Total flavonoid extract from <i>Gynura formosana </i>Kitam., preparation method thereof, and use of same in treating non-alcoholic fatty liver disease

Abstract

A total flavonoid extract from Gynura formosana Kitam., comprising 80-85% of rutin. A preparation method comprises selecting a complex enzyme consisting of a specific composition and ratio of enzymes for enzymatic hydrolysis, extracting and concentrating by a macroporous resin, and separating and purifying by a macroporous resin. The extract has therapeutic effect on non-alcoholic fatty liver disease.

Claims

1. A method for treating non-alcoholic fatty liver disease in a human in need thereof comprising administering an extract from Gynura formosana Kitam. to the human to effectively treat the non-alcoholic fatty liver disease, wherein the extract comprises 80% wt.-85% wt. of rutin, and is made by the method comprising the steps of: (1) extracting Gynura formosana Kitam., with an extraction solvent to obtain an extraction solution, and adjusting the extraction solution to a pH of 4-8 to obtain a reaction solution; (2) adding an enzyme complex comprising papain, cellulose and pectinase into the reaction solution to carry out enzymolysis through a forced circular reaction at a temperature of 30° C. to 50° C. for 1 hour to 4 hours, then carrying out suction filtration, and collecting a filtrate; (3) extracting the filtrate with a macroporous resin A to obtain an extracted solution, and concentrating the extracted solution to obtain a concentrated solution; wherein the macroporous resin A is selected from the group consisting of AB-8, DM-130, HZ841, ZH-00, ZH-01, ZH-02, ZH-03, CAD-40, CAD-45, BS-30 and mixtures thereof; and (4) centrifuging the concentrated solution, collecting a supernatant and carrying out elution by using a macroporous resin B, measuring absorbance at a wavelength of 510 nm, collecting the resulting eluate, concentrating and drying the eluate to obtain an extract from Gynura formosana Kitam; wherein the macroporous resin B is selected from the group consisting of D-101, D-140, D-141, XAD-3, XAD-4, HP-20, HP-21, LD-605, LSA-10 and mixtures thereof.

2. The method of claim 1, wherein the extract from Gynura formosana Kitam. as an active ingredient is mixed with a conventional auxiliary material and prepared according to a conventional process into a pharmaceutical preparation with clinically acceptable forms selected from the group consisting of tablets, capsules, powders, mixtures, pills, granules, syrups, plasters, suppositories, aerosols, ointments and injections.

3. The method of claim 1, wherein a weight ratio of the enzyme complex to the Gynura formosana Kitam. is 1:5 to 1:3.

4. The method of claim 1, wherein a weight ratio of papain to cellulase to pectinase in the enzyme complex is (0.5-1.5):(2-5):(1-3).

5. The method of claim 4, wherein the weight ratio of papain to cellulase to pectinase in the enzyme complex is 1:3:2.

6. The method of claim 1, wherein in the extraction in step 1, the extraction solvent is water, and a weight ratio of Gynura formosana Kitam. to water is 1:(20-60).

7. The method of claim 1, wherein in step 4, an ethanol aqueous solution with a volume concentration of 70-80% is adopted as an elution solvent, and the elution is performed at a rate of 3-15 m/h; and the concentrated solution comprises a total flavonoid from Gynura formosana Kitam. at a concentration of 0.5 mg/mL.

8. The method of claim 3, wherein a weight ratio of papain to cellulase to pectinase in the enzyme complex is (0.5-1.5):(2-5):(1-3).

9. The method of claim 8, wherein the weight ratio of papain to cellulase to pectinase in the enzyme complex is 1:3:2.

10. The method of claim 3, wherein in step 1, the extraction solvent is water, and a weight ratio of Gynura formosana Kitam. to water is 1:(20-60).

11. The method of claim 4, wherein in step 3, the extraction solvent is water, and a weight ratio of Gynura formosana Kitam. to water is 1:(20-60).

12. The method of claim 3, wherein: in step 4, an ethanol aqueous solution with a volume concentration of 70-80% is adopted as an elution solvent, and the elution is performed at a rate of 3-15 m/h; and the concentrated solution comprises total flavonoid from Gynura formosana Kitam. at a concentration of 0.5 mg/mL.

