ISOLATION AND CHARACTERIZATION OF BIOACTIVE CONSTITUENT OF N-BUTANOL SEED EXTRACT OF Azadirachta indica

Extraction of the seed powdered material of Azadirachta indica followed by an extensive column chromatography of the n-butanol portion on silica gel, purification over sephadex LH20 and subsequently HPTLC resulted in to the isolation of a pale yellowish solid coded as compound MND. The isolated bioactive constituent was found to have mp of 222-224oC. The structure was elucidated using a combination of 500 MHZ and 125MHZ 1-D and 2-D NMR techniques (COSY, NOESY, HSQC, DEPT and HMBC). Thus, the isolate (MND) was determined as 4a, 7 – dihydroxy – 7 – methyl – 1 – (3, 4, 5 – trihydroxy – 6– hydro methyl – tetrahydropyran – 2 – yloxy) – 1 -, 4a, 5, 6, 7, 7a, hexahydro – cyclopenta [C] pyran – 4 – carboxylic acid methyl ester (Ipolamiide). The Antimicrobial properties of the compound MND and partition portion of the extracts were tested against S. aureus, S. pyogenes, P. vulgari, P. aeruginosa, K. pneumoniae, E. coli, S. typhi, P. digitatum, C. albicans and P. nototum. The antimicrobial sensitivity test indicated that various partition portion of the extracts inhibited the growth of S. aureus , S. pyogenes P. aeruginosa, E. coli, S. typhi, K. pneumoniaer, P. digitatum, C. albicans and P. nototum with 42mm, 38mm, 31mm, 29mm, 22mm, 30mm, 26mm, 27mm , 21 mm and 20 mm while the highest activity of the isolate (MND) was exhibited against S. aureus, P. aeruginosa, E. coli and S. typhi with 42 mm, 38 mm, 31mm and 39mm respectively.


INTRODUCTION
Natural products research of plants with pharmaceutical value represents a key approach toward the development of new pharmaceutical products.The use of plants with therapeutic value for the treatment of diseases has been in practice for a long time and is documented (Twaij and Hasan, 2022).Humans, since primeval times have learnt to derive chemicals from plants and use them for therapeutic purposes (Jayawardene et al., 2021).The action of drugs in biological systems results from binding with receptors or enzymes.The biosynthetically altered active components of local plant extracts have enable them to bind successfully to human proteins in a process termed 'evolutionary molecular modeling'.Bioactive plant products can bind to enzymes and receptors thereby obstructing or stimulating them (Tsado et al., 2018;Abubakar et al., 2015a).Natural products today as in times of old are having great influence on treatments for ailments and health status of people.Natural products have made significant contributions to the evolution of the pharmaceutical industry, as many drugs have been developed from natural products as precursors (Twaij and Hasan, 2022).World Health Organization acknowledged herbal remedies and have not frowned at developing countries who have incorporate the use of safe herbal medicine in their health system (Benarba and Pandiella, 2020).The patronage for natural products is further enhanced by their availability, affordability 'perceived' efficacy in treatment of some ailments (Welz et al., 2018).Neem (Azadirachta indica) is one of those plants that have wide applications among locals especially in treatment of some ailments.
Azadirachta.indica is a fast-growing plant of the mahogany family (Meliaceae), it can reach can reach a height of 15-30 metres (49-98 feet).The leaves are evergreen toothed leaflets which drops amidst periods of drought.Several products from the plant, especially extracts have been used for medical purposes and in cosmetics.The plant's resilient nature allows it to grow well even in poor and rocky grounds.Neem is known to endure a wide variation in environmental conditions but does not grow well in waterlogged soils or freezing temperatures.The flowers are borne in clusters in the axils of the leaves with smooth yellow-green fruit (Petruzzello, 2022).Neem (A. indica) can be propagated from cutting suckers or from seed.It is grown for different purpose that include source of wood, provision of shade and reforestation; products obtained from A. indica have found applications in antiseptics, antipyretic and some in beauty products (Moin, et al., 2021;Adem et al., 2011).
Oil obtained from the seed have been used in the production detergent, soap and toothpaste.Other by-products have found application in the production of fertilizers.According to Imanuddin et al. (2020), A. indica contain about 300 secondary metabolites that are accountable for its properties.Research has shown over the years that neem seed is essentially non-toxic to vertebrate and it is the most potent growth regulator and feeding ever assayed (Wylie and Merrell, 2022).Neem seed is reported to be rich protolimonoids, fatty acid, cyclic tri-and tetrasulphides, alkyl sulphides and modified apoeuphol tetranortriterpenoids (Torres-Contreras, et al., 2022;Lin et al., 2021;Aarthy et al., 2018).In this research, a bioactive principle MND was isolated from the n-butanol seed extract of A. indica and the isolate was tested for its biological activities against the aforementioned microbes.

