RADICAL SCAVENGING COMPOUNDS FROM THE AERIAL PARTS OF SOLENOSTEMON MONOSTACHYS BRIQ (LAMIACEAE)

Background: Solenostemon monostachys Briq. (Lamiaceae) is a weed widely used in ethno-medicine to treat infections and inflammatory conditions but the active compounds are yet to be identified. This study isolated and identified the radical scavenging compounds from S. monostachys crude extract using diphenyl-1, 1-picryl hydrazyl (DPPH) to monitor the separation. Materials and Methods : The crude extract of the aerial parts of the plant was evaluated for antimicrobial activity using the agar diffusion test, anti-inflammatory test using carrageenan induced oedema of the rat paw and radical scavenging test using diphenyl-1, 1-picryl hydrazyl (DPPH). Repeated chromatographic separation of the ethyl acetate and n -butanol fractions on silica gel and Sephadex LH-20, monitored by DPPH bio-autographic assay resulted in the isolation of seven active compounds, which were identified using spectroscopic methods and comparison with literature data. Results: The ethyl acetate and n-butanol fractions were the most active fractions and contained apigenin, apigenin glucuronide, luteolin, caffeic acid, methyl caffeate, rosmarinic acid and methyl rosmarinate as the radical scavenging compounds with EC 50 values of 26.67 ± 0.31, 185.89 ± 1.02, 5.35 ± 0.31, 3.92 ± 0.06, 13.41 ± 0.18, 4.99 ± 0.10, 5.97 ± 0.08 µg/ml respectively compared with 2.32 ± 0.08 µg/ml for quercetin as the reference standard. Conclusion: The isolated compounds are reported for the first time in this plant. The presence of the compounds in S. monostachys P. Beauv. (Briq.) (Lamiaceae) may provide justification for some of the ethnomedicinal uses of the plant in infections and inflammatory conditions.


Introduction
Solenostemon monostachys (P.Beauv.)Briq (Lamiaceae) is an annual or perennial weed occurring in a wide range of habitats and widespread in West and Central Africa with numerous medicinal uses.The leaf sap is considered sedative and stomachic and is used to treat colic, convulsions, fever, headache and cough, especially in children, and externally against eyesight troubles and aphthae (Burkill, 1985).Also, the leaves are used to treat dysmenorrhoea, haematuria, female sterility, rheumatism, foot infections and snakebites.The roots are used to treat onchocerciasis (river-blindness).The plant has many ritual uses, especially related to pregnancy (Lemmens, 2004).It is eaten in Ghana as a leafy vegetable, while in South Western Nigeria; it is commonly employed in the recipe of many herbal medicines especially for infections and inflammatory diseases (Burkill, 1985).The dichloromethane and methanol extracts of the aerial parts of S. monostachys have been reported to have antibacterial and antioxidant activities (Ekundayo and Ezeogu, 2006;Jacques et al., 2010;Baba and Onanuga, 2011).The only constituents reported in literature are coleons, unusually rearranged diterpenes, from the aerial parts of the plant (Miyase et al., 1980).
As part of our ongoing investigations on antioxidants and antimicrobial agents from plants (Adeloye et al. 2005, Taiwo et al. 2006, Idowu et al. 2003;2010), we report the isolation of apigenin, apigenin glucuronide, luteolin, caffeic acid, methyl caffeate, rosmarinic acid and methyl rosmarinate for the first time from the aerial parts of S. monostachys.

General
Solvents used for extraction and chromatography were redistilled before use.Adsorption column chromatography was performed using Accelerating Gradient Chromatography (AGC).This is a modification of conventional medium pressure liquid chromatography (Dunstan, 1995).The ascending mode was employed using silica gel 60 (ASTM 230-400 mesh, Merck).The equipment for the AGC work station was from Baeckstrom Separo AB, Lidingo, Sweden.Size exclusion column chromatography was achieved using Sephadex LH-20 (Pharmacia) preswollen in the specified solvent before loading onto the column.All Thin Layer Chromatography (TLC) analysis were performed at ambient temperature using analytical silica gel 60 GF 254+366 pre-coated aluminum backed plates (Merck, 0.25 mm thick).The resulting spots on TLC plates were visualized under UV light (254 nm) and detected by the use of vanillin/H 2 SO 4 , anisaldehyde-sulphuric acid and DPPH spray reagents as appropriate.The UV spectra of isolated compounds were obtained in methanol on a Cecil UV/visible spectrophotometer (Cecil Instruments Ltd., Cambridge). 1 H NMR spectra were recorded at 200 MHz and 13 C NMR at 50 MHz, respectively, on a Varian 200MHz spectrometer at the Obafemi Awolowo University.Chemical shifts are expressed in ppm and referenced to the residual solvent signals.

