Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India

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Please cite this article as: Y.S. Biradar, S. Bodupally, H. Padh, Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India, Journal of Integrative Medicine (2019), doi: https://doi.org/10.1016/j.joim. 2019.11.001

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JIM-02-2019-OA-ER-0075 Original Research Article

Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India Yogesh Subhash Biradar1, Swathi Bodupally2, Harish Padh3 1. Department of Pharmacognosy and Phytochemistry, Bharat School of Pharmacy, Mangalpally, Ibrahimpatnam, Hyderabad 501510, Telangana, India 2. Department of Pharm D. Bharat School of Pharmacy, Mangalpally, Ibrahimpatnam, Hyderabad 501510, Telangana, India 3. Department of Biotechnology, B. V. Patel Pharmaceutical Education and Research Development Centre, Thaltej-Gandhinagar Highway, Thaltej, Ahmedabad 380054, Gujarat, India ABSTRACT Objective: The objective of this study was to evaluate the in vitro antiplasmodial properties against malaria parasite in 15 plants mentioned in Indian traditional medicine texts. Methods: In vitro antiplasmodial activity of methanolic extracts obtained from Indian traditional medicinal plants was evaluated on Plasmodium falciparum of FCK2 and INDO strains using schizont maturation inhibition assay and parasite lactate dehydrogenase inhibition assay. Results: Methanolic extracts of Adhatoda zeylanica, Embelia ribes, Piper nigrum and Plumbago zeylanica exhibited more than 50% inhibition in both the stains in schizont maturation inhibition asaay. Methanolic extracts of seven medicinal plants exhibited antiplasmodial activity at half maximal inhibitory concentration (IC50) ˂ 100 µg/mL, and methanolic extracts of five medicinal plants exhibited antiplasmodial activity at IC50 ˂ 50 µg/mL in P. falciparum lactate dehydrogenase (PfLDH) inhibition assay. A. zeylanica, E. ribes and P. nigrum exhibited promising antiplasmodial activity in PfLDH inhibition assay. A. zeylanica and E. ribes exhibited improved activity against resistant in comparison to sensitive strain. Conclusion: A. zeylanica and E. ribes were the most promising extracts from this study and deserve further investigation of their antiplasmodial properties. Please cite this article as: Biradar YS, Bodupally S, Padh H. Evaluation of antiplasmodial properties in 15 selected traditional medicinal plants from India. J Integr Med. 2019; Epub ahead of print. Received February 28, 2019; accepted May 12, 2019. Keywords: Medicinal plants; Plasmodium falciparum; Schizont maturation inhibition assay; Lactate dehydrogenase inhibition assay; Cytotoxicity Correspondence: Yogesh Subhash Biradar; E-mail address: [email protected] 1. Introduction Malaria is one of the most common parasitic diseases in the world, with 500 million new cases and 2 to 3 million deaths every year [1]. The number of clinical cases due to

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Plasmodium falciparum is thought to be 50% higher than that of the World Health Organization (WHO) estimates [1]. One reason for the increase in disease is due to progressive resistance to almost every drug used in malaria treatment, with loss of effectiveness of the newer antimalarial drugs occurring at an alarming rate [2]. Plants have always been an integral part of life in many indigenous communities, including those in India. Traditional medicines that have been employed for the treatment of malaria are a potential source for new antimalarial drugs [3,4]. Leaman et al. [5] showed that plants widely used as antimalarials by traditional healers are significantly more active in vitro against P. falciparum than plants that are not widely used, or not used at all, for the treatment of malaria. The molecular diversity and efficacy of antiparasitic plants, extracts, and herbal preparations have been intensively discussed in previous studies and reports [6–8]. Antimalarial properties of Cinchona bark have been known for more than 300 years, and recent development of artemisinin derivatives as effective antimalarial drugs showed that the majority of antimalarial drugs are derived from medicinal plants or are structures modeled on lead compounds from plants [9]. Resistance to artemisinin derivatives has also been reported [10,11]. To counter the drug resistance problem, combination therapy associating long and short acting compounds with different modes of action has been adopted. It offers efficient but expensive treatments. Hence, there is a clear need for a low-cost, efficient, curative (and possibly preventive) malaria treatment which does not induce resistance [10–13]. The WHO (2002) report states that although there is widespread use of traditional herbal remedies in the management of malaria, scientific understanding of these plants is largely unexplored [14]. In Indian systems of medicine, several plants have been mentioned as being potent in the treatment of malarial fever (Vishmajwara, in Ayurveda) [15]. Plants such as Triphaladiyogam, Trichatupanchadravya, Panchakolaghrutam, and Vardhamana pippali are some of the preparations used for the treatment of fever and malaria in Ayurveda. Ellagic acid and piperine are two of the major phytochemicals present in these preparations and these two compounds have been shown to have antimalarial activity [16,17]. This study is an effort to systematically collect information on antimalarial plants based on traditional treatments for Vishamajwara in Ayurveda [15] and to evaluate the efficacy of the extracts, fractions and compound(s) from selected plants for antimalarial activity. 2. Materials and methods 2.1. Information collection methodology of selected medicinal plants Information of selected plants for treatment of malaria and malaria-like symptoms such as fever was collected from traditional healers. In Ayurveda several plants have been mentioned for the treatment of Vishamajwara (interpreted to be malaria). For this study, a literature survey was conducted on the plants which appear to be widely used in Indian traditional medicine for malaria and its associated symptoms such as fever and joint pain, and plant species or families with previous interesting investigations as identified by traditional herbal practitioners and Ayurvedic physicians. For the present study, we selected fifteen plants (Table 1) based on traditional claims for Vishamajwara [15]. These plants were prepared using traditional methods including decoction, juice and dried powder.

