Free radicals are generated during the normal metabolic reactions in the body. These oxidants may be involved in the pathogenesis of diseases such as cancer, diabetes mellitus, cardiovascular and neurological diseases2. A number of antioxidant models (such as free radical scavenging, reducing power, iron chelating and lipid peroxidation inhibitory capacity) have been employed in the evaluation of antioxidant activities3. Similarly, the increase in microbial resistance to most antibiotics has attracted global interest4.
Livistona chinensis commonly known as Chinese Fan palm or Chinese Fountain palm is of the family, Arecaceae. Trunk is topped with an evergreen dense crown of palmate, or fan shaped leaves that droop downward creating fountain-like effect. The petioles are armed with sharp thorns5,6. Flowers are followed by small, oval green fruits of about 1 inch long which turn dark blue to blue-grey when ripe7. Recent analysis of molecular data uncovered the startling fact that L. rotundifolia constitute a distinct genus, Saribus8. L. chinensis is an ornamental and medicinal palm. The seeds and fruits of L. chinensis has long been used in China to clinically treat various types of cancer9,10. Extracts of the L. chinensis seed have been shown to inhibit the growth of several cancer cells11-13. Phenolic compounds isolated from the roots, fruits, and seeds of the Chinese fan palm has shown anti-osteoporotic, cell protective and antibacterial effects14-16.
Saribus rotundifolius (Arecaceae), round-leaf fountain palm is a medium-sized to large palm with a slender trunk bearing prominent leaf scars. Spiny stalks support rounded, dark green leaf blades divided into many linear, rigid, notched lobes. Panicles of cream flowers in summer are followed by spherical, bright-red fruit that ripen to black. S. rotundifolius bud is highly esteemed as a vegetable in the Philippines. Nuts are eaten when young and green. The rind is tasty when ripe17. S. rotundifolius is a medicinal plant employed to treat fresh wounds and diarrhoea18. The plant leaves extracts contain antibacterial compounds19.
Areca catechu (Arecaceae) is an erect, solitary palm that grows to a height of 20 m or more and diameter of 20-30 cm. Fruits, which are produced in bunches, are ovoid, smooth, orange or red when ripe20,21. A. catechu is widely used in ethno-medicine in most parts of Asia for the treatment of ailments such as urinary infections, diarrhoea, foot sore, and so on22. Traditionally, its powder has been used as a dentifrice in tooth paste and as taeniacide (kills tapeworms) especially in animals23.
Evidence suggests that compounds, especially from natural sources are capable of providing protection against free radicals24. The present study was conducted to determine the phytochemical content, antioxidant and antimicrobial activities of the palm nuts of L. chinensis, S. rotundifolius and A. catechu employed in herbal medicine.
2 Materials and methods
2.1 Plant materials and extraction
The mature ripe fruits of L. chinensis, S. rotundifolius and A. catechu were collected in the month of April – July, 2015 within Uyo metropolis, Akwa Ibom State, Nigeria. Plant samples were identified and authenticated by a taxonomist, M. E. Bassey, Department of Botany and Ecological Studies, University of Uyo, where voucher specimens were deposited. The fruits were peeled to expose the seeds. The seeds were sun-dried after which the endocarps were removed to obtain the kernels. The pulverized kernels of L. chinensis (701. 85 g), S. rotundifolius (816.80 g) and A. catechu (1208.08 g) were macerated in methanol (95%). The maceration process was repeated to obtain a good yield of extracts.
2.2 Phytochemical screening
Standard methods for phytochemical screening of alkaloids, flavonoids, saponins, tannins, carbohydrates, sterols and triterpenoids were employed. Alkaloids determination was done using Mayer’s and Dragendoff’s reagents25. The persistent frothing, sodium bicarbonate and carbonate tests, as described by Trease and Evans26 were used for saponins. The methods described by Trease and Evans26 and Harborne27 were used for the determination of flavonoids, phenols, cardiac glycosides, carbohydrates, sterols, triterpenes, tannins and phlobatannins.
2.3 Determination of total phenolics
The amount of total phenols in the palm nut extracts was determined with the Folin-Ciocalteu’s reagent using the method of Meda et al.28. 2.5 ml of 10% Folin Ciocalteu’s reagent was added to 0.5 ml of each concentration (2, 1.5, 1.0, 0.5 and 0.25 mg/ml) of the extract and then 2 ml of 2% w/v of Na2CO3 was introduced and incubated at room temperature (28ºC) for 30 minutes. The absorbance was measured at 760 nm using a uv/vis spectrophotometer (Unisio, Shanghai-China). Total phenol values were expressed in terms of gallic acid equivalent (mg/g of extract).
