In Vitro Antimicrobial Activity of Medicinal Plant Rumex Nervosus Against Selected Oral Pathogens.

Background: Rumex nervosus is a plant found and used in Saudi Arabia as traditional herbal medicine. Various types of plant extracts with inherent synergistic properties are being used against oral diseases. Thus, in the present study, the anti-microbial activity of Rumex nervosus extracts was studied against six common oral pathogenic bacterial strains and a pathogenic fungus. Materials and Method: Plants of R. nervosus were collected and air-dried, and extracts from various plant parts were obtained. Six clinical isolates of bacteria, namely Staphylococcus aureus, Streptococcus mutans, S. salivarius, S. sanguis, E. faecalis, and Lactobacillus acidophilus), and one pathogenic Candida (C. albicans) were obtained. Antibacterial and antifungal activity of R. nervosus was determined using the Kirby-Bauer agar disc diffusion method. Zones of inhibition were recorded after 48 h of incubation. Data collected were analyzed. A two-way analysis of variance (ANOVA) was applied. P < 0.05 was considered statistically significant.
Results: Methanol extract from leaves were highly effective against S. aureus, with a mean inhibition zone of 33 mm, followed by a 28-mm zone of inhibition using an extract from roots and a minimum inhibition zone using an extract from stems. Zones of inhibition using methanol extract from roots were effective against S. mutans, S. sanguinis, E. faecalis, and L. acidophilus, with mean inhibition zones being 19, 17, 33, and 31 mm, respectively.
Conclusion: The study has provided insight into a new potential herbal anti-microbial agent that may benefit dental care. Copyright:

INTRODUCTION

Nowadays, human pathogens have developed resistance against commonly used anti-microbial drugs. Thus, various medicinal plants have emerged for different medicinal purposes such as infectious diseases. They are the source of bioactive compounds that are advocated to be used for various medicinal purposes.[1] In the southern region of Saudi Arabia, the plant Rumex nervosus is found, which constitutes anthraquinones, gallic acid, and flavonoids.[2] Various researchers have investigated the medicinal importance of edible plants of the Rumex genus against different viral, bacterial, and chlamydial infections.[23] These plants are also used against tumor cells of the breast, colon, central nervous system ovary, and melanoma.[4]

The most infectious oral diseases such as dental caries and periodontitis are highly prevalent. Various types of plant extracts with inherent synergistic properties are used against oral infectious diseases. The most common bacterial strains causing oral diseases are Staphylococcus, Streptococcus, Enterococcus, Lactobacillus species, and Candida, a prominent fungus leading to oral infections.[5]

Because of the developing risk of multi-drug resistance, antibiotics are not being advised against oral infections. Thus, various medicinal plants are being advocated against oral diseases. Medicinal plants are being used as traditional medicines in rural areas of many developing countries.[6] They are the source of primary health care with a wide variety of biological and medicinal activities, low costs, and high safety margins. These herbal medicines are safe and overcome the resistance produced by oral pathogens. Resistance is overcome because these plant extracts exist in a combined or pooled form with more than one molecule in the plant cell's protoplasm.[78]

Rumex nervosus is being used in folk medicine by Saudi Arabian people and was thus selected for the study to determine its anti-microbial activity. In the present study, R. nervosus plants were collected from different locations in the Aseer region, Kingdom of Saudi Arabia. The anti-microbial activity of methanol and n-hexane extracts was studied against six common oral pathogenic bacterial strains (Staphylococcus aureus, Streptococcus mutans, S. salivarius, S. sanguis, E. faecalis, and Lactobacillus acidophilus) and one pathogenic fungus (C. albicans) by using the Kirby–Bauer agar disc diffusion method.

MATERIALS AND METHOD

Plants of R. nervosus were collected from different locations in Aseer province, Kingdom of Saudi Arabia, and were dissected into different parts such as roots, stems, leaves, and flowers. The plant samples were air-dried thoroughly at 24°C, away from sunlight, and then ground into a fine powder by using an electric grinder. To obtain R. nervosus extracts, 40 mL of methanol (polar) and n-hexane (non-polar) solvents were added to 7 g of ground parts of the plant in Falcon tubes. All the tubes were then placed into a rotary shaker instrument at 150 RPM at 25°C for 5 days to extract active compounds from various plant parts.

The extracts were filtered using Whatman paper and dried in the oven at 57°C for 4 days. The residue fraction of n-hexane and methanolic extracts was dissolved in 3 mL of sterile dimethyl sulfoxide. It was then placed in a rotary shaker at 150 RPM at 18°C for 72 h and kept in the refrigerator at 4°C for an anti-microbial activity test.

Six clinical isolates of bacteria, namely (Staphylococcus aureus, Streptococcus mutans, S. salivarius, S. sanguis, E. faecalis, and Lactobacillus acidophilus, and one pathogenic Candida (C. albicans) were obtained from the Microbiology Laboratory, Biology Department, Faculty of Science and Microbiology Laboratory, King Khalid University, Kingdom of Saudi Arabia. All the clinical isolates were subcultured first in nutrient broth and incubated at 27°C for 24 h for all bacterial isolates and 48 h for C. albicans.

Antibacterial and antifungal activity of R. nervosus was determined using the Kirby–Bauer agar disc diffusion method. The disc diffusion procedure by Bauer et al.[9] was followed. The discs (6.0 mm) were cut out of filter paper, sterilized, and dried overnight in a drying oven set at 40°C. Overnight grown microbial cultures were then diluted to 0.1 McFarland unit, and 50 μL of the inoculum was spread on BHIA and SDA plates for bacteria and fungus, respectively, by using a bent glass rod and a plate rotator. Plates were then left at room temperature for 30 min, and the pre-soaked disks with the test material were laid over the agar medium. Zones of growth inhibition were recorded after 48 h of incubation.