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Table 1. Selected plants being used in Indian traditional medicine to treat malaria and malaria-like symptoms. Plant name; family; part used Adhatoda zeylanica; Acanthaceae; leaf

Common name; chemical compounds Vasa; vasicine and vasicinone

Method of preparation

References

[18] [19]

Clerodendron phlomidis; Lamiaceae; leaf

Angimantha; serratosides A & B and serratumin A

1. Leaf is mentioned with parasiticidal properties. 2. Decoction of Tinospora cordifolia, Solanum xanthocarpum and Adhatoda vasica is taken orally for chronic fever. 3. A. vasica (Adusoge, Malabar nut; local name: Karnataka) leaf paste mixed with black pepper powder (Piper nigrum) made into pills taken to cure fever. 4. Decoction of leaves and root of A. zeylanica (Kawldawi; local name: Mizoram) taken orally for curing malaria in Mizoram. C. phlomidis is one of the components of Ayurverdic preparations Amrtarista and Agastya Haritaki Rasayana used for Vishamajwara.

Embelia ribes; Myrsinaceae; fruit Enicostemma littorale; Gentianaceae; whole plant

Vidanga; embelin

Dried fruits of E. ribes used for fever.

[18]

Nagajihva; swertiamarin

[18] [23]

Erythrina indica; Fabaceae; stem bark

Paribhadra; erythramine and erythrinine Ashwatha; lupeol

1. It is reported to be effective against malaria. 2. Leaf powder used as antimalarial in Chittorgarh district of Rajasthan. 3. The plant was useful against fever and malaria in Churu district in the Thar Desert, India. It is used as febrifuge.

No specific method mentioned.

[25]

Pilkhan; lupeol

No specific method mentioned.

[25]

Jatamansi; jatamansic acid and Jatamansone

It is used as antipyretic.

[18]

Gojihva

It is used as antipyretic.

[18]

Katuka; picrosides I and II

It is used in jwaram, fever, and malarial fever.

[26]

Maricha; piperine

1. P. nigrum is occasionally employed as antiperiodic in obstinate fevers either alone or with other drugs, preferably, quinine. In intermittent fever, black pepper is recommended to be given with the juice of the leaves of Ocimum sanctum or Leucas linifolia. 2. Fruits used as antiperiodic in malarial fever. 3. P. nigrum (Jhaluk; local name: Assam) taken along with Allium cepa orally for curing malaria in Assam.

[26]

Ficus religiosa; Moraceae; stem bark Ficus virens; Moraceae; stem bark Nardostachys jatamansi; Valerianaceae; rhizome Onosma bracteatum; Boraginaceae; leaf Picrorrhiza kurroa; Scrophulariaceae; rhizome P. nigrum; Piperaceae; fruit

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[20]

[21]

[22]

[24] [18]

[18] [21]

Plumbago zeylanica; Plumbaginaceae; root S. xanthocarpum; Solanaceae; whole plant

T. cordifolia; Menispermaceae; stem

Ventilago calyculata; Rhamnaceae; leaf

Chitraka; plumbagin

Kantakari; solasodine

Guduchi; tinosporoside

Raidhani; quercetin and lupeol

4. Decoction of Piper longum crushed fruit mixed with jaggery and ginger powder is taken for malaria. Root used in the treatment of intermittent fevers.

