Cytotoxicity screening of selected Indian medicinal plants using brine-shrimp lethality bioassay.
Indian medicinal plants have a great history of their utility as remedy for treatment of variety of ailments. The impact of medicinal plants and their isolated pure compounds is in variety of therapeutic areas but the major contribution is in the field of anti-infective and anticancer drug discovery (Newman, D.J. and Cragg, G.M., 2007). Although the ethno-medicine is widespread in India, most of the plants have not been investigated for toxicity. Our aim in the present work was to screen medicinal plants with different known therapeutic activities for their cytotoxic effect on Artemia salina and correlate toxicity results with their known ethnopharmacological activities.
There is great support provided by bench-top bioassays in discovery of bioactive compounds from plants. Several published whole-animal bioassays for assessment of chemical toxicity are reported in literature. Bacteria are commonly used for quantitative tests, and there are some procedures for qualitative tests on Blephisma (a one celled pond organism) and Daphnia (water fleas). Brine shrimp lethality assay have been used as a bench-top bioassay for the discovery and purification of bioactive natural products, and they are an excellent choice for preliminary assessment of toxicity of herbal drugs/ consumer products. Brine shrimp, Artemia species, also known as sea monkeys, are marine invertebrates about 1 mm in size. Freeze-dried cysts are readily available at aquarium stores. The cysts last for several years and can be hatched without special equipment. The assay involve killing brine shrimp thus there is a reasonable controversy over use of animals for such purposes, and some people have ethical or religious objections to killing even lower organisms (Lieberman, M., 1999). Brine shrimp (Artemia salina) assay has been suggested as a valid method to evaluate the cytotoxic activity of plant extracts (Alali, F.Q., et al., 1999, Lemos, T.L.G., et al., 2006, Lincoln, R.D., et al., 1996, Meyer, B.N., et al., 1982, Padmaja, R., et al., 2002, Pimenta, L.P.S., et al., 2003, Solis, P.N., et al., 1993, Wanyoike, G.N., et al., 2004). Some published data have suggested a good correlation between the toxic activity in the brine shrimp assay and the cytotoxicity against some tumor cell lines (Anderson, J.E., et al., 1991) and hepatotoxic activity (Kiviranta, J., et al., 1991). A plant Phyllanthus engleri with a brine-shrimp [LC.sub.50] of 0.47 [micro]g/mL (Moshi, M.J., et al., 2004) recently yielded Englerin A, a selective anticancer compound against kidney cancer cells (Ratnayake, R., et al., 2009) which provides further corroborative evidence on the potential of the brine shrimp test to predict the presence of anticancer compounds in plant extracts. The brine shrimp assay is, therefore, usually used as a low-cost and easily achievable cytotoxicity test replacing cell lines assays (Piccardi, R., et al., 2000). Brine shrimp tests are normally conducted to draw inferences on the safety of the plant extracts and further to depict trends of their biological activities.
Fourteen Indian medicinal plants were selected randomly, mainly based on their availability and apart from their traditional uses. The ethnobotanical information of selected fourteen Indian medicinal plants is provided in Table 1. In the present work, ethanolic extracts of these medicinal plants were tested for their cytotoxic effect on Artemia salina nauplii.
Materials and methods
Plant materials were collected and authenticated by Green Pharmacy Kothrud, Pune where the voucher specimens are preserved. Plant materials include Acorus calamus (Araceae) stem, Asparagus racemosus (Asparagaceae) root, Aegle marmelos (Rutaceae) leaves and fruits, Cassia fistula (Fabaceae) fruits, Gymnema sylvestre (Asclepiadaceae) leaves, Holarrhena antidysenterica (Apocynaceae) bark, Mimusops elengi (Sapotaceae) bark, Ocimum sanctum (Lamiaceae) leaves, Piper longum (Piperaceae) leaves, Sapindus trifoliatus (Sapindaceae) fruits, Terminalia arjuna (Combretaceae) bark, Terminalia bellerica (Combretaceae) fruits, Terminalia chebula (Combretaceae) fruits and Withania somnifera (Solanaceae) roots. All chemicals and solvents were procured from the local supplier. Soxhlet apparatus was used for extraction. Rotary evaporator (Euiptronics; Model: Evator) was used for evaporation of solvent from extracts. All materials for cytotoxicity bioassay (hatching tray, lamp, magnifying glass, Pasteur pipette) are procured from local supplier. Brine shrimp eggs were procured from Dr. Inder Pal Singh, NIPER, Mohali. Artificial sea water (ASW) was prepared using the reported protocol (Kester, D.R., et al., 1967).
