Study on the Effects of Reducing Blood Lipid by Flavonoids from Ampelopsis grossedentata on Hyperlipidemia Rats

ZUO Xue-mei, LIU Qian, LIAO Fang-fan, FU Ming, LI Sheng-hua, XIAO Qiang

College of Biological and Food Engineering, Huaihua University, Hunan Provincial Key Laboratory of Research and Utilization of Ethnic Medicinal Plant Resources, Key Laboratory of Medicinal Plants and Ethnobotany in Western Hunan Province, Huaihua 418008, PRC

Abstract In order to study the effects of Ampelopsis grossedentata on endogenous cholesterol biosynthesis and the effects of reducing blood lipid in hyperlipidemia rats, extraction of flavonoids from Ampelopsis grossedentata by using ethanol (TFAG), and the effects of TFAG on intracellular cholesterol synthesis were detected by amphotericin B- cell model； SD hyperlipidemia rat model was established by feeding high fat diet. A formulated medicine called Xuezhikang was used as a positive control, and TFAG of different doses were administered to the stomach for 30 d continuously to measure the indexes of heart, liver tissue homogenate and serum； part of the liver was taken for pathological observation. The results showed that TFAG could significantly inhibit the synthesis of cholesterol in cells. TFAG of different doses could significantly reduce the content of TC and TG in serum of hyperlipidemia rats, and increase the content of HDL； TC and TG in heart and liver were also decreased； besides, it could increase the content of SOD, CAT and GSH in the liver of hyperlipidemia rats, and reduce the content of MDA. The results of pathological section showed that TFAG could improve the damage degree of hepatocytes in hyperlipidemia rats, and the effect of high dose group was similar to that of Xuezhikang group. In general, TFAG has good antioxidant and reducing blood lipid effects, and can effectively inhibit liver steatosis.

Key words Flavonoids from Ampelopsis grossedentata； Rat； Hyperlipidemia； Cholesterol biosynthesis； Reducing blood lipid

1. Introduction

In recent years, with the improvement of living standards, the dietary pattern of human beings has gradually changed, and the intake of more high-fat foods has made the incidence rate of hyperlipidemia increase year by year. Hyperlipidemia is one of the important inducements of fatty liver, coronary heart disease and atherosclerosis[1]. In order to prevent fatty liver, coronary heart disease and atheroscl- erosis, timely treatment of hyperlipidemia is particularly important. It shows that flavonoids[2-3], polysaccharides, phenols and other substances[4-5]have better antioxidant and hyperlipidemia effects, which are the main components of hypolipidemic drugs, and most Chinese herbal medicines contain these substances. In order to avoid the side effects of chemical synthetic drugs, it has a bright future to develop efficient and low toxic drugs for lowering blood lipid by using natural Chinese herbal medicines.

Ampelopsis grossedentatais the tender leaf ofAmpelopsis grossedentata(handmazz) W.T.WANG. It is processed into a daily tea drink by native residents. It has the functions of clearing heat and removing dampness, reducing blood lipid and blood pressure of liver, activating blood circulation and unblocking collaterals.Ampelopsis grossedentatahas a high content of flavonoids[6], rich polysaccharides and phenolics[7], it is anti-inflammatory[8], bacteriostatic and a antioxidant[9], is hypoglycemic[10], antihypertensive[11]and it could reduce lipid[12],etc. In 2013,Ampelopsis grossedentatawas approved by National Health and Family Planning Commission as a new food raw material[13], which can be processed into antibacterial and antiseptic food additives and new flavor drinks. In order to study the antioxidative and hypolipidemic effects of total flavonoids fromAmpelopsis grossedentata(TFAG), a rat model of hyperlipidemia was established by feeding high-fat diet.

2. Materials and Methods

2.1. Test material

2.1.1. Material sources

Ampelopsis grossedentatasamples were collected from Huaihua City and identified as the tender leaves ofAmpelopsis grossedentata(hand Mazz) W.T.WANG by Professor ZENG Han-yuan of Huaihua University. The trial sample SD rat (Sprague Dawley rat) was purchased from the animal center of Xiangya School of Medicine (CSU) with the license number of SYXK (Xiang) 2011-0012.

