SYNOPSIS

Introduction and Need for the study:

Obesity and overweight are defined as a condition of excessive or abnormal fat

accumulation in adipose tissue, to the extent that health may be impaired1. Obesity is a

growing and costly health problem in many of the richest nations of the world and it is now

considered as a chronic disease that is reaching epidemic proportions in the developed

world2.

Obesity threatens to become the 21st century’s leading health problem. The

prevalence of obesity is increasing and the world health organization (WHO) estimates that

more than 1.5 billion adults are overweight worldwide and of these 200 million men and

nearly 300 million women are clinically obese.

Obesity, which affects up to 30% of the adult population in developed countries, is

associated with serious mortalities including a high incidence of type 2 diabetes,

hyperlipidemia, cardiovascular disease, and osteoarthritis as well as an increased risk of many

forms of cancer. It is suggested that obesity per se may induce systemic oxidative stress and

that increased oxidative stress in accumulated fat is at least in part, the underlying cause of

dysregulation of adipocytokines and development of obesity associated metabolic syndrome3.

It has been reported that, diet induced obesity in rat models showed an increased level of

oxidative stress in the liver due to excessive production of reactive oxygen species and/or

decreased antioxidant potentials.

As a consequence of anti-obesity research, our knowledge of the bodyweight control

system increased but, despite this, the pharmacological approaches to the treatment of obesity

have not resulted yet in effective drugs. Only sibutramine and orlistat are the currently

available drugs for the treatment of obesity4. However, these drugs are not only inaccessible

and unaffordable, but also possess many toxic adverse effects. Therefore, there is a great need

for the development of an effective, safe and cheap anti obesity agents from plants and other

alternative sources. A variety of natural products, including crude extracts and isolated

compounds from plants have been reported to induce body weight reduction and prevent diet

induced obesity5.

The rhizomes of Alpinia galanga (Zingiberaceae) and roots of Argyreia speciosa

(Convolvulaceae) are traditionally used for the treatment of obesity. Hence, the present study

was undertaken to investigate the possible beneficial effects of various solvent extracts of

Alpinia galanga rhizomes (AG) and Argyreia speciosa roots (AS) in cafeteria diet and

atherogenic diet induced obesity in rats. An attempt was also made to isolate and characterize

the active phytoconstituents responsible for the activity along with determination of possible

mechanism of anti-obese action.

Objectives of the study:

The primary objective of the study was to evaluate the anti-obesity potential of

various solvent extracts of AG rhizomes and AS roots in Cafeteria diet and

Atherogenic diet induced obesity in rats.

To determine in vitro antioxidant potentials of the ethanol extracts of plant materials

under study by using DPPH, Nitric oxide and Hydroxyl radical scavenging assay.

To determine total phenolic and flavonoid content of the ethanol extracts of Alpinia

galanga rhizomes and roots of Argyreia speciosa.

To isolate and characterize the active phytoconstituents responsible for antiobese and

antioxidant properties in Alpinia galanga rhizomes and Argyreia speciosa roots by

using various chromatographic and spectroscopic techniques.

To elucidate the possible mechanism of anti-obese property by determining the

influence of isolated compounds on pancreatic lipase enzyme activity.

Methodology:

The fresh rhizomes of AG and roots of AS were collected, identified, shade dried and

powdered. The Pet. Ether (40-60°C), chloroform, ethanol and aqueous extracts of the

powdered plant materials was prepared and subjected to preliminary phytochemical tests and

oral toxicity studies. The extracts were found to be safe even at the highest dose of 2000

mg/kg in albino rats. Hence, 1/4th of maximum tested dose i.e. 500 mg/kg was selected as

experimental dose to screen the anti-obesity effects in rats.

The cafeteria diet (CD) or atherogenic diet (AD) were given along with normal diet to

albino wistar rats daily for six weeks to develop obesity like condition6. The body weight and

food intake per day was determined on day 1 and then every week thereafter. On day 42,

blood was collected; serum was separated and subjected to estimation of glucose, lipids,

leptin, SGOT and SGPT concentration. Animals were sacrificed with excess of ether

anesthesia; liver and parametrial adipose tissues were removed and weighed immediately.

The liver tissue was subjected to estimation of triglycerides, lipid peroxidation and

concentration of antioxidant enzymes. The histopathological study of liver tissue was carried

out by staining with Haematoxylin-Eosin dye.

