Background

Due to increased prevalence of multi-antibiotic resistant bacteria, current antimicrobial agents have become insufficient to treat some microbial disease (Riffel et al., 2002). Traditionally, medicinal plants have been used for the treatment of various diseases from cultures worldwide, and novel compounds with use as drugs continue to be discovered through research from medicinal plants. There are more than 20,000 plant species used in traditional medicines throughout the world, and many are prospective reservoirs for the development of new pharmaceuticals. With the decline in the production of new antibiotics, traditional medicinal plants have received significant attention to explore unique phytochemicals as new drug molecules (Amor et al., 2009).

Currently, there has been an increased interest in the study of different traditional medicinal plant extracts containing phytocompounds with potential sources of new antimicrobial agents. The search for new antimicrobial agents has become an important line of research due to a steady increase in human diseases due to the emergence of multi-drug resistant pathogenic microorganisms (Riffel et al., 2002; Hossain et al., 2012). In many developing countries, people are dependent upon plant based traditional medicines to treat various diseases due to lack of affordable healthcare in these countries. Thus, plants used as traditional medicines are readily accessible and economical sources for the treatment of many human infections and pathogenic diseases. In recent years, medicinal plants have started to increase its appeal to pharmaceutical companies and the scientific research community (Savoia, 2012). Currently, the research is focused on extraction and characterization of active phytocompounds from plants, since they can lead to new high activity profile drugs (Vaghasiya et al., 2011).

India has a rich heritage of knowledge on plant based medicines. Indiscriminate use of commercial antimicrobial drugs both for use in preventive and curative medicine has led to the development of multidrug resistance (MDR). Because of its vast and wide variations in soil and climate, the Indian sub-continent is suitable for cultivation of a large number of medicinal and aromatic plants, which can be used as raw materials for the pharmaceutical, cosmetic, food and agrochemical industries. A large number of these plants, which have been used in traditional medicines for centuries, still grow wild and are used to treat modern human diseases. Some of these plants may contain phytocompounds for production of new drugs with high economic potential (Nair and Chanda, 2005).

The Indian Himalayan Region (IHR), is one of the richest source of flora and fauna biodiversity in the world (Myers et al., 2000). The flora in the IHR includes about 8,000 species of angiosperm (40% endemic), 44 species of gymnosperm (16% endemic), 600 species of pteridophyte (25% endemic), 1,737 species of bryophyte (33% endemic), 1,159 species of lichen (11% endemic) and 6,900 species of fungi (27% endemic) (Singh and Hajra, 1996; Samant et al., 1998). These include some 1,748 species of medicinal plants with various traditional and modern therapeutic uses (Samant et al., 1998), which includes: 675 species of wild edible plants (Samant and Dhar, 1997), 118 species of medicinal plants yielding essential oils, 279 species of fodder, 155 sacred plants and 121 rare-endangered plants (Nayar and Sastry, 1987, 1988, 1990; Samant and Pant, 2003).

Rheum emodi Wall. Ex Meissn (family Polygonaceae), commonly known as Himalayan rhubarb, is a stout perennial herb (Figure 1). This leafy perennial herb can reach 1.5-3.0 m in height, and grows naturally in humus rich soil in exposed areas of alpine and sub-alpine zones of the Himalayas. It grows predominantly in sub-tropical and temperate regions of Asian countries including India, Nepal, China and Bhutan. In the IHR, it is distributed in altitudes ranging from 2,800 to 3,800 m in the temperate and subtropical regions of the Himalayas (Nautiyal et al., 2003). Roots of R. emodi are widely used in Ayurvedic and Asian folk medicine as a stomachic, purgative, astringent and tonic. It is also used by traditional healers in certain skin diseases, fevers, ulcers, bacterial infections, fungal infections, jaundice and liver disorders (Babu et al., 2003; Huang et al., 2007). Compounds isolated from R. emodi are reported to have antiviral, antibacterial, antifungal, tumor cell-growth inhibitory and cytostatic activities (Agarwal et al., 2000). Anthraquinones, a type of polyphenolic compound with pharmaceutical importance are the major active constituents of this plant showing significant biological activities (Singh et al., 2005).

