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INTRODUCTION
The Melaleuca bracteata F. Muell. belongs to Myrtaceae family of plants which is diverse, with essential oils rich in various genera such as Melaleuca, Eucalyptus, Leptospermum, Myrtus, Pimenta, Plinia, Psidium, Eugenia Pseudocaryophyllus and Syzygium (Siddique et al., 2020). Melaleuca, a genus with around 230 species, is widely distributed, mainly in Australia and Tasmania and shows significant phenotypic diversity in different ecosystems (Joshi et al., 2021, Fatma et al., 2020). Essential oils from Melaleuca species exhibit antibacterial, anti-inflammatory, fungicidal, acaricidal, antioxidant, and antiviral properties. Major constituents such as methyl eugenol, methyl ether, 1,8-cineole, and terpinen-4-ol have been identified in the essential oils of various Melaleuca species from different regions. Melaleuca bracteata, also known as black tea-tree or river tea-tree, is widely distributed across eastern, central, and northern Australia. It is cultivated globally for its ornamental value and medicinal properties. It is a versatile plant, ranging from a large shrub to a medium-sized tree, typically reaching heights of up to 15 m. Its foliage consists of small, narrow, and hairy leaves measuring between 3 to 12 mm long. The plant produces small bottlebrush-like flowers, about 20 mm long, primarily at the tips of its twigs, with woody, cup-shaped capsules as fruits, measuring 2-3 mm long and 2.5-3 mm wide, appearing on branches (Babu et al., 2022). Previous studies have highlighted its medicinal properties, including reported antisecretory, antiulcerogenic, anti-HIV, cytotoxic, insecticidal, antifungal, and growth-inhibition activities. The essential oil of this plant varies in composition based on geographic region. Major components include methyl eugenol, elemicin, (E)-isoelemicin, and (E)-methyl isoeugenol. The essential oil from Malaysian origin, for instance, contained 76.0% methyl eugenol with significant fruit fly attractant activity. Betulinic acid, oleanolic acid, water-soluble betaines, and triterpene, betulinic acid, have been characterized in extracts from different parts of the plant, showing anti-HIV, cytotoxic, and antifungal activities (Goswami et al., 2017). Uttarakhand, known for its natural beauty in the Himalayas, is a treasure trove of plants and animals. It has different climates, soils, and landscapes that support many useful plants, both wild and grown. The region's height varies from 200 to 7817 meters above sea level, and it's divided into five parts based on its geology. These parts include areas like the Trans-Himalayas, Great Himalaya, Lesser Himalaya, and the Outer Himalaya, which includes the Tarai and Bhabhar. The highest parts are icy, while the lower areas are covered in forests and grasslands. Some major cities, such as Haldwani and Ramnagar, sit in a region called the Himalayan Bhabar belt, which is a stretch of land along the Himalayan foothills (Kholiya et al., 2023). The Kumaun Himalayan Bhabhar region spread over a geographical area of 51125 km2, 77034` to 81002`E longitude and 28043 to 31027` N latitude (Padalia et al., 2018). The flowing water from Shivalik Himalayas is submerged here due to lack of porosity nature of soils that leads to dry land surface over the area. Towards its south, the Tarai belt is running parallel to the Bhabar that composed with wet to marshy land through the reemergence of the river and regarded as a cap of sediments, silt etc. form of deposition as well (Devi, 2020) A thorough examination of existing literature uncovered a gap in the exploration of the medicinal and aromatic properties of M. bracteata found in Himalayan and bhabar belt of Kumaun Uttarakhand.
MATERIALS AND METHODS
Sample Collection
The Plant Material: Fresh plant material of Melaleuca bracteata including leaves and flowers were collected from, Haldwani (29.2183° N & 79.5130° E), Nainital, Uttarakhand.
Extraction Method: Essential oil was extracted from M. bracteata via the hydrodistillation technique through Clevenger type apparatus (Clevenger, 1928) at the Department of Chemistry, M B Govt. P. G. College Haldwani. The aerial parts, comprising stems and leaves (200 g) of the plant, were thoroughly cleaned before undergoing hydro-distillation using a Clevenger-type apparatus for approximately 4 hr. The weight of the dried samples was recorded before hydro-distillation. The essential oil contents (%) were expressed as volume of essential oil vs. weight of fresh leaves (v/w).
The obtained oil was dehydrated using anhydrous Na2SO4 and stored in a refrigerator until analysis by GC-FID/GC-MS.
