the drug was sacrificed under anesthesia, blood was collected, centrifuged at 3000rpm at 4 ℃ and serum Total Cholesterol (TC), low density lipoprotein (LDL-C), Triglyceride (TG), alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) were measured (see Table 7).

6)4) after the last administration, anesthesia was sacrificed, blood was taken, centrifuged at 3000rpm at 4 deg.C, and serum TNF-. alpha.content was determined (see Table 8).

7) After the last dose, fat (fig. 6) and liver (fig. 7) were removed and fixed in formalin and tissue sections were visualized (fig. 6-7).

The results of the experiments show that the compounds of formulae S9 and S24 have a significant effect on blood glucose in hyperglycemic mice and a significant reduction in glucose-loaded mice (tables 14 and 15).

TABLE 1 influence of Pachymaran on food intake in ob/ob diabetic obese mice

Grouping	Total food intake (g)	Spleen/body weight ratio	Kidney/body weight ratio
				(1)	108.10	0.59****	1.32****
(2)	121.172	0.22	0.66
				(3)	123.59	0.32**	0.72*
(4)	120.72	0.33*	0.70
				(5)	121.69	0.27*	0.63

P <0.05, P <0.01, compared to model group

TABLE 2 influence of pachyman on body weight of ob/ob diabetic obese mice

P <0.05, P <0.01, compared to model group

TABLE 3 influence of pachyman on blood glucose in ob/ob diabetic obese mice (fasting blood glucose)

P <0.05, P <0.01, compared to model group

TABLE 4 influence of pachymaran on glucose tolerance in ob/ob diabetic obese mice

P <0.05, P <0.01, compared to model group

TABLE 5 influence of pachymaran on insulin tolerance in ob/ob diabetic obese mice

P <0.05, P <0.01, compared to model group

TABLE 6 influence of pachyman on the insulin sensitivity index and glycated hemoglobin of ob/ob diabetic obese mice

Grouping	Insulin sensitivity index	HbA1c/Hb
			(1)	0.19**	0.98*
(2)	0.13	1.12
			(3)	0.21**	0.92**
(4)	0.19*	0.94*
			(5)	0.19**	0.91**

P <0.05, P <0.01, compared to model group

TABLE 7 influence of pachyman on blood lipid and liver function in ob/ob diabetic mice

P <0.05, P <0.01, compared to model group

TABLE 8 influence of pachymaran on TNF- α content in serum of ob/ob diabetic obese mice

Grouping	TNF-alpha content (ng/L) in serum
		(1)	401.35*
(2)	530.86
		(3)	443.34*
(4)	475.07
		(5)	470.13

As can be seen from the experimental results, the content of TNF-alpha in the serum of the pachyman high-dose group is significantly reduced compared with that of the model group.

Compared with a control group and a model group, the pachymaran can obviously improve the blood fat condition of a mouse, has good reversal effect on serum ALT and AST of the mouse, and prompts that liver damage caused by hyperlipidemia can be protected. The vacuole of fat in the liver cells of the mice in the pachymaran group becomes small, which suggests that pachymaran can effectively treat or relieve the non-alcoholic fatty liver disease complicated by diabetes.

(2) Example 1 Effect of pachyman on the intestinal flora of ob/ob mice:

1) determination of intestinal content 16sDNA

Extraction of microbial DNA reference is made to the instructions of the kit for extraction of DNA from intestinal contents. Total DNA quality control was then performed using Thermo NanoDrop 2000 ultraviolet microspectrophotometer and 1% agar gel electrophoresis. Then, the diluted genome DNA is used as a template, and KAPA HiFi Hotstart ReadyMix PCR kit high fidelity enzyme is used for PCR, so that the amplification accuracy and high efficiency are ensured. The PCR product was detected by 2% agarose gel electrophoresis and recovered by cutting with AxyPrep DNA gel recovery kit. After recovery, library quality testing was performed using a Thermo NanoDrop 2000 ultraviolet microspectrophotometer and 2% agarose gel electrophoresis. And after the quality of the library is qualified, quantifying the library by using the Qubit, and mixing according to the corresponding proportion according to the data volume requirement of each sample. The V3-V4 region of the intestinal flora 16s rDNA was subjected to differential sequencing using Illumina HiSeq PE 250. And finally, performing bioinformatics analysis on the sequencing result.

TABLE 9 alteration of the intestinal flora of ob/ob diabetic obese mice by pachyman

Belong to	Model set (Mod)	Pachyman high dose group (PCP-H)
			Bacteroides (Bacteroides)	10.92	14.90
Bacillus (Barnesiella)	16.39	11.27
			Lactobacillus (Lactobacillus)	10.19	6.57
Prevotella (Alloprovella)	2.06	5.84*
			Bacteroides paraguatus (Parabacteroides)	1.68	3.81*
Clostridium (Clostridium IV)	1.33	3.29
			Muspirillum (Lachnospiraceae)	0.13	2.88*
Saccharomycete (Saccharomyces)	1.58	0.71*
			Enterobacter (Enterobacter)	1.30	0.81
Proteobacteria (Proteus)	1.21	0.01
			Ruminococcus (Ruminococcus)	0.56	1.02*

