Isolation and Characterization of Bioactive Compound from Ethanol Extract of Root Bark of Grewia mollis (Dargaza’a) Widely Growing in the Wild in North Eastern Nigeria

: Grewia mollis is a plant, has been used as a source of food and in traditional medicinal preparations. Hence, the objective of the present study was to isolate and characterize the bioactive compound from ethanol extract of root bark of Grewia mollis (Dargaza’a) widely growing in the wild in North Eastern Nigeria using standard methods. The isolated compounds were also subjected to antimicrobial, antioxidant and cytotoxic assay. The isolation from the ethanol extract afforded a compound B that showed some degree of bioactivities. Compound B isolated showed good antioxidant activity by bioautography method. For the cytotoxic activity the result revealed that Compound B was moderately active with LC 50 value of 14.06 µg/ml. The result for the antimicrobial activity of the isolated compound B using bioautography showed moderate inhibition by inhibiting three of the test organisms. The structural elucidation of the isolated compound B were done using UV, IR,

African plants constitute a rich untapped pool of natural products.Scientific investigations of medicinal plants have been initiated in many countries because of their contribution to health care (Fatope, 1995).In developing countries it was estimated that about 80% of the population relies on plant based preparation used in their traditional medicinal system and as the basic need for human primary health care (WHO, 2000;Hamayun et al., 2006).The global demand for herbal medicine is growing (Muregi et al., 2003;Zowai ., 2003).Interest in medicinal plants reflects the recognition of the validity of many traditional claims regarding the value of natural products in health care (Hamayun et al., 2006).Medicinal plants are able to produce a large number of diverse bioactive compounds, particularly secondary metabolites.For this reason, extensive studies using different plant extracts have been reported by several scientists, to investigate the antibacterial, anti-inflammatory, analgesic, antioxidant and many other medicinal values of these extracts Mshelia et al., 2008.The Nigerian flora is rich in medicinal plants that are commonly used in folklore medicine for the treatment of various diseases.Grewia mollis (Tiliceae) commonly known in Northern Nigeria as dragaza'a is a shrub or small tree that grows up to 10.5m tall, and grows in tropical areas (Burkill, 2000;Sharma, 2002Katende et al., 1995).The fruit is edible and very Isolation and Characterization of Bioactive Compound from Ethanol Extract…..

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MSHELIA, E. H; TADZABIA, K; PINDAR, W; MOHAMMED, A. H;ASKA, S. A sweet (Person, 1982).It is popularly used in folk medicine to treat malaria fever (Fowler, 2006).The mucilaginous bark and leaves are applied to ulcer, cuts, sores and snake bites (Brink, 2007).The bark and root preparation are taken to treat cough.Extracts of stem bark and leaves are drunk to treat fever.The decoction of the stem bark is taken to treat diarrhoea, and maceration is taken to ease child birth.The mucilage is credited with laxative properties while an infusion of the bark is used to treat colic (Lockett et al., 2000).The pounded leave mixed with water are taken against stomach problems and also given by constipated domestic animals (Ruffo et al., 2002).The decoction of the leaves is used in baths and drinks against rickets in children and difficult birth (Kokwaro, 1993).A decoction of the roots is drunk in case of palpitation (Katenda et al., 1995).The sap from root shavings is placed under the eyelid to treat sore eyes, where as a liquid obtained by kneading the root bark in water is drunk to treat stomach ache, colic and poisoning by certain plants (Neuwinger, 2000).The paste of ground root is applied to rheumatic swellings and inflammation while the fruit is used as febrifuge and in treatment of malaria fever (Lockett et al., 2000;Fowler, 2006).Some findings demonstrated that the mucilage obtained from the stem bark can serve as a good binder in paracetamol formulations (Martins et al., 2008;Muazu, et al., 2009).A reports suggest that high concentration of stem bark in dietary may cause some adverse effects, especially liver injury (Wilson, 2010).Therefore the objective of the present study was to isolate and characterize the bioactive compound from ethanol extract of root bark of Grewia mollis (Dargaza'a) widely growing in the wild in North Eastern Nigeria

MATERIALS AND METHODS
Collection and preparation of plant material: The root bark of Grewia mollis was collected in Hawul local Government Area of Borno State.
The collection was done in April and the soil on the root was removed by gently washing it under running tap water and the bark removed.
The root bark was then air dried under a shade in the laboratory at room temperature and pulverized using motorized miller.

