INTRODUCTION:

The family Zingiberaceae is well known globally to be used as spices and remedy for medicinal uses in traditional system. The plants belonging to this family contains bioactive compounds as gingerol, kaempferol, curcumin, coronarin, zerumbone etc. Among all the metabolites present diterpenes is the group of structurally diverse molecules which is widely distributed in nature. They are very much effective as anticancer agent that has been isolated from cultivated and wild ginger plants which bears antitumoral effects and NF-kB inhibition have induced by other stimulis^1,2. Most medicinal plants belonging to Zingiberaceae have rhizomes which bear aromatic and volatile essential oil. The leaves of some ginger species are applied to relieve stiff, sore joints and its essential oil has been used in perfume industries as reported³. Hedychium coronarium Koen or white butterfly ginger is mostly used in pharmaceutical and cosmetics.

This plant is originated in Himalayas but also found in Japan, Nepal, Brazil and China⁴. These medicinal plants also bear an interesting character of antiproliferative effects which are reported for cervical and breast cancer treatment^5,6. Here also in this plant due to the presence of some natural bioactive compounds in its leaf and other plant parts it is highly exhibiting medicinal effects. One of the compound present its leaf essential oil is caryophyllene that bears anti-inflammatory properties to protect the brain from swelling and inflammation during a stroke to improve stroke outcomes. It also helps to improve cold tolerance at low ambient temperatures. Another is eucalyptol, a promising compound having anti-inflammatory and potent antioxidant effects which is used in treatment of diseases like respiratory diseases, pancreatitis, colon damage, cardiovascular and neurodegenerative diseases. Due to this strategy, research on important medicinal species and its novel compounds to be used as natural anticancer drug has become important. Always medicinal plant has history of being used as natural drugs for treatment of various diseases so has been used by local communities. H. coronarium has tremendous therapeutic uses as studied earlier for the cure of arthritis, diabetes, headache, hypertension etc. thereby its huge demand is increasing the plant as well as its essential oil cost⁷. Hence the current work were undertaken to select, multiply and finally conserve this high quality plant.

MATERIALS AND METHODS:

Plant material, regeneration and establishment of H. coronarium plantlets in different lines by PGR’s treatment:

The healthy base of the shoot was used as explant from the control plant and was washed under tap water thereby treated with 1% extran for 6 minutes and repeatedly four times washed. In the laminar flow hood the explants using 0.1% mercuric chloride were surface sterilized followed by washing thrice with sterile distilled water. H. coronarium, explant was kept on MS basal medium with sucrose (30g/L)⁸. Then regenerated plants was induce variation by phytohormonal treatment like kinetin (KIN), benzyladenine (BA), indole-3-acetic acid (IAA), indole-3-butyric acid (IBA), naphthalene acetic acid (NAA), gibberellic acid (GA) and adenine sulphate (ADS). Upto 5.7 the pH of the medium was maintained and agar (0.8%) was added for solidification. The autoclave was set at 121°C and 1.06kg/cm² pressure for 15 minutes for media sterilization. Then inoculation was done aseptically and cultures were kept at 25ºC under fluorescent light with 50μmole m^-2s^-1light intensity for 16h photoperiod. The replicas of in vitro treated lines with raised shoot and roots were transferred to greenhouse for growth and estimation of leaf essential oil alongwith its mother plant. The survival percent of each line after 1year in field condition was studied⁹.

Extraction of leaf essential oil and Gas chromatography-mass spectrometry analysis:

Leaves were collected from regenerated plantlets of different lines in H. coronarium, washed under tap water for removing soil and immediately were used for extraction of leaf oil from treated lines. 100g of leaves and 250ml of sterilized water were mixed for essential oil extraction by hydro-distillation process using Clevenger’s apparatus unit. A flask containing leaves was heated up to 3 hours for leaf oil at 30-50°C and the vapour condensed was separated in water-oil surface. At the upper most layers the oil present was collected in a micro centrifuge through collecting chamber and further stored at 4°C. The component identification was done by GC–MS analysis using HP 6890 series GC (Hewlett-Packard, USA) coupled with a mass selective detector (MSD), HP 5973 series (Hewlett-Packard). Helium as a carrier gas was used and sample was injected in split less mode in column HP5 phenyl methyl siloxane [25m (film thickness) 30 m (internal diameter) 9 30m (length of 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. The initial temperature was 60°C, ramping rate at 3°C, final temperature at 243°C and running time was 61 minutes. For this evaluation, 15 plants from each treated lines were used in random manner¹⁰.

