For Review Only - Prince of Songkla Universityrdo.psu.ac.th/sjstweb/Ar-Press/61-Jan/8.pdf · 2017. 12. 27. · For Review Only 2 1 simvastatin treated groups significantly increased

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Hypolipidemic effect of methoxyflavone-enriched extract of

Kaempferia parviflora in cholesterol-induced dyslipidemic

rats

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2017-0318.R1

Manuscript Type: Original Article

Date Submitted by the Author: 10-Oct-2017

Complete List of Authors: Somintara, Somsuda; Department of Anatomy, Faculty of Medicine, Khon

Kaen University, Khon Kaen, 40002 Thailand Sripanidkulchai, Bungorn; Khon Kaen university, Faculty of Pharmaceutical sciences Pariwatthanakun, Choowadee ; Department of Anatomy, Faculty of Medicine, Mahasarakham University Sripanidkulchai, Kittisak; Khon Kaen University, Department of Anatomy, Faculty of Medicine

Keyword: dyslipidemia, Kaempferia parviflora, methoxyflavones, simvastatin

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Songklanakarin Journal of Science and Technology SJST-2017-0318.R1 Sripanidkulchai

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Type of Article (Original Article) 1

Hypolipidemic effect of methoxyflavone-enriched extract of Kaempferia parviflora 2

in cholesterol-induced dyslipidemic rats 3

4

Somsuda Somintara1, Bungorn Sripanidkulchai2*, Choowadee Pariwatthanakun1 and 5

Kittisak Sripanidkulchai1 6

7

1Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen, 8

40002 Thailand 9

2Center for Research and Development of Herbal Health Products, Faculty of 10

Pharmaceutical Sciences, Khon Kaen University, Khon Kaen, 40002 Thailand 11

* Corresponding author, Email address: [email protected] 12

13

Abstract 14

Kaempferia parviflora is a Zingiberaceae plant that has been used as traditional 15

medicine in Asia. The pharmacological effects have been widely reported such as anti-16

inflammatory, anti-allergy, anti-obesity and suppression of adipocyte hypertrophy. This 17

study aimed to examine the hypolipidemic effect of methoxyflavone-enriched extract of 18

K. parviflora rhizome (MKE) in cholesterol-induced dyslipidemic rats. To develop the 19

hypercholesteroremic condition, the rats were fed with cholesterol (2g/kg) twice a day 20

for 6 weeks. Dyslipidemic rats were divided into 6 groups including three control 21

vehicle groups, two doses of MKE treatment groups, and a simvastatin positive control 22

group. Oral administration of MKE at daily doses of 150 or 300 mg/kg for 12 weeks 23

significantly decreased in the levels of cholesterol, triglyceride and LDL. The MKE and 24

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simvastatin treated groups significantly increased the levels of HDL. These results 1

suggest that MKE and its active components exhibit hypolipidemic effects in 2

dyslipidemic rats. 134 words 3

4

Keywords: dyslipidemia, Kaempferia parviflora, methoxyflavones, simvastatin 5

6

1. Introduction 7

Dyslipidemia is a metabolic disorder and a non-comunicable disease 8

characterized by elevation of plasma cholesterol, triglycerides (TG), or both, and a low-9

density lipoprotein (LDL) level. It is one of the highest risk factors associated with 10

atherosclerosis which finally leads to the development of cardiovascular diseases 11

(CVDs) including coronary artery disease, ischemic stroke, and peripheral arterial 12

disease (Catapano et al., 2016). CVDs are the first leading cause of death globally in 13

both males and females causing a major economic burden on private and public health 14

systems (Lozano et al., 2013; Mendis, Davis, & Norrving, 2015). An estimated 17.7 15

million people died from CVDs in 2015, representing 31% of all global deaths (Mendis 16

et al., 2015). Although the trend of mortality rates of CVDs declined in the 21st century, 17

CVD burdens come from patients living with the disease (Benjamin et al., 2017; Sidney 18

et al., 2016). In Thailand, cardiovascular death was an estimated 0.15 million people, 19

representing 29% of all causes of death (Organization, 2014). Simvastatin is in a group 20

of drugs called HMG-CoA reductase inhibitors, or statins. It is commonly used to 21

reduce blood cholesterol and triglyceride levels (Germershausen et al., 1989). 22

Numerous clinical controlled trials reported the safety of statins, however; their side 23

effects that include myopathy, liver enzyme and creatine kinase elevation have been 24

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observed (Baigent, Keech, Kearney, & Blackwell, 2005; Finegold, Manisty, Goldacre, 1

