WO2012094833A1 - Petroleum ether extract of traditional chinese medicine for prevention and treatment of sugar and lipid metabolism disorders and preparation method thereof - Google Patents

Abstract

The petroleum ether extract of traditional Chinese medicine for the prevention and treatment of sugar and lipid metabolism disorders, wherein the active ingredients of the extract are composed of 1-cerotol, β-sitosterol, n-hexacosanoic acid, atractylenolide III, oleanolic acid, berberine, jateorrhizine, salvianolic acid B, cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, ginsenoside Rb1. The preparative method comprises extracting crude drug Radix Salviae Miltiorrhizae, Fructus Ligustri Lucidi, Rhizoma Coptidis, Herba Cirsii Japonici, Cortex Eucommiae, Rhizoma Atractylodis Macrocephalae, Radix Notoginseng and Fructus Citri Sarcodactylis with C_1-3 alcohol and/or water, combining total extracts, extracting the total extracts with petroleum ether to obtain the petroleum ether extract.

Description

 Traditional Chinese medicine petroleum ether extract for controlling glycolipid metabolism disorder and preparation method thereof

Technical field

The invention relates to the field of traditional Chinese medicine, in particular to a petroleum ether extract of traditional Chinese medicine for preventing and controlling diseases related to disorders of glycolipid metabolism (hyperlipidemia, diabetes, metabolic syndrome, animal atherosclerosis and the like) and a preparation method thereof.

Background technique

Chinese Patent No. 200410051250.4 discloses a drug for treating hyperlipidemia, which is named "Fufang Qishu Tiaozhi Fang" (FTZ), which contains Ligustrum lucidum, Daphnia, Atractylodes, Salvia, Eucommia, bergamot, and can also join Sanqi. And Huanglian. The preferred weight percentage of each component is 20 to 25% of Ligustrum lucidum, 12 to 15% of Daphnia, 10 to 15% of Atractylodes, 10 to 15% of Salvia miltiorrhiza, 10 to 15% of Eucommia, 10 to 12% of bergamot, and 10 to 10 of bergamot. ~12%, berberine 6 to 10%. The prescription is used to treat hyperlipidemia and achieve ideal therapeutic effect. The total clinical effective rate is 91%.

Although Fufang Shuzhu Tiaozhi Recipe has good blood lipid regulation and anti-atherosclerotic function and clinical curative effect, but it is a compound preparation of traditional Chinese medicine, there are still many shortcomings: (1) its composition is more complicated, its active ingredients are not clear enough, It is not easy to quantify. In actual application, the quality of its products is not easy to obtain effective control, and ultimately it will affect the stability of clinical efficacy of the product; (2) The preparation process is relatively simple, the active ingredients in the prescription are not purified as expected, and the impurity components are large. Large (about 5 grams per day), patient compliance is not ideal; (3) due to the large amount of prescription extract, it is inconvenient to make various controlled release dosage forms, the preparation of pharmaceutical preparations can not be diversified, the application range is subject to various restrictions.

Summary of the invention

The object of the present invention is to overcome the above-mentioned deficiencies of the prior art, and to provide a traditional Chinese medicine petroleum ether extract which is effective in curative effect, easy to quantify the active ingredient, easy to control in quality, and small in dosage to prevent glycolipid metabolism disorder.

Another object of the present invention is to provide a process for the preparation of the above petroleum ether extract.

The present invention achieves the above objects by the following technical solutions:

A petroleum ether extract of traditional Chinese medicine for preventing and controlling disorders of glycolipid metabolism, the active ingredients thereof are wax alcohol, β-sitosterol, n-hexadecanoic acid, atractylenolide III, oleanolic acid, berberine, jatrorrhizine , salvianolic acid B, Cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, ginsenoside Rb1.

The above-mentioned active ingredients laurol, β-sitosterol, n-hexadecanoic acid, atractylenolide III, oleanolic acid, berberine, jatrorrhizine, salvianolic acid B, The weight ratio of cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, and ginsenoside Rb1 is from 1 to 5:1 to 8:1 to 8:1. 5: 1~8: 1 to 8:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5:1 to 6.

The preparation method of the above-mentioned traditional Chinese medicine petroleum ether extract adopts the formula of "Fufang Qishu Tiaozhi Fang" (FTZ) for treating hyperlipidemia disclosed in Patent No. 200410051250.4, and each component is expressed by weight percentage: Ligustrum lucidum 15～ 35%, Daphnia 10-25%, Atractylodes 5-20%, Salvia miltiorrhiza 5-20%, Eucommia 5-15%, Bergamot 5-15%, preferred components are: Ligustrum lucidum 15-35%, Dagu 10 ~ 25%, Atractylodes 5 to 15%, Salvia miltiorrhiza 5 to 15%, Eucommia 5 to 15%, Bergamot 5 to 15%, Sanqi 5 to 15%, and Huanglian 3 to 10%. The raw materials Danshen, Ligustrum lucidum, Rhizoma Coptidis, Daphnia, Eucommia, Atractylodes, Sanqi and Bergamot were extracted by C1-3 alcohol and/or extracted with water, and the total extract was combined, and the total extract was extracted with petroleum ether. A petroleum ether extract of Chinese medicine with disorder of glycolipid metabolism. The C1-3 alcohol is methanol, ethanol or propanol.

The above method includes the following steps:

(1) Carrying out C1-3 alcohol extraction from Sanqi and Ligustrum lucidum to obtain C1-3 alcohol extract, and extracting and concentrating Daxie, Atractylodes Rhizome, Salvia miltiorrhiza, Eucommia, Bergamot and Coptis, to obtain water extract, and merging C1-3 alcohol extract and water extract to obtain a total extract;

(2) adding the total extract obtained in the step (1) to 0.5 to 15 times, preferably 6 to 10 times by volume of petroleum ether, extracting 1 to 5 times, preferably 3 to 4 times, combining the extracts, and drying at a low temperature to obtain prevention and treatment A petroleum ether extract of Chinese medicine with disorder of glycolipid metabolism.

The C1-3 alcohol extract in the step (1) is preferably extracted with 30 to 95% by volume of ethanol for 1 to 5 times, preferably 2 to 4 times, and the volume of the C1-3 alcohol extracted per time is 1 to the mass of the medicinal material. 15 times, preferably 6 to 10 times, each extraction time is 5 Min ~ 5 h, preferably 2 ~ 3 h.

The water extraction in the step (1) is decocted by water 1 to 5 times, preferably 2 to 4 times, and the volume of water per time is 1 to 15 times, preferably 6 to 12 times, the mass of the medicinal material. 5 min ~ 5 h, preferably 2 to 3 h.

The concentration in the step (1) means that the concentrated liquid volume after concentration is 0.2 to 5 times, preferably 1 to 2 times the mass of the medicinal material.

The obtained petroleum ether extract is processed into an oral preparation or an injection preparation, such as a tablet, a capsule, a powder, a pill, a powder, a granule, a crystal, a solution, an extract, a suspension, according to a pharmaceutically acceptable carrier, using a general preparation method. In the form of agents, soups, syrups, tinctures, teas, oils, and the like. It is used for the preparation of a pharmaceutical preparation or a health food for preventing or treating a disease associated with disorders of glycolipid metabolism (hyperlipidemia, diabetes, metabolic syndrome, animal atherosclerosis and the like).

