Antibacterial, Antifungal, Antiviral, and Anthelmintic Activities of Medicinal Plants of Nepal Selected Based on Ethnobotanical Evidence

Joshi, Bishnu; Panda, Sujogya Kumar; Jouneghani, Ramin Saleh; Liu, Maoxuan; Parajuli, Niranjan; Leyssen, Pieter; Neyts, Johan; Luyten, Walter

doi:https://doi.org/10.1155/2020/1043471

Evidence-Based Complementary and Alternative Medicine

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Abstract Background Methods Results Discussion Conclusions Abbreviations Data Availability Conflicts of Interest Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2020 | Article ID 1043471 | https://doi.org/10.1155/2020/1043471

Antibacterial, Antifungal, Antiviral, and Anthelmintic Activities of Medicinal Plants of Nepal Selected Based on Ethnobotanical Evidence

Bishnu Joshi,^1,2Sujogya Kumar Panda ,³Ramin Saleh Jouneghani,¹Maoxuan Liu,¹Niranjan Parajuli,⁴Pieter Leyssen,⁵Johan Neyts,⁵and Walter Luyten³

Academic Editor: Carlos H. G. Martins

Received08 Dec 2019

Revised18 Mar 2020

Accepted25 Mar 2020

Published22 Apr 2020

Abstract

Background. Infections by microbes (viruses, bacteria, and fungi) and parasites can cause serious diseases in both humans and animals. Heavy use of antimicrobials has created selective pressure and caused resistance to currently available antibiotics, hence the need for finding new and better antibiotics. Natural products, especially from plants, are known for their medicinal properties, including antimicrobial and anthelmintic activities. Geoclimatic variation, together with diversity in ethnomedicinal traditions, has made the Himalayas of Nepal an invaluable repository of traditional medicinal plants. We studied antiviral, antibacterial, antifungal, and anthelmintic activities of medicinal plants, selected based upon ethnobotanical evidence. Methods. Ethanolic and methanolic extracts were tested (1) on a panel of microbes: two Gram-positive bacteria (Staphylococcus aureus and Listeria innocua), four Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Salmonella enterica, and Shigella sonnei), and one fungal species: Candida albicans; (2) against three different viruses: yellow fever, chikungunya, and enterovirus; and (3) on the nematode Caenorhabditis elegans. Also, cytotoxicity was assessed on human hepatoma (Huh), rhabdosarcoma (RD), and Vero (VC) cell lines. Results. Of 18 plants studied, Ampelocissus tomentosa and Aleuritopteris anceps inhibited S. aureus (MIC 35 μg/mL and 649 μg/mL, respectively) and Pseudomonas aeruginosa (MIC 15 μg/mL and 38 μg/mL, respectively). Rhododendron arboreum and Adhatoda vasica inhibited S. enterica (MIC 285 μg/mL and 326 μg/mL, respectively). Kalanchoe pinnata, Ampelocissus tomentosa, and Paris polyphylla were active against chikungunya virus, and Clerodendrum serratum was active against yellow fever virus (EC₅₀ 15.9 μg/mL); Terminalia chebula was active against enterovirus (EC₅₀ 10.6 μg/mL). Ampelocissus tomentosa, Boenninghausenia albiflora, Dichrocephala integrifolia, and Kalanchoe pinnata significantly reduced C. elegans motility, comparable to levamisole. Conclusions. In countries like Nepal, with a high burden of infectious and parasitic diseases, and a current health system unable to combat the burden of diseases, evaluation of local plants as a treatment or potential source of drugs can help expand treatment options. Screening plants against a broad range of pathogens (bacteria, viruses, fungi, and parasites) will support bioprospecting in Nepal, which may eventually lead to new drug development.

1. Background

Infectious diseases are the major cause of morbidity and mortality and thus a serious public health problem in developing countries. Despite the arsenal of antibiotics available, the situation is worsening due to emerging drug resistance. Antimicrobial resistance in a hospital setting is a major issue these days due to the extensive use or misuse of these drugs. Hence, there is an urgent need to find new molecules to treat infectious diseases [1, 2]. An attractive strategy for finding such molecules would be to test plants used to treat infectious diseases in traditional systems of healing, since medicinal plants have been a source of many pharmaceutical drugs for a range of diseases, including viral, bacterial, fungal, and protozoal infections, as well as for cancer [3, 4]. Over the past four decades, natural products were the direct or indirect source of approximately 50% of newly approved drugs [5, 6]. Plant products are often seen as natural, readily available, and having few side effects. Besides the popularity of herbal drugs, the use of herbal cosmetics and nutraceuticals has been increasing worldwide [7]. However, screening of different plant parts against a wide range of pathogens is cumbersome and requires significant resources. However, plant selection based on ethnobotany and traditional practices such as Ayurveda, Yunani, Siddha, Traditional Chinese Medicine, and Japanese Kampo medicine increases the probability of finding bioactive molecules that may be subsequently developed clinically [8, 9].

Asia represents one of the important centers of knowledge about plant species for the treatment of various ailments. Within this region, Nepal is rich in (medicinal) plant biodiversity, and linking this with the indigenous knowledge on medicinal and aromatic plants provides an attractive approach for the development of novel (e.g., anti-infective) drugs. “Medicinal plants have a long tradition, but ethnopharmacology as a well-defined field of research has a relatively short history, dating back about 50 years only” [10, 11]. Ethnopharmacological selection combined with testing on a range of pathogens (viruses, bacteria, fungi, and protozoa) is a more efficient way of identifying new bioactive molecules than testing randomly selected plants. Therefore, we tested the antiviral, antibacterial, antifungal, and anthelmintic activities of 18 medicinal plants of Nepal, selected based on ethnobotanical evidence for their use in infectious disease.

2. Methods

2.1. Plant Materials

Eighteen plant species were collected for evaluation of their antibacterial, antifungal, antiviral, and anthelmintic activity based on their uses in the scientific literature and indigenous knowledge. Bark, leaves, fruit, or roots of the selected plants (Table 1) were separately collected from different regions in Nepal from December 2013 to April 2014. The rules for plant collection and identification were followed according to the Department of Plant Resources, Ministry of Forest and Environment, Government of Nepal. All the collected plant materials were identified by professional taxonomists at the Department of Plant Resources, and voucher specimens [63] were deposited in the National Herbarium and Plant Laboratories, Department of Plant Resources, Ministry of Forest and Environment, Government of Nepal. The information on these plants with their official taxonomic name (according to the plant list: http://www.theplantlist.org), indigenous use, used part(s), literature availability, and voucher number is shown in Table 1. Among the collected plants, none are endangered or rare species.

2.2. Reagents

Ethanol, methanol, DMSO, growth media (components), ciprofloxacin hydrochloride, and miconazole were obtained from Sigma-Aldrich (Belgium). Water was obtained from a Milli-Q® Integral Water Purification System for ultrapure water (Millipore).

2.3. Extraction

The collected plant materials were washed thoroughly with tap water and shade-dried at ambient temperature. Dried samples were ground to a powder with an electric blender and subjected to Soxhlet extraction using polar solvents (ethanol and methanol). The extracts were evaporated on a rotary evaporator under reduced pressure till a solid mass was obtained. All the extracts were kept in sealed vials, labelled with appropriate code, and transported by airmail from Nepal to the KU Leuven, Belgium, for integrated in vitro screening. The dried extracts were kept at 4°C until analysis.

2.4. Microbial Strains

The in vitro antibacterial activity of the crude extract was evaluated using a panel of pathogenic strains including Gram-positive bacteria: Staphylococcus aureus (ATCC 6538, Rosenbach) and Listeria innocua (LMG 11387), Gram-negative bacteria: Escherichia coli (DH10B), Pseudomonas aeruginosa (PAO1), Salmonella enterica subsp. enterica (ATCC 13076), and Shigella sonnei (LMG 10473), and one fungal strain: Candida albicans (SC5314). The (frozen) storage, maintenance, and preparation of working culture suspensions were carried out following established procedures described in our previous studies [64].

