Effects of Lipid Regulation Using Raw and Processed Radix Polygoni Multiflori in Rats Fed a High-Fat Diet

Raw and processed Radix Polygoni Multiflori have been used in the prevention and treatment of nonalcoholic fatty liver disease (NAFLD), hyperlipidemia, and related diseases in Asian counties for centuries. The lipid regulation ability of raw and processed Poligoni Multiflori Radix were compared in high-fat diet fed rats in this research. Total cholesterol (TC) and low density lipoprotein-cholesterol (LDL-C) in blood and liver tissue were all significantly higher in model rats. However, triglyceride (TG) contents increased only in liver tissue, not in the blood samples. The rats fed the high-fat diets were considered the model of type IIa hyperlipidemia and early-stage nonalcoholic fatty liver disease. Both Radix Polygoni Multiflori (RPM) and Radix Polygoni Multiflori Praeparata (RPMP) revealed TC-lowing effects, and middling doses of RPMP displayed the most significant TC-lowing effects, as indicated by blood samples. Neither RPM nor RPMP was found to reduce LDL-C in rats' blood. Nevertheless, RPM showed dose-dependent TC- and TG-lowing effects in the liver tissue samples. In conclusion, RPM showed more pronounced effects on lipid regulation in liver samples in the treatment of early-stage NAFLD. RPMP, however, displayed better effects in regulating lipids in circulating blood for the treatment of hyperlipidemia.


Introduction
Fatty liver is a reversible condition in which large vacuoles of triglyceride fat accumulate in liver cells via the process of steatosis (abnormal retention of lipids within a cell). Considering the contribution that alcohol can make to this condition, fatty liver may be termed alcoholic steatosis or nonalcoholic fatty liver disease (NAFLD).
The development of nonalcoholic fatty liver disease comes from an imbalance between the influx and production of fatty acids and the use of fatty acids for oxidation or secretion. The progress of NAFLD is usually characterized by morphologic changes in the hepatocytes and hepatic triglyceride content (HTGC). However, early-stage NAFLD shows changes only in the fatty acid levels but no or few morphologic changes in hepatocytes [1]. In normal human livers, the mean values for total cholesterol (TC) and triglyceride (TG) are 3.9 and 19.5 mg/g wet weight, respectively. Hepatic steatosis (fatty liver) is a condition that is defined by fat accumulation within hepatocytes that exceeds 50 mg/g of the liver by weight [2,3].
Fatty liver disease was recently recognized as a feature of the metabolic syndrome; fatty liver has evolved as a key player in the pathogenesis of hyperlipidemia. Hyperlipidemia is characterized by elevated TG, TC, and low-density lipoprotein cholesterol (LDL-C) and decreased high-density lipoprotein cholesterol (HDL-C) levels. Hyperlipidemia is a major risk factor for cardiovascular disease.
Because of the effectiveness and acceptable prices, the prevention and treatment of NAFLD and hyperlipidemia by traditional Chinese medicine attract more and more attention worldwide [4,5]. Radix Polygoni Multiflori (RPM, heshouwu in Chinese) and Radix Polygoni Multiflori Praeparata (RPMP, zhiheshouwu in Chinese), originating from the root of Polygonum multiflorum Thunb., are used in the treatment of NAFLD and hyperlipidemia in oriental counties for centuries [6]. Although both RPM and RPMP have a history of use in the treatment of NAFLD and hyperlipidemia, RPMP is used more frequently in traditional Chinese medicine than RPM is. For example, the Pharmacopoeia of the People's Republic of China (2010 edition) lists three different prescription preparations containing RPMP for the treatment of hyperlipidemia [6]: Xuezhining Wan, Xuezhiling Pian, and Shouwu Wan. It lists only one preparation, Zhengxin Jiangzhi Pian, that contains RPM. In the meantime, few studies have compared the effects of RPM and RPMP in the treatment of NAFLD, hyperlipidemia, and related diseases. Our research group has performed previous studies including systematic comparisons of raw and processed RPM with respect to their antioxidative activities, adverse laxative effects, cytotoxicity, and in vitro lipid-regulation effects [7][8][9]. In our in vitro studies, raw RPM showed stronger abilities to regulate TG and TC than RPMP, indicating that RPM might be effective in the clinic treatment of NAFLD. However, in vivo results are required for corroboration. We compared the relative activities of raw and processed RPM in SD rats fed high-fat diets. Comprehensive studies of indexes of lipid metabolism in both the blood and liver tissue samples of the test animals were analyzed.

