Composition, antibacterial and antioxidant potentials of the essential oil of Hedychium matthewii
Abstract
Essential oils are known for their medicinal value since time immemorial and continue to be of vital importance until the present day. The present study describes the composition, antibacterial and antioxidant potential of rhizome oil of Hedychium matthewii. Thirty-five constituents of the oil were identified to account for 82.7%, of which 85.7% was monoterpes and the rest were sesquiterpnes. Most of the major constituents were alcohols and linalool was the prominent one (45.7%). The antibacterial assay showed the bactericidal effect of the essential oil and the most susceptible organism was Streptococcus hemolyticus with a zone of inhibition of 33.8 ± 1.7 mm. The reducing power and nitric oxide scavenging activity of the essential oil was far exceeding the reference compound ascorbic acid and it could be the sign of the potential antioxidant power of the oil isolated. The present study also revealed the prospective of H. matthewii as a new natural source of linalool, which has medicinal and various industrial applications.
Introduction
The genus Hedychium, an ornamental and ethnomedicinal plant comprises about 90 species, mostly distributed from India to South-East Asia (Raj et al., 2005; Thomas et al., 2015). Hedychium species are a good source of various essential oil compounds having medicinal and other industrial applications (Sakhanokho et al., 2013). The essential oil obtained from leaves of H. coronarium constituted beta-pinene as the major compound (Raj et al., 2013), whereas beta-ocimene and linalool as the major constituents in its flower oil (Baez et al., 2011) and 1,8 cineole as the major compound in rhizome oil (Joy et al., 2007).
Leaf and flower essential oils of H. gardnarianum reported the presence of alpha- and beta-pinenes as the major components (Medeiros et al., 2003). H. flavum was characterized by 1,8 cineole and linalool as major compounds in the essential oil obtained from the inflorescence (Baez et al., 2011). Rhizome oil of H. larsenii was dominated by linalool (Raj et al., 2005), whereas leaf and inflorescence oil dominated by arcurcumin and p-cymene, respectively (Raj et al., 2013). It has been reported that rhizome oil of H. thyrsiforme, H. flavum and H. bousigonianum were dominated by 1,8 cineole, whereas linalool was the major constituent in H. forrestii and H. coccineum. beta-Pinene was also found to be the major rhizome oil component in H. elatum and H. flavescens (Sakhanokho et al., 2013).
Essential oils obtained from H. spicatum, H. aurantiacum, H. coronarium and H. ellipticum exhibited good antioxidant potentials by quenching DPPH radicals and moderate to good Fe2+ chelating activity and may perhaps afford protection against oxidative damage (Joshi et al., 2008). Flower and leaf oils of H. gardnerianum showed good antimicrobial activity against Staphylococcus aureus and Staphylococcus epidermidis (Medeiros et al., 2003). The rhizome essential oil and solvent extracts of H. flavescens, H. venustum, H. ellipticum, H. aurantiacum and H. coronarium showed a broad spectrum antimicrobial activity (Joshi et al., 2008; Joy et al., 2007; Sabulal et al., 2007). Essential oil of H. spicatum is reported to possess antimicrobial and antioxidant activity (Bisht et al., 2006; Joshi et al., 2008). Therefore, Hedychium species not only have ornamental value but also have medicinal and industrial value.
Hedychium matthewii, a recently reported taxon, is known only from Idukki, Kerala (Thomas et al., 2015). There are no reports available on the essential oil composition and their biological property of this species. Therefore, the present study aimed at analyses the composition, antibacterial and antioxidant potential of the essential oil isolated from rhizomes of this least studied taxon.
Materials and Methods
Plant material
The rhizomes of H. matthewii were collected from two populations at Idukki and brought to the laboratory and washed to remove adhering soil and dust particles. The rhizomes were cut into pieces and dried at room temperature. A voucher specimen (RHT65200) deposited in The Rapinat Herbarium (RHT), St. Joseph's College, Tiruchirappalli, Tamilnadu.
Chemicals
1, 1- Diphenyl-2-picrylhydrazyl (DPPH), sodium nitrite, sulfanilamide and ascorbic acid (AA) were purchased from Sigma chemical co. (USA). Potassium ferricyanide, trichloroacetic acid (TCA), sodium nitroprusside, alpha-napthylethylenediamine and ferric chloride were purchased from Merck chemical supplies (Germany). All the chemicals used including the solvents, were of analytical grade.
