Antimicrobial Effectiveness on Selected Bacterial Species and Alkaloid and Saponin Content of Rosa nutkana C. Presl (Nootka Rose) and Urtica dioica L. (Stinging Nettle) Extracts

Nootka rose (Rosa nutkana C. Presl) and stinging nettle (Urtica dioica L.) have been traditionally used in the treatment of skin infection by Indigenous peoples of Vancouver Island, British Columbia, Canada. The main objective of this study was to examine the antibacterial efficacy of extracts of Nootka rose and stinging nettle against the common pathogenic skin bacteria Staphylococcus aureus, Micrococcus luteus, and Pseudomonas aeruginosa using Indigenous science and standard methods of analysis. The Indigenous science method of plant extraction by steeping as advised by the Traditional Knowledge keeper was performed to examine minimum inhibitory concentration (MIC) values and minimum bactericidal concentrations (MBC) by serial dilution and bacterial population counts. Soxhlet extractions and Kirby Bauer disc sensitivity testing showed that Nootka rose extracts possessed antibacterial effectiveness against all three bacterial species while stinging nettle extracts were effective against M. luteus. Results for MIC and MBC indicated antibacterial activity against M. luteus and S. aureus for the Nootka rose when using full-strength solutions; all three bacterial species exhibited growth when undiluted stinging nettle treatments were used. When considering bacterial population counts for S. aureus, results indicated that only the Nootka rose treatment offered effective inhibition. scientific methods of plant extract preparation. (Rosa C. Presl), L.),


Introduction
Indigenous peoples of North America have historically used plants such as Nootka rose (Rosa nutkana C. Presl) and stinging nettle (Urtica dioica L.) to heal skin infections by following traditional protocols for plant extract preparation and application. Medicinal plants are a largely unexplored drug repository and some have great potential for novel leads for inhibitors of microbial agents of disease. By the sharing of knowledge of plant medicines by Indigenous Knowledge keepers, Indigenous science has made important contributions to universal health and wellbeing. Statistics continue to show that a large proportion of the world's population continues to use medicinal plants as preventative maintenance of wellbeing, for their primary health care needs, and/or treatment of disease [1]. In this context, plants are consistently viewed as sources of useful medicines and are considered to have great potential in future applications for improving health, including the healing of infections as highlighted in this study. Recently, plants have been shown to treat infections that are difficult to manage with standard antibiotics, due in part to bacterial species that are becoming increasingly drug-resistant [2] [3]. Plants produce secondary bioactive compounds such as phenols, saponins and alkaloids that are inessential to the normal growth and reproduction of the plant. These compounds found in plant extracts have been analyzed by Western scientific methodologies and have revealed to be protective agents capable of deterring animal consumption and multidrug resistant bacterial species [4] [5]. Because the development of antibiotics resistance is a world-wide health concern, finding new plants to effectively treat infections can be especially helpful and timely.
Nootka rose is native to British Columbia, Canada, and ranges from Alaska to the northwestern states in the USA. Traditional use of the leaves includes application to painful bee stings and abscesses [6]. Studies on the Rosa family have shown the therapeutic potential of its antioxidant activity for the treatment of disorders such as cancer, rheumatoid arthritis, osteoporosis, diabetes, skin disorders, and infectious diseases [7]. Methanolic Nootka rose branch extracts have also exhibited antiviral activity and a high level of effectiveness against an enteric coronavirus [8]. Nootka rose has a significant concentration of total phenolic compounds in its seeds and a markedly higher concentration in its whole hips which show greater antioxidant activity compared to other British Columbia rose species [9]. Extracts of both plant structures positively correlated with an-  [9] and several methanolic extraction compounds of Nootka rose fruits were also found to be active against methicillin-resistant S. aureus [10].
Stinging nettle is a common plant found throughout North America, Northern Europe, and Asia at large. The genus designation Urtica comes from the Latin verb urere, which means "to burn", and refers to stinging hairs located on the surface structures of the stinging nettle plant. The stinging sensation of the leaf hairs is caused by several plant chemicals including formic acid, histamine, serotonin, and acetylcholine accompanying impalement of hairs into the skin [11]. In the Cree culture, stinging nettle is used in leaf decoctions to stop hemorrhaging following childbirth, to help reduce the incidence of diarrhea and rid individuals of infection caused by intestinal worms [12]. The plant has also been used as a diuretic, a therapy for joint pain, and as an anti-inflammatory aid for arthritis and rheumatism [13] [14]. Its anti-inflammatory actions in rheumatoid arthritis are attributed to its ability to inhibit the production and release of pro-inflammatory cytokines and T lymphocytes [15]. Stinging nettle shows promising results against inflammation [16], possesses immune-modulatory potential [17] and antimutagenic and radical-scavenging properties that may contribute to chemoprevention of cancers [18]. Water extracts of stinging nettle show antioxidant activity and antibacterial activity [19] and recently these extracts have shown antibacterial activity against multi-drug resistant strains of Mycobacterium tuberculosis [2] and methicillin-resistant Staphylococcus aureus [3].
In addition to antimicrobial activity, secondary bioactive compounds such as alkaloids and saponins also play a role in defending plants against consumption by herbivores [5] [20] [21] [22]. Alkaloids include unpalatable, bitter compounds such as chelerythrine, caffeine, quinine, and scopolamine. Saponins are high molecular weight glycosides that have detergent properties that create stable foams in aqueous solutions [23]: the soapwort plant's saponin content produces soap-like foam when agitated in water and has been effectively used in traditional liquid soaps [24]. Saponin compounds are usually found in the outer cell layers of plants and provide an effective barrier against establishment of microbial populations. These glycosidic compounds also reduce plant palatability due to the strong bitter flavour, thereby decreasing the frequency of foraging on the host plant [25].