Chemicals.
Simvastatin (Hangzhou MSD Pharmaceutical Co., Ltd., China) and fenofibrate (Laboratories Fournier S.A., France) were used as positive controls for lowering cholesterol and triglyceride levels, respectively. Lard oil was purchased from the Shuangliu Luxiao oil factory in Chengdu, Sichuan Province, China. Sodium chloride, methylthiouracilum (MTU), ether, and other reagents were of analytical grade.

Processing and Extraction of Radix Polygoni Multiflori.
Polygonum multiflorum Thunb. plants were collected in June of 2008 by the authors in Luquan County within Yunnan Province and identified by Professor Ronghua Zhao of Yunnan University of Traditional Chinese Medicine. No specific permits were required for the described field studies. This location is not privately owned or protected in any way and the field studies did not involve endangered or protected species. Voucher specimens were deposited in the Herbarium of Pharmacognosy, Yunnan University of Traditional Chinese Medicine. RPMP was steamed by the authors from RPM with black soybean decoction according to the procedure recorded in Pharmacopoeia of the People's Republic of China (2010 edition) (Figure 1) [6].
Extracts, 300 g RPM and 472 g RPMP, were decocted with water (10 times, 8 times, and then 6 times by volume) for three times, respectively. Extracts were combined, condensed, and lyophilized. The concentrations of RPM and RPMP were 0.6980 g/mL and 0.8580 g/mL, respectively. The recommended dosages of RPM and RPMP are 3-6 g and 6-12 g/per day according to the Pharmacopoeia of the People's Republic of China, 2010 edition [6]. We conversed the human dosage equivalently to rat dosage. The low, middle, and high dosages of RPM are 0.405, 0.810, and 1.62 g/kg body weight [10]. The low, middle, and high doses of RPMP are 0.810, 1.62, and 3.24 g/kg body weight.

Animals and Diets
. SD rats of both sexes were provided by Experimental Animal Center of Yunnan University of Traditional Chinese Medicine. They were aged 8 weeks and weighed 245 ± 20 g. Rats of the same sex were housed six to a stainless steel cage containing sterile paddy husk as bedding in ventilated animal rooms. They were acclimated in the controlled environment (temperature 22 ± 1 • C; 60 ± 10% humidity; and a 12 h/12 h light/dark cycle) with free access to water and a commercial laboratory complete food. All animal experiments were performed in compliance with the animal experimental ethics committee of Yunnan University of Traditional Chinese Medicine. All reasonable efforts were made to minimize the animals' suffering.
One hundred and twenty SD rats of both sexes were randomly divided into 10 groups of twelve each (Table 1). The male and female rats were all six for each group. Group A received normal diets only and served as vehicle. Group B received high fat diets only and served as the model group.
Other groups were fed high fat diets throughout the whole study periods. Eighteen days after the start of the study, the RPM group (Groups C, D, and E), RPMP group (Groups F, G, and H), and two positive control groups (simvastatin in Group I and fenofibrate in Group J) received corresponding treatments every day for another 24 days (Table 1). All rats were fasted for 2 h every day before administration of therapeutic agents.

Assessment of Total Cholesterol, Triglyceride, Lipoprotein, and Liver Marker Enzyme in Blood.
Samples of blood 1.5-2 mL in volume were collected from the retro-orbital venous plexus once every 6 days throughout the study. Blood Samples were collected under ether anesthetic condition, two hours after administration of therapeutic agents in the morning. Serum was centrifuged at 16,000 rpm for 15 min and analyzed immediately. Levels of AST, ALT, TG, TC, LDL-C, and HDL-C in serum were determined by enzymatic colorimetric method using commercial standard enzymatic assay kits (Biosino Bio-technology & Sience Inc.) by AB-1020 automatic biochemical analyzer (Sunostik Medical Technology Co., Ltd.). TG and TC were tested on days 0, 6, 18, 24, 30, 36, and 42. LDL-C and HDL-C were tested on days 0, 6, 18, 24, 30, and 42. AST and ALT were tested only on days 0, 18, and 42 day. VLDL was analyzed using enzyme-linked immunosorbent assay kits from Rapidbio (U.S.). VLDL was tested only on days 0, 18, and 42. All bioassays were carried out in duplicate.