2.4 Determination of flavonoids
The method of Meda et al.28 was used to determine the total flavonoid content. 2.5 ml of aluminium trichloride (AlCl3) in methanol were mixed with different concentrations (2, 1.5, 1.0, 0.5 and 0.25 mg/ml) of the palm extracts. Absorption readings at 415 nm using uv/vis spectrophotometer (Unisco, Shanghai – China) were taken after 30 minutes. The total flavonoid content was determined using a standard curve prepared with gallic acid and expressed as gallic acid equivalent (GAE)/g of extract.
2.5 Determination of tannins
The tannin content in each extracts was analysed using the method described by Kalpana et al.29. 3 ml of extract or standard solution of tannin acid (0.0625-0.5 mg/ml) were mixed with 1 ml of Folin-Ciocalteau reagent and 1 ml of 3.5% Na2CO3 solution was added. The volume was made to 5 ml with distilled water, and absorbance read at 725 nm after 30 minutes of incubation. Tannin content was expressed as mg tannic acid equivalent per g of extract.
2.6 DPPH radical assay
The free radical scavenging activity of the palm extracts was determined using the modified method of Blois30. 2.5 ml of different concentrations (0.25-2.0 mg/ml) of the extracts and standard drug (ascorbic acid) was separately measured into test tubes, and then 2.5 ml of 0.1 mM DPPH in methanol was added. The mixtures were incubated in a dark chamber for 30 minutes after which the absorbance was measured (in triplicates) at 517 nm against a DPPH control (containing reagents except test samples). Percentage scavenging activity was calculated using the expression:
2.7 Iron chelating activity
The reaction mixture containing 1 ml of o-phenanthroline (0.025 M), 2 ml of ferric chloride (0.05 M) and 2 ml of extract at various concentrations (0.0625-0.50 mg/ml) was incubated for 10 minutes at an ambient temperature. The absorbance at 510 nm was recorded. Ascorbic acid was added instead of extract and absorbance obtained was taken as equivalent to 100% reduction of all ferric ions. The readings were taken in triplicate31.
Where AC is the absorbance of the control and AS is the absorbance in the presence of the extracts or standards.
2.8 Hydrogen peroxide scavenging capacity
The ability of the palmextracts to scavenge hydrogen peroxide was determined according to the method of Ruch et al.32. A solution of hydrogen peroxide (2 mM) was prepared in 50 mM phosphate buffer (pH 7.4). Extracts (1 ml, 0.5 mg/mL) and standard drugs (ascorbic acid) were separately added to a hydrogen peroxide solution in phosphate buffer (3 ml, 40 mM). Absorbance of hydrogen peroxide at 230 nm was determined 10 minutes later against a blank solution. The percentage of hydrogen peroxide scavenging of bothextracts and standard compound were calculated:
% Scavenged [H2O2] = [(AC – AS)/AC] x 100
Where AC is the absorbance of the control and AS is the absorbance in the presence of the extracts or standards.
2.9 Molybdate ion reduction assay
The extracts were evaluated for their total antioxidant capacity following the spectrophotometric molybdate ion reduction assay of Prieto et al.33 with slight modifications. 1 ml of each extracts solution (0.5 mg/ml) was added to 3 ml of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The mixtures were incubated at 95 °C for 90 mins. After incubation, the mixture was cooled at room temperature, and the absorbance was measured at 695 nm against the blank. Ascorbic acid was used as a standard and a standard curve was obtained.
2.10 Collection of bacterial and fungus isolates
Clinical bacterial and fungus isolates were collected from St. Lukes Hospital, Anua, Uyo and University of Uyo Teaching Hospital, Uyo, Akwa Ibom State, Nigeria. These isolated were transported on slants to Microbiology Laboratory, University of Uyo, Nigeria. The test organisms were sub-cultured into nutrient broth and incubated for 48 hrs at 37 ºC. The microbes were sub-cultured on a nutrient agar slant for the isolation of pure culture. Isolates were identified using standard cultural, microscopic and standard biochemical methods such as motility test, gram staining, oxidase test, oxidation fermentation test, indole test, catalase test, gelatin liquefaction test, citrate utilization, esculin hydrolysis, urease activity, decarboxylase reactions and hydrogen sulphide production tests. The Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis) and fungus(Candida albicans)were serially diluted to factor three using 10 fold dilution. Gram negative isolates (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, Salmonella typhi and Shigella dysenteriae) were serially diluted to factor five using 10 fold dilution. The isolates were sub-cultured into their selective media based on their exhibited morphological characteristics. They were preserved in a refrigerator at 4 °C and later used for this work.
