Journal of Ethnopharmacology 128 (2010) 533–536 Contents lists available at ScienceDirect Journal of Ethnopharmacology journal homepage: www.elsevier.com/locate/jethpharm Ethnopharmacological communication Anticonvulsant activity of Benkara malabarica (Linn.) root extract: In vitro and in vivo investigation Nibha Mishra a,∗ , Awadesh Oraon a , Abhimanyu Dev a , Venkatesan Jayaprakash a , Arijit Basu a , Ashok K. Pattnaik a , Satya N. Tripapthi a , Mustari Akhtar a , Sadab Ahmad a , Shreyshri Swaroop a , Mahua Basu b a b Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra 835215, Ranchi, India Shri Shikshayatan College, 11, Lord Sinha Road, Kolkata 700071, India a r t i c l e i n f o Article history: Received 15 August 2009 Received in revised form 7 January 2010 Accepted 19 January 2010 Available online 28 January 2010 Keywords: Benkara malabarica Anticonvulsant GABA transaminase a b s t r a c t Aim of the study: To systematically investigate the anticonvulsant activity of methanol extract of Benkara malabarica roots and to provide a biochemical basis elucidating its mode of action. Methods: The median lethal dose (LD50 ) of Benkara malabarica extract was determined. The anticonvulsant activity of the extract was assessed in strychnine-induced and isoniazide-induced convulsion models; phenytoin (20 mg/kg) and diazepam (1 mg/kg) were used as standards, respectively. Percentage protection provided by the drug was accounted as decrease in the number of convulsions within 8 h of observation. Mechanism of action was studied by performing GABA transaminase (GABA-T) assay, isolated from rat brain. Active constituent was isolated and characterized from the plant extract. Results: The median lethal dose (LD50) of Benkara malabarica was found to be more than 500 mg/kg. It demonstrated 30% and 35% protection against strychnine-induced convulsions and 60% and 80% protection against isoniazide-induced convulsions, at doses of 25 mg/kg and 50 mg/kg, respectively. Enzyme assay results revealed that Benkara malabarica extract possesses GABA-T inhibitory activity (IC50 = 0.721 mg/ml). Scopoletin which was identified as the major constituent of the extract was found to be an inhibitor of GABA-T (IC50 = 10.57 ␮M). Conclusions: The anticonvulsant activity of the plant extract is predominantly GABA mediated and may be due to the action of scopoletin alone or is a result of synergy of different compounds in the extract in which scopoletin is the major constituent. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Benkara malabarica belongs to the family Rubiaceae. Its parts are used by folklore of Jharkhand, India for wide variety of illnesses, such as an emetic, as an astringent, as sedative and as nervine tonic (Nadkarnu, 2000). Tribes of Mesra, Jharkhand, India are using its root as an anticonvulsant for many years. In spite of these, the plant species is relatively unexplored with only few reports like used in arthritis (Bhakuni et al., 1971) and as antimicrobial (Jayasinghe et al., 2002). Its related species were reported to contain scopoletin (Anjaneyulu et al., 1965). Scopoletin was reported to have anticonvulsant property (Adesina, 1985) in some other plants. The use of this plant as folklore, together with the reports for the presence of scopoletin in related species, provided a strong basis to investigate and explore its anticonvulsant activity. In the current ∗ Corresponding author. Tel.: +91 9430358968. E-mail addresses: nmishra@bitmesra.ac.in, mailnibhamishra@yahoo.co.in (N. Mishra). 0378-8741/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2010.01.042 work anticonvulsant activity of the methanol extract of its root was extensively studied using isoniazide-induced and strychnineinduced models. Additionally, enzyme assay against gamma amino butyric acid transaminase (GABA-T) was performed to establish its mode of action. Finally, the extract was chemically characterized and the major constituent (scopoletin) was identified, isolated and assayed for GABA-T inhibitory activity. 2. Materials and methods Samples of root of Benkara malabarica were collected from Mesra, Ranchi, India. The plant was identified and authenticated by National Herbarium, Botanical garden, Kolkata, India (voucher specimen no. CNH/1-J (ii) 2001-Tech.II/321) was preserved in the Department of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, India. After collection, the roots were washed with distilled water. They were air dried under the shade until a constant weight was obtained. It was then powdered in wooden mortar and pestle, sieved with 20-mesh sieve size. The powdered root (100 g) was extracted with 1000 ml of methanol using Soxhlet extraction 534 N. Mishra et al. / Journal of Ethnopharmacology 128 (2010) 533–536 method. The extract was concentrated