PHYTOCHEMICALS, VITAMINS,MACRO AND MICRO ELEMENTS AND ANTIMICROBIAL ANALYSIS OF THE STEM BARK OF NAPOLEONA VOGELII(AKPAESU)

Sequel to the increasing application of plant materials in ethnomedicine and for nutritive purposes the phytochemicals, vitamins, macro and micro elements and antimicrobial analysis of the stem bark of Napoleonavogelii were undertaken using standard methods. The result shows that Napoleonavogelii contains bioactive phytochemicals such as tannin (0.45±0.25%), phytosterols (12.53±0.25%), alkaloids (4.42±0.23%), flavonoids (9.27±0.18%), saponins (4.27±0.25%), hydrogen cyanide (0.37±0.20%). The vitamin analysis shows that the stem bark of Napoleonavogelii contains vitamin A (1.20±0.03Mg/g), vitamin E (1.31±0.04 Mg/g), vitamin C (2.78±0.04 Mg/g), vitamin B1(7.20±0.05 Mg/g), vitamin B2 (0.90±0.31 Mg/g), vitamin B9(4.09±0.05 Mg/g), vitamin B3(1.27±0.25 Mg/g), vitamin B6 (12.50±0.04 Mg/g) and vitaminB12 (8.25±0.04Mg/g) in varying amounts while vitaminB5 and vitaminB7 wereabsent. Sodium(1247mg/kg), Calcium (2006mg/kg),Copper(1.23mg/kg),Phosphorous(410.22mg/kg), Iron(304.660mg/kg), Manganese(56.99mg/kg) and Zinc(2.69mg/kg) were the detectable mineral elements, Potassium (4996mg/kg) had the highest value while Arsenic and Nickel were below detection limit (0.001mg/kg).The result of the antimicrobial screening of the ethanol, butanol and chloroform extracts of the stem bark of Napoleonavogelii against five pathogenic microbes, Escherichia coli , pseudomonas aeruginosa, streptococcus sp , Staphylococcus aureus , and Candida albicans shows that the butanol extract showed higher antimicrobial activities compared to the other extracts. Results from this study have shown that the stem bark of Napoleonavogelii contains medicinal properties. The antimicrobial activity of the extracts were studied in different concentration (50,100,150,200) against four pathogenic bacterial strains, two Gram-positive (Staphylococcus aureus, Streptococcus sp.) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa ), and a fungus strain Candida albicans). Antibacterial and antifungal potential of extracts were assessed in terms of zone of inhibition of bacterial growth. The growth inhibition zone measured ranged from 7 to 19 mm for all the sensitive bacteria, and ranged from 9 to 21 mm for fungal strain. In this study, the results showed that the three extracts of Napoleonavogelii possess antimicrobial activities against the tested organisms at different concentrations. With the butanol extract, the highest antimicrobial activity of 18mm in E.coli and least activity of 8mm in streptococcus were recorded. With the ethanol extract, the highest antimicrobial activity of 17mm in pseudomonas and least activity of 8mm in staphylococcuswas recorded. With the chloroform extract, the highest antimicrobial activity of 15mm in streptococcus and least activity of 8mm in staphylococcus were recorded. Butanol extract demonstrated a better antifugal activity than the other extracts. The results of the present investigation clearly indicates that the antibacterial and antifungal activity vary with the different solvent used in extraction. Thus the study ascertains the value of solvents used for extraction which could be of considerable interest to the development of new drugs. The result of this study revealed the presence of most of the phytochemicals with various biological activities. This might be responsible for the observed antimicrobial activities against test organisms. The important minerals found in the stem bark may also be major contributors to the medicinal use of the plant. This data indicates that Napoleonavogelii stem bark contains potent bioactive compounds and justifies the claimed use of the stem bark in the traditional system of medicine to treat infectious diseases such as skin diseases caused by microbes


