*Corresponding Author:
Sinjumol Thomas
Department of Botany, Vimala College, Thrissur–680 009, India
E-mail: [email protected]
Date of Submission 15 November 2015
Date of Revision 24 February 2016
Date of Acceptance 03 July 2016
Indian J Pharm Sci 2016;78(4):452-457  

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Abstract

The essential oil constituents of rhizomes of Hedychium forrestii var. palaniense were analyzed by gas chromatography and gas chromatography-mass spectrometry. A total of 26 constituents comprising 68.20% of the oils were identified. The volatile fraction was characterized by monoterpene hydrocarbons (42.31%), oxygenated monoterpenes (38.46%), sesquiterpene hydrocarbons (15.38%) and oxygenated sesquiterpenes (3.85%). The predominant constituents identified were β-pinene (18.30%), β-linalool (17.80%), 1,8-cineole (12.00%), and 4-terpineol (5.50%). Sesquiterpenoids were present only in negligible quantities. The antibacterial activities of the essential oil was evaluated against ten bacteria and Pseudomonas aeruginosa was the most susceptible organism with a zone of inhibition of 42.16±0.76 mm. Additionally, the essential oil showed promising radical scavenging and electron donating activity.

Keywords

Hedychium forrestii var. palaniense, Zingiberaceae, β-pinene, β-linalool, 1,8-cineole, DPPH

The genus Hedychium J. Koenig comprises about 80 species, distributed from India to South East Asia [1]. This is considered to be the largest genus of Zingiberaceae in India with about 45 taxa [2]. They are generally used as ornamentals and are also used for their industrial (paper manufacture and perfumery) or medicinal properties [3-5].

Composition of essential oils from Hedychium species is characterized by the abundance of monoterpenes with major percentage of 1,8-cineole and pinene derivatives [6]. Essential oils from H. spicatum, H. aurantiacum, H. coronarium and H. ellipticum exhibited good antioxidant potentials by quenching 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and moderate to good Fe2+ chelating activity and could afford protection against oxidative damage [7]. Flower and leaf oils of H. gardnerianum showed good antimicrobial activity against Staphylococcus aureus and S. epidermidis [8]. The rhizome essential oil as well as solvent extracts of H. flavescens, H. venustum, H. ellipticum, H. aurantiacum and H. coronarium showed a broad antimicrobial spectrum [7,9-11]. Essential oil of H. spicatum is reported to possess antimicrobial and antioxidant activity [7,12]. Therefore, Hedychium species not only have ornamental value but also have medicinal and industrial value.

Hedychium forrestii var. palaniense, a recently reported taxon, is known only from four localities of Pulney Hills, Tamil Nadu, at an altitude of 1300-1600 m. It prefers to grow in grassy slopes and open rock cliffs as patches of 15-20 mature individuals [13]. There are no previous reports of the essential oil composition and its biological properties of this new taxon. Therefore the present study aimed to analyze the essential oil composition, antibacterial and antioxidant potential of the essential oil isolated from rhizomes of this least studied taxon.

Materials and Methods

The rhizomes of H. forrestii var. palaniense were collected from Perumalmalai (1520 m) region of Pulney Hills, Tamilnadu in the month of May, 2014. Voucher specimen (RHT65200) has been deposited in The Rapinat Herbarium (RHT), St. Joseph’s College, Tiruchirappally, Tamilnadu.

Chemicals

DPPH and ascorbic acid (AA) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Potassium ferricyanide, trichloroacetic acid (TCA), and ferric chloride were purchased from Merck Chemical Supplies (Damstadt, Germany). All the chemicals used including the solvents, were of analytical grade.

Essential oil extraction

Air dried rhizomes (25 g) of the plant were hydrodistilled for 3 h using a Clevenger-type apparatus. The distillated oil was dried over anhydrous sodium sulfate, and stored in tightly closed vials at 4° for analysis. The essential oil content was determined as percentage on dry weight basis as an average of three independent extractions.

