Evaluation of in vitro schizontocidal activity of plant parts of Calotropis procera—an ethnobotanical approach

Journal of Ethnopharmacology 68 (1999) 83 – 95 www.elsevier.com/locate/jethpharm

Evaluation of in vitro schizontocidal activity of plant parts of Calotropis procera—an ethnobotanical approach P. Sharma, J.D. Sharma * School of En6ironmental Sciences, Jawaharlal Nehru Uni6ersity, New Delhi 110067, India Received 6 January 1999; received in revised form 22 February 1999; accepted 23 March 1999

Abstract Calotropis procera (Ait.) R.Br. commonly known, as ‘Arka’ is a popular medicinal plant found throughout the tropics of Asia and Africa and is used in many traditional systems of medicine. Important factors of the various parts of this plant have been widely reported. Good record keeping of subjective and objectively recorded cures by practitioners of traditional medicinal system will help in the establishment of the use of C. procera as an antimalarial plant. It has been attempted to see the effect of crude fractions of its flower, bud and roof against a chloroquine sensitive strain, MRC 20 and a chloroquine resistant strain, MRC 76 of Plasmodium falciparum using the Desjardins method and the effectiveness of its fractions compare better with the CQ sensitive strain than the CQ resistant strain in vitro. © 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethnobotany; Rajasthan; Calotropis procera; P. falciparum; In-vitro; Chloroquine resistant strain; Chloroquine sensitive strain

1. Introduction Global spread of multiple drug resistant malaria has become a major health problem and efforts to search for new antimalarials are needed (Perez et al., 1997). Ethnobotanical studies have very often resulted in the discovery of important drug plants like the discovery of quinine from Cinchona bark in South America and artemisinin from Artemisia in China. India with its wide variety of vegetational types and secluded tribal populations affords ample scope for studies con* Corresponding author. E-mail address: [email protected] (J.D. Sharma)

cerning various aspects of folk medicine (Sebastian and Bhandari, 1984) Antimalarial plants mentioned in literature and the ones used in folk medicine, which were tested scientifically for their efficacy have been extensively reviewed and exhaustively compiled from the year 1911 to 1998 (Sharma and Sharma, 1998). Under the ‘Medicinal plants project’ at the Central Drug Research Institute, Lucknow (India), more than 3000 plant species have been screened for a wide range of antiprotozoal activities including, antimalarial (Aswal et al., 1996). Some of the Indian medicinal plants tested for their efficacy against only Plasmodium falciparum are shown in Table 1. The Desert Medicine Research Centre (DMRC), Jodh-

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pur, is also active in finding antimalarial plants with the changing incidence rates of malaria spreading along the newly formed water canals linked to the major Indira Gandhi Canal. Stagnation at leakage points along these have led to an explosion of the mosquito population (Jain, 1994). Rajasthan is the third largest and one of the important areas of arid zone biodiversity in India. Due to the constant association with the forest environment the tribals have accrued considerable knowledge of the plants and their utility, especially for medicinal purposes (Tripathi et al., 1996). This represents an incalculable but tangible benefit over the costs required to get western medicines from the market place (Sharma and Sharma, 1997). Calotropis procera is prevalent in the state in different ecotypic forms. C. procera (Ait.) R.Br. commonly known as ‘Ak’ or ‘Arka’ throughout the tropics of Asia and Africa is widely used in the Sudanese medicinal system (Ayoub and Kingston, 1981; Ayoub and Svendsen, 1981), Unani system of medicine (Anderson, 1988) and Arabic medicine system (Abulfatih, 1987). It is an erect much branched shrub 2 – 3 m high bearing purple spotted white flowers (Fig. 1) (Bhandari, 1977). It is worshipped by Hindus and is usually planted near temples of the Indian God, Lord Shiva. The wood is used in Havan and Yaggya (Sacred altar fires) as a sacred

Fig. 1. A desert ecotype of Calotropis procera as occurred in approximately 10 acres of sandy land along the road side. They stretch like a field upto the horizon as seen in this photograph.

wood (Shah, 1982). In the traditional Indian medicinal system, different parts of the plant have been advocated for a variety of disease conditions (Warrier et al., 1994; Sharma, 1997). Different parts of the plant have been reported to possess a number of biological activities as listed in Table 2. Regarding its antipyretic activity, a tincture of the leaves is used in the treatment of intermittent fevers (Chopra et al., 1960; Kapoor and Kapoor, 1980; Duke, 1985), flower buds mixed with black pepper and common salt are believed to possess antipyretic activity (Anis and Iqbal, 1986). The latex is given in malarial and low hectic fevers (Khory and Katrak, 1981) while flower buds and root bark are crushed to make tablets in ‘jaggery’ against malarial fever (Singh and Ali, 1994). A scientific evaluation of its antiplasmodial potential has been undertaken to find dose effective responses and quantify it using in vitro culture of P. falciparum. A comparison of this in vitro testing has been undertaken between CQ sensitive and CQ resistant strains.

