Methyl Eugenol, 1,8-Cineole and Nerolidol Rich Essential Oils with their Biological Activities from three Melaleuca Species Growing in Tarai Region of North India

Abstract

The essential oils from the fresh leaves of three Melalecua spp. viz; Melaleuca leucadendron (L.) Melaleuca linariifolia Sm. and Melaleuca bracteata F. Muell. growing in Tarai region of North India were analyzed by a combination of gas chromatography/mass spectrometry. The analysis revealed the presence of several constituents of industrial and pharmacological importance. M. leucadendron essential oil was found to be dominated by E-nerolidol (85.7%) rich chemotype. 1,8-cineole (61.1%) along with significant presence of α-terpineol (12.3%), α-pinene (4.0%), β-myrcene (3.8%), and E-caryophyllene (1.7%) were identified in the essential oil from M. linariifolia Similarly M. bracteata was dominated by the presence of phenylpropanoids viz; methyl eugenol (74.8%) and methyl cinnamate (8.0%). The essential oils were studied for their in-vitro antioxidant, anti-inflammatory and antimicrobial potential. All the oils revealed potential antioxidant activity with maximum in M. bracteata essential oil. All the oils exhibited significant antibacterial activity against Bacillus megaterium, Staphylococcus aureus, Escherichia coli,Salmonella typhimurium and anti-fungal activity against phytopathogenic fungi Fusarium oxysporum, Sclerotinia sclerotiorum, Exserohilum turcicum and Curvularia lunata. The observations from present study suggest further cultivation of Melaleucas and its commercialization as industrial crops.

Keywords:
Methyl eugenol; 1,8-cineole; (E)-nerolidol; Melaleuca Antioxidant; Antifungal; antimicrobial

HIGHLIGHTS

E-nerolidol, 1,8-cineol and methyl eugenol rich species of Melaleuca were identified in this study.

Essential oils exhibited strong antioxidant,antibacterial and Invitro anti inflammatory activity.

These marker constitutents impart valuable place of this crop in agriculture.

INTRODUCTION

The genus Melaleuca is an aromatic and medicinal shrub. The shrubs are generally found in open forest, woodland or shrubland, particularly along water courses and the edges of swamps [¹]. The Melaleuca genus belongs to the Melaleucae tribe, subfamily Myrtiodeae [²] and predominantly occurs in Australia. It comprises approximately 230 species of worldwide occurrence with 220 species endemic to Australia and Tasmania, but also occurring in Indonesia and New Papua Guinea [³,⁴]. The family Myrtaceae is well known for economically important timber trees, especially Eucalyptus spp., oils (e.g., Eucalyptus spp., Melaleuca spp.), and cultivated as ornamentals plants such as Callistemon (bottlebrush), Chamelaucium (wax-flower), Eucalyptus spp., Leptospermum (tea tree), and Myrtus (myrtle) [⁵]. The fleshy-fruited species include many economically important food plants, agricultural crops, and ornamentals, including the Mediterranean genus Myrtus (myrtle), spices such as clove (Syzygium aromaticum /L./ Merr. & L.M. Perry), allspice (Pimenta dioica /L./ Merr.), and bay rum (Pimenta racemosa / Mill./ J. W. Moore), and the fruits of Psidium (guavas), Myrciaria, Eugenia, Syzygium, Plinia and Luma[⁶]. The plants of genus Melaleuca are commonly known as tea tree and are rich source of commercially valuable volatile oils [⁷]. The essential oils of various Melaleuca species have been reported to show extensive compositional variability under different geographic and ecological conditions. Phenylpropanoids (methyl eugenol, (E)-methylisoeugenol), monoterpenoids (mainly, 1,8-cineole, terpinen-4-ol, terpinolene, along with p-cymene, α-terpinene, α-terpineol, α-pinene), and sesquiterpenoids ((E)-nerolidol, viridiflorol, ledol, β-caryophyllene) have been reported as marker constituents in the essential oils from most of the Melaleuca species[⁸-¹⁵]. Commercially useful essential oils have been sourced from the broad leaved M.quinquenervia (niaouli oil) and M. cajuputi (cajuput oil) and the small-leaved M. alternifolia-M. linariifolia complex[¹⁶]. These oil have been used mainly in the manufacture of cosmetics, germicides and as antiseptic agents. Studies also revealed the broad spectrum antimicrobial potential of essential oils [¹⁷,¹⁸]. The leaves and stem of several Melaleuca species are source of essential oils with strong aroma for medicinal application, with potential use for cancer treatment alongwith its use in traditional medicine against many pathological conditions like acne, wounds, sores, dandruff, and skin lesions [¹⁹-²¹]. Previous studies also suggest the allelopathic properties of Melaleuca species resulting in an inhibition of other species in the same ecosystem. The bare ground in Melaleuca forests was an excellent example of allelopathy in this genus [²²].

Literature survey revealed no pre-existing comparative study of Melaleuca species from different climatic zones of India. In present scenario people are again relying on natural resources for pharmaceutical and neutraceutical practices. Hence its need of time to screen these indigenous essential oil resources for their judicious exploitation, cultivation and commercialization. The present study was carried out to investigate the compositional diversity among the essential oils of three Melaleuca species viz; Melaleuca bracteata, Melaleuca linariifolia and Melaleuca leucadendron grown in Tarai region of North India. The bioactivity of these oils as antifungal, antimicrobial, and antioxidant agents was also assessed to evaluate their pharmaceutical and neutraceutical values.

