1. INTRODUCTION:

Traditional herbal medicine has a long history of serving mankind all over the world. Achyranthes aspera, family Amaranthaceae, is found throughout the tropical Asia, Africa, Australia and America from the seashore to 2,100 m height. It is an annual or perennial herb. Different parts of the plant are reported to have great medicinal importance for gynecological disorders¹, diabetes^2,3, different infections^4,5and other diseases. Decoction of the root of the plant are reported to be used as antibacterials⁶and different gynecological disorders^7,8. The aim of our present work is to identify the different phyto-constituents in different solvent extracts of roots of Achyranthes aspera by different phyto chemical tests. The toxicity study for the methanol extract of the root was performed for safety purpose. The traditional uses of the root extract as anti inflammatory, anti pyretic, anti diabetic and antimicrobial agent also have been established by using different in vivo and in vitro models.

2. MATERIAL AND METHODS:

2.1. Plant material and extraction procedure:

The roots of Achyranthes aspera were dried in a drier for two days at 40ºC, crushed in a mechanical grinder to fine powder of mesh 40. The powder (500 g) was then extracted successively with petroleum ether, methanol and water in a Soxhlet apparatus. The extracts were filtered and the solvents were evaporated to dryness under reduced pressure in an Eyela Rotary Evaporator (Japan) at 40–45 °C. The yields are 1.25% and 3.46% w/w respectively for methanol and aqueous extracts.

2.2. Phytochemical identification:

The predominant phytochemicals in the root extracts were identified as per the standard qualitative methods^{9, 10}.

2.3. Animal used:

Swiss albino mice weighing between 18-20 g were used for toxicity study. Adult albino rats of either sex weighing 180–200 g each were used for other pharmacological studies. The animals were housed in standard polypropylene cages at room temperature and provided with standard pellet diet and water ad libitum.

Animal experiments were conducted following the guidelines of the institutional animal ethics committee.

2.4. Micro organism used:

A total of 12 strains of bacteria comprising of 2 gram-positive and 10 gram-negative genera were tested. They were isolated mostly as human pathogens and subjected to stringent culture. Isolates were identified by the methods of Barrow and Feltham^11,12 and were preserved in the freeze-dried state as well as at 4⁰C in stab slant agar. All bacterial strains are preserved in the Division of Microbiology, Dept. of Pharmaceutical Technology, Jadavpur University, Kolkata-32, West Bengal, India.

2.5. In vivo toxicity study:

The mice were randomly divided into experimental groups and a control group, with 10 animals in each group, and kept in polypropylene cage, which received different doses of methanol root extract in intra-peritoneal (I.P) route. Control animals received aqueous tween80 solution 2 %( v/v) and the experimental groups received methanol extract at doses of 50-400 mg/kg body weight of mice, administered through IP route ¹³. After administration of the extract by the I.P route the animals were observed under open field condition for 72 hours and the number of deaths and signs of clinical toxicity were recorded.

2.6. In vivo anti pyretic activity study:

2.6.1. Study on normal body temperature

Four groups of six animals were taken. The body temperature of each rat was measured rectally at 0, 1.5 and 3 h after administration of distilled water (control) methanol and aqueous fractions at a dose of 100mg/kg body wt. orally.

2.6.2 Study on yeast-induced pyrexia

Rats were divided into four groups of six rats each. Fever was induced ¹⁴. Animals were given a subcutaneous injection of 10 mL/kg body wt. of 15 % w/v yeast suspended in 0.5% w/v methylcellulose solution. After 19 h of yeast injection, the animals were restrained in individual cages for the recording of their rectal temperatures by insertion of a thermocouple to a depth of 2 cm into rectum. Elevation of body temperature ensures the induction of pyrexia

19 hrs after yeast injection the different fractions of Achyranthes aspera root extracts were administered orally at the dose of 100 mg/kg body weight into two groups of animal. One group of animal was treated with distilled water (10ml/kg body weight) and serve as control group. The fourth group of rats received the standard antipyretic drug paracetamol at a dose of 150 mg/kg body weight orally. Rats were restrained for recording the rectal temperature, which was recorded at 19, 21 and 23 hrs.

