Scholarly article on topic 'Antioxidant and hepatoprotective effect of different extracts of Guizhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlipidemia rats'

Antioxidant and hepatoprotective effect of different extracts of Guizhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlipidemia rats Academic research paper on "Veterinary science"

CC BY-NC-ND
0
0
Share paper
OECD Field of science
Keywords
{Bidens / Antioxidants / "Drug-induced liver injury" / Hyperlipidemias}

Abstract of research paper on Veterinary science, author of scientific article — Xichao Xia, Yuhong Ma, Xiankun Xing, Chuanfeng Huang, Ling Li, et al.

Abstract Objevtive To investigate the antioxidant and hepatoprotective properties of the different extracts Guizhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlipidemia rats. Methods The rats were divided into 7 groups, with 10 rats in each. Rats were treated with high-fat diet for 18 weeks besides the normal control group, then rats in both normal control and model groups were received 5 mL/kg−1·day−1 of saline and those in the positive control group with 2 mg/kg−1·day−1 of lovastatin. Rats in the positive control group and different Guizhencao (Herba Bidentis Bipinnatae) extracts treatment groups (ethyl acetate extract group, n-hexane extract group, ethanol extract group, and aqueous extract group) were treated with corresponding extract at a concentration of 5 mL/kg−1·day−1. After 8 weeks treatment, all rats were sacrificed and total blood samples were collected. Histological analysis of liver was underdone by hematoxylin and eosin. The levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), glouse (GLU), cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol were measured according to standard procedure using auto-analyzer. The superoxide dismutase (SOD) and malondialdehyde (MDA) levels in liver were ananlyzed by procedure instruction. Results The histopathological analysis implied that the administration of Guizhencao (Herba Bidentis Bipinnatae) extracts resulted in hepatoprotective role compared with that of the model group. In addition, the high-fat diet caused a remarkable increase of ALT, AST, GLU, TC, TG, LDL-C and MDA levels. A decline in HDL-C and SOD concentrations and a reversal of effects were observed in different Guizhencao (Herba Bidentis Bipinnatae) extracts groups, especially in the aqueous extract and ethanol extract groups. Conclusions The different extracts of Guizhencao (Herba Bidentis Bipinnatae) can play a protecting role against liver injury in hyperlipidemia rats maybe through decreasing ALT, AST, GLU, TC, TG, LDL-C and MDA levels and enhancing the liver anti-oxidative ability.

Academic research paper on topic "Antioxidant and hepatoprotective effect of different extracts of Guizhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlipidemia rats"

JTCM

Online Submissions: http://www.journaltcm.com info@journaltcm.com

JTradit Chin Med 2013 August 15; 33(4): 518-523 ISSN 0255-2922 © 2013 JTCM. All rights reserved.

EXPERIMENTAL STUDY

Antioxidant and hepatoprotective effect of different extracts of Gui-zhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlip-idemia rats

Xichao Xia,Yuhong Ma, Xiankun Xing, Chuanfeng Huang, Ling Li, Gaixia Gui, Qingchun Liu, Shipeng Xue

Xichao Xia, Yuhong Ma, Xiankun Xing, Chuanfeng Huang, Ling Li, Gaixia Gui, Qingchun Liu, Shipeng Xue,

Basic Medicine College, Nanyang Medical University, Nan-yang 473061, China

Supported by the National Natural Science Foundation of Henan (No. 122102310103)

Correspondence to: Prof. Xichao Xia, Basic Medicine College, Nanyang Medical University, Nanyang 473061, China. xiaxichao8336@163.com Telephone: +86-377-63526123 Accepted: March 7,2013

Abstract

OBJEVTIVE: To investigate the antioxidant and hepatoprotective properties of the different extracts Guizhencao (Herba Bidentis Bipinnatae) against liver injury in hyperlipidemia rats.