1. Results and Discussions

1.1 Qualitative analysis of phytochemicals

Phytochemical screening of methanolic extract of the rhizome of R. emodi was carried out for the detection of phytoconstituents using standard conventional procedures (Table 1). The extract was subjected to the phytochemical screening assays for alkaloids, flavonoids, phenolic compounds and tannins, carbohydrates, glycosides, steroids, saponins, proteins and amino acids. The assays revealed the presence of phytochemicals such as phenolic compounds, tannins, flavonoids, carbohydrates, glycosides, phytosteroids. However, alkaloids and saponin were absent. These results were in agreement with the studies of Nazir et al. (2012); Ahmed and Salam (2015). Some of these phytocompounds are known to be biologically active and exert antimicrobial activity through different mechanisms.

1.2 Quantitative analysis of total phenolic contents and flavonoid content

Total phenolic content (TPC) were determined by using FC method and the values were expressed as GAE. Total flavonoid content was measured using the aluminium chloride method and the values were expressed in terms of RE (Figure 2). Rhizome of R. emodi was rich in total phenolic content (258 ± 6.87 mg/g GAE) as compared to flavonoid content (50 ± 2.56 mg/g RE). However, a study from Isbrahim et al. (1999) showed that total phenolic and flavonoid content of ethanolic extract of R. emodi were 111.5 mg/g GAE and 68.5 mg/g RE. Rhizome extracts of R. emodi showed total phenolic content of 124.64 ± 0.81 and 92.82 ± 0.23 µg GAE/mg, respectively. The flavonoid content in the leaves (165 ± 0.57 µg QE/mg extract) and fruits (137.96 ± 1.08 µg QE/mg) was higher as compared to that of rhizome (69.8 ± 0.25 µg QE/mg) (Singh and Chaturvedi, 2018).

1.3 Antimicrobial potency of rhizome of R. emodi

Antimicrobial activity assays were performed by using the agar well diffusion method. Methanolic extract showed inhibition to growth of both Gram positive (B. subtilis, St. aureus) and Gram negative bacteria (E. coli, K. pneumonia, and P. aeruginosa) (Figure 3A). Methanolic extract showed good inhibition to growth of fungal strains- C. albicans (MTCC277) and C. albicans (ATCC 90028) as compared to S. cerevisiae (H1086) (Figure 3B). However, rhizome showed more antifungal potential as compared to that of antibacterial activity. MIC of methanolic extract was found to be 12.5 µg/ml, 25 µg/ml, 25 µg/ml, and 25 µg/ml against B. subtilis, St. aureus, K. pneumonia and E. coli, respectively. However, MIC values were 6.25 µg/ml against S. cerevisiae (H1086), C. albicans (MTCC277) and C. albicans (ATCC 90028) (Table 2).

Ahmed and Salam (2015) also reported the antimicrobial potential of methanolic extract and aqueous extract of rhizome of R. emodi against Pseudomonas aeruginosa (MTCC 3541), Bacillus megaterium (MTCC 3784), and the fungi (Fusarium solani MTCC 3871 and Aspergillus flavus MTCC 3784). Aqueous extracts of R. emodi showed MIC values less than methanolic extract (0.4-1.5 mg/ml). This may be due to the solubility of the antimicrobial compounds in the respective solvents used for extractions. Emodin, aloe-emodin and rhein are the major anthraquinone derivatives isolated from rhubarb have been reported to possess significant antimicrobial activity against four strains of methicillin-resistant S. aureus. Rhein has also been shown to possess antibacterial activity against E. coli K12 (Hatano et al., 1999). Rhein, chrysophenol, physcion and aloe-emodin isolated from R. emodi have been reported to possess antifungal activity against Aspergillus fumigates, C. albicans and Cryptococcus neofarmans (Agarwal et al., 2000); Babu et al. (2003) showed that revandchinone-4 isolated from R. emodi exhibit good antibacterial activity against Gram positive (B. subtilis, B. sphaericus, St. aureus) and Gram negative (K. aerogenes, Chromobacterium violaceum, P. aeruginosa) bacteria, while revandchinone-1 and 3 had moderate antibacterial activity. Revandchinone-1, 3 and 4 also exhibited moderate antifungal activity against Rhizopus oryzae and Aspergillus nige (Babu et al., 2003). The ethanolic extract from R. emodi had been reported to possess promising activity against different strains of H. pylori isolated from gastric biopsy specimens of gastric carcinoma in both in vitro and in vivo studies (Ibrahim et al., 2006). A previous study from Jiang et al. (2018) also showed the antibacterial potential of hydroalcoholic extract of R. emodi against E. coli, Enterobacter aerogenes, Salmonella typhimurium and Salmonella infantis with zone of inhibition in mm ranges from 15.6-19.1 and MIC value from 25-125 μg/ml.

