Phytochemical and antibacterial properties of Diodia scandens and Phyllanthus amarus on staphylococci isolated from patients in tertiary hospitals in Nigeria

Herbal medicine is gradually gaining ground especially on the African continent because of recurrent antibiotic-resistant strains of bacteria that are prevalent within the hospital environment and the community owing to cross-infection (Ojo, Ogodo & Esumeh 2013a). The primary benefits of using plant-derived medicines are that they are safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment with minimal or no side effects in the treatment of infectious diseases (Robbers, Speedie & Tyler 1996).


Introduction
The Current and universally recognised effective health care delivery includes primary, secondary and tertiary health care systems.The Primary health care recognises the importance of alternative, complementary and traditional medicines both in developed and developing countries (WHO 2003).
Diversity, flexibility, easy accessibility, broad continuing acceptance in developing countries and increasing popularity in developed countries, relative low cost, low levels of technological input, relative low side effects and growing economic importance are some of the positive features of traditional medicine (WHO 2002).
Herbal medicine is gradually gaining ground especially on the African continent because of recurrent antibiotic-resistant strains of bacteria that are prevalent within the hospital environment and the community owing to cross-infection (Ojo, Ogodo & Esumeh 2013a).The primary benefits of using plant-derived medicines are that they are safer than synthetic alternatives, offering profound therapeutic benefits and more affordable treatment with minimal or no side effects in the treatment of infectious diseases (Robbers, Speedie & Tyler 1996).
Diodia scandens belong to the family Rubiaceae, a straggling herb with slender angular stems of 1-3 m in length and scabrid leaves.It is commonly dispersed in tropical Africa, tropical Asia and America.Studies showed its use as a vermifuge for children, in treating pregnant women and as an afterbirth treatment to clear the womb, antidotes (venomous stings, bites, etc.), as a pain killer, in treating venereal diseases, arthritis, rheumatism, cutaneous and subcutaneous parasitic Background: The rapidly growing use of herbal drugs or supplements in complementary and alternative medicine as substitute for orthodox medicine both in developed and developing countries is fast gaining ground.
Aim: This study evaluated both qualitative and quantitative phytochemical constituents of Diodia scandens and Phyllanthus amarus vis-à-vis their synergistic effects on clinically isolated staphylococci.
Methods: A total of 200 wounds and burns samples were obtained from patients in the accident and emergency unit of different tertiary hospitals.Staphylococci were isolated and characterised using standard microbiological procedures.Whole plants of D. scandens and P. amarus were Soxhlet extracted with absolute ethanol.The phytochemical analysis was carried out using standard methods.Also, the minimum inhibitory concentration and bactericidal effect of the combined extracts were determined.

Conclusion:
It is noteworthy that the traditional use of these plants was not only confirmed but the combination of D. scandens and P. amarus also proved more effective as antibacterial agent compared with a previous study on the same plants using single determination.
Plants offer a large range of natural compounds belonging to different classes of phytochemicals.These molecules possess interesting biological activities which have attracted several researchers to their elucidation to provide knowledge that will lead to advancement in medicine (Zabri et al. 2008).The phytochemical analysis of plants has revealed various bioactive ingredients and other phytonutrients such as saponin, alkaloid, tannin, phytin phosphorus, polyphenol (flavonoids), phytate, oxalate and others in varying quantities, thus conferring antimicrobial properties on plants and producing definite physiological action on the human body.Most of the phytochemicals are rich in antioxidant activity.Studies have shown that many of these compounds possess anti-inflammatory, anti-atherosclerotic, anti-tumour, anti-mutagenic, anti-carcinogenic, antibacterial, and antiviral activities (Cowan 1999;De Britto, Gracelin & Rathna Kumar 2013).
Traditional healing practitioners are known to combine different medicinal plants in the treatment of infectious diseases and physiological sickness, thus increasing the efficacy or potency of the herbal decoction.This, however, implies that synergistic interaction exists between or among the various medicinal plants employed in patients' treatment.
D. scandens and P. amarus showed great potential as antibacterial agents and their rate of killing on multidrugresistant staphylococci has been previously reported with continuous average logarithm reduction in viable cell count at 6-hour time interval (Ojo, Ejims-Enukwe & Esumeh 2013b).
This study was then designed to elucidate the essential bioavailable constituents present in D. scandens and P. amarus and the possibility of their synergism against clinical isolates (staphylococci), which could be incorporated into staphylococcal wound treatment in orthodox medicine.

Study site
Fresh whole plants of D. scandens and P. amarus were harvested from Novena University, Ogume (Amai campus), Delta State, and its environs and subsequently identified at the Herbarium section of Delta State University, Abraka, Delta State, Nigeria.

Collection of clinical isolates
A total of 200 samples from infected sites of wounds and burns of patients were obtained from different tertiary hospitals within Delta State with sterile cotton swabs and transported to the laboratory.The cotton swabs were applied to freshly prepared slants of nutrient agar and mannitol salt agar (oxoid) and were incubated at 37 °C for 24 h.

Isolation and identification
Colonies growing on slants were streaked on top of freshly prepared plates of mannitol salt agar and incubated again at 35 °C.Primary characterisation of isolates was based on Gram stain, morphological and cultural characteristics, growth on nutrient agar and DNase agar and fermentation on mannitol salt agar, catalase and coagulase tests and other biochemical tests.β-lactamase assay was performed using the method as described by Ako-Nai et al. (2005).