Barron, & Francis, 2014). Recently, many studies are searching for natural products that 2

have the potential to decrease blood lipids but cause fewer side effects. 3

Kaempferia parviflora has long been used as a folk medicine for many centuries 4

in Southeast Asia. The extracts of K. parviflora rhizomes have been demonstrated to 5

have various pharmacological activities such as anti-cancer (Banjerdpongchai, 6

Suwannachot, Rattanapanone, & Sripanidkulchai, 2008; Leardkamolkarn, Tiamyuyen, 7

& Sripanidkulchai, 2009), anti-inflammation (Sae-wong, Tansakul, & Tewtrakul, 2009), 8

anti-allergic (Tewtrakul, Subhadhirasakul, & Kummee, 2008), antiobesity (Akase et al., 9

2011), and vasorelaxation (Tep‐areenan, Sawasdee, & Randall, 2010). The ethanolic 10

extract of K. parviflora rhizomes contain many methoxyflavones such as 5,7-11

dimethoxyflavone (DMF), 5,7,4′-trimethoxyflavone (TMF) and 3,5,7,3′,4′-12

pentamethoxyflavone (PMF) (Mekjaruskul et al., 2013). In a model of the metabolic 13

syndrome, mixing K. parviflora powder-containing normal feed suppressed visceral fat 14

accumulation and hypertension in Tsumura Suzuki Obese Diabetes mice leading to 15

improved lipid metabolism (Akase et al., 2011). In addition, polymethoxyflavonoids 16

enhanced lipolysis and suppressed hypertrophy in mature adipocytes and the author 17

suggested this beneficial effect is important for preventing metabolic syndrome (Okabe 18

et al., 2014). Since much evidence has been found that the major component of K. 19

parviflora is involved in lipid metabolism, this study aimed to determine the 20

hypolipidemic effect of the methoxyflavone-enriched ethanolic extract of K. parviflora 21

rhizome in cholesterol-induced dyslipidemia in rats. 22

23

2. Materials and Methods 24

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Reagents and chemicals 1

Sodium carboxymethylcellulose (CMC) (Sigma Ltd, USA); simvastatin (Berlin 2

Pharmaceutical Company, Thailand); olive oil (Sabroso, Spain) and pentothal sodium 3

(Abbott laboratories, Italy) were obtained from the local distributors. All the other 4

chemicals used in the study were of analytical grade and were obtained commercially. 5

The standards of DMF, TMF and PMF were obtained from the K. parviflora extract 6

using column chromatography as previously described (Sutthanut, Sripanidkulchai, 7

Yenjai, & Jay, 2007). 8

9

Preparation of the plant extract 10

To prepare the methoxyflavone-enriched extract from Kaempferia parviflora 11

(MKE), the plant purchased from the local farm in Loei province was used to prepare 12

the ethanolic extract following the previously used procedure (License number 4048). 13

Briefly, the dried rhizome powder was macerated in 95% ethanol, filtered and dried 14

using a rotary evaporator and freeze dryer, which provided a 5.71% yield. Then the 15

extract was standardized by HPLC analysis for three major methoxyflavones which 16

were 13, 13, 11% of the final extract. For animal administration, MKE was suspended 17

in 0.2% CMC before use. 18

High performance liquid chromatographic analysis of MKE 19

The quantity of methoxyflavones in MKE was determined using a reverse phase 20

high performance liquid chromatographic (HPLC) system (Agilent 1200 series, 21

Germany) and Zorbax Eclipse plus C18 column (3.5 um, 4.6x150mm). The analytical 22

system included a quaternary pump with a VWD detector at a wavelength of 254 nm. 23

The column temperature was set at 30°C. The mobile gradient phases consisted of a 24

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mixture of water and acetonitrile (80:20 at 0-5 min, 70:30 at 10-25 min, 60:40 at 30-40 1

min and 0:100 at a 50-55 min run), at a flow rate of 1ml/min. The injection volume was 2

20 ul. To determine the area of three major methoxyflavones, 5,7-dimethoxyflavone, 3

5,7,4'-trimethoxyflavone and 3,5,7,3',4'-pentamethoxyflavone, the area peaks were 4

compared with the standard (Fig 1). 5

Experimental animals 6

Male Sprague-Dawley rats (200-300g body weight) were purchased from the 7

National Animal Center, Mahidol University, Thailand. They were housed at an 8

ambient temperature of 22±2°C with 12 h light/dark cycles and were acclimatized for 7 9

days prior to the experiment. The animals received standard rat food (C.P. rat food 10

082S.W.T Co. Ltd, Samutprakan, Thailand) and water ad libitum. All experiments were 11

conducted under the National Institute of Health Guide for the Care and Use of 12