2. Methodology

2.1. Collection of plant material Fieldwork was carried out in April 1996. Information on antimalarial plants was obtained by direct field interviews of local healers (Gunijis and Vaidya), old villagers, ladies and Barots, the professional singers who incorporate the knowledge in their songs in three different villages, Dhivera station, Khari and Bajju of Bikaner district of Rajasthan. The most commonly described plant was ‘Ak’ and the intact bud was used properly in cases of malaria. Roots burnt in mud-pots under buried conditions in sand are ashed and given along with crystal sugar and large quantities of milk as a remedy against malaria. Latex beads picked up from the sand and used similarly in malarial fevers. Entire plants were dug out from these areas. Dr C.R. Babu of the Botany Department of Delhi University identified the specimens as C. procera. A voucher specimen (No. 12853) has been prepared and deposited in the herbarium over there.

Table 1 Effective dose range of some Indian medicinal plants tested scientifically for their efficacy against Plasmodium falciparum based on their mention in literature and use in folk medicine Family

Part used

Fraction/ compound

Effective dose range

Plasmodium species

Ref.

Coptis teeta

Ranunculaceae

Rhizome

Aqueous

IC50 8.8 mg/ml

P. falciparum

Alstonia conacea

Apocyanaceae

Bark

Asteraceae

Roots

IC50 (nM) 5.71 5.45 Ethanolic extracts of A. japonica and A. nilegarica showed 100% schizont maturation inhibition at 40 mg/ml MED50 mg/ml

P. falciparum

Artemisia nilegarica Artemisia maritima Artemisia japonica

Alkaloids: Corialstonine Corialstonidine Ethanol

Sharma et al. (1993) Wright et al. (1993)

Azadirachta indica

Meliaceae

Leaf

Ethanol

Seeds

Xanthium mal6acearum Atlantia monophylla Eucalyptus Globulus Artemisia par6ifiora Nyctanthes arbortristis Echinops echinatus Ocimum sanctum

P. falciparum FDL-R1 P. falcipanum

25–75 200 0.01–0.05 MED50 mg/ml

FAN-5, FCK-2, FCK-3, FMN-17, FMN-33, MP-11

FAN-5, FMN-13, FMN-17, and MP-11, SO respectively

Asteraceae

Aerial parts

Ethanol

25, 25, 25, 50 & 50

Rutaceae

Aerial parts

Ethanol

50, 50, 50, 50 & 50

Myrtaceae

Aerial parts

Ethanol

150, 75, 100, 100 & 100

Asteraceae

Aerial parts

Ethanol

250, 100, 200, 150 & 200

Nyctaginaceae

Aerial parts

Ethanol

1000, 1200, 100, N.D. & N.D.

Asteraceae

Aerial parts

Ethanol

Lamiaceae

Aerial parts

Ethanol

\2000, \2000, \2000, N.D. & N.D. \2000, \2000, \2000, N.D. & N.D.

Valecha et al. (1994)

P. falciparum

Badam et al. (1987)

Badam et al. (1988)

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Table 2 The biological uses of different parts of Calotropis procera and dose range of their extracts/fractions S.No.

Extract/fraction

Compound isolated

Effective dose range

Test system for biological activity

Biological activity

Ref.