MATERIAL AND METHODS

Plant materials and isolation of essential oil

The fresh aerial parts of Melaleuca species were collected from Medicinal Plant Research and Development Centre (MRDC) of the G.B.Pant University, Pantnagar. (M.bracteata and M.linariifolia) and Forest Training Institute, Lalkuan (M.leucadendra) (Nainital, Uttarakhand). Voucher specimen and herbarium record of the plant have been retained in Herbarium. Freshly collected leaves were hydro-distilled separately in a Clevenger apparatus for 3-4 h for extraction of essential oils. The essential oils were collected, dehydrated over anhydrous Na₂SO₄ and stored in an amber color vials at 4 ⁰C in refrigerator for further analysis.

Analysis and identification of essential oil constituents

The GC/MS analysis was carried out using Agilent 6890 gas chromatograph fitted with Mass Selective Detector 5973 and Autosampler 7683. Column HP-5 (30 m× 0.25 mm I.D.; 0.25 µm of film thickness) was used. The injector temperature was 250 ⁰C. Helium was used as carrier gas with a flow rate of 1.0 mL/min. Detector temperature was 150 ⁰C, source temperature was 230 ⁰C, ionization energy was 70 eV. Chromatograms were recorded in a programmed regime of linear temperature which increase from 50 ⁰C to 320 ⁰C at the rate of 3 ⁰C/min. Compounds of the essential oil and relative percentage composition are compiled. Linear retention indices (RI) were calculated using a homologous series of n-alkanes (C₈-C₂₅) under the same temperature-programmed conditions. The compounds were identified with the help of NIST 14 and Wiley 9 mass spectra libraries and by comparing the experimental RI with those from literature [²³].

Antioxidant activity

DPPH Radical scavenging activity

2,2-diphenylpicrylhydrazil (DPPH) free radical scavenging activity was performed by following the reported protocols with modifications [²⁴,²⁵]. Briefly the reaction mixture contained 5 mL of 0.004% methanol solution of DPPH and different amount ( 5- 25 µg/mL) of essential oils. The solutions were rapidly mixed and scavenging capacity was measured spectrophotometrically(Thermo Scientific Evolution 201) by monitoring the decrease in absorbance at 517 nm. BHT was used as positive control while reaction mixture (DPPH radical solution) minus essential oil solution was taken as control. Inhibition of free radicals (IC %) was calculated by using the formula:

$IC\%=A_0-A_s/A_0\times 100$ (1)

Where, A₀ = Absorbance value of Control sample A_s = Absorbance value of Test sample IC = Inhibitory concentration.Percent inhibition was plotted against concentration and the standard curve was drawn using standard antioxidant (BHT) to calculate the IC₅₀ values for standard and essential oils.

Metal chelating activity

The metal chelating activity of essential oils were examined by the reported methods [²⁶]. It was based on the principle of the Fe⁺² chelating ability where the absorbance of the Fe⁺² - ferrozine complex was measured at 562 nm. 0.1 mL of 2 mM FeCl₂.4H₂O, 0.2 mL of 5 mM ferrozine and 4.7 mL of methanol was added to different amounts (5 - 25 μg/mL) of essential oils. After incubation, the absorbance of test samples were measured at 562 nm. The IC% was calculated as:

$IC\%=A_0-A_s/A_0\times 100$ (2)

Where, A₀ = Absorbance value of Control sample A_s = Absorbance value of Test sample IC = Inhibitory concentration. Percent inhibition was plotted against concentration and the standard curve was drawn using standard antioxidant (citric acid) to calculate the IC₅₀ values for standard and essential oils.

Reducing power

The reducing power of essential oils were analyzed by the method developed earlier and recently being used [²⁷]. In brief varying amount (5-25 μg/mL) of essential oils were mixed with 2.5 mL of phosphate buffer (200 mM, pH= 6.6) and 2.5 mL of 1% potassium ferricyanide, K₃[Fe(CN)₆]. After 20 minute incubation at 50±1ºC, 2.5mL of trichloroacetic acid was added to the mixtures, followed by centrifugation at 650 RPM for 10 min. The upper layer (1.0 mL) was mixed with 5.0 mL of distilled water and 1 mL of 0.1% ferric chloride.The resultant solutions were measured at 700 nm using UV spectrophotometer. All the readings were taken in triplicate and BHT was used as the standard.

The reducing power of samples was calculated using the formula given below:

$Reducingactivity\%=A_0-A_s/A_0\times 100.$ (3)

Where, A₀ = Absorbance value of Control sample A_s = Absorbance value of test sample.Percent inhibition was plotted against concentrations and the standard curve was drawn using standard antioxidant (BHT) to calculate the RP₅₀ values for standard and essential oils.

Nitric oxide scavenging activity

Sodium nitroprusside (SNP) was used to generate nitric oxide and was measured by the Griess reagent. It is based on the principle that SNP in aqueous solution at physiological pH spontaneously generates NO, which interacts with oxygen to produce nitrite ions that can be estimated by the use of Griess reagent. Scavengers of NO compete with oxygen leading to reduced production of NO. 2 ml of SNP (10 mM) in phosphate buffer saline (PBS) pH 7.4 was mixed with different amount (5-25 μg /mL) of essential oil and incubated at 25°C for two and half hours. The above were reacted with 1 mL of Griess reagent (1% sulphanilamide, 0.1% naphthylethylenediamine dichloride and 2 mL orthophosphoric acid). Finally absorbance was measured at 546nm. Ascorbic acid was taken as standard [²⁸].

Super oxide radical scavenging activity

1 mL of nitroblue terazolium (156 mM), 1.0 mL nicotinamide adenine dinucleotide (reduced) (468 mM) and 0.1 mL of phenazine methosulphate solution (PMS) in 0.1 M of phosphate buffer solution (pH 7.4) were added to different amounts (5- 25 μg/mL), of essential oils followed by incubation at 25 °C for 5 min and the absorbance was read at 560 nm against blank, containing all reagent except PMS. Ascorbic acid was taken as standard [²⁹].

