LU500041B1 - Preparation Method of Highly Active Total Flavonoids of Hedyotis Diffusa Willd and Applications Thereof in Peroxidative Liver Injury - Google Patents

Abstract

The present invention relates to the technical field of extraction of traditional Chinese medicinal materials and medicines, in particular to a preparation method of highly active total flavonoids of Hedyotis diffusa Willd, that is, total flavonoids of Hedyotis diffusa Willd is extracted by directly using a small amount of cellulose. Compared with other extraction methods, the method disclosed by the present invention has the advantages of simple operation, low energy consumption, higher feasibility, and higher biological activity of the extracted total flavonoids (higher content of isoflavones). In addition, three components that are not extracted by other extraction methods can be extracted by the method for preparing the total flavonoids of Hedyotis diffusa Willd, i.e., dihydrokaempferol, apigenin and chrysin; and six components with significantly increased content (up-regulated by more than 2 times) are obtained, i.e., naringin, vitexin, isovitexin, genistein, luteolin and 7-O-glucoside luteolin.

Description

DESCRIPTION Preparation Method of Highly Active Total Flavonoids of Hedyotis Diffusa Willd and Applications Thereof in Peroxidative Liver Injury

TECHNICAL FIELD The present invention relates to the technical field of extraction of traditional Chinese medicinal materials and medicines, in particular to a preparation method of highly active total flavonoids of Hedyotis diffusa Willd and applications thereof in peroxidative liver injury.

BACKGROUND Hedyotis diffusa Willd is the dried whole herb of Hedyotis diffusa Willd of the family Rubiaceae. The herb is bitter, flat and nontoxic, with meridian tropism in stomach, large intestine and small intestine. Hedyotis diffusa Willd was first seen in the Records of Guangxi Traditional Chinese Medicine, and is also known as Oldenlandia diffusa Roxb. Hedyotis diffusa Willd is an annual herb, and the whole plants can be used as herbal medicine. As a widely distributed medicinal plant, the wild type herb is mainly produced in Zhejiang, Jiangxi, Hunan, Hubei, Guangdong, Guangxi, Fujian and other places in China; and widely distributed in Yunnan and Guizhou; and has also been cultivated in Henan in recent years. With a variety of active components including flavonoids, anthraquinone, terpenoids, sterols, polysaccharides, organic acids, alkanes, minor elements and volatile oil, Hedyotis diffusa Willd has various pharmacological activities such as clearing away heat and toxic materials, eliminating carbuncle and removing stasis, promoting urination and removing dampness, resisting tumor, protecting liver, resisting inflammation, resisting oxidation and regulating immunity, and is thus widely used for clinical treatment of various inflammations and tumors, including hepatitis, tonsillitis, sore throat, appendicitis and urinary tract infection, and a variety of cancers such as liver cancer, lung cancer and gastric cancer. Flavonoids are one of the active components of Hedyotis diffusa Willd that have various pharmacological effects. Common extraction methods of plant flavonoids include solvent extraction, heating reflux extraction, ultrasonicassisted extraction, microwaveassisted extraction and supercritical CO. fluid extraction. The existing extraction methods have the technical problems of complex methods, high energy consumption and low activity of the extracted total flavonoids. In order to fully exploit and utilize total flavonoids of Hedyotis diffusa Willd, a simple, efficient and environment-friendly extraction method is to be further developed.

Liver injury is a common clinical disease that endangers human health, and is a complex biological process mediated by multiple factors. As an important detoxification organ of the human body, the liver is most vulnerable to toxic substances. Abnormal liver function can be seen clinically, and hepatic failure can occur in severe cases. Mechanisms that cause chemical damage to hepatocytes include oxygen radicals, intracellular calcium overload, abnormal lipid metabolism, cholestasis and metabolic disorders of minor elements. Liver injury can be divided into immune and non-immune liver injury. Common viral hepatitis (e.g., viral hepatitis B and C), autoimmune hepatitis and some drug-induced liver injury are immune-mediated hepatocyte injury. Non-immune liver injury is found mainly in peroxidative liver injury induced by chemical substances such as alcohol, chemical poisons and drugs. According to the survey, fatty liver disease has become the second largest liver disease after viral hepatitis, and is seriously threatening the health of the nation. Studies have shown that there are many causes of fatty liver disease. According to the history of long-term excessive alcohol consumption, fatty liver disease is broadly classified into alcoholic fatty liver disease and non-alcoholic fatty liverdisease or both, and fatty liver disease is mainly non-alcoholic fatty liver disease. With the economic development and social progress, the incidence of non-alcoholic fatty liver disease has reached 15% in economically developed regions of China.

The pathogenesis of non-alcoholic fatty liver disease is very complex. The “two-hit” theory proposed by Day and Janmes in 1998 has become the main theory to explain the pathogenesis of non-alcoholic fatty liver disease. The first hit mainly refers to hepatocyte steatosis caused by insulin resistance and lipid metabolism disorder, resulting in relatively low hepatocyte activity; and the second hit mainly refers to oxidative stress and lipid peroxidation damage for various reasons, resulting in inflammation, necrosis and even fibrosis of steatotic hepatocytes. Reactive oxygen species (ROS) plays a very important role in the second hit. Non-alcoholic fatty liver disease is peroxidative liver injury.

SUMMARY In order to solve the technical problems of complex operation, high energy consumption and low biological activity of extracted total flavonoids in the existing extraction methods of plant flavonoids, the present invention provides a preparation method of highly active total flavonoids of Hedyotis diffusa Willd.