2.7. In vivo anti-inflammatory activity study:

2.7.1. Carrageenan-induced rat paw edema:

Edema was induced by sub-planter injection of 0.1 ml of 1% freshly prepared suspension of carrageenan (Sigma Chemical Co., USA) to each animal of four groups. The different extracts were suspended in 0.03% tween80 solution. The test groups received the extract (100 mg/kg body weight; orally), standard group received Indomethacin (25 mg/kg body weight; orally) and control group received the vehicle only. All the doses were given orally 30 minutes prior to the injection of carrageenann. The volume of the injected and contra-lateral paws was measured after 4 hours of carrageenan administration. Normal paw volume also noted at 0th hour i.e. before carrageenan injection. Inflammation was expressed in terms of paw edema by volume of water displaced by the inflammated rat paw in Plethysmometer¹⁵.

2.7.2. Dextran-induced rat paw edema:

The dextran induced rat paw edema test was performed according to the standard method¹⁶. This study was carried out in a similar manner of carrageenan induced paw edema model. Dextran (0.1 ml, 1% w/v in normal saline) was used in place of carrageenan.

The percentage inhibition of paw edema was calculated for both models¹⁷.

2.8. Oral glucose tolerance test (OGTT):

The oral glucose tolerance test ¹⁸was performed in overnight fasted (18 h) normal rats. Rats divided into three groups (n = 6) were orally administered distilled water(10ml/kg body weight), methanol extract and aqueous extract of Achyranthes aspera (100 mg/kg body weight). Glucose (2 g/kg body weight) was fed orally, 30 min prior to the administration of extracts. Blood was withdrawn from the tail vein and the glucose level was measured at 0^th, 1^st, and 2^nd hour with the help of single touch glucometer.

2.9. In vitro screening for the detection of antibacterial activity:

MIC was determined by agar dilution method¹⁹ and disk diffusion method²⁰. For this study sterile nutrient agar base was taken to which dried methanol root extract was added at such concentrations that the final concentration contain 0 (control), 100, 200, 400, 800, 1200 and 1600 g of extract in 1.0 mL of medium. The volume of nutrient agar medium poured in 80 mm Petri dishes was 20 mL.

The standard inoculums for determination of MIC with respect to various bacteria consisted of one loop full of overnight peptone water culture of the test organism²¹. The plates were incubated at 37˚C and examined after 24 h and incubated further for 72 h, if necessary. The MIC was defined as the lowest concentration of an antibiotic or a chemotherapeutic agent that completely inhibited the growth of the organism. The MIC test was performed in triplicate for each organism and the experiment was repeated where necessary.

2.10. Statistical Analysis:

The results were expressed as mean±SEM. The significance was evaluated by Student’s t-test compared with control²².

3. RESULTS:

3.1. Phytochemical analysis:

Table-1 shows the petroleum ether extract of the root contain only the steroids and triterpenoid whereas the methanol and aqueous extract contains alkaloids, steroid, triterpenoid, flavonoid, tannin, saponin and reducing sugar.

3.2. Toxicity study:

Table-2 shows that the methanol extract of the roots is safe up to dose 400 mg/kg body weight when tested with Swiss albino mice.

Table 2: The toxicity study of the methanol extract of Achyranthes aspera administered Intra peritonealy in albino mice

Treatment	Dose (mg/kg)	No. of animals used	No. of survival	No. of death
Control	0	20	20	00
Methanol Extract	50	20	20	00
	100	20	20	00
	150	20	20	00
	200	20	20	00
	250	20	20	00
	300	20	20	00
	350	20	20	00
	400	20	20	00

3.3. Antipyretic activity:

From Table-3 and Table-4 it was found that the methanol extract of the root possesses a significant action in maintaining the normal body temperature and reduces the yeast induced elevated temperature in albino rats and their effect are comparable to that of the standard antipyretic drug, paracetamol.