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

Through the preparation method of the invention, a large number of ineffective chemical components in the traditional Chinese medicine compound of the patent 200410051250.4 can be removed, and an effective extraction site is obtained, which not only retains the characteristics of the traditional Chinese medicine, but also greatly increases the content of the active ingredient in the traditional Chinese medicine, and reduces the processing of the ineffective ingredients to the product. And the influence of the quality of the preparation makes the effective ingredients of the compound Chinese medicine clear, the preparation process is stable, the product quality is controllable, and it is beneficial to industrial production. Animal experiments have shown that the compound petroleum ether extract has significant lipid-lowering and hypoglycemic effects on experimental hyperlipidemia and hyperglycemia, and can lower cholesterol, improve lipid metabolism, and prevent arteriosclerosis. Obvious side effects were observed and the safety of the medication was increased.

The compound Chinese medicine petroleum ether extract provided by the invention further expands the application range of the active ingredient group of the compound phlegm and blood stasis prescription, and provides a new way for preventing or treating diseases related to glycolipid metabolism. Compared with the same drugs for controlling diseases related to glycolipid metabolism and lipid disorders, the invention has the advantages of exact curative effect, safe and no obvious side effects, and has good application prospects when used alone or in combination with other drugs.

The petroleum ether extract of Chinese herbal medicine prepared by us has the following advantages compared with the original one:

(1) The dosage is reduced from the original 6g extract to 100mg, which is reduced to 1/60 of the original;

(2) The content of the active ingredient of the drug is increased by about 60 times;

(3) The petroleum ether extract provided by the invention has good stability, can prepare various oral controlled release preparations, greatly broadens the application of the compound phlegm and blood stasis prescription in the field of pharmaceutical preparations, and significantly improves the compound phlegm and blood lipid regulating formula. Bioavailability of active ingredients, easy to use;

(4) In vivo experiments in animals showed that the extract of the present invention has a significant lipid-lowering effect on experimental hyperlipidemia, at the same time, can lower cholesterol, improve lipid metabolism, and has significant hypoglycemic effect on experimental hyperglycemia, and improve blood sugar. Metabolism, and no significant side effects were observed, increasing the safety of the medication. For the anti-blood cholesterol effect of high-fat diet hyperlipidemia, the petroleum ether extract is better than the original one, and the purpose of reducing the dosage is not reduced.

DRAWINGS

Figure 1: HPLC chromatogram of the compound Chinese petroleum ether extract of Example 3;

detailed description

The invention is further explained by the following examples, but the examples are not intended to limit the invention in any way.

Example 1 Preparation of total extract of traditional Chinese medicine compound

Will be female, Atractylodes, Eucommia, Sanqi, Datun, Salvia, Coptis, bergamot (weight ratio 2:1:1:1: 1:2:1:1) The three medicines and the scorpion smashed into coarse powder, and the extracts were extracted three times with 10 times, 8 times and 6 times of 60% by volume of ethanol, respectively, 2 times each time. h, combine the extract, recover the ethanol and make the alcohol extract part concentration is 1 ml equivalent to 1 g of crude drug; the other herbs in the square are 12 times, 10 times and 8 times the amount of water, respectively, decoction and extract 3 times, each time 2 h, 3 times of aqueous extracts were combined, concentrated under vacuum to 1 g of medicinal material/ml extract, and the water part and the previous alcohol extracting part were combined to obtain a total extract of the traditional Chinese medicine compound.

Example 2 Preparation of Petroleum Ether Extraction Site of Compound Total Extract

The petroleum ether layer was separated by extracting 10 times of petroleum ether from the total extract of the compound, and the petroleum ether layer was separated by extraction with 8 and 6 volumes of petroleum ether. The petroleum ether layer was separated and the petroleum ether extract was combined for 3 times. Petroleum ether, vacuum drying at 0.08 MPa, 65 ° C, to obtain petroleum ether extract (FTZEE);

According to the total amount of the traditional Chinese medicine compound and the amount of the extracted parts, the yield of the extract was calculated. The yield of the petroleum ether extract is from 0.3% to 0.5% (1 kg of crude oil extract of 3.0 kg to 5.0 g).

Example 3 Chemical Composition Analysis of Petroleum Ether Extraction Site of Compound Total Extract

Example 2 Compound petroleum ether extract (FTZEE), which was analyzed by HPLC, and the fingerprint of Fig. 1 was obtained.

UPLC chromatographic conditions for FTZ petroleum ether parts: Agilent HC-C18 column (4.6mm × 250mm, 1μm), guard column phenomenex KJO-4282-C18 column; mobile phase using acetonitrile (A)-water (containing 0.25% glacial acetic acid + 0.13% triethylamine) (B) binary gradient system. Gradient elution: 2~5% A (0~5 Min), 5%~20% A (5~35 min), 20% A (35~45 min), 20%~35% A (45~70 min), 35%~80% A (70~85 min), 80%~100%A (85~87 min), 100%A (87~89 min). Detection wavelengths are 254 nm, 300 nm, and 330 Nm, column temperature is 30 ° C, flow rate is 0.2 mL / min, injection volume is 10 μL.

Qualitative analysis results: FTZ petroleum ether parts mainly contain wax alcohol, β-sitosterol, n-hexadecanoic acid, atractylenolide III, oleanolic acid, berberine, jatrorrhizine, salvianolic acid B, UPLC-MS fingerprints of cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, ginsenoside Rb1, etc., see Figure 1, in which the components The weight ratio is 1 to 5:1 to 8:1 to 8:1 to 5: 1 to 8: 1 to 8:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5:1 to 6.

Example 4 Preparation method of extract capsule

The extract was combined in the following parts by weight: Example 2, 5 parts of petroleum ether extract, 5 parts of starch, and 5 parts of carboxymethylcellulose. The extracts of the above ratio, the petroleum ether extract and the equal amount of starch are uniformly mixed, and then mixed with the remaining starch, and finally mixed with carboxymethyl cellulose, and the three are uniformly mixed, then dried and granulated, and dried under reduced pressure. Granules, filled with No. 3 capsules, polished, quality checked packaging. Each grain contains FTZEE 30 mg. The dosage is 3 times a day, 1 capsule each time. The obtained capsules were determined by HPLC method, and the results of the contents of the respective active ingredients are shown in Table 1:

Table 1 HPLC results of FTZEE capsules

Test items	Content (mg/granule)	Test items	Content (mg/granule)
Oleanolic acid	1.02	Flavone	1.56
Salvianolic acid	0.58	9,12-octadecadienoic acid	0.67
N-hexadecanoic acid	0.55	Atractylenolide III	0.91
Berberine	0.57	Ginsenoside Rb1	0.50

The capsules obtained were used for clinical treatment of hyperlipidemia, 3 times a day, 1 capsule each time, and even for 60 days. As a result, the total cholesterol and triglyceride of blood lipids in the treatment group decreased significantly after treatment, and blood lipids decreased. The efficiency was 90.5%, which was significantly different from the control group (P<0.01).