Collection
The aerial parts of S. monostachys P. Beauv.(Briq.)(Lamiaceae) were collected from the Obafemi Awolowo University campus, in October, 2006.The plant was identified by Mr. Oladele A.T. and a voucher specimen with number UHI 16,399 was deposited at IFE Herbarium, Obafemi Awolowo University, Ile-Ife, Nigeria.The plant materials were air-dried at room temperature and powdered.

Extraction and Isolation
The powdered plant material (1.5 kg) was extracted successively with dichloromethane (4 L) followed by extraction of the marc with ethyl acetate (4 L) at room temperature and lastly n-butanol (2.5 L).The crude dichloromethane and ethyl acetate extracts were each concentrated to dryness in vacuo.The dichloromethane, ethyl acetate and n-butanol fractions were 17.6 g, 11.7 g and 6.8 g respectively.

Antioxidant Tests
TLC autography assay (Qualitative) was conducted as follows.A small amount of the sample was dissolved in an appropriate organic solvent and spotted on silica gel sheet, dried and developed using a suitable solvent system.The dried plate was sprayed with 0.2% methanolic solution of stable radical diphenyl picryl hydrazyl hydrate (DPPH).Zones that turned yellow immediately after spraying were deemed to be strongly antioxidant; zones that turned yellow within 15 min of spraying were categorized as moderately antioxidant, while those that turned yellow within 30 min of spraying were categorized as weakly antioxidant (Burits and Bucar, 2000).Quantitative hydrogen donating or radical scavenging properties of the isolated compounds was determined using the stable radical DPPH-(2, 2-diphenyl-2-picrylhydrazyl) as described by Menzor et. al. (2001).To 1 ml of different concentrations (10, 8, 6, 4, 2 and lg/ml) of the isolated compounds or standard (Quercetin: 6, 4, 2, and 1 µg/ml) in a test tube was added 1 ml of 0.3 mM DPPH in methanol.The mixture was vortexed and then incubated in a dark chamber for 30 min.After 30 min, the absorbance (Ab) values were measured at 517 nm and converted into the percentage antioxidant activity.The 50% inhibition concentration (IC 50 ) was obtained from a linear regression plots of concentration of each of the test compounds (M) against the mean percentage of the antioxidant activity (% inhibition) obtained from three replicate assays.

Statistical Analysis
Results are expressed as S.E.M.The results were analysed using the one-way analysis of variance (ANOVA) followed by the student Newman Keul-Test for comparison between the isolated and reference compounds.The level of significance was set at ⃰ P ≤ 0.05 (5%) for all measurements carried out compared to the reference compound.

Agar-diffusion Method
Antibacterial activity tests were carried out using agar-diffusion (hole-in-plate) method (Adesina et al., 2000) with 18 h broth cultures of the following organisms: Escherichia coli NCTC 10418, Staphylococcus aureus, NCTC 6571; P. aeruginosa ATCC 10145, B. subtilis NCTC 8236 and Candida pseudotropicalis NCYC 6.A stock solution concentration of 20.0 mg/ml in methanol-water (1:1) was used except otherwise stated.Streptomycin (l.0 mg/ml) and the solvent, aqueous MeOH (50%) were used as controls.Each sample was tested in duplicate.The diameters of the zones of inhibition were measured to the nearest mm.

