Medicinal plants are natural resources yielding valuable herbal products which are often used in the treatment of various ailments (Dulger and Gonuz, 2004). In recent years attempt have been taken to investigate the indigenous drug against infectious diseases to help developing safer antimicrobial drugs (Rahman et al., 2001). In the continuation of this strategy of new drug discovery we have studied the alkaloids of the ripe fruits of the plant, Amlaki (Emblica officinalis) to know their biological activity specially antimicrobial and cytotoxic activity.
The Amlaki also called Indian gooseberry (Emblica officinalis) is a deciduous tree of the Euphorbiaceae family. Amlaki has undergone preliminary research, demonstrating in vitro antiviral and antimicrobial properties (Saeed and Tariq, 2007). Another in vitro study shows that Amlaki extracts induce apoptosis and modify gene expression in osteoclasts involved in rheumatoid arthritis and osteoporosis (Letizia et al., 2008).
Experimental preparations of leaves, bark or fruit have shown potential efficacy against laboratory models of disease, such as for inflammation, cancer, age-related renal disease and diabetes (Ganju et al., 2003; Yokozawa et al., 2007; Rao et al., 2005).
A human pilot study demonstrated reduction of blood cholesterol levels in both normal and hypercholesterolemic men (Jacob et al., 1988). Another very recent study with alloxan-induced diabetic rats given an aqueous Amlaki fruit extract has shown significant decrease of the blood glucose as well as triglyceridemic levels and an improvement of the liver function caused by a normalization of the liver-specific enzyme alanine transaminase (ALT) activity (Qureshi et al., 2009).
Although, fruits are reputed to contain high amounts of ascorbic acid (vitamin C), 445 mg/100 g, (Tarwadi and Agte, 2007) the specific contents are disputed and the overall antioxidant strength of Amlaki may derive instead from its high density of tannins and other polyphenols. The fruit also contains flavonoids, kaempferol, ellagic acid and gallic acid.
Amlaki is one of the three ingredients of the famous ayurvedic preparation,
triphala, which is given to treat chronic dysentery, bilousness and other disorders.
Amlaki has antioxidant, cytoprotective (Bandyopadhyay et al., 2000)
hepatoprotective (Gulati and Agrawal, 1995) anti-hepatitis, anti-cancer, anti-tumor
activity (Jeena et al., 2001) and also have antimutagenic activity (Kaur
et al., 2002). It is used for constipation, peptic ulcer and scurvy (Joshi,
2000), immunomodulator (Xia et al., 1997). It is also useful for burning
sensation in abdominal and cardiac regions and epigastric pain (Singh and Sharma,
Herbal drugs could be scientifically modified for better pharmacological activity and to establish safe and effective drugs. Among various types of metabolites alkaloids are very important source of drugs. Here we have emphasized to extract alkaloids from the plant and to evaluate their biological activity especially antimicrobial and cytotoxic activity.
MATERIALS AND METHODS
Extraction of alkaloids from the ripe fresh fruits of Amlaki and experiments of the antimicrobial activity and cytotoxicity was conducted in the Phytochemical Research Laboratory, Faculty of Pharmacy, University of Dhaka from February to November, 2007.
Plant material: The Plant sample (ripe fresh fruit) of Amlaki was collected from Dhaka in February 2007. A voucher specimen had been deposited in Bangladesh National Herbarium, Dhaka for proper identification.
Extraction of alkaloids from plant material: Fruits of the plant (1 kg) were cut into small pieces and then soaked into methanol for two weeks at room temperature with occasional shaking and stirring. It was then filtered through a fresh cotton plug and finally with a Whatman filter paper. The volume of the filtrate was then reduced using a Buchii Rotavapor at low temperature and pressure. Through solvent-solvent partioning method alkaloids were extracted (Ortiz and Mukherjee, 1982). Initially the extract was shaken with n-hexane to remove non polar compounds from the extract and the aqueous portion was collected which actually contained the alkaloids as salt form. Then the aqueous portion was basified with 10 N NaOH to get free alkaloids. This fraction was partitioned with CHCl3. So the alkaloids came into the CHCl3 portion. This fraction was tested to know the presence of alkaloids with Dragendorffs reagent (Waldi, 1962) which gave positive test of alkaloids.
Antimicrobial activity: The crude chloroform extract was subjected to antimicrobial screening by disc diffusion method (Bauer et al., 1966). The sample solution of the materials to be tested was prepared by dissolving a definite amount of material in appropriate solvent to attain a concentration of 40 mg mL-1. The 10 μL-1 of such solution was applied on the sterile disc (6 mm diameter filter paper) and allowed the solvent to dry off in an aseptic hood. Thus such discs contain 400 μg of crude alkaloid extract. To compare the activity with standard antibiotics, kanamycin (30 μg dics-1) was used.
