CYTOTOXICITY ASSESSMENT OF HERITIERA LITTORALIS (AITON), MADHUCA LONGIFOLIA (KÖNIG) MACBR., NERIUM INDICUM MILL. AND SAPIUM INDICUM (WILLD.) LEAVES ON ARTEMIA SALINA (L.)

Petroleum ether, CHCl3 and CH3OH extracts of leaves of Heritiera littoralis (Aiton), Madhuca longifolia (König) Macbr., Nerium indicum Mill. and Sapium indicum (Willd.) were subjected to assess cytotoxicity against Artemia salina (L.) nauplii. The petroleum ether extract of H. littoralis leaves showed LC50 values 273.77, 97.27, 51.60, 37.12, 14.60 and 12.59 ppm after 12, 18, 24, 30, 36 and 42 h; the CHCl3 extract showed LC50 values 733.25, 105.51, 40.72 and 18.20 ppm after 6, 12, 18 and 24 h whereas CH3OH extract showed 73.05, 30.62, 24.56, 20.85, 16.21 and 6.71 ppm after 6, 12, 18, 24, 30 and 36 h of exposure respectively. The petroleum ether extract of M. longifolia leaves possess LC50 values 259.35, 115.17, 56.84 and 8.73 ppm after 12, 18, 24 and 30 h; the CHCl3 extract possess LC50 values 585.43, 205.86, 112.74, 75.62, 52.84and 47.34 ppm after 12, 18, 24, 30, 36 and 42 h but CH3OH extract possess LC50 values 185.87, 60.70, 30.11 and 15.39 ppm after 12, 18, 24 and 30 h of exposure respectively. The petroleum ether extract of N. Indicum leaves recorded LC50 values 249.82, 146.07, 80.23, 54.21 and 40.19 ppm after 18, 24, 30, 36 and 42 h; the CHCl3 extract gave LC50 values 36.13, 21.72, 19.03, 16.81 and 16.34 ppm after 12, 18, 24, 30 and 36 h but CH3OH extract recorded LC50 values 394.90, 129.69, 81.50, 73.10 and 37.51 ppm after 18, 24, 30, 36 and 42 h of exposure respectively. Similarly, the petroleum ether extract of S. indicum leaves showed LC50 values 24.79, 13.18 and 4.61 ppm after 12, 18 and 24 h; the CHCl3 extract were 50.45, 42.64, 21.20 and 14.93 ppm after 18, 24, 30 and 36 h of exposure and the CH3OH extract showed LC50 values 306.37, 217.18, 149.38, 73.52, 54.45 and 22.91 ppm after 12, 18, 24, 30, 36 and 42 h of exposure respectively. The intensity of efficacy of the extracts could be arranged in the following descending order of S. indicum (petroleum etroleum ether extract) >H. littoralis (CH3OH extract) >M. longifolia (petroleum ether extract) >N. indicum (CHCl3 extract).


Introduction
The plant Heritiera littoralis (Aiton) (Malvaceae) has medicinal properties and various traditional uses. It has anti-cancer (Ioannou et al. 2009), anti-inflammatory (Tewtrakul et al. 2010), antifungal (Bandaranayake 2002), antibacterial (Islam 2017) and larvicidal (Ali et al. 2012) properties. It has also been used to control mosquitoes and as a piscicide (Pattanaik et al. 2008, Bandaranayake 2002. Madhuca longifolia (König) Macbr. (Sapotaceae) has many beneficial uses and ethnomedicinal importance. It has antidiabetic and anti-inflammatory properties (Ghosh et al. 2009, Dahake et al. 2010, antifungal and antioxidant properties (Prashanth et al. 2010). It also has good larvicidal and ovicidal activities (Banerji et al. 1985). Nerium indicum Mill. (Apocynaceae) leaves have been applied externally in the treatment of scabies and to reduce swellings. The leaves and the flowers are cardiotonic, diaphoretic, diuretic, emetic, expectorant and sternutatory (Jawarkar et al. 2012). Leaves and bark are treated as insecticide, rat poison and parasitic (Dey andChaudhuri 2014, Url 1. 2019). The leaves also exhibited antioxidant (Vinayagam and Sudha 2011), analgesic (Shah et al. 2011) and antiviral (Rajbhandari et al. 2001) activities. Sapium indicum (Willd.) is an evergreen tree of Euphorbiaceae. Fruits of this plant have a significant antimicrobial (Chumkaew et al. 2003, Silprasit et al. 2011), insecticidal (Khanam et al. 2008, pesticidal (Khalil 1984, Chowdhury 1996 and antifungal (Miah et al. 1990) activities. Artemia salina (L.) (Anostraca: Artemiidae) is commonly known as Brine shrimp belongs to a genus of crustaceans (Crayfish). It is one of the standard organisms for testing the toxicity of chemicals (Ruebhart et al. 2008). The females of this crustacean can produce eggs either as a result of mating or via parthenogenesis. Eggs hatch into nauplii that are about 0.5 mm in length. Eggs can remain in a dormant state as cysts. These cysts can last for several years and will hatch when they are placed in saltwater (Sara 2012). The present investigation was designed to the screening of the crude extracts of the above plants on cytotoxicity of A. salina.