[27]

1. This plant, singly and in combination with T. cordifolia, is used in fever. 2. Decoction of S. xanthocarpum used for chronic fever. 3. Decoction of T. cordifolia, S. xanthocarpum and A. vasica used for chronic fever. 4. Decoction of S. xanthocarpum, Swertia chirata (chiretta), Zingiber officinalis (ginger) given as frbrifuge. 1. Decoction of T. cordifolia, S. xanthocarpum and A. vasica used for chronic fever. 2. Decoction of stem bark of Wrightia tinctoria, root bark of Echinops echinatus, seeds of Nigella hispanica and stem bark of T. Cordifolia used to treat fever. 3. Decoction of T. cordifolia used for malarial fever. 4. Decoction of 5 g of T. cordifolia stem, with 8 to 10 petioles of Azadirachta indica and 8 to 10 tuberous roots of Cyperus rotundus, to treat intermittent fever and chronic fever. 5. Decoction of T. cordifolia and S. xanthocarpum used to treat fever. 6. Decoction of T. cordifolia (Gibe; local name: Rajasthan) used to cure fever in tribal areas of Rajasthan. 7. Root and stem decoction of T. cordifolia is given to cure fever by tribals of Uttar Pradesh. 8. In Assam the juice of T. cordifolia (Gulanch; local name: Assam) mixed with honey and taken orally for curing malaria. It is used in malarial fever.

[26]

[26]

[19] [19] [18]

[19] [28]

[29] [30]

[31] [32]

[33] [21]

[18]

2.2. Identification and collection of plant materials Plants were collected from various localities of India. Their authentication was confirmed by the taxonomist of our department and voucher specimens were deposited at the Department of Pharmacognosy and Phytochemistry, B.V. Patel Pharmaceutical Education & Research Development (PERD) Centre, Ahmedabad, India. All the plant materials were stored in airtight containers at room temperature until use. 2.3. Extraction of plant material Plant parts selected for testing antimalarial activity were dried at room temperature and powdered. Approximately 10 g of each powdered sample was extracted with 25 mL methanol three times under reflux for 30 min at a maximum temperature of 50 ºC. The extract was filtered; the filtrates were pooled and concentrated to dryness by removing the solvent under reduced pressure at 50 ºC. Extracts were stored in airtight glass bottles at 4 C in a refrigerator. For testing, each extract was dissolved in dimethyl sulfoxide (DMSO) (Table 2).

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Table 2. Extractive values of selected medicinal plants. Name of the plant

Amount of plant material taken (g)

Amount of extract obtained (g)

Adhatoda zeylanica Clerodendron phlomidis Embelia ribes Enicostemma littorale Erythrina indica Ficus religiosa Ficus virens Nardostachys jatamansi Onosma bracteatum Picrorrhiza kurroa Piper nigrum Plumbago zeylanica Solanum xanthocarpum Tinospora cordifolia Ventilago calyculata