13. The method of claim 4, wherein: in step 4, an ethanol aqueous solution with a volume concentration of 70-80% is adopted as an elution solvent, and the elution is performed at a rate of 3-15 m/h; and the concentrated solution comprises total flavonoid from Gynura formosana Kitam. at a concentration of 0.5 mg/mL.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The present invention is described in detail with reference to examples and accompanying drawings, wherein:

(2) FIG. 1 shows an elution curve in Example 1 of the present invention;

(3) FIG. 2 shows an infrared spectrum of a total flavonoid extract from Gynura formosana Kitam. prepared in Example 1 of the present invention;

(4) FIG. 3 shows a HPLC chromatogram of a rutin control solution in Example 1 of the present invention;

(5) FIG. 4 shows a HPLC chromatogram of the total flavonoid extract from Gynura formosana Kitam. prepared in Example 1 of the present invention;

(6) FIG. 5 shows the effect on the liver histopathology of NAFLD rats by the total flavonoid extract from Gynura formosana Kitam. prepared in Example 1 of the present invention (40×, HE staining).

DETAILED DESCRIPTION OF THE EMBODIMENTS

(7) Gynura formosana Kitam. used in the following examples and experimental example of the present invention are taken from Dengke village, Longwen disctrict, Zhangzhou city, Fujian Province, and are identified as the Gynura formosana Kitam.

Example 1

(8) A total flavonoid extract from Gynura formosana Kitam. is prepared according to the following method:

(9) (1) Extraction: 100 g Gynura formosana Kitam. was added into water with a weight 30 times the weight of Gynura formosana Kitam. to carry out extraction, producing an extraction solution, which was then adjusted to a pH of 5, obtaining a reaction solution;

(10) (2) Enzymolysis: 25 g of complex enzyme consisting of papain, cellulase and pectinase with a weight ratio of 1:3:2 was added into the reaction solution to carry out enzymolysis through a forced circular reaction at 40° C. for 3 hours, and the resulted solution was suction filtered and a filtrate was collected;

(11) (3) Extraction and Concentration: The filtrate was added to an extraction tank containing AB-8 macroporous resin and stirred for 12 hours at 30° C. and 100 rpm, then filtered to obtain an absorbed AB-8 macroporous resin. An ethanol solution with a volume concentration of 75% was added to the absorbed AB-8 macroporous resin at an amount 20 times the weight of the absorbed AB-8 macroporous resin, then stirred for 12 hours at 30° C. and 120 rpm and then filtered to obtain an extracted solution. The extracted solution was vacuum concentrated to produce a concentrated solution which comprises a total flavonoid extract from Gynura formosana Kitam. at a concentration of 0.5 mg/mL;

(12) (4) Separation and Purification: The concentrated solution was centrifuged at 10,000 rpm for 10 minutes, and a supernatant was collected and placed into a chromatographic column filled with a macroporous resin D-101 for stationary adsorption for 60 min. Then the column was eluted with an aqueous solution of ethanol with a volume concentration of 75% at a rate of 5 m/h, and absorbance was measured at a wavelength of 510 nm. An elution curve was plotted with absorbance as Y-axis versus elution time as X-axis, as shown in FIG. 1. Eluate corresponding to the absorption peak area of the elution curve was collected, concentrated, and freeze-dried to obtain the total flavonoid extract from Gynura formosana Kitam.

(13) Through calculation, the extraction rate of the total flavonoid extract from Gynura formosana Kitam. is 2.0%.

(14) By referring to FIG. 1, the elution curve of the eluate shows a significant single absorption peak at 340 min, indicating the relatively pure flavonoid in the eluate.

(15) A. The total flavonoid extract from Gynura formosana Kitam. was identified with an infrared spectrum according to the following method:

(16) The method comprises the following steps: A certain amount of dried rutin standard was mixed with dried potassium bromide at a weight ratio of 1:100, ground and prepared into a solid pellet. The pellet was then tested with a Fourier infrared spectrophotometer within a scanning range of 4000 cm.sup.−1 to 400 cm.sup.−1, a resolution of 4 and a scanning number of 4, thereby obtaining an infrared spectrum. The total flavonoid extract from Gynura formosana Kitam. was tested in the same manner to obtain an infrared spectrum. The results are shown in FIG. 2.