Laboratory Animals (NIH Publications No 80-23) revised 1996 and approved by the 13

Ethical Committee (Animal Care and Use Committee) of Khon Kaen University 14

(Reference No AE 011/51). 15

To induce hyperlipidemia, the rats were fed with a diet containing 2g/kg 16

cholesterol in olive oil, twice per day. After 6 weeks of high cholesterol treatment, the 17

animals with blood cholesterol levels of more than 140 mg% (Hirunpanich et al., 2006) 18

were randomly divided into 6 groups of 8 animals each and orally treated for 12 weeks 19

as follows: Group1: hyperlipidemic control (control 1) received 0.7 ml distilled water ; 20

Group 2: olive oil control (control 2) received 0.7 ml olive oil; Group 3: vehicle control 21

(control 3) received 0.7 ml 0.2%CMC ; Groups 4-5: K. parviflora treated groups 22

(KD150, KD300) received K. parvoflora extract at the doses of 150 and 300 mg/kg 23

body weight, and Group 6: positive control with simvastatin treatment (SIM) received 24

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40 mg/kg body weight of simvastatin. All animals were treated daily and bi-weekly 1

monitored for the body weight and the blood lipid profile including total cholesterol 2

(TC), triglyceride (TG), high-density lipoprotein (HDL) (Coulter Synchron CX4, 3

Beckman, USA). Low-density lipoprotein (LDL) was determined as follows: LDL = 4

(TC-HDL) - TG/5. The atherogenic indices (AI) which are the significant plasma 5

markers of cardiovascular risk (Niroumand et al., 2015) was also determined using the 6

following equation reported previously (Gill et al., 1986). AI = (TC-HDL)/HDL). 7

Statistical analysis 8

All data are presented as the mean±S.E.M. Statistical analysis was done using 9

one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison 10

test using the SPSS program version 13.0. Significant differences were at p<0.05. 11

12

3. Results and Discussion 13

This study has shown the hypolipidemic effect of the methoxyflavone-riched 14

extract of K. parviflora rhizome (MKE) in hypercholesterol-induced rats. Giving 15

cholesterol (2g/kg) in olive oil twice a day for 6 weeks caused a significant 16

hyperlipidemic condition with high serum lipid profiles. When compared to the 17

baseline, at 6 weeks after the induction, the levels of cholesterol went from 99.05±0.92 18

to 144.60±0.51 mg%, triglycerides from 87.02±0.83 to 144.93±1.03 mg%, and low 19

density lipoprotein (LDL) from 31.46±0.62 to 111.44±0.91 mg% significantly 20

increased, whereas the level of high density lipoprotein (HDL) significantly decreased 21

from 85.00±0.82 to 62.15±0.70 mg% (Fig 2). Among lipid profile parameters, total 22

cholesterol (TC) was commonly used to reflect the lipid metabolism. In this study, the 23

level of TC >140% was used as a criteria of dyslipidemia in a hypercholesteremic rats 24

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with high cholesterol feeding as previously reported (Hirunpanich et al, 2006). Several 1

clinical and epidemiologic studies has demonstrated that dyslipidemia can cause 2

cholesterol accumulation at the vascular cells and the arterial wall, promoting 3

endothelial cell dysfunction, and the development of atherosclerosis (Aikawa and 4

Libby, 2004; Takahashi et al., 2005). These phenomena were demonstrated in both cell 5

culture and animal models (Anila & Vijayalakshmi, 2002; Matos et al., 2005; Takahashi 6

et al., 2005).The animals with serum cholesterol levels of more than 140 mg% were 7

then randomly divided into 6 groups (n=8). During the 6-week high cholesterol 8

induction period, the body weights of all animals were increased without any significant 9

differences among the groups (Fig 3) suggesting that the animals grew similarly and 10

that they were appropriate to be used as hyperlipidemic models in this study (Gosain et 11

al., 2010). To elucidate the hypolipidemic effect of the plant extract, simvastatin was 12

used as a positive control drug: two doses each of the extracts KD 150 and KD300, and 13

three control vehicle groups: control 1 distilled water; control 2: olive oil and control 14

3:CMC, were implemented for another 12-week duration. Administration of the plant 15

extract at both doses did not affect the body weights of the animals when compared to 16

the three control and simvastatin-treated groups (Fig4). During this 12-week period, the 17

animal body weights of these six groups were not significantly different. Despite that 18

higher body mass indices are significantly associated with the risk of dyslipidemia 19