Chemical analysis of essential oil through GC-MS
GC–MS was used to analyze and identify the phytochemical content of the essential oil using GCMS-QP 2010 Ultra equipment with helium as the carrier gas at a pressure of 73.3 kPa and a split ratio of 10:1. The overall flow rate was 16.3 mL/min during the study, with a column flow rate of 1.21 mL/min, 39.9 cm/sec linear velocity and 3 mL/min purge flow were maintained, respectively. Carrier gas saver, high-pressure injection, and splitter hold were all turned off, and the oven temperature was set to 50°C RAMP@ 3°C/min up to 220°C (isotherm for 2 min), then 6°C/min up to 270°C (isotherm for 2 min), then hold for 16 min, using Flame Thermionic Detector (FTD). The components were identified by comparison with linear Retention Indices (RI) from literature Adams (Adams, 2007). Mass spectra with those of NIST mass spectral library or coinjection with standards.
Biological Activities
Antioxidant Activity
DPPH (2,2-diphenyl-2-picrylhydrazyl) radical scavenging activity
To measure DPPH (2,2-diphenyl-2-picrylhydrazyl) radical scavenging activity, 5 μL of various test compound stocks (ranging from 0% to 5%) were added to 0.1 mL of 0.1 mM DPPH solution in a 96-well plate. The reaction was conducted in triplicate, with duplicate blanks containing 0.2 mL DMSO/Methanol and 5 μL of different compound concentrations. The plate was incubated in the dark for 30 min. Post-incubation, decolorization was measured at 495 nm using an iMark microplate reader (BioRad) (Abubak et al., 2020, Imam et al., 2011). A reaction mixture with 20 μL deionized water served as the control. The scavenging activity was expressed as '% inhibition' relative to the control, and IC50 was determined using Graph Pad Prism 6 software. The percentage inhibition (IC %) of free radical by DPPH was calculated using the formula:
Where, Abs control and Abs sample represent the absorbance value for control sample and test sample, respectively.
ABTS Radical Scavenging Ability: ABTS radicals were prepared by mixing 2.45 mM APS and 7 mM ABTS solution, then diluted 100-fold to create the ABTS free radical reagent. In a 96-well plate, 10 µL of various sample stocks (0 to 2.5%) were added to the standard (Ascorbic Acid, 5 mg/mL) and 200 µL of the ABTS free radical reagent. The mixture was incubated in the dark at room temperature for 10 min. After incubation, the absorbance of the decolorisation was measured at 750 nm using an iMark microplate reader (BioRad) (Cao et al., 1998, Gupta et al., 2009). Results were compared to the negative control, and the IC50 was calculated using Graph Pad Prism 6 software. The percentage inhibition (IC%) of free radical by DPPH was calculated using the formula:
Where, Abs control and Abs sample represent the absorbance value for control sample and test sample, respectively.
Hydroxyl Free Radical Scavenging Assay: A reagent mixture of 66 µL was prepared, consisting of 10 µL EDTA (0.5M), 24.14 mg deoxyribose, 88 µL FeCl3 (10 mg/mL), 28 µL H2O2 (6%), and water up to 33 mL. To the wells of a 96-well plate, 10 µL of the sample (0–5%), 24 µL of phosphate buffer (50 mM, pH 7.4), and 10 µL of ascorbic acid were sequentially added. The mixture was then incubated at 37°C for 1 hr. Gallic Acid (0-50 μg/ml) was used as the standard. After incubation, 50 µL of 10% TCA and 50 µL of 1% TBA were added to each well, resulting in the development of a pink chromogen. Absorbance was measured at 540 nm using a microplate reader (iMark, BioRad) (Rahman et al., 2015, Hazra et al., 2008). The IC50 was calculated using GraphPad Prism 6 software.
Where, Abs control and Abs sample represent the absorbance value for control sample and test sample, respectively.
Reactive Nitrogen Oxide Scavenging Assay: A reaction mixture was prepared containing 50 μL of 10 mM sodium nitroprusside (Fisher Scientific, Cat no.-27864), 40 μL of distilled water, and 10 μL of sample/standard (Gallic Acid – SRL, Cat no.- 13142)/blank. This mixture was pre-incubated at room temperature for 15 min in the presence of light. After incubation, 100 μL of Griess reagent was added to the test and control wells, followed by an additional incubation for 5-10 min at room temperature for chromophore development and stabilization. Absorbance was measured at 540 nm and 660 nm using a microplate reader (iMark, BioRad) (Rao, 2013). The IC50 was calculated using GraphPad Prism 6 software.
