Preliminary phytochemical screening and cytotoxic potentials from leaves of Sanchezia speciosa Hook. f

Background: Sanchezia speciosa Hook.f is an important medicinal plant with several ethnomedicinal properties. In the present experiment the leaves of this plant were screened for the presence of major phytochemical groups. The phytochemicals are the wide variety of compounds produced by plants manipulated widely in the pharmacognostic drug development and in the treatment of the major ailments. Methods: Phytochemical screenings were performed by various functional group tests. All the leaves extracts were subjected to brine shrimp lethality bioassay and standard anticancer drug vincristine sulphate was used as positive control. Results: Phytochemical investigation of the leaves extracts showed the presence of alkaloids, glycosides, flavonoids, triterpenoids, carbohydrates, steroids, phenolic compounds, saponins and tannins. The ethyl acetate fractionates of the leaves extracts were positive for all the phytochemicals tested while n-hexane fractionates were negative for flavonoids and phenolic compounds. The n-hexane and ethyl acetate soluble fractionates showed significant cytotoxic activities with median lethal concentrations (LC50) 19.95 μg/mL and 12.88μg/mL while LC50 for the vincristine sulphate was 10.96 μg/mL. Conclusion: This study shows that the leaves of Sanchezia speciosa Hook. f may have potentials in control of carcinogenesis in the field of pharmacology.


Introduction
Traditional medicine system is based on different plants for many years around the world and still provides remedies in mankind [1]. Active pharmaceutical compounds can be isolated from these plants which have good medicinal properties [2].
According to World Health Organization (WHO, 1993), from 119 plant derived pharmaceutical medicines, about 74% are used in modern medicine in ways that correlate directly with their traditional uses as plant medicine [3].
In Bangladesh, most of the rural people rely on different forms of traditional medicine for their primary health care. Majority of the plants in Bangladesh did not undergone studies to investigate their bioactive compounds [4]. Therefore screening of Ayurvedic medicinal plant has potential in our country. The most important of these bioactive constituents of plants are alkaloids, tannins, flavonoids and phenolic compounds [5]. For discovery and development of novel drugs, scientists are looking forward to the alternative sources and in last few decades, medicinal plants have been extensively studied for their bioactive principles to develop new lead molecules for pharmaceutical use.
Brine shrimp assay is very useful tool for the isolation of bioactive compounds from plant extracts [6]. The method is attractive because it is very simple, inexpensive and low toxin amounts are sufficient to perform the test in micro well scale. Brine shrimp Artemia salina, also known as sea www.ssjournals.com monkey, is a marine invertebrate about 1 mm in size [7]. It is used as "benchtop bioassay" for the discovery and purification of bioactive natural products and is an excellent choice for elementary toxicity investigations of consumer products. The shrimp lethality assay is based on the ability to kill laboratory-cultured Artemia nauplii (animal"s eggs) [8].
Sanchezia speciosa Hook.f (Family: Acanthaceae) is a stout erect shrub commonly known as Fire Fingers in English. Sanchezia is named for Jose Sanchez, a nineteenth-century professor of Botany at Cadiz, Spain [9]. The plant is native to Ecuador and Peru. It is cultivated for both its attractive orange flowers and green leaves with yellow veins and is very popular as a hedge, screen or border plant. The species is commonly planted as an ornamental plant andfound in wet and shady areas and in many Pacific islands, Hawaii, Fiji and New Caledonia [10]. Traditionally the most important part used in this plant is the leaves and these are applied externally for wounds.
The present study aims to investigate the presence of various phytoconstituents and cytotoxic potentials of leaves extracts of commonly used plant Sanchezia speciosa Hook.f.

Plant material collection
Fresh, mature leaves of Sanchezia speciosa Hook.f were collected during the month of December 2012 from Natore city of Bangladesh and identified by Dr. AHM Mahbubur Rahman, Associate Professor, Department of Botany, University of Rajshahi, Bangladesh.

Plant materials extraction and fractionation
The fresh leaves were washed, sun dried and ground. The ground leaves(300 gm) were extracted with ethanol (2 litres) at room temperature for 6 days through occasional shaking and stirring [11], [12]. The extract was then concentrated by using a rotary evaporator in vacuum at 40ºC-50ºC. A portion of concentrated ethanol extracts of leaves were fractionated by the modified Kupchan partitioning method [13] in to n-hexane and ethyl acetate.

Phytochemical screening
The individual n-hexane and ethyl acetate soluble fractionates of ethanolic leaves extracts of Sanchezia speciosa Hook. f were subjected to phytochemical studies for the identification and presence of different phytoconstituents (Table 1). Test solution was prepared at 10% (w/v) concentration in distilled water unless otherwise mentioned in individual test. This investigation was carried out by using the standard protocols [14]- [16].

Tests for alkaloids
Dragendorff's test: Alkaloids were given reddish brown precipitate with Dragendorff"s reagent (Potassium bismuth iodide solution). Mayer's test: Alkaloids were given cream colour precipitate with Mayer"s reagent (Potassium mercuric iodide solution). Wagner's test: Alkaloids were given reddish brown precipitate with Wagner"s reagent (solution of Iodine in Potassium Iodide).

Test for glycosides Keller Killiani test:
To plant extract, glacial acetic acid containing a trace amount of ferric chloride was added. Then it was transferred to a test tube where concentrated sulphuric acid was added carefully along the side of the test tube. Blue colour in the acetic acid layer indicated the presence of glycosides.