2.11 Preparation of antimicrobial discs
A 5 mm diameter plunger was used to punch a Whatman no.1 absorbent filter paper to obtain 5 mm diameter paper discs. The discs were properly labeled and then sterilized by autoclaving for 15 min at 121°C. The discs were impregnated with the plant extracts (100- 400 µg/ml), dried and stored off in sterile bottles.
2.12 Evaluation of antimicrobial activity
Antimicrobial activity was tested using a modified discs diffusion assay (DDA) method34,35. A loop of culture from the nutrient agar (NA) slant stock was cultured in Mueller Hinton medium overnight and spread with a sterile swab into Petri-plates. Each microbial swab was spread on separate plates. Sterile disc (5 mm in diameter) impregnated with the plant extracts were placed on the cultured plates. Control experiment was carried out using commercial antibiotics, antifungal and solvent (stock). The solvent loaded disc without extracts served as control in the study. Streptomycin was used for bacterial isolates and Nystatin for fungal isolate; plates were incubated for 24 hrs and 48 hrs respectively. The results were recorded by measuring the zones of growth inhibition. Clear inhibition zones around the discs indicated the presence of antimicrobial activity. All data on antimicrobial activity were average of triplicate.
2.13 Determination of minimum inhibitory concentration
The minimum inhibitory concentrations of the extracts were determined using tube dilution method36. The initial concentration of each of the plant extracts was diluted using double fold dilution and standard volume of each diluted isolate (0.1ml) was aseptically inoculated into different concentrations of the extract. Control experiment was carried out without the crude extracts. All tubes were incubated at 37 °C for 24 hrs. Minimum inhibitory concentrations (MIC) were determined as the lowest concentration without turbidity.
2.14 Cell culture
HeLa (Cervical Cancer) cells were cultured in Minimum Essential Medium Eagle (MEME), supplemented with 5% of fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml of streptomycin in 75 cm2 flasks, and kept in 5% CO2 incubator at 37oC. Exponentially growing cells were harvested, counted with haemocytometer and diluted with DMEM37.
H460 (Lung cancer) cells were cultured in RPMI medium, supplemented with 10% of fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml of streptomycin in 75 cm2 flasks, and kept in 5% CO2 incubator at 37oC. Exponentially growing cells were harvested, counted with haemocytometer and diluted with RPMI38.
MCF-7 Breast cancer (ATCC No. HTB-2)39 and PC-3 prostate cancer cells (ATCC No. CRL-1435)37 were cultured in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 5% of fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml of streptomycin in 75 cm2 flasks, and kept in 5% CO2 incubator at 37oC. Exponentially growing cells were harvested, counted with haemocytometer and diluted with DMEM.
2.15 Cytotoxicity screening
Cytotoxic activity of compounds was evaluated in 96-well flat-bottomed micro plates by using the standard MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) colorimetric assay37. HeLa cells were plated into 96-well cell culture plates at 6 x 104 cells per well, H460 cells (4 x 104), MCF-7 (8 x 103) and PC-3 cells at 1 × 105 cells per well. The volume in each well was 100 μL for all cell types. After overnight incubation, supernatant fluid was removed by suction and 200 µL of fresh medium containing extract/ compound was added in triplicate, giving a final concentration of 30 µg/mL or 30 µM. Standard drug used in the MTT assay was doxorubicin. After the addition of the sample, plates were incubated for 48 h at 37 °C; 200 µL MTT (0.5 mg/ml) was added to each well and incubated further for 4 hrs. Formazan crystals, formed by reduction of MTT were dissolved in 100 µL DMSO and absorbance was taken at 570 nm using micro-plate reader (Spectra Max plus, Molecular Devices, CA, USA). The % inhibitions were processed by using Soft- Max Pro software (Molecular Device, USA). If extracts/compounds showed 50% or more percent inhibition, they were further processed for IC50 calculation. The cytotoxicity was recorded as concentration causing 50% growth inhibition (IC50) for all cell lines. The percent inhibition was calculated using the following formula:
Where, OTC: mean of O.D of test compound; ONC: mean of O.D of negative control; OPC: mean of O.D of positive control