In order to solve the technical problems, the present invention is realized by the following technical solutions: a preparation method of highly active total flavonoids of Hedyotis diffusa Willd, comprising the following steps: a. sample pretreatment, drying and crushing Hedyotis diffusa Willd, sieving through a 60-mesh sieve (with an aperture of 0.25mm), ultrasonically treating with petroleum ether, discarding the petroleum ether, filtering the residue and evaporating the petroleum ether, repeating the operation once, drying and cooling for later use; b. adding cellulase to pretreated Hedyotis diffusa Willd samples from the step a, extracting total flavonoids with 50-70% ethanol as a solvent, filtering under vacuum, and merging filtrates to obtain a crude extract of total flavonoids of Hedyotis diffusa Willd, and c. purifying the crude extract of total flavonoids of Hedyotis diffusa Willd with a 20-60-mesh low-polar macroporous adsorption resin, washing with distilled water to remove watersoluble impurities, then eluting with 70% ethanol, collecting the eluent, concentrating under vacuum, freeze-drying and crushing to obtain refined total flavonoids of Hedyotis diffusa Willd.

An application of the total flavonoids of Hedyotis diffusa Willd in prevention of peroxidative liver injury induced by chemical substances including alcohol, chemicals and drugs.

Further, the addition of the cellulase in the step b is 0.5-1.0% of the mass of the pretreated Hedyotis diffusa Willd samples; and the material-liquid ratio is 1:30-1:50 g/mL.

Further, pH of the crude extract in the step b is 4.0-5.5.

Further, enzymolysis temperature in the step b is 50-60 °C.

Further, enzymolysis time in the step b is 1-1.5h.

Six components with significantly up-regulated content (up-regulated by more than 2 times), i.e., naringin, vitexin, isovitexin, genistein, luteolin and 7-O-glucoside luteolin, as well as three components obtained only by the method of the present invention, i.e., dihydrokaempferol, apigenin and chrysin, are identified by UPLC-MS of the refined total flavonoids of Hedyotis diffusa Willd prepared by the present invention. The nine components have anti-inflammatory, anti-oxidation, anti-tumor and hepatoprotective effects. The total flavonoids of Hedyotis diffusa Willd prepared by the present invention have significantly improved biological activity.

The results of in vitro cell experiments show that the refined total flavonoids of Hedyotis diffusa Willd prepared by the present invention can significantly lower elevated levels of ALT, AST, LDH and ALP caused by H202-induced HL-02 cell injury, and have a protective effect on peroxidative liver injury; and the effect is significantly better than that of total flavonoids of Hedyotis diffusa Willd prepared by other methods.

The results of in vivo animal experiments show that the refined total flavonoids of Hedyotis diffusa Willd prepared by the present invention are safe and low in toxicity, have a therapeutic effect on non-alcoholic fatty liver disease, and can also lower cholesterol and triglyceride levels in the body and lower blood lipid while protecting the liver.

In addition, the present invention further provides an application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method in treatment of peroxidative liver injury.

An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method in prevention of peroxidative liver injury induced by chemical substances including alcohol, chemicals and drugs.

An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method in prevention of H2Oz-induced HL-02 cell injury, and an application thereof in lowering of levels of ALT, AST, LDH and ALP and other liver injury indexes.

An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method in treatment and prevention of liver injury caused by fatty liver disease.

An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method in lipid-lowering effect for non-alcoholic fatty liver disease.

Compared with the prior art, the present invention has the following advantageous effects: The present invention provides a preparation method of highly active total flavonoids of Hedyotis diffusa Willd, by which active components not found by other methods can be extracted and the content of active components with biological activity can be increased. Highly active and less toxic total flavonoids of Hedyotis diffusa Willd can be obtained by extracting total flavonoids of Hedyotis diffusa Willd byenzymolysisin the present invention to prevent and treat peroxidative liver injury while loweringblood lipid to some extent. This provides a reference for the further exploitation and utilization of Hedyotis diffusa Willd, and the total flavonoids of Hedyotis diffusa Willd can be developed into new drugs and health food against liver injury. In addition, the refined total flavonoids of Hedyotis diffusa Willd provided by the present invention can be prepared into common clinical dosage forms, which is convenient for clinical application.

Compared with conventional extraction methods such as reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction, the content of naringin, vitexin, isovitexin, genistein, luteolin and 7-O-glucoside luteolin in the total flavonoids of Hedyotis diffusa Willd prepared by the method of the present invention is significantly increased (p<0.05) (up-regulated by more than 2 times), and another three components are identified, i.e., apigenin, chrysin and dihydrokaempferol. Compared with other preparation methods, the total flavonoids prepared by enzymolysis in the present invention contain higher contents of apigenin, chrysin, genistein, naringin, vitexin and isovitexin (more than 3.5 times) and other active components with hepatoprotective effects, so that the total flavonoids prepared by enzymolysis in the present invention have more efficient hepatoprotective activity.

BRIEF DESCRIPTION OF THE FIGURES Fig. 1 shows RSD distribution of QC samples in Example 2; 1: rutin; 2: naringin; 3: soybean agglutinin; 4: daidzin; 5: isoliquiritigenin; 6: genistin; 7: naringenin; 8: luteolin; 9: kaempferol; 10: dihydroquercetin; 11: myricetin; 12: quercitrin; 13: vitexin; 14: isovitexin; 15: isoquercitrin; 16: glycyrrhizin; 17: daidzein; 18: genistein; 19: eriodictyol; 20: quercetin; 21: apigenin; 22: chrysin;

23: isornamnetin; 24:7-O-glucoside luteolin.

Fig. 2 is a volcano plot drawn by comparing and analyzing all data using t-test and fold change (FC) analysis method in Example 2; A represents enzymolysis; B represents reflux extraction; C represents ultrasonic extraction; D representsenzymolysis-ultrasonic assisted extraction; AvsB, AvsC, AvsD, BvsC, BvsD and CvsD represent pairwise comparison of the four preparation methods respectively.