10.1 10.4 10.2 10.1 10.3 10.3 10.2 9.9 10.2 10.2 10.3 10.1 10.5 10.2 10.1

0.39 0.52 0.41 0.17 0.22 0.39 0.37 0.14 0.17 0.18 0.22 0.22 0.21 0.15 0.14

Extract (%) 3.8 5.0 4.0 1.7 2.1 3.8 3.6 1.4 1.7 1.8 2.1 2.2 2.0 1.5 1.4

2.4. In vitro cultivation of P. falciparum Chloroquine (CQ)-sensitive strain FCK2 and CQ-resistant strain INDO of P. falciparum were obtained from the Jawaharlal Nehru Centre for Scientific and Advanced research, Bangalore. These cultures were used for in vitro cultivation and maintenance of P. falciparum culture, and to evaluate antiplasmodial activity of the selected plant extracts. The culture was maintained at Biotechnology Department of B. V. Patel PERD Centre. Method demonstrated by Trager and Jensen was implemented for maintenance of P. falciparum culture [34]. 2.5. Continuous culture of P. falciparum 2.5.1. Initiation of culture A total of 50 % suspension of infected cells with complete media (with 15% serum) was prepared. Appropriate number of uninfected cells were added to get an initial parasitaemia of 0.5% to 1.0% and diluted with complete media to get 5% cell suspension (5% haematocrit). Culture was kept in CO2 incubator at 37 C. 2.5.2. Monitoring culture growth After every 24 h, the media was removed using a sterile Pasteur pipette without disturbing the cells that settled down. Fresh complete media (with 10% serum) was added, mixed properly, and placed back in the incubator. Then the cells were mixed without frothing; a drop of blood was placed on the slide and a thin film was made. The thin film was stained with Giemsa stain and examined for parasitaemia. It was then compared with initial parasitaemia. 2.5.2.1. Smear preparation Approximately 3.0 µL of culture was placed on a slide toward the left side. Another slide was place on top to smear the cells across the slide, to obtain a thin film, and the film was dried at room temperature. The slide was fixed by immersion in methanol for 30 s followed by drying, then stained with Giemsa stain. The slide was then dried to remove the excess stain, and observed under the microscope. 2.5.2.2. Estimation of percentage of erythrocytes infected with P. falciparum An area of stained thin blood film where the erythrocytes were evenly distributed was observed using 100 × objective (under oil immersion). Approximately 100 erythrocytes in this area were counted. Without moving the slide, the number of infected erythrocytes amongst the 100 erythrocytes was also counted. The slide was moved randomly to adjacent fields and counting was continued as mentioned above. An equivalent of 1000 erythrocytes was counted. The counting was repeated twice for a total examination of three different parts

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of the slide, i.e., 3 areas, 1000 cells. The mean number of infected red blood cells (RBCs) per 1000 RBCs was taken by dividing the total infected RBCs by 3. Percent infected erythrocytes (% parasitaemia) = number of infected erythrocytes/1000 erythrocytes/10. 2.5.3. Subculturing (passaging) Old media were removed; freshly washed RBCs and complete media (with 10% serum) were added. For example, to obtain 20 µL of infected RBCs with around 5% parasitaemia, 160 µL freshly washed RBCs (i.e., 80 µL RBCs) suspension (concentration of prasitaemia = 1%) and 2000 µL of complete media were added. Culture was dispensed to more vials. 2.6. In vitro antiplasmodial assays on P. falciparum in human RBC culture The selected plants were evaluated for antimalarial activity in schizont maturation inhibition assay and P. falciparum lactate dehydrogenase (PfLDH) inhibition assay. 2.6.1. Schizont maturation inhibition assay 2.6.1.1. Experimental protocol The test procedure for antimalarial screening was based on a previous study [35]. The cultured FCK2 and INDO strains were synchronized using sorbitol and parasitaemia was adjusted to 1%–1.5% by diluting it with fresh human erythrocytes. The cells were diluted with complete media to make 8% haematocrit. The extracts were dissolved in DMSO to obtain 10 and 25 µg/mL concentrations. All tests were done in triplicate. After 24 h of incubation, thin films of the contents of each well were prepared, stained with Giemsa stain, fixed with methanol, and oil immersion fields were examined under the light microscope. 2.6.1.2. Evaluation of activity Parasite count for each blood film was made. Each film was observed at three different visual fields. The number of schizonts with three or more nuclei per 200 parasites was noted while control and test wells were compared for the determination of the inhibition percentage. All doses were studied in triplicate cultures. The inhibition of parasite growth in the treated groups was calculated as follows: Inhibition (%) = (1 −

Number of schizonts in test wells ) × 100 Number of schizonts in control wells

2.6.2. Lactate dehydrogenase inhibition assay 2.6.2.1. Experimental protocol To test antiplasmodial activity, the method based on measurement of the parasite lactate dehydrogenase (pLDH) activity of Makler and Hinrichs [36] was used. Continuous cultures of P. falciparum FCK2 and INDO strains were used for the pLDH assay. For each experiment, samples of the stock parasite cultures were diluted in culture medium containing sufficient non-infected type O+ human erythrocytes to yield a final haematocrit of 2% and parasitemia of 0.5%–1.0%. This culture was used for addition to the microtiter plates. The plant extracts were dissolved in DMSO to produce a stock solution of 1 mg/mL. These stock solutions were subsequently diluted with culture medium containing 10% human serum before being transferred in triplicate of 10 µL each at 5 concentrations of two-fold dilutions into 96-well microtiter plates. Parasitised RBC suspensions (0.5%–1% parasitaemia) of 100 µL were then added to each well. The positive control wells contained parasitised RBCs, the negative control wells contained only non-parasitised RBCs, and neither held any plant extracts and compounds. The plates were incubated in CO2 incubator. After the incubation period, the plates were frozen at –20 °C overnight followed by thawing at room temperature to haemolyse the RBCs. The simultaneous control parasite cultures devoid of plant extracts or drug were referred to as having 100% pLDH activity. The plates were then allowed to reach room temperature, and 20 µL of the supernatant blood suspension was dispensed into a new microtitre plate containing 100 µL Malstat reagent, and 25 µL nitroblue tetrazolium and phenazine ethosulfate mixture (20:1) was added. Optical density of the complex was read at 650 nm using a microplate reader (Model 550, BIO-RAD, California, United States).

