Snigdha Saikia2, M. Bordoloi2,3 and Dipanwita Banik1,2*

Chemical Science and Technology Division, 1Biological Sciences and Technology Division, Council of Scientific and Industrial Research (CSIR)-North East Institute of Science and Technology, Jorhat, Assam 785006, 2Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201002, 3Department of Chemistry, Assam University, Silchar, Assam 788011, India

*Corresponding Author:
Dipanwita banik
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh 201002, India
E-mail: dipanwitabanik@neist.res.in
Date of Submission 16 March 2021
Date of Revision 20 September 2021
Date of Acceptance 05 August 2022
Indian J Pharm Sci 2022;84(4):1051-1062  

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DOI: 10.36468/pharmaceutical-sciences.999

Abstract

Antimicrobial resistance against Mycobacterium and Candida are vital problems in immunocompromised patients. The current study was aimed to investigate the antimicrobial efficacy of fifteen folk medicinal plants found in Assam. Ethanolic plant extracts by well diffusion assay showed antimicrobial activity of 7 species against Mycobacterium smegmatis (ATCC®607TM) and 9 species against Candida albicans (ATCC®90028TM). Nepenthes khasiana exhibited highest zone of inhibition 21±0.5 mm against Mycobacterium smegmatis and this is the first report of Nepenthes khasiana showing anti-Mycobacterium activity from North East India. Mesua ferrea leaf extract first time exhibited good anti-candidal activity than the other studied species with zone of inhibition 11.83±0.76 mm. The impact of morphological characters on mechanistic convergence of anti-Mycobacterium and anti-Candida activities among the studied species was also assessed. Approximately 27 discrete morphological characters were used to prepare data matrix. Majority rule consensus tree of morphological data matrix with branch support ≥50 % using Mesquite 3.61 found that nearly 66.67 % species exhibited mechanistic convergence of anti-Mycobacterium and anti-Candida activity in combination congruent to morphological clustering. However, the clades viz., rosids, Murraya, Crinum and Alpinia showed partial congruence with angiosperm phylogeny group IV classification. A larger dataset including more than one representative species of each genus of the studied family and use of extensive morphological characters may exhibit a better mechanistic convergence.

Keywords

Medicinal plants, morphological data matrix, mechanistic convergence, antimicrobial, Mycobacterium, Candida

Assam is covered with dense forests and catchment areas along the river Brahmaputra which nurture and sustain natural wealth of the state. The region is gifted with rich floral diversity due to the presence of favorable climatic condition, diversified physiography and absolute geographical location. Among the floral community medicinally and economically important flora are found in wild and home gardens. Plants with medicinal properties are utilized by various indigenous community for their primary health related problems[1-3].

Antimicrobial resistance has been playing a major concern all over the world. Diseases caused by drug resistant microbes failed to recover as the standard drugs did not work on respective pathogens. This increases the risk of patients to survive, sometimes lead to prolonged illness followed by excessive healthcare cost. The genus Mycobacterium has got prioritization as drug resistant bacteriumcausing Tuberculosis (TB) all over the world for which new treatments are urgently needed[4]. Recently, Non TB Mycobacteria (NTM) has been gaining importance as they are found to cause some respiratory problems in immunocompromised patients[5]. Moreover, Human Immunodeficiency Virus (HIV) victims are more vulnerable to the infections caused by NTM. Fast growingNTM are responsible for causing infections of joint, skin, soft tissue and lymph node etc.[6]. Further, Candida albicans (C. albicans) is an opportunistic pathogen commonly found in genitourinary and gastrointestinal tract[7]. Generally causes infections on skin, vagina, mouth etc. Hospitalized patients with weak immune system are prone to resistant Candida infections. In immunocompetent patients (undergoing anticancer and HIV treatments) Candida can invade into bloodstream and infects internal organ system. About 7 % bloodstream infections are severe because they are resistant to available drugs[8].

On the basis of traditional knowledge and medicinal properties we have selected fifteen plants of Assam commonly growing in home gardens including cultivated one for anti- Mycobacterium and anti-candidal activity study. Further, morphological characters of each species were studied to prepare morphological character-based data matrix to analyses the mechanistic convergence of anti-Mycobacterium and anti-candidal activity of the studied plants.