2.5. Antimicrobial Assay

Antimicrobial activity was assessed as described in our previous study [64, 65]. In brief, two-fold serial dilutions of the agents were prepared in DMSO using sterile 96-well microdilution plates with flat-bottom wells. Antibiotics ciprofloxacin and miconazole were used as positive control for bacteria and fungi, respectively. For bacteria, 5% DMSO was used as the solvent control for all the experiments, while for C. albicans, 2% DMSO was used. The MIC was calculated by IC₅₀/IC₉₀ Laboratory Excel Calculation Tools and expressed as IC₅₀ as developed at the University of Antwerp (http://www.leishrisk.net/leishrisk/userfiles/file/kaladrugr/sops/lab/labsop18_ic50tool.pdf).

2.6. Anthelmintic Test

The anthelmintic assay was carried out in a 96-well microplate with flat-bottom wells in a WMicrotracker (Phylumtech, Argentina) apparatus, as described in our previous study [66]. The movement of worms in each well was measured every 30 minutes for 16 h at 20°C and recorded by the WMicrotracker. The percentage of the average movement within 16 hours of worms exposed to samples, compared to a DMSO control, was used to calculate the relative anthelmintic activity. Levamisole (50 μM) was used on every plate as a positive control, and DMSO was used as solvent control.

2.7. Antiviral Test

Antiviral activity was tested as described earlier [65, 67]. The final, maximal DMSO concentration in the assay wells with the highest sample input (1%) was well tolerated by the cells. Rupintrivir was used as a positive control. All data were expressed as EC₅₀, CC₅₀, and SI. For cytotoxicity assays, plant extracts (only those that showed antiviral activity) were evaluated by the MTS method. In brief, the same experimental setup was used as for the antiviral assay; except for cytotoxicity determination, uninfected cultures were incubated with a serial dilution of plant extract for three days at 37°C. “The cytotoxic concentration was calculated as CC₅₀ or the concentration of plant extracts required to reduce cell proliferation by 50% relative to the number of cells in the solvent-treated controls. The formula used to calculate cytotoxic activity was % CPE = [OD_cc − OD plant extract]/OD_cc, where OD_cc corresponds to the optical density of the uninfected and untreated cell cultures, and OD plant extract corresponds to the OD of uninfected cultures, treated with the extract. In addition, the selectivity index (SI) was calculated as the ratio of CC₅₀ for cell growth to EC₅₀ (CC₅₀/EC₅₀)” [65, 67].

2.8. Thin-Layer Chromatography (TLC) and Phytochemical Analysis

Phytochemical analysis by means of TLC was carried out for all crude extracts, as previously described by Panda et al. [65]. The TLC plate (dimensions 2.5 × 7.5 cm, coated with silica gel 60 F254, Merck, Germany) was developed with methanol : dichloromethane (95 : 5, v/v) at ambient temperature (approximately 20°C) and dried in an oven at 90°C for 5 minutes to evaporate the solvent. The plate was visualized under ultraviolet (UV) light at 254 and 360 nm. Later, the same plate was used for visualization of the spots by spraying with 5% sulphuric acid in ethanol, followed by heating at 100°C for 5 minutes. Phytochemicals were tentatively identified by comparison of Rf values and spot colours with literature data.

3. Results

3.1. Antimicrobial Testing

Since there is a renewed interest in the search for plant-based medicines using traditional knowledge (Table 1), we tested eighteen crude ethanolic/methanolic plant extracts against a panel of microbes: two Gram-positive bacteria (S. aureus and L. innocua), four Gram-negative bacteria (E. coli, P. aeruginosa, S. enterica, and S. sonnei), and one fungal species: Candida albicans. Out of these 18 plant extracts, those of Clerodendrum serratum, Sapindus mukorossi, Ampelocissus tomentosa, Dicrocephala integrifolia, Boerhavia diffusa, Rhododendron arboreum, Justicia adhatoda, and Terminalia chebula were active against a wide range of pathogens as crude extract at concentrations of 1000 μg/mL (Table 2). Upon further serial dilution of active plant extracts, most of the plant extracts (especially of S. mukorossi and D. integrifolia) rapidly lost their activity. This might be due to low potency compounds, low concentrations of potent compounds, or coloured compounds in the extracts that interfere with spectrophotometric endpoints at high concentrations. However, some of the plant extracts, such as A. tomentosa and Aleuritopteris anceps, displayed noteworthy antimicrobial activity against S. aureus (MIC of 35 μg/mL and 649 μg/mL, respectively) and P. aeruginosa (MIC of 15 μg/mL and 38 μg/mL, respectively). Furthermore, R. arboreum and J. adhatoda were found to be active against S. enterica (MIC of 285 μg/mL and 326 μg/mL, respectively) (Table 3).

3.2. Anthelmintic Activity

We already reported the antiprotozoal activities of crude extracts of select medicinal plants of Nepal and found three active species: Phragmites vallatoria, Ampelocissus tomentosa (for which no antiprotozoal activity had been reported previously), and Terminalia chebula [63]. Moreover, nonselective antileishmanial activity was shown for P. polyphylla, K. pinnata, and Terminalia chebula [63]. Encouraged by these findings, and also by the traditional use of the plants as anthelmintics [63, 68], we tested the effects of these plant extracts on the motility of the L4 stage of C. elegans. We used this nonparasitic free-living nematode and model organism to test anthelmintic activity due to its low cost and easy availability of mutant and transgenic strains for functional genomics and proteomics studies as well as to investigate key signaling events and drug interactions and its amenability to laboratory culture methods. Furthermore, C. elegans is taxonomically closely related to the suborder Strongylida, which comprises several economically important animal parasites, such as Haemonchus contortus and Strongyloides stercoralis, that cause both human and animal disease [69]. Drug screening in the natural hosts for every helminthic disease is almost impossible and a costly process. So, based on the hypothesis that plant extracts affecting C. elegans may also have effects on other (including parasitic) nematodes, we screened our plant extracts on the surrogate parasitic nematode C. elegans. To monitor worm viability, we used the WMicrotracker, a device that monitors the movement of helminths in real time using infrared light beams. The advantage of using the WMicrotracker over other methods is that it enables screening of a large number of compounds in a fully automated way that is easy, fast, label-free, and highly reproducible. Interestingly, four plants were found to be active out of 18 plants tested: A. tomentosa, B. albiflora, K. pinnata, and D. integrifolia. These four plants significantly reduced C. elegans motility, comparable to the positive control (>50%) (Figure 1). Moreover, plants such as B. diffusa, P. polyphylla, and T. chebula showed moderate inhibition using the WMicrotracker, but under the microscope, these extracts showed clear paralytic activity. The average inhibition was lower; however, very good activity was seen during the last four hours of WMicrotracker movement recordings, revealing that many extracts appear to have a slow onset of action.

3.3. Antiviral Testing

All of the plant extracts (listed in Table 1) were tested against three different viruses i.e., yellow fever, chikungunya virus (CHIKV), and enterovirus 71. One-third of the plants were active against these viruses. Among them, K. pinnata (EC₅₀ = 6.1 μg/mL, SI = 4.29), A. tomentosa (EC₅₀ = 7.79 μg/mL, SI = 4.69), and P. polyphylla (EC₅₀ = 8.74 μg/mL, SI = 1.75) were active against CHIKV, C. serratum was active against yellow fever virus, with EC₅₀ of 15.9 μg/mL, and T. chebula (EC₅₀ = 10.6 μg/mL, SI = 5.94) was active against enterovirus 71 (Table 4).