Fig. 3 shows screening test results of hepatoprotective activity of cells in vitro in Example 3; Fig. 3(a) shows screening results of hydrogen peroxide modeling concentration; * (P<0.05) and ** (P<0.01) indicate significant difference from the control group; Fig. 3(b) shows screening results of hepatoprotective activity of total flavonoids of Hedyotis diffusa Willd by different preparation methods, in which A represents enzymolysis, B represents heating reflux extraction, C represents ultrasonic extraction, and D represents enzymolysis-ultrasonic assisted extraction. The positive drug is 5 mg/mL bifendate, * (P<0.05) indicates a significant difference compared with enzymolysis in group A; Fig. 3(c) shows the effects of six flavonoids on the survival rate of HL-02 cells,* (p<0.05) and ** (p<0.01) indicate significant differences compared with the model group; and ## (P<0.01) indicates a significant difference compared with the control group.

Fig. 4 shows measured results of AST, ALT, LDH and ALP indexes in Example 3; ## (P<0.01) indicates a significant difference compared with the control group; a* (P<0.05) and a** (P<0.01) indicate significant differences compared with the model group; b* (P<0.05) indicates a significant difference compared with enzymolysis; A enzymolysis; B reflux extraction; C ultrasonic extraction; D enzymolysis-ultrasonic assisted extraction; bifendate: 5 mg/mL.

Fig. 5 shows morphology of HL-02 cells in Example 3; Fig. 5(a): normal HL-02; Fig. 5(b): H2O2-damaged HL-02; Fig. 5(c): HL-02 pretreated with bifendate; Fig. 5(d): HL-02 pretreated with low-dose total flavonoids of Hedyotis diffusa Willd; Fig. 5(e): HL-02 pretreated with medium-dose total flavonoids of Hedyotis diffusa Willd, and Fig. 5(f): HL-02 pretreated with high-dose total flavonoids of Hedyotis diffusa Willd, all photos were taken under 10x microscope.

DESCRIPTION OF THE INVENTION The present invention will be further described in combination with specific examples.

Example 1 Method for extraction of highly active total flavonoids of Hedyotis diffusa Willd by enzymolysis

1. Experimental method A highly active total flavonoids of Hedyotis diffusa Willd for preventing and treating non-immune liver injury was prepared by the following preparation method: sample pretreatment: drying and crushing Hedyotis diffusa Willd, sieving through a 60-mesh sieve (with an aperture of 0.25 mm), ultrasonically treating with petroleum ether, discarding the petroleum ether, filtering the residue and evaporating the petroleum ether, repeating the operation once, drying and cooling for later use. The step was intended to remove chlorophyll and liposoluble components from Hedyotis diffusa Willd, and facilitate the dissolution of flavonoids in the extraction process.

Adding 0.5-1.0% cellulase to the pretreated Hedyotis diffusa Willd powder, adding 30-50X of 50% ethanol solution by volume, adjusting pH to 4.0-5.5, then extracting by enzymolysis at 50-60°C for 1-1.5h, inactivating in boiling water for 5min after enzymolysis, filtering under vacuum while hot, and merging filtrates to obtain a crude extract of total flavonoids of Hedyotis diffusa Willd.

Finally, purifying the crude extract of total flavonoids of Hedyotis diffusa Willd with an AB-8 macroporous adsorption resin, washing with distilled water to remove watersoluble impurities, then eluting with 70% ethanol, collecting the eluent, concentrating under vacuum, freeze-drying and crushing to obtain refined total flavonoids of Hedyotis diffusa Willd.

With the same technical parameters, four samples of total flavonoids of Hedyotis diffusa Willd were prepared by heating reflux extraction (without cellulase), ultrasonic extraction (without cellulase) and combined enzymolysis (taking enzymolysis-ultrasonic assisted extraction as an example) for subsequent analysis of components and activities.

2. Experimental results The extraction rate, yield and content of total flavonoids of Hedyotis diffusa Willd obtained by different preparation methods were compared in the experiment. The detailed results are shown in Table 1. It can be seen from the results that both the extraction rate and content of total flavonoids are higher by enzymolysis and combined enzymolysis compared with heating reflux extraction and ultrasonic extraction. No significant difference was observed between enzymolysis and enzymolysis-ultrasonic assisted extraction, but compared with enzymolysis-ultrasonic assisted extraction, the content of isoflavones (4.3+0.9 ug/g) in total flavonoids of Hedyotis diffusa Willd prepared by direct enzymolysis was significantly higher (p<0.05, see Table 4), the protective activity against liver injury was higher (see Fig. 3(b)), the content of naringin, vitexin, isovitexin, genistein, luteolin and 7-O-glucoside luteolin in the total flavonoids increased significantly (p<0.05) (up-regulated by more than 2 times), and another three components, i.e., apigenin, chrysin and dihydrokaempferol were identified, among which apigenin, chrysin, genistein, naringin, vitexin and isovitexin (more than 3.5 times) are active components with hepatoprotective effects (see Table 5, Fig. 3(c) and Fig. 4), and the operation was simpler and energy consumption was less, so that direct enzymolysis was more suitable for the needs of actual industrial production.