in vacuo and lyophilized to obtain an average weight. The extract was then stored in vacuum desiccator and solutions of the extract were prepared freshly for each study. The methanol extract of the root of Benkara malabarica was brown in color and had a pleasant smell. The crude yield was 25.87% (w/w). Preliminary phytochemical screening (Trease and Evans, 2002) of methanol extract showed presence of flavonoids, saponins and tannins. Male Wister albino rats (weighing 150–200 g) were obtained from animal house of the Department of Pharmaceutical Sciences, Birla institute of Technology, Mesra, India. The animals were maintained in a well-ventilated room, fed on standard pellets and water ad libitum. The animal experiment was performed according to the institutional ethical committee approval and guidelines Registration No. 621/02/ac/CPCSEA of Birla Institute of Technology, Mesra, India. For each model of convulsion 24 rats were divided into 4 groups: control; extract treated (25 mg/kg); extract treated (50 mg/kg) and standard. All drug samples were suspended in distilled water and gavaged. Strychnine, isoniazide, vigabatrin, diazepam, phenytoin and scopoletin were purchased from Sigma–Aldrich Chemical Co. (St Louis, MO, USA). 2.1. Acute toxicity studies The median lethal dose (LD50 ) determination was performed using the method of Lorke (1983) for oral routes in Swiss albino mice. Three groups of two mice each were administered orally with the Benkara malabarica extract at doses of 10, 100 and 500 mg/kg body weight and observed for signs of toxicity and death within 24 h. No signs of toxicity were observed for the highest dose (500 mg/kg). 2.2. Screening for anticonvulsant activity Strychnine-induced convulsion was employed using phenytoin sodium (20 mg/kg, p.o.) as standard (Bum et al., 2001) and isoniazide-induced convulsion was employed, where diazepam, 1 mg/kg, i.p. was used as standard (Raza et al., 2000). One hour after drug treatment, rats in all the groups were injected with strychnine 2 mg/kg i.p. or isoniazide 300 mg/kg s.c. and observed for number of convulsions within 8 h duration. Decrease in the number of convulsion designated percentage protection as compared to control. by Adesina (1985) with slight modification. Different combinations of n-hexane, ethyl acetate, chloroform and methanol were employed. The spots were monitored for fluorescence at 365 nM for the identification of scopoletin, obtained from an elute containing chloroform:methanol::8:2. This elute was then subjected to distillation under reduced pressure, resulting in a yellow solid (crude yield approximately 50 mg/kg) after complete evaporation. This was further purified using preparative TLC finally recrystallized with chloroform. The isolated compound was confirmed using spectrometric techniques like IR, FAB-MS and 1 H NMR. 2.5. Statistical analysis Results were expressed as percentage (%) protection and mean ± SEM where applicable. Statistical significance was tested using Student’s t-test. The difference was taken to be statistically significant at p < 0.05. 3. Results 3.1. Acute toxicity studies The median lethal dose (LD50 ) of the Benkara malabarica extract in mice was found more than 500 mg/kg, body weight (p.o). 3.2. Screening for anticonvulsant activity The methanol extract of Benkara malabarica showed no significant protection against strychnine-induced convulsions in rats, it provided only 30% and 35% protection at doses 25 mg/kg and 50 mg/kg, respectively (Fig. 1). In contrast, when the effect of Benkara malabarica was evaluated using isoniazide-induced convulsions in rats, it produced significant protection. It provided 60% and 80% protection at doses 25 mg/kg and 50 mg/kg, respectively (Fig. 1). Thus the effect of the extract was dose dependent. In order to elucidate the mechanism involved in protection provided by Benkara malabarica against isoniazide-induced convulsion, GABA-T inhibitory activity was assayed and compared with positive control, vigabatrin. The results showed that Benkara malabarica methanol extract possesses GABA-T inhibitory activity with 2.3. Enzymatic assay Rat brain GABA-T was partially purified (Ricci et al., 2006). Rat brain GABA-T activity was assayed using fluorimetric method as described by Salvador and Albers (1959). It was based upon the measurement of succinic semialdehyde (SSA) produced from GABA during incubation with the enzyme. A standard curve of SSA concentrations versus fluorescence was obtained at excitation and emission wavelengths of 405 and 492 nm, respectively. One unit (1 U) of enzyme activity is equivalent to formation of 1 nM of SSA per min. Protein concentration was determined by the method of Bradford (1976). For assaying the activity, partially purified GABA-T was incubated with various concentrations of extract (0.1–1 ␮g/ml), positive control vigabatrin (5–50 ␮M/ml) and scopoletin (5–50 ␮M/ml). 