Where, Abs control and Abs sample represent the absorbance value for control sample and test sample, respectively.
Antibacterial Activity
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 subtilis (MTCC 1133) and Staphylococcus aureus (MTCC 96) and two Gram negative Escherichia coli (MTCC 452) and Pseudomonas aeruginosa (MTCC 3541). 0.5 Mc Farland
Standard dilution of microbes to be used for the study. 100 µL diluted log cultures of bacteria was added to the micro centrifuge tube and added with 5 µL of prepared treatment dilutions of different concentrations 0, 6.25, 12.50, 25.00, 50.00, 100 to the defined tubes and incubated for 24 hr. After Incubation all content was transferred to the 96 well plate and turbidity reading was taken by Elisa Plate Reader (iMark Biorad) at 630 nm. Ciprofloxacin (10 µg) was used as Positive Control.
RESULTS
Essential oil composition of aerial part: A total of 32 compounds were identified, accounting for 99.95% and 99.31% of the essential oil from aerial parts and flowers respectively. The predominant compound in both oils was methyl eugenol, (90.8%-77.18%) of the total composition, followed by (E)- methyl cinnamate (6.42 %-13.26%), linalool (0.23%-2.21%) and α-terpineol (0.24%-1.20%). 4-terpineol, elemicine, spathulenol, cadin-4-en-10-ol, benzoic acid, 3,4,5-trimethoxy methyl ester, conifer aldehyde methyl ether are the other compounds common in both essential oils. Phenylpropanoids are the major constituents in the essential oils of both the oils marking 96.97 and 90.83 of the total constituents. The percentage composition of the compounds identified in both the essential oils is given in Table 1. Collation of the current results with earlier reports from India revealed noteworthy qualitative variations.
| Sl. No. | Compound Name | R.T. | RIexp | Percentage Concentration | |||||
|---|---|---|---|---|---|---|---|---|---|
| Aerial Part | Flower | Goswami et al., 2017 | Joshi et al., 2021 | ||||||
| 1 | α-thujene | 7.367 | 927 | - | t | 0.08 | 0.1 | ||
| 2 | α –pinene | 7.614 | 933 | - | t | - | 0.1 | ||
| 3 | Sabinene | 9.112 | 972 | - | t | - | - | ||
| 4 | β- pinene | 9.28 | 978 | - | t | t | 0.1 | ||
| 5 | Myrcene | 9.819 | 991 | - | t | 0.12 | 0.3 | ||
| 6 | α-phellandrene | 10.476 | 1007 | - | t | 0.16 | 1 | ||
| 7 | p-cymene | 11.281 | 1025 | t | 0.43 | 0.36 | 0.7 | ||
| 8 | Limonene | 11.466 | 1030 | - | t | 0.3 | 0.3 | ||
| 9 | 1,8-cineol | 11.569 | 1030 | - | 0.3 | 0.34 | 0.2 | ||
| 10 | α – terpinolene | 13.947 | 1086 | - | t | 0.26 | 1.1 | ||
| 11 | linalool | 14.796 | 1101 | 0.23 | 2.21 | 0.94 | 1.2 | ||
| 12 | Citronellal | 17.083 | 1152 | - | t | - | 0.1 | ||
| 13 | (-)-4-terpineol | 18.358 | 1174 | - | 0.17 | 0.4 | 0.2 | ||
| 14 | cymen-8-ol | 18.815 | 1197 | - | 0.19 | - | - | ||
| 15 | α- terpineol | 19.14 | 1201 | 0.24 | 1.2 | 0.32 | 0.4 | ||
| 16 | Citronellol | 20.65 | 1232 | - | 0.64 | t | 0.4 | ||
| 17 | Neral | 21.059 | 1243 | - | 0.13 | - | 0.1 | ||
| 18 | Geraniol | 21.724 | 1255 | - | 0.13 | 0.07 | 0.1 | ||
| 19 | α- citral | 22.424 | 1271 | - | 0.42 | - | - | ||
| 20 | methyl geranate | 24.753 | 1326 | - | 0.12 | - | - | ||
| 21 | citronellyl acetate | 25.993 | 1353 | - | 0.12 | - | - | ||
| 22 | Eugenol | 26.218 | 1357 | 0.24 | 0.39 | t | 0.5 | ||
| 23 | (E)-methyl cinnamate | 27.652 | 1384 | 6.42 | 13.26 | 4.12 | 8 | ||
| 24 | methyl eugenol | 28.969 | 1403 | 90.31 | 77.18 | 88.18 | 74.8 | ||
