Pharmacognostical studies on the root of Maerua longifolia

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Antipyretic activity studies of two botanical sources of the drug Murva / Asian Journal of Traditional Medicines, 2010, 5 (5)

Regular articles

Antipyretic activity studies of two botanical sources of the drug Murva

V. Madhavan a, Amit Kumar Shukla a, Anita Murali b, Usha M a, S. N. Yoganarasimhan a *a. Department of Pharmacognosy, M. S. Ramaiah College of Pharmacy, Bangalore 560054, Indiab. Department of Pharmacology, M. S. Ramaiah College of Pharmacy, Bangalore 560054, India

Abstract

Murva is one of the controversial drugs used in Ayurvedic medicine. In the present study two botanical sources of Murva, viz. Wattakaka volubilis and Maerua oblongifolia, were tested for antipyretic activity by yeast-induced pyrexia in Wistar albino rats. Alcohol and aqueous extracts of both species significantly reduced the elevated rectal temperature in febrile rats within 30 min of their administration. The results of these studies support the traditional use of these two botanical sources of the drug Murva in the treatment of fever.

Key words: Wattakaka volubilis; Maerua oblongifolia; Murva; yeast-induced pyrexia; roots

IntroductionMurva is considered a controversial drug in

ayurvedic medicine since more than one botanical source viz. Bauhinia tomentosa L. (Caesalpiniaceace), Chonemorpha fragrans (Moon) Als ton (=C . macrophylla (Roxb .) G. Don) (Apocynaceae), Clematis triloba Heyne ex Roth (Ranunculaceae), Helicteres isora L. (Stercul iaceae) , Maerua oblongifolia (Forsk.) A. Rich. (Capparaceae), Sanseviera roxburghiana Schult. & Schult. f. (=S. zeylanica Roxb.) (Agavaceae) and Wattakaka volubilis (L.f.) Stapf (Asclepiadaceae), are used in different regions of the country by physicians

practising ayurvedic medicine [1]. The accepted botanical source of Murva is Marsdenia tenacissima (Roxb.) Moon (Asclepiadaceae) [2]

. Murva is used in

diseases like anaemia (pandu), fever (jwara), diabetes (prameha), stomach disorders (udara roga), typhoid (visama jwara), urinary infections (asmari) and cough (ksaya) [3]. It is also used in the treatment of diarrhea, polyuria, boils, leprosy, eye diseases, vomiting and poisoning [4]. The present study describes the antipyretic activity of two sources of Murva, viz. W. volubilis and M. oblongifolia.

The phytoconstituents reported in W. volubilis are glycosides and alkaloids (root) [5], triterpenoids (leaves) [6], Drevogenin D (seeds) [7], β-sistosterol, pregnane glycosides and kaempferol (bark) [8] while, in M. oblongifolia, compounds like lupine triterpenoid, betulin and betulinaldehyde have been found [9].

W. volubilis is reported to possess a number of properties including hypoglycemic [10], anti-inflammatory, analgesic and anti-lipidperoxidative [11] effects; it also protects against selenite- induced cataract

* Author to whom correspondence should be addressed. Address: Department of Pharmacognosy, M.S. Ramaiah College of Pharmacy, M.S. Ramaiah Nagar, MSRIT-Post, Bangalore 560054, Karnataka, India; Tel: (+91) 080-23608942; Fax: (+91) 080-23607537; E-mail: [email protected]

Received: 2010-03-29 Accepted: 2010-07-16

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in the rat lens [12], and exhibits in vitro anti-leishmanial and anti-tumour activity [13], hepatotoxicity [14], inhibits proteolysis in the rat lens [15], and has an apoptosis-inducing potential [16] while M. oblongifolia stem bark possesses wound-healing activity [17, 18] and is used to treat toothache and intestinal disorders [19].

Materials and methods

Plant materials

Roots of W. volubilis, (Amit Kumar Shukla 031) (March 2009) and roots of M. oblongifolia (Usha 029) (November 2007) were collected from the forests of Kalakkad, Tirunelveli district, Tamil Nadu (India). The respective voucher specimens have been deposited in the herbarium and museum of M. S. Ramaiah College of Pharmacy, Bangalore, Karnataka, India. The plants were authenticated by Dr. S. N. Yoganarasimhan, Taxonomist and Research Coordinator at M. S. Ramaiah College of Pharmacy. The taxonomic identification was carried out using Flora of Hassan [20], Flora of Coorg [21], Flora of Presidency of Madras [22].

