ISOLATION AND CHARACTERIZATION OF A FLAVONE FROM SUDANESE VITEX DONIANA (SWEET)(VERBENACEAE) AND BIOLOGICAL ACTIVITY OF THE METHANOLIC FRACTION

Abdel Karim M. From the leaves of Vitex doniana (Sweet) , a flavone (compound I) was isolated and characterized. The isolate was purified by different chromatographic techniques and identified via a combination of spectral tools( IR, UV, HNMR and Mass spectroscopy). The methanolic fraction of Vitex doniana was evaluated(in vitro) for its antimicrobial potential against Gram negative (Escherichia coli, Salomonella typhi and Pseudomonasa eruginosa) and Gram positive (Bacillus subtilis, Bacillus aureus and Staphylococcus aureus) bacteria and the fungus Candida albicans .Promising results were obtained. In vitro antioxidant assay for the methanolic extract was conducted . Evaluation of the antioxidant activity was carried out by measuring the capacity of the extract against stable DPPH radical. The extract showed significant antioxidant activity.

The leaves are used as an infusion for cold and flue. Hot aqueous extracts of the leaves are used in the treatment of stomach and rheumatic pains, inflammatory disorders, diarrhoea and dysentery (Etta,1984;Irvine,1961) indicating that the leaves may possess anti-inflammatory and analgesic properties among others. Leaf sap is used as an eye drop to treat conjunctivitis and other eye complaints. Leaf decoction is applied externally against headache, stiffness, measles, skin rashes, fever, chicken pox and internally as tonic, febrifuge, and to treat respiratory tract diseases .The fruits are acidic, and is a source of essential minerals (Leung et.al.,1986) .The fruits are used as remedy for sores at the corners of the mouth and eyes, which are associated with vitamin A and B deficiencies . It is also used for diarrhea and dysentery. The twigs are used as chewing sticks for teeth cleaning.
Stem bark is used as remedy for leprosy, paralysis, epilepsy and convulsions. It is administered for ailments including diarrhea pulmonary troubles and skin rashes. Bark powder is added to water and taken to treat colic, and a bark extract is used to treat kidney diseases and to control bleeding after child-birth. The bark of V doniana is used in Kano traditional medicine as anti-epilepsy and against female sterility (Atawodi,2005).
Hexane, ethyl acetate and methanolic extracts of stem bark were tested in vitro for antimicrobial potential using the well diffusion technique. The extract showed broad spectrum activity with zones of inhibition ranging from 19 to 24mm(Eghareba,2010).The petroleum ether extract of the leaves gave the highest activity with a zone of inhibition of 20mm against Salmonella typhi. Other extracts had zones of inhibition ranging from 4mm-14mm against almost all the tested organisms (Dauda et.al.,2011). Extract of the leaves demonstrated (Mudi,2011) a remarkable antimalarial activity at all concentrations. The most interesting anti-plasmodial activity was obtained with the methanolic extract.
Vitex doniana extracts showed significant antimicrobial activity against S.typhi (Dawang et.al.,2012) . Laboratory tests demonstrated the presence of high concentrations of progestogen-like compounds in Vitex doniana , thus natural consumption of Vitex doniana was a likely cause of the observed increases in progestogen (James et.al.,2007). Ukwuani et. al.(2012) reported that the aqueous leaves extract of Vitex doniana has a significant antidiarrhoeal activity which supports its use in traditional herbal medicine practice and it was suggested (James et.al.,2010) that aqueous extract of Vitex doniana may exert anti-hepatotoxic effect against CCl 4 -induced liver injury in animal model and that this effect is concentration-dependent. Owolabi, et. al.(2011) suggested that the use of aqueous extract of Vitex doniana in the treatment of diabetes produce a significant (p < 0.05) antidiabetic and hepatoprotective effect.. Test organisms:-Methanolic extract of Vitex doniana leaves was screened for antibacterial and antifungal activities using the standard microorganisms shown in Table(1). Pseudomonas aeroginosa G-ve 5 Escherichia coli G-ve 6 Salomonella typhi G-ve 7 Candida albicans fungus Methods:-Phytochemical screening:-Preliminary phytochemical screening for secondary metabolites was conducted according to the method described by Harborne(1973).

Extraction of flavonoids:-
Powdered shade-dried leaves of Vitex doniana (2.5kg) were defatted with petroleum ether (40-60 O C) and macerated at room temperature with 70% aqueous methanol . The methanol extract was evaporated under reduced pressure and temperature to afford 95g of a solid mass.

Isolation of flavonoids:-
A sample (150 g) dissolved in 50 ml aqueous ethanol (70%) was carefully applied to the top of a column (150x3.5 cm) containing 750g of polyamide 6S. Gradient elution started with water followed by H 2 O/ EtOH mixtures of decreasing polarities at a flow rate of 1ml/ minute . The bands migrating along the column were traced under UV light during elution to note their characteristics and to control the fractionation process as well.Six fractionsl(each 100ml ) were collected, dried in vacuo at ≈ 40 o C and subjected to detailed investigations by two dimensional paper chromatography (2DPC).
Fraction IV was eluted with 60% EtOH and was found by (2DPC) to contain a mixture of flavonoids (dark purple spots on PC under UV light; turning yellowish with AlCl 3 ). Among the mixture one constituents (I) existed in appreciable concentration.
A sephadex column fractionation of fraction IV, using H 2 O/EtOH mixtures for elution led to desorption of subfraction (i) which was collected and dried in vacuo. 2DPC revealed the presence of component (I) . Repeated preparative PC of this sub-fraction on Whatman Paper( No. 3mm) and irrigation with BAW afforded chromatographically pure sample of compound (I) .
Antimicrobial assay:-Preparation of bacterial suspensions:-One ml aliquots of 24 hours broth culture of the test organisms were aseptically distributed onto nutrient agar slopes and incubated at 37°C for 24 hours.The bacterial growth was harvested and washed off with sterile normal saline, and finally suspended in 100 ml of normal saline to produce a suspension containing about 10 8 -10 9 colony forming units per ml. The suspension was stored in the refrigerator at 4°C until used. The average number of viable organism per ml of the stock suspension was determined by means of the surface viable counting technique.
Serial dilutions of the stock suspension were made in sterile normal saline in tubes and one drop volumes (0.02 ml) of the appropriate dilutions were transferred by adjustable volume micropipette onto the surface of dry nutrient agar plates. The plates were allowed to stand for two hours at room temperature for the drop to dry, and then incubated at 37°C for 24 hours.