METHODS: The rats were divided into 7 groups, with 10 rats in each. Rats were treated with high-fat diet for 18 weeks besides the normal control group, then rats in both normal control and model groups were received 5 mL/kg-1 • day-1 of saline and those in the positive control group with 2 mg/kg-1 • day-1 of lovastatin. Rats in the positive control group and different Guizhencao (Herba Bidentis Bipinnatae) extracts treatment groups (ethyl acetate extract group, n-hexane extract group, ethanol extract group, and aqueous extract group) were treated with corresponding extract at a concentration of 5 mL/kg-1 • day-1. After 8 weeks treatment, all rats were sacrificed and total blood samples were collected. Histological analysis of liver was underdone by hematoxylin and eosin. The levels of serum ala-

nine aminotransferase (ALT), aspartate aminotransferase (AST), glouse (GLU), cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (LDL-C) and high-density lipoprotein-cholesterol were measured according to standard procedure using auto-analyzer. The superoxide dismutase (SOD) and malondialdehyde (MDA) levels in liver were ananlyzed by procedure instruction.

RESULTS: The histopathological analysis implied that the administration of Guizhencao (Herba Bidentis Bipinnatae) extracts resulted in hepatoprotective role compared with that of the model group. In addition, the high-fat diet caused a remarkable increase of ALT, AST, GLU, TC, TG, LDL-C and MDA levels. A decline in HDL-C and SOD concentrations and a reversal of effects were observed in different Guizhencao (Herba Bidentis Bipinnatae) extracts groups, especially in the aqueous extract and etha-nol extract groups.

CONCLUSION: The different extracts of Guizhencao (Herba Bidentis Bipinnatae) can play a protecting role against liver injury in hyperlipidemia rats maybe through decreasing ALT, AST, GLU, TC, TG, LDL-C and MDA levels and enhancing the liver an-ti-oxidative ability.

© 2013 JTCM. All rights reserved.

Key words: Bidens; Antioxidants; Drug-induced liver injury; Hyperlipidemias

introduction

Liver plays an important role in regulating lipid metab-518 August 15, 2013 | Volume 33 | Issue 4 |

olism pathways, including fatty acid b-oxidation, lipo-genesis, as well as lipoprotein uptake and secretion in response to nutritional and hormonal signals.1'2 Meanwhile, the liver is also one of the most vulnerable organs and prone to be impaired by lipid over-accumula-tion.34 It has demonstrated that the oxidative stress of fat metabolism can cause liver disorder, ultimately results in hyperlipidemia, cardiovascular and chronic liver diseases.1,5 6 Therefore, the great efforts have been invested to exploit the perfect antioxidant drug protecting liver from damage.78 Among them, the studies of Traditional Chinese Medicine have aroused enormous interest in the world because of its multi-acted-targets and multi-regulated-approaches in the therapy.9 Guizhencao (Herba Bidentis Bipinnatae), a folk medicine in contemporary China, is highly priced to treat diabetes, hypertension and arteriosclerosis in duce to its obvious inhibitory effects on pathogenic microorganisms and strong antioxidant properties.1011 In some cases, it is showed that Guizhencao (Herba Bidentis Bi-pinnatae) has an obvious lipid-lowering role,12,13 but reports on live injury protection in hyperlipidemia are rear.14 Therefore, this study is performed to investigate the effects and mechanisms of aqueous extract, ethanol extract, ethylacetate extract and n-hexane extract of Guizhencao (Herba Bidentis Bipinnatae) on liver in hy-perlipidemia rats.

MATERIALS AND METHODS

Plant material collection and extraction

Guizhencao (Herba Bidentis Bipinnatae) was collected in Funiu mountain region of Nanyang in Henan province, and was identified and authenticated by Prof. Zongcai Liu, College of Life Sciences, Nanyang Normal University. The dried stems and leaves of Guizhencao (Herba Bidentis Bipinnatae) (4.0 kg) were ground to a fine powder and equally divided into 4 fractions, followed by successive extraction at room temperature with distilled water, ethanol, ethyl acetate and n-hex-ane, respectively. Briefly, filtration and evaporation of extracts of Guizhencao (Herba Bidentis Bipinnatae) were performed under reduced pressure at 45T!, followed by lyophilization, and stored at — 20T! until use. Finally, the solid form of the extract was dissolved with dimethyl sulfoxide for use in experiments.