The in vitro antioxidant potential of the ethanol extracts was determined by estimating

free radical scavenging abilities against DPPH, NO and OH free radicals and the ethanol

extracts were analyzed for their total phenolic content and total flavonoid content. The

extracts with exceedingly promising anti-obesity activity were subjected for isolation of

phytoconstituents by chromatographic methods and characterized by spectral studies. The

probable mechanism of antiobese action of isolated compounds was also determined by

measuring in vitro pancreatic lipase enzyme inhibitory activity.

Results and Discussion:

Administration of a CD or AD along with normal diet for six weeks in rats produced

obesity-like conditions characterized by increase in body weight, parametrial adipose tissue

weight, and serum lipid levels. Furthermore, it also induced a fatty liver with the

accumulation of hepatic triglycerides. Treatment with extracts at the dose of 500 mg/kg/day,

significantly reduced the increase in body weight induced by a CD/AD - a clear sign of an

anti-obesity effect. The calorie or food intake measured once in every week was significantly

reduced as compared to CD/AD control rats. This result suggests that the body weight

reducing effect of extracts in CD/AD fed rats may be produced due to the hypophagic

property. (Table 1 - 3)

Significant increase in serum lipids, such as total cholesterol, LDL-C, and

triglycerides is observed in obese animals and humans. In addition, there is a decrease in

HDL / LDL ratio. Thus, alteration of lipid profiles can be used as an index of obesity7.

Treatment with extracts caused significant changes in the blood parameters, including

decreased levels of TC, LDL-C, and TG, but increased HDL-C. However, effects produced

by ethanol extracts of both plants were highly significant. These results indicate a significant

improvement in lipid profile by the treatment with extracts. The atherogenic index of plasma

(AIP) correlates with the size of the pro- and anti-atherogenic lipoprotein particles and is

known to predict a cardiovascular risk. An AIP value of less than 0.10 predicts a low

cardiovascular risk, which was observed in animals treated with extracts and sibutramine.

(Table 4 and 5)

There was significant increase in concentration of SGPT and SGOT in CD and AD

control rats as compared to that in normal diet fed rats. Treatment with various extracts of

AG and AS markedly reduced these enzyme activities induced by high fat diets. However

effect produced by ethanol extracts was highly significant. These results suggest that liver

function was significantly changed due to high fat diet, which was effectively reversed by

treatment with ethanol extracts. (Table 6 and 7)

Leptin, a 167 amino acid protein, is synthesized and secreted mainly by the adipose

tissue, in approximate proportion to the fat stores. It is reported that consumption of high-fat

diet results in the development of leptin resistance in rodents, marked by an increased

circulating leptin level, and is measured as a failure of leptin either to inhibit food intake or to

induce weight loss8. In the present study, the serum leptin was raised in the CD/AD control

group, indicating that obese rats accompanied with leptin resistance; however, treatment with

extracts resulted in a significant reduction in serum leptin levels when compared to the

CD/AD control group, suggesting that, the extracts could improve leptin resistance induced

by obesity. (Table 6 and 7).

The extracts produced a significant decrease in the liver and parametrial adipose

tissue weight and the accumulation of liver triglycerides in comparison with the CD/AD

control group. The rate of reduction of body weight corresponded with that in the parametrial

adipose tissue weight (Table 8 and 9).

Obesity is a principle causative factor for coexistence of metabolic syndromes

(hyperglycemia, dyslipidemia and hypertension) in same individual. It has been reported that

obesity may induce systemic oxidative stress and that increased oxidative stress in

accumulated fat is associated with dysregulation of adipocytokines and development of

metabolic syndrome.

In order to investigate if oxidative stress was increased in high fat diet fed rats, we

measured lipid peroxidation in liver tissue by estimating the malondialdehyde content using

TBARS essay. The high concentration of liver tissue malondialdehyde observed in CD and

AD control rats was an indication of increased oxidative stress in high fat diet rats. However,

the extract supplemented animals showed significant decrease in malondialdehyde

concentration, thus reducing lipid peroxidation. The ability of the ethanol extracts to

significantly suppress lipid peroxidation could be due to the anti-free radical activities of its

phenolic components, known to act as free radical scavengers and to increase in the activity

of antioxidant enzymes regardless of the available lipids.

Obesity has been shown to be one of the conditions that decrease antioxidant capacity.

