Evaluation of antimycobacterial, leishmanicidal and antibacterial activity of three medicinal orchids of Arunachal Pradesh, India

Background The ethnic population of Arunachal Pradesh uses a number of orchids as such, or in decoction for various ailments. Three untapped orchids namely, Rhynchostylis retusa, Tropidia curculioides and Satyrium nepalense, traditionally used in tuberculosis, asthma and cold stage of malaria in folk medicine, were selected for the present study. Methods Dried material of each plant was divided into three parts. Solvent extraction and fractionation afforded altogether 30 extracts and fractions, which were evaluated against Mycobacterium tuberculosis (H37Rv and MDR strain) for antimycobacterial activity; promastigotes and amastigotes of Leishmania donovani for leishmanicidal activity and two gram positive and three gram negative clinical isolates for antibacterial activity. Results The most significant antimycobacterial activity was observed with n-hexane fraction of the flower of Satyrium nepalense with MIC of 15.7 μg/mL. The most promising leishmanicidal activity was observed with diethyl ether fraction of the roots of Rhynchostylis retusa with IC50 values of 56.04 and 18.4 μg/mL against promastigotes and intracellular amastigotes respectively. Evaluation of antibacterial activity identified S. nepalense flower n-hexane and R. retusa roots diethyl ether as potential fractions with MIC values of ≤100 μg/mL against selected clinical isolates. Conclusions This is the first report of the plants possessing antimycobacterial and leishmanicidal activity. The investigation resulted in identification of S. nepalense as the most promising plant, which possessed all three activities in significant proportion. This laboratory outcome could be translated to marketable pharmaceutical products and also to produce maximum benefits to the local of nearby area. Graphical abstract Antimycobacterial and leishmanicidal activity of medicinal orchids


Background
India harbors a rich repository of untapped medicinal plants, with plenty of associated knowledge that needs to be appropriately utilized. The vast degree of diversity present in the country is directly related to the highly divergent ecosystem and altitudinal variations [1]. Proper scientific investigation of the unexplored natural resources and subsequent commercialization could bring benefits to the stakeholders and also could play a central role in the drug development programs.
Arunachal Pradesh (AR) is a natural habitat of 5000 flowering species including 550 species of orchid plants. Further, it is interesting to note that nearly 300 species of the total orchids are rare whereas, only 37 species are of medicinal importance [2]. Medicinal orchids prefer tropical climate though, a few of them grow under extreme climatic conditions also. Considering serenity of the habitat of AR and traditional use of orchids by the locals, we selected three relatively unexplored plants including Rhynchostylis retusa (Rr), Tropidia curculioides (Tc), and Satyrium nepalense (Sn) for the present investigation. R. retusa of the genus Rhynchostylis is widely distributed all over North-eastern Himalayas and specially linked with the people of Assam, commonly called 'Kopu Phool' in Assamese language. It is a symbol of fertility and merriment [3]. The roots of R. retusa are used in folk medicine for rheumatism, asthma, tuberculosis, nervous twitchings, cramps, infantile epilepsy, vertigo, palpitation, kidney stone, and menstrual disorders [4]. T. curculioides is an endangered plant commonly found in Sikkim and Arunachal Pradesh. The decoction of roots of T. curculioides is used for cold stage of malaria and diarrhoea [5]. S. nepalense (Hathjadi), a long terrestrial herb (25-60 cm) is commonly found at 2400-5000 m height. The tubers of S. nepalense are consumed by the native people as food, tonic and aphrodisiac [6]. The plant is reported to have beneficial effects in the symptom of diarrhoea, malaria and dysentery [7]. Qualitative phytochemical analysis and antimicrobial screening had been conducted with the plant extracts of R. retusa [8,9]. However, any literature related to phytochemical investigation and biological activities of T. curculioides was not available. The presence of quercetin, had been reported from the extracts of S. nepalense [10]. The literature search report inspired us to investigate the medicinal property of the plants.
The folk medicines of this region are used quite extensively for a number of common ailments and communicable diseases [11]. Isolated living environment and poor access to healthcare is a threat for survival of the ethnic population all over India. Especially, the management of highly prevalent communicable diseases for e.g., tuberculosis (TB), visceral leishmaniasis (VL) and malaria is a great concern. TB in humans is caused by Mycobacterium tuberculosis, a facultative intracellular microbe belonging to M. tuberculosis complex. Further, the alarming rise of multi-drug-resistant (MDR), extensively drug-resistant (XDR) and totally drug resistant (TDR) strains of M. tuberculosis, emphasizes the need of new leads based on traditional knowledge [12]. Visceral leishmaniasis, also known as kala azar is a fatal vector-borne illness caused by Leishmania donovani. It is the second most dreaded parasitic disease after malaria, causing considerable morbidity and mortality. Interestingly, a number of plant extracts and plant derived compounds are reported to possess significant leishmanicidal activity [13] however, very few have reached to the stage of clinical trials. Also, the dramatic increase of new and emerging MDR bacterial strains emphasizes the need of fresh investigation with those natural resources, which has not been examined yet.