2.1.2. Main reagent

China hamster oocyte (CHO) cell line (Hunan Medical College)； Xuezhikang (batch No. 2014 0803, Beijing Beidaweixin Biotechnology Co., Ltd.)； lipoprotein free serum (LPDS, US hyclone), amphotericin B (Sigma, USA), mevalonate (Sigma, USA)； Catalase (CAT), superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA), high density lipoprotein (HDL), triglyceride (TG), total cholesterol (TC) kit (Nanjing Jiancheng Bioengineering Research Institute). High fat feed: 62.8% basic feed, 15% casein, 10% lard, 5% cholesterol, 5% sucrose, 2% vitamin, 0.2% bovine bile salt. Cell culture medium: solution A, RPMI-1640 medium containing 10% calf serum and 300 μg/mL glutamine (Gln)； solution B, RPMI-1640 medium containing 10% LPDS and 300 μg/mL Gln； solution C, solution B containing 0.25 mmo1/L mevalonate lactone plus Xuezhikang or TFAG of different concentrations or the same amount of culture medium； solution D, solution B containing 300 μg/mL amphotericin B (normal group without amphotericin B).

2.1.3. Instruments and equipment

CTXNW-10B ultrasonic extractor (Beijing Hongxianglong Biotechnology Co., Ltd.)； IB200W carbon dioxide incubator (GOLD-SIM)； FD-1C freeze dryer (Beijing Detianyou Technology Development Co., Ltd.)； RE-52 rotary evaporator (Gongyi Yuhua Instrument Co., Ltd.)； SL-200 grinder (Zhejiang Yongkang Songqing Hardware Co., Ltd.)； Epoch2 microplate spectrophotometer (BioTek, USA).

2.2. Test method

2.2.1. Preparation of flavonoids fromAmpelopsis grossedentata

The tender leaves of the newAmpelopsis grossedentatawere green removed, dried and grinded, and screened for 60 mesh sieve； 4 kg of tender leaves ofAmpelopsis grossedentatawere soaked in 70% ethanol for 24 h； the extraction was carried out at 45℃ for 45 min with ultrasonic circulation extractor； the extract was concentrated in a rotary evaporator and dried in vacuum to obtain total flavonoids fromAmpelopsis grossedentata(TFAG).

2.2.2. Effects of TFAG on cholesterol synthesis in CHO cells

According to the method of LI C Yet al.[14], XUE Jet al.[15], CHO was digested with 0.25% pancreatin to make cell suspension. The cell concentration was adjusted to 2×104cells/mL with solution A, and 100 μL per orifice was inoculated on 96 orifice plate. The cells were cultured at 37℃ in a 5% CO2incubator, and 200 μL solution B was added into each orifice after the cells adhered to the wall for 24 h. Then 200 μL of solution C was added into each orifice, and divided into normal group, negative control group (NC group, adding equal amount of culture solution), Xuezhikang group (adding 0.5 mg/mL of Xuezhikang) and TFAG low, medium and high dose group (adding 2.5, 5.0 and 10.0 mg/mL of TFAG respectively). Each group was set with 6 multiple orifice, and washed twice with PBS buffer with pH value of 7.4 after 24 h of culture. 200 μL solution D was added to culture for 8 h, and then washed twice with PBS buffer. 5 mg/mL MTT 10 μL was added, and cultured for 4 h, and then added 10% SDS-0.01 mol/L hydrochloric acid 100 μL. The A570value of each orifice solution was measured. The higher the A value is, the more cells survive, then it shows that the more effective the drug is in inhibiting cholesterol biosynthesis.