RESULTS AND DISCUSSIONS:

We used different concentration of growth regulators but best media for the increase in chemical constituents of Hedychium coronarium was found in MS+3mg/l kinetin combination among all the treated lines. The survival was 95% as noted. All regenerates grown in different lines were maintained in field for maturity for study of its leaf essential oil content (Figure 1). These data revealed that leaf oil content was lowest in P11 line and highest in P2 line as 0.18%-0.36% respectively (Table 1, Figure 2). Further the GC-MS analysis revealed the data of highest percentage of caryophyllene was present in P2 line as 33.60% followed by P4 line as 31.51% and in control as 21.05%. Thereby the eucalyptol percentage was found highest in P2 line as 24.21% followed by P4 line as 22.98% and in control as 12.96% (Table 2). In the control plant (P0) the presence of leaf oil was 0.25% and the major compound found was caryophyllene (Figure 3). There are few reports on H. coronarium leaf oil analysis which bears β-pinene as the major constituent (20.0%) followed by linalool (15.8%)¹¹. Other reports on H. coronarium leaf and rhizome oil analysis are available^12,13. It has been also studied in other medicinal species as Thymus vulgaris which has chemical variation in its leaf essential oil at different stages of the plant growth condition¹⁴. There are many reports on the successful establishment of in vitro medicinal plants of Zingiberaceae family using auxin and cytokinin in a combined manner. We also in the present study used combination in different lines of H. coronarium for terpenoid improvement in essential oil content. In our study eucalyptol and caryophyllene produced varies in exposure to different PGR’s in combined manner significantly. Similar work was reported using different growth regulators for essential oil production in callus derived regenerants of Curcuma longa L. as compared to its control¹⁵. There are studies on the production of essential oil from the in vitro culture of Caryopteris and compared with its in vivo plant¹⁶. Though terpenes were identified in this plant earlier, the composition pattern was found different in the present study as compared to previous study from other areas of the world. These changes might be due to the plant age, ontogenic development, environmental conditions or handling procedures etc. Similarly GC-MS method has been used for the validation and determination of residuals in certain drug substances¹⁷. There are reports on the fungal endophytes associated with Boerhaavia diffusa, having phytochemicals and antioxidants that can act as a source of secondary metabolite to be used in the preparation of medicines. They have also performed the GC-MS and FTIR in the plant extracts which revealed the presence of certain chemical groups¹⁸. This work is similar to ours in which we have estimated the presence of certain bioactive compounds by GC-MS analysis. Studies on the micropropagation of leaf explants of Scoparia dulcis has been done on MS medium supplemented with different concentrations of TDZ and IAA giving rise to multiple shoots with 90.6% survival¹⁹. Here also we have similarity to this work by the presence of different combinations of plant growth hormones with 95% survivality. There are certain studies done on the quantification of total phenolic and flavanoid content present in the leaves of Cucumis melovaragrestis that is consumed by rural people in Tamil Nadu to cure many diseases²⁰. Other studies on the determination of clonazepam, a scheduled drug which is present in chocolate using High Performance Liquid Chromatography and confirmation by Gas Chromatography- Mass Spectrometry have been done²¹. There are reports on the analysis of phytochemicals present in the ethanolic leaf extract of Ficus racemosa. Their study revealed some antimicrobial activities for which it can be used as antibiotics in the drug development process²². There are researches on Murraya koenigii leaf extract and its evaluation as herbal gels having antibacterial activities²³. Some other studies on the chloroform leaf extract of Mimosa pudica and its in vitro antioxidant activity has been done²⁴. Above all the reports are favourable to us by their use of leaves in the experiment to detect various medicinal activities for which it can be used in pharmaceuticals.