Characterization of essential oil

Gas chromatography-mass spectrometry (GC/MS) analysis was performed using Shimadzu GC-2010 gas chromatograph (GC-FID) equipped with QP 2010 mass spectrometer (MS). Approximately 0.1 μl of pure oil sample was subjected to GC and GC/MS analysis. The column used was DB-5. The oven temperature was programmed as follows; 70° for 5 min and then increased to 110° at the rate of 5°/min, then up to 200° at the rate of 3°/min and again up to 220° at the rate of 5°/min, at which the column was maintained for 5 min; injector temperature of 250°. Helium was used as carrier gas at a flow rate of 1 ml/min. Identification of the individual components was made by matching their recorded mass spectra and linear retention indices with the library (NIST and Wiley) provided by the instrument software, online database (http://www.flavornet.org) and by comparing their calculated retention indices with literature value [14].

Test microorganisms

The bacterial strains used in the study were three Gram positive bacteria namely, Streptococcus haemolyticus (MTCC442), Bacillus cereus (MTCC430) and Staphylococcus aureus (MTCC87) and seven Gram negative, namely, Vibrio parahaemolyticus (MTCC451), Vibrio cholerae (MTCC3904), Salmonella paratyphi (MTCC735), Enterobacter aerogens (MTCC111), Escherichia coli (MTCC433), Klebsiella pneumoniae (MTCC3384), and Pseudomonas aeruginosa (MTCC741). All the tested strains are reference strains, and were collected from Microbial Type Culture Collection (MTCC) and Gene Bank, Institute of Microbial Technology, Chandigarh, India.

Antibacterial activity test

The antibacterial activity of essential oil was carried out by disc diffusion method [15], recommended by Clinical and Laboratory Standards Institute (CLSI), using 25 μl of standardized suspension of test bacteria (1.5×108 CFU/ml) spread on Mueller-Hinton agar (MHA, pH 7.3±0.1) plates. The discs (6 mm in diameter) were impregnated with 20 μl of essential oil, followed by air-drying and were placed on seeded agar plates. Amoxicillin (30 μg/disc) was used as positive control to determine the sensitivity of bacterial strain. The plates were incubated at 37° for 24 h. Antimicrobial activity was evaluated by measuring the zones of inhibition against the tested bacteria. Each assay was carried out in triplicate.

DPPH radical scavenging assay

The free radical scavenging activity of essential oil was measured using the stable DPPH radical, according to the method of Blois [16] with minor modification. Briefly, 0.1 mM solution of DPPH in methanol was prepared and this solution (1 ml) was added to essential oil in methanol (3 ml) at concentrations ranging from 10-50 μl/ml. The mixture was shaken vigorously and left to stand for 30 min in the dark, and the absorbance was then measured at 517 nm. The capability to scavenge the DPPH radical was calculated using the following equation: %DPPH radical activity = ((A0−A1)/A0)×100, where A0 and A1 are absorbance of the control and of the sample, respectively. Ascorbic acid was used for comparison.

Determination of reducing power

The reducing power of the essential oil was measured by making use of the method described by Yen and Duh [17] with some modifications. Various concentrations (10, 20, 30, 40 and 50 μl/ml) of essential oil in methanol was taken separately and mixed with 2.5 ml of 0.2 M sodium phosphate buffer (pH 6.6). The dilute sample was then mixed with 5.0 ml of 1% potassium ferricyanide and the mixture was incubated at 50° for 20 min. 5.0 ml of 10% trichloroacetic acid was added to the mixture and was centrifuged at 3000 rpm for 10 min. 5.0 ml of the supernatant solution was mixed with 5.0 ml of distilled water and 1.0 ml of ferric chloride (1%). The absorbance was measured spectrophotometrically at 700 nm (Shimadzu, UV-150-02). Ascorbic acid standard was used for comparison.

Results and Discussion

The dried rhizomes (25 g) were hydrodistilled to obtain a pleasant smelling, pale yellow colored oil at a yield of 1.2% v/w. A gas chromatogram of the volatile components of the rhizome is shown in Figure 1. The GC indicates the presence of more than 50 compounds, of which 26 were identified.