Animals

Albino Wistar rats weighing 170-200 g of both sexes were used in this study. Animals were acclimatized to the experimental conditions in the animal house of the M.S. Ramaiah College of Pharmacy, for 2 weeks prior to the study. The animal house was carefully maintained under standard hygienic conditions, at a stable temperature (22±2 ºC), and room humidity (60 % ±10 %) with a 12 h day and night cycle, with food and water provided ad libitum. Pharmacological studies were carried out after obtaining the approval from the Institutional Ethics Committee of the M.S. Ramaiah College of Pharmacy.

Preparation of extracts

Alcohol extract

The shade dried roots of W. volubilis and M. oblongifolia were powdered and extracted

exhaustively with 90 % v/v ethanol in a soxhlet apparatus. The ethanol extract was concentrated to a small volume and evaporated to dryness under reduced pressure. The yield of the alcohol-soluble extract of W. volubilis was 3.2 % w/w while that for M. oblongifolia was 41.3 % w/w, with reference to the air dried drug.

Aqueous extract

The aqueous ex t rac t s were p repared by maceration of powdered drugs with chloroform-water for 12 h, followed by filtration and concentration of the extract to a small volume. Finally, the extracts were evaporated to dryness. The yield of the aqueous-soluble extract of W. volubilis was 8.26 % w/w while that for M. oblongifolia was 43.52 % w/w, with reference to the air dried drug.

For experimental purposes, W. volubilis extract samples were prepared in distilled water containing 2 % w/v gum acacia while those of M. oblongifolia were prepared in distilled water containing 2 % v/v Tween 80.

The alcohol and aqueous extracts of each plant were subjected to preliminary organic analysis and chromatographic [23] studies. HPTLC was performed using a Camag HPTLC system equipped with a Linomat V applicator, a TLC scanner 3, and a Reprostar, with a 12 bit CCD camera for photo documentation, controlled by WinCATS- 4 software. All the solvents used were of HPLC grade obtained from MERCK. The Rf values obtained were compared with those available in the literature for the detection of glycosides in W. volubilis and alkaloids in M. oblongifolia [24].

Acute toxicity studies

Acute toxicity studies were performed following standard protocols [25, 26].

Anti-pyretic activity studies

Experimental protocol

Wistar albino rats of both sexes weighing between

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170-200 g were used. Each group contained 6 rats per drug. The W. volubilis groups were treated as follows:

Group 1: Vehicle control (Distilled water containing 2 % w/v gum acacia) p.o.

Group 2: Standard group (Paracetamol 150 mg/kg body weight) p.o.

Group 3: Alcohol extract (200 mg/kg body weight) p.o.


Group 5: Aqueous extract (200 mg/kg body weight) p.o.


The M. oblongifolia groups were treated as follows:

Group 1: Vehicle control (Distilled water containing 2 % v/v Tween 80) p.o.

Group 2: Standard group (Paracetamol 150 mg/kg body weight) p.o.





Py rex i a was i nduced i n a l l an ima l s by

subcutaneous injection of a 20 % w/v solution of brewer’s yeast (10 ml/kg) in distilled water, in between the shoulder blades. The basal rectal temperature was measured before the injection of the yeast solution, by inserting a digital clinical thermometer to a depth of 2 cm into the rectum. The rise in rectal temperature was recorded 19 h after the yeast injection. The febrile rats were divided into different experimental groups each consisting of 6 animals. Test doses of the extracts were chosen based on the acute toxicity results. Paracetamol (150 mg/kg body weight) was used as the standard comparator drug. The rectal temperature was recorded at regular intervals following the respective treatments [27].

Statistical analysis

The data were expressed as Mean ± S.E.M values and tested with a one way analysis of variance (ANOVA) followed by the Tukey-Kramer multiple comparison test.

