Ethnomedicinal value , phytochemical composition and bioactivity of Butea monosperma ( Lam . )

Ethnomedicinal study on Butea monosperma (Lam.) Taub. revealed that native people of Netrokona district extensively use flower, bark and leaves of the plant to treat different kinds of diseases. People of the studied area used the plant to treat goiter, diabetes, painful menstruation, body swellings, intestinal worms, urinary stone, leucorrhoea and chronic fever. Application of root powder mixed with honey as an antidote for snake bite was recorded for the first time. Phytochemical screening of the methanolic extracts of flowers, leaves and stem of this plant showed the presence of carbohydrate, flavonoid, glycosides, saponins, terpenoids and steroids. Through qualitative assessment, flower was found to be rich in flavonoids compared to leaf and stem. Leaf extract of B. monosperma showed relatively higher cytotoxicity than flower and stem extracts. The highest free radical scavenging activity was observed in flower sample (73.49%) and the lowest in leaf sample (48.17%). The results of the present study may be a proof of a scientific basis for the use of B. monosperma in traditional medicine.


INTRODUCTION
Bangladesh is a home to a number of tribes or indigenous communities.Latest ethnographic research suggests that the number of tribes within the country approximates more than 100 instead of the previously estimated about a dozen tribes (Murmu, 2009).
Traditional medicine practices and ethnobotanical information play an important role in the scientific research, particularly when the literature and fieldwork data have been properly evaluated.The knowledge of ethnobotanical use of plants often results in the discovery of new biologically active molecules (Gurib-Fakim, 2006).According to WHO, medicinal plants would be the best source to obtain a variety of drugs.Systematic screening of folk medicines and plants may result in the discovery of novel effective compounds (Tomoko et al., 2002).
There are around 5,000 angiosperms distributed among 200 families in Bangladesh and approximately, 500 of these are being used in the traditional medicines for the treatment of different types of diseases (Rashid et al., 2014).Among them, Butea monosperma (Family: Fabaceae), popularly known as 'palas' in Bengali, has been found to display a wide variety of biological activities.It is a moderate sized deciduous tree and widely distributed throughout Bangladesh, Myanmar, Ceylon and India.The plant is traditionally reported to possess astringent, bitter, alterative, aphrodisiac, anthelmintic, antimicrobial, anthelmintic, antidiabetic, diuretic, analgesic, antitumor, astringent and anti-asthmatic properties (Neelam et al., 2015;Rana & Avijit, 2012;Shrestha & Dhillion, 2003).
Netrokona district is located in between 24°34' and 25°12' north latitudes and in between 90°00' and 91°07' east longitudes.It is bounded by the Meghalaya state of India on the north, Kishoreganj district on the south, Sunamganj district on the east and Mymensingh district on the west.Garo, Hajong, Hodi and Banai are the minor indigenous communities among the population of Netrokona district (BBS, 2007).Being a peripheral district most of the indigenous people are on the verge of disappearance because of decline in population, loss in tribal habitat, or because of merging with the mainstream Bengalispeaking population.Madan and Kendua Upozilla of Netrokona district is well known for its diverse inhabitants and cultural practice.Since ethnomedicinal surveys of various tribes and folk medicinal practitioners are still at an early stage in Bangladesh, the primary objective of the present study was to document the hitherto unreported traditional medicinal practices of B. monosperma in the villages of Madan and Kendua Upozilla of Netrokona district.Till to date no document came up with the information on ethnomedicinal use of B. monosperma in Netrokona district of Bangladesh.Thus, the main objectives of the present research aims to prepare a comprehensive documentation of indigenous knowledge on the utilization of B. monosperma by local inhabitants and Garo and Hazong ethnic communities, followed by determination of phytochemical composition, cytotoxicity and antioxidant potential of different plant parts.

MATERIALS AND METHODS
Ethnobotanical study and survey: Extensive field trips were conducted for ethnobotanical survey using a semi-structured questionnaire prepared following Martin (2008).Ten kavirajes of two upozilla (Madan and Kendua) of Netrokona District with the age ranging from 50 to 60 years were interviewed individually and they pointed out the use of B. monosperma to treat different ailments.
Collection and Identification: Samples of root, stem, leaf, flower and seed of B. monosperma plant were collected separately from Netrokona district and deposited in Plant Systematics and Biodiversity laboratory of Jahangirnagar University for preliminary identification.Finally the identification of the plant specimens were verified and authenticated by the Bangladesh National Herbarium (DACB), Mirpur, Dhaka, Bangladesh.The voucher specimen (accession no-DACB-39193) has been deposited in DACB for further reference.