INTRODUCTION
Plants are composed of vast array of phytochemicals and vitamins that characterize their pharmacologic properties and nutritional values [1]. Phytochemicals are naturally synthesized in all parts of the plant and may contain active components [2].Phytochemicals have been recognized as the basis for traditional herbal medicine practiced in the past and currently in parts of the world [3]. Plants and phytochemical compounds are used in folk medicine for the treatment of different types of diseases [4]. A typical example is the Napoleonavogelli whose methanol leave extract of is used in the treatment of peptic ulcer diseases [5] and the bark is used for treatment of skin diseases by the local folks [6]. Napoleonavogelii is an evergreen and widely distributed plant with alternate leaves, usually crowded towards the apex of the branches. About 20 genera and 450 species have been identified in the tropical regions of Africa, Asia and Australia. Its region of distribution is mostly in African countries of Nigeria, Ghana, Guinea, Togo, Benin and Ivory-coast [7]. Napoleonavogelii, (Lecythidaceae) is known locally as akpuruke, mkpodu, or odure by the Ibos of South-East Nigeria. Its common names are Akpako, Boribons, Burukwa. The plant is found mostly in rain forest, along the sea shores extending from Sierra Leone to Nigeria. It is an ornamental shrub or tree 2.5-5cm tall. The plant grows best in full sun but tolerates light shade. It prefers warm and humid climate and requires medium water/regular moderate drought tolerance. Its fruits are sweet and edible and the fruits ripe in dry season [8].

Sample Collection
Fresh sample of Napoleonavogelii stems were collected from Uturu, in Isuikwuato Local Government Area of Abia State. The plant was identified by Mr. IbeKaluNdukwe of Michael Okpara University of Agriculture, Umudike, Abia State.

Micro-Organisms Collections
Pure cultures of bacterial strains, Escherichia coli, pseudomonas aeruginosa, streptococcus sp, Staphylococcus aureus, and fungal strain of Candida albicans were obtained from vetenary diagnosing laboratory veterinary research institute out station, Umudike, Abia state.

Preparation of Plant Materials
Freshly collected Napoleonavogelii stem bark was washed and dried under the shade at normal room temperature for 14 days. Upon drying, the stem bark was blended to powder. The powdered sample was then stored in air tight containers and kept under normal room temperature until required.

Determination of Minerals
The method described by [9] was adopted. 1g of the sample was weighed into a beaker and 100ml of digestion mixture(nitric acid: sulphuric acid: perchloric acid) in the ratio 2:2:1 was added and the mixture was hated on a hot plate in a fume hood at 105ºC until the production of white fumes which indicates complete digestion. 10ml of the de-ionized water was added to the digestateand was allowed to cool down to room temperature and filtered using No 42 watman filter paper into 50ml volumetric flask. The digestate was made to mark with de-ionized water and analysis of heavy metals was performed using Atomic Absorption Spectrophotometer AAS-Biotech 896,UK.

Determination of Phytochemicals
The plant extracts were screened for the presence of some phytochemicals using standard methods as described by [10] and [11].

Antibacterial Activity
Agar well diffusion method as described by [9] was employed to assay for the antibacterial activity.

QUALITATIVE PHYTOCHEMICAL SCREENING
The result of the qualitative phytochemical screening of the stem bark of Napoleonavogelii is shown in table 1. The result for the qualitative phytochemical analysis is presented on Table 1. The result revealed the presence of different phytochemicals that slightly varied with the solvent used for the extraction [15]. The presence of alkaloids, saponins, and tannins in the stem bark extracts of Napoleonavogelii has medicinal implication. These phytochemicals are known to be biologically active. They possess some properties such as antibacterial properties (tannins and flavonoids) antiseptic properties (tannins),anti-inflammatory properties (flavonoids,saponins), analgestic properties(steroids) and anaesttheticproperties (alkaloids) [16]. The presence of tannins was found to play a role in the antifungal, antibacterial, astringent and antibiotic activities [16]. Phenolic acids and flavonoids have been the object of a great number of studies of their anti-oxidative activity which is mainly because of their capacity to act as free radical scavengers and/or metal chelators [17] [18]. Both compounds have attracted considerable interest in the past few years due to their many potential health benefits. As polyphe-nols, phenolic acids and flavonoids are powerful antioxi-dants and have been reported to demonstrate antibacterial, antiviral, anticarcinogenic, anti-inflammatory and vasiodilatory actions [19][20] The above result is similar to the findings of Christainet al., [6] who in his report of methanol and petroleum extracts of the stem bark of Napoleonavogelii stated the presence of tannins, alkaloids and saponin. Previous phytochemical studies indicated the presence of alkaloids, tannins and saponins on Napoleonavogelii stem bark [16]. Though few works have been done on the phytochemicsls of the stem bark, it was found out that the leaves of the plant contained tannin, alkaloid, saponin, flavonoid carbohydrate and cardiac glycoside.