Data collected were analyzed using SPSS version 23.0.0.0 software. Two-way analysis of variance (ANOVA) was applied to determine statistically significant differences among plant extracts. P < 0.05 was considered statistically significant.

RESULTS

Zones of inhibition (ZOIs) of microbial growth were measured, showing the susceptibility of microorganisms to each extract. Leaves, stems, and roots were used to make extracts such as hexane, dichloromethane (DCM), and methanol. ZOIs were observed with methanol extracts. No inhibition zones were observed with hexane and DCM extracts. Methanol extract from leaves was highly effective against S. aureus, with a mean inhibition zone of 33 mm, followed by a 28-mm zone of inhibition using an extract from roots and a minimum inhibition zone using an extract from stems [Figure 1]. ZOIs using methanol extract from roots were effective against S. mutans, S. sanguinis, E. faecalis [Figure 2], and L. acidophilus, with mean inhibition zones being 19, 17, 33, and 31 mm, respectively. No inhibition zone was observed with methanol extract from leaves and stems against S. Sanguinis. Moreover, no ZOI was found against C. albicans in either of the extracts [Table 1].

Figure 1

Methanol extract from leaves was highly effective against S. aureus

Figure 2

Zones of inhibition using methanol extract from roots were effective against S. mutans, S. sanguinis, E. faecalis, and L. acidophilus, with mean inhibition zones being 19, 17, 33, and 31 mm, respectively

Table 1

Zones of Inhibition (ZOI) of microbial growth measured in (mm) showing the susceptibility of microorganisms to each extract

Solvent Strain	Hexane extract			DCM extract			Methanol extract
	Leaves	Stems	Roots	Leaves	Stems	Roots	Leaves	Stems	Roots
S. aureus	-	-	-	-	-	-	33	22	28
S. mutans	-	-	-	-	-	-	13	12	19
S. sanguinis	-	-	-	-	-	-	-	-	17
E. faecalis	-	-	-	-	-	-	28	20	33
L. acidophilus	-	-	-	-	-	-	25	17	31
C. albicans	-	-	-	-	-	-	-	-	-

*F-statistics=2.887; P<0.05 (significant)

Two-way analysis of variance (ANOVA) was applied to determine statistically significant differences among plant extracts. A statistically significant difference was observed among inhibition zones between different plant extracts against various microorganisms.

DISCUSSION

Various ethnobotanical investigations revealed significant evidence in identifying and developing the traditionally available therapeutic plants into modern drugs.[10] Similarly, we also used a traditional herbal plant in our study and determined its efficacy against prevalent oral microorganisms.

Various studies have been conducted determining the antibacterial, antiviral, anthelmintic, antifungal, and anti-inflammatory properties of herbal plants.[1112] However, studies revealing the anti-microbial effect of herbal plants against oral microflora are very few. Thus, the present study was conducted to determine the anti-microbial effect of Rumex nervosus against oral microflora.

Rumex nervosus is traditionally used as an excellent dressing for orthopedic fractures, wounds, and inflammatory and painful conditions. Dubaie et al.[13] observed that this plant also has anti-malarial properties. Hussein et al.[14] found that Rumex nervosus is an active anti-microbial agent, and it can be a revolutionary herbal plant for the medical industry. They revealed anti-microbial activity of Rumex nervosus against bacterial and fungal pathogens, including Staphylococcus aureus, Bacillus subtilus, Pseudomonas aeruginosa, E. Coli, Klebsiella pneumonia, and Candida albicans. Similar to this study, we also observed the anti-microbial effect of this plant against various oral pathogens. However, on the contrary, we observed that this herbal plant was not active against candida species.

The extraction of biologically active compounds from plant material depends mainly on the solvent used in the extraction procedure. A study conducted by Dhiman et al.[15] revealed that different organic extracts can provide more potent anti-microbial activity than aqueous extracts. Among various organic extracts, alcoholic extracts displayed the best anti-microbial activity compared to acetonic extracts.[13] Thus, their study substitutes the findings of earlier workers who advocated methanol and ethanol as the best solvent for extracting anti-microbial compounds from plants, followed by acetone and water.

In a study by Tedila et al.,[10] ethanol and methanol were the best solvents, followed by acetone and least by diethyl ether and hexane. They used solvents to extract biologically active substances. Similarly, methanol was the best solvent over diethyl ether and hexane in our study.

In our study, the methanolic extract of Rumex nervosus showed a vigorous inhibitory action against the tested bacteria, which matches the results from previous studies conducted by Tedila et al.,[10] George et al.,[16] and Al-Nowihi et al.[6]

We found that the anti-microbial activity of methanol extract was maximum against S. aureus. Similar results were observed by Al-Asmari et al.,[1] who reported that the methanol extract of Remux nervosus had shown antibacterial activity against S. aureus and no activity on C. albicans. In contrast to our study, Kasimala et al.[17] found that methanol extract of Rumex nervosus had shown no antibacterial activity against E. coli and S. aureus.

The present study results should promote further studies to observe and evaluate the efficacy of other herbal plants and segregate the compounds responsible for the anti-microbial activity. Various modern drugs are being extracted from traditional therapeutic plants and used for indigenous cures.[181920]

CONCLUSION

The current study has provided an insight into a new potential herbal anti-microbial agent that may prove beneficial in dental treatment. Antibacterial activity tests showed a marked anti-microbial activity by methanolic extract fractions of different plant parts against the test bacteria. It has been observed that no anti-microbial activity of plant extracts was found against C. albicans.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.