Table 1 Comparison of different preparation methods of total flavonoids of Hedyotis diffusa Willd (n=3) Extraction Purification Yield of total Content of total Extraction method rate flavonoids flavonoids % method % ma/ Enzymolysis 1.08+0.04 AB-8 4.96+x0.02 170.33x2.19 Reflux extraction 0.74+0.02” macro Jorous 4.66+0.12" 154.01+1.19” Ultrasonic extraction 0.67+0.04” on 4330.17" 145.63+1.86™ Enzymolysis-ultrasonic 4 9.0 03 Adsorption 4.91:0.03 168.77+2.99 assisted extraction * (p<0.05) and ** (p<0.01) indicated significant differences compared with enzymolysis. Example 2 Analysis on components of total flavonoids of Hedyotis diffusa Willd prepared by different methods

1. Experimental method Components of total flavonoids of Hedyotis diffusa Willd prepared by different methods in Example 1 were analyzed by UPLC-MS, and the content of flavonoids was determined to find out different components between cellulose-assisted extraction and other extraction methods.

A Waters column (HSS T3 2.5um, 2.1mmx150mm column) was used, with phase A water (0.1% formic acid):phase B acetonitrile (0.1% formic acid) as the mobile phase; with a flow rate of 1 mL/min; a column temperature of 4°C; and a sample size of 5 pL. Gradient elution procedures were given in Table 2. An Sciex 5500 QTRAP mass spectrometer was used for MRM mass spectrometry in a positive and negative ion switching mode. Parameters of an ion source in a positive ion mode were as follows: Source temperature, 550°C; Gas 1, 55; Gas 2, 50; CRU, 30; ISVF, 5500V. Parameters of an ion source in a negative ion mode were as follows: Source temperature, 550°C; Gas 1, 55; Gas 2, 50; CRU, 30; ISVF, -4500V.

Table 2 Gradient elution procedures Time (min) Phase A water (0.1% formic Phase B acetonitrile (0.1% acid) formic acid) 0 95 5 3 80 20 43 80 20 9 55 45 11 2 98 13 2 98

13.1 95 5 95 5

2. Experimental results

All samples were mixed in an equal amount to prepare QC samples, and the precision and stability of detection instruments were investigated by using the QC samples. The RSD% results of the QC samples are shown in Fig. 1, and the RSD% of each of the component to be determined is less than 30%, indicating that the experimental data were stable and reliable, and the precision of the instruments was good.

In the experiment, the working curve and linear range of each of the components to be determined were determined. According to the results, the components to be determined had good linearity within the linear range, with correlation coefficient greater than 0.99. The detailed results are shown in Table 3 (only some of results are listed).

Table 3 Working curve and linear range (partial) No Sample Name Linear R LinearRange(pg/mL) 1 Naringin Y=1.31811X-2461.84809 0.99904 25-125000 2 Genistein Y=7.82968X+100786 0.99646 25-125000 3 Luteolin Y=4.19309X-15833.07716 0.99988 250-125000 4 Vitexin Y=8.40506X-26598.52129 0.99971 25-125000 Isovitexin Y=8.82798X-10218.14071 0.99959 12.5-125000 6 7-O-glucoside luteolin Y=10.73673X-21914.3645 0.99973 125-125000 7 Apigenin Y=12.62999X+74927.3 0.99844 12.5-125000 8 Chrysin Y=4.80997X+1997.92924 0.99957 62.5-125000 9 Dihydrokaempferol ~~ Y=1.16739X-11745.38093 0.99670 250-125000 In the experiment, the content of total flavonoids of Hedyotis diffusa Willd prepared by different methods was determined. A represents the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention, B represents heating reflux extraction, C represents ultrasonic extraction, and D represents enzymolysis-ultrasonic assisted extraction. The content determination results are shown in Table 4.

Table 4 Content determination results Content (ug/g) Category ; Reflux Ultrasonic Enzymolysis-ultrasonic Enzymolysis ; ; N ; extraction extraction assisted extraction Flavonoid „76 546.7 173.3+1.2 179.6+3.5 184.941.1 glycosides Isoflavones 4.3+0.9 2.8+0.2* 2.4+0.07* 2.6+0.05* Flavanes 3.60.6 5.0+1.3 4.5+0.5 4.3+0.8 Flavonoids 23.5+2.8 23.944 .5 19.0+1.7 19.2+3.1 Table 5 Content determination results of 9 flavonoids Compound Average content ng/g

P A B C D Naringin 280.8+40.7 77.241.5** 63.4+8.8** 125.8+6.9** Luteolin 1091.9+3554 363.2+71.0* 391.2+46* 394. 3+50.8* Vitexin 825.5+51.2 135.5+3.8** 131.249.2** 126.0+4.7** Isovitexin 806.51+54.5 116.9x4.6** 122.9+7.1** 109.946.2** Genistein 1359.5+3736 119.8x42.3"" 122.1x7.6** 140.2+19.0** 7-O-glucoside luteolin 986.5+139.9 389. 3+38.9°* 405.3+24.7** 379.7+3.7** Apigenin 555.4x22.7 - - - Chrysin 201.3+25.8 - - - Dihydrokaempferol 150.0+31.4 - - - * (p<0.05) and ** (p<0.01) indicated significant differences in content compared with enzymolysis.

Flavonoid glycosides, isoflavones, flavanes and flavonoids were obtained by the four preparation methods, among them, the content of isoflavones (4.3£0.9ug/g) obtained by enzymolysis was significantly higher than that obtained by reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction (p<0.05), while the content of other flavonoids (flavonoid glycosides, flavanes and flavonoids) had no significant difference among the four preparation methods. Common isoflavones include genistein, genistin, daidzein, daidzin and other active components with antioxidant activity, which can alleviate liver injury and hepatic fibrosis and play a role in liver protection.

Therefore, compared with reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction, the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention can effectively increase the content of isoflavones, thereby improving the antioxidant and hepatoprotective capabilities.

