saponin extract of achyranthes aspera prevents …

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Tartte et al. World Journal of Pharmaceutical Research

SAPONIN EXTRACT OF ACHYRANTHES ASPERA PREVENTS

OBESITY AND OXIDATIVE STRESS IN HIGH FAT DIET

FED MALE WISTAR RATS

Bobbu Pushpalatha1, Indireddy Ramamanohar Reddy1, Netala Vasudeva Reddy1,

Nagam Venkateswarlu2, Tartte Vijaya*2

1Department of Biotechnology, Sri Venkateswara University, Tirupati, A.P. India, 2Department of Botany, Sri Venkateswara University, Tirupati, A.P. India,

ABSTRACT

The aim of the present study was to investigate the effects of saponin

rich ethanol (ASE) extract of Achyranthes aspera on obesity and

oxidative stress (OS) in high fat (HF) diet fed male wistar rats. ASE

administration at a dose of 120mg/kg for eight weeks along with HF

significantly decreased (p<0.05) the increase in food intake(FI), body

weight gain (BWG), visceral organ weights (VOWs), serum total

cholesterol (TC), triglycerides (TG), very low density lipoproteins

(VLDL-C), low density lipoproteins (LDL-C), atherogenic index (AI),

hepatic TC and TG levels and elevated (p<0.05) the levels of serum

high density lipoproteins (HDL-C), fecal TC and TG when compared

to HF diet alone fed group. Elevated excretion of lipids in feces reveal

impaired digestion and reduced intestinal lipid absorption by ASE

which results in decreased BWG and VOWs. In HF diet alone fed group there was a

significant increase in lipid peroxidation (LPx) and decrease in SOD, CAT and GPx activities

accompanied by a huge accumulation of lipid droplets and macrophage infiltrations within

the hepatocytes and adipocytes that could enhance the release of pro-inflammatory cytokines

which in turn generate ROS and cause OS. But these alterations were not observed in ASE

treated group as there was no excessive fat accumulation. These results suggest that saponins

might prevent obesity and oxidative stress in HF fed rats.

Key words: Achyranthes aspera, saponins, high fat diet, obesity, oxidative stress.

World Journal of Pharmaceutical research

Volume 3, Issue 1, 1107-1120. Research Article ISSN 2277 – 7105

Article Received on 20 October2013 Revised on 23 November 2013, Accepted on 26 December 2013

*Correspondence for

Author:

Dr. Tartte Vijaya

Associate Professor,

Department of Botany,

Sri Venkateswara University,

Tirupati A.P- 517502, India.

[email protected]

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INTRODUCTION

Obesity is one of the greatest health threats of this century, which has an important impact on

life style-related diseases such as IR, diabetes, coronary heart diseases, dyslipidemia,

hypertension and some cancers[1]. Several factors, including lack of exercise, sedentary

lifestyles and the consumption of energy rich diets contribute to the etiology of obesity [2].

OS during obesity is due to the presence of excessive adipose tissue itself, because adipocytes

and preadipocytes have been identified as sources of proinflammatory cytokines which

generate reactive oxygen spieces (ROS) [3].

Two different types of weight reducing drugs are currently available in the market [4]. One of

them is orlistat (Xenical), which reduces intestinal fat absorption through inhibition of

pancreatic lipase [5]. The other is sibutramine (Reductil), which is an anorectic, or appetite

suppressant [6]. Both drugs have side-effects, including increased blood pressure, dry mouth,

constipation, headache, and insomnia [7]. A number of anti-obesity drugs are currently

undergoing clinical development, including centrally-acting drugs (e.g. radafaxine and

oleoyl-estrone), drugs targeting peripheral episodic satiety signals (e.g. rimonabant and

APD356), drugs blocking fat absorption (e.g. cetilistat and AOD9604), and human growth

hormone fragments [8]. At present, because of dissatisfaction with high costs and hazardous

side-effects, the potential of natural products for treating obesity is under exploration, and

this may be an excellent alternative strategy for developing future effective, safe anti-obese

drugs [9]. A variety of natural products, including crude extracts and isolated compounds

from plants, can induce body weight reduction and prevent diet-induced obesity. Therefore,

they have been widely used in treating obesity [10]. A wealth of information indicates

numerous bioactive components from nature are potentially useful in obesity treatments. A

good example of such is the saponins. These show strong anti-obesity activity via pancreatic

lipase inhibition [11] or cholesterol precipitation and bile acid conjugation [12] or appetite

suppression [13].