Super oxide radical scavenging activity was calculated by following equation.

$Superoxideradicalscavenged\left(\%\right)=IC\%=A_0-A_s/A_0\times 100.$ (4)

Where, A₀= Absorbance value of Control sample A = Absorbance value of Test sample IC = Inhibitory concentration. Percent inhibition was plotted against concentration. The standard curve was drawn using standard antioxidant (ascorbic acid) to calculate the IC₅₀ values for standard and essential oil.

Antifungal activity

Plant pathogenic fungi

The soil borne pathogenic fungi Fusarium oxysporum, Sclerotinia sclerotiorum, Curvularia lunata and Exserohilum turcicum were procured from the Department of Plant Pathology, College of Agriculture, G.B.P.U.A&T, Pantnagar, India. Cultures of each fungal species were maintained on Potato Dextrose Agar (PDA) and stored at 4⁰ C.

In-vitro antifungal activity

Antifungal activity of the oils was tested by the poisoned food technique using potato dextrose agar (PDA) medium against the test fungi. The essential oil samples were prepared by dissolving the requisite amounts in 10% DMSO and then added into 20 mL PDA to obtain different final concentrations [³⁰,³¹]. Mycelial plugs (2.0 mm in diameter) from the edges of each culture were incubated in the center of each PDA plate (85 mm diameter). The control sets were prepared using equal amounts of 10% DMSO in place of the oil. The prepared plates were inoculated aseptically with assay discs of the test fungi and incubated at 26 ± 2 ⁰C for 3-7 days until the growth in the control plates reached the edge of the plates. Growth inhibition of each fungal strain was calculated as the percentage inhibition of radial growth relative to the control. The plates were used in triplicate for each treatment [³²]. IC₅₀ values of constituents were graphically obtained from the dose response curves based on measurement at five different concentrations.

Antibacterial activity

Bacterial strain

Four bacterial strains of significant importance were used to test the antibacterial properties of the essential oils. The antimicrobial activity was tested against two Gram positive bacteria Bacillus megaterium andStaphylococcus aureus and two Gram negative Escherichia coliandSalmonella typhimurium. These strains were obtained from the Department of Veterinary Public Health, College of Veterinary, G.B.P.U.A.T, Pantnagar, India. All the strains were stored in the appropriate medium before use. The antibacterial activity was studied using standard protocol [³³].

Determination of the minimum inhibitory concentration (MIC)

The MIC was determined using standard protocol with the serial dilution of 5µg/mL,10 µg/mL,15 µg/mL, 20 µg/mL, 25 µg/mL. A 100 μL suspension containing 1 × 10⁶ CFU/mL of bacteria were spread on nutrient agar plates [³⁴]. The wells were filled with 50μL of essential oil solution in the inoculated nutrient agar plates. The bacterial plates were incubated at 37 ± 2°C for 24 hours. The MIC was defined as the minimum concentration of the oil inhibiting the visible growth of each bacterium on the agar plate, so the lowest concentration of each essential oil showing a clear zone of inhibition was taken as the MIC. DMSO was used as the negative control, while refamycin was used as positive control. Each experiment was performed in triplicate.

Determination of in-vitro anti-inflammatory activity

Inhibition of albumin denaturation

In vitro anti-inflammatory activity was performed by the method generally being practiced [³⁵]. The reaction mixture (5.0 mL) consisted of 0.2 mL of egg albumin (hen’s fresh egg), 2.8 mL of phosphate buffered saline (PBS, pH 6.4) and 2 mL of varying amounts (250-1000 ppm) of essential oils. Similar volume of double-distilled water was used as control. The mixtures were incubated at (37±2 °C) in an incubator for 15 min followed by heating at 70 °C for 5 min. After cooling, the absorbance was measured at 660 nm by using distilled water as blank. Diclofenac sodium was used as reference drug and treated similarly for determination of absorbance. The percentage inhibition of protein denaturation was calculated by using the following formula:

$\%inhibition=100x\left(Vt/Vc-1\right).$ (5)

Where, V_t = absorbance of test sample, V_c = absorbance of control. The oil concentration for 50% inhibition (IC₅₀) was determined by plotting percentage inhibition with respect to control against treatment concentration.

Statistical analysis

The mean values and standard deviations were calculated for all tests. The data were analyzed using SPSS 16.0 statistical software. The results were calculated by Analysis of Variance (ANOVA). The means were compared by Duncan tests at a level of significance of p < 0.05.

RESULTS

Chemical constituents of essential oils

The chemical composition of essential oils led to the presence of marker constituents previously recorded from Melaleuca species with slight qualitative and quantitative differences. GC-MS analysis of the essential oils led to the identification of over 90 constituents from three species. The oil yield for M.leucadendron was highest (1.02%) followed by M.bracteata (0.62%) and M.linariifolia (0.60%).The detailed composition has been presented in Table 1.

