UK Journal of Pharmaceutical and Biosciences Vol. 5(1), 59-67, 2017 RESEARCH ARTICLE

UK Journal of Pharmaceutical and Biosciences

Available at www.ukjpb.com

ISSN: 2347-9442

Phytoconstituents, Antioxidant and Antimicrobial Activities of Livistona chinensis (Jacquin), Saribus rotundifolius (Lam.) Blume and Areca catechu Linnaeus Nuts

Emmanuel E. Essien*, Bassey S. Antia, Esangubong I. Etuk

Department of Chemistry, University of Uyo, 520101, Akwa Ibom State, Nigeria

Article Information Abstract Received 1 November 2016 Received in revised form 20 Feb 2017 Livistona chinensis, Saribus rotundifolius and Areca catechu are palms primarily cultivated for Accepted 23 February 2017 ornamental purposes and also utilized in herbal remedies. The chemical and biological Keywords: Livistona chinensis, studies were conducted using standard procedures. Phytochemical screening revealed the Saribus rotundifolius, presence of alkaloids, saponins, cardiac glycosides, terpenes, de-oxy sugars, Areca catechu, anthraquinones, phenols, flavonoids and phlobatannins in varying amounts. The total Phytochemicals, Antioxidant activity phenolics content ranged from 52.0-245.0 mg/g, DPPH radical inhibition (87.30-90.55%, 2.0

Corresponding Author: mg/ml), iron chelating activity (27.3-65.0%, 0.5 mg/ml), hydrogen peroxide scavenging E-mail : [email protected] (93.18-98.46%, 0.5 mg/ml), and molybdate ion reduction capacity (15.93-58.30%, 0.5 mg/ml). Mob.: +234-80-3368-3424 L. chinensis extract showed notable antimicrobial activity against B. subtilis, P. aeruginosa DOI: 10.20510/ukjpb/5/i1/147026 and C. albicans (MIC = 75-100 µg/ml) while A. catechu and S. rotundifolius extracts were potent against all test pathogens (75-100 µg/ml), except S. typhi (150 µg/ml). However, no appreciable cytotoxic effects on human cervical (HeLa), lung (H460), breast (MCF-7) and prostate (PC-3) cancer cells at 30 µg/mL was observed for the extracts. The notable antioxidant and antimicrobial activities of the palm extracts are a function of their inherent bioactive constituents and a potential source of medicinally useful compounds. The chemical and biological assessment of S. rotundifolius palm nuts is reported for the first time.

