First report on leaf Essential oil of In vitro Kaempferia galanga L. from Eastern India

Reena Parida¹*, Sujata Mohanty², Sanghamitra Nayak¹

¹Centre for Biotechnology, SPS, Siksha ‘O’ Anusandhan University, Bhubaneswar, Odisha, India

²Ramadevi Womens University, Bhubaneswar, Odisha, India

*Corresponding Author E-mail: ms.reenas@gmail.com

ABSTRACT:

Today over 95% of the medicinal plants used by the industries in India is collected from forest and other natural resources. Due to the fast changing lifestyle globally, now there is demand of medicinal plants that are of great commercial value and wild origin. In the present study, the leaf essential oil analysis of Kaempferia galanga by Gas chromatography-mass spectrometery (GC-MS) has been studied. These oils were extracted through hydro-distillation process and its chemical composition was analyzed by Gas chromatography and mass spectrometry. A total of 5 compounds were identified from the micropropagated plants. The major compound identified in were ethyl p-methoxycinnamate as 72.23 % respectively. The medicinally beneficial compounds present in the oil is the first report on in vitro Kaempferia galanga leaves confirming the plant to be treated for various diseases in human and for commercial purposes.

KEYWORDS: Kaempferia galanga, essential oil, GC-MS, medicinal, ethyl p-methoxycinnamate.

INTRODUCTION:

Kaempferia galanga L. is an Indian medicinal herb belonging to family Zingiberaceae. There are earlier reports of this plant to be used in preparation of ayurvedic drugs, perfumery and cosmetic industries also as spice ingredient¹. K.galanga leaves are used in flavouring foods, as hair tonic, mouth wash and its juice is beneficial for pregnant women. It possesses antioxidant, antinociceptive and anti-inflammatory activities which would help in treatment of mouth ulcers, migranes and headache. The leave poultices and its tea are used for curing sore throat, rheumatism and swollen eye infections^2,3. K.galanga is rarely cultivated at some places for medicinal use, its large scale deforestation and over exploitation reduces the plant population. Since by conventional propagation method it is not sufficient to meet the demand for its commercial use so tissue culture method is adapted.

In the recent years, micropropagation techniques are being profitably used to overcome the present demand of aromatic and medicinal plants. Tissue culture techniques hold promise for rapid in vitro production of leaf biomass in K.galanga. In vitro production of leaf biomass has also been reported in few other species having commercial implications^4,5. There are few reports on essential oil analysis of K.galanga but yet no report on in vitro leaf oil analysis^6,7,8. The price of its essential oil varies in international market due to high exploitation by local people and pharmaceutical industries. So the present paper deals with a suitable protocol for extraction of leaf essential oil from rapidly micropropagated K.galanga.

Plant material collection and GC-MS analysis:

The rhizomes were collected from the wild forest areas of eastern ghats and were grown in the medicinal plant garden of Centre for Biotechnology, Siksha ‘O’ Anusandhan University, Bhubaneswar. The axillary bud explants from the rhizome collected from disease-free plants were thoroughly washed in running tap water with a neutral liquid detergent (Extran, Merck) for 5 minutes. The explants were surface sterilized with 0.1% mercuric chloride for 5 minutes in a laminar flow cabinet and rinsed 3 to 4 times in sterile distilled water to remove the traces of sterilants prior to inoculation.

Table 1: Chemical composition of leaf oil in micropropagated (MP) Kaempferia galanga

Compound Name	Retention Time (min)	Molecular formula	Area % (MP)
Borneol	7.75	C₁₀H₁₈O	7.38
2-Propenoic acid	10.53	C₁₁H₁₂O₂	7.68
Murolene	10.94	C₁₅H₂₄	4.29
Caryophyllene oxide	11.59	C₁₅H₂₄O	8.42
Ethyl p-methoxycinnamate	12.10	C₁₂H₁₄O₃	72.23

Fig. 1: Gas chromatography-mass spectrometry chromatogram of micropropagated Kaempferia galanga

Explant were inoculated on basal Murashige and Skoog (MS) medium supplemented with various combination of phytohormones and 0.8 % of agar following our earlier reports^9,10. In vitro regenerated plants with well developed shoot and roots were studied for leaf oil extraction. This essential oil was extracted by hydro-distillation using a Clevenger’s apparatus following the method¹¹. A flask containing 100gm of cut leaves in 500ml of distilled water was heated for 3 hours and the condensed vapour was separated in oil-water surface. The transparent oil present at the upper most layers was collected in the ependroff tube through the collecting chambers. The component identification was achieved by the GC–MS analysis using HP 6890 series GC (Hewlett-Packard, USA) coupled with a mass selective detector (MSD), HP 5973 series (Hewlett-Packard). Helium was used as a carrier gas and the sample was injected in split less mode in a column HP5 phenyl methyl siloxane [25μl film thickness × 320μm internal diameter × 30 m length of the column]. Mass spectra were acquired over a 40–400 atomic mass unit range. Compounds were identified by comparing the mass spectral data with those in the NIST library provided with software and with commercially available data. Temperature programming was: initial temperature 60°C, ramping rate 3°C, final temperature 243⁰C, run time 61 min.

RESULTS AND DISCUSSIONS:

The in vitro leaves yielded 0.05% of essential oil which was pale yellow in colour and its GC-MS analyzed ethyl p-methoxycinnamate to be the major compound. The compounds present in micropropagated leaf oil compounds were borneol, 2-propenoic acid, murolene, caryophyllene oxide and ethyl p-methoxycinnamate. In case micropropagated leaf oil there were 5 constituents representing approximately 100 % of the oil as shown in Table 1. The peak of the GC-MS analysis of micropropagated leaf oil has been shown (Fig. 1). Earlier scientists have reported on the leaf oil composition of K.galanga⁷. In their reports linoleoyl chloride (21.42%) were identified as the major compound in the leaf oil whereas in our reports ethyl p-methoxycinnamate was the major compound. Studies on the phytochemical analysis of naturally grown and tissue cultured K. galanga has been done¹². There are other reports on medicinal, agronomical, economical and pharmacological importance of the plant¹³. But for the first time, the composition of in vitro raised plantlets leaf essential oil of K.galanga was elucidated. However recent works has been done using GC-MS analysis in other medicinal plants for their phytochemical evaluation¹⁴. The quantitative composition and the relative proportions of the oil components are widely influenced by the factors such as nature and age of the plant genotype, environmental and growing conditions, handling procedures etc. These may largely be responsible for the observed compositional variations between the studied oil samples and previous results elsewhere. There are many scientific reports on K.galanga having great potential for the treatment of various infectious diseases. In the present study, GC-MS analysis shows the presence of bioactive compounds which may be responsible for the medicinal activity of K.galanga. Hence GC-MS profile could be used as a tool for identification of the plant and provide foundation for future studies on the molecular mechanism of aromatic scent production. The present paper is the first report on leaf essential oil analysis of micropropagated K.galanga.

ACKNOWLEDGEMENTS:

The authors sincerely thank to Dr. Manoj Ranjan Nayak, President and Dr. S.C Si, Dean of Centre for Biotechnology of Siksha ‘O’ Anusandhan University for providing facilities and encouragement throughout.

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Received on 17.06.2019 Modified on 01.08.2019

Research J. Pharm. and Tech. 2020; 13(1): 157-159.

DOI: 10.5958/0974-360X.2020.00031.1