The alkaloids extract was tested against some gram positive bacteria: Bacillus cereus, Bacillus megaterium, Bacillus subtilis, Staphylococcus aureus, Sarcina luteasome some gram negative bacteria: Escherichia coli, Pseudomonas aeruginosa, Salmonella paratyphi, Salmonella typhi, Shigella boydii, Shigella dysenteriae, Vibrio mimicus, Vibrio parahemolyticus and some fungi: Candida albicans, Aspergillus niger and Sacharomyces cerevacae.
The average zone of inhibition was 07-11 mm for chloroform extract at 400 μg disc-1. Standard antibiotic disc of kanamycin at 30 μg disc-1 was used for comparison purpose. The chloroform extract showed strongest inhibitory activity against Bacillus subtilis. The growth of S. typhi, B. cereus, P. aeruginosa, S. boydii, S. dysenteriae, S. aureus, S. lutea were moderately inhibited.
Cytotoxic activity: The lethality of the chloroform extract (CLF) of the fruits of Amlaki to brine shrimp was determined following the procedure of Meyer et al. (1982). It gave the results of the brine shrimp lethality after 24 h exposure to the sample and the positive control, vincristine sulfate. The positive control compared with the negative control (sea water) was lethal, giving significant mortality to the shrimp. The lethal concentration LC50 of the test sample after 24 h was obtained by a plot of percentage of the shrimps killed against the logarithm of the sample concentration (toxicant concentration) and the best-fit line was obtained from the curve data by means of regression analysis.
The LC50 value of chloroform fraction of the ripe fruits of Amalaki was found to be 10.257±0.770 μg mL-1.
RESULTS AND DISCUSSION
The result of antimicrobial activity: The chloroform fraction of Amlaki
containing alkaloids was tested for antimicrobial activity against some Gram
positive and Gram negative bacteria and some fungi. This fraction was used in
a concentration of 400 μg disc-1. This fraction showed significant
antimicrobial activity against some microorganisms. The chloroform extract showed
strongest inhibitory activity against Bacillus subtilis. This extract
showed moderate inhibitory activity against S. typhi, B. cereus,
P. aeruginosa, S. boydii, S. dysenteriae, S. aureus,
S. lutea, E. coli, S. paratyphi, V. parahemolyticus
and V. mimicus (Table 1). The test microorganisms were
again tested with kanamycin, a Diameter less than 8 mm was considered as inactive.
||Antimicrobial activity of the crude extract of alkaloids of
the ripe fruits of Amlaki
The zone of inhibition has been shown here as mean±SD (n=3). The test
was done in triplicate standard antibiotic to be ensured about the validity
of the method. There exist a correlation between chloroform extract group and
kanamycin group in this way that where the zones of inhibition by the chloroform
extract are higher a higher zone of inhibition has also been found in case of
kanamycin without a few cases.
The result of brine shrimp lethality bioassay: The cytotoxic activity of the crude extract of alkaloids of the ripe fruits of Amlaki was studied by brine shrimp lethality bioassay (Meyer et al., 1982) and have been shown in the Fig. 1 and 2. The extract exhibited cytotoxic activity having a LC50 of 10.257±0.770 μg mL-1. The standard vincristine sulfate showed a LC50 of 0.378±0.017 μg mL-1. LC50 was obtained from the best-fit line slope of the graph. The test was done in triplicate (n = 3) and the LC50 has been shown here as Mean±SD. LC50 has been calculated from the regression equation by putting the value of y = 50 and getting the value of x.
An approximate linear correlation was observed between logarithm of concentration and percentage of mortality.
The present study revealed that the plant Amlaki has got antimicrobial and cytotoxic effects due to the presence of some bioactive principles. The crude chloroform extract of the fruit showed antibacterial activity against both Gram positive and Gram negative bacteria.
Our objective in this research was to investigate the biological activity of the alkaloids of the plant. So there exist a correlation between the findings and the objective of our study.
||Determination of LC50 value for chloroform soluble
fraction of the ripe fruits of Amlaki containing alkaloids from linear correlation
between logarithms of concentration versus percentage of mortality. The
study was done in triplicate
||Determination of LC50 value for standard (vincristine
sulfate) from linear correlation between logarithms of concentration versus
percentage of mortality. The study was done in triplicate
This study is a general agreement with the results of earlier investigators (Jasril et al., 1999) these investigators showed the activity of crude n-hexane extract or crude methanolic extract of the fruit of this plant but our investigation has been focused on the bioactivity of the crude chloroform extract of alkaloids of the plant.
Our findings also support the previous results (Joshi, 2000) where it is declared that alkaloids are present in this plant but there is no indication of the biological activity of the alkaloids of this plant but this is shown here in our study. This study will encourage other researchers to study more including phytochemical and biological investigation.
It may be concluded from this study that the extract of alkaloids of the fruits of Amlaki are active against the tested microorganisms and also have cytotoxic effects. In addition the results confirm the use of the plant in traditional medicine. Now present study directed to explore the lead compounds responsible for aforementioned activity from this plant.
The authors wish to thank the Faculty of Pharmacy, University of Dhaka for providing opportunity to carry out the research.