1

Flowers

Ethanol

N.D.a

IC50 — 1.4 mg/ml

Cytostatic activity

Smit et al. (1995)

2

Flower

Rectified spirit

N.D.a

3

Flower

Human beings

Asthma

4

Flower

Fried in cow’s ghee after N.D.a removing gynostegium Ethanol N.D.a

5

Latex

–b

N.D.a

20 mg/animal on alternate day resulted in widespread testicular necrosis after 30 days A total of 196 flowers in 27 days 100 mg/kg resulted in 20% inhibition 9.30% average loss of weight of different woods as compared to 46.29% in control

COLO 320 (a human colorectal carcinoma cell line) Meriones hurrianae (desert male gerbil)

6

Latex

–

N.D.a

7

Latex

–

N.D.a

8

Latex

95% aqueous ethanol

Uscharin

9

Latex

Petroleum ether Methanol Residual aqueous Dried aqueous Dry latex

N.D.a

Rats

Cedrus deodara Mangifera indica Dalbergia sisso Pinus excelsa Tectona grandis 1 ml of latex per 100 ml Anopheles stephensi water resulted in 100% Aedes aegypti larvicidal activity Culex fatigan Dry latex produced 71% Male albino rats inhibition in case of carrageenin inducedoedema while around 32% inhibition for formalin induced oedema LD50 was 0.82 mg/snail Thepa pisana after 24 h Significant effect at 1000 Bacterial and fungal mg/ml cultures

Garg (1979)

Upadhyay (1979) Analgesic activity Mascolo et al. (1988) Antitermites Giridhar et al. property (1988)

Mosquito control Giridhar et al. (1984) Anti-inflammatory activity

Kumar and Basu (1994)

Molluscicidal activity Antimicrobial activity

Hussein et al. (1994) Desta (1993)

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Part used

Table 2 (Continued) Part used

Extract/fraction

Compound isolated

Effective dose range

Test system for biological activity

10

Latex

N.D.a

50 mg/kg for 37.25, 27.50, Male rats Wistar strains Antiinflammatory 53.00, 39.25, 59.25 and activity 22.00% respectively

11

Latex

Petroleum ether Acetone Methanol Double distilled water Residue –

N.D.a

12

Terminal leaves

Dried

N.D.a

13

Leaves

Dried and aqueous extract

N.D.a

14

Leaves

50% ethanol

N.D.a

15

Leaves

95% ethanol

N.D.a

Bacteriolytic activity at Micrococcus lysodeikticus 175 U/ml Pair of leaves before sun- Human beings rise for 2 days for 100% cure 10 000 ppm for 100% Limnoea luteola mortality Gyraulus con6exiusculus 150 mg/kg bw. for 20% Rats inhibition MIC= 2 mg/ml Candida albicans

16

Leaves

Aqueous

N.D.a

17

Leaves

Aqueous

N.D.a

18

Leaves

Powder mixed with medium

N.D.a

19

Leaves

Hot aqueous

N.D.a

Blood pressure diminished 30% relative to the basal value during 30 min observation 5% crude extract shows 60% mortality rate in larve 60 000 ppm resulted in 26.31% mortality after 2 days 5 mg/100 g b.wt.

20

Leaves

Methanol

N.D.a

Dilutions 1:5 and 1:10

Bacterial and fungal cultures

21

Roots

Chloroform

N.D.a

5–15 mg/kg

22

Root

Chloroform

N.D.a

0.15 mg/kg in gastric ulcers

Rats and Swiss albino mice Rats Guinea pigs

Wistar rats

Biological activity

Ref.

Majumdar and Kumar (1997)

Antibacterial Shukla and Kractivity ishnamurti (1961) Effect on migraine Prasad (1985)

Antimolluscicidal activity

Bali et al. (1985)

Anti-implantation Prakash et al. activity (1978) Antifungal activity Tanira et al. (1994) Hypotensive Carbajal et al. (1991)

Eutectona machaeralis Insecticidal activity Meshram (1995) walk (Teak skeletonizer) Tribolium confusum

Insecticidal activity Jahan et al. (1991)

White mice

Anti-inflammatory Jangde et al. activity (1994) Antibacterial Kumar and activity Chanhan (1992) Antifungal activity Hepatoprotective ef- Basu et al. fect (1992) Antiulcer activity Basu et al. (1997)

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S.No.

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88

Table 2 (Continued) Part used

Extract/fraction

23

Root

N.D.a Arka mula Tvarka (an ayurvedic preparation)

24

Roots

Chloroform

N.D.a

250 mg, thrice a day for Human beings 7 days and 15 days respectively for 67.1 and 44.7% cure 5–15 mg Male albino rats

25

Whole plant

N.D.a

0.1 ml

Bacterial cultures

26

Roots/flower

Chloroform methanol Methanol Aqueous –

Calotropin

25 mg/kg b.wt.