1 Introduction turn dark blue to blue-grey when ripe7. Recent analysis of molecular data uncovered the startling fact that L. rotundifolia There is an upsurge in the use of medicinal plants for their constitute a distinct genus, Saribus8. L. chinensis is an therapeutic values1. Free radicals are generated during the ornamental and medicinal palm. The seeds and fruits of L. normal metabolic reactions in the body. These oxidants may be chinensis has long been used in China to clinically treat various involved in the pathogenesis of diseases such as cancer, types of cancer9,10. Extracts of the L. chinensis seed have been diabetes mellitus, cardiovascular and neurological diseases2. A shown to inhibit the growth of several cancer cells11-13. Phenolic number of antioxidant models (such as free radical scavenging, compounds isolated from the roots, fruits, and seeds of the reducing power, iron chelating and lipid peroxidation inhibitory Chinese fan palm has shown anti-osteoporotic, cell protective capacity) have been employed in the evaluation of antioxidant and antibacterial effects14-16. activities3. Similarly, the increase in microbial resistance to most antibiotics has attracted global interest4. Saribus rotundifolius (Arecaceae), round-leaf fountain palm is a medium-sized to large palm with a slender trunk bearing Livistona chinensis commonly known as Chinese Fan palm or prominent leaf scars. Spiny stalks support rounded, dark green Chinese Fountain palm is of the family, Arecaceae. Trunk is leaf blades divided into many linear, rigid, notched lobes. topped with an evergreen dense crown of palmate, or fan Panicles of cream flowers in summer are followed by spherical, shaped leaves that droop downward creating fountain-like bright-red fruit that ripen to black. S. rotundifolius bud is highly effect. The petioles are armed with sharp thorns5,6. Flowers are esteemed as a vegetable in the Philippines. Nuts are eaten followed by small, oval green fruits of about 1 inch long which Essien et al., Bioactivities of Medicinal Palm Nut Extracts when young and green. The rind is tasty when ripe17. S. introduced and incubated at room temperature (28 ºC) for 30 rotundifolius is a medicinal plant employed to treat fresh wounds minutes. The absorbance was measured at 760 nm using a and diarrhoea18. The plant leaves extracts contain antibacterial uv/vis spectrophotometer (Unisio, Shanghai-China). Total compounds19. phenol values were expressed in terms of gallic acid equivalent (mg/g of extract). 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, 2.4 Determination of flavonoids which are produced in bunches, are ovoid, smooth, orange or The method of Meda et al.28 was used to determine the total red when ripe20,21. A. catechu is widely used in ethno-medicine flavonoid content. 2.5 ml of aluminium trichloride (AlCl3) in in most parts of Asia for the treatment of ailments such as methanol were mixed with different concentrations (2, 1.5, 1.0, urinary infections, diarrhoea, foot sore, and so on22. 0.5 and 0.25 mg/ml) of the palm extracts. Absorption readings Traditionally, its powder has been used as a dentifrice in tooth at 415 nm using uv/vis spectrophotometer (Unisco, Shanghai - paste and as taeniacide (kills tapeworms) especially in China) were taken after 30 minutes. The total flavonoid content animals23. was determined using a standard curve prepared with gallic Evidence suggests that compounds, especially from natural acid and expressed as gallic acid equivalent (GAE)/g of extract. sources are capable of providing protection against free 2.5 Determination of tannins radicals24. The present study was conducted to determine the phytochemical content, antioxidant and antimicrobial activities of The tannin content in each extracts was analysed using the the palm nuts of L. chinensis, S. rotundifolius and A. catechu method described by Kalpana et al.29. 3 ml of extract or employed in herbal medicine. 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 2 Materials and methods solution was added. The volume was made to 5 ml with distilled 2.1 Plant materials and extraction water, and absorbance read at 725 nm after 30 minutes of incubation. Tannin content was expressed as mg tannic acid The mature ripe fruits of L. chinensis, S. rotundifolius and A. equivalent per g of extract. catechu were collected in the month of April – July, 2015 within Uyo metropolis, Akwa Ibom State, Nigeria. Plant samples were 2.6 DPPH radical assay identified and authenticated by a taxonomist, M. E. Bassey, The free radical scavenging activity of the palm extracts was Department of Botany and Ecological Studies, University of determined using the modified method of Blois30. 2.5 ml of Uyo, where voucher specimens were deposited. The fruits were different concentrations (0.25-2.0 mg/ml) of the extracts and peeled to expose the seeds. The seeds were sun-dried after standard drug (ascorbic acid) was separately measured into test which the endocarps were removed to obtain the kernels. The tubes, and then 2.5 ml of 0.1 mM DPPH in methanol was pulverized kernels of L. chinensis (701. 85 g), S. rotundifolius added. The mixtures were incubated in a dark chamber for 30 (816.80 g) and A. catechu (1208.08 g) were macerated in minutes after which the absorbance was measured (in methanol (95%). The maceration process was repeated to triplicates) at 517 nm against a DPPH control (containing obtain a good yield of extracts. reagents except test samples). Percentage scavenging activity 2.2 Phytochemical screening was calculated using the expression:

Standard methods for phytochemical screening of alkaloids, 퐴푏푠 표푓 퐶표푛푡푟표푙 − 퐴푏푠 표푓 푆푎푚푝푙푒 % Scavenging activity = 푋 100 flavonoids, saponins, tannins, carbohydrates, sterols and 퐴푏푠 표푓 퐶표푛푡푟표푙 triterpenoids were employed. Alkaloids determination was done 2.7 Iron chelating activity using Mayer’s and Dragendoff’s reagents25. The persistent The reaction mixture containing 1 ml of o-phenanthroline (0.025 frothing, sodium bicarbonate and carbonate tests, as described M), 2 ml of ferric chloride (0.05 M) and 2 ml of extract at various by Trease and Evans26 were used for saponins. The methods concentrations (0.0625-0.50 mg/ml) was incubated for 10 described by Trease and Evans26 and Harborne27 were used for minutes at an ambient temperature. The absorbance at 510 nm the determination of flavonoids, phenols, cardiac glycosides, was recorded. Ascorbic acid was added instead of extract and carbohydrates, sterols, triterpenes, tannins and phlobatannins. absorbance obtained was taken as equivalent to 100% 2.3 Determination of total phenolics reduction of all ferric ions. The readings were taken in

31 The amount of total phenols in the palm nut extracts was triplicate . determined with the Folin-Ciocalteu’s reagent using the method AC − 퐴푆 % Iron chelating activity = 푋 100 of Meda et al.28. 2.5 ml of 10% Folin Ciocalteu’s reagent was 퐴퐶 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 UK J Pharm & Biosci, 2017: 5(1); 60 Essien et al., Bioactivities of Medicinal Palm Nut Extracts

Where AC is the absorbance of the control and AS is the cultured into their selective media based on their exhibited absorbance in the presence of the extracts or standards. morphological characteristics. They were preserved in a refrigerator at 4 °C and later used for this work. 2.8 Hydrogen peroxide scavenging capacity 2.11 Preparation of antimicrobial discs The ability of the palm extracts to scavenge hydrogen peroxide was determined according to the method of Ruch et al.32. A A 5 mm diameter plunger was used to punch a Whatman no.1 solution of hydrogen peroxide (2 mM) was prepared in 50 mM absorbent filter paper to obtain 5 mm diameter paper discs. The phosphate buffer (pH 7.4). Extracts (1 ml, 0.5 mg/mL) and discs were properly labeled and then sterilized by autoclaving standard drugs (ascorbic acid) were separately added to a for 15 min at 121°C. The discs were impregnated with the plant hydrogen peroxide solution in phosphate buffer (3 ml, 40 mM). extracts (100- 400 µg/ml), dried and stored off in sterile bottles. Absorbance of hydrogen peroxide at 230 nm was determined 2.12 Evaluation of antimicrobial activity 10 minutes later against a blank solution. The percentage of hydrogen peroxide scavenging of both extracts and standard Antimicrobial activity was tested using a modified discs diffusion compound were calculated: assay (DDA) method34,35. A loop of culture from the nutrient agar (NA) slant stock was cultured in Mueller Hinton medium % Scavenged [H2O2] = [(AC – AS)/AC] x 100 overnight and spread with a sterile swab into Petri-plates. Each