Table 1
Comparative Chemical compositions of Melaleuca species

S.N.	Compounds	RI	% Composition
			Present Study			Padalia et.al [36,37,49]
			M. leucadendron	M. linariifola	M. bracteata	M.leucadendron (2015)	M.linariifola (2015)	M.bracteata (2017)
1.	Methyl 2- methylbutanoate	780	0.1	0.1	t	-	-	-
2.	α-thujene	924	-	0.1	0.1	-	t	0.08
3.	α-pinene	932	0.4	4.0	0.1	t	1.99	-
4.	camphene	948	t	0.1	-	0.10	t	-
5.	benzaldehyde	960	1.2	-	t	-	-	-
6.	sabinene	969				0.64
7.	β-pinene	975	t	1.6	0.1	0.50	0.85	0.04
8.	6-methyl-5-hepten-2-one	985	0.1	-	-	-		-
9.	β-myrcene	990	t	3.8	0.3	0.10	1.78	0.12
10.	pseudolimonene	1002	-	0.2	-	-	-	-
11.	α-phellandrene	1002	-	-	1.0	-	-	0.16
12.	p-mentha -1(7),8-diene	1002					0.14
13.	α-terpinene	1014	-	0.3	0.1	-	-	-
14.	p-cymene	1021	-	-	0.7	-	0.21	0.36
15.	limonene	1025	0.4	-	0.3	-	0.15	0.30
16.	1,8-cineole	1030	0.1	61.1	0.2	0.30	77.40	0.34
17.	E-ocimene	1046	t	0.1	0.1		-	-
18.	γ-terpinene	1056	t	0.8	0.1	t	0.41	-
19.	α-terpinolene	1086	0.1	0.2	1.1	-	-	0.26
20.	Methyl benzoate	1093	t	0.1	t	-	-	-
21.	linalool	1099	0.7	0.2	1.2	0.52	0.19	0.94
22.	exo-fenchol	1120					0.10
23.	camphor	1141				0.38
24.	isopulegol	1142	0.4	-	t	-	-	-
25.	citronellal	1152	-	-	0.1	-	-	-
26.	δ-terpineol	1165	-	0.6	-	-	0.46	-
27.	Terpin-4-ol	1174	t	1.1	0.2	t	0.76	0.40
28.	p-cymen-8-ol	1183	-	-	0.1	-	-	-
29.	α-terpineol	1190	0.1	12.3	0.8	0.10	7.72	0.54
30.	Methyl chavicol	1197	-	-	0.4	-	-	0.32
31.	citronellol	1228	0.2	-	0.4	-	-	0.04
32.	neral	1239	-	-	0.1	0.22	-	-
33.	E-geraniol	1253	t	-	0.1	-	-	0.07
34.	linalyl acetate	1254				-
35.	Methyl citronellate	1260	-	-	0.1	-	-	-
36.	α-citral	1269	-	-	0.2	-	-	-
37.	geranial	1270						0.12
38.	bornyl acetate	1284				0.11
39.	S-methyl thiobenzoate	1290	0.1	-	-	-	-	-
40.	Z-methyl cinnamate	1304	-	-	0.1	-	-	-
41.	Methyl geranate	1323	-	-	0.1	-	-	-
42.	δ-elemene	1335				-
43.	α-terpinyl acetate	1346				0.53
44.	α-cubebene	1348	-	-	0.1	-	-	-
45.	Citronellyl acetate	1353	-	-	0.2	-	-	-
46.	eugenol	1356	0.1	0.3	0.5	-	t	0.04
47.	Neryl acetate	1364	-	0.1	-	-	t	-
48.	α-ylangene	1369					t
49.	isoledene	1371	-	0.1	-	-	-	-
50.	α-copaene	1374	-	-	0.1	-	t	-
51.	E-methyl cinnamate	1383	-	-	8.0	-	-	4.12
52.	Geranyl acetate	1383	-	0.1	-	-	t	-
53.	β-bourbonene	1387				-
54.	α-gurjunene	1408	-	0.1	-	-	-	-
55.	Methyl eugenol	1416	-	0.6	74.8	-	t	88.18
56.	E-β-caryophyllene	1417	0.9	1.7	-	1.52	2.88	-
57.	Z-β-copaene	1431	-	-	0.5	-	-	-
58.	α-guaiene	1432					0.46
59.	aromadendrene	1438	-	0.4	0.2	-	t	-
60.	Selina-5,11-diene	1441	-	0.1	-	-	-	-
61.	α-humulene	1452	0.2	0.3	0.2	0.22	0.31	-
62.	E-Muurola-3,5-diene	1453	-	-	0.1	-	-	-
63.	γ-gurjunene	1470	-	0.1	-	-	t	-
64.	Cabreuva oxide D	1475	0.1	-	-	-	-	-
65.	E-cadina-1(6),4-diene	1475	-	-	0.1	-	-	-
66.	D-germacrene	1480	-	-	1.1	-	-	0.60
67.	β-selinene	1484	-	0.1	-	-	-	-
68.	α-amorphene	1485					0.10
69.	δ-selinene	1489	-	0.1	-	-	-	-
70.	viridiflorene	1493	-	0.5	-	-	-	-
71.	bicyclogermacrene	1496	-	-	0.4	-	-	-
72.	E-methyl isoeugenol	1497						0.20
73.	α-muurolene	1499	-	0.1	0.1	-	0.18	-
74.	β-cadinene	1506	-	0.6	-	-	-	-
75.	E,E-α-farnesene	1508	0.1	-	-	-	-	-
76.	γ-cadinene	1513	-	0.1	0.1	0.10	0.12	-
77.	β-curcumene	1515					0.43
78.	E-calamenene	1522	-	0.2	-	-	0.10	-
79.	δ-cadinene	1522				-
80.	Z-calamenene	1524	-	-	0.7	-	-	-
81.	Z-nerolidol	1531				0.10
82.	Cadina-1,4-diene	1532	-	-	0.2	-	-	-
83.	α-calacorene	1542	-	1.1	t	-	-	-
84.	ledol	1557	-	0.2	-	-	-	-
85.	elemicin	1558	-	-	0.2	-	-	0.30
86.	germacrene-B	1558					0.99
87.	palustrol	1565	-	0.2	0.1	-	-	-
88.	E-nerolidol	1568	85.7	-	-	90.85	0.10	-
89.	spathulenol	1575	-	0.2	0.3	-	t	0.22
90.	Caryophyllene oxide	1581	3.7	-	-	0.99	0.10	0.14
91.	globulol	1582	-	1.2	0.3	0.70	-	-
92.	viridiflorol	1591	0.6	0.3	0.1	0.19	-	-
93.	Cubeban-11-ol	1592	-	0.5	0.1	-	0.52	-
94.	fokienol	1599	0.4	-	-	-	-	-
95.	juneperol	1599					t
96.	rosifoliol	1600	-	0.4	0.1	-	t	-
97.	Humulene epoxide II	1608	0.3	-	t	-		-
98.	1,10-di-epi-cubenol	1612	-	1.3	t	-	0.10	-
99.	10-epi-γ-eudesmol	1622				0.14
100.	epicubenol	1626	-	t	0.1	-	0.25	-
101.	Allo-aromadendrene epoxide	1632	0.1	-	-	-	-	-
102.	isospathulenol	1636	-	0.2	-	-	-	-
103.	Caryophylla-4(12),8(13)-diene-5-α-ol	1636	0.5	-	-	-	-	-
104.	τ-cadinol	1638	-	0.2	-	-	-	-
105.	epi-α-cadinol	1638				0.20	t
106.	τ-muurolol	1640	-	-	0.5	-	-	-
107.	α-muurolol	1643						0.20
108.	δ-cadinol	1644	-	0.1	0.1	-	-	-
109.	β-eudesmol	1649				t
110.	α-cadinol	1653	-	0.1	0.4	0.33	-	0.14
111.	E-14-hydroxy-9-epi caryophyllene	1670	0.1	-	t	-	-	-
112.	cadalane	1672	-	0.1	-		t	-
113.	β-bisabolol	1675				t
114.	(Z)-nerolidyl acetate	1679				t
115.	α-bisabolol	1685	0.2	-	-		-	-
116.	2Z,6Z-farnesol	1698	0.1	-	-		-	-
117.	2Z,6E-farnesol	1721	0.7	0.3	-		-	-
118.	Iso-longifolol	1732	0.1	-	-		-	-
119.	methylconiferylaldehyde	1777	-	-	0.3		-	-
120.	phytol	2112	-	0.3	-		-	-
121.	Total identified		97.8	99.0	98.1	98.84	98.90	98.24
122.	Monoterpene hydrocarbons		0.9	11.2	4.0	1.34	5.53	1.30
123.	Oxygenated monoterpenes		1.6	75.6	12.4	2.16	86.63	2.42
124.	Sesquiterpene hydrocarbons		1.2	5.7	3.9	1.84	5.67	0.60
125.	Oxygenated sesquiterpenes		92.2	5.3	2.1	93.50	1.07	0.70
126.	Others		1.9	0.6	75.7	-	-	93.14