Preparation of crude extracts:
The collected plant parts were separately sun-dried followed by drying in a hot air oven (Gallenkamp) at reduced temperature (<50ºC).About 200 g powder of each plant parts was digested with 1000 ml of methanol for three days accompanying with occasional shaking and stirring.The whole mixtures was filtrated by a piece of clean, white cotton material.The extract was concentrated at 45ºC under reduced pressure using a rotary evaporator.

Phytochemical screening:
The crude methanolic extracts of flower, leaves and stem were subjected to different qualitative tests to find out the presence of chemical constituents using standard procedure (Evans, 1989;Sofowara, 1993;Ghani, 1998 andDev, 2002).Molisch's and Fehling's reagents were used to investigate the presence of carbohydrates and reducing sugar, respectively.Hagger's reagent, Wagner's reagent, Mayer's reagent and Dragendroff's reagents were used to test the presence of alkaloids while FeCl 3 test and Keller Killiani's test (Khandelwal, 2008) were carried out for glycosides and cardenolides, respectively.Borntrager's test (Houghton & Rahman, 1998) was conducted to know the presence of anthraquinone glycosides; Lead acetate, Alkali, FeCl 3 and Conc.H 2 SO 4 were used for the detection of flavonoids.FeCl 3 , ammonia and lead acetate were used to test the presence of phenolic compounds.Concentrated H 2 SO 4 was used to detect terpenoids whereas acetic anhydride was used to check the presence of triterpene.The presence of phytosterols/steroids was indicated by the Salkowski's test (Kokate et al., 2008) while the presence of saponins was confirmed by foam test (Kokate et al., 2008).

Determination of antioxidant activity:
The antioxidant activities of the extracts were measured on the basis of the scavenging activity of the stable 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical following the method described by Blois (1958) and Aoshima et al. (2004).150 µl DPPH solution was added to 3 ml methanol and absorbance was taken immediately at 517 nm for control reading.50 µl of various concentrations of different fractions as well as standard compound (ascorbic acid) were taken and the volume was made uniformly to 150 µl using methanol.Each of the samples was then further diluted with methanol up to 3ml and to each 150 ml DPPH was added.Finally, absorbance at 517 nm was determined after 30 min.and the percent inhibition activity was calculated as Brine shrimp lethality bioassay: The lethality of a test sample in a simple zoological organism such as the shrimp (Artemia salina) has been utilized by Meyer et al. (1982) through the Brine Shrimp Cytotoxicity Test (BSCT).It has been well utilized to screen and fractionation of physiologically active plant extracts as well.This bioassay is indicative of cytotoxicity and a wide range of pharmacological activity of natural products.Brine shrimps (Artemia salina) lethality bioassay was followed by Meyer et al. (1982).