Table 1: Screening of leaf oil in different lines of micropropagated Hedychium coronarium

Media name	MS + Phytohormones(mg/l)	Leaf oil (%) ± SE
P0	Control	0.25 ± 0.1
P1	BA3	0.31 ± 0.2
P2	KIN3	0.36 ± 0.2
P3	BA3 +IAA1+ADS100	0.21 ± 0.1
P4	BA3+NAA0.5	0.32 ± 0.2
P5	KIN3+IBA1	0.23 ± 0.1
P6	KIN3+NAA 1+GA3	0.22 ± 0.3
P7	BA3+IAA0.5+GA3	0.23 ± 0.2
P8	KIN 1+IAA0.5	0.21 ± 0.3
P9	KIN3+IAA1+ADS100	0.20 ± 0.3
P10	BA3+IBA0.5+NAA1	0.19 ± 0.2
P11	BA3+ADS50+IBA1	0.18 ± 0.1
P12	BA3+IBA 1	0.19 ± 0.3

Data represents the mean of 15 replicates for each treatment

The phytochemical screening and effective in vitro anthelmintic activity of Adhatoda vasica suggests this plant to be used as medicine against human parasitic infections²⁵. The study on antioxidant activity and presence of phytonutrients in the essential oil of Coriandrum sativum has been done for its use as raw material in the preparation of active pharmacological products²⁶. The present study shows that the use of different phytoregulators in the basal media has the potential to increase in content of the secondary metabolite production. So also the consumption of leaf essential oil and its extracts plays a vital role in preventing human diseases as well as premature aging. The use of plant essential oils with potent antioxidants will also enhance the demand of tissue cultured plantlets to be used in cosmetics, foods and promote positive health benefits.

Table 2: Quality evaluation of leaf essential oil in different lines of Hedychium coronarium

Sl. No.	Oil constituents	P0 (Control)	P1	P2	P3	P4	P5	P6	P7	P8	P9	P10	P11	P12
Sl. No.	Oil constituents	Area %
1	Alpha pinene	15.02	4.17	5.13	3.47	6.22	5.10	4.90	4.88	5.10	3.21	3.85	4.32	2.92
2	Eucalyptol	12.96	18.52	24.21	17.29	22.98	19.60	16.13	15.93	13.87	11.69	12.31	13.11	15.61
3	Terpinen-4-ol	4.97	3.63	7.70	6.24	-	5.40	6.22	4.96	5.72	4.65	-	5.67	5.54
4	Gamma-elemene	2.76	2.96	-	3.98	-	3.40	3.85	3.79	3.42	3.02	-	1.31	2.34
5	trans-Isoeugenol	-	-	-	-	15.11	-	-	-	-	-	-	-	-
6	Eugenol	-	9.89	12.84	7.90	-	7.80	5.81	-	10.18	9.65	10.90	-	8.98
7	Caryophyllene	21.05	24.09	33.60	29.78	31.51	20.45	15.16	19.90	25.60	20.03	25.06	28.80	18.72
8	Humulene	-	4.50	7.31	8.09	5.51	6.70	4.92	6.72	-	-	6.56	8.68	3.20
9	(+)-b-elemene	2.41	9.41	-	7.80	-	4.32	4.67	-	5.23	4.23	4.90	2.20	3.42
10	(-)-beta-cadinene	2.07	0.91	-	-	-	-	-	-	-	-	-	0.43	-
11	Caryophyllene oxide	9.18	-	9.20	6.78	11.72	5.70	6.75	8.70	9.10	5.50	7.80	3.45	4.56
12	Calarene epoxide	-	4.90	-	-	6.97	4.98	3.95	6.75	8.90	5.90	5.80	5.67	6.91
13	Geranyl tiglate	3.37	-	-	3.42	-	-	4.12	2.92	-	-	3.93	4.51	2.43
14	Adamantane	3.23	1.75	-	-	-	-	5.71	-	2.75	2.75	2.40	-	-
15	(-)-spathulenol	3.85	2.34	-	-	-	5.63	-	-	-	5.61	-	1.91	0.90

Data taken from 15 plants of each somaclone

(a) (b)

Fig. 1: (a) In vitro grown Hedychium coronarium plantlets (b) In vitro plants maintained in field condition for essential oil extraction

Fig. 2: Graphical presentation of leaf oil in different lines of H. coronarium

(a)

(b)

(c)

Fig. 3: (a) Gas chromatography-mass spectrometry analysis of H. coronarium leaf essential oil in P0 line or control (b) P2 line (c) P4 line

ACKNOWLEDGEMENTS:

The authors are grateful to Prof (Dr.) M.R. Nayak, President and Prof (Dr.) S.C. Si, Dean, Centre for Biotechnology, Siksha ‘O’ Anusandhan University for providing facilities and encouraging throughout.

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Received on 10.04.2021 Modified on 21.09.2021

Research J. Pharm. and Tech 2022; 15(9):3866-3870.

DOI: 10.52711/0974-360X.2022.00648