Animals and experiment design

Adult male Sprague-Dawley rats (13-15 weeks, SPF, SCXKLU20080002) weighing 200-250 g were obtained from Henan Laboratory Animals Center for Medical Science and Research. The rats were conducted at a controlled temperature of 19^-25^ with a 12: 12 h light/dark cycle and fed a standard diet. The experiments were conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The rats were treated in accordance

with Committee of Ethicsin Animal Research of Nan-yang Medical University.

The rats were divided into following 7 groups, with 10 in each. Rats in normal control group were received a regular rodent chow (normal diet: 62.3% carbohydrate, 2.5% fat, 24.3% protein calories). The rest rats were treated with a high-fat diet (32.6% carbohydrate, 51.0% fat, 16.4% protein calories) for 18 weeks. When the total cholesterol level in serum of rats feed high-fat diet attained >3.87 mmol/L, those rats were selected and divided into six subgroups including model group, positive control group, and four different Gui-zhencao (Herba Bidentis Bipinnatae) extracts treatment groups (ethyl acetate extract group, n-hexane extract group, ethanol extract group, and aqueous extract group). Subsequently, rats in both normal control and model groups were received 5 mL/kg-1 • day-1 saline and those in positive control group with 2 mg/kg-1 • day-1 of lovastatin. Rats in different Guizhencao (Herba Biden-tis Bipinnatae) extracts treatment groups were treated with 5 mL/kg-1 • day-1 corresponding extract, respectively. All these treatments were given orally for 8 weeks, then the rats were killed by cervical dislocation and whole blood collected. Serum obtained by immediate centrifugation of blood samples using centrifuge at 3000 rpm for 15 min. Liver were dissected, immediately frozen in liquid nitrogen, and then stored at — 80T! until used.

Measurement of growth rate of body weight and Liver index

The body weight (BW) in each group was recorded on day 0 (the first day of experiment) and on the last day (the end time of experiment). The growth rate of body weight (BW%) was calculated by the following formula.

BW%= {[BW on the last day (g) — BW on day 0 (g)]/ BW on day 0 (g)}x100

The Liver index (LI) of rats was measured by the

Pooja's methods.14

LI=[(wet weight of liver/BW]x100

Histological analysis

A portion of liver tissue was fixed in phosphate-buffered 10% formalin, cut into 5 |xm sections and stained with hematoxylin and eosin (HE). Histological observations on liver tissues were underdone using light microscope (Olympus, Kanagawa, Japan).

Determination of biochemical parameters

Serum levels of the alanine aminotransferase (ALT), as-partate aminotransferase (AST) and glouse (GLU), as markers of hepatic damage and function, were measured according to standard procedure using AU2700 Automatic Biochemical Analyzer (Olympus, Kanaga-wa, Japan). The total cholesterol (TC), triglycerides (TG), low-density lipoprotein-cholesterol (HDL-C) and high-density lipoprotein-cholesterol were deter-

mined according to standard procedure (BOSTER, Wuhan, China).

Determination of oxidative stress markers

Livers were perfused with chilled normal saline to completely remove all the blood cells, then cut into small pieces, placed in 0.2 M phosphate buffer (pH 7.4), and homogenized using homogenizer to obtain 20% homogenate. The homogenate was centrifuged at 2500 rpm for 15 min. The lipid peroxidation was determined by malondialdehyde (MDA) level. In addition, the uperoxide dismutase (SOD) activity was measured spectrophotometrically using Superoxide dismutase Assay Kit (BOSTER, Wuhan, China).

Statistical analysis

The original datas were analyzed by Statistical Package for Social Sciences software (version 13.0) (SPSS, Inc., Chicago, IL, USA). The results were expressed as mean ± standard error of mean (SEM). The multiple sets of data comparison were carried out by one-way analysis of variance (ANOVA) and Brown-forsy way. The comparison of data between groups were underdone by least significance difference (LSD) combined with Tamhane's way. Values with P<0.05 were considered as statistically significant.

RESULTS

Growth rate of body weight and liver index

The growth rate of body weight and liver index in model group were significant increased compared with that in the normal group (Figure 1). In contrast with the model group, rats treated different extracts of Guizhencao (Herba Bidentis Bipinnatae) resulted in a statistical decline of the growth rate of body weight and liver index (Figure 1).