Plant materials
The plants were collected from the foothills of Tipi & Khellong, and Doimara and Sange district of Arunachal Pradesh. The details of geographical location, altitude and place of collection have been mentioned in Table 1

Extraction and fractionation
The dried plant material of R. retusa and T. curculioides were divided into root, stem and leaves while, S. nepalense was divided into pseudobulb, stem and leaves, and flowers. Dry weight of each part was noted and was extracted with methanol: water (9:1, 1 L× 24 h) for three consecutive days followed by extraction of the residue with H 2 O (0.5 L) at room temperature. The concentrated MeOH extracts of R. retusa and T. curculioides were suspended in water and was fractionated into diethyl ether (Et 2 O), n-BuOH and residual aqueous fractions successively. As, the plant material of S. nepalense was available in larger

Phytochemical screening of extracts
Phytochemical analysis for the presence of alkaloids, flavonoids, steroids, reducing sugars, cardiac glycosides, terpenoids, anthraquinones, tannins, phlobatanins and saponins were conducted with each fraction by using standard protocol [14].

Colorimetric redox indictor assay (CRI assay)
The pan sensitive strain of M. tuberculosis H37Rv TMC-102 (sensitive to streptomycin, isoniazide, rifampicin, ethambutol and pyrazinamide) and another MDR clinical isolate (Tb-14,348/16), resistant to rifampicin and isoniazid, was obtained as gift from Dr. V.M. Katoch, National JALMA Institute of Leprosy and other Mycobacterial Diseases, Agra, India. Briefly, the bacterium at log phase of growth (approximately 12 days), was transferred to a sterile vial containing glass beads and 8 mL of sterile 0.85% saline solution. The bacterial suspension was disaggregated by agitation and was allowed to stand for 15 min at room temperature. Turbidity of the suspension was compared with 1 McFarland tube standard and was adjusted with 7H9 broth to obtain a bacterial concentration of 3 × 10 8 CFU/mL. The working solution at 1:20 dilution of the suspension, in Middlebrook 7H9 broth was evaluated by Colorimetric Redox Indicator Assay (CRI assay) [15]. The extracts were dissolved in DMSO and were diluted appropriately to obtain final sample concentrations in the range of 100-500 μg/mL. The bacterial suspension at a concentration of 1.5 × 10 8 cells/mL was added to each well where, media along with bacterial suspension was considered as growth control and 0.5% DMSO with media and bacterial suspension were used as DMSO control. Besides, the well known antimycobacterial agent rifampicin was used as positive control. Sterile water was added to all perimeter wells to avoid evaporation. The microplates were incubated for 5-7 days at 37°C in an incubator followed by addition of 25 μL of resazurin (0.02% w/v) dye to each well. Then, the plates were re-incubated at 37°C for 24 h for color development. The minimum inhibitory concentration (MIC) was defined as the lowest drug/extract concentration that prevented color change of resazurin reagent from blue to pink. Blue color is interpreted as no mycobacterial growth and pink color as growth occurrence [16]. All the experiments were carried out in duplicate with three independent experiments.

