Antimicrobial activity of aqueous and organic extracts of a Saudi medicinal plant: Rumex nervosus
Abstract
Objective:
The antimicrobial effect of aerial part of Rumex nervosus obtained from the Southern region of Saudi Arabia was evaluated on bacterial strains Staphylococcus aureus, methicillin resistant S. aureus, Enterococcus faecalis, Escherichia coli, Pseudomonas Aeruginosa, and fungal strain Candida albicans.
Materials and Methods:
The solvents used for the extraction were aqueous, hexane, and methanol. The in vitro antimicrobial activity was performed by agar diffusion and disk diffusion methods and the confirmation of this activity was done by the enumeration of colony forming units (CFU).
Results:
The aqueous extract showed the growth inhibitory effect on Gram-positive bacteria while the Gram-negative P. aeruginosa was the most sensitive microorganism as determined by the agar diffusion technique. Surprisingly, the extract showed little antibacterial activity on other Gram-negative bacteria (E. coli) by this technique. Ethanolic extract was also found to be inhibitory to the growth of microorganisms. Hexane extract was relatively low in antimicrobial activity on Gram-negative E. coli and P. aeruginosa, while both the organic extracts were inhibitory to the growth of the fungus, C. albicans. Hexane gave no conclusive results with agar or disk diffusion methods, but showed the microbial growth inhibition in CFU enumeration. The antibacterial activity of active extracts was compared with vancomycin while antifungal activity of was compared with amphotericin B.
Conclusion:
The results obtained in the present study suggest that R. nervosus showed a marked antimicrobial activity with the test organisms.
The plant Rumex nervosus is abundantly found in the Southern region of Saudi Arabia. It contains flavonoids, anthraquinones, and gallic acid. The leaves of R. nervosus are used to treat the skin rashes and the young leaves with the tender shoots are eaten by the Shepard after they are roasted over open fire to reduce the acid content.[1] The charcoal made after the burning of stem is mixed with egg yolk to be used as a nursing for the burns and sometimes butter is added to save from flaking and drying of the burn wound. The genus Rumex includes many edible plants, and their medicinal importance was investigated by many investigators toward a number of bacterial, viral, and chlamydial infections.[234] Teklehaymanot et al. reported the use of this plant as anti-dysentery, cure for stomach ache, and effective treatment of warts.[5] The methanolic extract of the root and leaves of this plant was also found to be effective in the treatment of helminthiasis[6] and diarrhea induced in mice.[7] Other plants belonging to the genus Rumex are also used as anti-tumor agents in different tumor cell lines from colon, ovary, melanoma, breast, and central nervous system.[89]
The aim of this study was to investigate the antimicrobial activity of extracts from medicinal plant R. nervosus used in folk medicine. A comparative study of the antimicrobial properties of extracts obtained by three different methods was reported in order to assure the most potent extract.
Materials and Methods
Plant material
The aerial parts of the plant were procured from Asir region of Kingdom of Saudi Arabia. It was identified and a voucher specimen has been kept for future reference. The plant was air dried and made into a coarse powder in a grinder.
Preparation of aqueous extract
The aqueous extract was prepared by suspending 10 g of this powder in 100 mL of distilled water. The content was autoclaved (121°C; 15 min) and the supernatant was collected after centrifugation at 2000 × g for 15 min. Further dilutions were made in sterile phosphate buffer saline (PBS).
Preparation of organic extracts
The organic extracts (methanolic and hexane) were prepared by packing separately 60 g of the coarsely drug in a soxhlet apparatus and extracted at 60°C for 18 h using ethanol and hexane, respectively, as extraction solvents. The organic extracts obtained were concentrated in a rotary evaporator.