 Example 5 Preparation method of extract tablets

The petroleum ether extract prepared in Example 2 was 5 kg plus starch 5 Kg, carboxymethyl cellulose 5kg, mix, add 75% alcohol granules, dry at 80 ° C, add the amount of magnesium stearate, tablet, film coating, make about 1 million FTZEE tablets, each containing FTZEE 50 Mg. The dosage is 2 times a day, 1 tablet each time. The above tablets were determined by HPLC, and the results of determination of the content of each active ingredient in each tablet are shown in Table 2:

Table 2 HPLC results of FTZEE tablets

Test items	Content (mg/tablet)	Test items	Content (mg/tablet)
Oleanolic acid	1.56	Flavone	2.50
Salvianolic acid	0.90	9,12-octadecadienoic acid	0.97
N-hexadecanoic acid	0.85	Atractylenolide III	1.21
Berberine	0.91	Ginsenoside Rb1	0.80

Tablets were used for clinical treatment of hyperlipidemia, 2 times a day, 1 tablet each time, for 60 days. The results showed that the total cholesterol and triglyceride levels of blood lipids decreased significantly after treatment in the treatment group. The total effective rate of hypolipidemic was 93.3. % was significantly different from the control group (P<0.01).

Example 6 Preparation method of extract honey pill

Prepare FTZEE 5.0 kg and mix to make FTZEE honey pellets.

Above FTZEE plus starch 3 kg, dextrin 3.0 kg, honey 8. 0 kg panball into water honey pill, dry to get 15 Kg dry pellets, packaged into 100,000 pellets, that is, 0.15g / granules (each containing FTZEE 50 mg). Oral dose: 1 small capsule at a time, 2 times a day.

The prepared honey pill is used for clinical treatment of hyperlipidemia, 2 times a day, 1 capsule each time, even for 60 days. As a result, the total cholesterol and triglyceride of blood lipids in the treatment group decreased significantly after treatment. There was a significant difference (P<0.01). The total effective rate of lowering blood fat was 91.5%.

Example 7 Acute toxicity and long-term toxicity test

(1) Acute toxicity test in mice: 120 19-21 g NIH mice (male and female) were divided into 6 groups, 20 rats in each group, and administered with five different doses of FTZEE (prepared in Example 2) by oral gavage (p.o.), and normal saline (NS) negative control. Observe the response of the mice and record the number of deaths in each group of mice using Microsoft Excel's linear regression software calculates the LD50 based on the logarithmic dose and the empirical probability unit. The LD50 of FTZEE for oral gavage in mice is 450-751 Mg / kg (equivalent to 300 to 500 times the clinical dose of humans).

(2) Long-term toxicity test in rats: 120 male and female Wistar rats were divided into 4 groups, 30 in each group, respectively, p.o. Mg/kg, 90 mg/kg, 180 Mg/kg compound Chinese medicine petroleum ether extract, NS negative control p.o. physiological saline. After continuous administration for 180 days, the rats in the three dose groups (equivalent to 30 to 120 times of the clinical dose) showed no obvious blood, heart, lung, liver and kidney functions and no abnormalities in the nervous system, and no heart, lung, spleen and stomach. Important organs such as the brain and intestine have obvious pathological changes.

Conclusion: The petroleum ether extract of the compound Chinese medicine of the invention has low toxicity and high safety.

Example 8 Effect of FTZEE on Blood Lipid Metabolism in Dietary Hyperlipidemic Model Animals

Experimental material

Drugs and reagents: FTZEE, preparation method is the same as in Example 2; compound phlegm and fat-removing formula extract FTZ; total cholesterol, triglyceride, high density lipoprotein cholesterol assay kit.

Animals: clean grade SD rats, body weight (180 ~ 225 g), provided by the Experimental Animal Center of Southern Medical University; purebred New Zealand female rabbit, body mass (1.80-2.20 kg), provided by Guangdong Experimental Animal Center.

2. Experimental methods

2.1 Effects on blood lipids in rats with dietary hyperlipidemia

The effects of different doses of FTZEE on serum TC, TG, LDL-C and HDL-C in hyperlipidemia rats were observed.

According to the literature report method (Guo Yu, Bei Weijian, etc., the effect of Fufang Yishu Tiaozhi Decoction on hepatic lipase in rats with diet-induced hyperlipidemia, Chinese herbal medicine, 2009, 31(4): 582-585) The animal model of hyperlipidemia was confirmed to be successful.

Animals were randomly divided into groups: 96 rats were numbered and randomly divided into 8 groups, 12 in each group, 1 blank normal control group; 2 high fat model group; 3FTZ group; 4 lovastatin group; Fenofibrate group; 678 followed by FTZEE high, medium and low doses (15, 30, 60) Mg/kg) for each experimental group. In addition to the normal control group free access to ordinary feed, the other groups were given a high-fat emulsion by 10 ml/kg every morning on the basis of this, 4 After h, the corresponding drugs were given. The normal group and the model group were given the same amount of normal saline for 4 weeks, fasting for 1 day (not allowed for water), and the second day of the second day was administered 1 After h, blood was taken from the orbital venous plexus, and the serum was separated by centrifugation and determination of TC, TG, HDL-C, and LDL-C.

Statistical methods: Data were expressed as x±s, and analysis of variance and multiple comparisons between means were performed using SSP10.0 software.

3 results

3.1 Effects on blood lipids in rats with dietary hyperlipidemia

The results showed that FTZEE can significantly reduce serum total cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C) in diet hyperlipidemic mice, and significantly increase high-density lipoprotein (HDL- C), there is a role in regulating blood esters, see Table 3:

Table 3 Effect of FTZEE on blood lipids in diet hyperlipidemia model rats

Group	Dosage mg/kg	TC mmol/L	TG mmol/L	LDL-C mmol/L	HDL-C mmol/L
Normal control	0	2.18±0.29	1.02±0.29	0.70±0.09	1.31±0.12
High fat control	0	5.32±0.46**	3.08±0.78**	1.43±0.30**	0.71±0.30**
FTZ	500	3.43±0.43 ▲▲	1.71±0.64 ▲▲	0.97±0.43 ▲▲	1.26±0.41 ▲▲
Lovastatin	10	3.29±0.36 ▲	2.06±0.81 ▲	0.90±0.21 ▲▲	0.96±0.26 ▲▲
Fenonote	30	3.55±0.43 ▲▲	1.51±0.63 ▲▲	0.89±0.42 ▲▲	1.22±0.43 ▲▲
FTZEE	15	3.89±0.41 ▲	2.51±0.52	1.09±0.45	1.12±0.40
FTZEE	30	3.55±0.38 ▲▲	2.03±0.51 ▲	0.93±0.40 ▲▲	1.21±0.37 ▲▲
FTZEE	60	3.21±0.36 ▲▲	1.53±0.45 ▲	0.91±0.36 ▲▲	1.32±0.33 ▲▲

Note: Compared with the control group, **P<0.01; compared with the high-fat control, F=3.82, ^▲ P<0.05, ^▲▲ p<0.01.

[0039] FTZ and FTZEE have significant hypolipidemic effects on diet hyperlipidemia rats, and have a dose-effect relationship. The role of prevention and treatment of experimental dyslipidemia may include:

(1) The FTZEE of the present invention has been shown to have a significant effect on lipid-lowering, and has the dual functions of preventing and treating hyperlipidemia, in addition to affecting key enzymes of lipid metabolism, effectively regulating blood lipids, and improving blood rheology. Learning, anti-oxidation, anti-atherosclerosis and other multiple effects.

(2) The results of the study have shown that FTZEE can greatly enhance the serum CYP7A1 and HL enzyme activities in high-fat model rats.

Example 9 Effect of FTZEE on gene expression and activity of key enzymes in hepatocyte lipid metabolism

The effect of the petroleum ether extract of the present invention on the expression and activity of key enzymes of blood lipid metabolism was examined.