Where AC is the absorbance of the control and AS is the microbial swab was spread on separate plates. Sterile disc (5 absorbance in the presence of the extracts or standards. mm in diameter) impregnated with the plant extracts were placed on the cultured plates. Control experiment was carried 2.9 Molybdate ion reduction assay out using commercial antibiotics, antifungal and solvent (stock). The extracts were evaluated for their total antioxidant capacity The solvent loaded disc without extracts served as control in the following the spectrophotometric molybdate ion reduction assay study. Streptomycin was used for bacterial isolates and Nystatin of Prieto et al.33 with slight modifications. 1 ml of each extracts for fungal isolate; plates were incubated for 24 hrs and 48 hrs solution (0.5 mg/ml) was added to 3 ml of reagent solution (0.6 respectively. The results were recorded by measuring the zones M sulphuric acid, 28 mM sodium phosphate and 4 mM of growth inhibition. Clear inhibition zones around the discs ammonium molybdate). The mixtures were incubated at 95 °C indicated the presence of antimicrobial activity. All data on for 90 mins. After incubation, the mixture was cooled at room antimicrobial activity were average of triplicate. temperature, and the absorbance was measured at 695 nm 2.13 Determination of minimum inhibitory concentration against the blank. Ascorbic acid was used as a standard and a standard curve was obtained. The minimum inhibitory concentrations of the extracts were determined using tube dilution method36. The initial Absorbance of Test Sample % Antioxidant capacity = X 100 Absorbance of Ascorbic concentration of each of the plant extracts was diluted using double fold dilution and standard volume of each diluted isolate 2.10 Collection of bacterial and fungus isolates (0.1ml) was aseptically inoculated into different concentrations Clinical bacterial and fungus isolates were collected from St. of the extract. Control experiment was carried out without the Lukes Hospital, Anua, Uyo and University of Uyo Teaching crude extracts. All tubes were incubated at 37 °C for 24 hrs. Hospital, Uyo, Akwa Ibom State, Nigeria. These isolated were Minimum inhibitory concentrations (MIC) were determined as transported on slants to Microbiology Laboratory, University of the lowest concentration without turbidity. Uyo, Nigeria. The test organisms were sub-cultured into nutrient 2.14 Cell culture broth and incubated for 48 hrs at 37 ºC. The microbes were sub-cultured on a nutrient agar slant for the isolation of pure HeLa (Cervical Cancer) cells were cultured in Minimum culture. Isolates were identified using standard cultural, Essential Medium Eagle (MEME), supplemented with 5% of microscopic and standard biochemical methods such as motility fetal bovine serum (FBS), 100 IU/ml of penicillin and 100 µg/ml 2 test, gram staining, oxidase test, oxidation fermentation test, of streptomycin in 75 cm flasks, and kept in 5% CO2 incubator indole test, catalase test, gelatin liquefaction test, citrate at 37oC. Exponentially growing cells were harvested, counted utilization, esculin hydrolysis, urease activity, decarboxylase with haemocytometer and diluted with DMEM37. reactions and hydrogen sulphide production tests. The Gram H460 (Lung cancer) cells were cultured in RPMI medium, positive bacteria (Staphylococcus aureus and Bacillus subtilis) supplemented with 10% of fetal bovine serum (FBS), 100 IU/ml and fungus (Candida albicans) were serially diluted to factor of penicillin and 100 µg/ml of streptomycin in 75 cm2 flasks, and three using 10 fold dilution. Gram negative isolates o kept in 5% CO2 incubator at 37 C. Exponentially growing cells (Pseudomonas aeruginosa, Escherichia coli, Proteus mirabilis, were harvested, counted with haemocytometer and diluted with Salmonella typhi and Shigella dysenteriae) were serially diluted RPMI38. to factor five using 10 fold dilution. The isolates were sub- UK J Pharm & Biosci, 2017: 5(1); 61 Essien et al., Bioactivities of Medicinal Palm Nut Extracts

MCF-7 Breast cancer (ATCC No. HTB-2)39 and PC-3 prostate have been reported to contain flavonoids, phenolics, ceramides cancer cells (ATCC No. CRL-1435)37 were cultured in and glycerides13-15. Dulbecco’s Modified Eagle Medium (DMEM), supplemented Table 1: Phytochemical composition of L. chinensis, A. with 5% of fetal bovine serum (FBS), 100 IU/ml of penicillin and catechu and S. rotundifolius nuts 2 100 µg/ml of streptomycin in 75 cm flasks, and kept in 5% CO2 incubator at 37oC. Exponentially growing cells were harvested, Test L. A. S. counted with haemocytometer and diluted with DMEM. chinensis catechu rotundifolius 2.15 Cytotoxicity screening Saponins +++ + ++ Cytotoxic activity of compounds was evaluated in 96-well flat- Phlobatannins +++ ++ + bottomed micro plates by using the standard MTT (3-[4, 5- dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide) Tannins +++ +++ ++