Materials and Methods

Collection of plant material:

15 species were selected based on folk medicinal use with prospective antimicrobial activity vide published literature (Table 1)[9-40]. All the species were collected from adjoining areas of Council of Scientific and Industrial Research (CSIR)-North East Institute of Science and Technology (NEIST), Jorhat and Golaghat (Assam) with vegetative and reproductive parts (fig. 1 and fig. 2) and the field collection notes viz., name of the species, family, location, date of collection, collector’s name and number were recorded (Table 2). Herbarium sheets were deposited at the herbarium of CSIR-NEIST, Jorhat.

Scientific names Name of the user tribe/country Plant parts used Health ailments/Indications of use References
A. wilkesiana Mull. Arg. Nigeria, Mauritius Le Asthma, skin infections, antibacterial activity against E. coli, S. aureus, Klebsiella pneumoniae, antifungal activity and others [25-27]
A. nigra (Gaertn.) Burtt Assam, Manipur, Tripura Le, shoo, see, rhi Infections and others. Alpiniasp. is reported with anti-bronchitis, antibacterial and antifungal activity. [1], [28-30]
A. ficoidea (L.) P. Beauv. Lodha tribe in West Bengal, Pakistan w. pl. Herbal tribal medicine for asthma, cough, diarrhea and others. [31,32]
C. maxima (Burm.) Merr. Traditional Medicine, Asia, SE Asia, America Fr, le Cold, inflammation, leprosy, respiratory ailments, antifungal and antibacterial activity against Salmonella typhimurium and E. coliand others. [33,34]
C. cujete L. Mayan healers, Himalayan, peninsular India Ba, le, fr pulp, see, Fr decoction Cold, respiratory trouble, bronchitis, cough, asthma, activity against M. smegmatis, Mycobacterium tuberculosis (multi drug resistant isolate), Mycobacterium fortuitum and others. [35-38]
C. asiaticum L. Traditional Medicine, South Pacific islands, Asia Le, bulbs Infections, antimicrobial activity against E. coli, Klebsiella pneumoniae, P. aeruginosa, S. aureus and others. [39-41]
K. erratica (Hook. F. & Thomson) J. Sinclair India La Oral problems. [13]
M. ferrea L. Chirang Reserve, Assam, Meetei, Manipur Flw, le, see isolated coumarins Respiratory, dermal infection, anti-Mycobacteriumactivity, anti-Candida albicans activity and others. [24], [42-44]
M. micrantha Kunth Chorei tribe, (Assam) Malaysia Young le, W. pl. Respiratory problems, ulcers, itches, rashes on skin, healing wounds, antimicrobial activity viz., against E. coli, S. aureus, P. aeruginosa, Salmonella typhi, Streptococcus pneumoniae, Mycobacterium sp. and others. [1], [45,46]
M. koenigii (L.) Spreng. Ayurvedic use, Apatani (Arunachal Pradesh) Le, v. oil Inflammation, fungal infection, anti-M. smegmatis mc2155 activity, anti-Candida activity [12], [47-49]
M. paniculata (L.) Jack Thai Le Anti-Mycobacterium activity, anti-AIDS activity, anti-C. albicans activity [24], [48,50]
N. Khasiana Hook.f. Khasi, Garo tribes (Meghalaya, NE India) Pit., fluid of unopened pit. Leprosy and other ailments. [51]
O. gratissimum L. India, Malaysia, Africa Le v. oil Antimicrobial activity against common bacterial and fungal pathogens causing respiratory, dermal infections and others. [52]
O. corniculata L. Apatani, North Tripura, Bangladesh Shoo, le Dermal infection, antimicrobial activity against S. aureus, Salmonella typhiand others. [35], [47], [53]
P. guajava L. Khasi, Jayantia, Garo tribes, Tropical America Ba, le, fr Infections, wounds, inflammation, antibacterial activity and others. Leaf nano particle has anti-mycobacterial activity. [54-56]