3.4. TLC and Preliminary Phytochemical Screening

All the plant extracts were subjected to TLC, to generate fingerprints. Using methanol : dichloromethane (95 : 5, v/v) as mobile phase, numerous absorbing bands were observed under UV light (254 nm and 360 nm). Furthermore, after exposing the plates to 5% sulphuric acid in ethanol, spots were visualized, and the reaction products were compared under UV light. Interestingly, with or without derivatization, several extracts show similar fingerprints (observed colour as well as Rf), signifying the presence of similar chemical classes. Most of the plant extracts exposed to 254 nm UV light show orange, yellow, pink, green, or blue fluorescence, confirming the presence of flavonoids and polyphenols (Supplementary Figure 2). Select plant extracts such as A. anceps, A. tomentosa, and B. albiflora show similar Rf values and band patterns (blue or green fluorescence at 360 nm, converted to yellow, blue, or blue-green fluorescence after exposure to sulphuric acid) confirming the presence of coumarins (Supplementary Table 1). C. zeylanicum extract forms dark zones under UV 254 that disappear at 360 nm, suggesting sesquiterpene lactones. Several plant extracts such as C. zeylanicum, D. integrifolia, E. acuminate, J. adhatoda, K. pinnata, and S. mukorossi did not show any bands under UV initially but form clear bands of blue, light green, or brown colour after exposure to sulphuric acid, confirming the presence of saponins. Three plant extracts: C. wallichii, J. adhatoda, and P. polyphylla, show blue colour fluorescence turning to green or gray after exposure to sulphuric acid, confirming the presence of alkaloids. Only one extract, R. arboreum, shows the presence of tannins, while extracts such as B. albiflora, C. serratum, D. integrifolia, and P. tithymaloides contain sterols and glycosides. The TLC fingerprints suggest the presence of several groups of phytochemical constituents, but which of these constitute the bioactive compounds responsible for the detected activity requires further study.

4. Discussion

4.1. Antimicrobial Activity

In the area of infectious disease, of the 141 small molecules approved between 1981 and 2014 as antibacterial agents, 82, or 58%, originated from natural sources [6]. Thus, there is a huge potential for discovering new compounds with anti-infective properties from natural sources. Some of our findings are corroborated by Tantry et al. [70], who isolated oleanadien-3β-ethan-3-oate from Rhododendron species, and this compound inhibits Salmonella typhi (MTCC 53) (inhibition zone of 18.6 mm) at a concentration 0.1 μL/mL. Similarly, Pradhan et al. [71] observed an MIC of 78.12 μg/mL for a methanolic extract of J. adhatoda against S. enteritidis, in agreement with our findings. In our study, we could not find noteworthy antimicrobial activity of K. pinnata and Dichrocephala integrifolia. In contrast, Tatsimo et al. [72] investigated the antimicrobial activity of Bryophyllum pinnatum (synonym K. pinnata) on a wide panel of bacteria and yeast and found noteworthy antimicrobial activity in a crude methanolic extract, with MICs ranging from 32 to 512 μg/mL. Likewise, earlier researchers have shown inhibitory effects of D. integrifolia against S. aureus, E. coli, and S. typhi in a dose-dependent manner from all crude extracts, mostly in nonpolar solvents except ethanol [73]. Several factors may explain these discrepancies: differences in the plant part or variety, soil or climate, collection or postharvest treatment, extraction procedure, etc. Furthermore, in our study, we only found moderate activity of Clerodendrum serratum against S. enterica (MIC of 882 μg/mL). A large number chemical constituents (∼283 compounds) have been reported from the genus Clerodendrum (monoterpenes and derivatives, sesquiterpenes, diterpenoids, triterpenoids, flavonoids and flavonoid glycosides, phenylethanoid glycosides, steroids and steroid glycosides, cyclohexylethanoids, anthraquinones, and cyanogenic glycosides), showing a multitude of biological activities [74]. Arokiyaraj and colleagues measured the antimicrobial activity of Clerodendrum siphonanthus against Klebsiella pneumoniae, Proteus mirabilis, S. typhi, S. aureus, E. coli, and Bacillus subtilis and observed inhibition zones of 30, 16, 16, 12, 11.5, and 10 mm, respectively [75].

The present study finds broad-spectrum antimicrobial activity (extracts effective against a panel of test bacteria) with plants such as Ampelocissus tomentosa, Boerhavia diffusa, Dichrocephala integrifolia, Pedilanthus tithymaloides, Paris polyphylla, and Sapindus mukorossi. Indeed, plants such as Boerhavia diffusa [76], Dichrocephala integrifolia [73], Pedilanthus tithymaloides [77], Paris polyphylla [78, 79], and Sapindus mukorossi [80, 81] were previously reported for their antimicrobial properties. However, we report here for the first time that the methanolic extract of Ampelocissus tomentosa root is effective against all tested pathogens: Gram-positives, Gram-negatives, and Candida albicans. Similarly, plants such as Aleuritopteris anceps, Boenninghausenia albiflora, Cynoglossum zeylanicum, Clerodendrum serratum, Rhododendron arboretum, and Terminalia chebula were found to show narrow-spectrum activity towards either Gram-negatives or Gram-positives. Plants such as Boenninghausenia albiflora [56, 82], Clerodendrum serratum [83, 84], Rhododendron arboreum [85, 86], and Terminalia chebula [34, 76, 87] were already reported as having antimicrobial activity. To the best of our knowledge, Aleuritopteris anceps and Cynoglossum zeylanicum are shown for the first time as active against bacteria. Three plants, that is, Ampelocissus tomentosa, Dichrocephala integrifolia, and Paris polyphylla, were found to inhibit significantly the growth of Candida albicans.

Amongst our 18 plants, the family Asteraceae is represented the most, with three plants. Despite the discovery of numerous secondary metabolites in Asteraceae, this family has received little attention in ethnopharmacological research, resulting in few systematic explorations and few commercialized products [88]. However, in the present study, one Asteraceae plant: Cirsium wallichii, did not show antimicrobial property against any tested microbe when we fix our cutoff at 50% inhibition. In addition, two other plants do not show antimicrobial activity: Kalanchoe pinnata (Crassulaceae) and Phragmites vallatoria (Poaceae).

4.2. Antiviral Activity

Phytomedicines have been an integral part of the traditional health care system in most parts of the world for thousands of years [89]. Plants contain thousands of secondary metabolites that have a broad range of bioactivities. Hence, it is interesting to test antiviral properties of plant extracts that are active against infectious diseases. For the plants we selected, we did not find any prior reports on activity against yellow fever, chikungunya, or enterovirus. Nonetheless, from a member of the same genus (Kalanchoe gracilis), compounds such as ferulic acid, quercetin, and kaempferol have already been isolated, which exhibited antiviral effects against enterovirus 71 (EV71) and coxsackievirus A16 (CVA16).

Chikungunya virus (CHIKV) is one of the re-emerging vector-borne viral diseases and considered as a neglected tropical disease, mainly because the affected regions are Africa and Southeast Asia. The recent outbreak of CHIKV in Central and South America also raised the possibility of massive epidemics linked to the worldwide proliferation of the mosquito vectors that transmit this disease. Moreover, there are no currently approved vaccines or antiviral treatments available yet for the prevention of CHIKV infection; it is therefore imperative to look for new bioactive molecules, and we found that several plants (Kalanchoe pinnata, Ampelocissus tomentosa, and Paris polyphylla) show activity against CHIKV (Table 4). Taking into account the IC₅₀ values for inhibition of CHIKV replication and the SI value, further research is warranted on the structural elucidation of the active constituents. To identify novel inhibitors of CHIKV replication, researchers have selected Euphorbiaceae species, and upon bioassay-guided purification, they have isolated daphnane- and tigliane-type esters with anti-CHIKV activities. Euphorbia species are characterized by their ability to produce diterpenoids belonging to the tigliane, ingenane, and jatrophane chemical classes, along with other types of diterpene esters. In our study, we also found promising results of P. tithymaloides (Euphorbiaceae species) extracts against CHIKV; however, its antiviral action was not selective. The moderate cytotoxicity might be due to the presence of pedilstatin or eurphorbol, which have already been established as irritants and carcinogens. Likewise, a recently published manuscript by Shrestha et al. [90] reported that Paris polyphylla was the second most widely used plant in Eastern Nepal (use-value = 0.96, fidelity level = 77.42) to treat a wide range of ailments (Table 1). In earlier published work, we also found the promising activity of P. polyphylla on Leishmania; however, its action was nonselective due to its toxic effect on the MRC-5 cell line [63]. We also found activity from these plants against Candida and CHIKV. However, due to its toxic nature, caution is necessary since deleterious health consequences may occur if high doses are administered for a long time.