RESULTS AND DISCUSSION
Folk medicine is a traditional form of medicinal practice in Bangladesh, which is practiced by practitioners existing both among the mainstream Bengali-speaking population as well as among the various indigenous communities of Bangladesh.The mainstay of the folk medicinal formulations consist of medicinal plants, which are used directly or in the form of decoctions, juice, pastes and are administered either orally or topically, depending upon the ailments treated.The findings from the ethnomedicinal survey among the local inhabitants including a few ethnic communities (Garo and Hazong) of Madan and Kendua Upozilla of Netrokona district have been presented in Table 1.The Kavirajes were explained properly for providing the information that was wanted, the purpose for obtaining this information, and told that the survey results may be disseminated both nationally and internationally as they prefer to keep their knowledge and formulations, inherited from their forefathers, between themselves.
The survey revealed that stem and flower of B. monosperma were the most used parts for medicinal purposes in Netrokona district (Fig. 1).The next popular parts of the plant used were leaves and seeds, where each of them scored 17% of the total usage.Petiole and root of the plant showed least usage (8%).Interestingly, the use of B. monoperma root in medicinal purpose was not well recorded.In Bangladesh, this may be the first information on the medicinal use of B. monosperma root (Table 1).Patil et al. (2006) reported on the similar use of this plant from India.However, the findings on the usage of leaf, stem, flower and seeds of B. monosperma plant to treat different diseases of human are corroborated with that of Sharma & Deshwal (2011), Kirtikar & Basu (1935) and Kumar & Samanta (2012).The results of phytochemical screening of flower, leaves and stem of B. monosperma as presented in Table 2 showed the inconsistent presence of major secondary metabolites, viz.carbohydrates, glycosides, cardiac glycoside, flovonoids, phenolics tannins and terpenoids.Crude methanolic flower extract of B. monosperma showed strong presence of flavonoids and glycosides, moderate presence of carbohydrates, phenols, tannins and very weak presence of terpenoids and saponins.These results are corroborated with the findings of Ahmed et al. (2011).Stem and leaves showed moderate presence of carbohydrates, glycosides, flavonoids, phenols and tannins.Alkaloids, terpenoids and saponins were found to be very feebly present in them (Rajput et al., 2011).In the present experiment different plant parts of B. monosperma showed varying degree of cytotoxicity.In Brine Shrimp Lethality bioassay, the crude methanolic extract of leaves of B. monosperma showed LC 50 value 427.88 µg/ml after 6 hours followed by the LC 50 values 691.70 µg/ml for flowers and 809.57µg/ml for stem extracts.With the progression of hours the cytotoxicity gradually increased.After 12 hours, the LC 50 values for leaves, flowers and stem were 148.25 µg/ml, 223.03 µg/ml and 463.25 µg/ml respectively.However, after 24 hours the LC 50 values for flower, leaves and stem were 94.44 µg/ml, 26.06 µg/ml and 113.24 µg/ml respectively (Table 3).Vincristine sulphate was considered as the standard compound to assess the cytotoxicity which showed LC 50 values 0.069 µg/ml, 0.031 µg/ml and 0.009 µg/ml after 6 hours, 12 hours and 24 hours respectively.Among the plant parts, leaves extract showed greater cytotoxicity compared to flower and stem extracts.Kumar et al. (2009) reported on LC 50 value 502.41 µg/ml for Butea frondosa leaf extracts.Sehrawat & Sultana (2006) reported on the application of leaf extract of B. monosperma in treatment against 2-AAF induced hepatic toxicity and hyperproliferation.Considering the above facts plant samples may be regarded as moderate to poor cytotoxic.Panda et al. (2009) isolated a stigmasterol from the bark of B. monosperma and evaluated for its thyroid hormone and glucose regulatory efficacy in mice.Rekha (2011) reported that the ethanolic extract of B. monosperma showed significant anti-cancer activity in the tested animal models.However, the present findings may be the first comparative cytotoxicity profile for different parts of B. monosperma plant.
The antioxidant activities of flower, leaves and stem extracts were measured as percentage of DPPH free radical scavenging activity and have been presented in Fig. 2. The methanolic extract of the flower of B. monosperma showed maximum antioxidant or DPPH free radical scavenging activity (73.49%) followed by scavenging activities of stem (58.58%) and leaves (48.17%) respectively.Ascorbic acid, the reference compound to measure antioxidant potential of the samples, showed 87.07%DPPH free radical scavenging activity.Relatively higher concentrations of the samples showed higher antioxidant activity compared to low and moderate concentrations of the fractions.Present results on antioxidant potential of different parts of B. monosperma corroborates with the findings Edwin et al., 2009;Lavhale &Mishra, 2007 andSharma &Garg, 2009.The total phenolic content (TPC) was determined in comparison with standard Gallic acid and the results were expressed in terms of mg Gallic acid equivalent (GAE)/ 100 g dry sample.Among the B. monosperma samples, maximum amount of phenolic content was recorded as 85.88 mg/100 g in flower extract followed by 64.84 mg/100 g in stem extract and 60.48 mg/100 g in leaves extract (Fig. 3).These variations were found statistically significant when analyzed by DMRT at 5% level of significance.Salar & Seasotiya (2011) reported that antioxidant properties of bark of B. monosperma extracted according to increasing and decreasing solvent polarity.According to them significant variations were found in total phenolic content and antioxidant activity depending on the solvent and method of extraction.Phenolic compounds are widely distributed in plants (Li et al., 2006), which have gained great attention, due to their antioxidant activities and free radical-scavenging abilities, which potentially have beneficial implications for human health (Govindarajan et al., 2007).In the present investigation, other than phenols, stem and leaves extract showed the presence of sterols, steroids, terpenoids and glycosides which are known to have ameliorative effect on human health.Besides, COX-2, an enzyme responsible for inflammation and pain in human being are inhibited by aromatic-5-membered ring heterocycles.The COX-2 inhibitors have analgesic, antipyretic and inflammatory activity comparable to NSAIDs and are used therapeutically in acute pain, and primary dysmenorrhea (Rao et al., 2010).Moreover, epidemiological studies and associated metaanalyses strongly suggested that consumption of diets rich in plant polyphenols offered some protection against development of cancers, cardiovascular diseases, diabetes, osteoporosis and neurodegenerative diseases (Pandey & Rizvi, 2009).Thus, the results of the present study may be a proof of a scientific basis for the use of B. monosperma in traditional medicine and further studies are needed to focus on isolation of novel bioactive compounds from different parts of B. monosperma following extensive phytochemical and pharmacological studies.

Fig. 1 .
Fig. 1.Comparison among the usage of different parts of B. monosperma plant by the Kavirajes of Madan and Kendua upozilla of Netrokona district sulphate used as a positive control.