Experimental
Perkin-Elmer (Model 341 LC) spectrometer was used to measure optical rotations at room temperature. 1H NMR and  The purity of the isolates was checked and maintained in slants of nutrient agar and saborand dextrose for bacteria and fungi respectively.

Extraction and Isolation
The powdered sample was macerated using the cold maceration technique using MeOH 100 % (3.0 dm 3 ) at 45 o C for 48 hrs with intermittent shaking.The extract was concentrated at low pressure to dryness in order to obtain semi solid material.It was resuspended in water (800 cm 3 ) and exhaustively partitioned consecutively with n-hexane, (3×500 cm 3 ), chloroform(3×600 cm 3 ), ethyl acetate (3×400 cm 3 ) and n-butanol (5×400 cm 3 ).The various partition portions of the extracts were concentrated by use of rotary evaporator to obtain n-hexane, (5.92 g), chloroform (3.15 g) ethyl acetate (4.25 g), nbutanol (5.20 g) and aqueous (7.22 g) residues respectively.The different fractions of the extract obtained were subjected to preliminary phytochemical screening according standard methods (Sofi and Nabi, 2018;Chóez-Guaranda et al. 2022).
FA (72 mg) with two homogeneous spots was concentrated and further resubmitted for HPTLC analysis.The analysis was done using Fluka silica gel pre-coated glass plates 20×20 cm having thickness layer of 0.25mm.A thin line of about 1.5 cm was drawn with a pencil from the bottom of the plate.Pooled sample of FA (72 mg) was dissolved in MeOH to obtain concentration of 20 mg/cm 3 .It was then uniformly applied along the thin line with the aid of capillary tube.The plate was then left to dry before it was developed with an appropriate solvent system.The plate which developed was dried under air in fume cupboard, pencil was used to mark the position of the band of interest then scraped off the back of the plate onto a foil.The size of the scraped sorbent was reduced using pestle and mortar, then transferred onto a sintered glass funnel and repeatedly washed with Acetone, followed by evaporation of the solution obtained to give a pale yellowish isolate coded as compound MND (40.3 mg, Rf0.6).Elution progress was monitored with TLC using pre-coated plate in different solvent systems; n-hexane:Ethylacetate (80:20), chloroform:ethylacetate (65:45) and EtOAC:MeOH (70:30).The chromatogram obtained was spread with 10 % H2S04 and kept in an oven at of 105 o C for 5min then removed to ascertain the compound on the plate (Mohammed, 2022).

Ferric Chloride Test
About 5.0 % iron(III)chloride in 0.5 N hydrochloric acid was spurted on the chromatogram, fluka-silica gel precoated glass plate of compound MND.This test was to check for the presence of phenolic compounds (Abubakar et al., 2015b).

Vanillin/Sulphuric Acid Test
4.0 g solution of vanillin was dissolved in 100 cm 3 of H2SO4.This solution was then spread using spray canister on the chromatogram precoated glass plate of compounds MND in a fume chamber.The plate was then heated in an oven at 110 o C for 5 -10 minutes before it was removed from the oven to ascertain color formed (Mohammed et al., 2019).