Similarly, another important viral disease (yellow fever), transmitted by mosquito bites, causes sporadic outbreaks with a case fatality rate of 15–50%, with no known cure. Though effective vaccination is available against this disease, due to inadequate coverage of vaccination in African regions and threats of re-emergence in its endemic habitat, new drugs are urgently needed. In our study, we have found an interesting activity of C. serratum against the yellow fever virus for the first time. Though anticonvulsant, antidiabetic, and antimalarial activity had already been reported on C. serratum [45], no one examined its activity against the yellow fever virus thus far. The development of new anti-yellow fever drugs from bioactive compounds obtained from these plants and the topical use of bioactive compounds present in C. serratum such as lupeol and pellitorine might be explored for the prevention of virus infection through the bite of the mosquito vectors.

Only one plant extract (T. chebula) was found to be active against enterovirus. EV71 infections can cause mild hand, foot, and mouth disease but also lead (more rarely) to severe fatal neurological complications. The anti-enterovirus activity of T. chebula could be due to the presence of hydrolyzable tannin (chebulagic acid), since Yang et al. [91] showed that chebulagic acid treatment reduced the viral cytopathic effect on rhabdomyosarcoma cells with an IC₅₀ of 12.5 μg/mL. Furthermore, in mice challenged with a lethal dose of EV71, treatment with chebulagic acid substantially reduced mouse mortality and relieved clinical symptoms through the inhibition of viral replication. Dichrocephala integrifolia root and flower extracts are effective in the control of human herpes simplex virus [92]. Another study conducted by Ojha et al. [42] isolated (using bioassay-guided purification) luteolin, which was active against herpes simplex virus type 2 (HSV-2).

4.3. Anthelmintic Activity

A search for new antiparasitic drugs has been underway over the past several decades. However, despite the abundant literature, more work is needed to yield potent, commercially available drugs based on natural products [93]. To the best of our knowledge, no anthelmintic activity has been reported previously from A. tomentosa and B. albiflora; however, insecticidal compounds coumarin, murraxocin, jayantinin, and murralongin have already been isolated from B. albiflora [55, 94]. Consistent with our study, Takaishi et al. [95] have characterized different isoforms of coumarin from the plant Ageratum conyzoides via bioassay-guided purification, and they were found to be active against a phytopathogenic nematode: Bursaphelenchus xylophilus. Furthermore, in a later study, the same research group has also chemically synthesized different derivatives of coumarin and found 5-ethoxycoumarin having the highest nematicidal activity, which was comparatively harmless against both the brine shrimps, Artemia Salina, and the Japanese killifish, Oryzias latipes [69, 95].

Furthermore, Wang et al. [68] investigated the anthelmintic activity from Paris polyphylla and found two steroidal saponins: dioscin and polyphyllin D, obtained from its methanolic extract, that exhibited significant activity against Dactylogyrus intermedius with EC₅₀ values of 0.44 and 0.70 mg/L, respectively. These were more potent than the positive control, mebendazole (EC₅₀ value = 1.25 mg/L).

Wabo et al. [22] evaluated ethanolic extracts of D. integrifolia on the gastrointestinal nematode Heligmosomoides bakeri and concluded that they contain compounds that have ovicidal and larvicidal properties. The ethanolic extract of D. integrifolia inhibited 98.1% and 98% of L1 and L2 larvae, respectively, of Heligmosomoides bakeri at 5 mg/mL [22]. The earlier findings on P. polyphylla and D. integrifolia as anthelmintic [22, 68] are also supported by our study using the model organism C. elegans.

Of course, plants traditionally used as anthelmintics by local healers do not necessarily show anthelmintic activity in C. elegans. Although activity in C. elegans does not correlate perfectly with activity against parasitic nematodes, it remains a useful preliminary test. Some plant extracts that do not exhibit direct anthelmintic activity may act as an irritant of the gastrointestinal tract and expel worms from the intestines that way.

Interestingly, one plant extract (Sapindus mukorossi) increased the movement of C. elegans (Figure 1) but the mechanisms are unclear. Many plants contain antioxidants and are known to increase lifespan, healthspan, and resistance to stress in C. elegans [96]. In addition, this plant did not show any cytotoxicity when tested against cell lines such as Huh, RD, and VCL (CC₅₀ > 100 μg/mL, Table 4), providing further evidence of nontoxicity.

The major limitation while working with crude plant extracts is that they contain complex mixtures of phytoconstituents, which may act individually or synergistically, or sometimes in an antagonistic way, resulting in varying bioactivities. Nevertheless, screening anthelmintic activity of crude extracts will help to find new anthelmintic drug lead compounds, which is a pressing need at present as there are only a limited number of anthelmintic drug classes available, and parasitic diseases have major socioeconomic consequences for humans as well as for livestock. Furthermore, the excessive use of commercially available anthelmintics (benzimidazoles, imidazothiazoles/tetrahydropyrimidines, macrocyclic lactones, and amino-acetonitrile derivatives) has led to drug resistance, and these treatments are no longer as effective as they were initially.

Efforts have been made to develop new vaccines against parasitic platyhelminthes and nematodes. Nonetheless, an efficient vaccine has not been successful because it fails to provide protective immunity. Given that no new effective anthelmintic compounds are becoming available, combined with the lack of a vaccine and emerging resistance problems against the currently available antiparasitic drugs, research into alternative sources of drugs is urgently needed. Since no human anthelmintics have been developed from plant sources so far, there is hope that new anthelmintic molecules can be found in plants that exhibit broad-spectrum activity, excellent safety, and therapeutic profiles and also display significant activity against different developmental stages of parasites [97]. Therefore, finding a broad-spectrum drug that could treat multiple parasites or multiple life stages is desirable, especially in developing countries with limited resources.

Another major limitation in our study (due to time and financial constraints) is that we could only test methanolic/ethanolic plant extracts and single plant parts. Some bioactive constituents are preferably extracted by nonpolar solvents such as chloroform or hexane, or by mild polar solvents such as ethyl acetate. In our study, we only used one polar solvent (methanol) on D. integrifolia and this might explain why we could not observe significant antimicrobial activity against S. aureus and C. albicans, despite this plant being used traditionally in the treatments of wound infections and other infectious ailments. The choice of an appropriate solvent is crucial for bioactivity. Solvents used in the extraction techniques and the extraction scheme change the quantity and the efficacy of the extracted phytoconstituents. For example, in the case of the famous antimalarial drug artemisinin, the hot water extract of Artemisia annua was ineffective in rodent mouse models infected with P. berghei, while the ether extract did show promising results.

Moreover, plants showing no evidence of activity in our study may be worth studying further since the plants studied were tested at 1 mg/mL, and many crude extracts may only show activity at significantly higher concentrations. Also, the activity of the extract is affected by spatiotemporal variation, the plant part taken for screening purposes, and the environmental conditions under which the plant material was harvested. These factors may also explain some of our negative results or discrepancies with earlier reports.

5. Conclusions

The purpose of the current study was to investigate the antiviral, antibacterial, antifungal, and anthelminthic activity of select medicinal plants of Nepal. To the best of our knowledge, this is the first systematic report on the use of plants selected based on ethnobotanical surveys (information gathered from local healers or already available in the literature) and their testing for different anti-infective bioactivities. Further work will be needed to identify the bioactive compounds from the active extracts, as well as their drug target or mode of action.