A volcano plot was drawn by comparing and analyzing all data using t-test and fold change (FC) analysis method, as shown in Fig. 2. With log2 (FC) as the X-axis and -log10 (P-value) as the Y-axis, a point represents a variable. Two lines parallel to the Y-axis were X=1 and X =-1 respectively. Points on the left side of X=-1 were components down-regulated by more than 2 times, and points on the right side of X=1 were components up-regulated by more than 2 times. A dotted line Y=1.30 was parallel to the X-axis, i.e., -log10 (0.05), and points above the dotted line were components with significance less than 0.05. Components above the dotted line Y=1.30, on the right side of X=1 and on the left side of X=-1 were recorded as components with significant differences. The comparison between A and B, A and C, A and D revealed that there were 6 significantly up-regulated components, i.e., vitexin, isovitexin, naringin, genistein, luteolin and 7-O-glucoside luteolin. No component with significant difference was observed in samples B, C and D.

As shown in Table 5, compared with conventional extraction methods such as reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction, the content of isoflavones in the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention was higher, among them, the content of active hepatoprotective components, i.e.,

naringin, vitexin, isovitexin, genistein, luteolin and 7-O-glucoside luteolin was significantly increased (p<0.05), and another three components were identified, i.e., apigenin, chrysin and dihydrokaempferol. In conclusion, the nine different components were finally screened from the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis.

Example 3 Protective effects of total flavonoids of Hedyotis diffusa Willd prepared by different preparation methods on hydrogen peroxide-induced liver injury

1. Experimental method HL-02 cells were treated with H2O2 at concentrations of 50, 100, 150, 200, 400, 600 and 800umol/L for 4 h, the OD value was measured by MTT method, then the inhibition rate of H2O2 on the cells was calculated, and the optimal modeling concentration was determined according to the inhibition rate.

The HL-02 cells to be tested were divided into a drug group, a model group, a control group and a positive drug group, with at least three replicate wells in each group, for hepatoprotective activity screening assay. Control group: no treatment. Model group: HL-02 cells were cultured in an H2O2 medium with the optimal modeling concentration for 4 h. Drug group: HL-02 cells were added with the drug (the total flavonoids of Hedyotis diffusa Willd prepared by different methods in Example 1) for pretreatment for 12 h, and cultured for another 4 h in the H202 medium with the optimal modeling concentration. Positive drug group: HL-02 cells were added with bifendate for pretreatment for 12 h, and cultured for another 4 h in the H202 medium with the optimal modeling concentration. The OD value was determined by MTT method, and the protective rate of drugs was calculated.

Four components (vitexin, isovitexin, naringin and genistein) with fold change larger than 3.5 from the nine compounds screened in Example 2 and two active components (apigenin and chrysin) with higher content from the new components (dihydrokaempferol, apigenin and chrysin) were further selected for activity screening assay to explore whether they have hepatoprotective activity.

The cell supernatant was collected after administration of the total flavonoids of Hedyotis diffusa Willd prepared by different methods in Example 1 to determine liver injury indexes AST, ALT, LDH and ALP based oninstructions for kit operation.

The experimental data were expressed as mean + standard deviation, and statistical difference of the results was analyzed by t-test. When p<0.05, statistical difference was considered, and all experiments were repeated for 3 times in parallel.

2. Experimental results The cell morphology of normal HL-02, H2Oz-damaged HL-02 and drug-pretreated HL-02 is shown in Fig. 5.

According to the results in Fig. 3(a), the survival rate of HL-02 cells decreased gradually with the increase of H202 concentration. The cell inhibition rate was about 60% when the H202 concentration was 200 umol/L. Therefore, the modeling concentration was 200 umol/L H2O2.

The protective activities of samples A (enzymolysis in the present invention), B (reflux extraction), C (ultrasonic extraction) and D

(enzymolysis-ultrasonic assisted extraction) at 31.25, 62.5 and 125 pg/mL are shown in Fig. 3(b). With the increase of the concentration of total flavonoids prepared by different methods, the hepatoprotective rate increased gradually in a dose-dependent manner, and the maximum hepatoprotective rate was achieved at 125 pg/mL (59.30+0.81%, 39.26+6.18%, 27.26+0.75% and

42.44+6.35% respectively). The hepatoprotective activity of the total flavonoids prepared by enzymolysis in the present invention was significantly better than that of total flavonoids prepared by reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction at the same concentration (p<0.05).

According to the results in Fig. 3(c), the cell survival rate was found to increase in a dose-dependent manner after pretreatment with 4, 8 and 16 pmol/L naringin, vitexin, isovitexin, genistein, apigenin, chrysin and the positive control drug bifendate, respectively. The cell survival rate was significantly higher in the 8 and 16 pmol/L groups compared with the model group (p<0.05, p<0.01), indicating that naringin, vitexin, isovitexin, genistein, apigenin and chrysin had protective effects on H2Oz-induced HL-02 injury and increased the cell survival rate. Moreover, the cell survival rate after pretreatment with naringin, vitexin, isovitexin, genistein, apigenin and chrysin was not significantly different from that after pretreatment with the positive control drug bifendate at the same concentration (p>0.05), indicating that all the six compounds had protective effects on H202-induced HL-02 injury, and the hepatoprotective effect was comparable to that of bifendate.

The measured results of AST, ALT, LDH and ALP are shown in Fig. 4. Compared with the control group, the levels of ALT, AST, LDH and ALP in the cell supernatant of the model group were significantly higher (p<0.01), indicating that H2O2 treatment could cause HL-02 cell injury. Compared with the model group, the effects of 62.5 and 125 pg/mL total flavonoids of Hedyotis diffusa Willd samples A on lowering the levels of AST and LDH in the cell supernatant were significantly better than those of samples B, C and D (p<0.05), as shown in Figs. 4(a) and 4(c); while no significant difference was observed in lowering the levels of ALT and ALP (p>0.05), as shown in Figs. 4(b) and 4(d).