Obesity seems to decrease antioxidant defense by lowering the levels of antioxidant enzymes

such as superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase9.

Accordingly, in our study also, there was significant decrease in concentration of these

antioxidant enzymes in liver tissue of CD and AD control rats. However, the animals treated

with extracts showed an elevation in their antioxidant enzymes concentration. The ethanol

extracts were highly significant to improve antioxidant defense system by enhancing in vivo

antioxidant enzymes activities (Table 10 and 11).

In histopathological studies, rats fed with high fat CD or AD showed mild fatty

change surrounding central vein, and congestion in hepatic vein and dilated central vein with

kupffer cells hyperplasia and there was vacuolation seen. However, rats supplemented with

sibutramine or ethanol and pet. ether extracts of AG and AS significantly reversed these

pathological changes and exhibited almost normal architecture.

Since, the ethanol extracts of AG and AS significantly increased in vivo antioxidant

defensive mechanisms, the in vitro antioxidant potential of the these extracts was determined

by estimating free radical scavenging abilities against DPPH, NO and OH free radicals. The

ethanol extracts produced marked scavenging of these free radicals (Table 12).

Phenolic compounds especially flavonoids have been considered to play an important

role as dietary antioxidants for the prevention of oxidative damage in living systems10

. In this

study, the ethanol extracts were analyzed for their total phenolic compounds and total

flavonoids. Both the extracts contained approximately the same amount of phenolic

compounds and the total flavonoid content was relatively less in AS extract compared to AG

extract. Thus, the in vitro and in vivo antioxidant activities of the ethanol extracts of AG and

AS could be related to their high phenolic and flavonoid contents (Table 13).

The results of the present study suggests that, ethanol extracts of AG and AS produces

significant beneficial effects in treatment of high fat diet induced obesity in experimental rats,

which was comparable with standard antiobese drug, sibutramine. This could be mediated by

their hypophagic, hypolipidemic and antioxidant effects. Overcoming leptin resistance and

subsequent reduced adiposity would further add to the beneficial effects of these extracts in

reversing the changes of obesity.

The extracts with exceedingly promising anti-obesity activity were subjected for

isolation and characterization of phytoconstituents by chromatographic methods and spectral

studies. The phytochemical separation of the ethanol extracts of AG rhizomes and AS roots

led to isolation of two compounds from each plant. The spectral data revealed that, the

compounds isolated from ethanol extract of AG rhizomes are flavonoid derivatives with

substituted flavone structure namely – 3, 5, 7-trihydroxy flavone (Galangin) and 3, 5, 7-

trihydroxy 4-methoxy flavone (Kaempferide). The compound isolated from AS roots were

found to be a phenolic compound and sterol namely- 3, 4, 5 trihydroxy benzoic acid (Gallic

acid) and β-Sitosterol. Hence, in the present study, the isolated compounds present in the

extracts could be responsible for their beneficial effects in obesity.

It is well known that the dietary lipid is not directly absorbed from the intestine unless

it has been subjected to the action of the pancreatic lipase enzyme. The two products formed

by the hydrolysis of fat in the presence of pancreatic lipase enzyme are fatty acids and 2-

monoacylglycerols, which are absorbed. PL inhibition is one of the most widely studied

mechanisms for the determination of the potential efficacy of natural products as anti-obesity

agents11

. All the isolated compounds markedly inhibited the action of the pancreatic lipase

enzyme, except β-Sitosterol (12.32%). The maximum inhibition of enzyme activity was

observed with galangin (51.86%). This was further confirmed by reduction in plasma

triacylglycerol content after 2 h of oral lipid emulsion load in rats (Table 14 and 15).

Conclusions:

In conclusion, our study demonstrates that AG rhizomes and AS roots may modulate

over weight, obesity and associated complications by reducing the excess accumulation of

body fat due to inhibition of pancreatic lipase activity, altering the lipid profile, overcoming

the leptin resistance, and enhancing the antioxidant defensive mechanisms of the body.

The active components responsible for the activity were identified as flavonoids such as

galangin and kaempferide from AG extract, and a phenolic compound (gallic acid) and

phytosterol (β-Sitosterol) from AS extract.

Thus the present study clearly indicates that, ethanol extracts of both the plants

possess significant anti-obesity action against high fat diet induced obesity in rats and hence

validates their traditional usage. Hence, these plants might help in preventing obesity

complications and serve as a good source of nutraceuticals for the preparation of polyherbal

formulation for treatment of obesity and related metabolic disorders.