Preparation of plant extracts
Ethanolic extracts of all plant materials were prepared using Soxhlet extraction procedure. Briefly, extraction procedure involved packing a known amount of dried powdered plant material (25-50 g) in the Soxhlet extraction assembly using a filter paper thimble followed by addition of 70% ethanol (200-300 mL) to make one complete siphon. Additional solvent (200 mL) was then poured into the thimble. Extraction assembly was heated at 70-80 [degrees]C using heating mantle for 48-72 h till the completion of extraction. Completion of extraction was confirmed by visualizing a TLC spot of liquid coming out of siphon tube under UV. Collected extract was then concentrated under reduced pressure (below 40 [degrees] C). Ethanolic extracts of all plants were weighed (around 20-30 mg) and dissolved in appropriate amount of distilled water to make the final concentration of 10 mg/ml. This stock solution was used for making further dilutions during cytotoxicity screening.
Brine shrimp toxicity screening
The method of Meyer et al. (Meyer, B.N., et al., 1982) was used in this assay. A 24-h [LC.sub.50] bioassay was performed in a multi-well test plate using nauplii of the brine shrimp Artemia salina. The test was conducted according to the standard operating procedure (25 [+ or -] 1[degrees]C, 35% salinity) with three replicates for each treatment and ten nauplii per replicate. Artificial sea water was prepared using the composition reported by Kester, D.R. et al. (Kester, D.R., et al., 1967). All chemicals (sodium chloride, 23.9 g; sodium sulfate, 4 g; potassium chloride, 0.67 g; sodium bicarbonate, 0.20 g; potassium bromide, 0.98 g; boric acid, 0.026 g and sodium fluoride, 0.003 g) were weighed and dissolved in 1 L of distilled water to make ASW. Brine shrimp eggs (Artemia salina) were incubated in ASW in a specially designed two-compartment plastic tray under a 60 W lamp, providing direct light and warmth (24-26[degrees]C). After an incubation time of 24 h, the hatched nauplii were separated from the shells and remaining cysts using a Pasteur pipette and transferred to fresh ASW. This was facilitated by attracting the shrimps from one compartment to another compartment of the tray with a light source. The bioactivity of extracts was determined by the brine shrimp lethality test. The protocol used in this lethality test was as follows. To the six-well plate containing 3 mL of ASW, 10-15 nauplii were added using Pasteur pipette. To the wells containing nauplii, aliquots from stock solution of extracts was added to make three different concentrations viz. 100, 500 and 1000 [micro]g/mL. Ten nauplii added in 3 mL of ASW was used as a negative control while potassium dichromate was used as positive control during the experiment. All plates were incubated for 24 h at room temperature. Number of dead nauplii were counted after 4 and 24 h with the help of magnifying glass. The percentage mortality of brine shrimp nauplii was determined from the number of dead nauplii. [LC.sub.50] was calculated for selected extracts by treating nauplii with five different concentrations. All values are mentioned as mean [+ or -] SEM.
Results and discussion
All ethanolic extracts were screened for cytotoxicity using brine-shrimp bench-top bioassay. The assay was based on the ability of extracts/ pure compounds to kill laboratory cultured Artemia nauplii brine shrimp. Assay was done according to protocol reported by Meyer et al. (Meyer, B.N., et al., 1982) and Lincoln et al. (Lincoln, R.D., et al., 1996). Results of brine shrimp lethality assay are depicted in Table 2. Majority of the extracts tested showed good brine shrimp larvicidal activity according to Meyer et al. (Meyer, B.N., et al., 1982), who classified crude extracts and pure substances into toxic ([LC.sub.50] value < 1000 [micro]g/ml) and non-toxic ([LC.sub.50] value > 1000 [micro]g/ml).
All extracts were screened at three different concentrations viz. 100, 500 and 1000 [micro]g/mL and observed for their toxic effect on A. salina after 4 and 24 h. Potassium dichromate was used as a reference standard (Padmaja, R., et al., 2002). Most of the extracts were devoid of significant toxicity after 4 h of exposure, the most potent being Piper longum with 50% mortality at 100 [micro]g/mL. Results observed after 24 h of exposure showed promising toxicity profile. Complete (100%) mortality of brine shrimp nauplii was observed for most of the plant extracts at 1000 [micro]g/mL concentration after 24 h except A. racemosus, C. fistula, O. sanctum and W. somnifera. Ethanolic extracts of P. longum, H. antidysenterica and T. chebula showed most potent toxicity with 100, 75 and 85% mortality of A. salina nauplii at 100 [micro]g/mL. Piper longum extract and reference compound potassium dichromate showed 100% lethality at all three concentrations tested. Four extracts viz. A. racemosus, C. fistula, O. sanctum and W. somnifera were virtually non-toxic on the shrimps as they exhibited < 90% toxicity even at 1000 [micro]g/mL.