Extraction of Plant Material (Soxhlet extraction method):
The extraction was carried out using the soxhlet extraction with the following solvents hexane, ethyl acetate, acetone, ethanol and water in order of increasing polarity for about 6 to 8 hours (Vogel, 1979).
Isolation: The fraction F4 from column chromatography using silica gel (100-200mesh) and eluated with solvent system of increasing polarity (hexane: ethyl acetate: methanol) of the n-butanol fraction of solventsolvent separation of the ethanol extract form soxhlet extraction.Fraction FC4 and FC5 showed very good antibacterial activity using bioautography.FC4 was re-subjected for further separation using column chromatography on silica gel (60-120mesh) with solvent systems hexane: ethyl acetate (7:3) and then hexane: ethyl acetate (5:5).Four fractions were obtained after re-combining based on TLC profile, all the fractions showed some degrees of antibacterial activity using bioautography.Fraction FCC1 was washed with hexane in ethyl acetate (9:1) to obtain compound A. Fraction FCC2 was re-applied on silica (60-120mesh) and eluted with chloroform: hexane (8:2) to obtain four fractions after recombining and all of them showed some degree of antimicrobial and antioxidant activity.The fractions FCD1, FCD2 and FCD4 are too small in quantity and are not subjected for further purification.Fraction FCD3 was washed with hexane: chloroform (9:1) to obtain compound M after crystallization.FC5 was resubjected to column chromatography on silica gel (100-200) mesh with solvent system ethyl acetate: methanol (9:1) to obtain four fractions after recombining.Fraction FD2-FD4 were remixed and applied on column using sephradex and eluted with chloroform: methanol (7:3) to obtain two fractions FDD1 and FDD2.Fraction FDD1 is very small in quantity and shows relatively very low degree of antibacterial activity therefore it was not subjected for purification.The fraction FDD2 was washed with chloroform in acetone and allowed to crystallize to obtain compound B which was active on the test microorganism using bioautography.
Melting Point Determination using melting point apparatus and the electrothermal melting point apparatus.

UV-Vis Spectroscopy method:
The Genesys 10S UV-Vis spectrophotometer was used for the analysis and scans were performed over a wavelength range of 200 to 600 nm.
Fourier Transform Infrared Spectrophotometer (FTIR): For IR analysis, Perkin Elmer frontier MIR/NIR Fourier Transform Infrared spectrophotometer (FTIR) was used for the analysis.The spectra were recorded at room temperature between 4000 and 400 cm -1 with 4 cm -1 resolution running 10 scans.