(17) By referring to FIG. 2, both the infrared spectrums of the rutin standard and the total flavonoid extract from Gynura formosana Kitam. show a wide and strong absorption peak around 3685.455 cm.sup.−1 to 3018.177 cm.sup.−1, corresponding to telescopic vibration peak of-OH, indicating there are large amount of phenolic hydroxyl groups or sugar hydroxyl groups. A weak absorption peak occurs at 2914.036 cm.sup.−1, corresponding to a telescopic vibration peak of a carbon-hydrogen bond, indicating less hydrogen on saturated carbon. A strong peak appears at 1654.694 cm.sup.−1 in each spectrum, corresponding to a telescopic vibration of C═O. The peaks in the two spectrums appear at substantially same position and have substantially same shape, indicating that the extract is a flavonoid. Bending vibration peak of hydroxyl groups appears at 1371.88 cm.sup.−1 and 1362.89 cm.sup.−1. An absorption peak caused by ortho hydrogens of phenyl ring appears at 804.80 cm.sup.−1 and 810.56 cm.sup.−1. An absorption peak caused by the position of a substituent on the phenyl ring appears at 1010.07 cm.sup.−1 to 696.62 cm.sup.−1, but the peak position is different in the two spectrum, indicating the hydroxyl substitution position of the extract is different from that of the rutin standard. These results indicate that the extract contains hydroxyl, carbonyl, and other functional groups such as different position-substituted benzene rings, and the characteristic absorption peaks are substantially consistent. Thus, it can be determined that the extract is a flavonoid compound.

(18) B. The total flavonoid extract from Gynura formosana Kitam. was analyzed by liquid chromatography to determine the content of rutin therein according to the following method:

(19) B1. Liquid Chromatography Conditions

(20) Liquid Chromatography Conditions are as follows:

(21) Eclipse XDB-C18 AnalyticalGuard Column (4.6×12.5 mm, 5 μm) and ZOR BZX Eclipse XDB-C18 Column (4.6×150 mm, 5 μm) were used as a protection column. Flow rate is 0.5 mL/min; Column temperature is 35° C. Detection wavelength is 368 nm, 254 nm and 210 nm, respectively; Sample loading volume is 10 μL; Mobile phases consists of (A) 0.03% formic acid aqueous solution and (B) Acetonitrile; Gradient elution procedures are as follows: 0-10 min, 80% (A) and 20% (B); 10-12 min, 76% to 80% (A) and 20% to 24% (B); 12-20 min, 76% (A) and 24% (B); 20-25 min, 70% to 76% (A) and 24% to 30% (B); 25-48 min, 70% (A) and 30% (B).

(22) B2. Preparation of Control Sample Solution

(23) 0.001 g Rutin was weighed accurately and dissolved in 1 mL of methanol to prepare a single control sample solution of 1 mg/mL. The control sample solution was filtered with a disposable filter and then loaded into a small test tube for later use.

(24) B3. Determination

(25) The control sample solution and a test sample solution (1 μg/μL methanol solution of the total flavonoid extract prepared in Example 1) are respectively accurately sucked and injected to the liquid chromatography column to perform analysis according to the above mentioned liquid chromatography conditions.

(26) The HPLC chromatogram of the control sample solution is shown in FIG. 3, and the HPLC chromatogram of the test sample solution is shown in FIG. 4.

(27) By referring to FIG. 3, the rutin control solution can be completely isolated within 10 minutes. A substantially straight baseline is observed in the chromatogram of Rutin under the chromatographic conditions of this experiment, and peak tailing is not observed. There are no interfering peaks of impurities. The peaks appear earlier and the retention time is 2.745 min.

(28) By referring to FIG. 4, the amount of rutin in the extract was 81.29% as calculated by area normalization.

Example 2

(29) A total flavonoid extract from Gynura formosana Kitam. is prepared according to the following method:

(30) (1) Extraction: 100 g Gynura formosana Kitam. was added into water with a weight 20 times the weight of Gynura formosana Kitam. to carry out extraction, producing an extraction solution, which was then adjusted with diluted sodium hydroxide solution to a pH of 8, obtaining a reaction solution;

(31) (2) Enzymolysis: 20 g of complex enzyme consisting of papain, cellulase and pectinase with a weight ratio of 0.5:5:1 was added into the reaction solution to carry out enzymolysis through a forced circular reaction at 30° C. for 4 hours, and the resulted solution was suction filtered and a filtrate was collected;