Histopathologic analysis on liver sections

The liver sections of normal rats showed normal cell structure with distinct hepatic cells, sinusoidal spaces and a central vein (Figure 2A). The liver sections of the model group showed presence of destructive alterations in the parenchyma, extensive fatty changes, disarrangement of normal hepatic cells with high degree of damage, characterized by the centrilobular necrosis, focal necrosis, and loss of cell boundary bile duct proliferation (Figure 2B). The liver sections treated with different extracts of Guizhencao (Herba Bidentis Bipinnatae) and lovastatin exhibited less centrilobular necrosis and fatty changes compared with that in the model group. Meanwhile, the administration of Guizhencao (Herba Bidentis Bipinnatae) aqueous extract showed perfect hepatoprotective activity in rats, then the ethanol extract and ethyl acetate extract (Figure 2C-2E).

Biochemical parameters

The concentrations of ALT, AST and GLU in the model

1 II III IV V VI vn

Figure 1 Effects of different extracts of Guizhencao (Herba Bidentis Bipinnatae) on growth rate of body weight and liver index

A and B were the results of the growth rate of body weight and liver index, respectively. Bars were normal control group ( I), model group ( H), positive control group (M), ethylacetate extracts group (IV), n-hexane extract group (V), aqueous extract group (M) and ethanol extract group (W). n=9. aP<0.01 vs normal control group. bP<0.05, cP<0.01 vs model group.

group increased significantly in contrasted with those in the normal group. After treatment of Guizhencao (Herba Bidentis Bipinnatae) ethanol extract, the levels of ALT and AST decreased by 34.9% (P<0.05) and 52.8% (P<0.01), respectively, while in the aqueous extract group by 34.1% (P<0.05) and 46.6% (P<0.05) as compared with those in the model group. Among the 7 groups, the lowest level of GLU was detected in the ethylacetate extract group in which it reduced by 61.1% (P<0.01) as compared with that in the model group and 35.0% (P<0.05) in the positive control group (Table 1).

Compared with that in the model group, the levels of TC, TG and LDL-C in other groups were obviously decreased. After treatment with ethyl acetate extract, the contents of TC and LDL-C respectively decreased by 35.8% (P<0.05) and 45.3% (P<0.01), and HDL-C increased by 73.7% (P<0.01). The levels of TC and LDL-C in aqueous extract group was reduced by 29.5% (P<0.05) and 45.1% (P<0.01), respectively, but the HDL-C increased by 77.6% (P<0.01) (Table 1).

! 111 131 BBS

H H1 III

Figure 2 Histopathological changes occurred in liver (hematoxylin and eosin x200)

A: normal control group; B: model group; C: positive control group; D: ethylacetate extract group; E: n-hexane extract group; F: ethanol extract group; G: aqueous extract group.

Oxidative stress

In the ethys acetate extract, aqueous extract, and ethanol extract groups, SOD expression level increased by more than 21.7% (P<0.05) and the MDA level reduced by less than 18.8% (P<0.05) compared with that in the model group (Table 2).

DISCUSSION

Hyperlipidemia is considered to be one of the major risk factors for liver diseases, such as cirrhosis, hepatocellular carcinoma and liver failure.1,15,16 In the Chinese medicine, many plants are used as hypolipidemic drugs and played an important protecting role in liver dis-ease.3,17,18 Among them, Guizhencao (Herba Bidentis Bipinnatae) is attracting great interest in the oriental traditional medicine.12,13

Present results showed that oral administration of different Guizhencao (Herba Bidentis Bipinnatae) extracts resulted in decline of the growth rate of body weight and liver index, reduction of serum TC, TG and LDL-C levels, and evaluation of HDL-C content, indi-

cating the different extracts contributed to reduce the lipid accumulation in hyperlipidemia rats. It has been well established that high-fat diet plays an important role in the etiology of hyperlipidemia and atherosclerosis.19,20 The continuous administration of high-fat diet can lead to the elevation in major parameters of lipid profile including TC, TG and LDL-C, and reduction of HDL-C.