Results

Phytochemical and chromatographic studies

Preliminary phytochemical studies of the roots of W. volubilis revealed the presence of carbohydrates, glycosides, saponins, phenolic compounds, gums and mucilage while the roots of M. oblongifolia contained alkaloids, carbohydrates, glycosides, phytosterols,

Table 1. Phytoconstituent differences between W. volubilis and M. oblongifolia in alcoholic and aqueous extracts

Note: ++ - Present; -- Absent

PhytoconstituentW. volubilis M. oblongifolia

Alcohol extract Aqueous extract Alcohol extract Aqueous extractAlkaloids -- -- ++ ++

Carbohydrates & glycosides ++ ++ -- ++

Phytosterols -- -- ++ --Phenolic compounds & tannins ++ ++ -- --

Saponins ++ ++ ++ ++Gums & mucilage -- ++ -- --

Proteins & amino acids -- -- -- ++

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saponins, proteins and amino acids (Table 1). HPTLC profiles of the alcoholic and the aqueous extracts of glycosides, in case of W. volubilis, and alkaloids, in case of M. oblongifolia, were obtained and the Rf values were recorded.

W. volubilis: At 254 nm, the R f value 0.50 correspond to the presence of glycosides in the alcoholic extract and 0.40, 0.60, 0.67 and 0.68 in the aqueous extract. At 366 nm, the Rf values 0.44, 0.55, 0.61 and 0.72, exhibited a blue fluorescence indicating the presence of glycosides in the alcoholic extract while Rf values of 0.40, 0.60, 0.69 and 0.71 were observed in aqueous extract (Figs. 1-4).

M. oblongifolia: At 254 nm, the Rf values 0.29, 0.46, 0.53 (black color) correspond to the presence of alkaloids in the alcoholic extract. At 366 nm, 0.20 & 0.42 and 0.36 & 0.49 (blue fluorescence) corresponding to the presence of alkaloids in the alcoholic and aqueous extracts, respectively (Figs. 5-7).Acute toxicity studies

Acute toxicity studies showed that the alcoholic and aqueous extracts of both drugs were safe up to

2000 mg/kg body weight when administered orally to rodents. Antipyretic activity studies

A reduction in the rectal temperature of febrile rats, treated with different doses of the aqueous and alcoholic extracts of the drugs, was recorded at 30, 60, 120, 180 and 300 min post-administration. The reduction in the rectal temperature of the treated animals at each interval was compared with that exhibited by the untreated febrile rats. The alcoholic and aqueous extracts of both drugs produced significant antipyretic effects [W. volubilis (P<0.01; P<0.001) and M. oblongifolia (P<0.05)], within 30 min of administration. The maximum temperature reduction was observed at 30 min for W. volubilis and at 120 min for M. oblongifolia. The results were dose-dependent in M. oblongifolia, whereas this was not the case with W. volubilis. However, there was a significant reduction in the rectal temperature of febrile rats after all time intervals for both drugs.

DiscussionFever may be a result of infection, tissue damage,

Fig. 1. Chromatogram showing Rf values of phytoconstituents of the alcoholic extract of W. volubilis when scanned at 254 nm

Peak number

Start position

(Rf)

Start height (AU)

Max position

(Rf)

Max height (AU)

Max( %)

End position

(Rf)

End height (AU) Area Area

( %)

1 0.48 4.1 0.50 49.2 12.9 0.54 2.6 800.4 3.152 0.74 17.6 0.88 331.9 87.1 0.95 1.7 24639.0 96.85

Rf

AU

14

Fig. 1. Chromatogram showing Rf values of phytoconstituents of the alcoholic extract of W. volubilis when scanned at 254 nm

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

450

400

350

300

250

200

150

100

50

0

2

1

55


Fig. 2. Chromatogram showing Rf values of phytoconstituents of the aqueous extract of W. volubilis when scanned at 254 nm

Peak number

Start position

(Rf)

Start height (AU)

Max position

(Rf)

Max height (AU)

Max( %)

End position

(Rf)


( %)

1 0.03 47.4 0.04 531.00 52.36 0.06 1.0 448.6 1.182 0.57 2.2 0.40 15.90 1.57 0.42 11.9 24639.0 96.853 0.57 7.7 0.60 19.50 1.92 0.60 18.0 406.3 1.074 0.61 18.3 0.67 48.49 4.82 0.67 47.3 1841.5 4.845 0.68 47.3 0.68 50.00 4.93 0.72 38.6 1591.2 4.186 0.76 35.3 0.88 348.80 34.39 0.98 3.6 24802.0 70.47