QUANTITATIVE PHYTOCHEMICAL SCREENING
The result of the quantitative phytochemical screening of the stem bark of Napoleonavogelii is shown in table 2 The phytochemical result indicates that the flavonoid content of Napoleonavogelii stem bark is 9.27%. This value is relatively high compared to the value of 4.65 reported of Napoleonavogelii Hook fruit [22]. However, [23] reported that high amounts of flavonoid help protect blood vessels from rupture or leakage, enhance the power of vitamin C; protect cells from oxygen damage and prevent excessive inflammation. The value obtained for tannin in the stem bark of this plant was (0.45%). This value is lower than that reported forNapoleonavogelii Hook fruit [22] and the stem bark of Napoleonavogelii in methanol(9.5%) and in petroleum ether(1.8%) [6]. High tannin content in thesample implies severe nutritional challenge to animals or humans due to its affinity for certain digestive enzymes. The saponin value is 4.27%. This is quite high compared to the value of 0.75% reported for Napoleonavogelii Hook fruit [22] and lower than the value of (1.8%) in petroleum ether reported by [6]. A high Saponin diet can inhibit dental and platelet aggregation in treatment of Hypercalciuria in human (excessive urinary calcium excretion, an antidote against acute lead poisoning [24]. Saponin also decreases blood lipids, lower cancer risks and blood glucose response as well as posses antioxidant activity. Toxicology studies of saponin using relevant experimental models have established that even at an upper concentration of 3.5%, saponin was safe and failed to cause systemic side effect [25].The value obtained for hydrogen cyanide in NapoleonaVogelii stem bark is 0.37% and is lower than the 36mg/100g considered lethal dose for man [26]. The alkaloid content (4.27%) is low compared tothat reported by [6].Caution should be taken in the consumption of plant materials with very high concentration of Alkaloid because they could inhibit certain mammalian enzymes activities such as those of cyclic adenosine monophosphate (AMP) [27]. European Food Safety Authority [28] stated that since cooking only lowers alkaloid content of foods by 40 -50%, highly sensitive individuals should avoid this category of food entirely.

VITAMINS ANALYSIS
The result of the vitamins analysis of the stem bark of Napoleonavogelii is shown in table 3. The presence of vitamin C (ascorbic acid) in the diet enhances iron absorption, and iron to ascorbic acid ratio provides an index of iron availability in food sample in such a way that the lower ratio, the greater the relative bioavailability of iron [21] Vitamin C prevents many debilitating diseases, increases the body's immunity and is powerful antioxidant [30]. It also helps in recycling other antioxidants and it also aids in the formation of collagen. Collagen, tendons and ligaments depend upon Vitamin C to stay strong and healthy [31].
Vitamin E provides protection which might include its function as an antioxidant and its roles in anti-inflammatory processes, inhibition of platelet aggregation, and immune enhancement [23]. Vitamin E also may block the formation of nitrosamines, which are carcinogens formed in the stomach from nitrites consumed in the diet [32]It also may protect against the development of cancers by enhancing immune functions. Some evidence links higher intake of vitamin E to a decreased incidence of prostate and breast cancers [23]. The vitamin A content of the plant is 1.20mg/g. Vitamin A serves as an antioxidant and is essential for normal growth and for the formation of strong bones and teeth in children, for normal vision and cell structure, for protecting the lining of the respiratory, digestive, and urinary tracts against infection, and for healthy skin [33]. It also functions as a hormone, visual pigment of the vertebrate eye and regulates gene expression in the development of epithelial tissue, including skin [34].