Assessment of Total Cholesterol, Triglyceride, Lipoprotein,
and Liver Marker Enzyme in Liver Tissue. The rats were sacrificed by cervical dislocation two hours after the last administration of therapeutic agents in the morning. Tissue samples from their livers were immediately processed for biochemical analysis and morphologic observations. Singlegram tissue samples of liver were homogenized with 9 mL ice-cold 0.9% physiological saline. Homogenates of liver were centrifuged at 4000 rpm for 10 min at 4 • C. Five milliliters of supernatant was maintained at −80 • C until analysis. One hundred microliter samples of liver homogenate were diluted with 400 μL distilled water for determination [12]. AST, ALT, TG, TC, LDL-C, and HDL-C were tested in all liver tissue samples at the end of the study.

Morphologic Observations.
For light microscopic observations, samples from liver were fixed in formalin fixative and processed routinely for embedding in paraffin. Tissue sections 5 μm in thickness were stained with hematoxylin and eosin (H&E) and examined under a light microscope.

Statistical Analysis.
All data in this study are expressed in the form of mean ± SD. The data were evaluated by one-way analysis of variance (ANOVA), and the differences between means assessed using Duncan's test with a significance level of P < 0.05, <0.01, and <0.001.

General Condition of Rats. One hundred and twenty
Sprague-Dawley rats of either sex were randomly divided into 10 groups of twelve in each ( Table 1). The rats fed on normal diets showed about 43% weight gain in 42 days of experiment duration (Figure 2(a)). However, the body weights of rats fed high-fat diets were significantly lighter than those of rats fed normal diets groups beginning on day 18 day and persisted until the end of the experiment across all treatment groups (Figure 2(a)). We attributed the weight loss to the rats' displeasure with the taste of the high-fat diets. Indexes of liver, kidney, and spleen were recorded after the rats were killed (Figures 2(b), 2(c), and 2(d)). Liver and spleen indexes showed no significant differences between the control group and the high-fat-diet group. However, kidney indexes in the high-fat diet group were slightly lower than those of the in control group. Slight decreases in the kidney and spleen indexes were observed in the middle-dose RPM (Group D). The liver and spleen indexes of rats in the simvastatin group also decreased. Indexes of liver lipid levels Experiment days were increased in the high-dosage RPM group (Group E). No serious pathological alterations were observed in this study, as determined by organ indexes.

Biochemical Indexes in Blood Sample of Model Group Rats.
The TG contents in Group B were higher than those of Group A on day 6. After that, the TG contents dropped to a level lower than Group A ( Table 2). The TC contents in Group B increased acutely from the beginning of the experiment ( Table 3). The TC contents of Group B were increased to the highest level on day 24, 359.9 ± 61.90 mg/dL, almost 3 times than control group ( Table 3). The amplitude of TC contents started to be decrease after day 24 but remained very high, at about 300 mg/dL. LDL-C (Table 4) and HDL-C (Table 5) contents in Group B were higher than Group A from day 6 through the end of the study. Very low density lipoprotein (VLDL) content, as indicated by ELISA kits, showed no differences from the control group at the beginning of the study or on day 18 (Table 6). At the end of the experiment, the VLDL contents were even lower than those of control group.
Blood levels of aspartate aminotransaminase (AST) and alanine aminotransaminase (ALT) were evaluated on days 0, 18, and 42 (data not shown). No increases in AST or ALT   Low density lipoprotein cholesterol (LDL-C) contents were assayed by assay kits as described in the text.
were observed, which indicated that liver damage did not occur during the research period.

