Study of Antibacterial and Antifungal Efficacy of Alkaloid Isolated from Nutmeg (Myristica fragrans)

The present study included the isolation of alkaloids from the nutmeg fruit (Myristica fragrans) where one of the alkaloid compounds in the nutmeg fruit was found, which is the existence of the aromatic and has carbonyl and phenol groups in addition to nitrogen. The isolated alkaloid showed antibacterial activity against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli), while the alkaloid compound showed an antifungal effect against Candida albicans and Cryptococcous neoformans. No cytotoxicity of human blood solution in concentration (10-200 ppm) was shown in the isolated alkaloid compound. From this result, it was concluded that the alkaloid isolated from Myristica fragrans can be used in treatment of various disorders because of the effectiveness of the compound against bacteria and fungi.


INTRODUCTION
Myristica fragrans, is a tropical evergreen tree (family Myristicaceae), nutmeg was used as a sachet, and the Romans used it as incense native of the E. Moluccas. It is found only as a specimen tree in Botanical gardens. The seed of the plant is known as "nutmeg" and the spice made of its seed and the arillus of the seed is called "mace". Both nutmeg and mace contain many volatile oils. The contents of these oils have a variety of individual pharmacological effects, some of which disagree others . The fruit contains ethereal oil-cells often with phenolic and myristicin; the seed and the aril are used for flavouring food .
Nutmeg is diffuse as a spice and also possesses various therapeutic properties. It has a characteristic pleasant fragrance and a slightly warm taste. It is used to flavour many kinds of baked foods, confections, confectionery, meats, sausages, vegetables and drinks. Medicinally, Nutmeg is used as an anti-diarrheal agent for patients with medullar carcinoma of the thyroid. The effectiveness of the treatment may be due to the inhibition of prostaglandin synthesis in the mucosa and sub mucosa of the colon. It is sometimes used as a stomachic, tonic, carminative as well as for intestinal colic and catarrh, to stimulate appetite, to control flatulence, it also promotes and regulates the flow of menstruation and abortion (Olaleye et al., 2006).

Plant Materials & Chemical
The Nutmeg Myristica fragrans plant used in this study was obtained from the local markets. All the chemicals were buy from Sigma & Alderich Co.
The reagents were prepared in deionized distilled water to eliminate the pollution of metal ions.

Preparation of extracts Alkaloid extract
Twelve (12 g) of plant powder defending oil was mixed with 150 ml of ethanol (10% acetic acid) and left on continuous mixing under room temperature for 4 h. The mixture was filtered by using filter paper (Whatmann No, 31) and Buchner funnel; then the filtrate was concentrated to a quarter of its size by a rotary evaporate at 70°C. The sulfuric acid (2%) was added to the concentrated filtrate and the concentrated filtrate was treated with concentrated ammonium hydroxide until pH9,then the solution was filtered and separated by addition 20ml of chloroform. The precipitate was removed by filtration, through filter paper, then filtrates was concentrated under vacuum then dried at room temperature (Yubin et al., 2014), the weight of yellow amorphous compound was 1.935g.

Preliminary qualitative test
Preliminary tests were carried out on the alkaloid compound, as showed in the table (1).

Solubility test
Uses several polar and nonpolar solvent to solubility test to alkaloid compound.

Active groups test
After ensuring the one alkaloid compound, then uses active groups test to do it .

FTIR and UV-Visible spectroscopy
Fourier transform Infrared using PyE-UNICAM-30300S, and UV-Visible analysis using JASCOUV, were done to alkaloid compound.

Biological activity
Two bacterial isolates, namely Escherichia coli and Staphylococcus aureus were used to establishment the antibacterial activity, in addition to two isolates of yeasts, Candida albicans and Cryptococcous neoformans that were used to discernment of antifungal activity. All clinical bacterial with yeast isolates were obtained from microbiology laboratories of Biology Department, Science College, Basrah University.

Antibacterial and Antifungal tests
The minimum inhibitory concentration, (MIC s ) for extract of alkaloid was evaluated by broth microdilution method (CLSI, 1996 andCLSI, 2002). The extract was dissolved in dimethyl sulfoxide (DMSO) with concentrations ranging from 100-0.05 µg/ml, while the suspension for each isolate was prepared and incorporated to a turbidity of 0.5 McFarland scale (1x10 6 cfu/ml ) for yeasts and (1x10 8 cfu/ml) for bacteria, and diluted with broth microdilution technique to reach the final concentration 1-5x10 3 cfu/ml for yeasts and 1-5 x10 4 cfu/ml for bacteria. The test was performed by using microtiter plates and each concentration was tested in duplicate with positive and negative controls (the microorganism and pure media respectively). The plates were incubated at 37°C for 24 h for bacteria and at 30°C for 48 h for yeasts.
The MIC was measured by the lowest concentration of extract that yield an 80% reduction in observable growth was compared with the control, as well as, when MIC value was 100 µg/ml or less, the antimicrobial activity was considered significant.

Cytotoxicity test
Biocompatibility test was carried out for the prepared alkaloid extract against fresh human blood according to Nair et. al. (1989) method that including in briefly:-Different concentration of extract were prepared (200, 100, 50 and 10 ppm), then 100 µl of each tube of human blood solution. The tubes were left at room temperature and the formation of turbidity of blood solution was tested after 15, 30 and 60 min. as an indication for the cytotoxicity of extract.

RESULTS AND DISCUSSION
In the existing study, alkaloids extract was separated and purified with extraction percentage equal to (16.39%). Table (1) appears the qualitative chemical analysis of alkaloids segregate from Myristica fragrans. The results showed the presence of alkaloid only but carbohydrate, glycosides, phenols, saponins, and amino acids were not found. It is known that alkaloids are a lot in many medicinal plants. Table (2) show the thin layer chromatography (TLC) result of an alkaloid extracted from Myristica fragrans. There one spot was separated have rate of flow(Rf) value equal to 0.59 was tested and developed by Dragendroff reagent as a qualitative and specific developer for alkaloid, also by using iodine vapour the alkaloid Journal of Pure and Applied Microbiology separated showed brown colour and by using 40% H 2 SO 4 which showed the alkaloid was attached with sugar (Harborne, 1984).
For making sure of purification alkaloid compound isolated from Myristica fragrans and determination of chemical groups for it, where observed the isolated compound contains aromatic structure, carbonyl group, nitrogen and phenol groups as shown in table (3), Fig.(1) and table (5) shows the full scan of IR spectrum of alkaloid extract we deduce from the spectrum that the alkaloid extract is molecular that has an aromatic composition and contains a carbonyl group. The isolated alkaloid compound also showed solubility in ethanol, methanol and n-hexane as indicated in the table (4).
The UV-Spectrum shows maximum absorption at 298 nm due to (π-π * ) transition, which is the characteristic of the unsaturated double bond. The antibacterial and antifungal activities of Myristica fragrans alkaloid extract against two bacterial and two yeast isolates were investigated in this study. The result has reported the potency of alkaloid extract as antibacterial and antifungal activities with concentration ranging from 100-50 µg/ml ( Table 6).
The current study clearly indicates that Myristica fragrans alkaloid extract exhibited excellent antimicrobial activity against both gram positive and gram negative bacteria in addition to significant antifungal activity against yeast isolates (Fig. 3).
The present work was revealed that no any cytotoxic effect of Myristica fragrans alkaloid extract against human blood solution in all its concentrations that used in cytotoxicity assay conditions (Fig. 2).