2.4. Isolation and characterization Since the extract was showing blue fluorescence the HPTLC fingerprinting of the extract was carried out using Scopoletin as marker. After identification of the compound, the extract was subjected to Column Chromatography with normal silica gel using gradient elution technique. Scopoletin was isolated as carried out Fig. 1. DZ: diazepam; Ph: phenytoin; BM: Benkara malabarica. Protective effect of methanol extract of Benkara malabarica root against seizures produced after strychnine (2 mg/kg i.p.) or isoniazide (300 mg/kg s.c.) injection in rats. Percentage protection was calculated as number of convulsions within 8 h of observation. Results are expressed as mean ± SEM, n = 6. *p < 0.05, **p < 0.01 and ***p < 0.001, compared with control (no protection). N. Mishra et al. / Journal of Ethnopharmacology 128 (2010) 533–536 535 Fig. 2. Inhibitory effect of vigabatrin, scopoletin and methanol extract of Benkara malabarica on GABA-T. IC50 value 0.721 mg/ml and IC50 value of vigabatrin was found to be 26.6 ␮M (Gram et al., 1989). Whereas, scopoletin which was identified as the major constituent of the extract possesses an IC50 = 10.57 ␮M (Fig. 2). 3.3. Phyto constituent In search of the active compound(s) responsible for the biological activity HPTLC finger printing were carried out which revealed the presence of scopoletin. Guided by the observation the identified compound was isolated and it was characterized by different spectrometric methods as follows: yellow white crystalline powder, MP-208-210 ◦ C (uncorrected), FT-IR (3433 cm−1 , 1596 cm−1 ), FABMS (M+H 193, 95%), 1 H NMR (CDCl3 /DMSO 300 MHz), ı 3.83(s, 3H, –CH3 ), ı 6.2 (d, 1H, CH), ı 7.79 (d, 1H, CH), ı 6.70 (s, 1H, –Ar(H)), ı 6.83(s, 1H, –Ar(H)) ı 10.1 (br, 1H, –OH), the spectral data are in accordance with the literature values (Shapizadeh and Melnikoff, 1970; Carpinella et al., 2005). 4. Discussion In the present study, anticonvulsant activity of Benkara malabarica root’s methanol extract was extensively studied. Two different models (strychnine induced and isoniazide induced) were used. The convulsions produced by strychnine is primarily due to interference with postsynaptic inhibition and is mediated by glycine; an important inhibitory transmitter to motor neurons and inter neurons in the spinal cord (Rajendra et al., 1997). Whereas, the convulsions produced by isoniazide is due to inhibition of GABA mediated pathway (Schousboe and Waagepetersen, 2007). From this study, we have found that the Benkara malabarica root’s methanol extract was not showing any protection against strychnine-induced convulsion group. Interestingly, marked protection was observed against isoniazide-induced convulsion group. On the basis of these evidences, it was presumed that the anticonvulsant effect of Benkara malabarica (for which it is used among folklore of Jharkhand, India) may not be due to its effect on NMDA receptor (Larson and Beitz, 1988) but may be due to GABA mediated mechanism (Kevin and Wallace, 1997; Schousboe and Waagepetersen, 2007). To validate our claim, the extract was evaluated for its GABA-T inhibitory activity. GABA-T is an enzyme, which metabolizes GABA into succinic semialdehyde (␥-aminobutyrate + 2-oxoglutarate succinate semialdehyde + l-glutamate) (Wood and Peesker, 1973). GABA-T decreases the level of GABA in the brain and also increases the level of l-glutamate (which is an excitatory neurotransmitter) therefore, producing the excitation of neurons by dual mechanism. It has been reported earlier (White, 1999) that the molecules with GABA-T inhibitory property exhibits significant protection and play a central role in the management of epilepsy. The extract was found of inhibit GABA-T (IC50 = 0.721 mg/ml) thereby, supporting the hypothesis (exhibiting a GABA mediated action) and corroborating with its in vivo anticonvulsant property. In order to figure out the possible constituent(s) responsible for the anticonvulsant activity, the extract was subjected to preliminary phytochemical screening which shows prominent presence of flavonoids, and smaller amounts of saponins and tannins. Moreover, the extract was tested positive for hydroxycoumarins (a strong blue fluorescence under UV light). HPTLC fingerprinting and other spectrometric analysis confirmed the presence of scopoletin. Scopoletin, which was reported earlier as anticonvulsant (Adesina, 1985) tentatively, supports the anticonvulsant activity of the plant extract which may be due to scopoletin alone or is a result of synergy of many compounds in the fraction in which, scopoletin is the major constituent. 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