(Shamai et al., 2011), there is evidence that changes in blood lipid profiles are unrelated 20

to weight (Karam et al., 2016). In general, the animals gained 72-100 g of the baseline 21

body weight or 6-8.3 g per week, indicating the safety of the plant extract in terms of 22

the growth rate. 23

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Bi-weekly monitoring of the serum lipid profile indicated that there were 1

significant decreases in the levels of cholesterol, triglyceride and LDL with the 2

significant increase in the level of HDL of the plant extract treated-groups and as 3

observed in the simvastatin-treated group (Fig 5-8). These findings confirm those of an 4

earlier study which stated that mixing 3% of K. parviflora powder with normal feed 5

suppressed dyslipidemia in spontaneously obese type II diabetic mice (Akase et al., 6

2011). In the present study, the changes in these lipid parameters were seen as early as 2 7

weeks and then continuously observed through week 12 of the treatment. In contrast, 8

there were no significant changes in the lipid profiles of the animals in the three control 9

groups. The atherogenic index calculated from the described equation in the methods 10

demonstrated the clear hypolipidemic effect of the plant extract. Since the 2 week 11

period of treatment, the atherogenic indices of KD150 and KD300 and simvastatin- 12

treated groups were lower than that of the three control groups (Fig 9). These 13

atherogenic indices were much lower in the KD300 treated group than in the KD150 14

treated group; however, the lowest numbers occurred in the simvastatin-treated group, 15

indicating the dose-dependent effect of the plant extract. Atherogenic Index (AI) is a 16

significant marker to predict the risk of atherosclerosis and cardiovascular disease. The 17

epidemiological reports showed an association of AI with several risk factors such as 18

life style, exercise and diet and the states of diseases (Niroumand et al., 2015; 19

(Olamoyegun, Oluyombo, & Asaolu, 2016). The plasma level of AI in human at more 20

than 0.21 level was also used to monitor the increase risk of cardiovascular disease 21

(Dobiasova et al, 2011). 22

A previous study by the current authors reported that there are many types of 23

flavonoids, especially methoxyflavones presented in a K. parviflora extract (Sutthanut 24

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et al., 2007). In this study, the modified HPLC assay (Sutthanut, Sripanidkulchai, 1

Yenjai, & Jay, 2007; Mekjaruskul, Jay, & Sripanidkulchai, 2012) demonstrated the high 2

peaks of methoxyflavones in the K. parviflora extract. Three peaks were identified 3

following this analysis, including PMF, DMF and TMF by comparing their retention 4

times with those of the reference standards. The retention times of PMF, DMF and TMF 5

were 31.4, 33.2 and 34.2 min, respectively. Although these methoxyflavones display 6

many pharmacological effects as has already been mentioned, the exact mechanisms of 7

the effects of K. parviflora extract on dyslipidemia remain unknown. Adipose 8

triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) play a key role in 9

regulation of adipocyte lipolysis (Lafontan & Langin, 2009). It had been reported that 10

mRNA and protein expression levels of ATGL and HSL in mature adipocytes were up-11

regulated by the PMF- and TMF-enriched fractions (Okabe et al., 2014). Therefore, the 12

possible explanation for hypolipidemic effects of MKE may be to decrease intracellular 13

triglyceride content and enhance lipolysis in mature adipocytes by activation of ATGL 14

and HSL, leading to suppression of adipocyte hypertrophy (Okabe et al., 2014). 15

Simvastatin administration results in lowering of LDL cholesterol by inhibiting 16

the rate-limiting step in cholesterol biosynthesis by competitively inhibiting HMG-CoA 17

reductase in the liver (Istvan & Deisenhofer, 2001). The present study revealed that 18

MKE treatment reduced the elevated levels of LDL cholesterol in hyperlipidemic rats, 19

however, the simvastatin group exhibited better results. MKE containing several 20

methoxyflavones that may inhibit HMG-CoA reductase activity as previously reported 21

that ortanique peel polymethoxylated flavones inhibited the hepatic activity of HMG-22

CoA reductase in the hypercholesterolemic rats (Green et al., 2011). Nevertheless, the 23

exact mechanism of this action remains to be investigated. 24

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1

4. Conclusions 2

This study demonstrated the hypolipidemic effect of the methoxyflavones-3

enriched ethanol extract of K. parviflora in a dose-dependent manner in dyslipidemic 4

rats. Further studies would be required to elucidate the mechanisms of action and how 5

individual components contribute to the combined pharmacologic efficacy. 6

7

Acknowledgments 8

This work was supported by the research grant of the national Research Council 9

of Thailand (NRCT). The authors would like to acknowledge Prof. James A. Will for 10

editing the manuscript via Publication Clinic KKU, Thailand. 11

12

13

14

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parviflora by gas chromatography. Journal of Chromatography A, 1143(1), 227-1

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SJST MANUSCRIPT TEMPLATE FOR A FIGURE FILE

A figure caption list should precede all the figures.