P <0.05 in comparison with model group

The results of the PCoA analysis showed that there was a significant difference in the intestinal flora after administration of pachyman compared to the model group (FIG. 8). Sequencing results show that bacteria with 7 phyla exist, wherein Bacteroidetes (Bacteroidetes) and Firmicutes (Firmicutes) are absolute dominant bacteria in intestinal flora, and the abundance of Firmicutes is increased and decreased in obesity and diabetes states in the literature; the abundance of the model groups Bacteroides (Bacteroides) and Firmicutes (Firmicutes) was 58.06% and 28.17%, respectively, and the abundance of the pachyman groups Bacteroides (Bacteroides) and Firmicutes was 64.34% and 23.38%, respectively.

At the genus level, increased abundance of lachnospira (lachnospiraceae), Ruminococcus (Ruminococcus), Bacteroides (Bacteroides), prevotella (Alloprevotella), Parabacteroides (Parabacteroides) and Clostridium (Clostridium IV) after administration of pachyman in model mice; bacillus (Barnesiella), Lactobacillus (Lactobacillus), Saccharobacterium (Saccharomyces), Enterobacter (Enterobacter) and (Proteus) decreased abundance (FIGS. 9-10 and Table 9). Wherein the genera Lachnospira (Lachnospiraceae), Ruminococcus (Ruminococcus) and Prevotella (Alloprovellula) which are significantly increased after administration of Poria are short chain fatty acid producing bacteria, while the significantly decreased genera Barnesiella and Enterobacter (Enterobacter) are associated with obesity, insulin resistance development.

2) Detection of Short Chain Fatty Acids (SCFAs)

30mg of mouse feces after 28 days of administration is taken, ground, placed in a 1.5mL centrifuge tube, added with 1mL methanol solution, ultrasonically treated for 15 minutes, centrifuged for 5min (5000r/min), and the supernatant solution is taken for GC-MS analysis.

GC-MS chromatographic conditions:

a chromatographic column: Rtx-Wax-60m

Temperature programming: keeping at 60 deg.C for 0 min; raising the temperature to 100 ℃ at a speed of 5 ℃/min, and keeping the temperature for 1 min; heating to 150 deg.C at 5 deg.C/min, and maintaining for 5 min; raising the temperature to 225 ℃ at a speed of 30 ℃/min, and keeping the temperature for 20 min.

Carrier gas: he; flow rate: 1 mL/min; sample introduction volume: 2 mu L of the solution; no flow split.

Mass spectrum conditions: EI source: mass scan range: 50-800Da, scanning time 10/s; the temperatures of the sample inlet, the transmission line and the ion source are respectively as follows: 250 ℃, 250 ℃ and 220 ℃.

The results are shown in the following table:

TABLE 10 Effect of pachymaran on SCFAs in the feces of ob/ob diabetic obese mice

	Acetic acid	Propionic acid	Butyric acid
				Model set (Mod)	1	1	1
Pachyman high dose group (PCP-H)	0.91	0.98	2.11*

Model set SCFAs were set to "1" with model set as reference, P <0.05 compared to model set

As can be seen from the experimental results, the content of butyric acid was significantly increased after administration of pachyman.

In conclusion, pachymaran can be used for remarkably regulating the dynamic balance of intestinal flora, increasing the abundance of probiotics in the intestinal tract, reducing the abundance of harmful bacteria and increasing the generation of butyric acid in the intestinal tract. (several studies have shown that there is a close link between SCFAs and glucose homeostasis.some of the beneficial metabolic effects induced by propionate and butyrate are mediated by de novo synthesized glucose from the intestinal epithelium, which is sensed in the portal vein and signaled by the gut-cranial nerve circuit to increase insulin sensitivity and glucose tolerance, etc.)

Example 8 effect of pachyman obtained in example 1 on blood glucose and body weight in DIO mice:

c57BL/6J (4weeks, super-sex, SPF grade) mice were fed on high-fat diet for 4weeks, screened according to body weight, and selected mice were randomly divided into model group and administration group, and C57BL/6J mice (normal diet) were used as control. After 4weeks, mice with weight more than 20% are considered as successful in modeling, fasting blood glucose is measured, and the mice are averagely grouped according to blood glucose value and weight condition, wherein 10 mice are used in each group. The grouping situation is as follows: (1) c57BL/6J (distilled water given at the same dose as the group administered) fed with normal feed in the blank control group; (2) the model group was given the same dose of distilled water as the administration group; (3) the pachyman high dose group is 1 g/kg; (4) the pachyman low dose group is 500 mg/kg; (5) inulin (positive drug) group 5 g/kg. The administration was continued for 5 weeks.