MACRO AND MICRO ELEMENTS ANALYSIS
The result of the macro and micro elements analysis of the stem bark of Napoleonavogelii is shown in table 4 J u l y 1 8 , 2 0 1 5  [38].Sodium and Potassium are necessary to maintain osmotic balance in the body as well as the pH.. Iron is present in considerable amount (304.66mg/kg) and its necessary in formation of haemoglobin and normal functioning of the central nervous system. Phosphorus, a mineral in bone formation is also present (410.22mg/kg). Phosphorus is required for normal function of the body since it is a major structural component of bone and teeth and helps to maintain normal pH [39]. It is crucial for production of ATP and also plays important role in the growth, maintenance and repair of cells and tissues [21]. The other Micro minerals like Manganense, Copper and Zinc are also present and they play important roles in metabolic activities. These elements support human biochemical processes by serving structural and functional roles as electrolytes [34]

ANTIMICROBIAL ANALYSIS
The result of the antimicrobial activity screening of the stem bark of Napoleonavogelii is shown in table 5

±1.20
Values show means of triplicate analysis ±standard deviation.
The antimicrobial activity of the extracts of Napoleonavogelii were studied in different concentrations (50,100,150,200) against four bacterial staphylococcus aureus, streptococcussp, Escherichia coli, pseudomonas aeruginosa and a fungal strain candida albicans. Antibacterial and antifungal potential of extracts were assessed in terms of zone of inhibition of bacterial growth. The results of the antibacterial and antifungal activities are presented in the table. The antibacterial and antifungal activities increased linearly with increase in the concentration of extracts. The result of this study agrees closely with the work of [6] on antimicrobial activity of Napoleonavogelii. In both works, Pseudomonas was significantly inhibited. However, the result could possibly be due to the different extraction solvents used in the different works [15]. J u l y 1 8 , 2 0 1 5

DIAMETER OF INHIBITION ZONE
The result of the diameter of inhibition zone of the stem bark of Napoleonavogelii is shown in table 6  The antimicrobial activity of the extracts were studied in different concentration (50,100,150,200) against four pathogenic bacterial strains, two Gram-positive (Staphylococcus aureus, Streptococcus sp.) and two Gram-negative (Escherichia coli, Pseudomonas aeruginosa ), and a fungus strain Candida albicans). Antibacterial and antifungal potential of extracts were assessed in terms of zone of inhibition of bacterial growth. The growth inhibition zone measured ranged from 7 to 19 mm for all the sensitive bacteria, and ranged from 9 to 21 mm for fungal strain. In this study, the results showed that the three extracts of Napoleonavogelii possess antimicrobial activities against the tested organisms at different concentrations.
With the butanol extract, the highest antimicrobial activity of 18mm in E.coli and least activity of 8mm in streptococcus were recorded. With the ethanol extract, the highest antimicrobial activity of 17mm in pseudomonas and least activity of 8mm in staphylococcuswas recorded. With the chloroform extract, the highest antimicrobial activity of 15mm in streptococcus and least activity of 8mm in staphylococcus were recorded. Butanol extract demonstrated a better antifugal activity than the other extracts. The results of the present investigation clearly indicates that the antibacterial and antifungal activity vary with the different solvent used in extraction. Thus the study ascertains the value of solvents used for extraction which could be of considerable interest to the development of new drugs. J u l y 1 8 , 2 0 1 5

MINIMUM INHIBITION CONCENTRATION (MIC)
The minimum inhibition concentration of the stem bark of Napoleonavogelii is shown in table 7

CONCLUSION
The result of this study revealed the presence of most of the phytochemicals with various biological activities. This might be responsible for the observed antimicrobial activities against test organisms. The important minerals found in the stem bark may also be major contributors to the medicinal use of the plant. This data indicates that Napoleonavogelii stem bark contains potent bioactive compounds and justifies the claimed use of the stem bark in the traditional system of medicine to treat infectious diseases such as skin diseases caused by microbes