37 colorimetric assay . HeLa cells were plated into 96-well cell Anthraquinones ++ - + culture plates at 6 x 104 cells per well, H460 cells (4 x 104), Cardiac glycosides - - ++ 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 Terpenes - - +++ incubation, supernatant fluid was removed by suction and 200 Alkaloids ++ ++ + µL of fresh medium containing extract/ compound was added in triplicate, giving a final concentration of 30 µg/mL or 30 µM. Flavonoids ++ ++ + Standard drug used in the MTT assay was doxorubicin. After Deoxy-sugars +++ +++ + 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 Reducing sugars - - - incubated further for 4 hrs. Formazan crystals, formed by Phenols +++ ++ + reduction of MTT were dissolved in 100 µL DMSO and +++: High, ++: Moderate, +: Trace, -: Not detected absorbance was taken at 570 nm using micro-plate reader The concentration of antioxidant components (mg/g) in the palm (Spectra Max plus, Molecular Devices, CA, USA). The % nut extracts are shown in Table 2. In this study, total phenolics inhibitions were processed by using Soft- Max Pro software content ranged from 52.0-245 mg GAE/g, flavonoids (1.5-25.5 (Molecular Device, USA). If extracts/compounds showed 50% mg GAE/g, while tannins content was 21.5-78.5 mg/g. L. or more percent inhibition, they were further processed for IC50 chinensis constituted the highest amount of antioxidant calculation. The cytotoxicity was recorded as concentration compounds. The polyphenols content of A. catechu seed causing 50% growth inhibition (IC50) for all cell lines. The (114.14 mg/g)41 is similar to our report, but with higher flavonoid percent inhibition was calculated using the following formula: content (77.36 mg/g). Higher phenolic content (155.80 mg

OTC − ONC 42 % Inhibition = 100 − X 100 TAE/g) for A. catechu nut was shown by Hannan et al. . OPC − ONC Antioxidative properties of polyphenols arise from their high Where, OTC: mean of O.D of test compound; ONC: mean of reactivity as hydrogen or electron donors from the ability of the O.D of negative control; OPC: mean of O.D of positive control polyphenol derived radical to stabilize and delocalize the unpaired electron (chain-breaking function) and from their 3 Results and Discussions potential to chelate metal ions (termination of the Fenton The phytochemical screening indicated varying amount of reaction)43. Flavonoids and tannins exert their antioxidant alkaloids, saponins, cardiac glycosides, terpenes, de-oxy activities by different mechanisms, such as scavenging free sugars, anthraquinones, phenols, flavonoids and phlobatannins radicals, chelating metals and inhibiting lipid peroxidation. The – in the palm extracts (Table 1). The terpenes and saponins OH at C3 of the flavonoid structure plays a role in chelating and content were relatively high in S. rotundifolius and L. chinensis scavenging activity44,45. The variation in the concentration of respectively; tannins in all three samples. Reducing sugars phytochemicals (polyphenols) in extracts may be attributed to were not detected in the nut extracts. Currently, several the specie type, maturity of plant and the inherent nature of the compounds have been isolated and characterized from A. constituents. These factors also influence the biological catechu, such as alkaloids, tannins, flavones, triterpenes, activities exhibited by the plant extracts. Wetwitayaklung et al.46 40 steroids, and fatty acids . A. catechu seed contains several reported the change of the content of the phenols and tannin in alkaloids belonging to the pyridine group – arecoline, A. catechu at different growth stages. arecaidine, arecolidine, guvacine, guvacoline, isoguvacine, The results of the antioxidant activity models are displayed in norarecaidine and norarecoline41. L. chinensis fruit and root Fig. 1-4. The method widely used to predict the ability of flavonoids to transfer H atoms to radicals is based on the free UK J Pharm & Biosci, 2017: 5(1); 62 Essien et al., Bioactivities of Medicinal Palm Nut Extracts radical, 1,1-diphenyl-2-picrylhydrazyl in the DPPH assay. In the other studied palm extracts. Lee et al.47 showed that methanol DPPH radical assay, the palm nut extracts (0.25-2.0 mg/ml) extract of A. catechu nut strongly enhanced viability against significantly scavenged the DPPH radical in a concentration H2O2 induced oxidative damage in Chinese hamster lung dependent manner (Fig. 1). The results also showed that at 2.0 fibroblast (V79-4) cells where the extract exhibited higher mg/ml dose, L. chinensis, A. catechu and S. rotundifolius antioxidant activity than resveratrol known to protect cells extracts inhibited DPPH radical by 87.30, 90.55, and 90.40% oxidative damage (Fig. 3 and 4). Therefore, it is essential to use respectively compared to the standard, ascorbic acid (92.32%). more than one method to evaluate antioxidant capacity of plant This is an indication of the antioxidants potential in the extracts materials because of the complex nature of phytochemicals48. to reduce the stable, purple-coloured radical DPPH to the yellow coloured DPPH-H form. Li and Lin (2010) demonstrated that the absolute methanol extract of A. catechu nut showed DPPH activity (24.15% to 38.77%, 2.5-25 µg/ml); ascorbic acid and gallic acid (32.21 and 52.26 %, 25 µg/ml, respectively). This result is an indication of the high antioxidant potential of A. catechu nut.