1.4 Synergistic activity of methanolic extract of rhizome of Rheum emodi with various antibiotics against bacteria and fungi

It was found that methanolic extract showed synergistic effect with amoxyclav (against K. pneumonia, B. subtilis, St. aureus), chloramphenicol (against K. pneumonia and St. aureus), erythromycin (against E. coli) and tetracycline (against E. coli and K. pneumonia). Combination of chloramphenicol and tetracycline with extract was found to be bactericidal against K. pneumonia; whereas, combination of erythromycin and tetracycline with extract was found to be bactericidal against E. coli (Table 3).

Methanolic extract of R. emodi showed synergistic activity with fluconazole against fungal strains, whereas no synergistic effect was observed with amphotericin B. Combination of fluconazole with extract was found to be fungicidal against the three tested fungi, whereas the combination of amphotericin B with extract was fungicidal against S. cerevisiae (H1086) and fungistatic against C. albicans (MTCC277) (Table 4). Although several studies have been conducted on the antibacterial and antifungal activity of the rhizome of R. emodi, no work has been done on synergistic efficacy of rhizome extracts with various antibiotics. This study is the first to reveal that methanolic extract showed specific synergistic effects against bacteria and fungi when combined with antibiotics. Moreover, the extract could enhance the bacteriostatic nature of the synthetic antibiotics to bactericidal, thus making them more useful for treatment of microbial and fungal diseases. The Indian patent application for this study has been submitted (Dev et al., 2017).

1.5 Analysis of antioxidant potential in rhizome extract of R. emodi

The antioxidant potential was determined by using DPPH radical scavenging and the FRAP assay. There was a dose-dependent response of DPPH radical scavenging activity and the levels of Fe (II) equivalents in the FRAP assay observed from methanolic extracts of R. emodi in comparison to ascorbic acid (Figure 4). The methanolic extract demonstrated antioxidant activity as shown by IC₅₀ value [60.89 μg/ml with DPPH assay and 13.022 µM Fe (II) equivalents] with respect to standard ascorbic acid IC₅₀ value [43.84 μg/ml with DPPH assay and 2.682 µM Fe (II) equivalents with FRAP assay] (Table 5).

Previous studies from Raudsepp et al. (2013), and Singh and Chaturvedi (2018) also showed the antioxidant potential of different parts (roots, fruits and stems) of R. emodi. The activity-guided isolation revealed that eugenol, gallic acid, quercetin, rutin, epicatechin, desoxyrhapontigenin, rhapontigenin and mesopsin are the major phenolic compounds responsible for the antioxidant activity of the roots of R. emodi (Singh et al., 2013). Several reports have shown that antioxidant activity of medicinal plants may be due to their phenolic compounds (Petridis et al., 2012; Rakholiya et al., 2014; Tripathi et al., 2014; Chandel et al., 2016; Guleria et al., 2016; Kumar et al., 2016; Kumar et al., 2018) and compared different extracts of rhizome of R. emodi using DPPH radical scavenging method. It was found that methanolic crude extract (IC₅₀ 18.28 mg/mL) showed the highest levels of antioxidant activity, followed by ethyl acetate crude extract (IC₅₀ 19.37 mg/mL) and chloroform extract (IC₅₀ 20.27 mg/mL). In addition, all of the extracts showed a concentration dependent scavenging of DPPH radicals, and were comparatively less active than both quercetin hydrate (IC₅₀ 4.95 mg/L) and gallic acid (IC₅₀ 11.26 mg/L) in quenching DPPH radicals than standard antioxidants (Tripathi et al., 2014). The R. emodi extracts contained a higher number of phenolic compounds, which were found to have significant positive correlation with free radicals (DPPH and -OH) scavenging efficacies (Tripathi et al., 2014). The results were in agreement with those of Kumar et al. (2014), who showed that methanolic extract of R. emodi to be a more active radical scavenger than aqueous extract. The antioxidant potential of extracts of R. emodi may be due to presence of anthraquinone derivatives (Krenn et al., 2003). Chai et al. (2012) showed that stilbenoid such as Piceatannol-4’-O-β-D-glucopyranoside (PICG) was responsible for the antioxidant potential of R. emodi rhizome. Further studies are required to identify the phytocompounds responsible for synergistic effects with antibiotics and antioxidant potential.