Table 1: Folk Medicinal uses of 15 Studied Species

S. no. Names of the species Collection localities Voucher no.
1 A. wilkesiana Mull.Arg. CSIR-NEIST, Jorhat campus S. Saikia 1608
2 A. nigra (Gaertn.) Burtt CSIR-NEIST, Jorhat campus S. Saikia 1610
3 Alternanthera ficoidea (L.) P. Beauv. CSIR-NEIST, Jorhat campus S. Saikia 1838
4 C. maxima (Burm.) Merr. CSIR-NEIST, Jorhat campus S. Saikia 1612
5 C. cujete L. Lohpohia, Jorhat S. Saikia & D. Banik 1846
6 C. asiaticum L. CSIR-NEIST, Jorhat campus S. Saikia 1609
7 K. erratica (Hook.f. and Thomson) J. Sinclair Deoparbat, Golaghat, Assam S. Saikia and J. Saikia 1320
8 M. ferrea L. CSIR-NEIST, Jorhat campus S. Saikia 1611
9 M. micrantha Kunth Tezpur S. Saikia1831
10 M. koenigii (L.) Spreng. CSIR-NEIST, Jorhat campus S. Saikia 1837
11 M. paniculata (L.) Jack CSIR-NEIST, Jorhat campus S. Saikia 1830
12 N. khasiana Hook.f. CSIR-NEIST, Jorhat campus S. Saikia 1834
13 O. gratissimum L. CSIR-NEIST, Jorhat campus S. Saikia 1836
14 O. corniculata L. CSIR-NEIST, Jorhat campus S. Saikia 1833
15 P. guajava L. CSIR-NEIST, Jorhat campus S. Saikia 1832

Table 2: Collected Plant Specimens

IJPS-vegetative

Fig. 1: Vegetative and reproductive parts of A=P. guajava; B=C. asiaticum; C=M. paniculata; D=N. khasiana; E=C. maxima; F=A. nigra; G=A. wilkesiana and H=K. erratica

IJPS-micrantha

Fig. 2: Vegetative and reproductive parts of M. micrantha; J=A. ficoidea; K=C. cujete; L= M. ferrea; M=M. koenigii; N=O. corniculata and O=O. gratissimum

Study of macro and microscopic characters:

The morphological characters of the species were critically examined from live specimens and measurement was recorded in metric scale. Magnus-TZ trinocular stereo zoom microscope was used to study micro-morphological characters. Identification of all the specimens was determined by consulting taxonomic literature like national and regional flora, protologue, revisionary work[41-45] and by consulting authentic specimens deposited in the herbarium of Eastern Circle Botanical Survey of India, Shillong (Assam) and several other herbaria available online viz., royal botanic gardens, the natural history museum, royal botanic garden Edinburgh, the New York botanical garden, Conservatoire et Jardin botaniques de la Ville de Geneve. The acronyms of the herbaria were used vide Index Herbariorum[46,47]. The species names were verified vide viz., The Plant List (www.theplantlist.org), International Plant Name Index (www.ipni.org), Plants of the World Online, Kew Science (http://www.plantsoftheworldonline.org/) and JSTOR (www.plants.jstor.org).

Morphological data matrix and cluster analysis:

Morphological data matrix of 15 folk medicinal plant species was prepared using 27 discrete characters viz., 4 binary and 23 multistate characters where for quantitative characters the mean value of natural range was considered to assign the character state (Table 3). Michelia champaca (M. champaca)from Magnoliaceae was used as out group in the study. Altogether 432 data points were prepared including out group following standard procedure[48,49]. The morphological cluster analysis was conducted through reconstructing majority rule consensus tree where 174 most parsimonious trees were constructed using the software Mesquite 3.61 (build 927) to visualise the cladogram[50]. Mesquite’s heuristic search resulted 174 equally good trees based on tree length. For rearrangements of the trees, Sub-tree Pruning and Re-grafting (SPR) method was considered. The majority rule consensus tree was formed with branch support ≥50 % where the branches with frequency less than 0.5 were allowed to get collapsed. The Consistency Index and Retention Index (CI and RI) and parsimony tree length were obtained from tree analysis while characters were treated as un-weighted and unordered.