Essential oil extraction
The dried rhizomes (25 g) were ground and hydrodistilled for 3 hours using a Clevenger-type apparatus. The distillate was dried over anhydrous sodium sulfate and stored in tightly closed vials at 4°C for analysis. The essential oil content was determined as a percentage on dry weight basis as an average of three independent extractions of each sample.
Characterization and identification of essential oil constituents
GC/MS analyses were 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 gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) analysis. The column used was DB-5. The oven temperature was programmed as follows; 70°C for 5 min and then increased to 110°C at the rate of 5°C/min,
then up to 200°C at the rate of 3°C/min and again up to 220°C at the rate of 5°C/min, at which the column was maintained for 5 min; injector temperature of 250°C. 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 by comparing their calculated retention indices with literature value (Adams, 2009).
Test microorganisms
The bacterial strains used in the study were three gram positive, namely, Streptococcus haemolyticus (MTCC442), Bacillus cereus (MTCC430) and Staphylococcus aureus (MTCC87) and nine gram negative, namely, Vibrio parahaemolyticus (MTCC451), Vibrio cholerae (MTCC3904), Salmonella paratyphi (MTCC735), Enterobacter aerogens (MTCC111), Escherichia coli (MTCC433), Klebsiella pneumoniae (MTCC3384), Proteus vulgaris (MTCC426), Salmonella typhi (MTCC733) and Pseudomonas aeruginosa (MTCC741). All the tested strains are reference strains, and were collected from Microbial Type Culture Collection and Gene Bank, Institute of Microbial Technology, Sector 39-A, Chandigarh 160036, India.
Antibacterial activity test
The antibacterial activity of essential oil was carried out by disc diffusion method (Bauer, 1966), recommended by clinical and laboratory standards institute (CLSI), using 25 uL 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 uL of essential oil, followed by air-drying and were placed on seeded agar plates. Amoxicillin (30 ug/disc) was used as positive control to determine the sensitivity of bacterial strain. The plates were incubated at 37°C for 24 hours. Antimicrobial activity was evaluated by measuring the zones of inhibition against the tested bacteria. Each assay was carried out in triplicate.
Determination of reducing power
The reducing power of the essential oil was measured by making use of the method described by Yen and Duh (1993) with some modifications. Various concentrations (10, 20, 30, 40 and 50 uL/mL) of essential oil in methanol were 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°C 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). The ascorbic acid standard was used for comparison.
DPPH radical scavenging activity
The free radical scavenging activity of essential oil was measured using the stable DPPH radical, according to the method of Blois (Mani and Thomas, 2014) with minor modifications. 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 uL/mL. The mixture was shaken well 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 formula:
(%) = ((A0 - A1)/A0) x 100 (1)
Where, A0 and A1 are absorbance of the control and of the sample, respectively. Ascorbic acid was used as reference.
Nitric oxide (NO) scavenging activity
Nitric oxide scavenging effect was determined according to Griess Illosvoy reaction (Johnson, 1964) with slight modification. The reaction mixture contained: 10 mM SNP in 0.5 M phosphate buffer (pH 7.4) and various doses (10-50 uL/mL) of the essential oil in a final volume of 3 mL. After incubation for 60 min at 37°C, Griess reagent (0.1% alpha-napthyl-ethylenediamine in distilled water and 1% sulphanilamide in 5% H3PO4) was added. The pink chromophore generated during diazotization of nitrite ions with sulphanilamide and subsequent coupling with alpha-napthylethylenediamine was measured spectrophotometrically at 540 nm. Ascorbic acid was used as a positive control. Nitric oxide scavenging ability (%) was calculated by using the equation (1).
Statistical analysis
Experimental results were expressed as the mean ± standard deviation (SD) of the number of experiments indicated in the legends. Statistical significance was tested using a one way analysis of variance (ANOVA) followed by an LSD test, where p<0.05 was considered significant.
Results
Essential oil characterization
The dried rhizomes were hydrodistilled to obtain a pleasant smelling, pale yellow oil at a yield of 2.6%. The gas chromatogram (GC) of the rhizome essential oil of H. matthewii is shown in Figure 1. The GC profile indicates the presence of more than 50 compounds, of which 35 were identified by comparing and matching the mass spectra and GC retention index (RI) of the compounds with those of reference.