Similar to T. chebula, other two species of genus Terminalia viz. T. arjuna and T. bellerica showed promising toxicity profile with 47 and 59% mortality of A. salina at 100 [micro]g/mL. Cytotoxicity results depict the general trend among plants of genus Terminalia, which are known to contain cytotoxic compounds such as hydrolysable tannins. Saleem et al. (Saleem, A., et al., 2002) reported that a growth of cancer cell was inhibited by crude extract and the phenolics (gallic acid, ethyl gallate, luteolin and tannic acid) of T. chebula, which supports the potent brine shrimp toxicity exhibited by extracts of Terminalia species.
[LC.sub.50] values of three plant extracts viz. P. longum, H. antidysenterica and T. chebula were determined and were found to be 10 [+ or -] 11, 104 [+ or -] 28 and 107 [+ or -] 32 [micro]g/mL respectively (Table 3). Ethanolic extract of P. longum ([LC.sub.50] 10 [micro]g/mL) showed better toxicity than the reference standard (potassium dichromate [LC.sub.50] 32 [micro]g/mL) used in the bioassay. As well as this extract has higher toxicity compared with other reference compounds viz. emetine hydrochloride (Wanyoike, G.N., et al., 2004) and cyclophosphamide (Moshi, M.J., et al., 2004) reported in the literature. Ethanolic extract of P. longum fruit was earlier reported to show potent brine shrimp toxicity with [LC.sub.50] value of 6.9 [micro]g/mL. Piperine, a main chemical constituent of P. longum also exhibit potent toxicity with [LC.sub.50] of 2.4 [micro]g/mL which supports the potent lethality exhibited by crude extract of P. longum fruits and leaves (Padmaja, R., et al., 2002). Further, it is noteworthy to mention that ethanolic extracts of both fruits as well as leaves of P. longum have equivalent toxicity for Artemia salina shrimps. The potent cytotoxicity exhibited by P. longum was in correlation with its earlier reported antitumor activity against variety of cancer cell lines viz. Dalton's lymphoma ascites (DLA), Ehrlich ascites carcinoma (EAC) and L929 cells (Sunila, E.S. and Kuttan, G., 2004).
Ethanolic extract of H. antidysenterica also exhibited potent cytotoxicity for brine shrimps but there was no earlier report on anticancer activity of plant extract or its isolated compounds. Therefore, anticancer / antitumor effect of crude extract of this plant needs to be evaluated in order to discover potential anticancer agents, whereas this plant is widely used in treatment amoebiasias and other gastrointestinal infections. The exact link between brine shrimp toxicity and anti-amoebic activity of H. antidysenterica cannot be established until its anticancer profile is obtained.
Fourteen Indian medicinal plants selected randomly, mainly based on their availability and apart from their traditional uses were tested for cytotoxic activity using brine shrimp lethality test. Most of the extracts showed significant cytotoxic activity amongst which Piper longum, Holarrhena antidysenterica and Terminalia chebula possessed potent cytotoxic activity. These results warrant follow up through bioassay directed isolation of the active principles.
Authors are thankful to Prof. M. N. Navale, founder president, STES and Dr. K. S. Jain, Principal, Sinhgad College of Pharmacy for providing research facilities. SBB is thankful to Dr. Inder Pal Singh for providing brine shrimp eggs.
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Corresponding Author: Dr. Sandip B. Bharate, Associate Professor, Department of Pharmaceutical Chemistry, Sinhgad Technical Education Society's Sinhgad College of Pharmacy, Suvery no. 44/1, Off Sinhgad Road, Vadgaon (Budruk), Pune- 411041, Maharashtra, India. Tel. +91-9011995957; Fax. +91-20-24354720 E-mail: firstname.lastname@example.org
(1) Chaitali H. Ved, (1) Nikhil S. More, (2) Sonali S. Bharate and (1) Sandip B. Bharate
(1) Sinhgad Technical Education Society's Sinhgad College of Pharmacy, Suvery no. 44/1, off Sinhgad Road, Vadgaon (Budruk), Pune- 411041, Maharashtra, India.
(2) P.E. Society's Modern College of Pharmacy (For Ladies), Borhadewadi, At/Post- Moshi, Tal-Haweli, Dist- Pune, Pin--412105, Maharashtra, India.