Method

1.1 Effects on cholesterol 7-α-hydroxylase (CYP7A1) and hepatic lipase (HL) activity in L-O2 hepatocytes

L-O2 human normal hepatocytes were cultured as usual. After the cells were fused, various concentrations of FTZEE (prepared by the method of Example 2) and FTZ were added, and the culture was continued. After h, the cells were collected into cell homogenate, and the HL activity was detected by colorimetric assay. The effects of different concentrations of FTZEE on the activity of HL1 enzyme were studied, and the HL enzyme activity in hepatocytes under different concentrations of FTZEE was compared. .

Co-culture of FTZEE and liver LO2 cells 24 After h time, hepatocytes were collected, as reported by Hylemon et al. (see Hylemon, P. B.; Studer, E. J.; Pandak, W. M. et al) 1989. Simultaneous measurement of cholesterol 7 alpha-hydroxylase activity by Reverse-phase high-performance liquid chromatography using both endogenous and Exogenous [4-14C] cholesterol as substrate. Analysic Biochemistry 182(2): 212-216) Preparation of hepatocyte microsomes, detection of CYP7A1 activity by HPLC using the CYP7A1 assay kit (Hylemon et al., 1989) To study the effect of FTZEE on the activity of CYP7A1 enzyme, and compare the changes of CYP7A1 activity in hepatocytes under different concentrations of FTZEE.

1.2 inhibition test of HMGCoA reductase activity

In vitro HMGCoA reductase enzyme activity inhibition experiments were performed using the above FTZEE, and their IC50 for inhibition of HMGCoA reductase was determined and compared with pravastatin.

1.3 Effects on the expression of CYP7A1 and HL genes in L-O2 hepatocytes

L-O2 human normal hepatocytes were cultured as usual, and 1.0 μg/ml and 5.0 were added after cell fusion. FTZEE of μg/ml and FTZ of 25.0 μg/ml are three groups, continue to culture 24 After h, the cells were collected, total RNA was extracted from liver cells by Trigol reagent, and the expression of CYP7A1 and HL gene in hepatocytes was determined by RT-PCR. The effect of FTZEE on the expression of CYP7A1 and HL gene in hepatocytes was observed.

2 statistical methods

Data were expressed as mean ± standard deviation, and significance test was performed using the analysis of variance.

3 Experimental results

3.1 Effects on liver cell lipase

The hepatic lipase activity of hepatocytes was significantly increased after treatment with different doses of FTZEE and FTZ (P<0.01), and there was a dose-dependent effect. The results are shown in Table 4. It is indicated that the petroleum ether extract (FTZEE) of the traditional Chinese medicine has a significant effect on the hepatic lipase activity of hepatocytes, and is beneficial for enhancing the clearance of lipoproteins and lowering blood triglycerides and cholesterol.

3.2 Effect on hepatocyte CYP7A1

CYP7A1 activity was significantly increased after treatment with different doses of FTZEE and FTZ (P<0.01), and there was a significant dose-dependent enhancement. The results are shown in Table 4. It is indicated that the petroleum ether extract (FTZEE) of the traditional Chinese medicine has a significant effect on the activity of CYP7A1 in hepatocytes, and is beneficial for promoting the conversion of cholesterol into bile acid and excreting, thereby lowering blood cholesterol.

Table 4 Effects of different drugs on the activity of CYP7A1 and HL in L-O2 cells

Group	Concentration ( μg/ml )	n	CYP7A1 activity ( U/mg·min )	HL activity (μmol FFA/mL·h)
normal group	0	7	1.63±0.31	5.06±0.46
FTZ	25.0	7	2.65±0.52*	6.15±1.32*
FTZEE	1.0	7	2.98±1.02**	7.08±1.52*
FTZEE	5.0	7	3.38±0.61**	8.08±1.29**
Fenonote	5.0	7	1.65±1.02	9.71±1.02**

Note: *P<0.05; **P<0.01 compared with the normal control group.

 3.4 Inhibition of HMG-CoA reductase activity

The activity of HMG-CoA reductase solution and HMG-CoA reductase was determined by using rat hepatocyte microsomes by literature method (Kleinsek, DA, Ranganathan, S. and Porter, JW, 1977. Purification of 3-hydroxy- 3 -methylglutaryl-coenzyme A reductase from rat liver. Proceedings of the National Academy of Sciences USA 74, 1431-1435) respectively determine the enzyme activity at the specified concentration of each drug, and determine the activity of HMG-CoA reductase by each drug such as FTZEE. IC ₅₀ (μg/ml).

The results are shown in Table 5. The results show that FTZEE can significantly reduce the activity of HMG-CoA reductase in rats in a dose-dependent manner. FTZEE inhibits the activity of HMG-CoA reductase, inhibits the synthesis of HMG-CoA, a key ingredient in cholesterol synthesis, and inhibits the biosynthesis of cholesterol and produces cholesterol-lowering effects.

Table 5 IC ₅₀ (μg/ml) of FTZEE on -HMG-CoA reductase activity

Medicine	IC 50 (μg/ml)
FTZEE1	1.11±0.13
Pravastatin	1.05±0.13
FTZ	15.85±1.43

Figure 3 shows that FTZEE inhibits liver HMG-COA R-lowering enzyme activity, inhibits liver TC synthesis, and significantly reduces liver TC content.

Example 10 Effect of Chinese Herbal Petroleum Ether Extract on Islet B Cells

1.1 method

1.1.1 Effect on islet B cells

The rat islet B cells were cultured as usual. After the cells were fused, 200 μM H ₂ O ₂ was added for 1 h, and then various concentrations of FTZEE (prepared by the method of Example 2) and FTZ were added to continue the culture for 24 h. Collect cells to make cell homogenate, using superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) and glutathione (GSH) detection reagents The effects of different concentrations of FTZEE on the activities of SOD, CAT, GSH-Px and GSH in pancreatic islet B cells were studied by colorimetric assay for SOD, CAT and GSH-Px activities and GSH content.

1.1.2 Effect on Bcl-2, PPAR-a and Bax and Fas Gene Expression in Islet B Cells

The method of treating islet B cells was the same as 1.1.1. Total RNA was extracted from islet B cells by Trizol reagent. The expression of Bcl-2 and Bax and Fas genes in pancreatic islet B cells was determined by RT-PCR. The expression of Bcl- in islet B cells by FTZEE was observed. 2. Effects of PPAR-a and Bax and Fas gene expression.

1.2 Statistical methods: Data are expressed as mean ± standard deviation, and significance test is performed by analysis of variance.

1.3 Experimental results

1.3.1 Effects on SOD, CAT and GSH-Px activities and GSH content in islet B cells

The SOD, CAT and GSH-Px activities and GSH content of islet B cells were significantly increased after treatment with different doses of FTZEE and FTZ (P<0.01), and there was a dose-dependent enhancement. The results are shown in Table 6. It is indicated that the FTZEE of the present invention can significantly improve the activity of SOD, CAT, GSH-Px and GSH of pancreatic islet B cells, and is beneficial to anti-oxidative stress damage. Protect islet B cells.