Table 1: Mean difference in body weight between day 1 and day 42 in cafeteria diet fed rats

Groups Mean difference in body weight in g

between day 1 and 42

% reduction in body weight compared

to CD control group

Normal control 77.9 ± 2.06 -

CD Control 159.4 ± 2.56 (104.62%) -

Sibutramine 75.6 ± 1.86 (-2.95%) 52.58%

Pet. ether extract of AG 112.4 ± 2.62 (44.28%) 29.49%

Chloroform extract of AG 96.2 ± 2.34 (23.49%) 39.64%

Ethanol extract of AG 89.6 ± 2.24 (15.01%) 43.78%

Aqueous extract of AG 99.6 ± 2.45 (27.85%) 37.51%

Pet. ether extract of AS 96.6 ± 2.64 (24.0%) 39.39%

Chloroform extract of AS 110.4 ± 3.42 (41.72%) 30.74%

Ethanol extract of AS 80.4 ± 2.24 (3.20%) 49.56%

Aqueous extract of AS 115.4 ± 3.02 (48.13) 27.60%

Values in parenthesis indicate % change in body weight as compared to normal control group.

Table 2: Mean difference in body weight between day 1 and day 42 in atherogenic diet fed rats

Groups Mean difference in body weight in g

between day 1 and 42

% Reduction in body weight compared

to AD control group

Normal control 82.9 ± 2.52 -

AD Control 139.4 ± 3.24 (68.15%) -

Sibutramine 84.4 ± 2.14 (1.80) 39.45%

Pet. ether extract of AG 100.4 ± 3.02 (21.10%) 27.97%

Chloroform extract of AG 104.2 ± 3.26 (25.69%) 25.25%

Ethanol extract of AG 93.8 ± 2.68 (13.14%) 32.71%

Aqueous extract of AG 105.6 ± 2.84 (27.38%) 24.24%

Pet. ether extract of AS 99.8 ± 2.22 (20.38%) 28.40%

Chloroform extract of AS 104.4 ± 3.04 (25.93%) 25.10%

Ethanol extract of AS 90.0 ± 2.42 (8.56%) 35.43%

Aqueous extract of AS 108.0 ± 2.65 (30.27%) 22.52%

Values in parenthesis indicate % change in body weight as compared to normal control group.

Table 3: Effect of Alpinia galanga and Argyreia speciosa extracts on food/calorie intake in CD/AD fed rats.

Values in parentheses indicate amount of food intake in g. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001

considered statistically significant when compared to corresponding value of HFD control rats. cp<0.001 considered statistically significant when compared to

corresponding value of normal control rats.

Groups CD (Kcal/day/6 rats) AD (g/day/6 rats)

Normal control 386.10 ± 2.31 (137.89 ± 2.22)

140.40 ± 2.91***

HFD Control 474.00 ± 3.28c

(143.63 ± 2.45) 160.1 ± 1.63

Sibutramine 347.10 ± 4.00***

(105.18 ± 1.84)

130.12 ± 1.91***

Pet. ether extract of AG 425.70 ± 3.31

**

(129.02 ± 2.24)

151.6 ± 1.25*

Chloroform extract of AG 456.60 ± 3.53**

(138.36 ± 2.64)

157.12 ± 1.99

Ethanol extract of AG 359.90 ± 3.93***

(109.06 ± 2.35)

136.42 ± 1.54***

Aqueous extract of AG 427.44 ± 3.42**

(129.52 ± 3.15) 152.70 ± 2.59

Pet. ether extract of AS 389.14 ± 3.34**

(117.92 ± 2.56) 150.00 ± 1.30

**

Chloroform extract of AS 391.92 ± 3.48

**

(118.76 ± 2.84) 153.72 ± 1.62

Ethanol extract of AS 370.00 ± 3.00***

(112.12 ± 2.42) 134.72 ± 1.49

***

Aqueous extract of AS 402.90 ± 2.23**

(122.09 ± 2.38) 155.14 ± 1.99

Table 4: Effect of Alpinia galanga and Argyreia speciosa extracts on serum glucose and lipid parameters in CD fed rats.