Chaitali H. Ved, Nikhil S. More, Sonali S. Bharate and Sandip B. Bharate; Cytotoxicity Screening of Selected Indian Medicinal Plants using Brine-Shrimp Lethality Bioassay
Table 1: Ethnobotanical information about selected Indian Medicinal Plants Botanical name Family English name Acorus calamus Araceae Sweet flag Asparagus racemosus Asparagaceae Asparagus Aegle marmelos Rutaceae Stone apple Cassia fistula Fabaceae Golden Shower Gymnema sylvestre Asclepiadaceae Gymnema Holarrhena Apocynaceae Connessi bark antidysenterica Mimusops elengi Sapotaceae Spanish cherry Ocimum sanctum Lamiaceae Basil Piper longum Piperaceae Long pepper Sapindus trifoliatus Sapindaceae Soapnut Terminalia arjuna Combretaceae White Marudah Terminalia bellerica Combretaceae Ink nut, chebulic myrobalan Terminalia chebula Combretaceae Myrobalan Withania somnifera Solanaceae Winter cherry Botanical name Vernacular name Part used Acorus calamus Vekhand Rhizome Asparagus racemosus Shatavari Roots Aegle marmelos Bael Fruit Cassia fistula Suvarnaka, Fruit Root Amaltas Gymnema sylvestre Gudmar Fruit Holarrhena Kurchi Barks, Seeds antidysenterica Mimusops elengi Bakul Bark Ocimum sanctum Tulasi Leaves, seeds Piper longum Pippali Fruits Sapindus trifoliatus Ritha Roots Terminalia arjuna Arjun Bark Terminalia bellerica Behada Fruit Terminalia chebula Hirda Fruits Withania somnifera Ashwagandha Root Botanical name Uses / conditions treated Acorus calamus Cough, bronchitis, depression, dysentry Asparagus racemosus As uterine tonic, as a galactogogue Aegle marmelos Diarrhea, dysentery, common cold Cassia fistula Fruitpulp--as a mild laxative against fever, arthritis; root--strong purgative Gymnema sylvestre Inflammation, cold, diabetes, worms, liver disorder, headache, constipation, jaundice Holarrhena Amoebic dysentry, diabetes, antidysenterica diarrhoea, asthma, gastropathy, skin diseases Mimusops elengi Odontoparhy, ulemorrhagia, constipation Ocimum sanctum Swellings (antiseptic) Piper longum Longevity enhancer Sapindus trifoliatus Roots--epilepsy, hysteria and hemicrania; Fruits--asthma, gout, cough, food poisoning Terminalia arjuna As a constituent of the triphala churna--employed in number of diseases Terminalia bellerica As a constituent of the triphala churna--employed in number of diseases Terminalia chebula As a constituent of the triphala churna--employed in number of diseases Withania somnifera Chronic fatigue, dehydration, bone weakness, muscle weakness and tension Botanical name Pharmacological reports Acorus calamus Bronchodilator (Shah, A.J. and Gilani, A.-H., 2010), anti-inflammatory (Kim, H., et al., 2009) immunosuppressive (Mehrotra, S., et al., 2003) Asparagus racemosus Immunomodulator (Gautam, M., et al., 2009), antioxidant (Kamat, J.P., et al., 2000), antitussive (Mandal, S.C., et al., 2000), antidepressant (Singh, G.K., et al., 2009) Aegle marmelos Hypoglycemic (Kesari, A.N., et al., 2006), immunomodulatory (Patel, P. and Asdaq, S.M.B., 2010), anti- inflammatory, antipyretic, analgesic (Arul, V., et al., 2005) Cassia fistula Antimicrobial (Duraipandiyan, V. and Ignacimuthu, S., 2007), hepatoprotective (Bhakta, T., et al., 1999) Gymnema sylvestre Antimicrobial (Satdive, R.K., et al., 2003), antidiabetic (Baskaran, K., et al., 1990, Chattopadhyay, R.R., 1998, Shanmugasundaram, E.R.B., et al., 1990), anti-sweet (Yoshikawa, K., et al., 1989) Holarrhena Antibacterial, antidiarrhoeal (Kavitha, antidysenterica D.,et al., 2004), antiamoebic (Acton, H.W. and Chopra, R.N., 1933) Mimusops elengi Antiulcer (Shah, P.J., et al., 2003) Ocimum sanctum Immunomodulatory (Goel, A., et al., 2010), chemopreventive (Prakash, J. and Gupta, S.K., 2000) Piper longum Antiplatlet (Park, B.