Abbreviations

ATCC:	American Type Culture Collection, Manassas, Virginia, USA
CC_50-:	50% cytostatic/cytotoxic concentration
CHIKV:	Chikungunya virus
CPE:	Cytopathic effect
DMSO:	Dimethyl sulphoxide
EC₅₀:	50% effective concentration
EV71:	Enterovirus 71
HSV-2:	Herpes simplex virus type 2
Huh:	Human hepatoma cell line
MHA:	Mueller–Hinton agar
MIC₅₀:	Minimum inhibitory concentration required to inhibit the growth of organisms by 50%
MTS:	(3–[4,5,dimethylthiazol–2–yl]–5–[3–carboxymethoxy–phenyl]–2–[4– sulfophenyl]–2H–tetrazolium, inner salt
NGM:	Nematode growth medium
OD:	Optical density
RD:	Rhabdosarcoma cell line
SI:	Selectivity index
TI:	Therapeutic index
VCL:	Vero cell line
VC:	Virus controls
YPD:	Yeast extract peptone dextrose medium.

Data Availability

All data generated or analysed during this study are included in this published article and its supplementary material.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Authors’ Contributions

Bishnu Joshi and Sujogya Kumar Panda contributed equally to this work. BJ, PL, and SKP participated in the study design, performed experiments, contributed to obtaining the results, and drafted the manuscript. RSJ, ML, and NP contributed to the study design and performed experiments. JN and WL coordinated the work and critically reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgments

The authors are very much indebted to Dr. Jyoti Joshi Bhatta (Department of Plant Resources), Keshab Ranabhat, Kamal Prasad Regmi, and Lila Bahadur Magar for arranging the sample collection, voucher specimen, and shipment of plants to Belgium. The authors are also thankful to Purity Ngina Kipanga (KU Leuven) for technical help in the laboratory and Yipeng Ma (KU Leuven) for help with the antiviral section and some suggestions while preparing the manuscript. SKP is grateful for receipt of a postdoc fellowship from EMINTE (Erasmus Mundus, EU; http://eminte.eu/). BJ and WL largely supported themselves.

Supplementary Materials

Figure 1: TLC of crude extracts exposed to UV at 360 nm; mobile phase: methanol : dichloromethane (95 : 5, v/v). Figure 2: TLC of crude extracts exposed to UV at 254 nm; mobile phase: methanol : dichloromethane (95 : 5, v/v). Figure 3: TLC of crude extracts after spraying with 5% sulphuric acid. Figure 4: TLC of crude extracts exposed to UV at 360 nm after spraying with 5% sulphuric acid. Figure 5: TLC of crude extracts exposed to UV at 254 nm after spraying with 5% sulphuric acid. Table 1: interpretation of TLC results of crude extracts. (Supplementary Materials)