Groups Glucose

(mg/dl)

Total

cholesterol

(mg/dl)

HDL-C

(mg/dl)

LDL-C

(mg/dl)

TGs

(mg/dl)

AIP

Normal control 141.00 ± 2.96 96.83 ± 3.01 23.50 ± 1.17 50.67 ± 1.54 64.17 ± 3.37 0.076

CD Control 153.28 ± 2.56a 125.20 ± 2.83

c 25.50 ± 1.17 100.20 ± 1.40

c 100.3 ± 2.84

c 0.235

Sibutramine 134.24 ± 2.76***

103.92 ± 2.69***

35.83 ± 1.24***

69.67 ± 1.43***

76.50 ± 3.48***

-0.031

Pet. ether extract of AG 143.70 ± 2.92 115.50 ± 2.43 26.83 ± 1.13 85.33 ± 4.00* 86.67 ± 4.05* 0.107

Chloroform extract of AG 142.20 ± 3.08 114.30 ± 3.19 29.50 ± 1.17 85.33 ± 4.36* 86.47 ± 3.12* 0.149

Ethanol extract of AG 138.04 ± 3.15**

109.72 ± 2.40**

36.67± 1.14***

62.67 ± 2.60***

73.83 ± 2.73***

-0.056

Aqueous extract of AG 142.06 ± 3.05 113.02 ± 3.87 26.33 ± 0.98 89.33 ± 3.22 86.17 ± 3.22* 0.155

Pet. ether extract of AS 140.20 ± 3.08* 111.26 ± 3.65* 31.00 ± 1.52* 87.02 ± 4.09

* 84.67 ± 4.05* 0.076

Chloroform extract of AS 147.30 ± 2.71 113.72 ± 3.15 29.97 ± 1.04 84.67 ± 4.40* 84.67 ± 3.49* 0.091

Ethanol extract of AS 137.70 ± 2.55**

109.52 ± 3.08**

37.50 ± 1.17***

63.37 ± 2.30***

73.17 ± 2.27***

-0.070

Aqueous extract of AS 140.06 ± 3.05* 115.50 ± 3.34 28.33 ± 1.11 89.00 ± 3.49 85.50 ± 2.87* 0.120

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically

significant when compared to CD control rats. ap< 0.05,

c<0.001 considered statistically significant when compared to corresponding value of

normal control rats.

Table 5: Effect of Alpinia galanga and Argyreia speciosa extracts on serum glucose and lipid parameters in AD fed rats.

Groups Glucose

(mg/dl)

Total

cholesterol

(mg/dl)

HDL-C

(mg/dl)

LDL-C

(mg/dl)

TGs

(mg/dl)

AIP

Normal control 138.80 ± 3.14 98.67 ± 2.71 24.17 ± 1.04 52.00 ± 1.46 65.50 ± 3.31 0.073

AD Control 147.20 ± 3.69 144.22 ± 2.83c 25.83 ± 1.24 101.72 ± 1.90

c 97.00 ± 2.23

c 0.215

Sibutramine 135.02 ± 2.76* 103.24 ± 2.22

***

37.83 ± 1.49***

67.83 ± 1.99***

72.83 ± 3.68***

-0.076

Pet. ether extract of AG 145.8 ± 1.90 132.30 ± 2.01* 27.50 ± 1.08 89.17 ± 2.04

** 84.00 ± 4.16

* 0.068

Chloroform extract of AG 144.52 ± 3.22 133.00 ± 2.82* 30.33 ± 1.28 89.67 ± 2.45

** 81.33 ± 2.99

** 0.125

Ethanol extract of AG 134.0 ± 2.36* 113.32 ± 2.52

***

37.83 ± 1.62***

65.67 ± 2.51***

73.83 ± 2.99***

-0.070

Aqueous extract of AG 144.36 ± 3.20 131.30 ± 3.52* 29.50 ± 1.58 91.00 ± 1.91

** 87.83 ± 3.11 0.114

Pet. ether extract of AS 146.80 ± 1.83 131.20 ± 2.71**

32.50 ± 1.40* 89.33 ± 2.43

** 81.67 ± 3.28

** 0.040

Chloroform extract of AS 143.82 ± 3.28 133.72 ± 2.94* 32.00 ± 1.89

* 89.67 ± 2.37

** 82.33 ± 2.39

* 0.050

Ethanol extract of AS 134.20 ± 3.34* 109.50 ± 2.74

*** 39.33 ± 1.68

***

65.50 ± 2.11***

74.50 ± 2.23***

-0.083

Aqueous extract of AS 141.26 ± 3.06 130.50 ± 4.14** 30.50 ± 0.92 90.17 ± 1.93

** 88.67 ± 2.51 0.103

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when

compared to AD control rats. ap< 0.05,

c<0.001 considered statistically significant when compared to corresponding value of normal control rats.