27

Leaves Flower Root bark Leaves Stem Root

Ethanol Ethanol Ethanol Ethanol

Oil

0.02 ml

Male gerbils, rabbits, rats Bacterial cultures

N.D.a

Root bark showed 1Z 16 Enterobacter cloacae mm inhibition Stem showed IZ 17 mm Fusarium moniliformae inhibition

N.D.a

MIC (mg/ml)

28

Compound isolated

Effective dose range

Test system for biological activity

Biological activity

Ref.

Antidiarrhoeric Jain et al. (1985) and antidysenteric activity Anti-inflammatory activity Analgesic activity Antibacterial activity

Basu and Nagchaudhuri (1991) Almagboul et al. (1985)

Abortifacient activity Antibacterial activity

Gupta et al. (1990) Nawazisht et al. (1979)

Antimicrobial activity

Jain et al. (1996)

Fungal cultures

Antifungal activity

Larhsini et al. (1997)

Bulinus truncatus

Molluscicidal activity

Flower Fruit Root bark 29 Flower Leaves

Ethylacetate n-butanol Ethylacetate n-butanol

Latex Latex

Aqueous Ethanol Residual Aqueous extract

24–72 20–68 24–59 24–76 24–68 LC50 (ppm) 66 36 76

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S.No.

S.No.

Part used

Extract/fraction

30

31

32 33

a b

Compound isolated

Effective dose range

N.D.a

LD50 mg/kg b.wt.

Root

Ethanol

550

Leaves

Ethanol

550

Root

Chloroform–

N.D.a

Significant effect

Flower Leaves, branch, stem –

methanol 21% decoction

N.D.a

66.7% mortality rate

50% ethanol

N.D.a

Application of paste on ulcerated region for 9 days for 60% growth regression

N.D., not detected. –, not mentioned.

Test system for biological activity

Biological activity

Ref.

Bhakuni et al. (1969) Mongrel dogs or cats

Respiration and blood pressure Human epidermoid car- Tissue culture cinoma of nasopharynx Rats Antisperm activity Qureshi et al. (1991) Plecoptera reflexa Guen. Pesticidal activity Chaudhury (1992) (Noctudiae, Lepidoptera) Human beings (male and Antiulcerous Bhatnagar and female) activity Verma (1986)

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Table 2 (Continued)

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2.2. Extraction procedure Different parts namely flowers, buds, roots, stems and leaves were carefully separated, air dried and powdered. One kg dried powder of each part was extracted with absolute ethanol (7 l) for 4 days using a Soxhlet apparatus. The filtered ethanolic extracts were concentrated to dryness using a Rotary evoporator and stored in the dried state. The percentage yields (w/w) based on the dried starting material were 14.11, 12.02, 4.21, 8.79 and 16.67%, respectively. Furthermore, 30 g of flower, bud and root ethanolic extracts each were subjected to column chromatography on Si gel (60–120 mesh) and eluted successively with 250 ml ethylacetate, acetone and methanol. The percentage yields (w/w) were 2.6, 11.6 and 39.43% for flower, 9.77, 13.15 and 15.6% for bud and 9.63, 16.9 and 43.07% for root, respectively. The fractions were separately evaporated to dryness Thus three fractions for each extract were obtained which were tested for their antiplasmodial activity in vitro.

2.3. Antimalarial screening The test procedure for antimalarial screening was based on the method of Ang et al. (1995). Chloroquine sensitive P. falciparum, MRC 20 was obtained from the Malaria Research Centre, New Delhi. The culture was synchronized using sorbitol and parasitaemia was adjusted to 1 – 1.5% by diluting with fresh human erythrocytes. The cells were diluted with complete media to make 8% haematocrit. Five mg of each fraction was dissolved in 1 ml DMSO, 1 ml ethanol and 998 m1 RPMI-1640 to obtain a stock of 5 mg/ml (stock solution). A series of six concentrations were prepared from the stock solutions by 2-fold dilutions (0.0625–2 mg/ml) to give concentration around the range of 50% inhibition (IC50). All tests were done in triplicate. After 36 h, thick films of the contents of each well were prepared and examined under the microscope. Parasite count for each blood film was made under oil immersion with ×100 objective and ×10 eye piece. Each film was observed at three different visual fields. The number of schizonts with three or more nuclei per

200 parasites were noted and compared between control and test wells for the determination of the %age inhibition. All doses were studied in triplicate cultures and the mean was observed for purposes of inferences.

2.4. Preliminary phytochemical screening The ethanolic extracts of flower, bud and root were subjected to various preliminary phytochemical tests (Chhabra et al., 1984; Kokate, 1986) for the presence or absence of various classes of compounds as shown in Table 3.

