ISOLATION, CHEMICAL COMPOSITION, CHARACTERIZATION AND ANTI-BACTERIAL ACTIVITY OF ACRIDINE DIGLYCOSIDE FROM MORINGA OLIFERA Ikpa,

Moringa olifera (drumstick tree) is a medicinal plant commonly used in phytomedicine to cure and prevent diseases in Nigeria. The ethanolic extract of the leave sample showed alkaloid {(9.66 ± 0.20) %}, phenols {(0.75 ± 0.22) %}, flavonoids {(6.86 ± 0.20) %}, saponines {(8.46 ± 0.10) %}, and tannins {(1.10 ± 0.20) %}. The plant also contains nutritional nutrients such as ash {(6.08 ± 0.02) %}, protein {(23.65 ± 0.12)%}, carbohydrates {(57.01 ± 0.01)%}, fats {(2.43 ± 0.01) %}, crude fibre {(6.02 ± 0.13)%}, moisture {(9.50 ± 0.10)%} and energy {(3276 ± 0.14)%}. Antibacterial studies showed that the plant leave successfully inhibited staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Klebisiella pneumonia. Acridine diglycoside was elucidated using Bruker NMR spectroscopy in combination with FT-IR NATRIC and HREIMS (m/z) mass spectral data. This result authenticates the use of the plant leave in the treatment of infections, tumors and as an anti-inflammatory agent


Introduction
Moringa oleifera, the most widely cultivated species of moringa plants is a multipurpose tree cultivated throughout the tropics [1]. M.olifera, commonly known as drumstick tree, horseradish tree or benzoil tree [2] is used in herbal medicine and can also be used for water purification and hand washing [3]. M. oleifera is used as an alternative source for nutritional supplements and growth promoters in some countries [4]. Apart from nutritional benefits, M. Oleifera has been reported to be used for the treatment of rheumatism, ascites, infection, hiccough influenza and internal abscess [5].
The leaf extract is capable of reducing hyperglycaemia, cholesterol lowering [9], and dyslipidemia [6]. Important medicinal properties of the M. olifera which have been recently reported include antipyretic, antiepileptic, anti-inflammatory, anti-ulcerative [7], anti-hypertensive [8], anti-oxidant [10], anti-diabetic, hepatoprotective [11], anti-bacterial and anti-fungal activities [10]. In addition, M. oleifera seed possesses water purifying power [12,13]. The plant species have long been recognized by folk medicine practitioners as having value in the treatment of tumours [14]. The edible Moringa leaves contain essential provitamins, including ascorbic acid and carotenoids [15]. Various parts of the plant such as the leaves, roots, seed, bark, fruit, flowers and immature pods act as cardiac and circulatory stimulants, and also possesses anti-tumour agents [16].
Extraction and isolation of the plant sample was done by drying the leaves at room temperature for two weeks. The leaves were grounded to powder and stored in an amber bottle. Phytochemicals and proximate analysis were done with some of the powdered leave sample, while the rest were percolated with 2L of ethanol for four days. The extract was filtered and concentrated with rota at 35 0 C-40 0 C.

Proximate analysis.
Moisture content of the sample was determined using the method described by AOAC [17]. One gram of sample in pre-weighed crucible was placed in an oven (105°C) for 24 h, cooled, and reweighed. The percentage moisture was calculated as follows: Moisture (%) = w 2 -w 3 ⁄ w 2 -w 1 × 100 (w 1 = weight of the crucible, w 2 = weight of the crucible and sample after drying, and w 3 = weight of the crucible and the sample after cooling in airtight desiccators. The ash content was determined using a weighed (1.0g) sample which was subjected to dry ashing in a well cleaned proclaim crucible at 550°C in a muffle furnace for 2 hours. The ash percentage was calculated as follows: Ash (%) = w2-w3⁄ w2-w1 × 100. Where, w1 = weight of the crucible, w 2 is the weight of the crucible and sample ash at 550 0 C, and w 3 = weight of the crucible and the sample ash after cooling in airtight desiccators [18].
Crude protein was determined using the micro-Kjeldahl method as reported by Fahey [18]. The crude protein was calculated by multiplying nitrogen with the conversion factor of 6.25 [P% = TN x 6.25]. Fat content was determined using the method described by Folch, and Stanley [19]. Homogenized tissue (10g) was progressively added to small amounts of a chloroform/methanol 2:1 (v/v) mixture (up to 200 ml), with vigorous shaking, and then the extraction was carried on for a further 2 hours, using an electromagnetic stirrer. The mixture was filtered and the filter was rewashed with fresh solvent and pressed. Fifty millilitres of 0.88% potassium chloride were added and the mixture was shaken. The aqueous layer (upper) was removed by aspiration and the washing procedure was repeated. The extract was then dried by adding anhydrous sodium sulphate, which was filtered again before the solvent was removed using a rotary evaporator. The extract was then placed in desiccators overnight and weighed.
Crude fibre was determined using 1.0g of sample (W 2 ) which was transferred directly into a filter bag and sealed with a heat sealer. Sample and blank bags were immersed in enough amount petroleum ether for 10 minutes to extract fat content from samples. All bags were air dried and transferred to a Ankom 2000 Fiber Analyzer using H 2 SO 4 and NaOH and the crude fibre was calculated according to the following equation: % Crude fibre = 100 x (W 3 -(W 1 x C 1 )) ⁄W 2 Where: W 1 =Bag tare weight W 2 =Sample Weight W 3 =Weight of Organic matter (Loss of weight on ignition of bag and fibre) C 1 =Ash corrected blank bag factor (running average of loss of weight on ignition of blank bag/original blank bag) [20].
Carbohydrate content was determined by difference, that is, addition of all the percentages of moisture, fat, crude protein, ash, and crude fibre were subtracted from 100%, while the sample energy value was estimated (in KCal/g) by multiplying the percentages of protein, lipid, and carbohydrate [21].
Phytochemical evaluation.The M. olifera plant leave sample was evaluated for alkaloids and phenols according to Harbon [22]. While flavonoids were evaluated as described by Boham [23]. The method described by Van-burden and Robinson was adopted for the determination of tannins [24]. Saponines were determined as reported by Obadoni [25].