Table I depicts the volatile components indentified and those 35 compounds accounting for 82.7% of the oil hydrodistilled from the dried rhizome. 85.7% of the oil consisted of monoterpenes whereas rests (14.3%) accounted for sesquiterpenes. The constituents indentified consisted of fourteen monoterpene hydrocarbons (40%), sixteen oxygenated monoterpenes (45.7%), two sesquiterpene hydrocarbons (5.7%) and three oxygenated sesquiterpenes (8.6%). The analyzed oil contained esters, oxides, alcohols, phenols, aldehydes and ketones. The majority of essential oil constituents were found in the monoterpene class where the most prominent compounds were linalool (45.6%) followed by β-pinene (6.5%), borneol (6.3%), 4-terpineol (5.6%), camphene (3.3%) and alpha-pinene (2.5%).
Figure 1: Total ion chromatogram (TIC) of the volatile compounds from the dried rhizomes of two populations (A and B) of H. matthewii
Table I: Chemical composition of the essential oil from H. matthewii
| SL. No. | Component | RIa | RIb | Composition(%) |
|---|---|---|---|---|
| 1 | alpha-Thujene | 930 | 931 | 0.4 ± 0.1 |
| 2 | alpha-Pinene | 938 | 939 | 2.5 ± 0.5 |
| 3 | Camphene | 953 | 953 | 3.3 ± 0.9 |
| 4 | Sabinene | 972 | 972 | 1.2 ± 0.5 |
| 5 | beta-Pinene | 981 | 980 | 6.5 ± 0.8 |
| 6 | beta-Myrcene | 992 | 992 | 0.5 ± 0.04 |
| 7 | alpha-Phellandrene | 1007 | 1005 | 0.5 ± 0.2 |
| 8 | 3-Careen | 1111 | 1011 | 0.2 ± 0.1 |
| 9 | alpha-Terpinene | 1012 | 1012 | 0.4 ± 0.05 |
| 10 | o-Cymene | 1026 | 1027 | 1.4 ± 0.1 |
| 11 | D-Limonene | 1031 | 1031 | 1.2 ± 0.03 |
| 12 | 1,8-Cineole | 1033 | 1033 | 0.1 ± 0.02 |
| 13 | trans-beta-Ocimene | 1050 | 1050 | 0.1 ± 0.1 |
| 14 | gamma-Terpinene | 1059 | 1059 | 1.2 ± 0.1 |
| 15 | alpha-Terpinolene | 1063 | 1063 | 0.5 ± 0.2 |
| 16 | trans-Linalool oxide | 1065 | 1065 | 0.7 ± 0.5 |
| 17 | Linalool | 1098 | 1098 | 45.6 ± 3.9 |
| 18 | Fenchol | 1112 | 1112 | 0.02 ± 0.0 |
| 19 | p-Menth-2-en-1-ol | 1120 | 1121 | 0.2 ± 0.05 |
| 20 | L-Camphor | 1140 | 1139 | 0.1 ± 0.01 |
| 21 | Borneol | 1163 | 1162 | 6.3 ± 1.7 |
| 22 | 4-Terpineol | 1180 | 1180 | 5.6 ± 1.5 |
| 23 | alpha-Terpineol | 1189 | 1189 | 0.7 ± 0.1 |
| 24 | Myrtenal | 1190 | 1190 | 0.2 ± 0.03 |
| 25 | alpha-Phellandrene epoxide | 1192 | 1192 | 0.1 ± 0.1 |
| 26 | Piperitol | 1199 | 1198 | 0.2 ± 0.1 |
| 27 | Bornyl fomate | 1232 | 1232 | 0.1 ± 0.01 |
| 28 | Bornyl acetate | 1283 | 1283 | 1.1 ± 1.0 |
| 29 | Thymol | 1284 | 1284 | 0.1 ± 0.02 |
| 30 | alpha-Terpinyl acetate | 1352 | 1352 | 0.03 ± 0.0 |
| 31 | beta-Caryophyllene | 1418 | 1418 | 0.1 ± 0.1 |
| 32 | alpha-Cedrene | 1433 | 1433 | 0.2 ± 0.1 |
| 33 | Caryophyllene oxide | 1583 | 1583 | 0.1 ± 0.1 |
| 34 | Guaiol | 1597 | 1597 | 0.5 ± 0.2 |
| 35 | 10-epi-gamma-Eudesmol | 1619 | 1619 | 0.5 ± 0.2 |
| Values are mean ± SD of two samples (n = 2); RIa and RIb: Experimental and reference retention indices | ||||
Antibacterial assay
The results of the antibacterial studies, of the essential oil obtained, by the disc diffusion technique are given in Table II. By analyzing the antibacterial activity of essential oil, we observed that all the bacteria tested were susceptible and the essential oil showed hopeful antibacterial activity. A volume 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 Streptococcus haemolyticus with a zone of inhibition of 33.8 ± 1.7 mm. The bacteria such as S. haemolyticus and Vibrio cholerae were more susceptible towards essential oil than antibiotic tested. The results also showed that the essential oil was found to be effective antibacterial agent as the standard antibiotic used in the study. Moreover, the essential oil inhibited the growth of both gram positive and gram negative bacteria; consequently it may be used as a broad spectrum natural antibacterial agent.