(32) (3) Extraction and Concentration: The filtrate was added to an extraction tank containing DM-130 macroporous resin and stirred for 24 hours at 30° C. and 80 rpm, then filtered to obtain an absorbed DM-130 macroporous resin. An ethanol solution with a volume concentration of 95% was added to the absorbed DM-130 macroporous resin at an amount 10 times the weight of the absorbed DM-130 macroporous resin, then stirred for 24 hours at 30° C. and 80 rpm and then filtered to obtain an extracted solution. The extracted solution was vacuum concentrated to produce a concentrated solution which comprises a total flavonoid extract from Gynura formosana Kitam. at a concentration of 0.5 mg/mL;

(33) (4) Separation and Purification: The concentrated solution was centrifuged at 6,000 rpm for 8 minutes, and a supernatant was collected and placed into a chromatographic column filled with a macroporous resin HP-21 for stationary adsorption for 60 min. Then the column was eluted with an aqueous solution of ethanol with a volume concentration of 80% at a rate of 3 m/h, and absorbance was measured at a wavelength of 510 nm. An elution curve was plotted with absorbance as Y-axis versus elution time as X-axis. Eluate corresponding to the absorption peak area of the elution curve was collected, concentrated, and freeze-dried to obtain the total flavonoid extract from Gynura formosana Kitam.

(34) Through calculation, the extraction rate of the total flavonoid extract from Gynura formosana Kitam. is 1.82%.

(35) The total flavonoid extract from Gynura formosana Kitam. was analyzed by liquid chromatography to determine the content of rutin therein according to the method as described in section B of example 1. According to the resulted HPLC chromatogram, the amount of rutin in the total flavonoid extract was 80% in this example.

Example 3

(36) A total flavonoid extract from Gynura formosana Kitam. is prepared according to the following method:

(37) (1) Extraction: 100 g Gynura formosana Kitam. was added into water with a weight 60 times the weight of Gynura formosana Kitam. to carry out extraction, producing an extraction solution, which was then adjusted with a diluted hydrochloric acid to a pH of 4, obtaining a reaction solution;

(38) (2) Enzymolysis: 32 g of complex enzyme consisting of papain, cellulase and pectinase with a weight ratio of 1.5:2:3 was added into the reaction solution to carry out enzymolysis through a forced circular reaction at 50° C. for 1 hour, and the resulted solution was suction filtered and a filtrate was collected;

(39) (3) Extraction and Concentration: The filtrate was added to an extraction tank containing ZH-01 macroporous resin and stirred for 6 hours at 30° C. and 150 rpm, then filtered to obtain an absorbed ZH-01 macroporous resin. An ethanol solution with a volume concentration of 70% was added to the absorbed ZH-01 macroporous resin at an amount 30 times the weight of the absorbed ZH-01 macroporous resin, then stirred for 6 hours at 30° C. and 150 rpm and then filtered to obtain an extracted solution. The extracted solution was vacuum concentrated to produce a concentrated solution which comprises a total flavonoid extract from Gynura formosana Kitam. at a concentration of 0.5 mg/mL;

(40) (4) Separation and Purification: The concentrated solution was centrifuged at 8,000 rpm for 5 minutes, and a supernatant was collected and placed into a chromatographic column filled with a macroporous resin XAD-3 for stationary adsorption for 60 min. Then the column was eluted with an aqueous solution of ethanol with a volume concentration of 70% at a rate of 15 m/h, and absorbance was measured at a wavelength of 510 nm. An elution curve was plotted with absorbance as Y-axis versus elution time as X-axis. Eluate corresponding to the absorption peak area of the elution curve was collected, concentrated, and freeze-dried to obtain the total flavonoid extract from Gynura formosana Kitam.

(41) Through calculation, the extraction rate of the total flavonoid extract from Gynura formosana Kitam. is 1.91%.

(42) The total flavonoid extract from Gynura formosana Kitam. was analyzed by liquid chromatography to determine the content of rutin therein according to the method as described in section B of example 1. According to the resulted HPLC chromatogram, the amount of rutin in the total flavonoid extract was 85% in this example.

Experiment Example 1

(43) Study on treatment of NAFLD rats by the total flavonoid extract from Gynura formosana Kitam.