Table 2: Phenolics content of L. chinensis, A. catechu and S. rotundifolius nuts extracts

Phytochemical L. A. S. (mg/g) chinensis catechu rotundifolius Fig. 2: Iron chelating activity of L. chinensis, A. catechu Polyphenols 245.0±0.02 106.0±0.11 52.0±0.05 and S. rotundifolius nuts

Flavonoids 25.5±0.15 1.5±001 10.5±0.02

Tannins 78.5±0.18 65.0±1.0 21.5±0.03 Values are mean & SEM

Fig. 3: Hydrogen peroxide scavenging activity of L. chinensis, A. catechu and S. rotundifolius nuts

Fig. 1: DPPH scavenging activity of L. chinensis, A. catechu and S. rotundifolius nuts

Similarly, high antioxidant activity was observed for iron chelating activity (Fig. 2) in a concentration dependent pattern. The percentage inhibition by this mechanism at 0.5 mg/ml for L. chinensis, A. catechu, S. rotundifolius and ascorbic acid were 42.6, 27.3, 65.0 and 71.3% respectively. The polyphenols in S. rotundifolius showed better metal chelating ability compared with the other studied palm extracts. L. chinensis, A. catechu and S. roundifolius extracts also exhibited significant hydrogen Fig. 4: Molybdate ion reduction activity of L. chinensis, A. peroxide scavenging (93.18, 98.46 and 96.47 respectively, 0.5 catechu and S. rotundifolius nuts. mg/ml) and molybdate ion reduction capacity (58.3, 23.72 and 15.93 respectively, 0.5 mg/ml) compared to the standard The antibacterial and antifugal activities of L. chinensis, A. compound. The polyphenols in L. chinensis demonstrated catechu and S. rotundifolius extracts displayed significant stronger molybdate (VI) reduction ability compared with the antimicrobial activity (Table 3a & 3b). The results revealed that UK J Pharm & Biosci, 2017: 5(1); 63 Essien et al., Bioactivities of Medicinal Palm Nut Extracts all the extracts tested showed varying degree of microbial while a highly active antimicrobial agent gives a relatively low inhibition (6.0-12.5 mm) against the test microbial strains. The MIC35. zones of inhibition varied with the extract and the organism L. chinensis extract exhibited strong antimicrobial property tested. It was observed that the zones of inhibition increased against B. subtilis, P. aruginosa and C. albicans (MIC = 75-100 with increase in concentration as improved antimicrobial activity µg/ml); A. catechu was potent against all test pathogens (75- was concentration dependent. 100 µg/ml) while S. rotundifolius showed notable antimicrobial The antibacterial activity was highly comparable to the standard action against all but one studied microbe, S. typhi (150 µg/ml). drugs, streptomycin, except against S. aureus and C. albicans. It has been documented that gram-positive bacteria are more Minimum inhibitory concentration (MIC) which is the lowest sensitive to chemical compounds than gram-negative bacteria concentration of an extract that inhibits completely the growth of due to differences in the structures of their cell walls49. This is micro-organism in 24 hours ranged from 75-250 µg/ml on tested obvious by the sensitivity of the gram-positive bacteria to the bacteria and fungus for various extracts (Table 4). Antimicrobial tested extracts. agents with low activity against an organism have a high MIC Table 3a: Zones of inhibition of palm nut methanol extracts in millimetres (mm)