Microbial cultures and growth condition
Freeze dried cultures of Staphylococcus aureus ATCC 25923 (S. aureus), Enterococcus faecalis ATCC 29212 (E. faecalis), Escherichia coli ATCC 25922 (E. coli), Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa), and Candida albicans ATCC 66027 (C. albicans) were purchased from microbiologics, Inc., (St. Cloud, MN. USA). Methicillin resistant S. aureus (MRSA) strain 12,498 was obtained from the Department of Laboratory Medicine, Prince Sultan Military Medical City in Riyadh, Saudi Arabia. The culture media like Brain Heart Infusion agar (BHIA) and Brain Heart Infusion broth (BHIB) were purchased from Scharlau Diagnostics, Barcelona, Spain, while the Sabouraud dextrose agar (SDA), Sabouraud dextrose broth (SDB) and BHIA prepared plates were obtained from Watin-Biolife, Inc., in Riyadh, Saudi Arabia.
All of the microbial cultures with the exception of C. albicans were grown in BHIB and the stocks prepared in 50% glycerol were stored in −40°C freezer. The C. albicans culture was grown in SDB and stored as a glycerol stock at −40°C.
Antimicrobial assay by agar diffusion method
Agar diffusion procedure was conducted by melting the presterilized BHIA and keeping in a water bath at 45°C. Inoculum from overnight grown bacterial cultures was added to BHIA agar in separate tubes and after mixing the content, Petri plates were poured. After the agar was solidified, plates were transferred to the refrigerator for 2–3 h. A dedicated agar punch was used to cut 6.0 mm holes, and 50 µL of neat and diluted extracts were added to the wells in triplicate. The diluent was used as a control.
Plates were left on the bench for 30 min and transferred to the incubator. Zones of microbial growth inhibitions were recorded at 24 and 48 h. The identical procedure was used to conduct the agar diffusion procedure for C. albicans, but SDA was used as culture medium.
Antimicrobial assay by disc diffusion method
The disc diffusion procedure was conducted as a slight modification by Bauer et al.[10] The discs (6.0 mm) were cut out of filter paper and sterilized in an autoclave. Discs were dried overnight in a drying oven set at 40°C. Overnight grown microbial cultures were diluted to 0.1 McFarland unit and 50 µL of the inoculum was spread on BHIA and SDA plates for bacteria and fungus, respectively, using a bent glass rod and a plate rotator. Plates were left at room temperature for 30 min and the presoaked disks with the test material were laid over the agar medium. Zones of growth inhibition were recorded after 48 h of incubation.
Determination of minimum biocidal concentration
Microbial cultures were grown for 24 h and adjusted to 0.5 McFarland standard. Cultures were incubated in presence and absence of aqueous and organic extracts and after 24 h of incubation, 0.1 mL of inoculum was spread on BHIA plates. Presence and absence of growth were recorded after 48 h of incubation. In parallel, vancomycin was used as a positive control for bacteria while amphotericin B was employed for C. albicans. Negative control values were obtained by using PBS.
Results and Discussion
The extracts of R. nervosus were tested in different concentrations for antimicrobial effect against Gram-positive (S. aureus, MRSA and E. faecalis), Gram-negative (E. coli and P. aeruginosa) bacteria, as well as antifungal activity against C. albicans strain. When aqueous extracts were tested by agar diffusion method, the results showed that among Gram-positive microorganisms, R. nervosus has dose-dependent growth inhibitory activity of S. aureus and MRSA while E. faecalis was found to be resistant. With Gram-negative strains, only P. aeruginosa was found sensitive. The zones of growth inhibition on the three Gram-positive bacteria and P. aeruginosa, which is a Gram-negative bacteria, showed the maximum growth inhibition [Figure 1]. No antibacterial activity was observed in E. coli. The antimicrobial activity of these extracts is shown in the form of bar diagram [Figure 2].
Effect of Rumex nervosus plant aqueous extract on the growth of microorganisms showing positive growth inhibition in agar diffusion procedure (organic extracts figures are not shown due to presence of intense color)
Antimicrobial effect of Rumex nervosus extracts in agar diffusion procedure (the organic extracts were used in mg/mL, while the aqueous extract is used as neat and its dilutions)
The numerical values of the microbicidal effect of R. nervosus extracts were recorded [Table 1]. Microorganisms were incubated with several concentrations/dilutions of the plant extracts and after 24 h of incubation; the quantitative determination of microbicidal effect was conducted by the dilution procedure. Normalized values are used to populate in Table 1.