Table 6: Effect of Alpinia galanga and Argyreia speciosa extracts on serum enzymes and leptin levels in CD fed rats.

Groups SGPT (U/L) SGOT (U/L) Leptin (ng/L)

Normal control 31.02 ± 1.52 145.3 ± 1.74 230. 30 ± 3.11

CD Control 61.33 ± 2.66c 214.2 ± 3.00

c 619.3 ± 5.40

c

Sibutramine 41.00 ± 1.52***

136.7 ± 2.57***

291.3 ± 3.28***

Pet. ether extract of AG 54.33 ± 2.02 178.3 ± 4.63* 446.7 ± 6.29

***

Chloroform extract of AG 52.00 ± 2.30* 157.0 ± 2.42

** 444.30 ± 8.30

***

Ethanol extract of AG 43.67 ± 1.33***

130.0 ± 2.30***

364.0 ± 4.71***

Aqueous extract of AG 51.02 ± 2.23**

150.0 ± 5.29**

464.0 ± 8.21***

Pet. ether extract of AS 49.52 ± 2.77**

161.5 ± 5.21**

456.8 ± 9.50***

Chloroform extract of AS 51.50 ± 1.74* 158.3 ± 3.82

** 455.5 ± 8.28

***

Ethanol extract of AS 42.02 ± 1.48***

133.3 ± 2.53***

343.7 ± 7.53***

Aqueous extract of AS 49.17 ± 2.88**

154.5 ± 5.15**

448.80 ± 9.13***

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when

compared to CD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control rats.

Table 7: Effect of Alpinia galanga and Argyreia speciosa extracts on serum enzymes and leptin levels in AD fed rats.

Groups SGPT (U/L) SGOT (U/L) Leptin (ng/L)

Normal control 32.02 ± 1.65 147.5 ± 1.45 231.80 ± 3.35

AD Control 63.03 ± 3.17c 209.7 ± 3.67

c 597.70 ± 8.99

c

Sibutramine 42.50 ± 1.47***

138.8 ± 3.10***

301.32 ± 3.84***

Pet. ether extract of AG 55.67 ± 2.02 184.7 ± 5.99**

456.30 ± 9.62***

Chloroform extract of AG 53.33 ± 1.97* 185.7 ± 5.53

** 436.00 ± 7.23

***

Ethanol extract of AG 44.83 ± 1.60***

139.5 ± 3.18***

357.30 ± 6.12***

Aqueous extract of AG 52.50 ± 2.29* 184.3 ± 3.29

** 465.55 ± 7.86

***

Pet. ether extract of AS 51.00 ± 3.34**

183.0 ± 2.78**

435.80 ± 6.28***

Chloroform extract of AS 51.17 ± 3.09**

184.0 ± 2.56**

457.55 ± 9.56***

Ethanol extract of AS 43.52 ± 1.58***

141.0 ± 4.73***

333.70 ± 7.53***

Aqueous extract of AS 52.50 ± 1.54* 186.5 ± 2.88

** 457.20 ± 8.11

***

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when

compared to AD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control rats.

Table 8: Effect of Alpinia galanga and Argyreia speciosa extracts on liver weight, PAT weight and liver TGs in CD fed rats.

Groups Liver weight (g) PAT weight (g) Triglycerides(mg/g)

Normal control 5.99 ± 0.18 4.02 ± 0.23 5.71± 0.25

CD Control 12.46 ± 0.41c 10.65 ± 0.48

c 12.45 ± 0.44c

Sibutramine 5.92 ± 0.19***

6.56 ± 0.16***

7.14 ± 0.27***

Pet. ether extract of AG 10.08 ± 0.25** 9.05 ± 0.31

** 10.38 ± 0.36**

Chloroform extract of AG 8.95 ± 0.20**

9.05 ± 0.24** 10.48 ± 0.35

**

Ethanol extract of AG 07.04 ± 0.25***

6.24 ± 0.29***

7.90 ± 0.32***

Aqueous extract of AG 09.08 ± 0.25**

8.90 ± 0.39** 10.54 ± 0.37

**

Pet. ether extract of AS 9.01 ± 0.26**

8.94 ± 0.31** 10.56 ± 0.34

**

Chloroform extract of AS 09.09 ± 0.18**

9.30± 0.31* 10.46 ± 0.37**

Ethanol extract of AS 6.88 ± 0.23***

6.42 ± 0.46***

8.44 ± 0.36***

Aqueous extract of AS 9.93 ± 0.27** 8.99 ± 0.28

** 10.46 ± 0.39**

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when

compared to CD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control rats.