IJPS-Schematic-representation

Figure 1: GC-MS chromatogram of oil.
Total ion chromatogram (TIC) of the volatile compounds from dried rhizomes of Hedychium forrestii var. palaniense.

Table 1 lists the volatile components identified and those 26 constituents accounted for 68.20% of the oil hydrodistilled. The analyzed oil contained monoterpene hydrocarbons (42.31%), oxygenated monoterpenes (38.46%), sesquiterpene hydrocarbons (15.38%) and oxygenated sesquiterpenes (3.85%). The majority of the essential oil components were monoterpenes and the major constituents were β-pinene (18.30%), β-linalool (17.80%), 1,8-cineole (12.0%), 4-terpineol (5.50%), α-pinene (4.9%), γ-terpinene (1.90%), borneol (1.60%), α-terpineol (1.60%) and camphene (1.40%). The sesquiterpene compounds were present in least concentration and germacrene B (0.20%) was the leading one.

Component RIa RIb Composition (%)
α-thujene 936 938 0.5
α-pinene 938 939 4.9
Camphene 953 953 1.4
β-pinene 994 994 18.3
α-phellandrene 1007 1006 0.3
3-careen 1009 1009 tr
α-terpinene 1011 1012 0.8
1,8-cineole 1029 1030 12
cis-β-ocimene 1036 1038 tr
4-thujanol 1072 1073 0.2
γ-terpinene 1074 1074 1.9
α-terpinolene 1086 1086 0.5
Linalool 1098 1100 17.8
Fenchol 1139 1139 tr
L-camphor 1140 1139 0.3
p-menth-2-en-1-ol 1145 1145 0.1
Pinocarvone 1158 1160 0.1
Borneol 1163 1162 1.6
4-terpineol 1180 1179 5.5
α-terpineol 1192 1195 1.6
Bornyl acetate 1294 1287 tr
δ-elemene 1338 1335 tr
β-caryophyllene 1466 1467 0.1
β-sesquiphellandrene 1558 1560 0.1
Germacrene B 1562 1562 0.2
Caryophyllene oxide 1574 1573 tr
<0.1%

Table 1: Chemical Composition of Essential Oil Extracted from Dried Rhizomes

The present study showed that qualitative and quantitative differences were found among the oils of H. forrestii var. palaniense Sanoj and M Sabu and H. forrestii Diels which is a close relative of the taxon studied [18]. Sakhanokho et al. [18] reported that the major components in the essential oil from dried rhizomes of H. forrestii were characterized by linalool (56%), β-pinene (14%) and 1,8-cineole (10.1%) and found that the oil differs quantitatively from those of H. forrestii var. palaniense. It is interesting to see that, though the oils of both taxa contained monoterpenes (87.5% in H. forrestii Diels) and sesquiterpenes (8.33% in H. forrestii Diels), hydrocarbon sesquiterpenes were not detected in H. forrestii Diels. However, a diterpene was found in the rhizome essential oil of H. forrestii Diels [18].

Monoterpene constituents detected in the present study such as 4-thujanol, fenchol, p-menth-2-en-1- ol, camphor, pinocarvone and bornyl acetate were absent in the earlier reports [18]. At the same time the monoterpenes detected in the essential oil of H. forrestii like, sabinene, myrcene, limonene, cymene, translinalool oxide and cis-linalool oxide were not found in the present study. Earlier reports also showed that major essential oil constituents are common among various species of Hedychium [9,18-21] whereas minor components, which reveal the specific differences between taxa. Finally, we suggest that the differences in essential oil composition, both qualitative and quantitative, may depend on factors such as genotype, season and environment.

The results of the antibacterial studies of the essential oil obtained by the disc diffusion technique are given in Table 2. It was interesting to see that all the bacteria tested were susceptible and the essential oil showed hopeful antibacterial activity. A concentration of 20 μl of essential oil showed nearly equal and effective inhibition against all the strains used in the present study. The most susceptible bacterium tested was P. aeruginosa with a zone of inhibition of 42.16±0.76 mm. The results also showed that the essential oil was found to be more effective antibacterial agent than the standard antibiotic used in the study. Moreover, the essential oil inhibit the growth of both Gram-positive and Gram-negative bacteria, consequently it may be used as a broad-spectrum natural antibacterial agent.