Table II: Antibacterial activity of the essential oil from H. matthewii
| Species | Zone of inhibition (mm) | |
|---|---|---|
| Essential oil | Amoxicillin | |
| Gram positive | ||
| Bacillus cereus | 19.3 ± 0.9 | 28.3 ± 0.6 |
| Staphylococcus aureus | 29.2 ± 0.8 | 30.2 ± 0.3 |
| Streptococcus hemolyticus | 33.8 ± 1.7 | 31.5 ± 0.9 |
| Gram negative | ||
| Enterobacter aerogens | 20.6 ± 1.5 | 27.8 ± 0.3 |
| Salmonella paratyphi | 23.8 ± 1.2 | 30.5 ± 0.5 |
| Vibrio cholerae | 30.6 ± 1.04 | 27.8 ± 0.3 |
| Salmonella typhii | 28.0 ± 1.7 | 34.3 ± 0.8 |
| Escherichia coli | 21.0 ± 1.0 | 30.5 ± 0.9 |
| Vibrio parahaemolyticus | 26.3 ± 1.5 | 32.3 ± 0.6 |
| Proteus vulgaris | 28.2 ± 1.7 | 32.2 ± 0.3 |
| Klebsiella pneumoniae | 24.2 ± 1.04 | 26.0 ± 0.9 |
| Pseudomonas aeruginosa | 26.2 ± 1.6 | 28.3 ± 0.6 |
| Values are presented as mean ± SD of triplicate experiments | ||
Antioxidant assay
Reducing power: The reducing power of a compound or extract 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 2). The oil exhibited significantly higher activity than the standard ascorbic acid. The results of the present study suggest that essential oil isolated from H. matthewii has potent reducing power and promising antioxidant activity.
Figure 2: Reducing power of rhizome oil of H. matthewii and reference compound ascorbic acid (Values are mean ± SD of two samples analyzed individually in triplicate)
DPPH radical scavenging
DPPH radical scavenging assay, commonly used for analyzing the radical scavenging ability of a compound or extract, is simple and highly sensitive (Miguel, 2010). The antioxidant potential of the essential oil obtained from H. matthewii was studied by analyzing the radical scavenging capacity and electron donating ability of the constituents in the essential oil. The DPPH radical scavenging activity of the essential oil and standard antioxidative compound (ascorbic acid) is shown in Table III. The result showed a dose-dependent inhibition of DPHH radical by both oil and the standard and there was no significant difference exists between the scavenging activity of the oil and the reference compound ascorbic acid. Concentration at which the oil decreased DPPH radical by 50% (IC50 value) was 31.5 µL/mL. Correspondingly, IC50 value for ascorbic acid, used as a standard, was 30.5 µg/mL.