Anti-inflammatory Test
The method used was the carrageenan induced oedema of the rat paw (Winter et al., 1962).The level of inflammation was assessed by measuring the volume of the paw, using a plethsysmometer.The paw volume was measured at time 0, 0.5, 1, 2, 3, 4, 5 and 6 hours after the carrageenan injection.The acute inflammatory activities in the animals that received the crude extract (100 mg/ kg body weight) were compared with those of the vehicle and indomethacin (5 mg / kg body weight).The percentage inhibition of edema was normalized with the vehicle control animals.

Results and Discussion
Repeated chromatography of the ethyl acetate fraction of S. monostachys on silica gel and Sephadex LH-20 yielded compounds 1-7 (Fig. 1).Compound 1 was isolated as an amorphous brown solid with an AB coupling pattern characteristic of a trans-stilbenoid skeleton in the 1 H NMR. The compound was identified as rosmarinic acid by comparison of the recorded spectra with those reported for it in literature (Wang et al., 2000;Mehrabani et al., 2005).The spectra of compound 2 were almost the same with those of 1 except for a singlet at 3.60 ppm for a methoxy group in the 1 H NMR spectrum and an additional signal observed at 51.6 ppm in the 13 C NMR spectrum.This signal is characteristic of a methyl group of an ester.On comparison of the spectral data with literature compound 2 was identified as methyl rosmarinate.(Wang et al., 2000, Mehrabani et al., 2005).
Compounds 3 and 4 showed the presence of an aryl ABX system and trans-olefinic protons identified by the large J values (16 Hz) at 7.50 and 6.25 ppm, with the occurrence of a signal at 167.8 ppm for an αβ unsaturated carbonyl carbon suggestive of the existence of a caffeoyl group with an additional signal at 3.8 ppm in the proton spectrum and 52.13 ppm in the 13 C NMR indicative of an ester methyl group in compound 4. Comparison of the spectral data of compounds 3 and 4 with those in the literature (Wang et al., 2000), enabled the compound to be identified as caffeic acid and methyl caffeate respectively.Compounds 5-7 are flavones and their structures were established from UV shift reagent studies, 1 H and 13 C NMR spectroscopic data, inclusive of 2D NMR data and comparison with those reported in literature.Compounds 5, 6 and 7 were identified as apigenin (Xiao et al., 2006), luteolin (Markham, 1982) and apigenin-7-O--D-glucuronide and respectively.

Biological Activity
The crude extract showed activity against G +ve organisms only, as shown in Table 1 and demonstrated anti-inflammatory activity in the carrageenan induced oedema of the rat paw compared with indomethacin (reference compound) at 5 mg/kg (Table 2).The crude extract also showed spots with strong radical scavenging activity in the rapid DPPH thin layer autographic assay used in the preliminary screening and the process was employed to monitor the isolation of the compounds.The EC 50 values for the isolated pure compounds from the plant are as shown in Table 3.The compounds (1-7) conformed to the structural requirements for strong radical scavenging activity as described by Seyoum et al., (2006).Flavonoids have been reported in literature to have good anti-inflammatory activities (Cody et al., 1986;Middleton and Kandaswami, 1992;Gonzalez-Gallego et al., 2007).Caffeic acid derivatives especially rosmarinic acid (RA) and methyl rosmarinate (MR) have also been reported to have a number of remarkable biological activities e.g antiviral, antibacterial, anti-inflammatory and antioxidant activities (Peake et al., 1991, Sahu et al., 1999, Abedini et al., 2013, Inatani et al., 1983, Kuhnt et. al., 1995, Dyke et al., 1986).
This study reported, for the first time in the plant, the presence of luteolin, apigenin, apigenin glucoronide, caffeic acid, methyl caffeate, rosmarinic acid and methyl rosmarinate as compounds responsible for the radical scavenging activity of S. monostachys.The literature reports on the anti-inflammatory and antibacterial activity activities of these compounds may additionally justify the ethno-medicinal uses of this plant species in infections and inflammatory conditions.

Table 2 :
Anti-inflammatory activities S. monostachys expressed in mean paw volume and percentage inhibition of oedema formation at the 3 rd hour.

Table 3 :
DPPH radical scavenging activity of compounds isolated from S monostachys