S. No. Character state number with names of diagnostic morphological characters
1 Habit: 0, Herb; 1, Rhizomatous herb, bulbous herb; 2, Climber, creeper; 3, Subshrub, undershrub; 4, Shrub; 5, tree.
2 Roots: 0, Tap root; 1, Adventitious root; 2, Tap and adventitious root.
3 Twig indumentums: 0, Glabrous; 1, Puberulous; 2, Velutinous; 3, Villous; 4, Strigose; 5, Stellate.
4 Leaf petiole: 0, Sessile; 1, Pseudo petiolate; 2, Petiolate; 3, Wing petiolate.
5 Petiole indumentum: 0, Glabrous; 1, Slightly pubescent; 2, Pubescent.
6 Petiole size: 0, 0-0.35 cm; 1, 0.7-1.65 cm; 2, 2.25 cm; 3, 4.55-9.25 cm; 4, 0.7 cm; 5, 5.3 cm.
7 Leaf lamina shape: 0, Ovate; 1, Cordate; 2, Oblong; 3, Elliptic; 4, Lanceolate; 5, Obcordate; 6, Spathulate.
8 Leaf arrangement: 0, Alternate; 1, Alternately fascicled with tubercle; 2, Opposite; 3, Opposite decussate; 4, Radical.
9 Leaf apex: 0, Acute; 1, Acuminate; 2, Acute to acuminate; 3, Cuneate; 4, Cuspidate; 5, Caudate; 6, Obtuse; 7, Emerginate; 8, Acuminate with pitcher.
10 Leaf lamina margin: 0, Entire; 1, Crenulate; 2, Serrate; 3, Dentate.
11 Leaf lamina size (Length): 0, 0.85 cm; 1, 2.25-7.45 cm; 2, 12.0-13.2 cm; 3, 14.5-15.75 cm; 4, 23.5 cm; 5, 35.0 cm; 6, 49.5 cm; 7, 80.0 cm.
12 Leaf lamina size (Width): 0, 0.85 cm; 1, 2.15-2.45 cm; 2, 3.0-3.75 cm; 3, 4-5.0 cm; 4, 6-7.0 cm; 5, 9.6 cm; 6, 12.0-12.5 cm.
13 Inflorescence: 0,Solitary; 1, Solitary cauline; 2, Raceme; 3, Panicle; 4, Spike; 5, Corymb, corymbose panicle; 6, Umbel, cymose umbel; 7, Head; 8, Verticillaster; 9, Tubercle.
14 Colour of flower: 0, White; 1, Greenish white; 2, Pink; 3, Reddish; 4, Greenish red; 5, Yellow; 6, Brown.
15 Fragrance of flower: 0, Absent; 1, Present.
16 Flower sex:0, Bisexual; 1, Unisexual.
17 Filament no.:0, 5; 1, 10; 2, 13-13.5; 3, 20, 7.5; 4, 4; 5, 6; 6, 1; Many, 7.
18 Anther shape: 0, Linear; 1, Ellipsoid; 2, Reniform; 3, Vermiform; 4, Saggitate; 5, Appendiculate; 6, Linear curved; 7, Rotundus; 8, Ecrestate.
19 Stigma shape: 0, Capitate; 1, Truncate; 2, Discoid; 3, Peltate; 4, Laciniate; 5, Bifid; 6, Bilipped each ellipsoid; 7, Lobed and lobulate.
20 Style length: 0, 0-0.5 mm; 1, 3.5-5 mm; 2, 6-6.5 mm; 3, 8-13 mm; 4, 32 mm; 5, 46 mm; 6, 95 mm.
21 Ovary position: 0, Hypogynous; 1, Epigynous.
22 Fruit type: 0, Drupe; 1, Capsule; 2, Berry; 3, Hesperidium; 4, Utricle; 5, Carcerulus; 6, Achenes.
23 Fruit stalk length: 0, Sessile; 1, 1.5 mm; 2, 5.5-7.5 mm; 3, 9 mm; 4, 12 mm; 5, 15 mm; 6, 60 mm.
24 Fruit indumentums: 0, Glabrous; 1, Puberulous; 2, Sparsely pubescent; 3, Valutinous; 4, Hispid.
25 Seed shape: 0, Globose, rotundus, subglobose; 1, Triangular transversely globose; 2, Ovoid; 3, Obovoid; 4, Linear obovoid; 5, 3-4 tri- tetragonal; 6, Spindle shaped, winged; 7, Irregularly angular; 8, Unequally pyriform.
26 Leaf type: 0, Simple; 1, Unifoliate compound; 2, Trifoliate compound; 3, Pinnately compound.
27 Androecium character: 0, Ployandrous (stamens many & free); 1, Stamens in 2 whorls; 2, Stamens united at base; 3, Stamens epiphyllous in 2 whorls, stamens in 2 whorls united at base; 4, Monadelphous; 5, Polyadelphous; 6, Didynamous, epipetalous; 7, One stamen fertile rest modified into petaloid staminodes; 8, Syngenesious; 9, Filaments modified as columnar disc, stamens connate.