2.2.3. Rat models

The temperature of the feeding room is kept at 25±2℃, the relative humidity is 50%±5%, 12 h of light every day, and the tested rats were feed with standard animal feed and drinking water. During the test, the regulations on the administration of experimental animals promulgated by theNational Scienceand Technology Commissionshall be observed, and the ethical issues of experimental animals shall be paid attention to. After feeding 80 SD rats with basic diet for one week, they were randomly divided into two groups, 10 of them were in normal group (group I), and they were fed with basic diet continuously； in addition, 70 rats were fed with high-fat diet for 15 d. Blood was collected from the tail of the experimental rats and the contents of TG and TC in the serum were determined. If the serum TG and TC content of the model rats were significantly higher than that of the normal group, the difference was extremely significant (P＜0.01), indicating that the model of hyperlipidemia rats was successful.

2.2.4. The effects of TFAG on the indexes of hyperlipidemia model rats

50 hyperlipidemia rats were selected to test the flavonoid content ofAmpelopsis grossedentata. The rats with hyperlipidemia were randomly divided into 5 groups(group Ⅱ-Ⅵ) and fed with high-fat diet. The second group is the model group, the stomaches of the rats in this group were filled with distilled water of equal volume； the third group was Xuezhikang group, which was infused with the same volume of Xuezhikang (30 mg/kg)； the stomaches of the rats in Ⅳ, Ⅴ and Ⅵ groups were filled with 100, 200 and 400 mg/kg TFAG respectively. After 30 d of continuous gavage, the rats were fasted for 12 h after the last gavage and weighed. Then the rats were killed after anaesthesia with ether. The blood was collected from the orbit and centrifuged to prepare the serum for use. The contents of TG, TC, HDL and phospholipid in serum were determined according to the instructions of the kit, and the arteriosclerosis index AI= (TCHDL)/HDL was calculated. Then the heart and liver of the experimental rats were taken, and washed with 4℃ normal saline, they were weighed, and stored at -40℃； The contents of TG, TC, phospholipid, GSH, SOD, CAT and MDA were measured according to the instructions of the kit. In addition, the liver tissue at the same position in the right lobe of the liver was dried, fixed with 10% formalin, embedded in paraffin, sectioned, stained with hematoxylin eosin (HE), and observed under the microscope.

2.2.5. Data statistics and processing

SPSS 10.0 software was used for data statistics and processing. The results were expressed as±s.

3. Results and Analysis

3.1. Effects of TFAG on cholesterol synthesis in CHO cells

As shown in Table 1, the A570value of the negative control group was significantly lower than that of the normal group (P＜0.001)； compared with the negative control group, the A570value of Xuezhikang group increased significantly (P＜0.001), The A570value of TFAG in different dose groups also increased significantly (P＜0.01), in particular, the difference between the A570value of the middle and high dose groups treated with TFAG and the negative control group reached 0.001 level, which was equivalent to the effect of Xuezhikang. This indicated that TFAG had a strong inhibitory effect on cholesterol biosynthesis.

Table 1 Effects of TFAG on cholesterol synthesis in CHO cells

3.2. The effects of TFAG on lipid in serum of hyperlipidemia model rats

It can be seen from Table 2 that compared with the normal group, the contents of TC and TG in the serum of rats in the hyperlipidemia model group increased significantly (P＜0.001), whereas the contents of HDL and phospholipid decreased significantly (P＜0.001)； compared with the model group, the contents of TC and TG in serum of Xuezhikang group were significantly decreased (P＜0.001), and the contents of HDL and phospholipid were significantly increased (P＜0.001)； the contents of TC, TG in serum of rats in TFAG low, middle and high dose groups were also significantly decreased, the contents of HDL and phospholipid were significantly increased, and showed a dose-dependent manner. The effect of TFAG in high dose group was similar to that of Xuezhikang (P＜0.001).

It can be seen from Fig. 1 that the ratio ofcholesterol to phospholipid in the model group was the largest, showing a very significant difference (P＜0.001), whereas the ratio of the two in different treatment groups were significantly reduced (P＜0.01)； the atherosclerosis index (AI) of the model group was the highest, and the difference between the model group and the normal group was very significant, while the AI of each treatment group was significantly decreased, especially AI decreased significantly after high dose treatment of Xuezhikang and TFAG (P＜0.001).