Table 6 Effects of different drugs on SOD, CAT, GSH-Px activity and GSH content in islet B cells under oxidative stress injury

Group	Concentration (μg/ml)	n	SOD activity ( U/mg·min )	CAT activity (U/g·min)	GSH-Px activity ( U/mg·min )	GSH content (μmol/mg)
normal group	0	7	3.63±0.53	4.06±0.67	2.36±0.41	3.16±0.43
Model group	0	7	2.03±0.41 ☆☆	3.13±0.61 ☆☆	1.53±0.45 ☆☆	2.56±0.51 ☆☆
FTZ	10.0	7	4.65±0.59*	6.18±1.12*	3.15±0.43*	4.15±0.52*
FTZ	50.0	7	5.61±1.01**	7.36±1.25**	3.88±0.45**	4.68±0.65**
FTZEE	1.0	7	5.18±1.10*	7.08±1.32*	3.38±0.52*	4.28±0.51*
FTZEE	2.0	7	6.65±1.32**	7.68±1.31**	3.98±0.56**	4.88±0.50**
FTZEE	5.0	7	7.01±1.41**	8.01±1.49**	4.08±0.59**	5.06±0.69**
BBR	5.0	7	5.95±1.02**	7.41±1.06**	3.71±0.42**	4.71±0.62**

Note: (1) Compared with the normal control group, ^{☆ ☆} P <0.01; compared with the model group, *P<0.05;**P<0.01.

(2) FTZ: compound 贞 调 调 方 方, FTZEE: compound 贞 调 调 方 石油 石油 petroleum ether extract, BBR: berberine hydrochloride.

1.3.2 Effect of Bcl-2 and Bax and Fas Gene Expression in Islet B Cells

The expression of Bcl-2 and Bax and Fas genes in normal islet B cells was normal. The expression of Bcl-2 gene was significantly increased in islet B cells after different doses of FTZEE treatment (P<0.01), while the expression of Bax and Fas genes was significantly decreased (P<0.05). <0.01), indicating that FTZEE significantly promoted Bcl-2 gene expression (P<0.01), and inhibited the expression of apoptosis genes Bax and Fas, thereby protecting islet B cells from oxidative stress damage and apoptosis. The results are shown in Table 7.

Table 7 Effects of different drugs on the expression of Bcl-2, PPAR-a and Bax and Fas genes in islet B cells

Group	Concentration ( μg/ml )	n	Bcl-2	PPAR-a	Bax	Fas
normal group	0	7	53.6 ± 6.3	55.1 ± 6.6	33.6±6.1	35.0 ± 6.4
Model group	0	7	31.6±5.1 ☆☆	46.6±6.4 ☆☆	75.1±9.6 ☆☆	85.6±9.8 ☆☆
FTZ	10.0	7	66.6±8.5**	66.2±7.3**	56.1 ± 8.3**	66. 5±8.3**
FTZ	50.0	7	76.62±10.5**	87.38±9.5**	47.38±7.5**	47.38±8.1**
FTZEE	1.0	7	75.9 ± 12.1**	77.08±8.8**	52. 8±8.5*	52.8±8.5**
FTZEE	2.0	7	87.6±10.2**	97.88±13.3**	46. 8±8.3**	46.8±7.3**
FTZEE	5.0	7	98.2±11.5**	108.08±13.9**	38.08±6.9**	41.0±6.9**
BBR	5.0	7	84.9±11.2**	89.71±10.2**	9.71±1.02**	53.7±7.0**

Note: (1) Compared with the normal control group, ^{☆ ☆} P <0.01; compared with the model group, *P<0.05;**P<0.01.

(2) FTZ: compound 贞 调 调 方 方, FTZEE: compound 贞 调 调 方 石油 石油 petroleum ether extract, BBR: berberine hydrochloride.

Example 11 Effect of Chinese Herbal Petroleum Ether Extract on Blood Glucose in Normal Mice

Animals: 60 healthy NIH mice, half male and half female, weighing 18-22 g.

Grouping: randomly divided into 5 groups according to gender: blank control group, glibenclamide group, compound total extract group, petroleum ether extract group 1, 2, 3 (prepared by the method of Example 2), 10 in each group.

Experimental method: After the animal was fasted for 12 h, blood was taken from the orbital venous plexus, and the blood sample was centrifuged at 3000 rpm. Min, serum was separated and operated according to the instructions of the glucose kit to determine the blood sugar level. Immediately after blood collection, the corresponding test drug is administered by gavage, wherein glibenclamide 50 Mg/kg, the blank control group was administered with the same volume of normal saline, and the dosage volume was 0.2 mL/10 g body weight. 3 h, 6 h and 9 after administration At h, the blood glucose was measured by the above method, and the experimental results are shown in Table 8.

Results The t test was used for statistical analysis to compare the differences between the drug-administered groups and the control group.

 Table 8 Effect of Chinese herbal petroleum ether extract on blood glucose in normal mice (n=10 x ± s)

Group	dose	Pre-dose blood glucose level (mmol/L)	3h blood glucose level after administration (mmol/L)	6h post-dose blood glucose level (mmol/L)	Blood glucose content (mmol/L) 9 h after administration
Blank control group	/	6.72±1.27	6.67±1.38	7.06±1.51	6.86±1.42
Glyburide	50 mg/kg	6.56±1.27	4.68±1.57*	4.46±1.45**	5.16±1.32*
Total extract group	500 mg/kg	6.68±1.30	5.64±0.85*	5.16±1.03**	5.54±0.95*
FTZEE	3 mg/kg	6.23±1.28	5.52±1.09*	5.08±0.82**	5.46±1.06*
FTZEE	6 mg/kg	6.46±1.22	5.43±1.08*	4.86±1.07**	5.34±1.02*
FTZEE	12 mg/kg	6.46±1.22	5.14±1.03**	4.68±0.91**	5.09±1.22*

Compared with the control group, * P < 0.05.

The experimental results showed that compared with normal mice, after 3, 6, and 9 hours of administration, the positive control drug glibenclamide group, total extract group and petroleum ether extraction group significantly reduced the blood glucose of normal mice. The hypoglycemic effect was most significant after 6 hours of administration in each group.

Example 12 Effect of Chinese Herbal Petroleum Ether Extract (FTZEE) on Blood Glucose in Diabetic Model Mice

Drugs and reagents: FTZEE (Chinese herbal petroleum ether extract, prepared by the method of Example 2); blood glucose kit (Beijing Beihua Kangtai Reagent Co., Ltd.); total cholesterol, triglyceride, high-density lipoprotein cholesterol determination kit; Zuomycin (STZ), the product of American Sigma Company; Xiaokean Capsule, Tonghua Baishan Pharmaceutical Co., Ltd. products.

Blood glucose was measured using an Advantage electronic blood glucose meter and a supporting strip (product of Roche, Switzerland).

 The subjects were clean grade NIH mice provided by the Experimental Animal Center of Southern Medical University, male and female, weighing 18-22 g.

Effects of blood glucose and blood lipids on streptozotocin (STZ) type diabetic mice

Eighty healthy NIH mice were numbered and randomly divided into 8 groups, 10 in each group, and 1 group was taken as the normal control group. After the other groups of mice were fasted for 16 hours, the freshly prepared 1% streptozotocin solution 150 was intraperitoneally injected. Mg/kg, 72 hours later, blood was taken from the eye, and blood glucose was measured using an Advantage electronic blood glucose meter and supporting paper (product of Roche, Switzerland). The blood sugar level was greater than 16.7. The mmol/L was determined to be a diabetic model rat. Diabetes model mice were randomly divided into 7 groups according to blood glucose values, and FTZEE 3 treatment groups (30, 60, 120) Mg/kg), FTZ2 group (250,500 mg/kg), Xiaokean Capsule treatment group (500 Mg/kg), model control group. Each group was administered intragastrically once a day for 30 days, and the animals were fasted 12 hours before the last administration, and blood was taken from the iliac venous plexus 2 hours after the administration. Blood sample 3000 Centrifuge at rpm for 10 min, separate the serum, and measure the blood glucose level according to the glucose kit instructions.