Achyranthes aspera Linn (Amaranthaceae), popularly known as apamarga, is a commonly

available plant in India and had claims in the treatment of obesity in ayurveda, an Indian

system of medicine. The ayurvedic herbal powder, obeloss in which A.aspera is the principle

component was found to be effective in the treatment of obesity [14]. A.aspera

possesses chemical constituents like flavonoids, triterpenoids, polyphenolic compounds,

steroids and saponins [15,16].

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Therefore the present study was aimed to investigate the protective effect of saponin enriched

extracts of A. aspera in high fat diet induced obesity and OS in male wistar rats.

MATERIALS AND METHODS

Collection of plant material

A.aspera was collected from Tirumala hill region and the identification was confirmed with

Taxonomist, Department of Botany Sri Venkateswara University, Tirupati, Andhra Pradesh,

India. Voucher specimen was deposited for future reference.

Extraction of saponin fraction

Saponins were extracted as per the method described by Sivaramakrishna et al [17]. In brief

the powdered whole plant material (500 g) was extracted with 70% ethanol by soxhlet

method and was concentrated under vacuum. The dark colored residue was refluxed with n-

butanol for 2 h and n-butanol soluble constituents were separated by filtration. The n-butanol

layer was sequentially washed with distilled water, alkali (2% KOH) and distilled water.

Then evaporated and dried under vacuum to obtain a dark green color powder. Charcoal

treatment was given to the powder and the residue was further dried under vacuum to give

saponin rich fraction. The fraction was then tested for saponins by Libermann- Burchard test

[18].

Acute toxicity study

The acute toxicity of ASE was evaluated in rats using the up and down procedure. Male rats

weighing 150±10 g, received ASE starting at 100mg/kg orally by gavage. The animals were

observed for toxic symptoms continuously for the first 4 h after dosing. The number of

survivors was noted after 24 h, and the animals were then maintained and observed daily for

next 13 days for any further toxicity.

Experimental protocol

Male wistar rats weighing from 150±10 g obtained from Sri Venkateswara Animal Agency at

Bangalore, India. The rats were allowed free access to feed and tap water under strictly

controlled pathogen free conditions with room temperature 25±2ºC, relative humidity 60-

70% and 12h light/dark cycle. The rats were fed on standard rodent pellet chow and

acclimatized to the lab conditions for two weeks before commencement of the experiment.

After acclimatization the rats were randomly assigned into five groups (n=6/group); NDC

(normal standard diet control); ND+ASE (normal diet+ saponin rich aqueous extract of A.

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aspera); HFDC (High fat diet control); HF+ASE (High fat diet+saponin rich ethanol extract

of A. aspera) and HF+OL (High fat diet+orlistat). While the control rats were maintained on

standard chow obtained from Hindustan Liver limited, Bombay, the experimental groups

were fed on HF diet (carbohydrates 39%, fat 21.5%, protein 34.5% and vitamin & mineral

mixture 5% AIN 93), obtained from NIN, Hyderabad, India. The treatment groups were

orally administered with ASE at 120 mg/kg or OL at 25 mg/kg for 8 weeks once a day

(between 8:00 AM to 10:00 AM). The animals were weighed at the start of the experiment

and then every week thereafter. Quantity of FI in all groups was measured every day. Fecal

samples were collected during the last 3 days of the experiment, weighed, and freeze-dried.