References

A. J. Alanis, “Resistance to antibiotics: are we in the post-antibiotic era?” Archives of Medical Research, vol. 36, no. 6, pp. 697–705, 2005.
View at: Publisher Site | Google Scholar
E. D. Brown and G. D. Wright, “Antibacterial drug discovery in the resistance era,” Nature, vol. 529, no. 7586, pp. 336–343, 2016.
View at: Publisher Site | Google Scholar
A. Gurib-Fakim, “Medicinal plants: traditions of yesterday and drugs of tomorrow,” Molecular Aspects of Medicine, vol. 27, no. 1, pp. 1–93, 2006.
View at: Publisher Site | Google Scholar
A. L. Harvey, “Natural products as a screening resource,” Current Opinion in Chemical Biology, vol. 11, no. 5, pp. 480–484, 2007.
View at: Publisher Site | Google Scholar
D. J. Newman and G. M. Cragg, “Natural products as sources of new drugs over the Last 25 years,” Journal of Natural Products, vol. 70, no. 3, pp. 461–477, 2007.
View at: Publisher Site | Google Scholar
D. J. Newman and G. M. Cragg, “Natural products as sources of new drugs from 1981 to 2014,” Journal of Natural Products, vol. 79, no. 3, pp. 629–661, 2016.
View at: Publisher Site | Google Scholar
R. M. Kunwar and R. W. Bussmann, “Ethnobotany in the Nepal himalaya,” Journal of Ethnobiology and Ethnomedicine, vol. 4, no. 1, p. 24, 2008.
View at: Publisher Site | Google Scholar
P. Cos, A. J. Vlietinck, D. V. Berghe, and L. Maes, “Anti-infective potential of natural products: how to develop a stronger in vitro “proof-of-concept”,” Journal of Ethnopharmacology, vol. 106, no. 3, pp. 290–302, 2006.
View at: Publisher Site | Google Scholar
B. Patwardhan and R. A. Mashelkar, “Traditional medicine-inspired approaches to drug discovery: can ayurveda show the way forward?” Drug Discovery Today, vol. 14, no. 15-16, pp. 804–811, 2009.
View at: Publisher Site | Google Scholar
M. Heinrich and A. K. Jäger, Ethnopharmacology, John Wiley & Sons, Hoboken, NJ, USA, 2015.
A. W. K. Yeung, M. Heinrich, and A. G. Atanasov, “Ethnopharmacology—a bibliometric analysis of a field of research meandering between medicine and food science?” Frontiers in Pharmacology, vol. 9, p. 215, 2018.
View at: Publisher Site | Google Scholar
A. R. Joshi and K. Joshi, “Indigenous knowledge and uses of medicinal plants by local communities of the Kali Gandaki Watershed Area, Nepal,” Journal of Ethnopharmacology, vol. 73, no. 1-2, pp. 175–183, 2000.
View at: Publisher Site | Google Scholar
I. Shrestha and N. Joshi, “Medicinal plants of the lele village of Lalitpur district, Nepal,” International Journal of Pharmacognosy, vol. 31, no. 2, pp. 130–134, 1993.
View at: Publisher Site | Google Scholar
U. P. Claeson, T. Malmfors, G. Wikman, and J. G. Bruhn, “Adhatoda vasica: a critical review of ethnopharmacological and toxicological data,” Journal of Ethnopharmacology, vol. 72, no. 1-2, pp. 1–20, 2000.
View at: Publisher Site | Google Scholar
S. Vijaylakshmi, M. J. Nanjan, and B. Suresh, “In vitro anti-tumour studies on Cnicus wallichi DC,” Ancient Science of Life, vol. 29, no. 29, pp. 17–19, 2009.
View at: Google Scholar
A. Hassan, M. Ajaib, M. Anjum, S. Siddiqui, and N. J. B. Malik, “Investigation of antimicrobial and antioxidant activities of Cirsium wallichii DC,” Biologica, vol. 62, pp. 297–304, 2016.
View at: Google Scholar
I. E. Jordon-Thaden and S. M. Louda, “Chemistry of Cirsium and Carduus: a role in ecological risk assessment for biological control of weeds?” Biochemical Systematics and Ecology, vol. 31, no. 12, pp. 1353–1396, 2003.
View at: Publisher Site | Google Scholar
J. R. S. Tabuti, K. A. Lye, and S. S. Dhillion, “Traditional herbal drugs of Bulamogi, Uganda: plants, use and administration,” Journal of Ethnopharmacology, vol. 88, no. 1, pp. 19–44, 2003.
View at: Publisher Site | Google Scholar
N. P. Manandhar, “Medico botany of Gorkha district, Nepal -an elucidation of medicinal plants,” International Journal of Crude Drug Research, vol. 28, no. 1, pp. 17–25, 1990.
View at: Publisher Site | Google Scholar
T. Morikawa, O. B. Abdel-Halim, H. Matsuda, S. Ando, O. Muraoka, and M. Yoshikawa, “Pseudoguaiane-type sesquiterpenes and inhibitors on nitric oxide production from Dichrocephala integrifolia,” Tetrahedron, vol. 62, no. 26, pp. 6435–6442, 2006.
View at: Publisher Site | Google Scholar
G. J. M. K. Wanda, S. Djiogue, F. Z. Gamo, S. G. Ngitedem, and D. Njamen, “Anxiolytic and sedative activities of aqueous leaf extract of Dichrocephala integrifolia (Asteraceae) in mice,” Journal of Ethnopharmacology, vol. 176, pp. 494–498, 2015.
View at: Publisher Site | Google Scholar
P. J. Wabo, V. Payne, T. G. Mbogning et al., “In vitro anthelminthic efficacy of Dichrocephala integrifolia (Asteraceae) extracts on the gastro-intestinal nematode parasite of mice: Heligmosomoides bakeri (Nematoda, Heligmosomatidae),” Asian Pacific Journal of Tropical Biomedicine, vol. 3, no. 2, pp. 100–104, 2013.
View at: Publisher Site | Google Scholar
J. Espinosa-Rivero, E. Rendón-Huerta, and I. Romero, “Inhibition of Helicobacter pylori growth and its colonization factors by Parthenium hysterophorus extracts,” Journal of Ethnopharmacology, vol. 174, pp. 253–260, 2015.
View at: Publisher Site | Google Scholar
G. Sharma and K. Bhutani, “Plant based antiamoebic drugs; part II1. Amoebicidal activity of parthenin isolated from Parthenium hysterophorus,” Planta Medica, vol. 54, no. 2, pp. 120–122, 1988.
View at: Publisher Site | Google Scholar
N. P. Manandhar, “A survey of medicinal plants of Jajarkot district, Nepal,” Journal of Ethnopharmacology, vol. 48, no. 1, pp. 1–6, 1995.
View at: Publisher Site | Google Scholar
M. Anitha, E. D. Daffodil, S. Muthukumarasamy, and V. R. Mohan, “Anti-inflammatory activity of whole plant of Cynoglossum zeylanicum (vahl ex hornem),” Journal of Advanced Pharmacy Education & Research, vol. 3, no. 1, pp. 25–27, 2013.
View at: Google Scholar
M. Anitha, G. Sakthidevi, S. Muthukumarasamy, and V. R. Mohan, “Evaluation of anti-fertility activity of ethanol extract of Cynoglossum zeylanicum (Vahl Ex Hornem) Thunb. Ex. Lehm (boraginaceae) whole plant on male albino rats,” Journal of Current Chemical and Pharmaceutical Sciences, vol. 3, no. 2, pp. 135–145, 2013.
View at: Google Scholar
M. Anitha, E. Daffodil, S. Muthukumarasamy, and V. R. Mohan, “Antitumor activity of Cynoglossum zeylanicum (vahl ex hornem) thunb. Ex. Lehm. against dalton ascites lymphoma in Swiss albino mice,” International Journal of Applied Biology and Pharmaceutical Technology, vol. 3, no. 4, pp. 457–462, 2012.
View at: Google Scholar
M. Anitha, E. Daffodil, S. Muthukumarasamy, and V. R. Mohan, “Hepatoprotective and antioxidant activity of ethanol extract of Cynoglossum zeylanicum (Vahl ex Hornem) Thurnb ex Lehm in CCl4 treated rats,” Journal of Applied Pharmaceutical Science, vol. 2, no. 12, p. 99, 2012.
View at: Google Scholar
S. Choudhury, P. Sharma, M. D. Choudhury, and G. D. Sharma, “Ethnomedicinal plants used by Chorei tribes of Southern Assam, North eastern India,” Asian Pacific Journal of Tropical Disease, vol. 2, pp. S141–S147, 2012.
View at: Publisher Site | Google Scholar
Y.-C. Wang and T.-L. Huang, “Screening of anti-Helicobacter pylori herbs deriving from Taiwanese folk medicinal plants,” FEMS Immunology & Medical Microbiology, vol. 43, no. 2, pp. 295–300, 2005.
View at: Publisher Site | Google Scholar
IUCN, IUCN 2000: National Register of Medicinal Plants, IUCN, Kathmandu, Nepal, 2000.
X.-M. Zhang, D.-P. Yang, Z.-Y. Xie, Q. Li, L.-P. Zhu, and Z.-M. Zhao, “Two new glycosides isolated from Sapindus mukorossi fruits: effects on cell apoptosis and caspase-3 activation in human lung carcinoma cells,” Natural Product Research, vol. 30, no. 3, pp. 1459–1463, 2015.
View at: Publisher Site | Google Scholar
A. Bag, S. K. Bhattacharyya, and R. R. Chattopadhyay, “The development of Terminalia chebula Retz. (combretaceae) in clinical research,” Asian Pacific Journal of Tropical Biomedicine, vol. 3, no. 3, pp. 244–252, 2013.
View at: Publisher Site | Google Scholar