Effect of adrenaline on blood glucose in hyperglycemic mice

Sixty healthy NIH mice were weighed and randomly divided into 6 groups, 10 in each group. One group was taken as normal control group, adrenaline group, FTZEE size dose (120, 60 mg/kg) group, FTZ. 250 mg/kg group, Xiaoke'an capsule treatment group (500 mg/kg). Each group was intragastrically administered once a day, and the normal control group and the adrenaline group were given an equal volume of normal saline for 7 days, at the last administration 2 h, the control group ip equal volume of normal saline, the other groups of ip adrenaline (240 mg / kg) solution, respectively, after ip adrenaline 0.5 h, 1 h Blood was taken from the iliac venous plexus of the mouse, serum was separated, and blood glucose was measured.

Effect of glucose on blood glucose in hyperglycemic mice

Sixty healthy NIH mice were numbered and randomly divided into 6 groups, 10 in each group, normal control group, glucose high glucose model group, FTZEE size dose (120, 60 mg/kg) group, FTZ. 250 mg/kg group, Xiaokean Capsule treatment group (500 Mg/kg). Each group was intragastrically administered once a day, and the normal control group and the glucose group were given an equal volume of normal saline (NS) for 7 days of continuous administration. After h, the control group was intraperitoneally injected (ip) with the same volume of normal saline, and the other groups of ip glucose (2 g/kg) solution were 0.5, 1, 2 after ip glucose. h Blood was taken from the iliac venous plexus of the mouse, serum was separated, and blood glucose was measured.

The experimental results are shown in Table 9, Table 10, and Table 11.

Table 9. Effect of FTZEE on blood glucose in streptozotocin (STZ) type diabetic mice (n=10, x±s)

Group	Dosage mg/kg	Pre-dose blood glucose concentration (mmol/L)	Blood glucose concentration after administration (mmol/L)	Hypoglycemic rate %
Control group	0	8.01±3.19	8.89±1.28	__
STZ+NS	0	23.32±6.49 **	24.28±3.79 *	__
STZ+ Xiaokean Capsule	500	23.59±6.62 **	16.38±5.82 △△▲	31.57
STZ+FTZEE	3	22.95±5.41 **	18.78±4.56 △△	18.17
STZ+FTZEE	6	23.61±6.21 **	16.40±5.53 △△▲	30.54
STZ+FTZEE	12	23.32±5.36 **	14.90±6.41 △△▲▲	36.10
STZ+FTZ	250	23.49±5.06 **	20.38±4.83 △▲	14.28
STZ+FTZ	500	23.09±5.16 **	18.63±4.56 ▲	19.32

Note: Compared with the normal group, ^* P<0.05, ^** P<0.01; compared with the model group, ^△ P<0.05, ^△△ P<0.01; compared with before treatment ^▲ P<0.05, ^▲▲ P< 0.01.

Table 10. Effect of FTZEE on blood glucose in adrenaline-induced hyperglycemic mice (n=10, x±s)

Group	Dosage mg/kg	Blood glucose concentration (mmol/L) 0.5h	Blood glucose concentration (mmol/L) 1h
Control group	0	6.01±2.15	6.80±2.38
Adrenaline +NS	0	13.02±3.49 **	14.08±2.97 *
Adrenaline + Xiaokean Capsule	500	8.59±3.26 ▲▲	9.33±3.86 ▲▲
Adrenaline +FTZEE	6	9.01±3.12 ▲▲	9.84±2.35 ▲▲
Adrenaline +FTZEE	12	8.38±3.03 ▲▲	8.59±2.43 ▲▲
Adrenaline +FTZ	250	8.98±3.03 ▲▲	10.59±2.43 ▲▲

Note: Compared with the normal group, *P<0.05, **P<0.01; compared with before treatment, ▲▲P<0.01.

Table 11. Effect of FTZEE on blood glucose in glucose-induced hyperglycemic mice (n=10, x±s)

Group	Dosage mg/kg	0.5h after glucose administration (mmol/L)	1h after glucose administration, blood glucose concentration (mmol/L)	2h after glucose administration, blood glucose concentration (mmol/L)
Control group	0	6.80±1.31	6.78±1.39	6.93±1.43
Glucose +NS	0	12.30±2.43 **	10.02±2.13 **	9.30±1.33 **
Glucose + Xiaokean Capsule	500	9.51±2.53 ▲▲	8.85±2.06 ▲	7.50±1.96 ▲▲
Glucose +FTZEE	6	9.86±2.11 ▲▲	9.06±2.12 ▲	8.06±1.56 ▲
Glucose +FTZEE	12	8.93±3.15 ▲▲	8.32±2.16 ▲▲	7.52±1.51 ▲▲
Glucose +FTZ	250	10.08±3.25 ▲▲	9.19±2.03 ▲	8.25±1.60 ▲

Note: Compared with the normal group, **P<0.01; ▲P<0.05, ▲▲P<0.01 compared with before treatment.

Example 13 Effect of FTZEE on hydrocortisone-induced insulin resistance in mice

Forty mice were weighed and randomly divided into 4 groups, 10 in each group. One group was given subcutaneous injection of normal saline (NS) as a normal control group and a group of subcutaneously injected hydrocortisone (36). Mg/kg), the other two groups were injected subcutaneously with hydrocortisone (36 mg/kg) and then administered with FTZEE size (60, 120). Mg/kg, prepared by the method of Example 2). Each group was intragastrically administered once a day, the normal control group and the hydrocortisone group were given an equal volume of normal saline for 10 days, at the last administration 2 h, the control group ip equal volume of normal saline, the other groups of ip insulin (0.5 g / kg), respectively, after ip glucose 0.5, 2 h Eyes were taken for blood, serum was separated, and automatic blood biochemistry analyzer was used to measure blood sugar. The results are shown in Table 12 and Table 13.

Table 12 Effect of FTZEE on hydrocortisone-induced insulin resistance in mice (n=10, x±s)

Group	Dosage mg/kg	Change in blood glucose concentration 0.5 h after sugar administration (%)	1h after glucose administration, blood glucose concentration change (%)	2h after glucose administration, blood glucose concentration change (%)
Control group	0	20	33	62
Hydrocortisone +NS	0	65	67	70
Hydrocortisone +FTZEE	6	40	45	68
Hydrocortisone +FTZEE	12	32	38	63

Table 13 Effect of FTZEE on hydrocortisone-induced insulin resistance in mice (n=10, x±s)

Group	Dosage mg/kg	0.5h after glucose administration, blood glucose concentration mmol/L	1h after sugar supply, blood glucose concentration mmol/L	2h after glucose administration, blood glucose concentration mmol/L
Control group	0	6.80±1.31	6.78±1.39	6.93±1.43
Hydrocortisone +NS	0	12.30±2.43 **	10.02±2.13 **	9.30±1.33 **
Hydrocortisone +FTZEE	6	9.86±2.11 ▲▲	8.06±2.12 ▲	8.06±1.56 ▲
Hydrocortisone +FTZEE	12	8.93±3.15 ▲▲	6.32±2.16 ▲▲	7.52±1.51 ▲▲

 Note: Compared with the normal group, **P<0.01; ▲P<0.05, ▲▲P<0.01 compared with hydrocortisone+NS group.

Example 14 Effect of FTZEE on streptozotocin-type diabetic rats

Drugs and reagents: Streptozotocin (STZ), American Sigma product; sodium citrate, Shantou Guanghua Chemical Factory, batch number 20000116.