Species Zone of inhibition (mm)
Essential oil Amoxicillin
Vibrio parahaemolyticus(-) 36.33±0.57 32.33±0.57
Bacillus cereus (+) 36.17±0.28 34.00±0.50
Enterobacteraerogens(-) 34.20±0.23 32.17±0.28
Salmonella paratyphi(-) 34.33±0.60 32.67±0.57
Vibrio cholera (-) 37.83±0.80 36.33±0.60
Staphylococcus aureus(+) 37.83±0.76 36.17±0.28
Escherichia coli (-) 36.00±0.86 30.33±0.30
Streptococcus haemolyticus(+) 37.16±0.77 34.16±0.29
Klebsiella pneumonia (-) 38.20±0.28 32.33±0.57
Pseudomonas aeruginosa(-) 42.16±0.76 32.17±0.29

Table 2: Antibacterial Activity Ofextracted Essential Oil From Rhizomes

Monoterpenes are good antibacterial agents [22] in particular the oxygenated compounds [23]. The major components in the essential oil of H. forrestii var. palaniense were β-pinene, linalool, 1,8-cineole and 4-terpineol, which were studied for their antibacterial properties previously. The earlier studies showed that β-pinene, linalool and 4-terpineol were found to be possess good antibacterial properties [22-24] whereas 1,8-cineole was least effective [23,25]. However, recent studies showed that that 1,8-cineole alone or in combination with other essential oils showed good antibacterial activity [10,26] and this might be due to synergism [27]. Essential oil of H. forrestii var. palaniense showed a promising antibacterial activity and it might be attributed not only by the major constituents but also by the action of minor components. Hence, essential oil from rhizomes of this plant can be used in the antibacterial formulations, which in turn broadens its applications in the medicinal and industrial fields.

The antioxidant potential of the essential oil obtained from H. forrestii var. palaniense was studied by analyzing the radical scavenging capacity and electron donating ability of the constituents in the essential oil. The DPPH radical scavenging activities of the essential oil and standard antioxidative compound (ascorbic acid) are shown in Figure 2. The result showed a dose-dependent inhibition of DPPH radical by both oil and the standard and the scavenging capacity of the essential oil was significant when compared with that of standard. Concentration at which the oil decreased DPPH radical by 50% (IC50 values) was 34.0 μl/ml. Correspondingly, IC50 value for ascorbic acid, used as standard, was 30.50 μg/ml. The reducing power of a sample was related to its electron transfer ability and might, therefore, served as an indicator of its potential antioxidant activity. The reducing power of the essential oil and ascorbic acid increased with the concentration (Figure 3). The oil exhibited significantly higher activity than the standard ascorbic acid. The results of the present study suggest that essential oil isolated from H. forrestii var. palaniense has promising radical scavenging ability and potent reducing power. Antioxidant properties of essential oils such as lipid peroxidation, scavenging of free radicals, chelating metal ions, and reducing power are often come from their monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpenes [28,29], which means that the strong antioxidant activity of essential oil from the rhizomes of H. forrestii var. palaniense may be related to the sum of the effects of constituents in the essential oil. Therefore, the high antioxidant activity of this essential oil strengthens their application for possible use as natural antioxidants.

IJPS-Schematic-representation

Figure 2: DPPH radical scavenging activity.
DPPH radical scavenging activity of essential oil (μl/ml,

image) isolated from dried rhizomes of H. forrestii var. palaniense and reference compound ascorbic acid (μg/ml,image). Values are mean±SD of triplicate experiments.

IJPS-Schematic-representation

Figure 3: Reducing power.
Reducing power of essential oil (μl/ml,image) isolated from dried rhizomes of H. forrestii var. palaniense and reference compound ascorbic acid (μg/ml,image). Values are mean±SD of triplicate experiments.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

References