Preparation of fungal suspensions:-
Fungal cultures were maintained on dextrose agar incubated at 25°C for four days. The fungal growth was harvested and washed with sterile normal saline, and the suspension was stored in the refrigerator until used.

Testing for antibacterial activity:-
The cup-plate agar diffusion method was adopted , with some minor modifications, to assess the antibacterial activity. (2ml) of the standardized bacterial stock suspension were mixed with 200 ml of sterile molten nutrient agar which was maintained at 45°C in a water bath. (20 ml) Aliquots of the incubated nutrient agar were distributed into sterile Petri dishes. The agar was left to settle and in each of these plates which were divided into two halves, two cups in each half (10 mm in diameter) were cut using sterile cork borer (No 4), each one of the halves was designed for one of the test solutions. Separate Petri dishes were designed for standard antibacterial chemotherapeutics(ampicillin and gentamycin).
The agar discs were removed, alternate cups were filled with 0.1 ml samples of test solution using adjustable volume microtiter pipette and allowed to diffuse at room temperature for two hours. The plates were then incubated in the upright position at 37°C for 24 hours.
The above procedure was repeated for different concentrations of the standard chemotherapeutics. After incubation, the diameters of the resultant growth inhibition zones were measured in triplicates and averaged.

Results and Discussion:-
Phytochemical screening:-Results of phytochemical screening of Vitex doniana leaves are depicted in Table 2.
The sodium methoxide spectrum (Fig.2) revealed a 50nm bathochromic shift in band I without decrease in intensity indicating a 4`-OH function (Harborne,1998).  (Figures 3 and 4 ).Such hydroxylation pattern is further confirmed by the boric acid spectrum .Boric acid spectrum of compound I (Fig.5) revealed a 25nm bathochromic shift in band I indicating a B ring catechol moiety. Since band II in the methanolic spectrum ( Fig.1) consists of two peaks the catechol is located at C 3`, C 4` ( Harborne,1998;Mabry et.al.1972) .   (Fig.6) when sodium acetate was added to a methanolic solution of compound I indicating a free 7-OH (Mabry et.al.,1972). The UV spectral data of compound I are summarized in Table (3). Table 3:-UV spectral data of compound I The 1 HNMR spectrum (Fig.7) gave a resonance at δ3.40(5H) which accounts for sugar protons . The anomeric proton resonated downfield relative to the rest of the sugar protons at δ4.65ppm.No signal for the vinylic proton of C 3 was observed in the spectrum and this postion seems to be the site of glycosylation. Acid hydrolysis followed by UV studies using the shift reagent sodium methoxide confirmed a C 3 -glycosylation. Acid hydrolysis followed by paper chromatography using authentic samples indicated the presence of glucose.The signal at δ1.10(3H) accounts for a methyl group,while the resonance at δ3.46(6H) was attributed to two methoxyl groups.On the basis of the retro Diels-Alder fission (Scheme I), one of these methoxyls was assigned to A ring , the other one exists in B-ring on the same basis(see scheme I). The double doublets at δ6.21 and δ 6.81ppm are characteristic of C 6 -and C 8 -protons respectively. The resonance at δ7.49ppm was assigned for C 2` and C 6`-protons which usually appear as a singlet in case of a C 3`,4`,5`-oxygenation (Harborne,1989) .The mass spectrum (Fig.8) gave m/z360 for the molecular ion of the aglycone .The glycoside rarely give discernible molecular ions (Mabry et.al. ,1973;Harborne,1989) .Other important fragment (m/z137,149 and m/z181)resulting from the retro Diels-Alder fission (scheme I) of the aglycone were also recorded in the electron beam.These fragments which result from intact A and B rings site evidence for the suggested substitution pattern in the flavone nucleus.  622 Comparison of the above cumulative data with literature data resulted in the following structure for compound I: Compound I The C 6 methylation was dictated by HSQC data which indicated direct connectivity of the methyl function to C 6 .The assigment of the methoxyl functions at C 5 and C 5` was based on 1 H-1 H COSY NMR experiments which indicated long range coupling between a methoxyl function and C 6methyl group ,and another long range coupling between a methoxyl group and C 6` -proton . The retro Diels-Alder cleavage of the aglycone is shown in scheme I.  Diameters of the growth inhibition zones for standard chemotherapeutic agents are depicted in Tables (5) and (6). The methanolic extract exhibited significant antibacterial activity against Gram negative bacteria Pseudomonas aeruginosa.It also showed activity against Gram negative bacteria Salmonella typhi and the fungus Candida albicans. Thus this extract is a promising candidate for future optimization.   Antioxidants activity of Vitex doniana methanolic extract:-Evaluation of the antioxidant activity was carried out by measuring the capacity of the methanolic extract against stable DPPH radical.The methanolic extract of Vitex doniana showed high value for absorbance inhibition at a concentration of 64.2 μg/ml (Table 7 and Fig.9). It could be concluded from Figure 9 that the Vitex doniana extract showed high antioxidant capacity which is comparable to the value exhibited by the positive reference.