Table 3: Character States of diagnostic morphological characters of studied plants

Solvent extraction of plant material and study of antimicrobial activity:

The collected species and plant parts as listed in Table 2 were shade dried, powdered in a Willy Mill and macerated in ethanol (95 %) for 48 h. Filtrate was taken through Whatman no. 1 filter paper and was evaporated at 40° under reduced pressure using rotary evaporator (Buchi, Switzerland). The concentrated extracts were completely dried in lyophilizer (Delvec pumps Pvt. Ltd., India) at -50°. Strains of Mycobacterium smegmatis (M. smegmatis)(ATCC®607TM) and C. albicans (ATCC®90028TM) were procured from HiMedia. M. smegmatis was cultured on sterile brain heart infusion agar and broth media and C. albicans was cultured on yeast malt agar and potato dextrose broth media. Sterile Mueller Hinton Agar (MHA) media was poured on petriplates to solidify and inoculum (ca.1×108 Colony Forming Unit (CFU)/ml) was spread on it. CFU concentration was determined comparing with McFarland solution (absorbance 0.12±0.003 at 625 nm)[51]. Well diffusion assay was used to evaluate antimicrobial activity[52]. Dimethyl Sulfoxide (DMSO) was used to prepare stock solution of the ethanol extract and was used as negative control. Isoniazid (1.5 mg/ml) was used as positive control for M. smegmatis and fluconazole (60 µg/ml) for

C. albicans. Petriplates were incubated at 37° for M. smegmatis and at 30° for C. albicans. Experiment was carried out in triplicates (n=3), Zone of Inhibition (ZOI) was measured using zone scale (HIMEDIA), data were presented as mean±Standard Deviation (SD) and statistical analysis was carried out in Microsoft Excel 2007.

Results and Discussion

The reconstructed consensus tree obtained with Mesquite 3.61 (Build 927) exhibited the tree length 199, CI 0.73869347 and RI 0.43478261. The tree showed monophyletic and paraphyletic group in two distinct clades including all the 15 species (fig. 3). Psidium guajava (P. guajava), Mesua ferrea (M. ferrea) and Citrus maxima (C. maxima) showed polytomy. According to recent classification of Angiosperm Phylogeny Group IV (APG IV), Mesua, Psidium and Citrus belong to the orders Malpighiales, Myrtales and Sapindales respectively and are found in the same clade rosids[53] which is congruent in the morphological matrix-based cladogram. M. ferrea, P. guajava and C. maxima share certain common characters viz., tree with tap root system, white flowers, fragrant and bisexual, leaf lamina entire and fruit glabrous. The subclade Mikania and Ocimum share 8 common morphological characters viz., simple leaf, lamina dentate, flower white, fragrant, bisexual, stigma bifid, average style length 6-6.5 mm and glabrous fruit indumentum and the subclade is supported with frequency 0.93. Crescentia clade is supported with frequency 0.57 and includes the subclade of Ocimum+Mikania. The other clade supported with a frequency 0.65 comprises 3 subclades viz., Alternanthera+Oxalis with subclade frequency 0.93, Alpinia+Crinum with subclade frequency 0.93 and Murraya subclade with frequency 0.93. Knema, Acalypha and Nepenthes with frequency 0.58, 0.51 and 0.58 exhibited paraphyly to the same. In APG IV classification, Oxalis and Alternanthera belong to two distinct orders viz., Oxalidales and Caryophyllales, but in the present morphological matrix-based cluster analysis they appeared within the same clade with frequency 0.93. The subclade with Oxalis corniculata (O. corniculata) and Alternanthera ficoidea (A. ficoidea) shares the common characters viz., herbaceous habit with adventitious roots, velutinous twig and entire lamina, bisexual flower with 10 filaments, hypogenous ovary and ovoid seeds. However, phylogenetically, the taxa Crinum and Alpinia belong to Asparagales and Zingiberales respectively and are very close to each other[53] which is congruent to morphological cluster analysis. Similarly, the genus Murraya with two species Murraya paniculata (M. paniculata)and M. koenigii appeared within the same subclade. M. champaca used in the study as out group was supported with frequency 1.00 in the majority rule consensus tree. Thus, the morphological cluster analysis could discriminate the identity of the genus Murraya, clustering of Crinum and Alpinia through morphological characters. However, a larger morphological matrix with more numbers of taxa i.e., including more than one species from each genus of the studied plant families as well as extensive use of morphological characters may help to better understand the congruence with APG IV system of classification[53].