(44) 1. Experimental Objectives

(45) This example was carried out to study the treatment effect of the total flavonoid extract from Gynura formosana Kitam. on high-fat feed-induced non-alcoholic fatty liver disease (NAFLD) model rats.

(46) 2. Materials and Methods

(47) 2.1 Experimental Animals

(48) 62 healthy male Spague-Dawlay (SD) Rats of SPF grade, each weighed 210±10 g, were provided by Shanghai Sippr BK laboratory animal Co. Ltd. (Certification No: 2007000531494; animal license No.: SCXK (Hu) 2007-0005).

(49) 2.2 Test Drugs and Experimental Reagents

(50) The total flavonoid extract from Gynura formosana Kitam. prepared in Example 1 was diluted with distilled water to form test drugs of different concentrations: a low dose of 6 mg/10 mL, a medium dose of 12 mg/10 mL, and a high dose of 24 mg/10 mL.

(51) Kits for total triglyceride (TG), total cholesterol (TC), alanine aminotransferase (ALT), aspartic acid transferase (AST), high-density lipoprotein (HDL-C), and low-density lipoprotein (LDL-C) were purchased from NanJing JianCheng Bioengineering Institute. High-fat feed (comprising lard 10%, cholesterol 2%, pig bile salt 0.7%, base feed, 87.3%) was provided by Fuzhou Minhou Zhuqi Animal Service Center. Common feed was provided by Experimental Animal Center of Fujian University of Traditional Chinese Medicine.

(52) 2.3 Experimental Instruments

(53) The following instruments were used: Electronic Balance (Ohaus International Trading (Shanghai) Co., Ltd.), BX51T-PHD-J11 Microscope (Olympus, Japan), Low Speed Centrifuge (Thermo, US), Biological Tissue Paraffin Embedding Machine (Yaguang Medical Electronic Technology Co., Ltd, Xiaogan, Hubei), Biological Tissue Automatic Dehydrator (Yaguang Medical Electronic Technology Co., Ltd), Full-Automatic Paraffin Slicing Machine (Leica, Germany), BS-120 Full-Automatic Biochemical Analyzer (Mindray Biomedical Electronics Co. Ltd., Shenzhen).

(54) 3. Experimental Methods

(55) 3.1 Animal Grouping and Model Establishment

(56) After one week adaptive feeding, 62 male SD rats of SPF-grade were randomly divided into two groups by body weight: a control group (16 rats) and high-fat feed group (46 rats), respectively. Rats in the control group were fed with the base feed, and rats in the high-fat feed group were fed with the high-fat feed. The rats were given free access to water and feed, and exposed to a 12-hour light/dark cycle. The rats were weighed once every week.

(57) After six weeks, 6 rats were randomly selected and sacrificed in each of the control group and the high-fat diet group, and liver tissue was taken to prepare pathological slices. Pathological observation of the slices showed that hepatocyte steatosis was serious in liver of rats in the model group, and hepatocyte steatosis exceeded more than ⅔ per unit area. Serious diffuse large-bubble hepatocyte steatosis, hepatic cell point-like necrosis and moderate fibrosis hyperplasia were observed in the liver tissues, indicating that the non-alcoholic fatty liver disease model was successfully established.

(58) After the model is successfully established, the remaining 40 rats in the high-fat feed group were randomly divided into a model group, a low dose group of the total flavonoid extract, a medium dose group of the total flavonoid extract and a high dose group of the total flavonoid extract, with 10 rats in each group. Starting from the 7th week, rats in the control group and the model group were administered via lavage with saline, and rats in other groups were administered with different doses of the total flavonoid extract, wherein rats in the low dose group was administered with the total flavonoid extract at a dose of 6 mg/10 ml, rats in the medium dose group was administered with the total flavonoid extract at a dose of 12 mg/10 mL, and rats in the high dose group was administered with the total flavonoid extract at a dose of 24 mg/10 mL. At the end of the 10th week, the rats fasted for 12 hours overnight, and next day were anesthetized intraperitoneally with pentobarbital sodium at a dose of 40 mg/kg. Blood was collected from abdominal aortic and centrifuged at 3000 r/min for 15 min to obtain a serum which is then stored in a refrigerator at −80° C. for use in subsequent detection.

