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Cytotoxicity screening of selected Indian medicinal plants using brine-shrimp lethality bioassay.

Introduction

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

Materials

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.

4. Conclusion

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.

Acknowledgments

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: sandipbharate@gmail.com

(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.
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Title Annotation:Original Article
Author:Ved, Chaitali H.; More, Nikhil S.; Bharate, Sonali S.; Bharate, Sandip B.
Publication:Advances in Natural and Applied Sciences
Article Type:Report
Geographic Code:9INDI
Date:Sep 1, 2010
Words:4609
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