Leishmanicidal assay
Pan sensitive strain of L. donovani (DD8) was obtained from Department of Laboratory Medicine (AIIMS), New Delhi, India. The culture was routinely maintained at 24°C in M-199 (GIBCO®, USA) medium supplemented with penicillin (100 U/mL), streptomycin (100 μg/mL) (Invitrogen, USA) and 10% heat inactivated fetal calf serum (FCS; GIBCO®, USA). Promastigotes at logarithmic phase were seeded (1 × 10 6 cells/mL) in 96-well microtiter plate in the presence of different concentrations of samples and incubated at 24°C for 48 h. Thereafter, 100 μl of MTT (5 mg/mL) solution was added to each plate and incubated for 4 h at 24°C. Finally, 100 μl of DMSO was added in each well to dissolve the formazan produced, followed by 18 h of incubation. The absorbance was measured at 570 nm and DMSO (0.5%) was considered as control while, amphotericin B and miltefosine was used as reference. Each assay was performed in duplicate with three independent experiments [17].
To produce intracellular amastigotes, J774G8 (5 × 10 5 cells/mL) macrophage cells were plated onto 13-mm coverslips in 24-well plates for 1 h at 37°C in a CO 2 incubator. Non adherent cells were removed and the cells were further incubated overnight. Adherent cells were infected with L. donovani promastigotes at a parasite: macrophage ratio of 10: 1 and further incubated for 1 h. Unbound promastigotes were removed by extensive washing with PBS (pH 7.2). The infected macrophages were incubated with various dilutions of samples and the mean percentage of viable amastigotes was calculated relative to control and the results were expressed as concentration inhibiting the parasitic growth. The leishmanicidal effect of each sample was expressed in IC 50 values.

Cell cytotoxicity assay
The cell cytotoxicity was assessed against J774G8 murine macrophage cells (1 × 10 6 cells/mL) with different concentrations of active fractions in the range of 15.62-250 μg/mL. The cell viability was determined by MTT assay [18] as well. The results were expressed as percentage reductions in cell viability, compared to untreated control wells. The cytotoxic concentration required to kill 50% of the cells (CC 50 ) was calculated.

Antibacterial assay
SnF n -Hex  Dr. Kumardeep Dutta Choudhary, Department of Medical Oncology, Rajiv Gandhi Cancer Research Institute, Delhi, India. All bacterial strains were revived in nutrient broth to conduct antibacterial screening [19]. Briefly, nutrient agar plates were inoculated with 0.1 mL of each organism (1 × 10 8 CFU/mL) and were treated with 50 μL of samples. The plates were incubated at 37°C for 24 h. The antimicrobial activity was expressed as the mean diameter of inhibition zones (mm) with standard deviation produced by the tested fractions. Tetracycline and gentamycin were used as positive controls for disc diffusion assay. Minimum inhibitory concentrations (MIC) values were determined for the most potent extract by tube dilution method [20]. A series of two fold dilutions of each extract ranging from 1 mg/mL to 0.1 mg/mL were done in Muller Hilton broth and were inoculated with 0.1 mL of suspension of the test organism. The tubes were incubated at 37°C for 24 h and checked for turbidity. Minimum inhibitory concentration was determined as highest dilution of the extract that showed no visible growth.