Figure legends

Fig 1 HPLC chromatograms of standard methoxyflavones (A) and MKE (B). The retention

times of PMF, DMF and TMF were 31.4, 33.2 and 34.2 min, respectively.

Fig 2 The changes of serum lipid profile level during 6 weeks of the course of

hypercholesterolemic induction.

Fig 3 Body weight of animals during the 6-week duration.

Fig 4 Body weight of animals during the 12-week duration of treatment with the

methoxyflavone-enriched extract of K.parviflora.

Fig 5 Level of serum cholesterol of hyperlipidemic rats after treatment with methoxyflavone-

enriched extract of K. parviflora a, b, c, d, e, f = significant differences from control 1,

control 2 (olive oil), control 3 (CMC), KD150, KD300, Simvastin at < 0.05, respectively, * =

differences from baseline (week 0) at p <0.05.

Fig6 Level of triglyceride of hyperlipidemic rats after treatment with methoxyflavone-

enriched extract of K. parviflora, a, b, c, d, e, f = significant differences from control 1,



Fig7 Level of serum HDL of hyperlipidemic rats after treatment with methoxyflavone-




Fig8 Level of serum LDL of hyperlipidemic rats after treatment with methoxyflavone-




Fig9 Level of atherogenic of hyperlipidemic rats after treatment with methoxyflavone-




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Fig 1 HPLC chromatograms of standard methoxyflavones (A) and MKE (B). The retention

times of PMF, DMF and TMF were 31.4, 33.2 and 34.2 min, respectively

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Fig 2 The change of serum lipid profile level during 6 weeks of the course of

hypercholesterolemia induction.

0

20

40

60

80

100

120

140

160

Total cholesterol Triglyceride HDL LDL

baseline

2 week

4 week

6 week

*

* *

*

Serum

lipid profile le

vel (mg%

)

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Fig 3 Body weight of animals during the 6-week duration.

0

50

100

150

200

250

300

350

400

450

Control 1 Control 2 Control 3 KD150 KD300 Simvastin

Bod

y weigh

t (g)

Groups

baseline

2 week

4 week

6 week

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Fig 4 Body weight of animals during the 12-week duration of treatment with the

methoxyflavone-enriched extract of K.parviflora.

0

100

200

300

400

500

600

Control 1 Control 2 Control 3 KD150 KD300 Simvastin

Bod

y weigh

t (g)

Groups

baseline

2 week

4 week

6 week

8 week

12 week

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Fig 5 Level of serum cholesterol of hyperlipidemic rats after treatment with methoxyflavone-




0

20

40

60

80

100

120

140

160

180

Control 1 Control 2 Control 3 KD150 KD300 SIM

baseline

2 week

4 week

6 week

8 week

12 week

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, f, *

a, b, c, f, *

Total cho

lesterol le

vel (mg%

)

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Fig 6 Level of triglyceride of hyperlipidemic rats after treatment with methoxyflavone-



differences from baseline (week 0) at p <0.05

0

20

40

60

80

100

120

140

160

180


baseline

2 week

4 week

6 week

8 week

12 week

a, b, c, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, f, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

Trig

lyceride (m

g%)

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Fig 7 Level of serum HDL of hyperlipidemic rats after treatment with methoxyflavone-




0

10

20

30

40

50

60

70

80


baseline

2 week

4 week

6 week

8 week

12 week a, b, c, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, e, f, *

HDL (m

g%)

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Fig 8 Level of serum LDL of hyperlipidemic rats after treatment with methoxyflavone-




0

20

40

60

80

100

120

140

160

180


baseline

2 week

4 week

6 week

8 week

12 week

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, e, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, d, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, d, f, *

a, b, c, f, *

a, b, c, e, f, *

a, b, c, e, f, *

a, b, c, f, *

LDL (m

g%)

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Fig 9 Level of atherogenic index of hyperlipidemic rats after treatment with methoxyflavone-




0

1

2

3

4

5


2 week

4 week

6 week

8 week

12 week

a, b, c, d

a, b, c, d

a, b, c, d

a, b, c,

a, b, c,

a, b, c,

a, b, c,

a, b, c, f

a, b, c, f

a, b, c, f

a, b, c, f

a, b, c, f

Atherog

enic in

dex

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For Review Only - Prince of Songkla Universityrdo.psu.ac.th/sjstweb/Ar-Press/61-Jan/8.pdf · 2017. 12. 27. · For Review Only 2 1 simvastatin treated groups significantly increased