IJPS-numerical

Fig. 3: Cluster visualization in Mesquite 3.61 (build 927) for 15 plant species by morphological data matrix. The numerical values on the colored branches represent the values of branch support (≥50). Studied plants are grouped into clades which are presented by different colors. M. champaca is considered as out group with branch support 100 %. The name of species colored red are both M. smegmatis and C. albicans positive, species name with dark blue colour are only M. smegmatis positive, species name with pink color are only C. albicans positive and species showed no activity are colored orange in the circular tree

The genera studied in the experiment have been reported with folk medicinal use. Ethanolic extracts of lesser explored plant parts of 15 species were tested against M. smegmatis and C. albicans. Among the 15 species viz., M. koenigii, M. ferrea, Alpinia nigra, (A. nigra) A. ficoidea and Acalypha wilkesiana (A. wilkesiana) have showed activity against both the pathogens. On the other hand, Ocimum gratissimum (O. gratissimum), Knema erratica (K. erratica), C. maxima and Crescentia cujete (C. cujete) didn’t show any activity. The study confirmed the anti-Mycobacterium activity of 7 species and anti-Candida activity of 9 species out of 15 studied species (Table 4). Among the tested plant species tender shoot with leaves of Nepenthes khasiana (N. khasiana) exhibited highest ZOI against M. smegmatis and extracts of M. koenigii, M. ferrea and Crinum asiaticum (C. asiaticum) exhibited good antifungal activity against C. albicans. Earlier reports showed that Acalypha indica and Alpinia galanga possess anti-TB activity[54,55] and seed extracts of M. ferrea was inactive against C. albicans[56]. In this experiment, the plants N. khasiana, A. nigra, A. ficoidea and A. wilkesiana are reported first time with anti-M. smegmatis activity and M. ferrea leaf extract with anti-C. albicans activity. ZOI study exhibited that the anti-Mycobacterium activity was stronger than anti-Candida activity in the studied species (Table 4). As antimicrobial activity is commonly studied against Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) the antimicrobial activity of these 15 species were also reviewed against E. coli and S. aureus where A. wilkesiana, C. maxima, A. nigra and P. guajavashowed promising activity i.e., inhibition zone 17.00±0.00-25.10±0.2 mm against E. coli and 22.00±0.23-30.0±0.1 mm against S. aureus (Table 5)[57-71]. Similarly, the bioactive molecules and chemical constituents of these species were also reviewed where Saikia et al., 2020 showed abundance of (-)-Isoledene, (±)-Debromofliformin, δ-Cadinene, t-Caryophyllene in the floral essential oil of Mikania micrantha (M. micrantha) which exhibited cytotoxicity with IC50 <6 µg/ml against HeLa and <11 µg/ml against PA1 cell lines and antimicrobial activity with ZOI approximately 10-18 mm against Pseudomonas aeruginosa(P. aeruginosa), C. albicans, S. aureus and M. smegmatis (Table 6)[72-85]. Therefore, after the experimental validation of traditional use of these 15 folk medicinal species, further extensive studies are required to investigate the efficacy of these medicinal plants along with the bioactive molecules for the translational use and development of antibacterial and antifungal treatments to replace the resistant drugs.