Table 9: Effect of Alpinia galanga and Argyreia speciosa extracts on liver weight, PAT weight and liver TGs in AD fed rats.

Groups Liver weight (g) PAT weight (g) Triglycerides(mg/g)

Normal control 6.18 ± 0.25 4.00 ± 0.22 5.79± 0.24

AD Control 12.66 ± 0.19c 10.81± 0.51

c 12.68 ± 0.36c

Sibutramine 6.09 ± 0.22***

6.67 ± 0.19***

7.26 ± 0.27***

Pet. ether extract of AG 10.61 ± 0.32** 9.16 ± 0.34

** 10.66 ± 0.35**

Chloroform extract of AG 10.06 ± 0.31**

9.11 ± 0.25** 10.81 ± 0.32

**

Ethanol extract of AG 07.21 ± 0.19***

6.33 ± 0.28***

8.04 ± 0.41***

Aqueous extract of AG 10.02 ± 0.31**

9.13 ± 0.16** 10.75 ± 0.38

**

Pet. ether extract of AS 09.88 ± 0.34**

9.16 ± 0.20** 10.66 ± 0.33

**

Chloroform extract of AS 10.46 ± 0.29** 10.04 ± 0.24 10.76 ± 0.28

**

Ethanol extract of AS 7.11 ± 0.15***

6.51 ± 0.27***

8.65 ± 0.38***

Aqueous extract of AS 9.95 ± 0.32**

9.12 ± 0.36** 10.67 ± 0.33

**

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when

compared to AD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control rats.

Table 10: Effect of Alpinia galanga and Argyreia speciosa extracts on liver lipid peroxidation and antioxidant enzymes in CD fed rats.

Groups Malondialdehyde

(µM/mg of protein)

SOD

(U/mg protein)

Catalase

(U/mg protein)

Glutathione peroxidase

(nM of NADPH/min/mg protein)

Normal control 0.84 ± 0.01 6.27 ± 0.19 7.35 ± 0.14 193.3 ± 5.12

CD Control 1.66± 0.09c 3.32 ± 0.17

c 4.24 ± 0.18

c 140.8 ± 4.15

c

Sibutramine 0.95 ± 0.02***

5.88 ± 0.18***

6.85 ± 0.13***

183.7 ± 3.91***

Pet. ether extract of AG 1.40 ± 0.04* 3.98 ± 0.10

* 4.49 ± 0.15 161.0 ± 2.67

**

Chloroform extract of AG 1.35 ± 0.03**

4.14 ± 0.23**

5.23 ± 0.14**

155.5 ± 2.27*

Ethanol extract of AG 0.96 ± 0.02***

5.80 ± 0.16***

6.65 ± 0.15***

184.3 ± 3.40***

Aqueous extract of AG 1.33 ± 0.06**

4.20 ± 0.12**

4.82 ± 0.19 157.5 ± 2.27*

Pet. ether extract of AS 1.38 ± 0.04**

4.29 ± 0.10**

5.26 ± 0.26**

161.2 ± 1.79**

Chloroform extract of AS 1.35 ± 0.05**

4.36 ± 0.16**

4.85 ± 0.22 159.8 ± 1.90**

Ethanol extract of AS 0.94 ± 0.02***

5.76 ± 0.16***

6.77 ± 0.13***

184.3 ± 3.85***

Aqueous extract of AS 1.36 ± 0.05**

4.28 ± 0.15**

4.80 ± 0.19 157.8 ± 1.90**

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically

significant when compared to CD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control

rats.

Table 11: Effect of Alpinia galanga and Argyreia speciosa extracts on liver lipid peroxidation and antioxidant enzymes in AD fed rats.