In summary, the effect of the total flavonoids prepared by enzymolysis in the present invention on lowering the levels of AST and LDH was significantly higher than that of reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction (p<0.05), and the protective effect on H202-induced liver injury was also significantly higher than that of reflux extraction, ultrasonic extraction and enzymolysis-ultrasonic assisted extraction (p<0.05). Compared with other preparation methods, the total flavonoids prepared by enzymolysis in the present invention contain higher contents of apigenin, chrysin, genistein, naringin, vitexin and isovitexin (more than 3.5 times) and other active components with hepatoprotective effects, so that the total flavonoids prepared by enzymolysis in the present invention have more efficient hepatoprotective activity.

Example 4 Experimental study on acute toxicity of total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention

1. Experimental method

Preliminary experiment: twenty ICR mice, half male and half female, were allowed to acclimate for one week, and then randomly divided into 4 groups, with 5 mice in each group, i.e., a blank control group, a high dose group (100 mg/mL), a medium dose group (50 mg/mL) and a low dose group (25 mg/mL). The mice were fasted with waterfor 12 h before administration, and administered by gavage at the maximum dose of 0.4mL/10g, while the control group was given the same amount of distilled water. After administration, the mice were observed for 7 consecutive days,during which the mice were fed normally, with conditions recorded. If a dose at which all mice are dead or not dead can be found, a formal experiment will be designed to find out the median lethal dose LDso. If any mice in the high dose group die, the concentration of the high dose group can be reduced, and an experiment will be further designed to determine the maximum tolerance dose (MTD). If there is no death in the high dose group, the maximum dose test can be carried out in accordance with the Technical Requirements for Pharmacological & Toxicological Research on New Traditional Chinese Medicine.

Twenty ICR mice were randomly divided into two groups, i.e., a drug group and a control group, with 10 mice in each group. The drug group was administered with 0.4mL/10g (ie., the maximum dose and maximum concentration of total flavonoids of Hedyotis diffusa Willd for gavage) total flavonoids by gavage once a day, while the control group was given the same amount of distilled water, and fasted with water for 12 h before gavage. The mice were observed for 14 consecutive days, during which the mice were fed normally, with conditions recorded.

2. Experimental results In the preliminary experiment, the mice in the high, medium and low dose groups did not die within 7 days; and no obvious abnormalities in weight, appearance, behavior, mental state, diet and excretion were observed compared with the control group. The LDso and the MTD could not be measured, so the maximum dose test was carried out.

The mice were administered at the maximum dose concentration and the maximum dose volume once a day, the mice in both the control group and the drug group showed signs of depression, less activity and squinting at the initial stage of administration, but no abnormal toxic reactions such as piloerection, convulsion and tumbling, and the symptoms recovered gradually one hour after administration. After observation for 14 consecutive days, no mouse was found dead, and no significant abnormalities in weight, appearance, behavior, mental state, diet and excretion were observed compared with the control group. The results of weight change are shown in Table 6.

Table 6 Changes in weight of mice (n=10) - Weighug Group At the On Day 7 after On Day 14 after beginning administration administration Blank control group 22.99+2.07 26.72+1.81 30.99+3.33 Flavonoids dosing group 21.8+1.83 26.95+1.95 32.37+1.82 (49 kg”-d"") Compared with the blank control group, there was no significant difference in weight of the mice treated with total flavonoids of Hedyotis diffusa Willd (p>0.05). Therefore, it can be considered that the maximum dose of the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention is 4 g"-kg"-d"", and the total dose of Hedyotis diffusa Willd is 80.65 g*-kg"-d" when converted into crude drugs (according to the results in Example 1, the yield of total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention is 4.96+0.02%, therefore, the total dose of Hedyotis diffusa Willd (g) = flavonoid dose (g)/yield of total flavonoids), which is equivalent to 161.3 times the clinical daily dose for an adult based on the clinical daily dose of Hedyotis diffusa Willd (the daily oral dose for a 60 kg adult is 30 g). Therefore, it can be considered that the acute toxicity of total flavonoids of Hedyotis diffusa Willd is low, and the conventional dose is safe and reliable.

Example 5 Study on the effect of total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention on non-alcoholic fatty liver disease

1. Experimental method Sixty SPF male SD rats with weigh of 170-220 g were provided by Experimental Animal Center of Shanxi Medical University. The sixty rats were randomly divided into 6 groups, with 10 rats in each group, i.e., a control group, a model group, a positive control group (30 mg/kg fenofibrate) and low, medium and high dose groups of total flavonoids of Hedyotis diffusa Willd (60 mg/kg, 90 mg/kg, 120 mg/kg). The control group was given a normal maintenance diet, the other groups were given high-fat diets (45% fat for energy supply), and the control group and the high-fat diet groups were fed with respective diets for 3 weeks. Blood was collected from orbits of the rats 3 weeks later, serum was separated, and the levels of TC and TG in the serum were measured to determine whether the modeling was successful. After successful modeling, the positive control group was given fenofibrate (30 mg/kg), and the drug group was given low, medium and high doses (60 mg/kg, 90 mg/kg, 120 mg/kg) of total flavonoids of Hedyotis diffusa Willd prepared by the method of the present invention in Example 1.

The rats were administered by gavage at 10:00 am. every day. Fenofibrate and total flavonoids of Hedyotis diffusa Willd were dissolved in 0.9% normal saline respectively and prepared into suspensions, and the rats were administered by gavage at respective doses once a day for 2 weeks, then weighed regularly and records were made.