• Mode of identification: RI (retention index), MS, t = trace (<0.1%)

The most abundant component of essential oil of M.leucadendron was E-nerolidol (85.7%). Other major components identified were caryophyllene oxide (3.7%), benzaldehyde (1.2%), 2Z,6E-farnesol (0.7%), viridifloral (0.6%) and caryophyllene (0.9%) etc. The major constituents identified in M.linarifolia essential oil were 1,8-cineole (61.1%) along with significant presence of α-terpineol (12.3%), α-pinene (4.0%), β-myrcene (3.8%), E-caryophyllene (1.7%), α-calacorene (1.1%), globulol (1.2%), 1,10-di-epi-cubenol (1.3%), β-pinene (1.6%) while eugenol methyl ether (74.8%) was identified as a principal component in M.bracteata essential oil along with methyl cinnamate (7.97%). The RI and percentage contribution are shown in Table 1. The essential oils from M.bracteata and M. lineriifolia showed quantitative variability in chemical composition when compared with the earlier reports [³⁶,³⁷]. The chemical composition of essential oil of Melaleuca leucadendron was mostly composed of oxygenated sesquiterpenes (92.2%), whereas Melaleuca linariifolia essential oil consisted of oxygenated monoterpenes (75.6%). The Melaleuca bracteata essential oil was characterized by dominance of phenylpropanoid methyl eugenol (74.8%).

The compounds α-phellandrene (1.0%), p-cymene (0.7%), methyl chavicol (0.4%), α-citral (0.2%), methylcinnamate (8.0%), β-copane (0.5%), D-germacrene (1.1%), bicyclogermacrene (0.4%), calamenene (0.7%), cadina-1,4-diene (0.2%), elimicin (0.2%), τ- muurolol (0.5%), methyl coniferylaldehyde (0.3%) were found in M. bracteata essential oil but were missing in M. leucadendron and M. lineriifolia essential oils, whereas caryophyllene oxide (3.7%), fokienol (0.4%), caryophylla-4(12),8(13)-dien-5-alphaol (0.5%), epi-α-bisabolol (0.1%), α-bisabolol (0.2%), 2Z,6Z-farnesol (0.1%), iso-longifolol (0.1%), the minor constituents of M. leucadendron were absent in the other two essential oils. Similarly, psuedolimonene (0.2%), δ-terpineol (0.6%), viridiflorene (0.5%), β-cadinene (0.6%), E-calamene (0.2%), ledol (0.2%), isospathulenol (0.2%), τ-cadinol (0.2%), phytol (0.3%) were identified in M.lineriifolia essential oil but were absent in M.leucadendron and M.bracteata essential oils. Previously 1,8-cineole has been reported as the major component in the essential oil of M. leucadendron grown in Cuba, Ivory Coast, India, Indonesia[⁷,³⁸,³⁹]. Another report from Thiland revealed terpinolene (29.21%), α-terpinene (22.55%), 2-carene (8.53%) and α-phellandrene (7.61%) as the major compounds of M. leucadendron essential oil. Recently Siddique and coauthors,reported eugenol methyl ether (95.4%) as the dominant component of M. leucadendron essential oil [⁴⁰]. Our findings corroborate the earlier report , where E-nerolidol (90.85%) was the major component although the quantity of E-nerolidol (85.7%) was smaller than reported in previous findings[⁴¹]. Caryophyllene oxide (3.7%) in the present study is higher in amount than that of earliar report[⁴¹].