RESULT AND DISCUSSION
The Percentage Recovery, Colour and Texture of Solvent-Solvent Extraction of Ethanol Extract (14.0000gm) is presented in table 1.The result also showed a moderate cytotoxic effect with LC50 value of 14.06 µg/ml and a good antioxidant activity of (+++) by bioautography method.The antimicrobial activity showed moderate activity using bioautography by inhibiting three of the test organism.Figure 1 and Table 4 showed the UV spectrum and UV spectrum data of compound B with two major absorption bands in (MeOH) at 363nm and 262nm with another weaker absorption at 298nm. Figure 2 and Table 5 shows the Fourier transform infrared spectroscopy FTIR spectra and data of compound B.
The FTIR showed frequencies at 3395.26 cm -1 and 1662.92cm - indicating the presence of hydroxyl group and keto group in conjugation and the absorption peaks at 1520.06 cm -1 and 1165.67 cm -1 indicating the presence of ethylenic double bond and aromatic ring respectively.Figure 3 and 4 showed the 1H-NMR spectrum of compound B. The spectrum in fogure 3 showed the complete spectrum showing all peaks from delta 0 to 12.5 ppm, while that of figure 4 showed peaks from delta 6.0 to 8.1ppm presumably the most important peaks.C-CO-C stretch and bending in ketone 841.17, 794.89, 671.31, 655.64 C-H out of plane bending of aromatic hydrocarbon Figure 5 and 6 showed the 1 H-1 H COSY Spectrum of compound B. Figure 3 showed the whole spectrum for delta 0-12 ppm, while figure 5 showed the spectrum with the peaks between 4.0 to 8.2pmm on the x-axis and 5.5 to 9.5ppm on the y-axisFigure 5: Figure 4: 1 H-1 H COSY Spectrum of compound B. Figure7 and 8 showed the 13 C-NMR Spectrum of compound B. Figure 7 showed the 13 C-NMR peaks of compound B with the chemical shift between 0-220 ppm, while figure 8 showed the 13 C-NMR Spectrum of compound B showing the peaks between delta 90.0 to 210ppm.Figure 9 and 10 showed 13C-DEPT Spectrum of compound B. the DEPT spectrum in figure 8 showed all the peaks that falls between delta 0 to 220ppm including the solvent peak.Figure 9 showed the presumably the most inportant peaks of compound B tha falls between delta 90.0 to 210ppm.156.9180, 135.9272, 175.7608, 161.4106, 164.3174, 156.5770, 103.2515, 120.6464, 145.0757 and 146.2216ppm.Based on the correlation data for ( 1 H -1 H COSY), it shows that there are two coupling between δH 6.2231 ppm (H -6) and δH 6.4750 ppm (H -8) and δH 6.9491 (H -5') and δH 7.6454 (H -6').

Fig 11 :
Fig 11: Compound B: 2-(3', 4'-Dihydroxyphenyl)-3, 5, 7trihydroxy-1-benzopyran-4-one (quercetin)Conclusion:The plant Grewia mollis contain a bioactive compound that have been previously isolated from the plant that had some degree of antimicrobial, cytotoxicity and antioxidant activities.The isolated bioactive compound was elucidated to be 2-(3', 5, Table 2 showed the Brine Shrimp Lethality Test of Compounds B from the Root bark of Grewia mollis the result indicate that compound B showed moderate cytotoxic activity with LC50 value of 14.06 µg/ml.

Table 3
shows the result for the Melting Point, Rf Values, LC50, Antioxidant and Antimicrobial Activity of Compound B. the isolated compound B had a melting point of 316 o C with the RF values of 0.9342 in EMW, 0.6308 in CBM and 0.8382 in CEF mobile systems.

Table 1 :
Percentage Recovery, Colour and Texture of Solvent-Solvent Extraction of Ethanol Extract (14.0000gm)

Table 2 :
Brine Shrimp Lethality Test of Compounds B from the Root bark of Grewia mollis

Table 3 :
The Melting Point, Rf Values, LC50, Antioxidant and Antimicrobial Activity of Compound B Key: SA= Staphylococcus aureus, SD =Shigella dysenteriae, ST=Salmonella typhi, EC= Escherichia coli, AO= Antioxidant activity Fig 1: UV Spectra of compound B

Table 4 :
UV spectroscopy of compound B * Fig 2: Fourier transform infrared spectroscopy FTIR Spectra of compound BTable 5: Fourier transform infrared spectroscopy FTIR spectra of compound B Frequency (cm -1 ) Group Isolation and Characterization of Bioactive Compound from Ethanol Extract….. 95 MSHELIA, E. H; TADZABIA, K; PINDAR, W; MOHAMMED, A. H; ASKA, S. A Isolation and Characterization of Bioactive Compound from Ethanol Extract…..