Feces were ground to produce a homogeneous powder and stored at -20 ºC until analysis. At

the end of the feeding period, blood was drawn from the heart under sodium pentobarbital (60

mg/kg, i.p.) anesthesia following 4–5 h of food deprivation and the blood was centrifuged at

3000g for 15 min at 4 °C. All serum samples were frozen at -70 °C, and these samples were

used to measure the biochemical parameters. After collecting the blood samples, the liver,

kidney, perirenal fat and peritoneal fat pads were immediately excised and weighed. The

animal experimentation was carried out according to Institutional Animal Ethical Committee

Guidelines (CPCSEA), Sri Venkateswara University, Tirupati, A. P., India.

Lipid profile and AI

Serum lipid parameters such as TC, TG and HDL-C levels were measured by enzymatic

colorimetric methods using kits purchased from Kamineni life sciences, PVT, Hyderabad,

India. VLDL-C and LDL-C levels were calculated as per Friedewalds formula [19]. Hepatic

and fecal lipids were extracted in chloroform: methanol (2:1) mixture and dried according to

Folch et al., (1957). Dried lipid extract was dissolved in 1% triton X 100 as per the

methodology Thounaojam et al.[20] and TC, TG were analyzed using above mentioned kits.

AI was calculated as per Kayamori and Igarashi formula [21].

Hepatic anti-oxidant enzymes and LPx

Tissue from control and Experimental rats were isolated and homogenized in 50mM

phosphate buffer (pH 7.0). The homogenized sample was centrifuged at 10,500x g for 60

minutes. The supernatant (cytosol) fraction was used for assay of the enzyme activity.

Superoxide dismutase activity was determined by using the Epinephrine assay as per Misra

and Fridovich [22]. Catalase activity was measured as per Chance and Machly [23].

Glutathione peroxidase activity was determined by Rotruck et al[24]. The level of LPx in the

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liver was measured in terms of malondialdehyde content and determined by using the

thiobarbituric acid (TBA) reagent. The reactivity of the TBA is determined with minor

modification of the method adopted by Hiroshi et al [25].

Liver and Adipose tissue histology

The isolated livers and adipose tissue were fixed in 10% buffered formaldehyde (pH 7.4) and

embedded in a paraffin block for microtome slicing into 5-µm thick sections. The tissue

sections were deparaffinized and stained with hematoxylin-eosin. The stained sections were

then photographed using a microscope (model CX31, Olympus, Tokyo, Japan) with a digital

camera.

Statistical analysis

All values are expressed as mean ± SD. Data was analyzed by one-way ANOVA, and then

differences among means were analyzed using the bonferroni multiple comparisons post hoc-

test. Differences were considered significant at P<0.05.

RESULTS

Toxicity study

Over the study duration of 14 days, there were no deaths recorded up to 1.2g/kg of the 70%

ethanol extract of A.aspera orally. During this period, animals did not produce any variations

in the general appearance. The acute toxicity study does not show any toxic symptoms,

changes in behavior, or mortality at 1.2g/kg dose. One tenth of this dose was used for the

present study. All animals survived until the scheduled euthanasia and no gross pathological

alteration was found in the internal organs.

Table 1. Effect of ASE or OL on BWG and FI in HF diet fed rats.

Values represent mean ± S.D of 6 rats. Values that share same superscript do not differ significantly from each other (P<0.05).

Parameters NDC ND+ASE HFDC HF+ASE HF+OL Initial weights(g)

148.83±8.81a

148.5±6.74a

149±9.42a

148.5±5.71a

149.83±6.58a

Final weights(g)

242.5±5.68a

234.83±4.57a

316.0±5.29b

256 ± 5.45c

260 ± 5.26c

Bodyweight gain (g/ 8weeks)

93.6± 3.72a

87.8 ± 7.27a

167±7.32b

108±8.36c

110.13±5.74c

Food intake(g/week)

15.82±0.41a 15.06±0.43a 18.56±0.57b 17.515±0.4c 17.88± 0.44c

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Food intake

In HFDC group there was a significant increase in FI when compared to NDC group whereas

there was a significant decrease in HF+ASE or HF+OL group when compared to HFDC

groups. The average food consumption measured for 8 weeks being 15.82±0.41, 15.06±0.43,

18.56±0.57, 17.51±0.4 and 17.88±0.44g/week for NDC, ND+ASE, HFDC, HF+ASE and

HF+OL respectively. (p<0.05, table 1).