Result and discussion
Solvent extraction and fractionation of the plants afforded 30 components, dry weight of which is demonstrated in Table 2. The results of qualitative analysis for different classes of phytochemicals viz., alkaloids, flavonoids, steroids, reducing sugar, cardiac glycosides, terpenoids, anthraquinones, tannins, phlobatanins and saponins had been presented in Table 3. The phytochemical analysis data provides an overview of the chemical classes and their relative proportion in a fraction. Further, this knowledge could serve as a background to select the isolation strategy of the active ingredients. The antimycobacterial activity was determined by colorimetric redox indicator assay and the results were expressed in MIC values (Table 4). An analysis of the results showed that n-hexane fraction of the flower of S. nepalense (n-Hex SnF) exhibited most significant antimycobacterial activity against H37Rv and MDR strains with MIC of 15.7 and 42.5 μg/mL followed by Et 2 O of roots of R. retusa (Et 2 O RrR) with MIC of 62.5 and 125 μg/mL respectively. It is well documented that MIC <100 μg/mL is considered as potent while, 100-625 μg/ mL represent moderate [21] antimycobacterial activity. Few more fractions including, Et 2 O fraction of RrS and TcR; EtOAc fraction of SnP and SnF; and n-hexane fraction of SnL exhibited moderate antimycobacterial activity (MIC 125 μg/mL) against H37Rv strain. Also, better performance demonstrated by non polar fractions might be involving the lipophilic constituents, which causes disturbance to the lipid portion of the plasma membrane, leading to a loss of permeability and leakage of intracellular materials [22]. Though, in vitro biological activities, not necessarily be transposed to clinical trials, but in vitro experiments clarify the safety aspects of a sample, to determine whether a drug candidate possesses scientific merit for further investigation.
In the second phase, the fractions were evaluated against promastigotes and intracellular amastigotes of L. donovani (DD8). The most efficient screening strategy   Data is presented as the mean ± SD of three independent experiments IC 50 inhibitory concentration of 50% parasites, CC 50 cytotoxicity concentration of 50% cells activity in the range of 50-100 μg/mL against promastigotes and 18-25 μg/mL against intracellular amastigotes (Table 5). To determine cell cytotoxicity of the active fractions, J774G8 murine macrophages were treated with different concentrations of the fractions. After 48 h, the viability was checked by MTT assay which showed negligible cytotoxic effect against macrophages at a concentration of CC 50 values (97.2 ± 1.2, 89.4 ± 4.7 and 100.7 ± 1.7 μg/mL), which was higher than the IC 50 values ( Table 6). The cell cytotoxiciy results clearly demonstrated that the active fractions have least harmful effect on the normal cells however; the parasitic cells were affected drastically at those concentrations.
In commensurate with previous observations, the IC 50 values of the active fractions were lower against amastigotes than promastigotes [24]. In this connection it is worthy to mention that screening of plants by bioassay guided fractionation only provides primary knowledge, which could be processed further by isolating compounds and evaluating their biological activity individually.
The present study was further extended to assess the best predictor of antibacterial activity against two gram positive and three gram negative MDR clinical isolates. The zone of inhibition values >10 mm was considered as active (Table 7). Interestingly, n-hexane fraction of all three parts of Sn demonstrated good antibacterial activity against S. aureus while, n-BuOH RrL and Et 2 O TcR showed significant efficiency against Enterococcus sp. However, most of the fractions failed to exhibit any significant activity against Serratia sp. Moreover, n-Hex SnF and Et 2 O TcR exhibited good antibacterial activity against S. aureus and E. coli with MIC values of 62.5 μg/mL and 125 μg/mL respectively while, only one fraction RrR Et 2 O showed substantial activity against Acinetobacter sp. with MIC of 104.16 μg/mL ( Table 8). The results also revealed that aqueous extracts did not possess any antimicrobial activity against the tested strains. Similar observations were also reported from other plant species [25]. The results showed the efficacy of the plants as traditional medicine. Moreover, the active fractions were evaluated on pre-coated TLC plates with increasing concentration of ethyl acetate: n-Hex at 1: 9; 3: 7; and 1:1 proportions to generate the chemical profile. Maximum resolution was achieved at 3:7 proportion for Rr Et 2 O and TcR Et 2 O fractions and at 1:9 proportion for SnF n-Hex and SnP n-Hex (Fig. 1) fractions. The active fractions showed the presence of a number of distinct spots, which could be useful as a chemical profile and also in isolation of compounds.

Conclusion
The present study involving antimycobacterial, leishmanicidal and antibacterial activity of three orchids demonstrated S. nepalense as the most promising plant followed by R. retusa and T. curculioides. More specifically, the overall screening results identified n-Hex SnF as the most potent fraction possessing significantly high activities. Also, the fractions exhibited cell cytotoxicity well within the permissible limit. As proper phytochemical investigation of the plants has not been conducted so far, isolation and characterization of compounds could lead potential drug candidates for the experimental diseases in future.