The experimental subjects were 36 female Wistar rats, weighing 220-250. g. (Provided by the Experimental Animal Center of the First Military Medical University, Certificate No.: Guangdong Inspection Certificate No. 2004B023)

After one week of adaptive feeding in all rats, fasting for 12 h, 7 rats were taken as normal control group (abbreviated as control group), and other rats were injected intraperitoneally 50 times. Mg/kg STZ (STZ was prepared with 0.1 mol/L, pH 4.5 sodium citrate buffer before use, and used within 10 min), while the control group was injected with the same amount of sodium citrate solution. 72 After h hg blood glucose FBG is greater than 16.7 Methyl/L is a hyperglycemic diabetic model rat. Diabetic rats were randomly divided into a diabetic model group and a FTZEE size dose group (prepared by the method of Example 2). The large dose group is equivalent to human and rat doses of 40 mg per day. Mol / kg administration, 20 mg / 5 ml / kg daily in the small group, 5 per day in the model group and the normal group Ml/kg distilled water, once in the morning, and treated for 15 days. Blood samples (FBG) and serum insulin (Ins) were taken before, after, and after treatment in each group. Finally, rat pancreas was taken for HE staining and immunohistochemical staining.

Blood glucose was measured using an Advantage electronic blood glucose meter and a supporting strip (product of Roche, Switzerland). Glucose oxidase method for blood glucose and radioimmunoassay for serum insulin.

The experimental data were expressed as mean ± standard deviation (x ± s), and the significance test was performed by analysis of variance.

The results of blood glucose were shown in Table 14. Compared with the normal group, the FBG was significantly increased in the model group (P<0.01). After the large-dose treatment, the FBG was significantly decreased compared with the model group (P<0.01), indicating that FTZEE has significant hypoglycemic effect. .

Table 14 Table of blood glucose changes in rats in each group of FTZEE versus STZ model (mmol/L)

Group	n	Dosage (mg/kg)	Before modeling	Before treatment	After treatment
normal group	7		5.56±0.53	5.61±0.52	5.67±0.67
Model group	7		5.61±0.56	20.28±2.63 **	22.86±2.81 *
FTZEE small group	7	6	5.65±0.69	20.90±2.49 **	15.21±2.13 **△△▲▲
FTZEE large group	7	12	5.57±0.78	21.25±2.61 **	12.50±3.39 **△△▲▲
FTZ	7	250	5.68±0.64	20.79±2.34 **	17.01±2.33 **△▲▲

Note: Compared with the normal group, *P<0.05, **P<0.01; compared with the model group, △P<0.05, △△P<0.01; compared with before treatment ▲P<0.05, ▲▲P< 0.01.

The results of the effect on serum insulin are shown in Table 15. Compared with the normal group, the Ins was significantly lower in the model group (P<0.01). The serum Ins level was significantly higher than that in the model group after the large-dose treatment (P<0.05), indicating that FTZEE was secreted by Ins. There is a certain promotion.

Table 15 Table of serum insulin changes (MU/L) of FTZEE in STZ model rats

Group	n	Dosage (mg/kg)	Before modeling	Before treatment	After treatment
normal group	7		29.25±2.37	29.16±2.48	29.32±2.31
Model group	7		29.37±2.80	19.03±2.59 **△△	16.04±3.63 **
FTZEE small group	7	6	29.27±3.80	19.31±4.11 **△△	20.03±3.18 ▲▲
FTZEE large group	7	12	28.95±3.30	18.95±3.47 **△△	23.13±3.92 ▲▲
FTZ	7	250	29.26±2.64	18.39±2.84 **△△	19.98±2.39 ▲▲

Note: *P<0.05, **P<0.01 compared with the normal group. Compared with before modeling, △P<0.05, △△P<0.01; ▲P<0.05, ▲▲P<0.01 compared with the model group.

Histological observation of HE staining showed that the normal group of islets were round and elliptical cell clusters with clear boundary, the number of islets and the number of cells in the island were more, and the cytoplasm was abundant. The number of islets and the number of cells in the island decreased in STZ model group. Irregular, nuclear size varies, the shape is different, part of the nucleus is pyknosis, a few cells are vacuolized; the number of islets and the number of cells in the island are significantly increased in the high-dose group, the cells are evenly distributed, the nuclear size is basically equal, and the nuclear-free pyknosis The number of islets and the number of cells in the island increased in the low-dose group, the morphology was relatively regular, and a small part of the nucleus was pyknosis. This indicates that FTZEE has a significant protective effect on islet cell tissue damaged by STZ model and can promote insulin secretion.

Example 15 Clinical validation of prevention and/or treatment of hyperglycemia/diabetes

FTZEE tablets (Chinese herbal petroleum ether extract tablets, prepared in Example 5) for the treatment of 60 cases of diabetes

The test is based on the editor-in-chief of Zheng Yi, the guiding principle of clinical research on new Chinese medicine (Trial), China Medical Science and Technology Press, 1st edition, 2002, 233-237: The guiding principle of clinical research for the treatment of diabetes with new Chinese medicine.

1. Clinical data

Sixty patients were diagnosed as non-insulin-dependent hospitalized patients according to the 1993 diagnostic criteria, including 40 outpatients and 20 hospitalized patients; they were randomly divided into two groups. Among the 30 patients in the treatment group, 16 were male and 14 were female; 39 to 65 years old, average age (51.3 ± 8.6) years old. The duration of diabetes was 3 to 15 years, and the average duration of disease was (6.8±2.9) years; 19 patients with hyperlipidemia, 10 patients with coronary heart disease, and 18 patients with hypertension; in 30 patients in the control group, 15 males and 15 females; Age 38~65 years old; mean age (51.5±6.8) years; duration of diabetes 2.5-16 years, mean disease duration (6.9±3.1) years; 20 cases with hyperlipidemia, 9 cases with coronary heart disease, 17 cases with hypertension . The two groups were statistically treated in terms of gender, age, duration of disease, and complications. There was no significant difference (P>0.05), which was comparable.

2. Diagnostic criteria

Western diagnostic criteria (according to the 1999 WHO expert consultation report): fasting blood glucose (FPG) ≥ 7.0 Meng/L and 2 hours postprandial blood glucose (2 HPG) ≥ 7.0 mmol / L, random blood glucose ≥ 11.1 mmol / L.

High-pressure blood was developed in accordance with the 1999 World Health Organization/International Hypertension League classification method for hypertension (1999 World Health Organization/International Hypertension League guidelines on treatment of hypertension, Journal of Hypertension, 1999, 7(2): 9).

The diagnostic criteria for Chinese medicine refer to the clinical research guidelines for the treatment of diabetes mellitus (diabetes) by the Chinese medicine of the Ministry of Health of the People's Republic of China (1993): Anyone who has or has dry mouth, tiredness, fatigue, hunger, thirst, and shortness of breath Words, five upset fever, palpitations, insomnia, chest pain, urinary red constipation, red tongue or blush, pale purple or ecchymosis, fine pulse or string sputum, TCM syndrome differentiation is qi and yin deficiency, blood stasis Pulse card holder.

3. Treatment

The treatment group took the traditional Chinese medicine petroleum ether extract tablets (FTZEE tablets for short), one tablet each time, twice a day, two months for one course of treatment, during which other hypoglycemic drugs were stopped.