BWG and VOWs

Intake of HF diet for 8 weeks led to an approximate increase of 78% in BWG when

compared to NDC group. Significant reduction in BWG was observed in HF+ASE or

HF+OL group when compared to HFDC group. (p<0.05, table 1).

Consumption of HF diet resulted in a gain of 52.67%, 31%, 271% and 208% in liver, kidney,

peritoneal and perirenal adipose weights respectively when compared to NDC group. But in

HF+ASE or HF+OL group significant reduction in VOWs was observed when compared to

HFDC group. (p<0.05, table 2). In case of ND+ASE group there was a non-significant

decrease in BWG and VOWs when compared to NDC group (p<0.05, table 2).

Table II. Effect of ASE or OL on VOWs in HF diet fed rats.

Values represent mean ± S.D of 6 rats. Values that share same superscript do not differ

significantly from each other (P<0.05).

Serum lipids and AI

Consumption of HF diet substantially increased the levels of serum TC, TG, VLDL-C, LDL-

C and decreased the levels of HDL-C when compared to NDC group whereas in HF+ASE or

HF+OL group significant decrease in the levels of TC, TG, VLDL-C, LDL-C and significant

Parameters NDC ND+ASE HFDC HF+ASE HF+OL

Liver (g) 9.91±0.44a 9.66±0.43a 15.13±1.99b 10.15±0.94a 10.43±1.02a

Kidney (g) 2.39±0.25a 2.23±0.15a 3.13±0.36b 2.23±0.24a 2.37±0.22a

Peritonealfat(g) 3.75±0.72a 3.44±0.72a 13.93±1.27b 4.23±0.64a 4.81±0.79a

Perirenalfat (g) 3.26±0.47a 3.02±0.46a 10.07±0.79b 3.82±0.84a 4.22±0.97a

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increase in the levels of HDL-C was observed when compared to HFDC group (p<0.05, table

3).

Intake of HF diet led to significant raise of AI in HFDC group when compared to NDC

group. But in HF+ASE or HF+OL group significant reduction in AI was observed when

compared to HFDC group (p<0.05, table 3). In case of ND+ASE group there was a non-

significant decrease in serum levels of TC, TG, VLDL-C, LDL-C, AI and increase in HDL-C

levels when compared to NDC group (p<0.05, table 3).

Table III. Effect of ASE or OL on serum lipid profile and AI in HF diet fed rats.

Parameter NDC ND+ASE HFDC HF+ASE HF+OL

TC(mg/dl) 75.10±3.89a 72.60±2.03a 165.23±4.49b 90.95±4.53c 99.32±5.36d

TG(mg/dl) 83.02±4.52a 77.88±3.18a 164.89±7.11b 90.70±5.67a 91.43±5.24a

VLDL-C (mg/dl) 16.60±0.90a 15.57±0.63a 32.97±1.42b 18.14±1.13a 18.28±1.04a

LDL-C(mg/dl) 18.50±3.81a 16.09±1.89a 110.56±4.19b 35.30±3.33c 44.05±6.21d

HDL-C (mg/dl) 39.99±2.98a

40.94±1.85a 21.71±1.74b 37.04±2.62a 36.23±2.77a

AI 0.88±0.12a 0.77±0.079a 6.63±0.49b 1.46±0.16c 1.77±0.27c



Hepatic and fecal lipids

Significant increase in hepatic TC and TG content was observed in HFDC group when

compared to NDC group whereas there was a significant decrease in hepatic TC and TG

content in ASE or OL treated group when compared to HFDC group. Fecal lipid analysis

revealed that there was a significant elevation in TC and TG in the feces of HF+ASE or

HF+OL group when compared to HFDC group. In case of ND+ASE group there was a

significant decrease in hepatic TC and TG levels whereas there was a significant increase in

fecal TC and non-significant increase in fecal TG levels when compared to NDC group

(p<0.05, table 4).