The histological analysis imply that Guizhencao (Her-ba Bidentis Bipinnatae) extracts treatment may reduce hepatocellular damage, especially in ethanol extract and aqueous extract groups, reflecting the different extracts may play an protecting role in liver damage caused by high-fat diet. It is further indicated that the ways and/or targets acted by different extracts in the protection are not similar. The high level of serum ALT and AST is interpreted as a result of hepatocellular damage.21-23 In the present study, the different extracts treatment resulted in a notable decrease of ALT and AST levels as compared with those in the model group, and the significant decline mainly occurred in the etha-nol extract and aqueous extract groups. The results are

Table 1 Effects of different extracts of Guizhencao (Herba Bidentis Bipinnatae) on biochemical parameters (x ±s)

Group n ALT AST GLU TC TG LDL-C HDL-C

(U/L) (U/L) (mmol/L) (mmol/L) (mmol/L) (mmol/L) (mmol/L)

Normal control 9 30.025±3.525 104.364±7.627 2.225±0.158 2.818±0.189 0.373±0.073 0.835±0.093 1.952±0.115

Model 9 57.130±9.332" 197.521±34.051" 3.992±0.624d 4.411±1.114" 0.780±0.155" 2.279±0.124' 1.037±0.405d

Positive control 9 30.088±4.504b 110.665±8.202c 3.009±0.533 3.006±0.511b 0.501±0.041c 1.013±0.119b 1.093±0.221

Ethylacetate extract 9 52.876±5.150 136.813±5.788 1.956±0.705be 3.022±0.578c 0.547±0.027 1.241±0.135bf 1.794±0.323be

N-hexane extract 9 48.924±8.106c 124.681±29.591 2.628±0.326 3.913±0.447 0.515±0.036 2.242±0.192 1.534±0.216

Ethanol extract 9 36.496±5.474c 93.064±9.323b 2.606±0.354 3.775±0.821 0.445±0.069c 2.243±0.177 1.255±0.127

Aqueous extract 9 37.651±7.332c 105.418±10.795b 3.737±0.437 3.143±0.192c 0.598±0.055 1.218±0.376bf 1.839±0.264be

Notes: rats in normal control group and model group were treated with 5 mL/kg-1 ■ day- 1 of saline. Rats in positive control group were re-

ceived 2 mg/kg-1 ■ day-1 of lovastatin. Rats in ethylacetate extract group, n-hexane extract group, ethanol extract group and aqueous extract group were treated with corresponding extract of Guizhencao (Herba Bidentis Bipinnatae) at a concentration of 5 mL/kg-1 ■ day-1. ALT: alanine aminotransferase; AST: aspartate aminotransferase; GLU: glouse; TC: cholesterol; TG: triglycerides; LDL-C: low-density lipopro-tein-cholesterol; HDL-C: high-density lipoprotein-cholesterol. aP<0.01, P<0.05 vs normal control group; bP<0.01, P<0.05 vs model group; eP<0.05, fP<0.01 vs positive control group.

able 2 Effects of different extracts of Guizhencao (Herba Bidentis Bipinnatae) on oxidative stress ( x ±s)

Group n SOD (unit/mg) MDA (nmol/mg)

Normal control 9 40.819±3.497 0.644±0.068

Model 9 30.840±4.953a 0.912±0.072a

Positive control 9 39.674±3.411ь 0.661±0.053c

Ethylacetate extract 9 38.453±3.633b 0.697±0.056c

N-hexane extract 9 34.737±4.220 0.785±0.061

Ethanol extract 9 37.552±4.128ь 0.748±0.087bd

Aqueous extract 9 37.987±3.423ь 0.725±0.052ь

Notes: rats in normal control group and model group were treated with 5 mL/kg-1 • day-1 of saline. Rats in positive control group were received 2 mg/kg-1 • day-1 of lovastatin. Rats in ethylacetate extract group, n-hexane extract group, ethanol extract group and aqueous extract group were treated with corresponding extract of Guizhencao (Herba Bidentis Bipinnatae) at a concentration of 5 mL/kg-1 • day-1. SOD: superoxide dismutase; MDA: malondialdehyde. aP<0.01 vs normal control group; bP<0.05, CP<0.01 vs model group. dP<0.05 vs positive control group.