Our results of M. linariifolia essential oil characterized it as a rich source of monoterpenoides such as 1,8-cineole and α-terpineol, α-pinene, β-pinene, β-myrcene. As per earlier reports, the essential oil composition of M. linariifolia from Australia was characterized as ‘terpinen-4-ol’ and ‘1,8-cineole’ type based upon the dominance of these monoterpenoids[¹⁶,⁴²-⁴⁴]. However, the volatile oil of M.linariifolia from Brazil was shown to be characterized with a high content of methyleugenol (86.8%) [¹³]. Our results show similarity with the results published by Padalia and coauthors, 2015a where 1,8-cineole (77.40%) and α-terpineol (7.72%) were the major components with some differences in quantitative makeup [³⁶]. Aboutabl and coauthors investigated the essential oils from three Melaleuca species growing in Egypt and reported that eugenol is the major component of M. bracteata (97.7%)[⁸]. Pino and coauthors isolated the leaf essential oil of M. leucadendron from Cuba and identified it as a Virdifloral type chemotype with major presence of viridiflorol (28.2%) and 1,8-cineole (21.3%) [⁴⁵].In another report, Pino and coauthors analyzed the phytochemical composition and antioxidant activity of leaf and fruit essential oil of M. leucadendron from Cuba[⁴⁶]. A total of fourty one and sixty four compounds were identified and the major portion of leaf and fruit essential oil was composed of 1,8-cineole (43.0%) and virdifloral (47.6%). The other major compounds detected were viridiflorol (24.2%), α-terpineol (7.0%), α-pinene (5.3%), and limonene (4.8%) in the leaf oil, whereas the fruit essential oil consisted of globulol (5.8%), guaiol (5.3%), and α-pinene (4.5%)[⁴⁶].

The composition report of the essential oil of M.bracteata was in agreement with the previous reports where methyl eugenol was the major component of the leaf essential oil [⁸,⁴⁷,⁴⁸].Based on the quantity of nerolidol, 1,8-cineole and methyl eugenol in present study it can be concluded that these shrubs can be good natural source of these neutaceutically important terpenoids by developing the agricultural practices to cultivate these species in wastelands which can be also a good source of revenue generation for local residents.

Antioxidant activity

DPPH Radical scavenging activity

All the essential oils showd scavenging activity in a dose dependent manner (Table 2). In this study, the antioxidant activity of essential oils were compared with BHT, a reference standard antioxidant compound. It was found that the essential oils of Melaleuca species exhibited good antioxidant activity. The essential oil from M. bracteata was found most effective antioxidant (IC₅₀ = 4.06 ± 0.02 µg/mL) followed by M.linariifolia (IC₅₀ = 10.96 ± 0.00 µg/mL) compared to BHT (IC₅₀ = 9.29 ± 0.09 µg/mL), whereas M.leucadendron essential oil revealed moderate potential (IC₅₀ = 16.24 ± 0.05 µg/mL) towards radical scavenging activity(Table2).

Metal chelating activity

All the essential oils exhibited dose dependent metal chelating activity. Melaleuca leucadendron and Melaleuca linariifolia essential oils showed remarkable metal chelating activity with IC₅₀ of 9.56 ± 0.31 µg/mL and 10.51 ± 0.39 µg/mL compared to positive control citric acid (IC₅₀ = 4.04 ± 0.63 µg/mL) while Melaleuca bracteata essential oil with IC_{50 of} 8.80±0.44 µg/mL exhibited highest chelating activity among all the tested oils(Table2).

Reducing power

The Melaleuca bracteata essential oil was found most effective in reduction of Fe^{+ 3} (IC₅₀ = 2.11 ± 0.39 µg/mL) depicting comparable potential with standard reducing agent BHT (IC₅₀ = 2.46 ± 0.25 µg/mL).The Melaleuca linariifolia essential oil demonstrated significant reducing capability (IC₅₀ = 4.18 ± 0.33 µg/mL) whereas moderate activity was observed in M.leucadendron essential oil (Table2).

Super oxide radical scavenging activity

M.leucadendron (IC₅₀ = 20.92 ± 0.31 µg/mL) and M.bracteata (IC₅₀ = 21.79 ± 0.85 µg/mL) essential oils demonstrated significant superoxide radical scavenging activity compared to standard ascorbic acid (IC₅₀ = 9.85 ± 0.16 µg/mL) as indicated by their IC₅₀ values. A moderate radical scavenging activity was observed for M.linariifolia essential oil (IC₅₀ = 25.83 ± 0.19 µg/mL).

Nitric oxide(NO) radical scavenging activity

All the tested essential oils exhibited strong nitric oxide (NO) radical scavenging activity. Remarkably strong nitric oxide radical scavenging potential was observed in Melaleuca bracteata essential oil (IC₅₀ = 11.59 ± 0.09 µg/mL) compared to ascorbic acid (IC₅₀ = 10.36 ± 0.45 µg/mL). M. linariifolia (IC₅₀ = 12.68 ± 0.09 µg/mL) and M. Leucadendron were the least active among the three tested essential oils (IC₅₀ = 16.17 ± 0.06 µg/mL). All the tested oils demonstrated radical scavenging activity in a dose dependent manner with maximum scavenging at highest concentration (Table 2).

