INTRODUCTION:

Bryophytes, the non-tracheophytes are comparatively less explored for taxonomy and phytochemical constituents, at the same time shares significant amount in non-flowering plant diversity and possess greater range of biologically active compounds such as polyphenols, terpenoids, organic acids, sugar, alcohols, fatty acids, aliphatic compounds, acetogenins, phenylquinones, and aromatic and phenolic substances^15,4. Among the bryophytes, Marchantiophyta (Liverworts) are the rich sources of lipophilic terpenoides and aromatic compounds with biological properties such as antifungal, anti-microbial, antiviral, antioxidant, muscle relaxant, cytotoxic, antitumor and insecticidal activities^2,3,5,18.

The liverworts generally preferring shady moist habitats and are most common in moist tropical areas¹⁶.

The Western Ghats, India is one of the biological hotspots in tropics. During conservation biological studies of Atuna indica (Chrysobalanaceae), a Critically Endangered tree of Southern Western Ghats, located at Malabar Wildlife Sanctuary, Kerala, the first author came to experience an unusual, pleasant and nice smell from the leaves of the A. indica. The leaves of the species was collected and specimen was identified as Leptolejeunea balansae Steph. of the family Lejeuneaeae (Fig. 1). Daniels and Daniel and Pocs et al. has reported L. balansae from the Western Ghats. Leptolejeunea is a pan-tropical genus characterized by minute size and deeply bilobed under leaves with dentate margins. L. balansae nearly an epiphyllous leafy liverwort, with sparse distribution in Western Ghats⁶. Several species produce monoterpenes that emit fragrance⁹. Toyota et al. reported pleasant smell from L. elliptica, an allied species.

Figure 1: Leptolejeunea balansae on Atuna indica leaf.

The GC-MS analysis of L. balansae assumed to have the same phytochemical constituent as reported in L. elliptica which is responsible for pleasant odor. Antimicrobial activity of the species was also analyzed.

MATERIALS AND METHODS:

Plant Collection and Sample Preparation:

The plant material was collected from the Malabar Wildlife Sanctuary located along the Kerala part of the Western Ghats, South India during October, 2016. The collected sample was subjected to cold extraction. About 5g of sample was soaked in 30ml of chloroform for about one week. Then the filtered extract was poured into a petridish and air dried to get a solvent free sample. Dried sample was then weighed and redissolved in chloroform (1mg/ml) for GC-MS analysis.

Taxonomic description:

Leptolejeunea balansae Steph:

(Hedwigia 35: 105. 1896):

Leptolejeunea balansae Steph. is characterized by obovate, slipper-shaped leaves, 2-3 ocelli with large basal ocellus, apex acute, margin entire, oblong-ovate to rectangular leaf lobule, underleaves with 3-4 celled lobes with wide sinus, plants dioecious, androecia with 3-6 pairs of bract, perianth with five keels and margin of the dorsal keel with 2-3 projecting rounded cells.

Habitat:

Epiphyllous on Atuna indica (Bedd.) Kosterm. (Chrysobalanaceae) (fig.1); Evergreen forests

Specimen examined:

Kerala, Kozhikode district, Malabar Wildlife Sanctuary (11° 33'13.48" N, 75° 55' 14.10"E; 756 m asl.)

Table 1: Phytocomponents identified in extracts of L. balansae by GC-MS

RT	Name of compound	Area (%)	Important activity
6.37	Anisole, p-ethyl-	78.45	Anisole is a precursor to perfumes, insect pheromones, and pharmaceuticals.
7.06	Phenol, p-ethyl-	0.53	Food additives (Flavoring Agents)
7.46	Nonylcyclopropane	0.14	-
8.47	4-Ethylphenyl acetate	2.34	-
10.29	1-Tetradecene	2.15	1-Tetradecene is used as a specialty solvent in perfumes, flavors, medicines, dyes, oils and resins.
11.15	1,5-Cyclooctadiene, 3-t-butyl-	0.36	-
11.28	Delta-guajen (alpha-bulnesen)	0.55	-
11.62	(-)-Germacrene d	0.57	Antimicrobial ¹² and insecticidal ¹³ properties, though they also play a role as insect pheromones.
11.80	1,1-dimethylethyl	1.92
12.83	1-Hexadecene	2.29	As a surfactant in lubricating fluid, a drilling fluid in the boring and drilling industry, and in paper sizing.
12.92	Pentadecane	0.15	Treatment of Plantar Keratosis with Medicinal Plant in Diabetic Patients. Flavouring agent, Antibacterial ¹⁷
14.87	1-methylethenyl	0.63	-
15.10	1-Octadecene	2.21	Antifungal agent for plant and human pathogens ¹⁸
15.18	Heptadecane	0.14	-
15.56	Neophytadiene	0.45	-
15.82	E, E-2,13-Octadecadien-1-ol	0.12	Macroscelesia japona Female pheromone ¹⁹
16.02	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	0.15	-
16.36	beta-Springene	0.13	-
17.16	1-Heneicosanol	2.53	-
17.64	Gamolenic Acid	0.4	Medication against breast pain and eczema
18.27	T-phytol	0.68	Antimicrobial, anticancer, anti inflammatory
18.78	Ethyl 9.alpha.-linolenate	0.6	It has a role as a human metabolite and a plant metabolite. It derives from an alpha-linolenic acid.
19.96	4,8,13-Duvatriene-1,3-Diol	0.12	also seen in tobacco rhizome
20.38	O o'-biphenol, 4,4',6,6'-tetra-t-butyl-	0.19	Causes damage to organs through prolonged or repeated exposure
20.82	n-Tetracosanol-1	0.69	It has a role as a plant metabolite.
23.13	Eicosyltrifluoroacetate	0.35	-
34.30	Celidoniol, deoxy-	1.17	Antibacterial ¹⁴ Anti inflammatary and Anticancer ¹⁵ Pheremone of Orgyia leucostigma ¹⁶