Biochemical Indexes in Liver Tissues of Model-Group Rats.
Levels of TG, TC, LDL-C, HDL-C, AST, and ALT contents in liver tissue were tested after execution at the end of the research (Table 7). TC, TG, and LDL-C levels in the highfat diet group were significantly higher than in the control group, but other indexes showed no difference. The control and high-fat diet groups showed similar AST and ALT levels, indicating normal liver function. Morphologic observations were carried in every group. Livers in the high-fat diet groups were more paler than normal groups (figure not shown), probably due to the intracellular edema, However, no fatty deposits were observed in the liver biopsy slides of the model group ( Figure 3). Actually, no fatty deposits were observed in normal diet group, high fat diet group, positive groups, RPM group, and RPMP groups (figure not shown). That is probably due to the detention of morphologic changes compared to the biochemistry changes. For these reasons, the model group rats were considered to have early-stage NAFLD.
According to the Fredrickson's classification of hyperlipidemia, type IIa hyperlipidemia is characterized by elevation of total cholesterol and LDL-C, while type IIb hyperlipidemia is characterized by elevation of total cholesterol, LDL-C, and VLDL-C. Judging from the lipid indexes listed above, the rats fed the high-fat diets were diagnosed with type IIa hyperlipidemia and early-stage non-alcoholic fatty liver disease.

Antihyperlipidemic Effects of RPM and RRPM.
The TG levels in the blood samples were significantly higher in Group B after 6 days on the high-fat diet than those of control rats. TG did not continue to increase. TG levels were lower than those of control rats during the rest of the study. Considering the unremarkable increases in TG in the model group, we did  Very low density lipoprotein (VLDL) contents were assayed by assay kits as described in the text.
Values were mean ± SD (n = 12) and expressed in μmol/L. The * indicates a significant difference compared with control group, * P < 0.05, * * P < 0.01, * * * P < 0.001. The # indicates a significant difference compared with model group, # P < 0.05. The † indicates a significant difference compared with PMR group (same dosage level, F compared with C, G compared to D), † P < 0.05, † † † P < 0.001. not conclude that there was a relationship between different treatments, dosages, and durations of high-fat diet. The RPM, RPMP, and positive control treatments began on day 19 of the study. No TC melioration effects were observed in the low-dosage raw RPM group. Both the middle (Group D) and high dosage (Group E) RPM groups showed significant changes in TC regulation in blood samples. The most pronounced TC regulation effects of RPMP were observed in the middle-dosage RPMP group (Group G). Time-dependent TC regulation activities were observed in Group G. At the end of the experiment, the TC levels in Group G were reduced to 188.0 ± 50.93 mg/dL, significantly lower than that of model group (P < 0.001).
Similar TC-lowing effects were observed between Group I (simvastatin group), Group D, and Group G during the early stages of treatment (from day 19 through day 30). However, the rats given traditional Chinese medicines showed better results than those given simvastatin at the end of the study (Group D, E, F, and G).
Dosage relationships were observed between the LDL-C lowing activity in RPM groups, although no significant differences were observed across different groups (Table 4). Simvastatin (Group I) and fenofibrate (Group J) were found to remarkably downregulate the LDL-C content by the end of the study (P < 0.05).
HDL-C contents were generally higher in rats fed a highfat diet than those fed a normal diet no matter what kinds of treatment were administered (Table 5).

Anti-Nonalcoholic Fatty Liver Disease Effects of RPM and
PMPR. Liver tissue samples were homogenized and analyzed in order to evaluate the lipid regulation activities of raw and processed RPM in the liver. The RPM showed significant abilities to reduce levels of hepatic TG and TC. High doses of RPM were found to thoroughly control the progress of fatty accumulation in the liver. The TG and TC contents dropped to 153.6 ± 27.34 mg/dL and 57.18 ± 6.754 mg/dL, respectively, which were similar to the levels observed in the control group. Dose activity relationships were observed in the RPM groups. However, PRMP dosage was not found to be directly related to lipid regulation effects. Low and high doses of RPMP showed better effects than middle doses.
No remarkable increases in AST or ALT were observed in any of the treatment groups, indicating that liver function remained normal.
Simvastatin (Group I) and fenofibrate (Group J) showed excellent abilities to reduce TC levels, but simvastatin even showed more pronounced abilities to reduce TG than fenofibrate.
LDL-C contents were increased in RPM and RPMP treatment groups, especially the RPMP treated group. Simvastatin (Group I) and fenofibrate (Group J) were both found to downregulate LDL-C content.

Discussion
Hyperlipidemia and NAFLD are major risk factors for cardiovascular disease. Certain frequently used lipid-lowing drugs (fibrates, stains, and bile acid sequestraints) used for the treatment of hyperlipidemia and NAFLD have numerous