β-lactamase assay
Strips of starch paper measuring 4 cm × 7 cm were cut and sterilised with 70% ethanol.These were then soaked for 10 min in a solution of 0.1 g benzyl penicillin dissolved in a sufficient amount of phosphate buffer (pH 7.0, 0.067 mol/L) to make up 100 mL.They were spread evenly onto sterile Petri dishes.A culture of 24-h-old test isolates grown on nutrient agar was then inoculated on the surface of the paper and spread over an area of 2-3 mm.Each test paper was then used to test the staphylococcal isolates at a time with the inocula placed at least 1.5 cm apart.The Petri dishes were then inoculated for 30 min at 37 °C, after which the plate was flooded with Gram's iodine solution, which was immediately drained off.This caused the starch paper to turn uniformly black within 30 s of application.Colonies with decolourised zones were indicative of β-lactamase production.Results were read within 5 min, as black background tends to decolourise, making interpretations more difficult (Ako-Nai et al. 2005).

Processing of plants
Fresh whole plants of D. scandens and P. amarus were properly washed in tap water and then rinsed in sterile distilled water and left to air-dry for several weeks.The whole plants were micronised to powdered form using an electric blender (Magic Blender -Nakai Japan; Model number -SG-KIPN).
The pulverised plants were stored in air-tight containers until required.

Sample preparation and extraction
The extraction of the two plants was carried out using absolute ethanol as extracting solvent.The extraction of the active ingredients of the plants was performed using the method described by Harbone (1994) with slight modification.Seventy-five grams of the pulverised plant was Soxhlet extracted using 500 mL of the absolute ethanol.
The volatile oil obtained was purified by filtration through Whatman No.1 filter paper (Atata, Sani & Ajewole 2003), and further sterilised by filtration through Millipore membrane filter of 0.45 µm pore size (Sule & Agbabiaka 2008) and then concentrated by evaporation using water bath at 100 °C.The sterile extracts obtained were stored in sterile capped bottles and refrigerated at 4 °C until required for use.The extracts were re-dissolved in 5% dimethyl sulphoxide to achieve different concentrations of 400 µg/ mL, 200 µg/mL and 100 µg/mL.

Microbe-free proof of the extracts
The extracts were tested for presence or absence of turbidity using Millipore filtration technique by introducing 2 mL of these extracts into 10 mL of sterile Mueller-Hinton broth and incubating at 37 °C for 24 h.A microbe-free extract was indicated by the absence of turbidity or clearness of the broth after the incubation period (Sule & Agbabiaka 2008).

Standardisation of microbial cell suspension
Each of the 24-h-old pure culture was prepared to McFarland standard using the method described by Clinical Laboratory Standards Institute (CLSI 2008).This was performed as follows: 0.5 mL of 1.0% (w/vol) anhydrous BaCl 2 was added to 99.5 mL of 1% (vol/vol) H 2 SO 4 solution, which was stirred to maintain a suspension and thoroughly mixed immediately before the next step; 5 mL of the 0.5 McFarland Standard was distributed into screw-cap tubes.The diameter of these tubes was the same as those used for adjusting the density of culture suspensions prior to inoculation.When these standards are thoroughly shaken, the turbidity equals that of a culture containing about 1.5 × 10 8 cells.The tubes containing the 0.5 McFarland standards were stored in the dark at room temperature.

Antibacterial susceptibility test
The samples obtained were cultured on Mueller-Hinton agar supplemented with 2% of NaCl and incubated at 35 °C for pure isolates.The antibacterial susceptibility profiles of the isolates were determined using broth dilution method as described by the CLSI (2008), Wayne, PA, USA.Reference-type strains of Staphylococcus aureus ATCC 25923 were included as positive control.

Results
The rate of isolation of staphylococci from 200 wounds and burns sources revealed a high prevalence of S. aureus (85%) and coagulase-negative staphylococci (CoNS) (15%).DNase characterisation showed 74% DNase-positive staphylococci and 26% DNase-negative staphylococci.The β-lactamase assay produces 30% β-lactamase-producing S. aureus and β-lactamase-producing CoNS (17%) (Table 2).The MIC value for P. amarus was 100 µg/mL, while for D. scandens it was 200 µg/mL (Table 3).The combined plant extracts revealed synergism with fractional inhibitory concentration (FIC) of D. scandens at 0.1563 and P. amarus at 0.3125, showing no turbidity (no cell growth) in all the isolates tested (Table 1).FICI was 0.4688 (≤ 0.5).It was observed from this study that the FIC has a lower value indicating synergism when the extracts were combined at lower concentrations.

Discussion
The high prevalence of pathogenic S. aureus and emerging infectious CoNS from clinical patients within the tertiary hospitals in Nigeria and around the world are becoming a life-threatening condition, which calls for urgent medical and research attention if the life expectancy of the entire populace is to be attained.This research which was a follow-up study to those conducted by Ojo et al. (2013a) and Ojo, Sargin and Esumeh (2014) et al. 2013;Tamil et al. 2010;Van-Burden & Robinson 1981;Young & Greaves 1940;Zabri et al. 2008) as in this study.By implication, the combination of these medicinal plants produces a greater efficacy in the treatment of staphylococcal wound infection as regularly experienced in orthodox medicines.This is because the anti-nutrients are equally present in all fruits, which have been found to have antioxidant and radical-scavenging properties alongside antimicrobials.

Conclusion
Research in ethnobotanical uses of medicinal plants in combined form has validated its claim in complementary alternative medicine owing to the presence of some bioactive constituents, which have similar mechanism of action with antibiotics.Therefore, the prophylactic and curative treatment of these plants should be determined for further study.

TABLE 4 :
Quantitative analysis on the phytochemical constituents of Diodia scandens and Phyllanthus amarus.