Table3. Effect of Achyranthes aspera root extract on normal body temperature in albino rats

Treatment	Rectal temperature (º F) before and after treatment
Treatment	0 h	1.5 h	3h
Normal (0mgl/kg)	98.2±0.4041	98.2±0.2309	98.7±0.1155
Methanol (100 mg/kg)	98.3±0.4910*	97.46±0.5696*	97.5±0.2887*
Aqueous (100 mg/kg)	98.26±0.9821*	96.73±0.5925*	96.86±0.4096*
Paracetamol (150 mg/kg)	98.26±0.1764*	97.13±0.1764*	97.2±0.1155*

Each value represents mean ± S.E.M. (n=6), * p< 0.05, with respect to control

Table 4. Effect of Achyranthes aspera root extracts on Yeast induced pyrexia in albino rats

Treatment	Rectal temperature (º F) after yeast injection
Treatment	19 h	21 h	23h
Control (0mgl/kg)	100.93±0.6566	99.63±0.088	98.96±0.260
Methanol (100 mg/kg)	99.23±0.0881*	97.933±0.260*	97.166±0.523*
Aqueous (100 mg/kg)	99.2±0.4163*	98.16±0.233*	98.066±0.348*
Paracetamol (150 mg/kg)	98.53±0.1453**	96.266±0.536**	95.93±0.290**

Each value represents mean ± S.E.M. (n=6). ** p< 0.01, with respect to control; *p<0.05, with respect to control

3.4. Effect on Carrageenan-induced and Dextran-induced rat paw edema:

The results obtained for methanol and aqueous extracts of root of Achyranthes aspera on carrageenan and dextran induced rat paw edema are shown in Table-5 and Table-6 respectively. The extracts significantly (p<0.001) inhibited the inflammatory edema at a dose of 100mg/ kg body weight of rat. The inhibition was highest for methanol extract. The percentages of inhibition of methanol extract for Carrageenan and Dextran induced paw edema are 66.99 and 69.81 respectively. Results were compared to that of the Indomethacin a non- steroidal anti-inflammatory drug.

3.5. Oral glucose tolerance test:

In oral glucose tolerance test, the extract, at 1st hr, showed significant reduction in plasma glucose level indicated in Table-7.

In the present study, oral glucose tolerance test (OGTT) was performed in normoglycemic rats. It is observed that methanol extracts and aqueous extracts reduce the blood glucose level more than the control (0.9% Nacl; 5 mL/kg.body weight) when administered orally.

3.6. In vitro antimicrobial study:

From Table-8 it was seen that out of 12 strains tested the MIC values of Achyranthes aspera against Shigella dysenteriae 6 and Shigella dysenteriae 8 were 1.2mg/mL and 1.6mg/mL respectively. Remaining 10 bacterial strains were resistant to the methanol root extracts up to the concentration of 1.6mg/mL.

4. DISCUSSION:

In our study we investigated on different phyto chemical, pharmacological and antibacterial properties of root extracts of plant Achyranthes aspera (Amaranthaceae). Table1. Indicates phytochemicals in the petroleum ether, methanol and aqueous extracts revealed differences in their constituents. Phytochemical compounds such as alkaloids, steroid, triterpenoid, flavonoid, tannin, saponin and reducing sugar are present

The in vivo studies established the profound anti pyretic, anti inflammatory and anti diabetic activity of methanol root extract of Achyranthes aspera with a dose of 100 mg/mL which was found to be safe for the animals by toxicity study (Table 2).

Table 3, 4 shows the effectiveness of the methanolic extract to maintain the normal body temperature and reduce the yeast induced pyrexia to a better extent than the aqueous extracts.

Table 5: Carageenan induced anti-inflammatory model

Treatment	Dose (mg/kg body weight) Orally	Paw volume in ml	% Inhibition of edema
Control	0.03% v/v aqueous tween80 solution	3.03±0.318	0
Methanol extracts	100mg/kg	1±0.115*	66.99%
Aqueous extracts	100mg/kg	1.43±0.145*	52.80
Indomethacin	25mg/kg	0.83±0.145*	72.60

Each value represents mean ±SEM (n=6), *P< 0.01 with respect to control

Table 6: Dextran induced anti-inflammatory model

Treatment	Dose (mg/kg body weight) Orally	Paw volume in ml	% Inhibition of edema
Control	0.03% v/v aqueous tween80 solution	3.2±0.208	0
Methanol extracts	100mg/kg	0.966±0.218	69.81
Aqueous extracts	100mg/kg	1±0.152	68.75
Indomethacin	25mg/kg	0.866±0.176	72.93

Each value represents mean ±SEM (n=6), *P< 0.01 with respect to control

Table 7: Effect of methanol extract of Achyranthes aspera on oral glucose tolerance test (OGTT)