Table 2 Effects of TFAG on lipid in serum of hyperlipidemia model group

3.3. Effects of TFAG on lipid in heart and liver of rats with hyperlipidemia model

Fig. 1 Effects of TFAG on the ratio of cholesterol to phospholipid in serum and AI(a in the figure indicates that the difference between the normal group and the other groups is 0.001 level； b, c and d indicate that the difference between the model group and the other groups is 0.05, 0.01 and 0.001 level, respectively. The same as below.)

As can be seen in Table 3, compared with the normal group, the TC and TG contents in the heart and liver of the model group increased significantly, whereas the phospholipid contents decreasedsignificantly, with a very significant difference (P＜ 0.001)； compared with the model group, TC and TG in the heart and liver of rats in Xuezhikang group decreased significantly, whereas the content of phospholipid increased significantly (P＜0.001)； TFAG treatment with different doses could significantly reduce the content of TC and TG in heart and liver, and increase the content of phospholipid (P＜0.05)； especially in the high dose TFAG group, the effect was similar to that of Xuezhikang group (P＜0.001).

Table 3 Effects of TFAG on lipid in heart and liver of rats

3.4. Antioxidant effects of TFAG in hyperlip- idemia model rats

As shown in Fig. 2, compared with the normal group, the content of GSH, SOD and CAT in the liver of the hyperlipidemia model group decreased significantly (P＜0.001), and the content of MDA increased significantly (P＜0.001)； compared with the model group, the contents of GSH, SOD and CAT in the liver of Xuezhikang group increased to a extremely high level (P＜0.001), whereas the contents of MDA decreased significantly (P＜0.001)； the contents of GSH, SOD and CAT in the liver of TFAG group increased significantly (P＜0.05), whereas the contents of MDA decreased significantly (P＜0.05). The antioxidant effect of TFAG group was similar to that of Xuezhikang group (P＜0.001).

3.5. Histopathological analysis of hepatocytes in hyperlipidemia rats

It can be seen from Fig. 3 that the hepatocytes (Fig. 3A) of normal group were closely arranged, with complete structure and clear boundary. The hepatocyte cords were radial, without fat vacuole or fatty degeneration； in the model group, the hepatocytes (Fig. 3B) were significantly larger, with more fat vacuoles, the nucleus being squeezed to the edge or missing, and the cell structure was not complete； in the Xuezhikang group, the number of fat vacuoles in hepatocytes (Fig. 3C) was decreased, and the cell volume was also decreasing, which was not significantly different from the normal cells.

4. Discussion

Fig. 2 Effects of TFAG on GSH, SOD, CAT and MDA in the liver

Fig. 3 HE staining sections of rat liver tissue(A: Normal group； B: Model group； C: Xuezhikang group； D: 100 mg/kg TFAG group； E: 200 mg/kg TFAG group； F: 400 mg/kg TFAG group； the image was enlarged 200 times)

The content of flavonoids inAmpelopsis grosse-dentatais as high as 41.25%～43.61%[16-17], and the content of dihydromyricetin alone can reach 39.50%[18]. CHEN X Jet al.[12]found that the total flavonoids ofAmpelopsis grossedentatacan reduce the content of TC, TG and AI value in the serum of egg yolk type hyperlipidemia rat and experimental hyperlipidemia quail. This is consistent with the author's research results. In addition, the author also studied the effects of TFAG on cholesterol biosynthesis in CHO cells, and established a rat model of hyperlipidemia with mixed high-fat diet. The study not only measured the content of TC and TG in serum, but also measured various biochemical indexes in liver.

HDL can transport cholesterol in blood to liver, promote its decomposition, reduce cholesterol content in blood, while LDL can promote cholesterol content in blood. After gavage of TFAG and Xuezhikang to hyperlipidemia rats, the serum indexes were improved. The effect of high dose TFAG group was similar to that of Xuezhikang.