(59) 3.2 Experimental Data Observation and Detection

(60) During the experiment, the activity, hair gloss, appetite, death and the like of the rats were recorded. Weights of the rats were recorded weekly. Liver and visceral fat pad were collected and weighed. Photos were taken to observe the general morphology of the liver of rats in each group. Then, a portion of the right lobe of liver was fixed with 4% paraformaldehyde solution and routinely embedded in paraffin for pathological slicing. The obtained slices were routinely stained with HE, and observed with an optical microscope. Tissue hepatocyte steatosis, inflammation and necrosis degree were evaluated by referring to the NAFLD diagnosis standard established by the fatty liver and alcoholic liver diseases unit of the Chinese Society of Hepatology. A full-automatic biochemical analyzer was used to determine contents of alanine aminotransferase (ALT), aspartate aminotransferase (ASL), cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL-C), and low density lipoprotein (LDL-C) in serum.

(61) 4. Experimental Data Processing

(62) All experimental data were analyzed using software SPSS 18.0. The results were presented in the form of mean value±standard deviation (x±s). Comparison between data in a plurality of groups was carried out using single-factor variance analysis, and comparison between data of two groups was carried out using a T-test. Count data was analyzed using rank test. P<0.05 was considered statistically significant.

(63) 5. Experimental Results

(64) 5.1 Effect of the Total Flavonoid Extract From Gynura formosana Kitam. on Liver General Morphology of NAFLD Rats

(65) Rats in the control group manifested bright red livers with normal size, regular shape, soft texture and smooth surface. Rats in the model group showed obviously swelled livers with a cream-yellow color and an obtuse and thick edge. Diffuse fine particles-like ridges were observed on the liver surface. The slice of the liver was greasy and the texture is relatively brittle. Local yellow white denaturing lesions were observed. Rats in each dose group of the total flavonoid extract manifested slightly swelled livers compared with the normal group. The color is slightly reddish than that of the model group, and is close to the normal color. The slice of the liver was not obviously greasy.

(66) 5.2 Effect of the Total Flavonoid Extract From Gynura formosana Kitam. on Liver Histopathology of NAFLD Rats

(67) Effect of the total flavonoid extract from Gynura formosana Kitam. on liver histopathology of NAFLD rats is shown in FIG. 5.

(68) By referring to FIG. 5, rats in the control group shows intact structure of hepatic lobule, with clear cellular outline. The central vein is large with thin walls. The hepatic cords are radially arranged. No hepatocyte steatosis and inflammatory cell infiltration were observed. In rats of the model group, diffuse large fat vacuoles were widely observed in cytoplasm of the liver tissue, and the liver cells were swelled. The hepatic cords became disordered and the liver sinuses were narrowed. Cell nucleus was squeezed towards one side. The most obvious lesions were observed around the central vein, including inflammatory cell infiltration which was also involved in hepatic lobule. Main lesions were large fat vacuoles. For rats in each dose group of the total flavonoid extract, the amount and degree of hepatocyte steatosis were obviously reduced compared with the model group, and only small fat vacuoles were observed under the high power microscope.

(69) 5.3 Effect of the Total Flavonoid Extract From Gynura formosana Kitam. on Liver Weight and Visceral Fat Pad of NAFLD Rats

(70) The effect of the total flavonoid extract from Gynura formosana Kitam. on liver weight and visceral fat pad of NAFLD rats is shown in Table 1.

(71) TABLE-US-00001 TABLE 1 Effect of the total flavonoid extract from Gynura formosana Kitam. on liver weight and visceral fat pad of NAFLD rats (n = 10, x ± s) Dose Liver weight Visceral fat pad Group (mg/10 mL) (g) (g) Control group — 12.88 ± 2.25  16.37 ± 4.69  Model group —  23.90 ± 3.52.sup.## 19.84 ± 5.94.sup.#  Low dose group 6 17.70 ± 2.58* 13.27 ± 3.71* Medium dose group 12 17.00 ± 2.25* 13.64 ± 2.98* High dose group 24 14.18 ± 1.38* 13.66 ± 3.48* Note: Compared with the control group, .sup.#P < 0.05, .sup.##P < 0.01, and compared with the model group *P < 0.05.