DPR, Medicinal Plants of Nepal, Government of Nepal, Ministry of Forests and Soil conservation, Department of Plant Resources, Thapathali, Kathmandu, Nepal, 2nd edition, 2016.
M. F. Muzitano, L. W. Tinoco, C. Guette, C. R. Kaiser, B. Rossi-Bergmann, and S. S. Costa, “The antileishmanial activity assessment of unusual flavonoids from Kalanchoe pinnata,” Phytochemistry, vol. 67, no. 18, pp. 2071–2077, 2006.
View at: Publisher Site | Google Scholar
S. Matthew, A. K. Jain, M. James, C. Matthew, and D. Bhowmik, “Analgesic and anti-inflammatory activity of Kalanchoe pinnata (lam.) pers,” Journal of Medicinal Plants, vol. 1, no. 2, pp. 24–28, 2013.
View at: Google Scholar
K. S. Patil and S. R. Bhalsing, “Ethnomedicinal uses, phytochemistry and pharmacological properties of the genus Boerhavia,” Journal of Ethnopharmacology, vol. 182, pp. 200–220, 2016.
View at: Publisher Site | Google Scholar
M. R. Bhandary and J. Kawabata, “Antidiabetic activity of Laligurans (Rhododendron arboreum Sm.) flower,” Journal of Food Science and Technology Nepal, vol. 4, pp. 61–63, 2008.
View at: Google Scholar
M. B. Rai, “Medicinal plants of Tehrathum district, eastern Nepal,” Our Nature, vol. 1, no. 1, pp. 42–48, 2003.
View at: Publisher Site | Google Scholar
N. Verma, A. P. Singh, A. Gupta, P. Sahu, and C. V. Rao, “Antidiarrheal potential of standardized extract of Rhododendron arboreum Smith flowers in experimental animals,” Indian Journal of Pharmacology, vol. 43, no. 3, p. 689, 2011.
View at: Publisher Site | Google Scholar
D. Ojha, R. Das, P. Sobia et al., “Pedilanthus tithymaloides inhibits HSV infection by modulating NF-κB signaling,” PLoS One, vol. 10, no. 9, Article ID e0139338, 2015.
View at: Publisher Site | Google Scholar
W. Mongkolvisut and S. Sutthivaiyakit, “Antimalarial and antituberculous poly-O-acylated jatrophane diterpenoids from Pedilanthus tithymaloides,” Journal of Natural Products, vol. 70, no. 9, pp. 1434–1438, 2007.
View at: Publisher Site | Google Scholar
CRC Press, CRC World Dictionary of Medicinal and Poisonous Plants: Common Names, Scientific Names, Eponyms, Synonyms, and Etymology, CRC Press, Boca Raton, FL, USA, 2012.
J. J. Patel, S. R. Acharya, and N. S. Acharya, “Clerodendrum serratum (L.) Moon.—a review on traditional uses, phytochemistry and pharmacological activities,” Journal of Ethnopharmacology, vol. 154, no. 2, pp. 268–285, 2014.
View at: Publisher Site | Google Scholar
B. Malla, D. P. Gauchan, and R. B. Chhetri, “An ethnobotanical study of medicinal plants used by ethnic people in Parbat district of western Nepal,” Journal of Ethnopharmacology, vol. 165, pp. 103–117, 2015.
View at: Publisher Site | Google Scholar
K. P. Devkota, M. T. H. Khan, R. Ranjit, A. Meli Lannang, Samreen, and M. Iqbal Choudhary, “Tyrosinase inhibitory and antileishmanial constituents from the rhizomes of Paris polyphylla,” Natural Product Research, vol. 21, no. 4, pp. 321–327, 2007.
View at: Publisher Site | Google Scholar
Z.-H. Li, J.-Y. Wan, G.-Q. Wang, F.-G. Zhao, and J.-H. Wen, “Identification of compounds from Paris polyphylla (ChongLou) active against Dactylogyrus intermedius,” Parasitology, vol. 140, no. 8, pp. 952–958, 2013.
View at: Publisher Site | Google Scholar
M.-S. Lee, J. Y.-W. Chan, S.-K. Kong et al., “Effects of polyphyllin D, a steroidal saponin in Paris polyphylla, in growth inhibition of human breast cancer cells and in xenograft,” Cancer Biology & Therapy, vol. 4, no. 11, pp. 1248–1254, 2005 1.
View at: Publisher Site | Google Scholar
T. Namba, Medicinal Resources and Ethnopharmacology in Sri Lanka and Nepal, Research Institute for Wakan-Yaku (Oriental Medicines), Toyama Medical and Pharmaceutical University, Toyama, Japan, 1983.
A. Krishna, M. P. Saradhi, B. N. Rao, P. Mohan, M. Balaji, and L. Samuel, “Evaluation of wound healing activity of Phragmites vallatoria leaf ethanol extract in stz-induced diabetic rats,” International Journal of Pharmacy and Pharmaceutical Sciences, vol. 4, pp. 393–395, 2012.
View at: Google Scholar
N. K. Radhika, P. S. Sreejith, and V. V. Asha, “Cytotoxic and apoptotic activity of Cheilanthes farinosa (Forsk.) Kaulf. against human hepatoma, Hep3B cells,” Journal of Ethnopharmacology, vol. 128, no. 1, pp. 166–171, 2010.
View at: Publisher Site | Google Scholar
R. Lamichhane, S.-G. Kim, A. Poudel, D. Sharma, K.-H. Lee, and H.-J. Jung, “Evaluation of in vitro and in vivo biological activities of Cheilanthes albomarginata Clarke,” BMC Complementary and Alternative Medicine, vol. 14, no. 1, p. 342, 2014.
View at: Publisher Site | Google Scholar
P. Basnet, S. Kadota, K. Manandhar, M. Manandhar, and T. Namba, “Constituents of Boenninghausenia albiflora: isolation and identification of some coumarins,” Planta Medica, vol. 59, no. 4, pp. 384–386, 1993.
View at: Publisher Site | Google Scholar
R. Sharma, D. S. Negi, W. K. P. Shiu, and S. Gibbons, “Characterization of an insecticidal coumarin from Boenninghausenia albiflora,” Phytotherapy Research, vol. 20, no. 7, pp. 607–609, 2006.
View at: Publisher Site | Google Scholar
I. Liaqat, N. Riaz, Q. U. Saleem, H. M. Tahir, M. Arshad, and N. Arshad, “Toxicological evaluation of essential oils from some plants of rutaceae family,” Evidence-Based Complementary and Alternative Medicine, vol. 2018, 7 pages, 2018.
View at: Publisher Site | Google Scholar
C. Burlakoti and R. M. Kunwar, “Folk herbal medicines of Mahakali watershed Area, Nepal,” M. K. Chettri and C. B. Tapa, Eds., pp. 187–193, Shrestha, Publisher: Ecological Society (ECOS), Kathmandu, Nepal, 2008.
View at: Google Scholar
N. Verma, G. Amresh, P. Sahu, N. Mishra, A. P. Singh, and C. V. Rao, “Antihyperglycemic activity, antihyperlipedemic activity, haematological effects and histopathological analysis of Sapindus mukorossi Gaerten fruits in streptozotocin induced diabetic rats,” Asian Pacific Journal of Tropical Medicine, vol. 5, no. 7, pp. 518–522, 2012.
View at: Publisher Site | Google Scholar
N. P. Manandhar, “An inventory of some herbal drugs of Myagdi district, Nepal,” Economic Botany, vol. 49, no. 4, pp. 371–379, 1995.
View at: Publisher Site | Google Scholar
M. C. Tamilarasi, U. Subasin, M. S. Kavimani, and M. B. Jaykar, “Phytochemical and pharmacological evaluation of Ampelocissus latifolia,” Ancient Science of Life, vol. 30, no. 4, p. 95, 2011.
View at: Google Scholar
G. R. Pettit, V. J. R. V. Mukku, G. Cragg et al., “Antineoplastic agents. 558.Ampelocissussp. cancer cell growth inhibitory constituents(1),” Journal of Natural Products, vol. 71, no. 1, pp. 130–133, 2008.
View at: Publisher Site | Google Scholar
K. Theng and A. Korpenwar, “Phytochemical analysis of ethanol extract of Ampelocissus latifolia (Roxb.) Planch tuberous root using UV-vis, FTIR and GC-MS,” International Journal of Pharmaceutical Sciences and Research, vol. 6, no. 9, p. 3936, 2015.
View at: Google Scholar
B. Joshi, S. Hendrickx, L. Magar, N. Parajuli, P. Dorny, and L. Maes, “In vitro antileishmanial and antimalarial activity of selected plants of Nepal,” Journal of Intercultural Ethnopharmacology, vol. 5, no. 4, pp. 383–389, 2016.
View at: Publisher Site | Google Scholar
S. K. Panda, Y. K. Mohanta, L. Padhi, and W. Luyten, “Antimicrobial activity of select edible plants from Odisha, India against food-borne pathogens,” LWT, vol. 113, Article ID 108246, 2019.
View at: Publisher Site | Google Scholar
S. K. Panda, R. Das, P. Leyssen, J. Neyts, and W. Luyten, “Assessing medicinal plants traditionally used in the Chirang reserve forest, Northeast India for antimicrobial activity,” Journal of Ethnopharmacology, vol. 225, pp. 220–233, 2018.
View at: Publisher Site | Google Scholar
S. K. Panda, L. Padhi, P. Leyssen, M. Liu, J. Neyts, and W. Luyten, “Antimicrobial, anthelmintic, and antiviral activity of plants traditionally used for treating infectious disease in the similipal biosphere reserve, Odisha, India,” Frontiers in Pharmacology, vol. 8, p. 658, 2017.
View at: Publisher Site | Google Scholar
A. Ledoux, M. Cao, O. Jansen et al., “Antiplasmodial, anti-chikungunya virus and antioxidant activities of 64 endemic plants from the Mascarene islands,” International Journal of Antimicrobial Agents, vol. 52, no. 5, pp. 622–628, 2018.