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2.6.2.2. Evaluation of the activity The inhibition of each extract was calculated as compared to the untreated control. ( Control − Test) Inhibtion (%) = × 100 Control 2.6.3. Cytotoxicity assay The cytotoxic effects of plant extracts were evaluated by functional assays using HeLa cells cultured in RPMI containing 10% fetal bovine serum, 0.21% sodium bicarbonate (Sigma, St. Louis, United States) and 50 µg/mL gentamicin. 104 cells/200 µL for each well were seeded in 96-well flat bottom tissue culture plates in complete medium. Plant extract solutions were added after 24 h of seeding and the cells were incubated for 48 h in a humidified atmosphere at 37 ̊C and 5% CO2. Twenty microliters of a stock solution of 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (5 mg/mL in 1× phosphate-buffered saline) was added to each well, gently mixed, and the cells were incubated for another 4 h. After spinning the plate at 1500 × g for 5 min, supernatant was removed and 100 μL of DMSO (stop agent) was added. Formation of formazan was read on a microtiter plate reader (Versa max tunable multi-well plate reader) at 570 nm. Fifty percent cytotoxic concentration (TC50) of drug was determined by the analysis of dose-response curves. 2.7. Statistical analysis Results were presented as inhibition percentage in the form of mean ± standard deviation which was calculated from three replicates. The half maximal inhibitory concentration (IC50) was calculated using GraphPad Prism statistical software (version 5, San Diego, United States). 3. Results Schizont maturation inhibition assay was used for the assessment of inhibition of growth of P. falciparum by selected plant extracts (Table 3). Methanolic extract of Adhatoda zeylanica leaf exhibited highest schizont inhibition at 68.75% ± 6.25% and 70.20% ± 7.85% in FCK2 and INDO strains at 25 g/mL respectively. Embelia ribes fruit exhibited 54.16% ± 3.60% and 70.35% ± 5.25% schizont inhibition in FCK2 and INDO strains at 25 g/mL respectively. Table 3. Effects of methanolic extracts of selected medicinal plants on schizont maturation of Plasmodium falciparum. Name of the plant Inhibition (%) Dose (g/mL) FCK2 INDO Adhatoda zeylanica 10 45.83 ± 9.54 55.22 ± 8.21 25 68.75 ± 6.25 70.20 ± 7.85 Clerodendron phlomidis 10 8.33 ± 3.60 15.20 ± 4.52 25 16.66 ± 3.60 22.31 ± 5.54 Embelia ribes 10 41.66 ± 3.60 60.21 ± 6.21 25 54.16 ± 3.60 70.35 ± 5.25 Enicostemma littorale 10 18.75 ± 6.25 14.69 ± 4.58 25 27.08 ± 3.60 15.21 ± 5.21 Erythrina indica 10 27.08 ± 3.60 22.27 ± 4.87 25 43.75 ± 6.25 30.21 ± 5.78 Ficus religiosa 10 6.25 ± 1.27 5.28 ± 1.02 25 14.58 ± 3.60 11.28 ± 2.28 Ficus virens 10 18.75 ± 6.25 13.28 ± 5.24 25 35.41 ± 3.60 20.28 ± 4.81

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Nardostachys jatamansi

10 8.33 ± 3.60 25 16.66 ± 7.21 Onosma bracteatum 10 12.50 ± 1.12 25 22.91 ± 3.60 Picrorrhiza kurroa 10 14.58 ± 3.60 25 27.08 ± 3.60 Piper nigrum 10 39.58 ± 3.60 25 56.25 ± 6.25 Plumbago zeylanica 10 39.58 ± 3.60 25 52.08 ± 3.60 Solanum xanthocarpum 10 33.16 ± 7.07 25 45.83 ± 3.60 Tinospora cordifolia 10 12.50 ± 6.25 25 27.08 ± 3.60 Ventilago calyculata 10 31.25 ± 6.25 25 45.66 ± 3.75 Data are expressed as mean ± standard deviation (n = 3).