The results showed that the contents of TC and TG in the heart of hyperlipidemia model rats were 1.98 times and 2.69 times of that of normal rats, whereas the content of phospholipid was only 43.13% of that of normal rats, and the ratio of TC and phospholipid was 4.55 times of that of normal rats； the content of TC and TG in the liver of hyperlipidemia model rats was 2.91 times and 3.06 times of that of normal rats, while the content of phospholipid was only 46.49% of that of normal rats, and the ratio of TC and phospholipid was 6.16 times of that of normal rats. Compared with the model group, the contents of TC and TG in liver and heart of rats in different treatment groups were significantly reduced, the content of phospholipid increased significantly, and the ratio of TC to phospholipid decreased obviously. This indicated that the extract ofAmpelopsis grossedentatacan effectively reduce the contents of TC and TG, and increase the content of phospholipid in the body of hyperlipidemia rats.

Under the action of ·OH、O2-· the fatty acids on the cell membrane were peroxidized and cause a series of chain reactions, which eventually led to cell damage or even death. MDA was the end product of a series of lipid peroxidation reactions. GSHin vivocan scavenge free radicals and their derivatives by directly supplying H+； SOD can catalyze O2-· to generate H2O2and O2, while CAT can catalyze H2O2to decompose into H2O and O2； the synergistic effects of multi-stage antioxidant system of GSH, SOD and CAT can jointly inhibit lipid peroxidation and enhance the antioxidant capacity of the body. After administration of high dose TFAG, GSH, SOD and CAT in hyperlipidemia rats were 2.12 times, 2.61 times and 2.30 times higher than those in model rats, respectively, whereas MDA was only 39.27% of that in model rats. The level of GSH, SOD and CAT in hyperlipidemia rats was similar to that in Xuezhikang group, and it showed that the extract ofAmpelopsis grossedentataplays an antioxidant role in the body of hyperlipidemia rats.

Hyperlipidemia can cause fatty degeneration of the liver, cause liver lesions, and further develop into liver cirrhosis and liver cancer. In the experiment, the fatty degeneration of the liver of rats in the hyperlipidemia model group was serious. The pathological section results of the liver of rats in the different dose of TFAG group and Xuezhikang group showed that the damage degree of the liver cells was improved. The effect of the high dose group was equivalent to that of Xuezhikang group.

Lipid lowering drugs can reduce blood lipid by preventing the synthesis of endogenous cholesterol, inhibiting the absorption of exogenous cholesterol and promoting the metabolism of lipid transport. In the amphotericin B cell model, the higher the A value is, the more the number of living cells is, the less the damage of amphotericin B to cells is, that is to say, the synthesis of endogenous cholesterol in cells is inhibited without providing exogenous lipids[15]. It was found that TFAG had a strong inhibitory effect on cholesterol biosynthesis in CHO cells in different dose groups, and the inhibitory effect in high dose group was similar to that of Xuezhikang group.

In conclusion, the flavonoid ofAmpelopsis grossedentatacan prevent the synthesis of endogenous cholesterol, significantly reduce the level of blood lipid in hyperlipidemia rats, and effectively inhibit the formation of fatty liver； by improving the antioxidant capacity of the body, the lipid peroxidation in the cells can be reduced； it is helpful to reduce the level of blood lipid, prevent atherosclerosis, and prevent cardiovascular and cerebrovascular diseases. It can be used as a safe and reliable research material of natural lipid-lowering drugs.

YANG P Let al.[19]found that citrus grandis peel flavonoid (PTFC) can reduce serum TC, TG, LDL-C, LDL-C/HDL-C and AI index, increase LDL-C/TC, and reduce tissue fatty degeneration and inflammation in hyperlipidemia rats. Their research results showed that PTFC is related to fat catabolism through the influence ofPPAR-αgene expression and PPAR-α, LPl, LiPc protein expression, so as to achieve the regulation of lipid metabolism. It showed that the regulation of blood lipid is a multi-channel regulation, and there are various connections between different pathways, and the mechanism is complex. In addition, the author used a hyperlipidemia SD rat model, and the synthesis of endogenous cholesterol was quite different from that of human. Therefore, the specific mechanism of reducing blood lipid and antioxidant ofAmpelopsis grossedentataflavonoid is not clear, which needs further study.