2.5. Statistical calculations All reported values are expressed as percentage growth inhibition. Dose response curves of the fractions were obtained by plotting percentage inhibition (on y-axis) against log concentration (x-axis). The values of the fractions provided a mid-point value where parasite growth would be 50%. Linear regression analysis was applied to the linear portion of the sigmoidal curve obtained using the computer program, Microsoft Word Excel and IC50 values were derived for each fraction. One way analysis of variance (ANOVA) was done to test the statistical significance between the different values for various parts for two strains of P. falciparum.

3. Results The results of primary screening of antimalarial activity of nine crude fractions from the three parts namely flower, bud and root against P. falciparum, MRC 20 and MRC 76 have been summarized in Table 4a and b. The statistical analysis by one way ANOVA shows that the most significant variations are:

3.1. For MRC 20 1. The fractions of flower showed significant variation between means at conc. 0.25 mg/ml (PB 0.001), at conc. 0.5 mg/ml (PB 0.01) and 1 mg/ml (PB 0.05).

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Table 3 Preliminary phytochemical screening of ethanolic extracts of flower, bud and root of Calotropis procera a S.No.

Class of compounds

Plant part

Test performed

Flower

Bud

Root

1

Alkaloids

+

+

+

2

Carbohydrates

+

+

+

3 4

Glycosides Phenolic compounds/Tannins

+ +

+ +

+ +

5 6 7

Proteins and amino acids Flavonoids Saponins

+ + +

+ + +

+ + +

8

Sterols

+

+

+

9

Acidic compounds

+

+

+

10

Resins

+

+

+

11 12

Peroxides Polyuronoids

− −

− −

− −

a

Dragendorff’s test Mayer’s test Molish test Fehling’s test Keller Killiani test Ferric chloride test Lead acetate test Gelatin test Xanthoprotein test Ammonia test With water With Sodium bicarbonate Liebermann–Burchard test Salkowski reaction Hesse’s reaction With sodium bicarbonate With litmus paper With double distilled water With acetone and conc. Hydrochloric acid Potassium iodide test Haematoxylin test

+, indicates the presence, and −, indicates the absence.

2. The fractions of bud were significant at conc. 0.25 mg/ml (PB0.001), and other conc. Between range 1–0.0625 mg/ml (P B 0.05). 3. The fractions of root showed non-significant variation between means.

3.2. For MRC 76 1. The fractions of flower showed significant variation at conc. 0.25 mg/ml (PB 0.05). 2. The fractions of bud were significant at conc. 0.5 and 1 mg/ml (P B0.001), and for conc. range 2, 0.25 and 0.125 mg/ml (P B 0.05). 3. The fractions of root showed significant variation at conc. 0.25 mg/ml (P B0.001), at conc. 0.5 mg/ml (PB 0.01) and at conc. 0.0625 mg/ ml (PB0.05). IC50 for bud acetone was least of all (0.105 mg/ml) followed by root ethylacetate (0.111 mg/ ml) and IC50 was least for flower ethylacetate (0.213 mg/ml) followed by root acetone (2.528

mg/ml) against P. falciparum MRC 20 (Table 4a). For P. falciparum MRC 76, IC50 was least for root ethylacetate (0.303 mg/ml) followed by root acetone (0.308 mg/ml) and IC90 showed an effective value for bud ethylacetate (2.138 mg/ml) followed by root ethylacetate (2.168 mg/ml) as shown in Table 4b.

4. Discussion In one of the earlier studies, ethanolic extract of whole plant C. procera excluding root showed 35.57% inhibition at 100 mg/ml in vitro and nil under in vivo condition against P. berghei NK 65 (Mishra et al., 1991). In the present study a comparable data set for the fractions of flower, bud and root within a dose range of 62–125 mg/ml has been obtained and the percentage inhibition varied from 7.51 to 61.38% between the various fractions against MRC 20 and for MRC

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76, the percentage age inhibition varied from 3.437 to 41.08% between the various fractions as shown in Table 4a and b. Furthermore, the inhibition of around 35% in Mishra’s study coincides well with the bud acetone, root ethylacetate and root acetone against MRC 20 and with flower methanol and root acetone against MRC 76. At the lower dose range the root extracts of C. procera seems to be the most effective for both P. falciparum MRC 20 and MRC 76. The traditional uses of its parts have been known and well documented in the subjec-

tive relief from fever and malaria as well as objective clinically acceptable cure by traditional practitioners of alternate medicine. Since records are sometimes available with Gunijis (Local Vaidyas), they should be encouraged to follow documentation along medically accepted norms. The preparation of ashes, concoctions, powders, pastes, decoctions are individual preferences of these practitioners and their patients. The root of C. procera is ashed and most commonly used (Fig. 2).