L. chinensis (µg/ml) A. catechu (µg/ml) Organism 100 200 300 400 100 200 300 400

S. aureus - 7.0 8.5 9.5 8.0 8.5 9.5 11.0

B. subtilis 7.0 8.0 8.5 11.0 7.0 8.0 10.0 11.0

E. coli - - 7.0 8.5 7.0 9.0 10.0 11.0

P. aeruginosa 6.0 7.5 8.0 10.0 7.0 9.0 10.0 10.0

P. mirabilis - 7.0 8.5 10.0 7.0 7.0 10.0 11.0

S. chlor - 7.5 8.0 10.5 8.0 8.0 11.5 12.0

S. typhi - 7.0 8.0 9.5 7.0 9.0 10.0 11.0

C. albicans 8.0 9.0 10.0 12.5 7.0 7.5 10.0 12.0

- : No observed inhibition; NT: Not tested

Table 3b: Zones of inhibition of palm nut methanol extracts in millimetres (mm)

S. rotundifolius (µg/ml) Streptomycin Nystatin Organism 100 200 300 400

S. aureus 8.0 9.0 9.5 11.0 20.0 NT

B. subtilis 8.0 9.5 10.0 12.5 12.0 NT

E. coli 7.0 9.0 10.5 12.0 10.0 NT

P. aeruginosa 7.5 8.5 10.0 11.0 15.0 NT

P. mirabilis 7.0 7.5 8.5 9.0 12.0 NT

S. chlor 7.0 7.5 8.0 10.0 11.0 NT

S. typhi - 7.0 8.5 9.5 10.0 NT

C. albicans 7.0 7.5 8.5 11.0 NT 24.0 - : No observed inhibition; NT: Not tested

The observed antimicrobial property can be attributed to the also showed that the concentration needed for 100% inhibition antimicrobial compounds inherent in each extract, especially the of growth of human and veterinary isolates was 3.3-7.0 µg/ml high amount of phenolic compounds. Anthikak and Michael50 (gram negative bacterial), 16 µg/ml for gram positive bacteria

UK J Pharm & Biosci, 2017: 5(1); 64 Essien et al., Bioactivities of Medicinal Palm Nut Extracts and 16.67 µg/ml for antifungal51 using hot water areca nut The authors declare no conflict of interest. extracts. S. rotundifolius leaf extract was shown to inhibit the 6 Author Contributions growth of E. coli, S. aureus, P. aeruginosa and S. typhi19. Furthermore, Kaur and Singh16 reported that the presence of EEE and BSA conceived and designed the experiments; EEE high concentration of phenolic compounds with astringent and EIE performed the experiments; EEE and BSA wrote the properties in L. chinensis fruit resulted in S. aureus DNA, manuscript. enzyme and protein denaturing. Tannins have astringent 7 References properties, hasten the healing of wounds and inflamed mucous membrane52. 1. Alonso-Castroa AJ, Balleza-Ramosb S, Hernández- Moralesb A, Zapata-Moralesa JR, González-Chávezc Table 4: Minimum inhibitory concentrations (g/ml) of palm MM, Carranza-Álvarezb C. Toxicity and antinociceptive nut methanol extracts effects of Hamelia patens. Revista Brasileira de Farmacognosia 2015; 25: 170-176. L. A. S. Organisms chinensis catechu rotundifolius 2. Srinivasan RM, Chandraseka JN, Nanjan MS, Suresh B. Free radical scavenging activity of Spomoea obscura (L.) S. aureus 150.0 75.0 75.0 Ker-gawl. J. Nat. Remed. 2007; 7(2): 184-188. B. subtilis 75.0 75.0 75.0 3. Beris H. Anti-oxidant affects bases of drug selection. E. coli 250.0 100.0 75.0 Drugs 1991; 42: 569-605.

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