10.21 ± 1.25 17.28 ± 4.37 14.80 ± 1.31 15.26 ± 3.32 20.26 ± 2.12 32.26 ± 2.26 41.26 ± 4.22 58.36 ± 5.64 41.65 ± 5.84 51.26 ± 4.80 39.25 ± 6.52 49.26 ± 6.29 11.28 ± 2.58 25.68 ± 3.86 28.68 ± 5.69 39.56 ± 3.98

In order to find a prospective antiplasmodial plant, methanolic extracts of 15 plants were screened in vitro against lactate dehydrogenase (LDH) of the FCK2 and INDO strains of P. falciparum. According to WHO guidelines and previous reports [37,38], antiplasmodial activity of plant extract is classified as follows: highly active at IC50 < 5 μg/mL, promising at 5–15 μg/mL, low at 15–50 μg/mL and inactive at > 50 μg/mL. In the present study a dose-dependent inhibition of the parasites as reflected in the inhibition of PfLDH was observed in vitro (Table 4). Of the plants screened A. zeylanica leaf (IC50 at 5.80 and 4.20 µg/mL) and E. ribes fruit (IC50 at 13.10 and 9.65 µg/mL) showed promising antimalarial activity in sensitive and resistant P. falciparum strains in inhibiting PfLDH with IC50 value below 15 µg/mL. Amongst the other plants tested, Piper nigrum, Plumbago zeylanica, Solanum xanthocarpum and Ventilago calyculata also exhibited good antimalarial activity with IC50 values of 16.25 and 20.26, 24.05 and 30.87, 48.48 and 47.25, 50.13 and more than 100 µg/mL in sensitive and resistant P. falciparum strains respectively. Table 4. Effects of methanolic extract of the selected medicinal plants on Plasmodium falciparum lactate dehydrogenase. IC50 (µg/mL) Name of the plant FCK2 INDO Adhatoda zeylanica 5.80 4.20 Clerodendron phlomidis  100  100 Embelia ribes 13.10 9.65 Enicostemma littorale 100.19  100 Erythrina indica  100  100 Ficus religiosa  100  100 Ficus virens 57.66 90.54 Nardostachys jatamansi  1000  1000 Onosma bracteatum  100  100 Picrorrhiza kurroa  100  100 Piper nigrum 16.25 20.26 Plumbago zeylanica 24.05 30.87

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Solanum xanthocarpum Tinospora cordifolia Ventilago calyculata Quinine dihydrochloride IC50: half maximal inhibitory concentration.

48.48  500 50.13 0.01

47.25  500  100 0.01

Since A. zeylanica, E. ribes, P. nigrum and P. zeylanica exibited good antiplasmodial properties, these extracts were evaluted for cytotoxicity potential and selectivity index (SI). A. zeylanica, E. ribes, P. nigrum and P. zeylanica exhibited low toxicity against human cell lines and high SI index (Table 5). Table 5. Cytotoxicity and selectivity index of methanolic extracts of medicinal plants Name of the plant FCK2 INDO Cytotoxicity SI of FCK2 SI of INDO Adhatoda zeylanica 5.80 4.20 54.20 9.34 12.90 Embelia ribes 13.10 9.65 134.66 10.28 13.95 Piper nigrum 16.25 20.26 144.3 8.88 7.12 Plumbago zeylanica 24.05 30.87 326.59 13.58 10.57 SI: selectivity index. 4. Discussion Schizont maturation inhibition assay is low-cost and relatively simple to perform [35]. The percentage of mature schizonts was always above 20% in the control wells. The control culture exhibited normal growth but the treated cultures had inhibited growth. Parasites of all stages were observed in the control culture wells. Microscopic observation of uninfected erythrocytes incubated with the methanol extracts of these plants showed no morphological differences after 24 h of incubation. Even at higher concentrations, erythrocytes showed no deformation. The in vitro bioassays play a very important role in natural product screening. In any screening program, the concentration of the sample to be tested is very important. Many of the drugs have a very narrow therapeutic window. In such cases we may lose potentially useful treatment candidates if an ineffective dose is selected for the test. The LDH activity of P. falciparum was determined in relation to density. The measurement of LDH activity was based on the biochemical reaction leading to formation of pyruvate from L-lactate in the presence of malarial LDH and acetylpyridine-adenine dinucleotide (APAD) coenzymes. The reaction results in the formation of reduced APAD, which in turn reduces nitroblue tetrazolium, forming a blue formazan product that can be detected both visually and by spectrophotometry at 650 nm [36]. The enzymatic activity detected in infected erythrocytes was at least twice that of uninfected erythrocytes. Even though A. zeylanica, E. littorale, P. nigrum, S. xanthocarpum and T. cordifolia are mentioned in many references (Table 1) for fever and malaria from different parts of India, only A. zeylanica and P. nigrum exhibited promising antimalarial activity in this study. A literature survey has shown that out of the 15 plants screened, only A. zeylanica had previously been investigated for antiplasmodial activity [39]. In the present study A. zeylanica showed higher antiplasmodial activity than that of earlier results, which might be due to the different extraction method adopted. A. zeylanica contains quinazoline alkaloids [40]. According to WHO, a synthetic chemical derived from febrifugine is one of the two most powerful antimalarials out of 4700 screened compounds. Quinazolinone ring is responsible for the chemical activity of febrifugine [41], which supports our results.