Sample collection and index determination: blood was collected from orbits of the rats, centrifuged at 3000 r/min for 15 min, then the supernatant was collected and stored at -20 °C for measurement to determine biochemical indexes related to fatty liver and liver injury, i.e., the levels of TG, TC, y-GT, GSH, SOD, MDA, LDH, AST, ALT and ALP, by the corresponding kit instructions. After blood collection, the rats were sacrificed by breaking their necks, then the heart, liver, spleen and kidneys were separated quickly, cleaned with normal saline, and weighed after drying with filter paper to calculate the organ coefficient, organ coefficient (mg/g)=wet weight of organs/weight of rats.

2. Experimental results The liver tissues of rats in the control group had normal morphology, bright red color, smooth surfaces, sharp edges, and ruddy and soft textures. The livers of rats in the model group were enlarged and significantly heavier than those of rats in the normal control group, with a brownish-yellow color,

yellow bands, blunt and rough edges and brittle textures.

The difference in weight change and liver coefficient of rats are shown in Table 7. Compared with the blank control group, the weight gain and liver index of rats in the model group were significantly higher (p<0.01), and compared with the model group, the weight gain and liver coefficient of rats treated with total flavonoids of Hedyotis diffusa Willd were significantly lower (p<0.01). The results showed that total flavonoids of Hedyotis diffusa Willd could significantly control the weight gain of rats on high-fat diets (p<0.01), and prevent obesity and liver fat deposition due to excessive weight gain. The weight gain and liver coefficient of rats in the medium and high dose group were significantly lower than those in the positive control group (p<0.01), indicating that the effect of total flavonoids of Hedyotis diffusa Willd on controlling weight gain was better than fenofibrate.

Table 7 Difference in weight change, wet liver weight and liver coefficient of rats ~ Group Dose Weight difference (g) Liver coefficient (mg/kg) (mg/g) Blank control group - 60.32+4.14 57.78+1.98 Model group - 114.54+3.03# 82.80+1.72% Low dose group 60 90.19+2.02** 72.61£2.67* Medium dose group 90 82.53+1.43**** 64.06+1.66**** High dose group 120 74.49+1.37**** 62.18x2.53**** Positive control group 30 100.41+1.88** 77.65+2.28* # indicates p<0.05 compared with the control group; ## indicates p<0.01 compared with the control group; * indicates p<0.05 compared with the model group, ** indicates p<0.01 compared with the model group; and ** indicates p<0.01 compared with the positive control group.

The effects of total flavonoids of Hedyotis diffusa Willd on blood lipid indexes of rats are shown in Table 8. The total cholesterol (TC) and triglyceride (TG) in serum of rats in the model group were significantly higher than those in the blank control group (p<0.05). After treatment with 90 and 120 mg/kg total flavonoids of Hedyotis diffusa Willd, the levels of TC and TG decreased significantly compared with those in the model group (p<0.05), indicating that total flavonoids of Hedyotis diffusa Willd may have a therapeutic effect on non-alcoholic fatty liver disease by regulating lipid metabolism in vivo and reducing excessive lipid deposition in the liver. No significant differences (p>0.05) were observed in the levels of TC and TG in the drug group compared with the positive control group, indicating that the effect of total flavonoids of Hedyotis diffusa Willd on reducing the levels of TC and TG was comparable to that of the positive drug fenofibrate.

Table 8 Effects of total flavonoids of Hedyotis diffusa Willd on blood lipid indexes of rats (n=10) Group Dose (mg/kg) TC (mmol/L) TG(mmol/L)_ Blank control group - 5.45+0.43 0.88+0.12 Model group - 8.62+0.50#* 2.46+x0.47*# Low dose group 60 7.07x0.67 1.82+0.14 Medium dose group 90 5.55+0.88* 0.78x0.12* High dose group 120 4.41+1.29* 0.75+0.07* Positive control group 30 5.54+0.53* 1.43+0.24 # indicates p<0.05 compared with the control group; * indicates p<0.05 compared with the model group.

The effects of total flavonoids of Hedyotis diffusa Willd on liver injury indexes are shown in Table 9. Compared with the blank control group, the levels of ALT, AST, LDH, ALP and y-GT in the model group were significantly higher (p<0.05, p<0.01). After treatment with total flavonoids of Hedyotis diffusa Willd, the levels of ALT, LDH and y-GT in the low, medium and high dose groups were statistically different compared with the model group (p<0.05, p<0.01); and the levels of AST and ALP in the medium and high dose groups were statistically different compared with the model group (p<0.05, p<0.01), indicating that total flavonoids of Hedyotis diffusa Willd had some protective effects on non-alcoholic fatty liver injury induced by high-fat diets, and could alleviate liver injury. Compared with the positive control group, the effect of total flavonoids of Hedyotis diffusa Willd on lowering the levels of ALT and AST in the high dose group was significantly better than that of fenofibrate (p<0.05), and the effect on improving the level of LDH was comparable to that of fenofibrate (p<0.05), but the effect on improving the levels of ALP and y-GT was lower than that of fenofibrate (p<0.05).

Table 9 Effects of total flavonoids of Hedyotis diffusa Willd on liver injury indexes of rats (n=10) Gro ALT(U/L AST(U/L LDH(U/L ALP GT(U/L up (UL) (UL) UL (Units/t00mL) | Y-CTUL) Control 2.11+0.61 8.44+2.02 60.15+7.16 77.53+6.07 5.96+1.41 group Model 16.43+1.47% 54 8515.17" 151.97+16.81# 130.76x11.47# 13.26+1.38# group Low 9.31+1.40* 27.75x7.74 84.10+8.24* 89.14+10.47 5.28+0.87** dose group Medium 8.71+0.39* 25.25+5.60* 64.0414 .99* 76.04+6.07* 5.16x0.37** dose group High 2.56x1.04*** | 5.74+2.72*** 60.80+3.75* 44.03+12.38* 3.70+0.43** dose group Positive 9.90+1.67 28.35+2.69* 62.8+6.91* 17.69+3.57** 3.720,41" control group # indicates p<0.05 compared with the control group; ## indicates p<0.01 compared with the control group; * indicates p<0.05 compared with the model group, ** indicates p<0.01 compared with the model group; the low, medium and high dose groups were 60, 90 and 120 mg/kg total flavonoids of Hedyotis diffusa Willd respectively; the positive control group was 30 mg/kg fenofibrate; and * indicates p<0.05 compared with the positive control group.