responding to the obvious protecting role of ethanol extract and aqueous extract in liver cells. Further research showed a remarkable induction of SOD expression and decrease of MDA levels were detected in aqueous extract and ethanol extract, suggesting the protecting role on liver of hyperlipidemia rat model is associated with enhancing antioxidant ability of rats. It was well known that MDA is considered as a biomarker to assay the lipid peroxidation which is prone to cause damage and failure of antioxidant defense capacity,24 that bring about the formation of excessive toxic species which in turn accelerate the cell damage.25,26 Similar studies on other Chinese herbs have been reported.27,28 The SOD possesses stronger scavenging properties to decompose these toxic spe-cies.4,6 Therefore, the lower MDA concentration and higher SOD level are attributed to preventing hepato-cellular damage. It is worth noting that ethylacetate extract treatment resulted in drop of GLU and MDA concentration and increase of SOD level, showing the different approaches were involved in the antioxidant event among different Guizhencao (Herba Bidentis Bi-pinnatae) extracts. It is also supposed that the complex components may be existed in ethylacetate extract of Guizhencao (Herba Bidentis Bipinnatae) which plays a potential role in lowering GLU level. Those works need to be further explored in the future.

ACKNOWLEDGEMENTS

We gratefully acknowledge the many contributions of Prof. Zongcai Liu to this research program.

REFERENCES

1 Gong WH, Zheng WX, Wang J, et al. Coexistence of hyperlipidemia and acute cerebral ischemia/reperfusion induces severe liver damage in a rat model. World J Gastroenterol 2012; 18(35): 4934-4943.

Diao Y, Zhao XF, Lin JS, Wang QZ, Xu RA. Protection of the liver against CCl4-induced injury by intramuscular electrotransfer of a kallistatin-encoding plasmid. World J Gastroenterol 2011; 17(1): 111-117.

Fang YZ, Li WJ. Free Radical and enzyme-basic theory and its applications in biology and medicine. Beijing: Science Press, 1989: 90-133.

Ganie SA, Haq E, Hamid A, et al. Carbon tetrachloride induced kidney and lung tissue damages and antioxidant activities of the aqueous rhizome extract of Podophyllum hexandrum. BMC Complement Altern Med 2011; 10(3): 11-17.

Yoshioka K, Kunitomo M, Yanai K, et al. Hepatocyte nuclear factor 1ß induced by chemical stress accelerates cell proliferation and increases genomic instability in mouse liver. J Recept Signal Transduct Res 2011; 31(2): 132-138. Zhang YB, Guo J, Dong HY, et al. Hydroxysafflor yellow a protects against chronic carbon tetrachloride-induced liver fibrosis. Eur J Pharmacol 2011; 660(2): 438-444. Feng Y, Wu ZH, Zhou XZ, Zhou ZM, Fan WY. Knowledge discovery in Traditional Chinese Medicine: State of the art and perspectives. Artif Intell Med 2006; 38(3): 219-236.

Chen JX, Zhao HH, Yang Y, Liu B, Ni J, Wang W. Lip-id-lowering and antioxidant activities of Jiang-Zhi-Ning in Traditional Chinese Medicine. J Ethnopharmacol 2011; 134(3): 919-930.

Wang XJ, Sun H, Zhang AH, Sun WJ, Wang P, Wang ZG. Potential role of metabolomics apporoaches in the area of Traditional Chinese Medicine: as pillars of the bridge between Chinese and Western medicine. J Pharm Biomed Anal 2011; 55(5): 859-868.

Nakama S, Ishikawa C, Nakachi S, Mori N. Anti-adult T-cell leukemia effects of Bidens pilosa. Int J Oncol 2011; 38(4): 1163-1173.

Abdou R, Scherlach K, Dahse HM, Sattler I, Hertweck C. Botryorhodines A-D antifungal and cytotoxic depsi-dones from Botryosphaeria rhodina an endophyte of the medicinal plant Bidens pilosa L. Phytochemistry 2010; 71 (1): 110-116.

Sun YB, Zhou QX, Wang L, Liu WT. Cadmium toler-

ance and accumulation characteristics of Bidens pilosa L. as a potential Cd-hyperaccumulator. J Hazard Mater 2009; 161(2): 808-814.