Collection and preparation of the tested plants
The leaves of the selected plants H. littoralis, M. longifolia, N. indicum and S. indicum were collected from Khulna, Bagerhat and Rajshahi Districts of Bangladesh. The leaves of the collected plant were cut into small pieces, spread out in wooden trays and kept in a well-ventilated room to dry them at room temperature. Well dried leaves were ground to powder with a blender, weighed, kept in separate conical flasks and then extracted with a sufficient amount of solvent (petroleum ether, CHCl3 or CH3OH) (100 g × 300 ml × 2 times) for 48 h. Filtration was done by Whatman No. 40 filter paper and after evaporation, the extracts were collected in glass vials and kept in a cool place at 4ºC with appropriate labeling.

Hatching of Brine shrimp cysts
Fresh cysts in vials were purchased from the Pet and Aquarium market of Nilkhet, Dhaka, Bangladesh. Eggs of Brine shrimp were hatched in an aquarium using 1 liter of 1 M NaCl brine solution (pH 8.5). The eggs were incubated for 48 h under fluorescent light and the nauplii were hatched within 24-36 h at 30-35°C. After hatching, the nauplii were transferred to test tubes. Ten nauplii were transferred to each tube. 1.5 ml NaCl solution was added to each test tube.

Test sample preparation and Brine shrimp lethality test
Test samples at different concentrations were considered as doses prepared in test tubes by addition of a calculated amount of DMSO (dimethylsulfoxide). Then water was added to fill the pre-marked (up to 10 ml) test tubes with the help of a pipette. The nauplii were counted by visual inspection and were released in test tubes containing 10 ml of water and the test tubes were kept at room temperature along with a control batch. Observation of mortality was made after 6, 12, 18, 24, 30, 36 and 42 h of exposure.  CHCl3 180,90,45,22.5 and 11.25 and in CH3OH were 100.00, 50.00, 25.00, 12.50 and 6.25 ppm. The doses for all the three extracts of S. indicum leaves were in Petroleum ether 100.00, 50.00, 25.00, 12.50 and 6.25 ppm; in CHCl3 174.00, 87.00, 43.50, 21.75 and 10.86 ppm and CH3OH 100.00, 50.00, 25.00, 12.50 and 6.25 ppm in artificial seawater containing 1% DMSO (v/v) used for this assay. Ten nauplii were used in each of the test tubes and three replicates were used for each concentration. Test tubes later had 1 ml of NaCl solution added to them. After 6, 12, 18, 24, 30, 36 and 42 h incubation, the number of survivors and count of the dead nauplii was done using a dissection microscope and the percentage of the mortality (%M) for each of the doses was calculated as compared with the control. DMSO (1%) served as the negative control and the final concentration of DMSO in the assay volume was kept always below 1% to prevent possible false effects originating from DMSO toxicity. LC50 value is the concentration of the sample required to kill 50% of the Brine shrimp population and this was calculated from the plot of % inhibition against the log concentration of sample extract. According to Meyer et al. (1982), an LC50 value of less than 1 mg/ml is considered toxic while an LC50 value greater than 1 mg/ml is deemed to be non-toxic.

Brine shrimp cytotoxicity
The cytotoxic activity of the test plants had been shown in Table 1 According to the intensity of activity, the extracts of the test plants could be arranged in the following descending order: S. indicum (petroleum ether extract) >H. littoralis (CH3OH extract) >M. longifolia (petroleum ether extract) >N. indicum (CHCl3 extract).  Table 1 are given in Fig. 1 (a-p), Fig. 2 (a-n), Fig. 3 (a-o) and Fig. 4 (a-m)

Discussion
The findings of this investigation on the four medicinal plants H. littoralis, M. longifolia, N. indicum and S. indicum have got support from the works of previous researchers. Patra and Mohanta (2014) reported the antimicrobial activities of the mangrove plant H. littoralis. Salini (2015) found this plant used as a mosquito control agent, cure for diarrhoea and as a fish toxicant. Bark, leaves, roots and stems are used by rural people for the treatment of diabetes and goitre, gastrointestinal disorders, skin diseases and hepatic disorders (Ali et al. 2011, Hossain et al. 2013, Patra and Thatoi 2013 (Rajbhandari et al. 2001) and analgesic (Shah et al. 2011) properties. Very strong support was reported from previous researchers on the potentiality of the plant S. indicum. Azis et al. (2015) described that S. indicum has good wound healing properties. They also mentioned the cytotoxic effects of S. indicum. The findings of Rahman and Monowar (2014) revealed that the petroleum ether extract of the fruit is more toxic than organochlorine compounds. The intravenous and oral administration of the extract kills laboratory animals, rats and mice that can be used as an alternative source of bio-pesticides (Rahman and Monowar 2014). Thus, the test plants H. littoralis, M. longifolia, N. indicum and S. indicum need further investigations to be attempted to reveal their total usefulness in the field of health and pest control technology.

Conclusion
By analyzing the results of cytotoxicity tests of H. littoralis, M. longifolia, N. indicum and S. indicum leaf extracts in petroleum ether, CHCl3 and CH3OH against A. salina it could be concluded that the plants have some bioactive potentials and that could be used in controlling pest organisms in aquatic media. Further studies on isolation and identification of the biologically active compounds of these plants are necessary.