S. no. Name of plants Plant parts used for ethanol extraction M. smegmatis C. albicans
(ZOI in mm) (ZOI in mm)
1
  1. wilkesiana
 Leaves 13.66±0.57 9.66±0.57
2 A. nigra Leaves 15.83±0.28 9.83± 0.28
3 A. ficoidea Whole plant with flower 10.33±0.57 10±0
4 C. maxima Fruit pulp -- --
5 C. cujete Fruit pulp -- --
6 C. asiaticum Pseudo shoot -- 11.16±0.28
7 K. erratica Leaves -- --
8 M. ferrea Leaves 10.83±0.28 11.83±0.76
9 M.micrantha Leaves and stem -- 10±0
10 M. koenigii Leaves 14.16± 0.28 11.83±0.28
11 M. paniculata Leaves -- 10.33±0.57
12 N. khasiana Tendershoot with leaves 21±0.5 --
13 O. gratissimum Leaves andshoot -- --
14 O. corniculata Whole plant -- 10.33±0.28
15 P. guajava Leaves 11.33±0.57 --
  Fluconazole NA 15.83±0.28
  Isoniazid 21.76±0.25 NA

Table 4: antimicrobial activity of the 15 plant species against m. Smegmatis and c. Albicans

S. no. Name of plants Plant parts used for solvent extraction E. coli S. aureus References
(ZOI in mm) (ZOI in mm)
1 A. wilkesiana Leaves (EtOH) 25.10±0.2 30.0±0.1 [57]
2 A. nigra Leaves (MeOH) 18.25±0.68 22.00±0.23 [58]
3 A. ficoidea Whole plant (Aqueous) -- n/r [59]
4 C. maxima Fruit pulp and seed (EtOH) 22 30 [60]
5 C. cujete Fruit pulp (EtOH) -- 3.75 [61]
6 C. asiaticum Bulb (EtOH) 11.8 7.1 [62]
7 K. erratica Leaves n/r n/r
8 M. ferrea Leaves (EtOH) 17.5±0.5 17.0±0.5 [63]
9 M.micrantha Leaves (EtOH) 8.01±0.01 9.33±0.01 [64]
10 M. koenigii Leaves (EtOH) 22.3 10.5 [65]
11 M. paniculata Leaves (EtOH) -- -- [66]
12 N. khasiana AuNP Leaves (Aqueous) 8 n/r [67]
13 O. gratissimum Leaves (EtOH) 5.00±0.09 8.00±0.11 [68]
14 O. corniculata Whole plant (EtOH) 14±1.64 n/r [69]
15 O. corniculata Whole plant (MeOH) 11±1.31 8.07±0.49 [70]
16 P. guajava Leaves 17.00±0.00 26.00±0.00 [71]

Table 5: antimicrobial activity of the 15 plant species reported against e. Coli and s. Aureus

S. no. Name of plants Marker chemical compounds References
1 A. wilkesiana Ethyl gallate, pyrogallol [72]
2 A. nigra β-caryophyllene, α-pinene, β-pinene [73]
3 A. ficoidea 3,7,11,15-tetramethyl-2-hexadecen-1-ol, 3-ethoxy-1,1,1,7,7,7-hexamethyl-3,5,5-tris, 9,12,15-octadecatrienoic acid, methyl ester [74]
4 C. maxima β-sitosterol, marmin, naringenin, naringin [75]
5 C. cujete Naphthoquinones [76]
6 C. asiaticum Lycoriside, pahnilycorine, [77]
7 K. erratica n/r
8 M. ferrea α-Humulene, β-caryophyllene oxide, t-caryophyllene [78]
9 M.micrantha (-)-Isoledene, (±)-Debromofliformin, δ-Cadinene, t-caryophyllene [79]
10 M. koenigii 1-Methyl-pyrrolidine-2-carboxylic acid, Ethyl à-d-glucopyranoside [80]
11 M. paniculata β-Caryophyllene, (E,E)-Geranyl linalool, isospathulenol, methyl palmitate [81]
12 N. khasiana 5-O-methyl droserone, droserone, naphthoquinones, plumbagin [82]
13 O. gratissimum Eugenol, germacrene D, terpinolene [83]
14 O. corniculata 5-Hydroxy-6,7,8,4’-tetramethoxyflavone, 5,7,4’ -Trihydroxy-6,8-dimethoxyflavone [84]
15 P. guajava β-sitosterol, guajanoic acid, oleanolic acid, ursolic acid, uvaol [85]