-S., et al., 2007), immunomodulatory, antitumor (Sunila, E.S. and Kuttan, G., 2004) Sapindus trifoliatus Antimigraine (Arulmozhi, D.K., et al., 2004, Arulmozhi, D.K., et al., 2005) Terminalia arjuna Anti-ischemic (Gauthaman, K., et al., 2005), antiulcer (Devi, R.S., et al., 2007) Terminalia bellerica Antispasmodic, bronchodilatory (Gilani, A.H., et al., 2008) Terminalia chebula Anti-inflammatory (Reddy, D.B., et al., 2009), anticancer (Saleem, A., et al., 2002), antioxidant (Pfundstein, B., et al., 2010) Withania somnifera Immunomodulatory (Davis, L. and Kuttan,G.,2000), antibacterial (Owais, M., et al., 2005) Table 2: Brine shrimp toxicity of ethanolic extracts of fourteen Indian medicinal plants Plant Plant part % Lethality [+ or -] SEM 100 [micro]g/mL Acorus calamus Stem 34 [+ or -] 4 Asparagus racemosus Root 45 [+ or -] 9 Aegle marmelos Fruits 68 [+ or -] 4 Aegle marmelos Leaves 25 [+ or -] 3 Cassia fistula Fruits 36 [+ or -] 13 Gymnema sylvestre Leaves 50 [+ or -] 12 Holarrhena antidysenterica Bark 75 [+ or -] 4 Mimusops elengi Bark 48 [+ or -] 8 Ocimum sanctum Leaves 0 Piper longum Leaves 100 Sapindus trifoliatus Fruits 52 [+ or -] 4 Terminalia arjuna Bark 47 [+ or -] 8 Terminalia bellerica Fruit 59 [+ or -] 5 Terminalia chebula Fruit 85 [+ or -] 3 Withania somnifera Root 33 [+ or -] 9 Potassium dichromate 100 500 [micro]g/mL Acorus calamus Stem 89 [+ or -] 3 Asparagus racemosus Root 56 [+ or -] 10 Aegle marmelos Fruits 89 [+ or -] 6 Aegle marmelos Leaves 67 [+ or -] 5 Cassia fistula Fruits 43 [+ or -] 8 Gymnema sylvestre Leaves 69 [+ or -] 10 Holarrhena antidysenterica Bark 100 Mimusops elengi Bark 66 [+ or -] 3 Ocimum sanctum Leaves 18 [+ or -] 4 Piper longum Leaves 100 Sapindus trifoliatus Fruits 81 [+ or -] 1 Terminalia arjuna Bark 74 [+ or -] 4 Terminalia bellerica Fruit 72 [+ or -] 6 Terminalia chebula Fruit 85 [+ or -] 5 Withania somnifera Root 56 [+ or -] 10 Potassium dichromate 100 1000 [micro]g/mL Acorus calamus Stem 100 Asparagus racemosus Root 87 [+ or -] 12 Aegle marmelos Fruits 100 Aegle marmelos Leaves 100 Cassia fistula Fruits 73 [+ or -] 22 Gymnema sylvestre Leaves 100 Holarrhena antidysenterica Bark 100 Mimusops elengi Bark 100 Ocimum sanctum Leaves 56 [+ or -] 12 Piper longum Leaves 100 Sapindus trifoliatus Fruits 100 Terminalia arjuna Bark 100 Terminalia bellerica Fruit 100 Terminalia chebula Fruit 100 Withania somnifera Root 89 [+ or -] 10 Potassium dichromate 100 Table 3: [LC.sub.50] values of selected plant extracts Plant extract [LC.sub.50]([micro]g/mL) [+ or -] SEM (a) Acorus calamus stem nd; 217[+ or -] 67 (Padmaja, R., et al., 2002) (b) Holarrhena antidysenterica bark 104 [+ or -] 28 Piper longum leaves 10 [+ or -] 11 Piper longum fruits nd; 6.9 (Padmaja, R., et al, 2002) (a) Terminalia chebula fruits 107 [+ or -] 32 Piperine nd; 2.4 (Padmaja, R., et al, 2002) (b) Potassium dichromate (c) 32 [+ or -] 11 [Lit. 28.7 [+ or -] 8.8 (Padmaja, R., et al, 2002)] (b) Emetine hydrochloride (c) nd; 20.1 [+ or -] 0.2 (Wanyoike, G.N., et al., 2004) (b) Cyclophosphamide (c) nd;16.3 [+ or -] 6.0 (Moshi, M.J., et al, 2004) (b) nd: not determined; (a) [LC.sub.50] is the lethal concentration of extract at which 50% mortality (lethality) of brine shrimp nauplii was observed; (b) mentioned [LC.sub.50] values for Acorus calamus, piperine and reference standards (Emetine HCl and Cyclophosphamide) have been taken from literature; (c) positive standards.