Liebermann Buchard's Test.
1 cm 3 of CH3COOH was added to 1 cm 3 of chloroform and cooled in a test tube to 0 o C. Then drops of conc.H2SO4 were added to the test tube which contain solution of compound MND (Mohammed, 2022).

Determination of Sugar in Compound MND
Compound MND (3.5 mg) was dissolved in 2.5 cm 3 of water; 2N solution of CH2F2-COOH (2.5 cm 3 ) was added and then refluxed on a water bath for 3 hours, after which the mixture was diluted with10 cm 3 of water then extracted with CH2Cl2 (2 x 5 10 cm 3 ).The extracts of CH2Cl2 were washed with water before evaporation to dryness in vacuo.The concentrated aqueous layer then was passed through Amberlite column (short) before evaporating to dryness in order to give sugar fraction (1.5 mg).These were analyzed with HPLC using CH3CN/H2O (85:15).Co-TLC of the sample was carried out and the sugar was analyzed with silica gel TLC in comparison to standard sugar using solvent system (Tsado et al. 2019).

Antimicrobial Assay
0.8g of the various partition portion of the extract were weighed separately and dissolved in 10.0 cm 3 of DMSO to get a concentration of 80.0 mg/cm 3 .This concentration was the initial for the extract used to determine the antimicrobial activities of these extracts (Abu-Reidah and Taamalli, 2022).Mueller Hinton agar medium was prepared using method in the manufacturer's Instruction; sterilized at temperature of 121 o C for 15 min, the medium sterilized was poured into sterile Petri dishes, the plates were then allowed to solidify on cooling.The extracts were screened using diffusion method.The medium was seeded with 0.1 cm 3 of standard inoculums of test microbes.The inoculums were evenly spread on the surface of the medium using sterile swab.After setting, the use of standard cork borer of a number 4 sterile cork borer of 6 mm in diameter were gotten, a well was cut at the middle of each inoculated plate medium.The medium inoculated was incubated at a temperature of 37 o C for 24 hrs.Each plate was observed for zone of inhibition of growth.Transparent ruler was used to measure the zone and the result recorded in millimeters (Adamu and Sajo, 2021).
The minimum inhibition concentration of the various partition portions of extracts were obtained with the use of broth dilution method.10 cm 3 of prepared Mueller Hinton broth was dispensed into a test tube, then sterilized at 121 o C for 15 min and left to cool.Mcfarlands turbidity standard number 0.5 was prepared to give turbid solution.10 cm 3 of prepared normal saline was dispensed into sterile test tubes and the test microbes inoculated and incubated at a temperature of 37 o C for 6 hrs.Test microbes in the normal saline were diluted until turbidity marched that of the Mc-Farland's scale using visual comparison; at this point the test microbe's concentration was about 1.5 x 10 8 cfu/ml (Tsado et al., 2018a).
Two fold serial dilution of the extract in the sterile broth was done to obtain concentrations of 80 mg/cm 3 , 40 mg/cm 3 , 20 mg/cm 3 , 10 mg/cm 3 and 5 mg/cm 3 respectively.0.8 g of the extract was dissolved in 10 cm 3 each of the sterile broth to obtain the initial concentration.0.1 cm 3 of standard inoculums of the test microbe was inoculated into different concentrations of the extract in the broth.Incubation was done at a temperature of 37 o C for 24 hrs, thereafter the test tubes were observed for growth (turbidity).The lowest concentration of the extract in the broth that did not show turbidity was noted as the minimum inhibition concentration (Ohikhena et al., 2017).
Minimum bactericidal and fungicidal concentrations were determined to check if the test microbes have been killed or only growth was inhibited.Prepared Mueller Hinton agar was poured into sterile Petri dishes, allowed to cool and solidify.Contents of the MIC in the serial dilutions were subcultured onto the prepared medium; incubation was made at temperature of 37 o C for 24 hrs, then each of the plates was observed carefully for colony growth.Plate having the lowest concentration of extract without colony growth can be considered the MBC for the bacteria and MFC for the fungi (Ohikhena et al., 2017).
The method of Mohammed (2022) was employed for the analysis of the isolated compound, MND.Organisms used were the same as those above.The medium of choice was Tryptic say Agar (Merck KGa A), it was prepared according to instructions of manufacturer.This was dispensed in sterile plates in 20 cm 3 aliquots after gelling and drying, the plates were seeded with the test microbes by streaking evenly in a cotton swab.The inoculums were allowed 5 min to dry, sterile filter paper disks (4 mm) earlier soaked with the isolated compound MND in (4µl/disk) placed and pressed down gently to ensure contact.