Tests for flavonoids Alkaline reagent test:
To the test solution few drops of sodium hydroxide solution was added, formation of an intense yellow colour which turned to colourless on addition of few drops of dilute acetic acid indicated the presence of flavonoids. Ferric chloride test: To the test solution few drops of ferric chloride solution was added, intense green colour indicated the presence of flavonoids.

Tests for steroids and triterpenoids
Salkowski's test: Leaves extracts were treated in chloroform with few drops of concentrated sulphuric acid, shaken well and allowed to stand for some times, appearance of red colour in the lower layer indicated the presence of steroids and formation of yellow colour at the lower layer indicated the presence of triterpenoids. Libermann-Burchard's test: Extract was treated with few drops of acetic anhydride, boiled and cooled, concentrated sulphuric acid was added along the side of test tube, brown ring at the junction of the two layers and the upper layer turned green, showed the presence of steroids and formation of deep red colour indicated the presence of triterpenoids.

Cytotoxicity screening
Brine shrimp lethality bioassay is widely used in the bioassay for the bioactive compounds by Meyer method [17]. Here simple zoological organism (Artemia salina Leach) was used as a convenient monitor for the cytotoxicity screening. The eggs (Carolina, Biological Supply Company, Burlington, NC, USA) of the brine shrimp were collected and hatched in artificial seawater (3.8% NaCl solution) for 48 hours to mature shrimp called nauplii. Dissolution of n-hexane and ethyl acetate soluble fractions of leaves extracts were performed in artificial sea water using DMSO. Each 5 ml solution of different concentrations (5, 10, 20, 40, 80 µg/mL) of the two separate solvent fractionates were taken in different vials where brine shrimp nauplii were placed and observed for mortality for 24 hours. Artificial sea water medium containing DMSO was considered as negative control while standard vincristine sulphate was used as positive control. The resulting concentration-mortality data were transformed to Microsoft Excel statistical analysis for the determination of LC 50 values of the plant extracts [18]- [21].

Results
In phytochemical investigation ethyl acetate fractionates from crude ethanol extracts of Sanchezia speciosa Hook.f leaves showed the presence of active biomolecules; alkaloids, glycosides, flavonoids, steroids, triterpenoids, phenolic compounds, carbohydrates, saponins and tannins as major phytochemical groups while the n-hexane soluble fractionates revealed the negative results for flavonoids and phenolic compounds ( Table 1). The results of brine shrimp lethality bioassay on nhexane and ethyl acetate soluble fractionates of crude ethanol extracts from Sanchezia speciosa Hook.f leaves (% mortality at different concentrations and LC 50 values) are depicted in Figure 1 and Table 2. The percentage of mortality increased with an increase in concentration. LC 50 values of n-hexane and ethyl acetate fractionates were found to be 19.95 µg/mL and 12.88 µg/mL in compared with positive control vincristine sulphate having significant LC 50 values of 10.96 µg/mL. Thus the ethyl acetate fractionates were more toxic to brine shrimp than the corresponding n-hexane fractions.

Discussion
Phytochemical screening is the first step in herbal medicine research to identify bioactive and novel lead compounds. Plant material consists of many different kinds of natural products with nature of different polarities leading to a different mode of solubility [22]. These secondary metabolites are secreted by the plants for their defense which are being used by peoples for various purposes and also are the key candidates in the medicinal value of plants [16] [23].
The variation in results of brine shrimp lethality may be due to the difference in the amount and types of cytotoxic substances (e.g flavonoids, triterpenoids or tannins) present in the crude extracts of this plant leaves [24]. Moreover, McLaughlin et al., 1993 also reported that alkaloids have been associated with medicinal uses for centuries and one of their common biological properties is their cytotoxicity. Thus this significant lethality of the crude leaves extracts to Artemia salina Leach was attributed to the presence of potent cytotoxic compounds which needs further investigation.
Most of the pharmacologically active compounds are obtained from the natural sources. Because of the traditional use, most of them are IJASR|VOL 01|ISSUE 03|2015 www.ssjournals.com safely utilized nowadays. But when we move towards new discoveries from the medicinal plants, toxicity profile of the respective plant must also be checked in order to enhance safety [25]. Pharmacological evaluation of plants provides a good source for the development of novel and safe medicinal plants. The brine shrimp lethality bioassay has been established as a safe, practical and economic method for the determination of the toxicity of synthetic compounds [26] as well as higher plant products [27]. The brine shrimp nauplii has been used for a number of bioassay systems in which natural product extracts, fractions or pure isolates are tested at various concentrations [17]. In the present study, the two derived fractions of plant extracts showed LC 50 values in safety range following the statement of Peteros and Uy [24] with LC 50 values> 100 µg/ml. Literature survey reveals that methanolic extract of Sanchezia speciosa Hook.f leaves had antioxidant and anticancer effects [28]. Rafshanjani et al., 2014 also reported in vitro antibacterial, antifungal and insecticidal activities of ethanolic extract and its fractionates from this plant [29]. To the best of our knowledge, it was our first attempt for phytochemical screening and determination of cytotoxic potentials using brine shrimp lethality test from leaves of Sanchezia speciosa Hook.f.

Conclusion
The n-hexane and ethyl acetate fractionates of Sanchezia speciosa Hook.f leaf can be regarded as a promising candidate for a plant derived antitumor agent in the field of pharmacology. Moreover, phytochemical screening and brine shrimp cytotoxicity assay results may be used for other researchers as a guide on which plant extracts/fractions will get priorities for further fractionation and isolation of these bioactive compounds.