| 25 | germacrene D | 31.376 | 1480 | 0.12 | - | 0.6 | 1.1 | ||
| 26 | cis-calamenene | 33.04 | 1537 | 0.1 | t | - | 0.7 | ||
| 27 | Elemicine | 34.242 | 1550 | 0.18 | 0.17 | 0.3 | 0.2 | ||
| 28 | Spathulenol | 35.264 | 1576 | 0.57 | 0.24 | 0.22 | 0.3 | ||
| 29 | Viridiflorol | 35.581 | 1594 | 0.24 | - | 0.1 | - | ||
| 30 | t- murrolol | 37.827 | 1645 | 0.1 | - | - | 0.5 | ||
| 31 | cadin-4-en-10-ol | 38.255 | 1659 | 0.41 | 0.13 | 0.14 | 0.4 | ||
| 32 | coniferaldehyde methyl ether | 42.912 | 1777 | 0.51 | 1.78 | - | 0.3 | ||
| Total Composition | 99.95 | 99.31 | 97.13 | 93.2 | |||||
| Monoterpene hydrocarbons | t | t | 0.96 | 3 | |||||
| Oxygenated hydrocarbons | 0.47 | 5.63 | 2.11 | 2.7 | |||||
| Phenylpropanoid | 96.97 | 90.83 | 92.34 | 83.3 | |||||
| Sesquiterpene hydrocarbons | 0.12 | t | 0.6 | 1.1 | |||||
| Oxygenated sesquiterpenes | 1.5 | 0.54 | 0.76 | 1.4 | |||||
| Others | 0.89 | 2.31 | 0.36 | 1.7 |
In vitro antioxidant activity
DPPH Radical scavenging activity
In this study, the antioxidant activity of essential oils was compared with ascorbic acid, a reference standard antioxidant compound. A good inhibition action on DPPH free radicals produced in the reaction was observed at all tested concentrations of the oil. The IC50 value for Melaleuca bracteata is 0.3831 compared to 1.773 of Ascorbic acid.
ABTS Radical Scavenging Ability
In this study, the antioxidant activity of essential oils was compared with ascorbic acid, a reference standard antioxidant compound. A good inhibition action on ABTS Radical produced in the reaction was observed at all tested concentrations of the oil. The IC50 value for Melaleuca bracteata is 0.06937 compared to 0.8237 of Ascorbic acid.
Hydroxyl Free Radical Scavenging Assay
The antioxidant activity of essential oils was compared with Gallic Acid, a reference standard antioxidant compound. A good inhibition action on Hydroxyl free radicals produced in the reaction was observed at all tested concentrations of the oil. The IC50 value for Melaleuca bracteata is 0.8192 compared to 14.71 of Gallic Acid.
Reactive Nitrogen Oxide Scavenging Assay
The antioxidant activity of essential oils was compared with Gallic Acid, a reference standard antioxidant compound. A good inhibition action on Nitrogen Oxide produced in the reaction was observed at all tested concentrations of the oil. The IC50 value for Melaleuca bracteata is 2.766 compared to 12.28 of Gallic Acid. Table 2 represents IC50 value of essential oil of Melaleuca bracteata against different radicals.
| Oil/Standard | Antioxidant activity in terms of IC50 (μg/mL ± SD) | |||
|---|---|---|---|---|
| DPPH radical scavenging activity | ABTS Radical Scavenging Ability | Hydroxyl Free Radical Scavenging Assay | Nitrogen Oxide Scavenging Assay | |
| MB (L) | 0.3831 ± 0.27 | 0.06518 ± 0.07 | 0.8192 ± 0.28 | 2.766 ± 0.18 |
| Ascorbic Acid | 1.773 ± 0.76 | 0.8237 ± 0.17 | - | - |
| Gallic Acid | - | - | 14.71 ± 0.28 | 12.28 ± 0.19 |
Anti-bacterial activities
The zone of inhibition diameters and Minimum Inhibitory Concentrations (MICs) of the essential oils against the tested microorganisms are presented in Table 3. In the study by Goswami et al., (2017), the essential oil showed no activity against P. aeruginosa and E. coli strains. However, in our study, the essential oil was found to be effective against these two pathogens, as well as S. aureus and B. subtilis, with zone of inhibition values ranging from 6.66 to 12.00 mm.