Table 2 present results of the phytochemical screening of partitioned portion of the seed extract.The results of the various extracts indicated the presence of tannins, alkaloids, flavonoids, carotenoids and glycosides in the methanol and water fractions but were not detected in the hexane extract.The distribution of the studied phytochemicals in the solvents used was dependent upon their polarities and those of the extracting solvents (Ndamitso et al., 2013).These phytochemicals are ubiquitous in plants and are usually common in human diet.They are known to inhibit microbes which could be resistance to orthodox antibiotics.Flavonoids' free -radical scavenging property has given rise to multiple biological functions that include anti-bactericidal, anti-inflammatory, anti-carcinogenic, immune stimulatory, vasodialotory, anti-allergic and anti-viral functions (Agidew, 2022).Consequent, the presence of flavonoids in the ethyl acetate and n-butanol extract in this study has shown the therapeutic efficacy of the extract and has given credence to the folkloric use of the plant for treatment of ailments especially inhibitory effect on S. aureus, K. pneumoniaer, S. typhi, E. coli and P. aeruginosa.Tannins have been reported to inhibit the growth of microorganisms by precipitating out the microbial protein hence depriving them growth (Deghima, et al., 2021;Huang, et al., 2018;Mujeeb et al., 2014).Tannins were present in the methanol, ethyl acetate and n-butanol fractions of the extract.This explains the good antimicrobial activity of the extract on tested pathogens (Huang et al., 2018).The presence of carbohydrate and reducing sugars in the extract of A. indica seed indicate that energy content was high and could be a source of raw material for food and drug industries that utilized carbohydrates reducing sugars (Deghima, et al., 2021).Saponin was detected in some portions of the extract and was found to produce antifungal activity against C. albican (Porte et al., 2022;Działo et al., 2016).
The antibacterial activity of the n-butanol fraction of the seed extract was found to be fairly good against gram (+) bacteria e.g.The antimicrobial effects of the extracts observed on the micro-organisms could be attributed to the presence of aforementioned secondary metabolites present in the plant (Huang, et al., 2018).Tannins are known to exhibit great potential in phytomedicines, as astrigent as well as anti-parasitic propertry (Deghima, et al., 2021).Terpenes have been reportedly used as anti-tumor and an antiviral agent; some are known to be cytotoxic to tumor cells.The eusdesmane sesquiterpenes are reported to exhibit high antibacterial properties (Działo, et al., 2016;Tanko, et al., 2020).Saponins, have anti-oxidant, anticancer, anti-inflammatory and anti-viral properties (Agidew, 2022) while flavonoids are known to exhibit anti-inflammatory properties (Panche et al., 2016).The large zone of inhibition exhibited by the MND against S. aureus, S. pyogenes, P. aeruginosa, E. coli,Kleb.P. and S. typhi justifies the use of A. indica by traditional healers.The presence of alkaloids also add to the medicinal importance of A. indica as significant quantities have been used locally as analgesic, anti-malarial and as stimulants (Sharifi-Rad et al., 2019).Phenolic compounds which represent varieties of natural antioxidants, have been used as nutraceuticals and also in control of human pathogenic (Mutha et al., 2021).
E.coli have been known to be a common cause of diarrhea, infant death and other diarrheagenic infections in humans.The moderate growth inhibition against E. coli might be attributed for the use of the seed portion of the extract to treat diarrhea and dysentery.(Diggle and Whiteley, 2020;Kumar and Goel, 2019).The high MIC of S. aureus is important in the health care sector, this is because it could be used as an alternative to orthodox antibiotics for treatment of infections caused by the microbes (Gnanamani et al., 2017).S. aureus is also known to play an important role in causing skin diseases including superficial and deep follicular lesion.The MIC exhibited by MND extract against S. aureus can be important in health care delivery, since it can be used as alternative to Orthodox antibiotics in the treatment of infections caused by these microbes (Tong et al., 2015).The use of these extracts and MND against S. aureus, E. coli, K. pneumoniea and S. typhi would reduce the cost of healthcare, since A. indica seeds are readily available and affordable (Shaba et al., 2013).