2. Material and Methods

2.1 Collection and preparation of extract

The rhizome of R. emodi was collected from the Chanshal valley of Distt. Shimla, Himachal Pradesh (India) in month of August, 2016. The Chanshal valley (Longitude 31.20 and latitude 77.99) is located at 4,520 meters above the sea level (Figure 1). The rhizome sample was thoroughly washed with running tap water followed by 70% ethanol and finally washed with sterilized distilled water. Afterwards, the rhizome was completely dried in an hot air oven at 40°C and then ground to fine powder using an electric grinder. The powdered material was then stored in air tight jars until further use.

The extract of dried powder of rhizome of R. emodi was prepared in methanol using a hot continuous method using a Soxhlet extractor apparatus. The extract was filtered through Whatmann filter paper no. 1 and the filtrate was dried at 40°C. The dried crude extracts were stored at 4°C until further use.

2.2 Qualitative analysis of phytochemicals

The crude methanolic extract of rhizome of R. emodi was tested for the presence of various secondary metabolites such as phenolics, flavonoids, tannins, saponins, alkaloids, glycosides, phytosteroids and carbohydrates by using standard protocols (Harbourne, 1984; Khandelwal, 2008).

2.3 Quantification of total phenolic contents and flavonoids

The total phenolic content (TPC) of methanolic extract of rhizome of R. emodi were determined by using the Folin-Ciocalteau reagent method as described by Singleton (1999). The total flavonoid content (TFC) of methanolic extract of rhizome of R. emodi was quantified by using the aluminium chloride (AlCl₃) method (Zhishen et al., 1999). The phenolic and flavonoid content was calculated from standard curve of gallic acid (GAE)/rutin (5-100 µg/ml) and expressed as GAE/rutin equivalents (RE) per g of the extract.

The total phenolic/flavonoid content was calculated using the following equation:

Where ‘C’ is total content of phenolic/flavonoid content in mg/g plant extract in GAE/RE, ‘c’ is the concentration of gallic acid/rutin estimated from the calibration curve (mg/ml), ‘V’ is the volume of extract in ml and m is the weight of crude plant extract in grams.

2.4 Analysis of antimicrobial and antioxidant activity of methanolic extract of rhizome of R. emodi

Antibacterial activity was assayed against both Gram positive (Staphylococcus aureus and Bacillus subtilis) and Gram negative (Escherichia coli, and Klebsiella pneumonia). Antifungal activity was tested against Saccharomyces cerevisiae (H1086), Candida albicans (ATCC90028) and Candida albicans (MTCC277). All the bacterial and fungal strains were obtained from Yeast Biology Lab, Shoolini University, Solan, Himachal Pradesh, India.