Treatment Control Standard Paracetamol

Alcohol extract (150 mg/kg)

Alcohol extract (200 mg/kg)

Aqueous extract (200 mg/kg)

Aqueous extract (400 mg/kg)

Basal rectal temp. (ºC) 37.46 ± 0.06 38.00 ± 0.11 37.17 ± 0.04 36.98 ± 0.15 37.21 ± 0.03 37.14 ± 0.07

Rectal tem. at 19 h of yeast administration

39.30 ± 0.08 38.65 ± 0.10 38.7 ± 0.09 38.03 ± 0.17 38.51 ± 0.09 38.59 ± 0.06

Rectal temp.at 30 min. (ºC) 39.30 ± 0.08 38.65 ± 0.10*** 38.71 ± 0.09** 38.08 ± 0.17*** 38.54 ± 0.10*** 38.62 ± 0.06**

Rectal temp.at 60 min. (ºC) 39.31 ± 0.09 38.59 ± 0.08*** 38.70 ± 0.08** 37.94 ± 0.15** 38.45 ± 0.11*** 38.57 ± 0.05***

Rectal temp.at 120 min. (ºC) 39.36 ± 0.11 38.19 ± 0.14** 38.46 ± 0.09*** 37.78 ± 0.13*** 38.30 ± 0.10*** 38.08 ± 0.07***

Rectal temp.at 180 min. (ºC) 39.28 ± 0.10 37.56 ± 0.19** 38.11 ± 0.21*** 37.48 ± 0.07*** 38.08 ± 0.13*** 37.53 ± 0.07***

Rectal temp.at 300 min. (ºC) 39.23 ± 0.10 37.16 ± 0.24*** 37.59 ± 0.10*** 37.20 ± 0.02*** 37.50 ± 0.09*** 37.16 ± 0.03***

Table 2. Effects of W. volubilis extracts on yeast induced pyrexia in rats

The values are expressed as Mean ± SEM; n= 6 animals in each group.Tukey-Kramer multiple comparison test***P<0.001, **P<0.01, in comparison with positive control

AU

15

Fig. 2. Chromatogram showing Rf values of phytoconstituents of the aqueous extract of W.volubilis when scanned at 254 nm

700

600

500

400

300

200

100

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00 Rf

2 34 5

6

1

56


inflammation, graft rejection, or other disease states and antipyretics are drugs which reduce an elevated body temperature. Regulation of body temperature requires a delicate balance between the production and loss of heat, and the hypothalamus regulates the point at which body temperature is maintained. In fever, this point is elevated, and drugs like paracetamol do not affect body temperature when it is increased by factors such as exercise or an increase in ambient temperature; the standard drug used for treatment is paracetamol which produces its antipyretic effect by inhibition of prostaglandin biosynthesis [28]. Yeast-induced pyrexia is called pathogenic fever and is due to the production of prostaglandins (PGE2) which set the thermoregulatory centre at a higher temperature[29]. The present study indicated that the antipyretic

effects of both W. volubilis and M. oblongifolia were comparable with that of the standard drug paracetamol (Tables 2 and 3). However, it was observed that W. volubilis produced results comparable with paracetamol from 30 min onwards whereas M. oblongifolia produced comparable results from 60 min only indicating that W. volubilis is preferable to M. oblongifolia.

The phytochemical studies of W. volubilis and M. oblongifolia showed the presence of many important phytoconstituents like phenols, alkaloids, carbohydrates, glycosides, phytosterols and saponins, which could all have contributed to the antipyretic effect [30, 31, 32]. Further investigation is needed to see whether the mechanism involved is inhibition of prostaglandin synthesis. The results of this study

Peak number

Start position (Rf)

Start height (AU)

Max position (Rf)

Max height (AU)

Area( %)

End position (Rf)

End height (AU)

Area Area( %)