The effects of total flavonoids of Hedyotis diffusa Willd on oxidative stress indexes are shown in Table 10. Compared with the blank control group, the level of MDA in the model group increased significantly (p<0.05), while the activity of SOD and the level of GSH decreased significantly (p<0.05, p<0.01). After treatment with total flavonoids of Hedyotis diffusa Willd, the activity of SOD increased significantly and there was statistical difference between the medium and high dose groups and the model group (p<0.05); the level of GSH increased after administration, and there was statistical difference between the high dose group and the model group (p<0.05); and the level of MDA decreased significantly after administration (p<0.01) compared with the model group, indicating that in rats with non-alcoholic fatty liver disease, the oxidation resistance and free radical scavenging rate decreased, resulting in lipid peroxidation and liver injury, while total flavonoids of Hedyotis diffusa Willd can exert therapeutic effects by regulating free radicals to keep them in metabolic equilibrium and inhibiting lipid peroxidation. Total flavonoids of Hedyotis diffusa Willd were not significantly different from fenofibrate in lowering the content of MDA (p>0.05), while the effects of improving the activity of SOD and the level of GSH were significantly better than those of fenofibrate (p<0.05).

Table 10 Effects of total flavonoids of Hedyotis diffusa Willd on oxidative stress indexes of rats (n=10) Group Dose (mg/kg) SOD(U/L) GSH(umol/L) MDA(nmol/mL) Blank control group - 139.89+10.02 27.91+0.57 2.54+0.2 Model group - 85.01+9.01# 4.37+0.94% 8.27+0.43% Low dose group 60 107.46+5.66* 4.62+1.13 5.72+0.21** Medium dose group 90 121.72+5.41** 6.28+1.50 4.85+0.22** High dose group 120 128.06+7.93** 12.30+1.04** 4.17+0.13** Positive control group 30 79 44+4 22 1.5+0.42 8.07+0.43 ## indicates p<0.01 compared with the control group; * indicates p<0.05 compared with the model group, ** indicates p<0.01 compared with the model group; and * indicates p<0.05 compared with the positive control group.

According to the acute toxicity test of the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention on mice and studies on the effect thereof on non-alcoholic fatty liver disease, it is known that the total flavonoids of Hedyotis diffusa are a safe and less toxic natural plant extract that has lipid-regulating, antioxidant and hepatoprotective effects, and has a therapeutic effect on non-alcoholic fatty liver disease.

In conclusion, the total flavonoids of Hedyotis diffusa Willd prepared by enzymolysis in the present invention havea therapeutic effect on peroxidative liver injury, and has good prospects for exploitation and utilization.

Claims (10)

1. A preparation method of highly active total flavonoids of Hedyotis diffusa Willd, characterized by comprising the following steps: a: sample pretreatment, drying and crushing Hedyotis diffusa Willd, sieving through a 60-mesh sieve, ultrasonically treating with petroleum ether, discarding the petroleum ether, filtering the residue and evaporating the petroleum ether, repeating the operation once, drying and cooling for later use; b: adding cellulase to pretreated Hedyotis diffusa Willd samples from the step a, extracting total flavonoids with 50-70% ethanol as a solvent, filtering under vacuum, and merging filtrates to obtain a crude extract of total flavonoids of Hedyotis diffusa Willd, and c: purifying the crude extract of total flavonoids of Hedyotis diffusa Willd with a 20-60-mesh low-polar macroporous adsorption resin, washing with distilled water to remove water soluble impurities, then eluting with 70% ethanol, collecting the eluent, concentrating under vacuum, freeze-drying and crushing to obtain refined total flavonoids of Hedyotis diffusa Willd.

2. The preparation method of highly active total flavonoids of Hedyotis diffusa Willd according to claim 1, characterized in that the addition of the cellulase in the step b is 0.5-1.0% of the mass of the pretreated Hedyotis diffusa Willd samples; and the material-liquid ratio is 1:30-1:50 g/mL.

3. The preparation method of highly active total flavonoids of Hedyotis diffusa Willd according to claim 1 or 2, characterized in that pH of the crude extract in the step b is 4.0-5.5.

4. The preparation method of highly active total flavonoids of Hedyotis diffusa Willd according to claim 3, characterized in that enzymolysis temperature in the step b is 50-60 °C.

5. The preparation method of highly active total flavonoids of Hedyotis diffusa Willd according to claim 3, characterized in that enzymolysis time in the step bis 1-1.5h.

6. An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method according to claim 1 in treatment of peroxidative liver injury.

7. An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method according to claim 1 in prevention of peroxidative liver injury induced by chemical substances including alcohol, chemicals and drugs.

8. An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method according to claim 1 in prevention of H202-induced HL-02 cell injury, and an application thereof in lowering of levels of ALT, AST, LDH and ALP and other liver injury indexes.

9. An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method of claim 1 in treatment and prevention of liver injury caused by fatty liver disease.

10. An application of the highly active total flavonoids of Hedyotis diffusa Willd prepared by the preparation method according to claim 1 in lipid-lowering effect for non-alcoholic fatty liver disease.