Table 4 Effect of different fractions of Calotropis procera on CQ sensitive strain, MRC 20 and MRC 76of Plasmodium falciparum in vitro Plant part

Solvent for extraction

(a) MRC 20 Flower Ethylacetate Acetone Methanol F

Observed % inhibition of schizont maturation at various extract conc. (mg/ml)

Statistically derived % inhibitory conc.

2

1

0.5

75.5 79.3 64.0 2.2

73.1 61.5 57.9 6.6*

72.5 40.0 54.2 20.4**

0.25

0.125

0.0625

IC50 (mg/ml)

IC90 (mg/ml)

66.1 27.4 39.4 41.8***

21.3 18.2 31.4 3.7

10.4 8.1 22.5 4.6

0.3 0.6 0.5

2.2 4.2 13.2

Bud

Ethylacetate Acetone Methanol F

79.7 80.3 56.4 3.8

61.1 75.0 52.9 7.0*

57.2 70.4 43.7 5.0*

39.5 68.3 29.8 14.8***

17.6 54.5 21.1 7.9*

7.5 36.8 13.6 8.0*

0.5 0.1 1.0

3.2 3.3 19.9

Root

Ethylacetate Acetone Methanol F

76.2 83.8 81.4 1.5

69.4 76.0 66.7 1.7

63.6 65.6 49.9 3.2

61.7 50.8 44.3 4.5

61.8 36.5 43.2 4.9

34.3 28.9 22.9 0.3

0.1 0.2 0.3

7.0 2.5 4.5

70.8 68.0 66.4 0.4 78.3 70.0 36.3 7.8*

66.8 56.2 59.3 4.2 74.8 59.2 23.7 21.1**

59.3 53.9 52.3 2.0 67.5 47.5 10.8 16.2**

42.8 41.9 47.7 0.2 55.4 18.5 7.0 10.8*

14.9 19.8 41.1 6.3* 29.7 15.2 8.4 9.0*

8.1 6.2 15.4 2.6 4.5 12.6 3.4 3.9

0.5 0.6 0.4

3.6 5.6 9.7

77.4 77.2 68.0 2.2

72.8 68.5 61.7 4.2

70.2 61.5 57.3 52.7 31.2 17.0 19.7** 182.5***

32.0 37.9 11.5 3.5

5.1 19.5 5.6 8.5*

0.3 0.3 0.9

(b) MRC 76 Flower Ethylacetate Acetone Methanol F Bud Ethylacetate Acetone Methanol F Root

Ethylacetate Acetone Methanol F

* PB0.05; ** PB0.01; *** PB0.001.

0.3 0.7 18.9

2.1 7.0 1765.7

2.2 3.8 6.6

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Pharmacognosy and Phytochemistry, Faculty of Pharmacy, Jamia Hamdard University, New Delhi.

References

Fig. 2. A traditional practitioner of medicine (Guniji) with his certificate of appreciation in updated record keeping. Also seen are his collection of different traditional medicines from plants and their parts.

Preliminary phytochemical screening shows the presence of various compounds in specific parts of the plant. Extensive studies in the isolation of its compounds and their detailed chemical characterization have been undertaken for pharmacotoxicological studies. The possibilities of finding active compounds and correlating with specific dose effective antimalarial activity, from those parts of the plant, which are used separately or together could be further pursued. The study of such individual plants and their medicinal ecology would bring the knowledge from experiences of traditional practitioners of medicine into the mainstream of established medical practice and contribute to the greater understanding of both.

Acknowledgements We thank the staff of URMUL, an NGO in Rajasthan for assisting in field work and collection of plant samples. We deeply thank Dr C.R. Pillai, Research Officer, Malaria Research Centre for providing Plasmodium cultures and also the staff over there for training in maintenance of culture and parasite counting. We thank Dr C.R. Babu, Department of Botany, Delhi University, Delhi for identifying the plant specimen. The phytochemical screening was performed with the advice of Dr Mohhamed Ali, Department of

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