1 0.02 66.0 0.03 285.6 24.42 0.06 10.8 5042.60 14.012 0.06 111.7 0.08 129.7 11.09 0.11 34.9 4220.50 11.723 0.11 95.0 0.13 117.6 10.06 0.16 75.7 3064.80 18.514 0.16 75.8 0.16 77.3 6.61 0.19 61.4 1862.20 5.175 0.19 61.6 0.26 390.6 33.39 0.34 45.8 163332.50 45.376 0.34 46.0 0.35 50.5 14.32 0.38 41.0 1361.40 3.787 0.40 40.6 0.44 66.6 5.70 0.48 22.1 2868.70 7.418 0.53 24.5 0.55 28.3 2.42 0.60 11.4 0.61 12.409 0.60 11.5 0.61 12.4 1.06 0.64 0.2 163.60 0.4510 0.69 1.3 0.72 10.8 0.93 0.74 0.0 224.00 0.62

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Fig. 3. Chromatogram showing Rf values of phytoconstituents of the alcoholic extract of W. volubilis at 366 nm

AU

600

500

400

300

200

100

0 0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

Rf

23

5

6 7

89 10

1

4

Fig. 3. Chromatogram showing Rf values of phytoconstituents of the alcoholic extract of W. volubilis at 366 nm

57


Peak number

Start position (Rf)

Start height (AU)

Max position (Rf)

Max height (AU)

Max( %)

End position (Rf)


( %)

1 0.05 14.1 0.06 235.3 42.71 0.07 0.09 1844.60 9.242 0.19 0.2 0.20 14.3 2.60 0.21 1.90 118.40 0.593 0.23 5.03 0.26 13.6 2.48 0.27 9.90 307.00 1.544 0.69 1.3 0.72 10.8 0.93 0.74 0.00 224.00 0.625 0.58 12.4 0.60 16.6 3.01 0.62 5.60 437.90 2.916 0.65 4.4 0.69 18.1 3.29 0.71 11.50 678.40 3.407 0.71 11.8 0.71 13.1 2.38 0.76 0.30 312.60 1.578 0.53 24.5 0.55 28.3 2.42 0.60 11.40 0.61 12.40

Fig. 4. Chromatogram showing Rf values of phytoconstituents of the aqueous extract of W. volubilis at 366 nm

17

Fig. 4. Chromatogram showing Rf values of phytoconstituents of the aqueous extract of W.volubilis at 366 nm

Rf

AU

200

150

100

50

0

0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

300

250

2 3 4 5 6 7

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Fig. 5. Chromatogram showing the HPTLC profile of the alcoholic extract of M.oblongifolia at 254 nm

Peak number

Start position (Rf)

Start height (AU)

Max position (Rf)

Max height (AU)

Max( %)

End position (Rf)


( %)

1 0.08 2.5 0.10 40.5 5.00 0.11 1.8 422.00 1.072 0.13 0.5 0.15 15.1 2.20 0.16 0.2 0.19 0.683 0.17 0.2 0.19 31.4 4.57 0.21 3.3 405.00 1.894 0.21 3.5 0.23 21.6 3.14 0.25 19.6 385.70 1.805 0.25 19.7 0.29 41.4 6.02 0.33 0.0 1318.40 6.146 0.42 0.7 0.46 35.4 5.15 0.47 26.0 686.00 3.207 0.47 25.5 0.53 485.1 70.61 0.58 5.4 17369.10 81.418 0.71 5.7 0.75 16.6 2.42 0.80 2.1 625.60 2.9218

Fig. 5. Chromatogram showing the HPTLC profile of the alcoholic extract of M.oblongifolia at 254nm

Rf

AU

700

600

500

400

300

200

100

00.00 0.05 0.25 0.45 0.65 0.85 1.05

12 3 4 5 6

7

8

58


Peak number

Start position

(Rf)

Start height (AU)

Max position

(Rf)

Max height (AU) Max %

End position

(Rf)


( %)

1 0.08 0.0 0.12 69.8 7.35 0.16 50.5 2213.9 12.512 0.16 50.5 0.20 719.8 75.84 0.26 30.8 11940.7 67.493 0.37 24.6 0.42 159.5 16.81 0.46 21.2 3539.1 20.00

Table 3. Effects of extracts of M. oblongifolia on yeast-induced pyrexia in rats

Treatment Control Standard Paracetamol(150 mg/kg)

Aqueous extract(250 mg/kg)

Aqueous extract(500 mg/kg)

Alcohol extract(250 mg/kg)

Alcohol extract(500 mg/kg)