Group	Treatment	Plasma glucose concentration (mg/dl)
Group	Treatment	0 hr	1hr	2hr
I	Normal control	91.66±1.66	86.66±2.90	89±2.08
II	Normal + methanol extracts (100mg/kg)	96.66±1.20*	68±1.15*	78.33±1.66*
III	Normal + aqueous extracts (100mg/kg)	91.66±10.17*	63.66±1.20*	80.66±1.33**

Values are expressed as mean ± SEM from n=6, * p < 0.05 compared with normal group, **P< 0.01 compared with normal group

Table 8. Effect of methanol root extract of Achyranthes aspera on different groups of bacteria

Bacterial strain tested	*Growth in nutrient agar containing different concentration of methanol root extract of Achyranthes aspera* (µg/mL)**
Bacterial strain tested	0(control)*	100 µg/mL	200µg/mL	400 µg/mL	800 µg/mL	1200 µg/mL	1600 µg/mL
*Vibrio cholerae* DN6	+	+	+	+	+	+	+
*Psedomanas* 25619	+	+	+	+	+	+	+
*Escherichia coli* 55	+	+	+	+	+	+	+
*Klebsiella pneumonia* J/114	+	+	+	+	+	+	+
*Shigella dysenteriae* 6	+	+	+	+	+	–	–
*Shigella flexneri* Eo3429	+	+	+	+	+	+	+
*Shigella sonnei* 288	+	+	+	+	+	+	+
*Shigella boydii* 10	+	+	+	+	+	+	+
*Salmonella typhi* NCTC74	+	+	+	+	+	+	+
*Shigella dysenteriae* 8	+	+	+	+	+	+	–
*Staphylococcus aureus* 25923	+	+	+	+	+	+	+
*Bacillus subtilis* UC 564	+	+	+	+	+	+	+

*, control plate without drug; +, growth; –, no growth

Table 5, 6 indicated the profound anti inflammatory activity of the methanol and aqueous extracts when compare with the standard drug. The carrageenan induced paw edema is believed to be biphasic, of which the first phase is mediated by early release of histamine and 5HT followed by the release of kinin in later phase ²³. Dextran mediated inflammation (edema) was also mediated by both histamine and serotonin²⁴. The exact mechanism of anti-inflammatory action of root extracts is not evaluated till now but there are many evidences of plant extracts which have this type of effects^{25, 26}. Flavonoids present in the extracts may be responsible for the anti-inflammatory and antipyretic properties due to their inhibitory effects on enzymes involved in the production of the chemical mediators of inflammation and pyrexia ²⁷.

Table 7 reveals the ability of extracts to reduce the normal blood glucose level so they may have a desirable antidiabetic effect consequently. The anti-diabetic effects may be due to the reduction of the intestinal absorption of glucose or may be able to increase the utilization of glucose by tissues²⁸. The results proved the anti-diabetic effect by the administration of methanol and aqueous extracts. This suggests that the possible mechanism of action may resemble as glibenclamide due to a direct stimulation of insulin secretion or delayed glucose absorption.

The in vitro screening for antibacterial activity proved effectiveness of methanol root extract against dysentery at a concentration comparable with other plant extracts containing tannins, flavonoids, saponins, sterols, triterpene and reducing sugars²⁹.

Isolation of active pure compounds and further analytical research on them will help to find out the exact mechanism of action for the pharmacological and antibacterial effects of the root extract of Achyranthes aspera.

ACKNOWLEDGEMENTS:

We are thankful to All India Council for Technical Education (A.I.C.T.E.) for the necessary financial assistance to conduct the work.

We are also grateful to “Botanical Survey of India”, Botanic Garden- Howrah,West Bengal, India. Vide No. CNH/1– 1(44)/2006/Tech.II/996 for the identification of the plant.

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Received on 10.03.2010 Modified on 24.03.2010

Research J. Pharm. and Tech.3 (3): July-Sept. 2010; Page 910-914

Extracts	Alkaloid	Anthraquinone glycoside	Steriod	Triterpenoid	Flavonoid	Tannin	Saponin	Reducing Sugar
Petroleum ether (60-80°C)	-	-	+	+	-	-	-	-
Methanol	+	-	+	+	+	+	+	+
Aqueous	+	-	+	+	+	+	+	+