CONCLUSION
The presence of the studied metabolites in the seed portion of A. indica suggests great potential in the extract as source of phytomedicines.The zone of inhibition shown by the various extracts and MND against S. aureus, P. pyogenes, K. Pneumoniea, E. coli and S. typhi, justifies its use in traditional medical practice for the treatment of sores, boils and dysentery.The strong activity of the isolate and n-butanol extract on the test microbes, shows that, the seed extract of A. indica can be a good source of compounds that are effective against some infectious diseases causative agents.The isolated MND have exhibited impressive activity against some of the test microbes, especially when the isolate is less combine with other secondary metabolites.Therefore, the observed antimicrobial properties of the seed extract of A. indica corroborate its use in the ethno medicine.
Plate I: A. indica tree Plate II: A. indica seed MATERIALS AND METHODS Sample Collection and Treatment Seeds of Azadirachta indica (Meliaceae) plant were obtained from a farm in Lapai town in Niger State, Nigeria.The plant was identified at the Department of Biological Sciences, Ahmadu Bello University (A.B.U.), Zaria, Nigeria.The seeds were decorticated and sliced into small pieces, then dried for 7 days at room temperature after which they were ground into powder.

Figure 1
Figure1present TLC results of isolate MND in different solvent system.Extraction of the seed portion of A. indica followed by column chromatography of n-butanol portion of the extract on silica gel, then purification over sephadex LH-20 folloed by (HPTLC) lead to the isolation of compound MND.The isolate gave positive colour when tested with ferric chloride indicating the compound is phenolic(Mottaghipisheh and Iriti, 2020).It also gave

Table 2 :
The results of phytochemical screening of the partition portions of the seed extract of the plant Azadirachta indica S. aureus and gram (-ve) bacteria e.g.K. pneumonia and E. coli.The antifungal activity of ethyl acetate and n-butanol fraction of the seed extract was found to have little activity against P. notatum.The antimicrobial sensitivity test indicated that, the extracts inhibited the growth of S. aureus S. pyogenes P. aeruginosa, E. coli, S. typhi, C. albicans and P. digitali.The extracts did not inhibit the growth of B. subtilis, A. niger and F. oxysorum.The increase in concentration of the extract also increases the zone of growth inhibition of some of the micro-organism.The highest growth inhibition of 42mm,38 mm,31 mm mm,39 mm diameter, was exhibited by (4µl/ disk), 80mg/ml of compound MND against S. aureus, S. pyogenes P. aeruginosa S. typhi and E. coli respectively.The lowest zone of growth inhibition was observed with 20 mm and 21 mm diameter as against C. albicans and P. notatum from the MND (4µl/disk) and (80mg/ml).The highest minimum inhibition concentrations of the extracts on the test isolate was exhibited against P. aeruginosa, E. coli and S. aureus from n-butanol (10 mg/cm 3 ).The lowest MIC recorded was against the test Microbes from the n-hexane fraction of the methanol extract.

Table 3 :
Antimicrobial Assay of some microorganisms against MND and various seed extracts of A. indica with their zone of inhibition (mm)