View at: Publisher Site | Google Scholar
G.-X. Wang, J. Han, L.-W. Zhao, D.-X. Jiang, Y.-T. Liu, and X.-L. Liu, “Anthelmintic activity of steroidal saponins from Paris polyphylla,” Phytomedicine, vol. 17, no. 14, pp. 1102–1105, 2010.
View at: Publisher Site | Google Scholar
S. Nakajima, K. Takaishi, Y. Nagano, Y. Alen, K. Kawazu, and N. Baba, “Nematicidal compound from Ageratum conyzoides,” Scientific Reports of the Faculty of Agriculture-Okayama University (Japan), vol. 93, pp. 1–4, 2004.
View at: Google Scholar
M. A. Tantry, R. Khan, S. Akbar, A. R. Dar, A. S. Shawl, and M. S. Alam, “An unusual bioactive oleanane triterpenoid from Rhododendron campanulatum D. Don,” Chinese Chemical Letters, vol. 22, no. 5, pp. 575–579, 2011.
View at: Publisher Site | Google Scholar
B. Pradhan, D. Bhatt, S. Mishra, and S. Sahoo, “Antimicrobial potential of leaves of Adhatoda vasica Nees. against human pathogens causing Infections of UT, GIT and skin,” Pharmaceutical and Biological Evaluations, vol. 2, no. 1, pp. 36–39, 2015.
View at: Google Scholar
S. J. Tatsimo, J. D. Tamokou, L. Havyarimana et al., “Antimicrobial and antioxidant activity of kaempferol rhamnoside derivatives from Bryophyllum pinnatum,” BMC Research Notes, vol. 5, p. 158, 2012.
View at: Publisher Site | Google Scholar
T. Mohammed and C. Teshale, “Preliminary phytochemical screening and evaluation of antibacterial activity of antibacterial activity of Dichrocepala integrifolia (Lf) O. Kuntze,” Journal of Intercultural Ethnopharmacology, vol. 1, no. 1, pp. 30–34, 2012.
View at: Publisher Site | Google Scholar
J. H. Wang, F. Luan, X. D. He, Y. Wang, and M. X. Li, “Traditional uses and pharmacological properties of Clerodendrum phytochemicals,” Journal of Traditional and Complementary Medicine, vol. 8, no. 1, pp. 24–38, 2018.
View at: Publisher Site | Google Scholar
S. Arokiyaraj, N. Sripriya, R. Bhagya, B. Radhika, L. Prameela, and N. K. Udayaprakash, “Phytochemical screening, antibacterial and free radical scavenging effects of Artemisia nilagirica, Mimosa pudica and Clerodendrum siphonanthus–an in–vitro study,” Asian Pacific Journal of Tropical Biomedicine, vol. 2, no. 2, pp. 601–604, 2012.
View at: Publisher Site | Google Scholar
S. Panda, Y. Mohanta, L. Padhi, Y.-H. Park, T. Mohanta, and H. Bae, “Large scale screening of ethnomedicinal plants for identification of potential antibacterial compounds,” Molecules, vol. 21, no. 3, p. 293, 2016.
View at: Publisher Site | Google Scholar
G. J. Vidotti, A. Zimmermann, M. H. Sarragiotto, C. V. Nakamura, and B. P. Dias Filho, “Antimicrobial and phytochemical studies on Pedilanthus tithymaloides,” Fitoterapia, vol. 77, no. 1, pp. 43–46, 2006.
View at: Publisher Site | Google Scholar
X.-J. Qin, D.-J. Sun, W. Ni et al., “Steroidal saponins with antimicrobial activity from stems and leaves of Paris polyphylla var. yunnanensis,” Steroids, vol. 77, no. 12, pp. 1242–1248, 2012.
View at: Publisher Site | Google Scholar
X.-J. Qin, M.-Y. Yu, W. Ni et al., “Steroidal saponins from stems and leaves of Paris polyphylla var. yunnanensis,” Phytochemistry, vol. 121, pp. 20–29, 2016.
View at: Publisher Site | Google Scholar
M. Ibrahim, A. A. Khan, S. K. Tiwari, M. A. Habeeb, M. Khaja, and C. Habibullah, “Antimicrobial activity of Sapindus mukorossi and Rheum emodi extracts against H pylori: in vitro and in vivo studies,” World Journal of Gastroenterology, vol. 12, no. 44, pp. 7136–7142, 2006.
View at: Publisher Site | Google Scholar
A. Sharma, S. C. Sati, O. P. Sati, M. D. Sati, and S. K. Kothiyal, “Triterpenoid saponins from the pericarps of Sapindus mukorossi,” Journal of Chemistry, vol. 2013, Article ID 613190, 5 pages, 2013.
View at: Publisher Site | Google Scholar
K. Khulbe and C. S. Sati, “Antibacterial activity of Boenninghausenia albiflora reichb. (Rutaceae),” African Journal of Biotechnology, vol. 8, no. 22, pp. 6346–6348, 2009.
View at: Publisher Site | Google Scholar
S. K. Panda, “Ethno-medicinal uses and screening of plants for antibacterial activity from Similipal biosphere reserve, Odisha, India,” Journal of Ethnopharmacology, vol. 151, no. 1, pp. 158–175, 2014.
View at: Publisher Site | Google Scholar
M. T. Rashid, A. Yadav, A. Bajaj, and A. Lone, “An assessment of antibacterial potency of aqueous leaf extract of Clerodendrum serratum Linn. against pathogenic bacterial strains,” Indo American Journal of Pharmaceutical Research, vol. 3, pp. 1637–1644, 2013.
View at: Google Scholar
M. Nisar, S. Ali, N. Muhammad et al., “Antinociceptive and anti-inflammatory potential of Rhododendron arboreum bark,” Toxicology and Industrial Health, vol. 32, no. 7, pp. 1254–1259, 2016.
View at: Publisher Site | Google Scholar
P. K. Sonar, R. Singh, S. Khan, and S. K. Saraf, “Isolation, characterization and activity of the flowers of Rhododendron arboreum (ericaceae),” E-Journal of Chemistry, vol. 9, no. 2, pp. 631–636, 2012.
View at: Publisher Site | Google Scholar
S. K. Panda, N. Patra, G. Sahoo, A. K. Bastia, and S. K. Dutta, “Anti-diarrheal activities of medicinal plants of similipal biosphere reserve, Odisha, India,” International Journal of Medicinal and Aromatic Plants, vol. 2, no. 1, pp. 123–134, 2012.
View at: Google Scholar
S. K. Panda, L. C. N. da Silva, D. Sahal, and M. Leonti, “Ethnopharmacological studies for the development of drugs with special reference to Asteraceae,” Frontiers in Pharmacology, vol. 10, p. 955, 2019.
View at: Publisher Site | Google Scholar
S. K. Rath, N. Mohapatra, D. Dubey, S. K. Panda, H. N. Thatoi, and S. K. Dutta, “Antimicrobial activity of Diospyros melanoxylon bark from similipal biosphere reserve, Orissa, India,” African Journal of Biotechnology, vol. 8, no. 9, pp. 1924–1928, 2009.
View at: Google Scholar
N. Shrestha, S. Shrestha, L. Koju, K. K. Shrestha, and Z. Wang, “Medicinal plant diversity and traditional healing practices in eastern Nepal,” Journal of Ethnopharmacology, vol. 192, pp. 292–301, 2016.
View at: Publisher Site | Google Scholar
Y. Yang, J. Xiu, J. Liu et al., “Chebulagic acid, a hydrolyzable tannin, exhibited antiviral activity in vitro and in vivo against human enterovirus 71,” International Journal of Molecular Sciences, vol. 14, no. 5, pp. 9618–9627, 2013.
View at: Publisher Site | Google Scholar
A. H. Hadun, J. M. Mbaria, G. O. Aboge, M. O. Okumu, and A. L. Yiaile, “Antiviral activity of Dicrocephala integrifolia (kuntze) against herpes simplex type-1 virus: an in vitro study,” Pharmacognosy Communications, vol. 8, no. 2, pp. 81–85, 2018.
View at: Publisher Site | Google Scholar
S. K. Panda and W. Luyten, “Antiparasitic activity in asteraceae with special attention to ethnobotanical use by the tribes of Odisha, India,” Parasite, vol. 25, 2018.
View at: Publisher Site | Google Scholar
P. C. Joshi, S. Mandal, and P. C. Das, “Jayantinin, a dimeric coumarin from Boenninghausenia albiflora,” Phytochemistry, vol. 28, no. 4, pp. 1281–1283, 1989.
View at: Publisher Site | Google Scholar
K. Takaishi, M. Izumi, N. Baba, K. Kawazu, and S. Nakajima, “Synthesis and biological evaluation of alkoxycoumarins as novel nematicidal constituents,” Bioorganic & Medicinal Chemistry Letters, vol. 18, no. 20, pp. 5614–5617, 2008.
View at: Publisher Site | Google Scholar
E. M. Vayndorf, S. S. Lee, and R. H. Liu, “Whole apple extracts increase lifespan, healthspan and resistance to stress in Caenorhabditis elegans,” Journal of Functional Foods, vol. 5, no. 3, pp. 1236–1243, 2013.
View at: Publisher Site | Google Scholar
P. Wangchuk, P. R. Giacomin, M. S. Pearson, M. J. Smout, and A. Loukas, “Identification of lead chemotherapeutic agents from medicinal plants against blood flukes and whipworms,” Scientific Reports, vol. 6, no. 1, p. 32101, 2016.
View at: Publisher Site | Google Scholar

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Copyright © 2020 Bishnu Joshi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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