Table III:DPPH radical and nitric oxide scavenging activityof rhizome oil of H. matthewii
| Concentration (uL/mL) | |||||
|---|---|---|---|---|---|
| 10 | 20 | 30 | 40 | 50 | |
| DPPH radical | |||||
| Ascorbic acid (µg/mL) | 12.6 ± 0.1a | 30.4 ± 0.1a | 49.2 ± 0.1a | 72.5 ± 0.1a | 97.4 ± 0.1a |
| Essential oil (µL/mL) | 10.8 ± 0.1a | 27.5 ± 0.2a | 46.7 ± 0.1a | 70.1 ± 0.2a | 91.7 ± 0.1a |
| Nitric oxide | |||||
| Ascorbic acid (µg/mL) | 10.3 ± 0.2a | 21.2 ± 0.1a | 32.6 ± 0.1a | 41.1 ± 0.2a | 52.2 ± 0.1a |
| Essential oil (µL/mL) | 16.8 ± 0.1b | 35.3 ± 0.1b | 54.1 ± 0.1b | 72.8 ± 0.1b | 94.3 ± 0.3b |
| Values are mean ± SD of two samples analyzed individually in triplicate. Superscripts with the same letters within each column are not significantly different at p<0.05 | |||||
Nitric oxide (NO) scavenging activity
Antioxidant activity of the essential oil was further determined by inhibition of nitric oxide radicals is shown in Table III. The nitric oxide scavenging capacity of extracts and the reference compound ascorbic acid was increased with increasing concentration. The results of the study showed that a significant difference (p<0.05) was observed between the two experimental conditions. A total of 50% (IC50) of the nitric oxide radicals was scavenged by essential oil at a concentration of 27.5 µL/mL, whereas that of the ascorbate was 48 µg/mL. The experimental analysis showed that, the essential oil of H. matthewii had good nitric oxide scavenging effect.
Discussion
The antibacterial activities of essential oils are well-known. Recently, the antibacterial activity of essential oils of Buddleja asiatica, Caesalpinia bonducella, Ginkgo biloba and Ligustrum lucidum are reported (Bajpai et al, 2015; Bajpai et al, 2016; Khan et al., 2015; Shukla et al, 2016). Previous studies showed that multiplication of pathogenic Salmonella spp., Escherichia coli (O157:H7), Listeria monocytogenes, Helicobacter pylori and Mycoplasma pneumoniae were inhibited by essential oils (Burt, 2004). Monoterpenes are good antibacterial and antifungal agent (Jirovetz et al., 2005; Sokovic et al., 2010) in particular the oxygenated compounds (Kotan et al., 2007). The major component in the essential oil of H. matthewii was linalool an oxygenated monoterpene, followed by 4-terpineol, β-pinene, camphene, borneol and α-pinene, together constitute 73% of the essential oil isolated. The earlier studies showed that linalool, β-pinene and 4-terpineol were found to be possessing good antibacterial properties against several microorganisms (Barel et al., 1991; Kotan et al., 2007; Park et al., 2012; Sokovic et al., 2010). Essential oil of H. matthewii showed a promising antibacterial activity might be attributed to these major constituents. Hence, essential oil from rhizomes of this plant can be used in the antibacterial formulations, which in turn broadens its applications in the pharmaceutical and other industries. Moreover, the broad-spectrum antibacterial activity of the essential oil of H. matthewii may perhaps due to the liposolubility of its constituents. It is a good indication of the antiseptic power of the essential oil isolated (Marino et al., 1999; Porter and Wilkins, 1999).
The antioxidant activity of essential oils is another biological property of great interest because they may have various industrial applications (Aruoma, 1998; Kamatou and Viljoen, 2010; Maestri et al., 2006). Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the two major classes of free radicals which oxidize various biomolecules and in turn lead to cellular damages and being responsible for diverse diseases even though they have important role in host defence mechanisms (Miguel, 2010). Again, the damaging effects are being increased by generating the peroxinitrite anion (ONOO-) after reacting with the superoxide anion radicals (Gomes et al., 2008; Kostka, 1995; Miyasaka and Hirata, 1997; Nagano, 1999). Therefore, molecules or agents which can inhibit the generation of NO have potential applications in the pharmaceutical industry.
Present investigation showed that the essential oil isolated from H. matthewii is a potent inhibitor and/or scavenger of DPPH and NO radicals. Linalool, a monoterpenoid possessing biological properties such as antibacterial and antioxidant activities (Liu et al., 2012), might be the major active molecule present in the oil of H. matthewii. Earlier studies showed that, linalool found to be a potent scavenger of DPPH radical, inhibit the production of NO radical and also showed bactericidal activities (Liu et al., 2012; Peana et al., 2006).
Antioxidant properties of essential oils such as reducing power, scavenging of free radicals, lipid peroxidation, chelating metal ions etc. are often come from their monoterpene hydrocarbons, oxygenated monoterpenes and sesquiterpenes (Loizzo et al., 2010; Tepe et al., 2005), hence the strong antioxidant activity of essential oil isolated from the rhizomes of H. matthewii may not only be related to linalool but also the sum of the effects of constituents in the essential oil.
Conclusion
Therefore, the high antioxidant activity of this essential oil strengthens their application for possible use as natural antioxidants.
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