Table 6: Some Reported Marker Chemical Compounds of the 15 Plant Species

The consensus tree generated by Mesquite 3.61 (Build 927) using morphological data matrix of 15 folk medicinal species was used to visualize the mechanistic convergence of antimicrobial activity where anti-Mycobacterium and anti-Candida activities were manually mapped on the consensus tree (fig. 3). The study showed that the clade I comprising 3 subclades viz., Alternanthera+Oxalis, Alpinia+Crinum and Murraya sub-clade exhibited mechanistic convergence of anti-Mycobacterium and anti-Candida activities where A. ficoidea, A. nigra and M. koenigii exhibited activity against both M. smegmatis and C. albicans and their adjacent members in the respective subclades exhibited activity against C. albicans. Further, immediate adjacent paraphyly exhibited to the clade I, by A. wilkesiana with activity against both the test organisms and N. khasiana with activity only against M. smegmatis. Thus, mechanistic convergence of antimicrobial activity was found congruent in clade I. In clade II, M. ferrea exhibited activity against both M. smegmatis and C. albicans whereas, P. guajavaonly against M. smegmatis, though the other clade member C. maxima did not show any activity. The clade III comprising C. cujete, O. gratissimum and M. micrantha did not exhibit any activity against M. smegmatis and only M. micrantha exhibited anti-Candida activity. Thus, nearly 66.67 % (out of 15 species nearly 10) species exhibited mechanistic convergence of antimicrobial activity against both M. smegmatis and C. albicans in combination as well as individually which are in congruence with their morphological clustering in above 3 clades. Absence of activity in C. cujete and O. gratissimum was also observed in the clade III. Thus, none of the clades reflected mechanistic convergence of individual antimicrobial activity of M. smegmatis and C. albicans but mechanistic convergence of activity was exhibited in combination against both M. smegmatis and C. albicans in clade I, partially in clade II and also through paraphyly by A. wilkesiana and N. khasiana to clade I irrespective of their status as per APG IV. However, a larger dataset with more than one representative species from each genus of studied plant families using extensive morphological characters may have reflected better mechanistic convergence.

The cladogram reconstructed from morphological matrix did not show congruence in clade support in respect to their individual antimicrobial activity. Phylogenetic analysis was conducted with morphological data matrix of more than 35 taxa including more than 115 characters to assign the generic circumscription of the genus Bromelia of the subfamily Bromelioideae and showed critical insights on paraphyly of the genus and the evolution among the subfamily[48]. Likewise, the current study exhibited the impact of morphological characters of 15 folk medicinal species in morphological data matrix-based phylogeny. Morphological clustering through reconstructing majority rule consensus tree using Mesquite 3.61 (build 927) with branch support ≥50 % showed congruence with APG IV for rosids, the clade comprising Mesua, Psidium and Citrus of Malpighiales, Myrtales and Sapindales, clustering of Murraya, Crinum and Alpinia.

Among the studied 15 folk medicinal plants 7 species exhibited activity against M. smegmatis and 9 species against C. albicans. Moreover, this is the first report of N. khasiana with prominent anti-Mycobacterium activity along with 3 more species. Among the studied plant species M. ferrea leaf extract showed best activity against C. albicans. The study validated the traditional uses of a few plants as antimicrobial agents and will initiate further translational research on anti-Mycobacterium and anti-Candida activity of folk medicinal plants used among cross cultural ethnic tribes. Further, the morphological matrix partly showed mechanistic convergence of anti-Mycobacterium and anti-candidal activity in combination among 66.67 % of the studied species. A larger dataset with more than one representative species from each genus of the studied plant families and use of extensive morphological characters may exhibit a better understanding of mechanistic convergence of anti-Mycobacterium and anti-Candida activity.

Acknowledgements:

We thank the Director, CSIR-North East Institute of Science and Technology, Jorhat, Assam 785006 (NMN 202142) for providing laboratory facilities to smoothly carry out the experiment.

Conflict of interests:

The authors declared no conflict of interests.

References