Table 2
Antioxidant activity (IC₅₀) of essential oils of Melaleuca Species.

Essential oil/Standard	Antioxidant activity(IC50)
	DPPH radical Scavenging	Metal chelating	Reducing power	NO Radical scavenging	SO Radical Scavenging
M.leucadendron	16.24±0.05	9.56±0.31	12.89±1.72	16.17±0.06	20.92±0.31
M.linerifolia	10.946±0.00	10.51±0.39	4.18±0.33	12.68±0.09	25.83±0.19
M.bracteata	4.06±0.02	8.80±0.44	2.11±0.39	11.60±0.09	21.79±0.85
Citric acid*	-	4.04±0.63	-	-	-
BHT*	9.29±0.09	-	2.46±0.25	-	-
Ascorbic Acid*	-	-	-	10.36±0.45	9.85±0.16

• Values are mean ± S.D.,*Standard antioxidant

Antifungal activity

The essential oils exhibited moderate to good antifungal potential by inhibiting the mycelial growth of pathogenic fungi. The inhibitory effect of the oil varied from 7.08% to 90% (Table 3). At the concentration of 500 ppm, the oils showed highest inhibitory effect on the radial growth of S. sclerotiorum. S. sclerotiorum and E.turcicum were found to be the most inhibited fungal pathogens by the oils. Essential oil of M. bracteata exhibited a strong antifungal effect against all the tested phytopathogens (up to 90%) as compared to other oils.

Table 3
Antifungal activity of essential oils of Melaleuca Species

Essential oil	Conc. (ppm)	Mycelial growth inhibition (%)
		Fusarium oxysporum	Sclerotinia sclerotiorum	Curvularia lunata	Exserohilum turcicum
M.leucadendron	500	18.95±1.301a	80.83±1.30a	39.66±3.53a	56.66±1.11a
	250	12.91±0.72b	77.70±0.95ab	38.75±2.5a	55.18±0.64ab
	100	12.08±1.90bc	75.83±3.20b	35.41±2.00ab	54.81±0.64b
	50	11.33± 1.01bc	75.16±0.314b	32.91±3.14b	52.59±1.28c
	25	10±1.25c	71.66±1.44c	32.91±1.57b	51.11±0.96c
IC50(ppm)		2332.65	1465.22	1136.35	318.57
M.linariifolia	500	28.95±2.00a	58.58±0.88a	38.16±0.57a	59.07±1.15a
	250	20.37±1.51b	51.87±1.08b	35.62±1.65a	55.55±1.11b
	100	9.79±1.57c	50.87±3.68b	31.25±1.25b	54.62±3.61bc
	50	9.16±1.57c	25±1.25c	27.91±3.14b	54.44±2.22bc
	25	7.70±0.368c	7.08±1.44d	23.54±3.55c	52.40±2.50c
IC50(ppm)		938.50	318.81	883.13	253.02
M.bracteata	500	46.87±1.65a	82.91±0.36a	75.20±0.36a	90±1.11a
	250	36.12±1.59b	76.04±0.95b	60.20±0.954b	88.51±0.64a
	100	31.87±2.86c	63.87±0.45c	32.29±2.81c	65.18±0.64b
	50	17.29±1.57d	47.08±2.00d	31.08±1.44c	58.14±1.69c
	25	7.29±0.95e	27.70±0.36e	20.12±0.66d	54.62±3.61c
IC50(ppm)	25	498.11	85.88	239.97	89.70
Carbendazim		100.00±0.00	100.00±0.00	100.00±0.00	100.00±0.00

• Values are Mean ± S.D. , Within a column means followed by the same letter are not significantly different (P > 0.05). Means followed by a different letter are significantly different (P < 0.005).

Antibacterial activity

The zone of inhibition around the well diameters and minimum inhibitory concentrations (MICs) of the essential oils against the microorganisms tested are shown in Table 4. The results obtained from the agar well diffusion method for the essential oils revealed S. typhimurium to be the most sensitive microorganism with the largest inhibition zones (20.67, 20.67 and 23.67 mm against M. leucadendron, M. linariifolia and M. bracteata essential oils respectively), while the minimum zone of inhibition were exhibited by S.aureus (16.33, 14.00, 15.67mm) against M.leucadendron, M.linariifolia and M.bracteata essential oils respectively.

Table 4
Antibacterial potential of essential oils of Melaleuca Species

S.N.	Sample	Zone of inhibition(mm)
		B.megaterium	S.typhimurium	E.coli	S.aureus
1.	M.leucadendron 10 15 20 25
		11.33 ± 0.58c	12.67±0.58d	16.33±1.15b	10.33±1.52c
		13.33 ±0.58b	14.33±0.58c	17±0.00b	13.67±0.58b
		14.33±0.58b	16.33±0.58b	18.33±0.58a	15.67±0.58a
		18.33±0.58a	20.67±0.58a	19.00±0.00a	16.33±1.15a
	MIC(µg/mL)	10	10	5	10
2.	M.linariifolia 10 15 20 25
		10.33±0.58c	11.67±0.58c	13.33±0.58c	8.67±1.15d
		12.67±0.58b	15.33±0.58b	14.67±0.58b	10.33±0.58c
		13.67±0.58b	16.33±0.58b	16±1.00a	12.33±0.58b
		19.67±0.58a	20.67±1.15a	17±0.00a	14±0.00a
	MIC(µg/mL)	10	10	5	10
3.	M.bracteata 10 15 20 25
		11±0.00c	16.67±0.58c	13.33±0.58d	10.67±1.15c
		11.67±0.58c	17.67±0.58c	15.67±0.58c	12.67±1.15b
		13.33±0.58b	20.67±0.70b	18.67±1.15b	13.67±1.15b
		14.67±0.58a	23.67±0.58a	20.67±0.58a	15.67±0.58a
	MIC(µg/mL)	10	5	5	10
4.	Refamycin 25 µg/mL
		31.33±1.22a	35±1.00a	32.67±1.15a	27±1.00a

• Values are Mean ± S.D. Within a column means followed by the same letter are not significantly different (P > 0.05). Means followed by a different letter are significantly different (P < 0.005).