Plant Material
The fresh above-ground parts of Nootka rose and stinging nettle were collected
Plant extract material was obtained by conducting Soxhlet extraction procedures using dried plant material [28], followed by application of a Buchi vacuum The freshly prepared plant extract solutions (10% sterile DMSO) were filter sterilized using 0.45 µL filter syringes. Sterile filter discs (6 mm) were then saturated with 40 µL of plant extract solution, placed into sterile, covered petri dishes and stored at room temperature in the dark and overnight to remove excess methanol [29]. The filter discs were applied to Mueller Hinton agar plates, following the uniform bacterial swabbing procedures. Plant extracts for each plant

Bacterial Population Counts
Bacterial population counts (CFU) were conducted using standard procedures [33]. Each plant trial included three S. aureus colonies, three replicates per colony, when using 20 g/200mL plant treatment of Nootka rose and stinging nettle.

Soxhlet Extraction for Determination of the Plant Biological Compounds
Chemicals used throughout these procedures were of analytical grade (hexanes,

Determination of Alkaloids by Soxhlet Extraction
Alkaloids were determined following the method of [34] [35].

Determination of Saponins by Soxhlet Extraction
Saponins were determined following the method of [34] [35].

Statistical Analyses
One-way ANOVA and Tukey's HSD were used to determine statistical differences between the diameters of zones of inhibition using the Kirby Bauer disc diffusion test. Statistical analyses were carried out with the statistical analysis software R (version 3.1.2) and values of p < 0.05 were noted as statistically significant.

Kirby Bauer Disc Sensitivity Testing
Results for zones of inhibition showing equal to or greater than 7 mm (6 mm being the diameter of the filter discs) were considered active against the bacterial populations tested [36].

Discussion
The antibacterial activity of Nootka rose and stinging nettle was considered for Plant preparations of stinging nettle prepared using the Traditional Knowledge keeper's advisement showed a lack of effectiveness against S. aureus. This agrees with the results observed when this bacterium was treated with Soxhlet plant extracts during Kirby Bauer disc sensitivity testing. Motamedi et al. [38] found that ethanolic and methanolic extracts of stinging nettle leaves showed M. luteus is a Gram-positive bacterium which is commonly found in the oral cavity and the respiratory tract. Stinging nettle showed a high level of effectiveness against M. luteus in the current study, which agrees with a previously conducted study by Gülçin et al. that reported that M. luteus growth was inhibited by water extract of stinging nettle [19]. Stinging nettle's mode of action against Gram-positive bacteria may include the disruption of the peptidoglycan cell wall layer [38].
In our study, the Nootka rose plant extract obtained by Soxhlet extraction using methanol inhibited the growth of the Gram-negative P. aeruginosa. A lack of effectiveness against P. aeruginosa became apparent when using the Nootka rose plant preparation and Indigenous science protocols for plant preparation to conduct MIC and MBC testing. One study reported that when the methanol extract of Nootka rose sepals was used, the growth of P. aeruginosa was inhibited [37]. Similarly, the rose R. rugosa showed growth inhibitory activity against the Gram-negative bacterium Acinetobacter baumannii and the phenol antioxidant ellagic acid was identified as the compound shown to possess selective antibacterial activity based on bacteria cell wall differences [9].
Similar to our data, ethanol extracts of stinging nettle leaves showed no antibacterial activity against P. aeruginosa [39]. Motamedi et al. [38] also found that ethanolic and methanolic extracts of stinging nettle leaves were not active against P. aeruginosa [38]. The resistance of Gram-negative bacteria toward stinging nettle extract can be explained by their outer membrane with lipopolysaccharides that acts as a barrier to the entrance of some substances including antibiotics [38].
In this study, Nootka rose showed elevated saponin levels and a greater level of effectiveness against the bacteria tested, when compared with the stinging nettle. In one study with Cyclamen persicum tuber extracts, saponins have shown inhibition of the Gram-positive S. aureus [22]. Saponins extracted from Anabasis articulata (a salt tolerant xerophyte) also showed greater antibacterial activity against both Gram-positive and Gram-negative bacteria compared with alkaloid extracts [40]. Several mechanisms have been proposed to explain the inhibitive effect of saponins on, especially, Gram-positive bacterial growth [21].
It is possible that saponins bind with sterols, causing an increased bacterial membrane permeabilization and thus inhibit bacterial growth by disrupting cell membrane integrity and allowing entry of antibiotic agents into the cytoplasm when used in combination with clinical drugs [21].
A higher percentage for alkaloids was found in the stinging nettle, which  [42]. However, the composition of stinging nettle may differ, potentially caused by differences in planting, climatic, seasonal, and experimental conditions [43]. The extracting solvent may also influence the antibacterial activity of its bioactive components [36]. Other compounds such as terpenes, fatty acid esters and phenols were reported to be the antibacterial compounds in stinging nettle leaves against bacteria [36] [43]. While the previous studies have examined leaves and stems, Kan et al. reported that it was linoleic fatty acid and oleic fatty acid that contributed to its antibacterial activity when S. aureus and P. aeruginosa were exposed to stinging nettle seed oils [44].
It would be helpful to compare the result of this study to those focusing on is of great concern by the medical community. Heightened interest in the usefulness of plants for treating infections should also come with assurances of appropriate protection of these valuable natural resources.