Effects on body weight, food intake and blood glucose: body weight was measured on days 0, 7, 14, 21, 28, and 35 of the administration, and free diet blood glucose and fasting blood glucose (after 4 hours fasting) were measured by blood sampling from the tip of the tail. (tables 9-11)

Glucose Tolerance Test (OGTT): glucose tolerance was measured on day 30 of administration, and animals were given blood (0 hr) after fasting for 12 hours and orally administered with glucose (2.0g/kg), blood glucose values were measured by blood taken from the tail tips at 30, 60, and 120 minutes, respectively, and blood glucose curves with time were plotted to calculate the area under the curve. (watch 12)

Insulin Tolerance Test (ITT): on day 27 of dosing, animals were fasted and bled 2 hours after dosing (0), injected subcutaneously with insulin (0.4IU/kg b.w.,0.1ml/20g B.W.), blood glucose was measured at 40 and 90 minutes after insulin injection, respectively, and blood glucose was plotted against time and the area under the curve was calculated. (watch 13)

TABLE 9 influence of Poria Cocos homogeneous component polysaccharide on food intake in DIO mice

Grouping	Total food intake (g)
		(1)	106.23
(2)	124.56
		(3)	123.47
(4)	125.90
		(5)	122.69

P <0.05, P <0.01, compared to model group

TABLE 10 influence of pachyman on body weight in DIO mice

P <0.05, P <0.01, compared to model group

TABLE 11 influence of pachyman on blood glucose in DIO mice (fasting blood glucose)

P <0.05, P <0.01, compared to model group

TABLE 12 influence of pachymaran on glucose tolerance in DIO mice

P <0.05, P <0.01, compared to model group

TABLE 13 influence of pachymaran on insulin tolerance in DIO mice

P <0.05, P <0.01, compared to model group

From the experimental results, it can be seen that pachyman can significantly reduce the blood glucose and weight gain rate of DIO mice compared to the model group. Similar to the positive drug, but the dose of pachyman is 1/5 of the positive drug inulin.

Example 9 the effect of pachyman obtained in example 1 on alcoholic liver injury mice:

c57BL/6J (9weeks, plus SPF grade) mice were randomly divided into three groups of 12 mice each, and the grouping was as follows: (1) normal control group Lieber-DeCarli control liquid feed (same dose of distilled water as administered group); (2) the model group was fed with Lieber-DeCarli alcohol liquid diet (given the same dose of distilled water as the administration group); (3) pachyman low dose group Lieber-DeCarli alcohol liquid feed (1g/kg pachyman).

Body weight was weighed every three days and changes in body weight were recorded. After the fifth week of administration (day 35), the night when the molding was completed (last feed) was fed 3 times at intervals of 1/3 feeding amounts every 8 hours in accordance with the intake of the alcohol liquid feed for the first 24 hours.

(1) Anesthesia was sacrificed and blood was collected, centrifuged at 4 ℃ and 3000rpm, and AST and ALT were measured in the blood (Table 14).

(2) Liver tissue was collected and 10% liver homogenate was prepared. The contents of SOD, malondialdehyde, TG and TC in the liver were measured (tables 15-16).

TABLE 14 inhibitory Effect of Pachymaran on alcohol-induced AST and ALT elevation

Grouping	AST content in serum (IU/L)	ALT content (IU/L) in serum
			(1)	18.48**	10.17*
(2)	22.42	17.94
			(3)	18.41**	9.84*

P <0.05, P <0.01, compared to model group

TABLE 15 inhibitory Effect of Pachymaran on alcohol-induced increase in TG and TC

Grouping	TG content in liver (mg/g)	TC content in liver (mg/g)
			(1)	22.52*	0.42*
(2)	30.67	0.67
			(3)	18.62**	0.38*

P <0.05, P <0.01, compared to model group

TABLE 16 influence of pachyman on alcohol-induced oxidative stress

Grouping	SOD content in liver (U/mgprot)	Malondialdehyde content in liver (mmol/mgprot)
			(1)	320.77***	3.86**
(2)	282.88	4.65
			(3)	317.79***	3.37*

Comparing to model group, P <0.05, P <0.01, P <0.001

From the experimental results, compared with the model group, pachymaran can obviously inhibit the increase of AST and ALT in the blood of mice induced by alcohol, improve the lipid metabolism abnormality induced by alcohol, reduce the contents of Triglyceride (TG) and Total Cholesterol (TC) in the liver, improve the oxidative stress of the liver induced by alcohol, increase the content of superoxide dismutase (SOD) in the liver and reduce the content of malondialdehyde. In conclusion, pachymaran prevents and treats alcohol-induced liver injury in mice.

Claims

1. The application of pachyman in preparing medicine for regulating intestinal flora is characterized by that said pachyman is extracted by adopting alkali extraction method.

2. The use according to claim 1, the alkaline extraction process comprising the steps of:

(1) pulverizing Poria sclerotium, adding 3-10 times of anhydrous alcohol, reflux extracting, and collecting alcohol extraction residue;

(2) adding distilled water with the weight 3-10 times of the filter residue into the filter residue, performing reflux extraction, and collecting water extraction filter residue;

(3) adding the water extraction filter residue into NaOH aqueous solution which is 10-60 times of the weight of the water extraction filter residue and is 0.75mol/L-2mol/L of the water extraction filter residue, leaching at room temperature, and collecting alkali extraction filtrate;

(4) adding HCl into the alkali extraction filtrate to neutralize until the pH value is 6-7, and obtaining a precipitate.