Antimicrobial activity of methanolic extract of rhizome of R. emodi was conducted using the agar well diffusion method (Perez et al., 1990). To determine the antimicrobial potential, for bacterial cultures nutrient agar (NA) and yeast peptone dextrose (YPD) agar plates were used for fungi. The bacterial/fungal culture of 0.5 McFarland Standard was uniformly spread on the surface of either NA YPD preset agar medium using sterile cotton swabs for bacterial cultures and yeast cultures, respectively. Test wells were punched with a sterilized cork borer (6 mm) in the agar and 50 μl of methanolic extract of R. emodi (10 mg/ml) were loaded in the wells. After 18 h incubation at 37ºC (for bacteria) and 48 h at 30ºC (for yeast), the zone of inhibition was measured using HiAntibiotic Zone scale-C (Himedia Biosciences, Mumbai, India). Amoxyclav (for bacteria) and Fluconazole (for yeast) were used as a positive control and DMSO (solvent) was used as negative control in antibacterial and antifungal assays. The tests were performed in triplicate and results were recorded as mean ± SD.

The minimum inhibitory concentration (MIC) of the extract was evaluated by broth dilution method described under (CLSI, 2012) guidelines using 2, 3, 5-triphenytetrazolium chloride. The methanolic extracts were dissolved in DMSO and geometric dilutions ranging from 12-0.025 mg/ml of extract were prepared in a 96-welled micro titer plate, including one growth control (nutrient broth/YPD broth containing DMSO) and a positive control (NB/YPD broth inoculated with bacterial culture and containing Amoxyclav/Fluconazole). Plates were incubated at 37°C for 24 h (for bacteria) and 48 h at 30°C (for fungi). After incubation, resazurin dye was added to each well and further incubated for 2 h. The color change was then observed visually. The growth was indicated by changes in color from blue to purple pink or colorless. The lowest concentration at which color change appeared was taken as the MIC value.

2.5 The same method to measure synergistic activity of methanolic extract of rhizome of R. emodi with various antibiotics and cidal/static activity of extract alone, antibiotics alone and combination of extract with antibiotics against bacteria and fungi

To perform the synergistic assay for antibacterial/antifungal activity, amoxyclave (10 µg), chloramphenicol (5 µg), erythromycin (5 µg), tetracycline (5 µg), fluconazole (100 µg) and amphotericin B (100 µg) were used alone and in combination with methanolic extract of R. emodi (50 µg/ml). To assay the cidal or static activity of extracts/antibiotics and in combination, cells were carefully taken from the zone of clearance around the well and streaked on nutrient agar plates and observed for the growth after 24 h of incubation at 37°C (for bacteria) and  48 h at 30°C (for fungi). Appearance of growth was considered as static, whereas no growth was considered as cidal effect (Figure 5).

2.6 Analysis of antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) method

The methanolic extract of rhizome of R. emodi were dissolved at a contraction of 1 mg/ml in methanol and then further diluted to a range of different concentrations (5-20 µg/ml) for antioxidant activity assays. The efficiency of the extract was evaluated in terms of the IC₅₀ value. The DPPH assay shows the ability of the test compound to act as a free radical scavenger, and this method is based on the ability of DPPH, a stable free radical, to decolorize in the presence of antioxidants (Jao and Ko, 2002; Kumarasamy et al., 2007). DPPH, a protonated radical, has characteristic absorbance maxima at 517 nm, which decreases with the scavenging of the proton radicals. DPPH radical scavenging activity of the extract was measured by the method described by Barros et al. (2007). The capability of scavenging DPPH radical was calculated using the following equation:

Where A _(control) is the absorbance of the control and A _(sample) is the absorbance of the test/standard.

2.7 Ferric-reducing ability of plasma (FRAP) assay to measure antioxidant activity

The ability to reduce ferric ions was measured using the method described by Benzie and Strain (1996). The antioxidant capacity based on the ability to reduce ferric ions of extract was calculated from the linear calibration curve of FeSO₄ (2.5-20 μM). The FRAP activity was expressed as μM FeSO₄ equivalents per gram of extract.

Authors’ contributions

RR, AS and VK conducted the research. AS, DJB and KD designed experiments, analyzed data, drafted, revised, read and approved the final manuscript.

Acknowledgements

The authors acknowledge Shoolini University, Solan, for providing infrastructure support to conduct the research work. Authors also acknowledge the support provided by Yeast Biology Laboratory, School of Biotechnology, Shoolini University, Solan, India.

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