13 Silva FL, Fischer DC, Tavares JF, Silva MS, Athayde-Filho PF, Barbosa-Filho JM. Compilation of Secondary Metabolites from Bidens pilosa L. Molecules 2011; 16(2): 1070-1102.

14 Gupta P, Mehla J, Gupta YK. Antiobesity effect of Safoof Mohazzil, a polyherbal formulation, in cafeteria diet induced obesity in rats. Indian J Exp Biol, 2012; 50(11): 776-784.

15 Tomczykowa M, Leszczynska K, Tomczyk M, Tryniszews-ka E, Kalemba D. Composition of the essential oil of Bidens tripartita L roots and its antibacterial and antifungal activities. J Med Food 2011; 14(4): 428-433.

16 Rao JY, Yeriswamy MC, Santhosh MJ, et al. A look into Lee's score: perioperative cardiovascular risk assessment in non-cardiac surgeries usefulness of revised cardiac risk index. Indian Heart J 2012; 64(2): 134-138.

17 Adeneye AA, Adeyemi OO, Agbaje EO. Anti-obesity and antihyperlipidaemic effect of Hunteria umbellata seed extract in experimental hyperlipidaemia. J Ethnopharmacol 2010; 130(2): 307-314.

18 Gao HL, Liu ZC, Wan WJ, Qu XL, Chen MH. Aqueous extract of Yerba Mate Tea lowers atherosclerotic risk factors in a rat hyperlipidemia model. Phytother Res 2012; 102(9): 48-56.

19 Hsu YJ, Lee TH, Chang CL, Huang YT, Yang WC. An-ti-hyperglycemic effects and mechanism of Bidens pilosa L water extract. J Ethnopharmacol 2009; 122(2): 379-383.

20 Lee JH, Oh JH, Lee YJ. Effects of experimental hyperlipidemia on the pharmacokinetics of Tadalafil in Rats. J Pharm Pharmaceut Sci 2012; 15(4): 528-537.

21 Miranda-Nantes CC, Fonseca EA, Zaia CT, et al. Hypo-

glycemic and hypocholesterolemic effects of botryosphaer-an from botryosphaeria rhodina MAMB-05 in diabetes-induced and hyperlipidemia conditions in rats. Mycobiology 2011; 39(3): 187-193.

22 Zhao LY, Wei H, Yuan QX, et al. Hypolipidaemic effects and mechanisms of the main component of Opuntia dillenii Haw polysaccharides in high-fat emulsion-induced hyperlipidaemic rats. Food Chem 2012; 134(2): 964-971.

23 Adaramoye O, Amanlou M, Habibi-Rezaei M, Pasalar P, Ali MM. Methanolic extract of African mistletoe (Viscum album) improves carbohydrate metabolism and hyperlipidemia in streptozotocin-induced diabetic rats. Ya Tai Re Dai Yi Yao Za Zhi 2012; 5(6): 427-433.

24 Kviecinski MR, Felipe KB, Schoenfelder T, et al. Study of the antitumor potential of Bidens pilosa L (Asteraceae) used in Brazilian folk medicine. J Ethnopharmacol 2008; 117(1): 69-75.

25 Yuan LP, Chen FH, Ling L, et al. Protective effects of total flavonoids of Bidens pilosa L (TFB) on animal liver injury and liver fibrosis. J Ethnopharmacol 2008; 116(3): 539-546.

26 Costa Rde J, Diniz A, Mantovani MS, Jordäo BQ. In vitro study of mutagenic potential of Bidentis Bipinnatae-Linne and Mikania glomerata Sprengel using the comet and micronucleus assays. J Ethnopharmacol 2008; 118(1): 86-93.

27 Huang X, Kong L, Li X, Chen X, Guo M, Zou H. Strategy for analysis and screening of bioactive compounds in Traditional Chinese Medicines. Analyt Technol Biomed Life Sci 2004; 812(1): 71-84.

28 Li DC, Zhong XK, Zeng ZP, et al. Application of targeted drug delivery system in Chinese medicine. J Control Release 2009; 138(2): 103-112.