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Table IV. Effect of ASE or OL on hepatic and fecal lipids in HF diet fed rats.

Parameters NDC ND+ASE HFDC HF+ASE HF+OL

Hepatic lipids

TC (mg/g) 14.16±0.64a 12.22±0.72b 29.70±1.17c 17.03±1.25d 18.34±0.78d

TG(mg/g) 24.39±0.72a 20.49±1.28b 53.05±1.63c 25.27±0.83a 28.97±0.93d

Fecal lipids

TC (mg/g) 7.35±0.95a 9.263±0.62b 27.57±1.42c 55.37±1.24d 52.71±1.21e

TG (mg/g) 8.47±0.71a 9.27±0.43a 35.13±1.41b 67.38±1.50c 62.84±1.85d



OS markers

Intake of HF diet for 8 weeks led to significant decrease in the activities of SOD, CAT, GPx

and significant increase in LPx when compared to NDC group whereas in HF+ASE or

HF+OL group there was significant increase in the activities of SOD, CAT, GPx and LPx

when compared to HFDC group (p<0.05,table 5). In ND+ASE there was a non significant

decrease in the activities of SOD, CAT, GPx and LPx when compared to NDC group.

(p<0.05, table 5).

Table V. Effect of ASE or OL on LPx and hepatic antioxidant enzymes in high fat fed

rats

Parameters ND ND+ASE HFDC HF+ASE HF+OL

SOD(units/mg ptn) 7.38±0.65a 7.16±0.59a 3.31±0.40b 5.56±0.58c 5.24±0.91c

CAT(µm/mg ptn/min) 26.40±1.5a 25.82±0.92a 13.39±1.43b 21.94±0.99c 19.90±1.10c

GPx(µm/mg ptn/min) 7.96±0.751a 7.42±0.62a 2.91±0.34b 5.15±0.12c

4.57±0.44c

LPx(µm/gtissue) 2.77±0.60a 2.72±0.35a 7.51±0.81b 3.71±0.6a 4.17±0.64c



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Liver and Adipose tissue histology

There was a huge accumulation of lipid droplets within the hepatocytes of HFDC group when

compared to NDC group. In addition there were prominent dark nuclei and dense cytoplasm

suggesting cellular degeneration leading to necrosis. Necrotic foci were accompanied by

mononuclear cell infiltrations, fibrotic areas and many macrophages (Fig1). Adipose tissue

sections of HFDC group also revealed huge accumulation of lipid droplets, mononuclear cell

infiltrations and macrophages when compared to NDC group (Fig 2). Such alterations were

not observed in liver and adipose sections of HF+ASE or HF+OL group (Fig 1 & Fig 2).

Figures 1. Effect of ASE or OL on liver histology in high fat diet fed rats.

Light micrographs of livers (hematoxylin and eosin stain) from the NDC (normal standard

diet control), ND+ASE (Normal standard diet + saponin rich ethanol extract of A.aspera),

HFDC (High fat diet control HF+ASE (High fat diet + saponin rich ethanol extract of

A.aspera) and HF+OL (High fat diet + Orlistat) groups. CV, central vein; Asterisks indicate a

necrotic focus containing fibrotic areas and mononuclear cells; White arrows show lipid

droplets within the hepatocytes.

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Figures 2. Effect of ASE or OL on adipose tissue histology in HF diet fed rats.

Light micrographs of adipose tissues (hematoxylin and eosin stain) from the NDC (normal

standard diet control), ND+ASE (Normal standard diet + saponin rich ethanol extract of

A.aspera), HFDC (High fat diet control), HF+ASE (High fat diet + saponin rich ethanol

extract of A.aspera) and HF+OL (High fat diet + Orlistat) groups. White arrows show

mononuclear infiltrations and macrophages.