(72) Table 1 shows that, rats in the model group exhibited significantly increased liver weight compared to the control group, and the difference is statistically significant (P<0.01). The difference between each dose group of the total flavonoid extract and the model group is statistically significant (P<0.05). The weight of visceral fat pad (perinephric and epididymal fat) of rats in the model group was significantly increased compared to the control group. Rats in each dose group exhibited reduced liver weight and reduced weight of visceral fat pad, and the difference is statistically significant (P<0.05). However, the difference in the weight of visceral fat pad among the experimental groups is not statistically significant (P>0.05).

(73) 5.4 Effect of the Total Flavonoid Extract From Gynura formosana Kitam. on Biochemical Indexes in Serum of NAFLD Rats

(74) The effect of the total flavonoid extract from Gynura formosana Kitam. on biochemical indexes in serum of NAFLD rats is shown in Tables 2 to 4.

(75) TABLE-US-00002 TABLE 2 Effect of the total flavonoid extract from Gynura formosana Kitam. on TG and TC contents in serum of NAFLD rats (n = 10, x ± s) Dose TG TC Group (mg/10 mL) (mmol/L) (mmol/L) Control group — 0.54 ± 0.08  1.79 ± 0.11  Model group — 0.85 ± 0.15.sup.#  2.11 ± 0.22.sup.#  Low dose group 6 0.62 ± 0.22* 1.90 ± 0.08* Medium dose group 12 0.51 ± 0.13* 1.84 ± 0.21* High dose group 24 0.54 ± 0.10* 1.62 ± 0.25* Note: Compared with the control group, .sup.#P < 0.01, and compared with the model group *P < 0.05.

(76) TABLE-US-00003 TABLE 3 Effect of the total flavonoid extract from Gynura formosana Kitam. on ALT and AST contents in serum of NAFLD rats (n = 10, x ± s) Dose ALT AST Group (mg/10 mL) (U/L) (U/L) Control group — 30.69 ± 4.33  176.58 ± 38.25  Model group — .sup. 62.00 ± 13.15.sup.# 268.55 ± 44.71.sup.#  Low dose group 6 31.11 ± 8.07* 203.00 ± 35.77* Medium dose group 12  27.43 ± 13.93* 185.88 ± 42.86* High dose group 24 36.00 ± 7.23* 180.83 ± 18.06* Note: Compared with the control group, .sup.#P < 0.05, and compared with the model group *P < 0.05.

(77) TABLE-US-00004 TABLE 4 Effect of the total flavonoid extract from Gynura formosana Kitam. on HDL-C and LDL-C contents of NAFLD rats (n = 10, x ± s) Dose HDL-C LDL-C Group (mg/10 mL) (mmol/L) (mmol/L) Control group — 0.79 ± 0.09 0.95 ± 0.13  Model group — .sup. 0.52 ± 0.06.sup.# 1.78 ± 0.14.sup.#  Low dose group 4.375 0.49 ± 0.05 1.83 ± 0.10  Medium dose group 8.75 0.46 ± 0.07 1.27 ± 0.27* High dose group 17.5 0.55 ± 0.07 1.53 ± 0.22* Note: Compared with the control group, .sup.#P < 0.05, and compared with the model group *P < 0.05.

(78) Tables 2 and 3 show that, rats in the model group exhibited a significant increase in TG, TC, ALT and AST contents in serum compared to the control group, and the difference is statistically significant (P<0.05). Rats in each dose group of the total flavonoid extract exhibited a significant decrease in TG, TC, ALT and AST contents in serum compared to the model group, and the difference is statistically significant (P<0.05).

(79) Table 4 shows that, rats in the model group exhibited a significantly increased LDL-C content and a significantly decreased HDL-C content, both differences of which are statistically significant (P<0.05). Rats in each dose group of the total flavonoid extract did not show statistically significant difference in HDL-C content compared to the model group. Rats in medium and high dose groups of the total flavonoid extract showed significantly decreased LDL-C content compared to the model group (P<0.05).

(80) 6. Experimental Conclusions

(81) The total flavonoid extract from Gynura formosana Kitam. has a good treatment effect on non-alcoholic fatty liver disease, and can obviously improve the blood lipid disorder and hepatocyte steatosis of non-alcoholic fatty liver disease rats.

(82) It is to be understood that the above-described examples are merely illustrative of the embodiments and are not intended to limit the embodiments. It will be apparent to one of ordinary skill in the art that other different forms of changes or variations can be made on the basis of the above description. It is to be understood that various changes or modifications may be made herein without departing from the scope of the invention as defined by the appended claims.