Groups Malondialdehyde

(µM/mg of protein)

SOD

(U/mg protein)

Catalase

(U/mg protein)

Glutathione peroxidase

(nM of NADPH/min/mg protein)

Normal control 0.86 ± 0.01 6.33 ± 0.17 7.54 ± 0.13 197.3 ± 3.98

AD Control 1.68 ± 0.12c 3.42 ± 0.28

c 4.37 ± 0.20

c 141.0 ± 2.40

c

Sibutramine 0.97 ± 0.03***

5.89 ± 0.19***

7.08 ± 0.24***

185.5 ± 4.40***

Pet. ether extract of AG 1.42 ± 0.05* 4.26 ± 0.23

* 4.46 ± 0.18 158.5 ± 2.71

**

Chloroform extract of AG 1.37 ± 0.04**

4.40 ± 0.21* 4.53 ± 0.20 160.3 ± 3.40

**

Ethanol extract of AG 0.99 ± 0.02***

5.97 ± 0.17***

6.64 ± 0.14***

186.7 ± 3.49***

Aqueous extract of AG 1.35 ± 0.07**

4.25 ± 0.13* 4.58 ± 0.23 161.0 ± 2.67

**

Pet. ether extract of AS 1.37 ± 0.04**

4.44 ± 0.23**

5.28 ± 0.16* 159.7 ± 2.33

**

Chloroform extract of AS 1.42 ± 0.05* 4.37 ± 0.14

* 5.19 ± 0.27

* 158.5 ± 3.57

**

Ethanol extract of AS 0.98 ± 0.02***

5.96 ± 0.26***

6.90 ± 0.16***

185.2 ± 5.79***

Aqueous extract of AS 1.41 ± 0.06* 4.31 ± 0.13

* 4.56 ± 0.13 159.2 ± 3.60

**

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and *p<0.05,

**p<0.01,

***p<0.001, considered statistically

significant when compared to AD control rats. c<0.001 considered statistically significant when compared to corresponding value of normal control

rats.

Table 12: Effect of Alpinia galanga and Argyreia speciosa extracts on in vitro antioxidant activity.

Table 13: Total phenolic and flavonoid content of ethanol extracts of AG rhizomes and AS roots.

Extracts Total phenolic content

(mg GA/g)

Total flavonoid content

(mg Quercetin/100g)

Ethanol extract of AG rhizomes 40.7 ± 1.20 219.30 ± 3.24

Ethanol extract of AS roots 51.4 ± 1.09 138.25 ± 2.68

Sample IC50 value for

DPPH scavenging

IC50 value for

NO scavenging

IC50 value for

OH scavenging

Standard 23.12 μg 19.23 μg 22.23 μg

Ethanol extract of AG 28.47 μg 31.42 μg 32.45 μg

Ethanol extract of AS 38.67 μg 33.07 μg 42.42 μg

Table 14: % Inhibitory values for pancreatic lipase activity

Compounds % Inhibition of lipase activity

Gallic acid 46.24

β-sitosterol 12.32

Galangin 51.86

Kaempferide 49.56

Table no. 15: Effect of isolated compounds on plasma triacylglycerol content in lipid

emulsion (LE) overloaded rats.

Values are mean ± SEM. Data were analyzed by ANOVA followed by Dunnett’s test and

*p<0.05,

**p<0.01,

***p<0.001, considered statistically significant when compared to respective

lipid emulsion control values.

Treatment

Plasma triacylglycerol content (mg/dl)

0 h 1 h 2 h 3 h 4 h

LE control 72.57 ± 3.54 143.36 ± 6.10 173.45 ± 6.09 207.08 ± 6.14 247.79 ± 6.15

LE +

Galangin 74.34 ± 4.42 111.47 ± 5.12

* 109.73 ± 5.10

*** 84.96 ± 4.15

*** 72.57 ± 3.16

***

LE +

Kaempferide 66.37 ± 5.08 129.20 ± 6.15

* 116.81 ± 4.14

*** 92.04 ± 4.12

*** 79.65 ± 4.10

***

LE +

Gallic acid 79.65 ± 2.65 134.51 ± 5.16 127.43 ± 5.12

*** 102.65 ± 4.64

*** 92.04 ± 4.65

***

LE +

β-sitosterol 72.57 ± 5.31 145.13 ± 6.12 161.06 ± 6.18 159.29 ± 5.18

** 150.44 ± 4.16

***

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