In-vitro anti-inflammatory activity

As part of the investigation on the mechanism of the anti-inflammatory activity, ability of essential oils to inhibit protein denaturation was studied. All the tested oils were found effective in inhibiting heat induced albumin denaturation (Table 5). The IC₅₀ for M. leucadendron essential oil was 27.82 ± 0.24 µg/mL whereas that of M. linariifolia was 23.43 ± 0.25 µg/mL. Melaleuca bracteata essential oil was found most effective in inhibiting protein denaturation with IC₅₀ of 20.68 ± 0.004 µg/mL compared to the standard diclofenac sodium (IC₅₀ = 11.93 ± 0.03 µg/mL).

Table 5
Anti-inflammatory activity of essential oils of Melaleuca Species

Anti inflammatory activity	Essential oil/Standard
	M.leucadendron	M.linerifolia	M.bracteata	Diclofenec sodium
IC50 Value	27.82±0.242	23.43±0.247	20.68±0.004	11.93±0.033

DISCUSSION

This study was undertaken to compare the essential oil composition, antioxidant and biological activity of the three Melaleuca species: Melaleuca leucadendron, Melaleuca linariifolia and Melaleuca bracteata from Northern plains of India. Differences and similarities in the groups of terpenes were established between the three Melaleuca species based on the present studies. The major findings from the three species were (E)-nerolidol, 1,8-cineole and methyl eugenol. M.bracteata exhibited highest antioxidant activity followed by M.linariifolia and M.leucadendron essential oil. Oils exhibited significant antibacterial activity. The microorganisms selected in our studies are mostly human pathogens whose effects are noticeable in skin, intestinal and respiratory infections. The essential oils revealed remarkable antimicrobial potential against all the tested organisms. This observation is particularly noteworthy because essential oils are known to be more active against gram-positive than gram-negative bacteria but in our study essential oils exhibited noticeable activity against all the bacterial species tested [⁵⁰]. All the oils showed moderate to strong anti-inflammatory potential within the tested range of concentrations. The results also revealed differential ability of oils in inhibiting the phytopathogenic fungal growth.

(E)-nerolidol a sesquiterpene alcohol with pleasant odour have several reported biological and pharmacological activities such as antimalarial, antileishmanial, antiulcer, antibacterial, antifungal, and as a topical skin penetration enhancer whereas 1,8-cineole is extensively used in food-flavor, pharmaceutical, and cosmetic industries [⁵¹-⁵⁴]. Several pharmacological activities including anti-microbial, anticancer, anti-inflammatory, antioxidant, bactericidal, herbicidal, insecticidal have been reported for 1,8-cineole [¹³,⁵⁵,⁵⁶]. In addition 1, 8-cineole possess strong larvicidal, insecticidal, fumigant, repellent and antifeedent and anti-inflammatory activities [⁴³, ⁵⁶-⁵⁸]. Methyl eugenol has been reported to possess antiepileptic and anti-inflammatory activity [⁵⁹, ⁶⁰]. Eugenol was found to inhibit the growth of various multi drug resistant pathogenic bacteria such as E. coli, Staphylococcus, Proteus, Klebsiella, Enterobacter, H. pylori and Pseudomonas isolated from human subjects [⁶¹, ⁶²]. Thus various reports supports our claim that the antioxidant, antimicrobial and antinflammatory activity of essential oils could be due to their major components such as 1.8-cineole, methyl eugnol, E-nerolidol. Nevertheless, the presence of minor component could also play a role in the biological activity.

CONCLUSION

The increasing population pressure and constraint of agriculture lands due to several developmental activities in India have raised alarm for food security and naturally the essential oil crops may limit their cultivation. Melaleuca species can grow in wild and are perennial species which have less agriculture cost inputs for their commercial production and can be grown in wastelands and used for filling of damp areas. The high content of (E)-nerolidol in the essential oil of M. leucadendron suggests this species as a new potential source of (E)-nerolidol which have a widespread use in the food-flavor, perfumery, cosmetics and pharmaceutical industries. The oil yield , 1,8-cineole content, and the biological activity of essential oil from M. linariifolia favours the cultivation and promising use of this species for isolation of 1,8-cineole and in pharmaceutical industry. Another species Melaleuca bracteata was found as a rich source of methyl eugenol, the commercially important molecule. All the oils showed significant in-vitro antifungal and antibacterial activity against tested fungal phytopathogens and bacterial strains responsible for plant diseases and several chronic conditions. The anti-inflammatory potential gives additional remarks to these species. Conclusively the outcomes of chemical composition and various activities suggested the cultivation and development of these species as commercial crops in agro forestry systems.

Acknowledgments

We thankfully acknowledge Dept. of Veterinary Public Health (VPH) for providing experimental facilities and Department of Biological Sciences, G.B., Pant University of Agriculture and Technology, Pantnagar for taxonomic identification and providing voucher specimen

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• Funding:

  This research received no external funding.

Edited by

Editor-in-Chief:

Alexandre Rasi Aoki

Associate Editor:

Jane Manfron Budel

Publication Dates

• Publication in this collection
  05 Jan 2022
• Date of issue
  2021

History

• Received
  26 Mar 2021
• Accepted
  10 June 2021