| Sl. No. | Concentration | E. coli | S. aureus | P. aeruginosa | B. subtilis |
|---|---|---|---|---|---|
| 1 | 0 | 0 | 0 | 0 | 0 |
| 2 | 6.25 | 06.66±0.56 | 06.66 ± 0.57 | 08.66 ± 0.57 | 09.00 ± 0.00 |
| 3 | 12.5 | 10.00±0.00 | 10.05 ± 0.00 | 10.00 ± 0.00 | 10.00 ± 0.00 |
| 4 | 25 | 11.11±0.00 | 11.00 ± 0.00 | 11.00 ± 0.00 | 07.50 ± 0.70 |
| 5 | 50 | 11.66±0.57 | 11.66 ± 0.57 | 10.66 ± 0.57 | 09.50 ± 0.71 |
| 6 | 100 | 12.00±0.00 | 12.00 ± 0.00 | 08.66 ± 0.57 | 12.00 ± 0.00 |
| MIC (MB) | 1000 | 500 | 500 | 500 | |
| MIC (Standard) | 0.06 | 0.4 | 0.02 | 0.05 |
DISCUSSION
The results of the current study show very high percentage of Phenylpropanoid in essential oil of Melaleuca braceata. The methyl eugenol constitutes almost 90.8% of the total compostion of leaves oils which is higher than the previous studies done by the Goswami et al., 2017 (88.18% methyl eugenol) and Joshi et al., 2021 (74.80 % methyl eugenol) from Tarai region of Kumaun, Uttarakhand and less than the study done by the Yasin et al., 2021 from Faisalabad (96.02% methyl eugenol). Compared to leaves oils the flower oils have lower percentage of methyl eugenol, 77.18% but higher percentage of (E)-methyl cinnamate 13.26%.
The antioxidant assays (DPPH, ABTS, Hydroxyl, and Reactive Nitrogen Oxide) demonstrated that M. bracteata oil possesses a remarkable ability to neutralize free radicals across different radical systems. The IC₅₀ values obtained in all assays were substantially lower than those of standard antioxidants such as ascorbic acid and gallic acid, indicating stronger antioxidant potential. In the DPPH assay, the oil exhibited an IC₅₀ value of 0.3831 compared to 1.773 for ascorbic acid, highlighting its high hydrogen-donating capacity. Similarly, in the ABTS assay, the oil showed an exceptionally low IC₅₀ value of 0.06937, suggesting superior electron transfer ability. The hydroxyl and reactive nitrogen oxide scavenging assays further confirmed the oil’s strong radical quenching efficiency, with IC₅₀ values of 0.8192 and 2.766, respectively, both notably lower than their respective standards. These findings suggest that M. bracteata essential oil contains potent antioxidant constituents capable of combating oxidative stress, possibly due to the presence of phenolic or terpenoid compounds reported in previous studies (e.g., Goswami et al., 2017; Adekunle et al., 2019).
The antibacterial results also underscore the broad-spectrum efficacy of M. bracteata essential oil. Contrary to the observations by Goswami et al., 2017 who reported no activity against Pseudomonas aeruginosa and Escherichia coli, our study demonstrated measurable inhibition zones against these strains, along with Staphylococcus aureus and Bacillus subtilis. The zone of inhibition values ranging from 6.66 to 12.00 mm indicates that the oil possesses effective antibacterial components, possibly acting through disruption of microbial cell membranes or inhibition of essential enzymatic processes. The observed differences may be attributed to variations in the chemical composition of essential oils due to geographical, seasonal, and environmental factors influencing the plant’s secondary metabolite profile.
CONCLUSION
This investigation into the essential oil derived from the aerial parts and flowers of Melaleuca bracteata in the Bhabar belt of Uttarakhand reveals a distinctive chemical profile, predominantly featuring Phenylpropanoid. The pronounced antioxidant activity demonstrated by the oil, effectively scavenging DPPH, ABTS, hydroxyl, and nitrogen oxide radicals, underscores its potential as a natural antioxidant agent. Additionally, the oil exhibits significant antimicrobial properties against various bacterial strains, aligning with findings from other Melaleuca species.
Given these bioactive properties, the Melaleuca oil from this region holds promising applications in the medical and cosmetic industries. Its antioxidants and antimicrobial activities suggest potential uses in developing therapeutic agents and natural preservatives. Furthermore, the high methyl eugenol content offers opportunities for its incorporation into fragrances and skincare products, catering to the growing consumer preference for natural ingredients.
Future research should focus on comprehensive toxicological assessments and clinical evaluations to ensure the safety and efficacy of this oil in various applications. Additionally, exploring sustainable harvesting and production methods will be crucial to meet industrial demands while preserving the ecological balance of the region.