Basal temperature (ºC) 34.83 ± 0.11 35.14 ± 0.22 35.70 ± 0.20 35.69 ± 0.31 36.37 ± 0.18 36.12 ± 0.26

Rectal temp. at 19 h of yeast administration (ºC)

38.40 ± 0.24 37.93 ± 0.23 37.81 ± 0.22 38.08 ± 0.19 37.82 ± 0.15 37.90 ± 0.14

Temperature at 30 min (ºC) 38.40 ± 0.29 37.16 ± 0.15** 37.42 ± 0.25 37.37 ± 0.22* 37.32 ± 0.14* 37.22 ± 0.24*

Temperature at 60 min (ºC) 38.53 ± 0.21 37.08 ± 0. 23** 37.08 ± 0.18** 37.12 ± 0.23** 37.18 ± 0.14 * 37.01 ± 0.44**

Temperature at 120 min (ºC) 38.52 ± 0.20 36.69 ± 0.32** 37.04 ± 0.09* 36.62 ± 0.21*** 36.61 ± 0.23*** 36.59 ± 0.50***

Temperature at 180 min (ºC) 38.50 ± 0.20 36.05 ± 0.41*** 36.77± 0.09* 36.33 ± 0.26** 36.40 ± 0.56** 36.18 ± 0.41***

Temperature at 300 min (ºC) 38.49 ± 0.20 35.43 ± 0.53 *** 36.51± 0.12* 35.97± 0.26** 36.19 ± 0.58** 35.87 ± 0.44***

The values are expressed as Mean ± SEM; n = 6 animals in each group.Tukey-Kramer multiple comparison test***P<0.001, **P<0.01, *P< 0.05, compared with the positive control

19

Fig. 6. Chromatogram showing the HPTLC profile of the alcoholic extract of M.oblongifoliaat 366 nm

Rf

AU

900

800

700

600

500

400

300

200

100

00.00 0.10 0.20 0.30 0.40 0.50 0.60

2

1

3

Fig. 6. Chromatogram showing the HPTLC profile of the alcoholic extract of M.oblongifolia at 366 nm

59


Peak number

Start position (Rf)

Start height (AU)

Max position (Rf)

Max height (AU)

Max( %)

End position (Rf)


( %)

1 0.01 7.36 0.05 90.1 12.75 0.10 55.9 4435.0 16.792 0.10 56.0 0.12 71.8 10.16 0.14 67.8 1458.9 5.52

3 0.14 67.6 0.15 70.6 9.99 0.18 51.9 1563.0 5.924 0.18 52.0 0.20 65.9 9.32 0.22 60.3 1635.4 6.195 0.30 57.5 0.36 76.3 10.79 0.43 66.4 5730.7 21.70

6 0.46 55.7 0.49 58.3 8.24 0.62 0.3 3785.0 14.33

7 0.62 0.6 0.66 51.0 17.22 0.69 0.1 769.1 2.91

8 0.70 0.4 0.77 41.7 5.90 0.85 13.1 2603.3 9.86

9 0.88 25.8 0.93 80.2 11.35 0.95 47.3 2148.7 8.1410 0.95 48.6 0.96 101.0 14.29 1.00 0.2 2282.3 8.64

should help stimulate further research, to isolate the bioactive principle(s) responsible for the antipyretic activity and to identify the exact mechanism(s) of action.

ConclusionThe alcoholic and aqueous extracts of both

botanical species of the drug Murva exhibited antipyretic activity, thus supporting their traditional use. It was also found that W. volubilis is preferable to M. oblongifolia in terms of antipyretic activity. This study should encourage the use of these species for their antipyretic activity and helps in preparing standardized therapeutic formulations.

AcknowledgementsThe authors thank Gokula Education Foundation

for the support and help provided which allowed this research to be carried out. They are also thankful to V. Chelladurai for supplying authentic plant material.

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Fig. 7. Chromatogram showing the HPTLC profile of the aqueous extract of M.oblongifoliaat 366nm

Rf

AU

150

100

50

00.00 0.05 0.25 0.45 0.65 0.85 1,05

250

200

12 3 4

56

7 8

9

10

Fig. 7. Chromatogram showing the HPTLC profile of the aqueous extract of M.oblongifolia at 366 nm

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