** Duke’s phytochemical and ethno-botanical database and other internet resources.

Figure 2: Chromatogram of Leptolejeunea balansae by GC-MS. a- Anisole, p-ethyl-, b- 1-Hexadecene, c- 1-Octadecene, d- 1-Heneicosanol

Gas chromatography-Mass spectrometry (GC-MS):

The chemical composition of the extracted sample in chloroform was analyzed using a Shimadzu QP-2010 GC–MS equipped with Rxi-5Sil MS capillary column of 30m in length, 0.25mm in internal diameter, and 0.25μm in thickness. Helium (99.9995%) was used as the carrier gas, at a constant flow rate of 1mL/min. Injection port temperature was adjusted at 260°C and the injection was performed in split less mode with a split ratio of 100. A 1μl of the syringe filtered (0.22μm) sample was analyzed with the column held initially at 80°C for 2 min and then increased at the rate of 10°C/min to 260°C, which is held constant for 10 min. At the end of this period, the oven temperature was raised up to 280°C, at the rate of an increase of 5^°C/min, and maintained for 6 min. For GC–MS spectral detection, EI mode was adopted at 70 eV with 0.5 sec of scan time and fragments were recorded in the 50 to 500 m/z range. The ion source temperature was maintained at 200°C. The components of the sample were identified by comparing the retention time of chromatographic peaks with NIST and Wiley mass spectra libraries.

Antimicrobial activity:

Bryophyte extract was analyzed for the antimicrobial activity via disc diffusion method. Bacterial cultures namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus and Bacillus subtilis were obtained from KFRI microbial culture repository. Different concentrations viz., 5, 10, 15, 20µl of the extracts were pre-inoculated with the sterile discs for 24 hrs so as to achieve the desired concentrations. Nutrient Agar (NA) medium was prepared and the 48 hrs old bacterial cultures were swabbed. Sterile discs with desired concentrations were placed at an equidistant and were incubated at 37°C for 48 hrs in an incubator for the observation of any activity.

RESULT AND DISCUSSION:

The GC-MS analysis of L. balansae resulted presence of phytochemicals with medicinal and economic importance. Major components present are viz. p-ethyl Anisole (RT 6.37), 1- Hexadecane (RT 12.83), 1- Octadecane (RT 15.10), 1- Heneicosanol (RT 17.16), T-Phytol (RT 18.27) (Fig.2). Anisole (1-ethy-4-methoxybenzene) is the most prominent constituent with area of 78.45% (Table 1) and responsible for intense fragrance¹⁸. (-)- Germacrene d reported antimicrobial¹³ and insecticidal⁸ properties, E, E-2,13- Octa decadien-1-ol as a pheromone in a moth (Macroscelesia japona). T- Phytol has reported with antimicrobial, anticancer and anti-inflammatory properties. Celidoniol, deoxy has antibacterial¹², anti-inflammatory and anticancer¹¹ activity and act as pheromone in a moth (Orgyia leucostigma)¹⁰. 1-Octadecene is an antifungal agent for plant and human pathogens¹. The interaction of L. balansae on the leaves of Atuna indica is to be studied thoroughly. The antimicrobial analysis for the studied concentrations exhibited no activity against the bacterial species stressing on standardizing the concentrations.

The volatile constituents of L. balansae shown various properties useful in medicine and other industries. The species doesn’t show any antimicrobial activities. The extended distribution of liverwort in the evergreen forest ecosystems indicating its probable association in many other host plants in the Western Ghats.

ACKNOWLEDGEMENTS:

First three authors are thankful to the Director, KSCSTE-Kerala Forest Research Institute, Peechi for facilities provided. The last author is thankful to the authorities of the Zamorin’s Guruvayurappan College, Kozhikode for support. The authors are grateful to the Kerala Forests and Wildlife Department, Thiruvananthapuram for granting permission to collect the samples and logistic support in the field. Sri. Roshan Lal (Central Instrumentation Unit, KFRI) for assisting sample preparation for GC-MS analysis. The first author extends a gratitude to University Grant Commission as UGC- JRF.

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Received on 09.05.2021 Modified on 20.05.2021

Res. J. Pharmacognosy and Phytochem. 2021; 13(3):115-118.

DOI: 10.52711/0975-4385.2021.00019