The control group was treated with Xiaoke'an Capsule (0.4g/granule, produced by Tonghua Baishan Pharmaceutical Co., Ltd.), 3 capsules each time, 3 times a day, and 2 months as a course of treatment for type 2 diabetes.

Both groups of patients underwent diabetes education, diet control, and moderate exercise. All cases were observed for 2 months.

4. Observation indicators

Blood pressure, fasting blood glucose (FBG) and 2 hours postprandial blood glucose before and after treatment (2 HFBG) (glucose oxidase method), glycosylated hemoglobin (HbALc), liver and kidney function, urine routine, blood pressure was detected simultaneously at 2, 4, and 6 weeks during treatment.

5. Clinical efficacy

The evaluation is based on the clinical research guidelines (1993) for the treatment of diabetes mellitus (diabetes) by the Ministry of Health of the People's Republic of China. Significant effect: Symptoms disappeared, laboratory tests were normal; effective: main symptoms and related laboratory tests improved; invalid: no change in symptoms and related laboratory tests.

The changes in blood glucose and hemorheology before and after treatment were shown in Tables 16 and 17.

Table 16 Comparison of blood glucose and HbALc before and after treatment in both groups

Project	FTZEE treatment group (n=30)	FTZEE treatment group (n=30)	Control group (n=30)	Control group (n=30)
	Before treatment	After treatment	Before treatment	After treatment
Blood glucose FBG (mmol/L)	10.3±2.4	6.8±1.7★★	10.2±2.5	7.01±1.8★★
2HFBG (mmol/L)	13.8±3.1	9.2±2.1★★	13.6±3.0	9.3±2.4★
HbALc (%)	8.8±1.6	7.0±1.3★	8.7±1.7	7.2±1.5★

Note: Compared with before treatment in this group, ★P<0.05, ★★P<0.01.

As can be seen from Table 16, the blood glucose and glycosylated hemoglobin in the two groups were significantly decreased after fasting and 2 hours after the treatment; there was a significant difference compared with the control group (P<0.01), and the control group also had similar effects after treatment, indicating that FTZEE Treatment of type 2 diabetes has a significant effect in improving blood sugar and urine protein.

Table 17 shows the efficacy statistics of FTZEE tablets on diabetes and metabolic syndrome, indicating that FTZEE tablets have the effect of lowering blood glucose in diabetic patients.

Table 17 Statistic of the efficacy of FTZEE tablets on diabetes and metabolic syndrome

Medicine	n	痊 愈	痊 recovery rate %	Significant example	Efficacy rate %	Effective example	Efficient %	Invalid	no efficiency %	Total efficiency %
FTZEE film	30	0	0	18	60.0	9	30.0	3	10.0	90.0
Xiaokean Capsule	30	0	0	16	53.4	10	33.3	4	13.3	86.7 *

Note: *P<0.05 compared with Xiaokewan group.

Therefore, the total effective rate of the traditional Chinese medicine petroleum ether extract (FTZEE) tablet for diabetes is 90.0%, which is markedly effective by 60.0%, which is slightly higher than the total effective rate of 87% of the commonly used drugs, and no significant side effects are observed.

Conclusion: FTZEE is confirmed by modern Chinese medicine pharmacology research to promote insulin secretion, increase serum insulin content, reduce blood sugar, improve blood sugar metabolism. Its mechanism of action may include:

(1) FTZEE can enhance B cell superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GSH-Px) activity and GSH content, and can inhibit lipid peroxidation and Clears hydroxyl radicals, resists oxidative stress damage, and promotes islet B cell regeneration.

(2) FTZEE can increase the expression of anti-apoptotic factor Bcl-2 gene in islet B cells and inhibit the expression of Bax and Fas genes in pancreatic islet B cells, thereby reducing the occurrence of apoptosis and stimulating the secretion of insulin by normal islet B cells.

(3) FTZEE can increase the expression of anti-inflammatory factor PPAR-a gene in islet B cells, antagonize the role of inflammatory factors, and reduce islet B cell damage. Increase serum insulin levels by protecting and repairing islet B cells.

The results of clinical research show that the traditional Chinese medicine petroleum ether extract FTZEE, which is harmful to the prevention and treatment of glycolipid metabolism, has significant hypoglycemic and blood sugar metabolism effects, and is clinically effective in treating hyperglycemia and diabetic blood glucose metabolism disorders.

Claims (9)

1. A petroleum ether extract of traditional Chinese medicine for preventing and controlling disorders of glycolipid metabolism, the active ingredients thereof are wax alcohol, β-sitosterol, n-hexadecanoic acid, atractylenolide III, oleanolic acid, berberine, jatrorrhizine , salvianolic acid B, Cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, ginsenoside Rb1
2. The petroleum ether extract of traditional Chinese medicine according to claim 1, characterized in that the alcohol, the β-sitosterol, the n-hexadecanoic acid, the atractylenolide III, the oleanolic acid, the berberine, the jatrorrhizine, the dan Phenolic acid B, The weight ratio of cyclotetracosane, 9,12-octadecadienoic acid, 5,7-dimethoxycoumarin, and ginsenoside Rb1 is from 1 to 5:1 to 8:1 to 8:1. 5: 1~8: 1 to 8:1 to 5:1 to 5:1 to 5:1 to 5:1 to 5:1 to 6
3. The method for preparing a petroleum ether extract of a traditional Chinese medicine according to claim 1, wherein the raw materials Danshen, Ligustrum lucidum, Rhizoma Coptidis, Daphnia, Eucommia, Atractylodes, Sanqi and Bergamot are extracted and/or extracted with water by C1-3. The total extract was combined, and the total extract was extracted with petroleum ether to obtain a petroleum ether extract of a traditional Chinese medicine for controlling glycolipid metabolism disorder.
4. The method of claim 3 including the steps of:

   (1) Alcohol extraction of C _1-3 and Ligustrum lucidum to obtain C _1-3 alcohol extract, water extraction and concentration of Datun, Atractylodes Rhizome, Radix Salvia, Eucommia, Bergamot and Rhizoma Coptidis to obtain water extract , combining C _1-3 alcohol extract and water extract to obtain total extract;

   (2) adding the total extract obtained in the step (1) to 0.5 to 15 volumes of petroleum ether, extracting 1 to 5 times, combining the extracts, and drying at a low temperature to obtain a petroleum ether extract of a traditional Chinese medicine for controlling glycolipid metabolism disorder.
5. The preparation method according to claim 4, wherein the C _1-3 alcohol extraction in the step (1) is carried out by using 30 to 95% by volume of C _1-3 alcohol for extraction 1 to 5 times, each time of extracting C. _The volume of _1-3 alcohol is 1 to 15 times the mass of the medicinal material, and the extraction time is 5 min to 5 h.
6. The preparation method according to claim 4, wherein the water extraction in the step (1) is decocted 1 to 5 times with water, and the volume of the water is 1 to 15 times the mass of the medicinal material, and each time the decocting is performed. Time is 5 Min~5 h.
7. The preparation method according to claim 4, wherein the concentration of the concentrated liquid in the step (1) is 0.2 to 5 times the mass of the medicinal material.
8. The process according to claim 4, wherein the obtained petroleum ether extract is processed into an oral preparation or an injection preparation in accordance with a pharmaceutically acceptable carrier.
9. The preparation method according to claim 8, wherein the oral preparation is a tablet, a capsule, a powder, a pill, a powder, a granule, a crystal, a solution, an extract, a suspension, a soup, a syrup, an elixir, a tea or oil.

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