Antimicrobial effect of aqueous and organic extracts of R. nervosus (enumeration of CFU/mL)
The minimum biocidal concentration of R. nervosus extracts was determined by incubating various concentrations of extracts with a standard inoculum of microbial cultures. After 24 h of incubation, an aliquot was removed from each test samples and the dilutions were plated on agar plates to determine the presence/absence of microbial colonies. The data [Table 2] shows that the activity of aqueous extracts of this plant was very much comparable to the bactericidal effect of vancomycin. R. nervosus and other species of this plant have been used for centuries in various parts of the world to counter the infective agents and other medical ailments. These microbial agents vary from bacteria like S. aureus, Streptococcus mutans, E. coli, P. aeruginosa and few species of fungi. Viruses such as human immunodeficiency virus, herpes and influenza, and sexually transmitted diseases from genital herpes, genital warts including the chlamydial infections are also treated by the extract of few species of genus Rumex.[24] Getie et al. described the antimicrobial effect of a crude extract of R. nervosus and Rumex abyssinicus on Staphylococcus and Streptococcus strains while the antiviral effect on Coxsackie and influenza was very much pronounced.[11] The organic extracts of these plants are also used in physiological disorders such as antidiarrheal agent,[7] antioxidant, and cytotoxicity studies.[12] Present study complemented the previous findings that the crude extracts of R. nervosus were indeed inhibitory to the growth of microorganisms. Extracts were highly effective on Gram-positive bacteria while showing no activity on the C. albicans. Trials must be made underway to see the effect of these extracts in vivo to counter the effect of certain infections initiated in rodents.
The MBC of R. nervosus extracts, vancomycin, and fungizone at different concentration
Conclusion
The results of the study concluded that the polar extracts of the medicinal plants have comparative antimicrobial activity with the standard. However, the organic extract specifically hexane extract had less efficacy against Gram-negative bacteria.
Financial support and sponsorship
Nil.
Conflicts of interest
There are no conflicts of interest.
References
- 1. Some Common Medicinal and Poisonous Plants Used in Ethiopian Folk Medicine1976EthiopiaAmare Getahun
- 2. Herbal medicines for sexually transmitted diseases and AIDSJ Ethnopharmacol2002804966[PubMed][Google Scholar]
- 3. Herbs as a food and medicine source in PalestineAsian Pac J Cancer Prev200564047[PubMed][Google Scholar]
- 4. Antiviral activity and cytotoxicity of the lipophilic extracts of various edible plants and their fatty acidsFood Chem20091157015[Google Scholar]
- 5. Knowledge and use of medicinal plants by people around Debre Libanos monastery in EthiopiaJ Ethnopharmacol200711127183[PubMed][Google Scholar]
- 6. Evaluation of anthelmintic activities of Rumex abyssinicus jacq and Rumex nervosus vahl.(Polygonaceae)Int J Pharm Sci Rev Res20105557[Google Scholar]
- 7. Antidiarrheal activity of methanolic extract of Rumex nervosusJ Pharm Res2004315[Google Scholar]
- 8. Antimutagenicity and cytotoxicity of the constituents from the aerial parts of Rumex acetosaBiol Pharm Bull200528215861[PubMed][Google Scholar]
- 9. Two novel naphthalene glucosides and an anthraquinone isolated from Rumex dentatus and their antiproliferation activities in four cell linesMolecules20121784350[PubMed][Google Scholar]
- 10. Antibiotic susceptibility testing by a standardized single disk methodAm J Clin Pathol1966454936[PubMed][Google Scholar]
- 11. Evaluation of the anti-microbial and anti-inflammatory activities of the medicinal plants Dodonaea viscosa, Rumex nervosus and Rumex abyssinicusFitoterapia20037413943[PubMed][Google Scholar]
- 12. In vitro cytotoxic screening of selected Saudi medicinal plantsJ Nat Med20126640612[PubMed][Google Scholar]