DISCUSSION

Obesity is characterized by an increased OS, which results in an imbalance between the

production of ROS and the levels of antioxidant defense. Increased OS leads to IR and other

metabolic disorders associated with obesity. In the present study, the protective effects of

ASE against high fat diet induced obesity and OS were evaluated in wistar rats.

The current results showed a significant increase in FI in HFDC group when compared to

NDC group. This was due to high palatability and energy density of HF diet which would

contribute to hyperphagia through reduced satiation signaling [26]. Increased FI and

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accompanied raise in caloric intake resulted in increase of BWG and VOWs in HFDC group.

ASE treated rats showed significant decrease in FI, BWG, and VOWs. Reduction in FI could

be attributed to appetite suppressing activity of saponins mediated through the reduced

expression of hypothalamic neuropeptide Y or serum leptin levels[13]. Decrease in BWG,

and VOWs were due to lowered lipid absorption in intestinal tract by the saponin [12] and

reduction in effective caloric intake. These results suggest that ASE has both hypophagic as

well as weight reducing properties. These results coincide with the observations made by

Anil et al [14].

In the present study, HF diet resulted in dyslipidemic changes as illustrated by increase in

serum FFAs, TC, TG, VLDL-C and LDL-C and a decrease in HDL-C levels a finding in

accordance with that of Woo et al [27]. ASE supplementation produced significant decrease

in serum TC, TG, VLDL-C, LDL-C and significant increases in HDL-C levels. These results

are in agreement with those of Khannal [28]. This desirable effect was due to saponins which

could reduce intestinal lipid absorption by precipitating cholesterol from micelles and

interfering with enterohepatic circulation of bile acids making them unavailable for intestinal

absorption of lipids [12]. The increase in the concentration of LDL-C and a decrease in the

concentration of HDL-C in HFDC group indicate increased atherogenicity and therefore the

higher incidence of coronary heart diseases. The clinical complications of atherosclerosis

could be diminished when lipid concentration is lowered by hypocholesterolemic agents[29].

In the present study decreased serum lipids and AI in ASE treated group implies that the

saponins has protective role against coronary heart diseases.

The consumption of the HF diet led to increase in hepatic lipid levels and decrease in fecal

lipid levels in HFDC group whereas reduced intestinal lipid absorption by saponins in ASE

treated group resulted in decreased accumulation of lipids in hepatic tissue and elevated

excretion of lipids in the feces when compared to HFDC group.

In the present study, there was significant increase in LPx and significant decrease in the

activities of SOD, CAT and GPx in HFDC group when compared to NDC group. In general,

SOD is the first line of defense against oxidative stress [30] and play a pivotal role in

dismutation of super oxide anions to H2O2. CAT and GPx neutralize hydrogen peroxides to

molecular oxygen and water [31]. The decrease in these enzymes in HFD group clearly

postulates improper dismutation of superoxides and improper decomposition of H2O2. In ASE

treated rats, there was no significant change in the activities of SOD, CAT, GPx and in LPx

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when compared to NDC group. In agreement with the above results, light microscopy of the

liver sections revealed accumulation of lipid droplets within the hepatocytes of HFDC group.

In addition there were prominent dark nuclei and dense cytoplasm suggesting cellular

degeneration leading to necrosis. Necrotic foci were accompanied by mononuclear cell

infiltrations, fibrotic areas and many macrophages implicating OS through inflammatory

response in liver. OS in adipose tissue was revealed by huge accumulation of lipid droplets,

mononuclear cell infiltrations and macrophages. No such alterations were observed in liver

and adipose sections of ASE or OL treated group. This implies that in ASE treated group,

there was no excessive serum FFAs and tissue fat accumulation that could enhance OS via

the release of ROS.

In summary, the saponin rich extract of A.aspera is competent in preventing obesity and OS

in HF diet fed rats by decreasing intestinal lipid absorption and increasing fecal lipid

excretion.

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saponin extract of achyranthes aspera prevents …

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