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In the present study, another plant that showed promising antimalarial activity was E. ribes. To the best of our knowledge this is the first report of antiplasmodial activity described for this plant. Major chemical compounds reported in E. ribes fruit are quinones [42]. Antimalarial activity of a quinone plumbagin was reported by Likhitwitayawuid et al. [43]. Quinones present in E. ribes fruit may be responsible for its antimalarial activity. E. littorale and T. cordifolia were found in most of the documented references [18,19,21,23,24,28–31,33] but in our screen these two plants did not show good antimalarial activity in vitro. A number of the plants selected did not display in vitro antiplasmodial activity, despite strong associations with their use for malaria in traditional medicine. A possible explanation for this could be that these plants might act as antipyretics or immune stimulants to relieve the symptoms of malaria, rather than having direct activity on the malaria parasite. Such plants need to be further studied for their possible clinical and immune system benefits in the infected host. In inhibition of PfLDH, A. zeylanica leaf, E. ribes fruit, and P. nigrum fruit were found to be the most active extracts. Piperaceae family is widely used in traditional medicine. Antimalarial activity has already been reported from many Piper spp., e.g. P. capense, P. umbellatum, P. pyrifolium, P. cumanense and P. hispidum [44,45]. In the present study, we report antimalarial activity of P. nigrum for the first time. Simonsen et al [39] reported antiplasmodial activity of P. zeylanica leaf. This is the first report of antiplasmodial activity of P. zeylanica root. As mentioned earlier, plumbagin isolated from Nepenthes thorelii showed good antimalarial activity [43]. Plumbagin is reported to be present in P. zeylanica root up to 4% and may be responsible for its antimalarial activity [46]. Even though S. xanthocarpum whole plant and V. calyculata leaf showed low antimalarial activity, S. xanthocarpum can be evaluated in combination with other plants such as T. cordifolia and A. vasica for antiplasmodial activity, since they are given in combination in traditional preparations (Table 1). The SI can be helpful in the evaluation of cytotoxic effect of plant extracts against human cell lines in comparison to the toxicity against the malaria parasite. It facilitates evaluation of selectivity of plant extracts for the malaria parasite. The SI values were calculated in terms of ratio between the cell line cytotoxic TC50 values and P. falciparum FCK2 and INDO IC50 values. The results indicate a possibility for discovering useful active natural products for antimalarial activity from A. zeylanica leaf and E. ribes fruit. 5. Conclusion Indian traditional medicine has a long history of using plants for the treatment of malaria. However, the traditional treatment methods should be supported by scientific validation in laboratory. This study has highlighted two promising plants, A. zeylanica and E. ribes, for further investigations on their antimalaria properties and elucidating their active constituents, with a focus on optimizing their utilization. These are particularly important objectives because malaria is a disease with wide-reaching negative impact that currently lacks effective chemotherapeutic agents. The present study provided laboratory evidence for the exploration of indigenous Indian medicinal plants as a source of antiplasmodial agents. Acknowledgements The authors thank the Indian Council of Medical Research, New Delhi, India for financial support.

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