FRACTIONAL SEPARATION OF THE COMPOUNDS
The concentrated ethanol was dried to obtain dark green gummy crude extract and a portion was partitioned between chloroform and water to obtain chloroform and water fractions. The dried chloroform fraction was partitioned between methanol and petroleum ether. The possible number of compounds in each fraction was determined using TLC. Methanol fraction showed the highest zone of inhibition in antibacterial activity and was subjected to column chromatography over silica gel Merck grade (60-120 mesh). The labelled eluted fractions were analysed with TLC and fraction M6

International Journal of Pharmacology, Phytochemistry and Ethnomedicine
Vol. 2 which is a dark green oil gave one spot with R f value of 0.74. The pure dark green oil was further analysed and found to be acridine diglycoside using IR Perkin Elmer model 760. The mass was determined with HREIMS positive ion mode using Bruker multinuclear NMR experiments of 1 H and 13 C.

EVALUATION OF ANTIBACTERIAL ACTIVITY OF M. OLEIFERA LEAVES
Preparation of the inoculums. The standard clinical isolated organisms of Pseudomonas aeruginosa (P. Aeruginosa), Staphylococcus aurous (Staph), Escherichia coli (E-coli), klebsiella pneumonia (Kleb) and Proteus mirabilis (Proteus) were obtained from Federal Medical Centre, Owerri and the analysis was carried at Department of Medical Science Laboratory Imo state University. The strains of the organisms were propagated on nutrient agar plates and maintained at 4 0 c. The isolates were sub-cultured in nutrient broth at 37 0 C for 8h prior to antibacterial testing.
Antibacterial sensitivity testing of compounds. Agar well diffusion technique was used to determine the antibacterial activity of the fractions of the leave sample. Sensitivity test of the agar plates were by inoculating with 0.1ml of an overnight culture of each bacteria strain (equivalent to 10 8 CFU/ml -1 ). The inoculated agar plates were allowed to dry and were appropriately labelled. Using a plastic cork borer of 6mm in diameter uniformed wells was bored in the inoculated nutrient agar. With a micropipette, 300µl of 20mg/ml of each fraction of the leave sample was delivered into each well. The plates were left on the bench for 30 minutes to allow the compound to diffuse into the agar. Thereafter, the plates were incubated at 37˚C for 24 hours. After incubation, the plates were observed for inhibition zones around the wells. The diameters of the zones were measured with metre rule to the nearest whole millimetre [26].

RESULTS AND DISCUSSION
The results of proximate analyses (in %) as shown in table 1 indicates that the Moringa oleifera leaves contains carbohydrate (57.01±0.01), protein (23.65±0.12), fats (2.43±0.01), moisture (9.50±0.10), crude fibre (6.20±0.13), ash (6.08±0.02) and energy (3276.34±0.14) revealing an excellent source of nutrition and natural energy which can be used as a good nutritional supplements.  The result of phytochemical analysis of M.oleifere leaves is presented in table 2. The high percentage of alkaloids, flavonoids and saponini contents of M.Oleifera supports the use of the plant leaves for the treatment of bacterial infections, allergies, inflammation, tumour, viruses and cancer [27]. Results of anti-bacterial activities of M.oleifera is presented in table 3.The anti-bacterial activity showed that the ethanol and methanol fractions showed higher zone of inhibition (in mm) than other fractions. The result supports the use of M.oleifera for the treatment of wound [28], which justifies the traditional use of M.oleifera in herbal treatments [3].  The presence of acridine derivatives which acts as anti-bacterial, anti-parasitic and anti-cancer agents [29], supports the use of M.Oleifera in the treatment of bacterial, parasitic infection [10], cancer and viral attacks [7,14]. The isolated compound Acridine diglycoside can be used by pharmaceutical firms for drug formulation.

Conclusion
This work showed that M. Olifera is an all-purpose plant. The leaves have very high nutritional value and a good source of protein, fiber, carbohydrate, fats and energy. M olifera is very important for its many impressive ranges of medicinal uses. Antibacterial studies of the plant leave proved that it could successfully inhibit the growth of staphylococcus aureus, escherichia coli, pseudomonas aeruginosa, proteus mirabilis and klebisiella pneumonia. The isolated compound Acridine diglycoside, which was elucidated with Bruker NMR spectroscopy in combination with FT-IR NATRIC and HREIMS (m/z) mass spectral data could be used by pharmaceutical by firms for drug formulation