Research Article |
Corresponding author: Lien-Thuong Thi Nguyen ( thuongntl@tdmu.edu.vn ) Academic editor: Rumiana Simeonova
© 2023 Danh Duc Nguyen, Hieu Nguyen-Ngoc, Hieu Tran-Trung, Dang-Khoa Nguyen, Lien-Thuong Thi Nguyen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Nguyen DD, Nguyen-Ngoc H, Tran-Trung H, Nguyen D-K, Thi Nguyen L-T (2023) Limonene and eucalyptol rich essential oils with their antimicrobial activity from the leaves and rhizomes of Conamomum vietnamense N.S. Lý & T.S. Hoang (Zingiberaceae). Pharmacia 70(1): 91-96. https://doi.org/10.3897/pharmacia.70.e96946
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Conamomum vietnamense, a new species of Zingiberaceae family, has been discovered and described from Tay Nguyen (Central Highlands, Vietnam) in 2022. The present study described the preparation of essential oil from leaves and rhizomes of the plant by hydrodistillation process. Then, the chemical composition of these essential oils was analyzed by Gas Chromatography-Mass Spectrometry (GC-MS), which indicated that limonene (18.74 and 26.20%) and eucalyptol (40.47 and 49.49%) were the main components, respectively. The essential oils also showed moderate antimicrobial activities against Gram-positive bacterial strains (Enterococcus faecalis ATCC 299212, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579), Gram-negative bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 13076), and a pathogenic yeast (Candida albicans ATCC 10231) in the MIC range of 32–256 μg/mL, which was comparable to those of positive controls, streptomycin and cycloheximide. For the first time, the chemical composition and antimicrobial activity of the essential oil of C. vietnamense were studied.
Conamomum vietnamense, GC-MS, essential oil, limonene, eucalyptol, antimicrobial activity
The genus Conamomum Ridl. is a small genus in the Zingiberaceae family with only 12 accepted species worldwide, mainly occurring in evergreen lowland and montane forests in Southeast Asian countries, such as Vietnam, Cambodia, Thailand, Malaysia, Singapore, Sumatra and Borneo (
The fresh leaves and rhizomes of Conamomum vietnamense were collected from Loc Bac, Bao Lam District (11°47'31.9"N, 107°35'47.2"E), Lam Dong Province, Vietnam in September 2022. The plant was identified by the first author based on the protologue (
The scraps of leaves (500 g) and rhizomes (500 g) of Conamomum vietnamense were hydro-distilled for 3 h (beginning from the water boiling point) using a Clevenger-type apparatus, according to the Vietnamese Pharmacopoeia (
The essential oils of Conamomum vietnamense were analyzed for their constituents via Gas chromatographic/mass spectrometry using an Agilent GC-7980 linked to an Agilent MS 5977C system working in EI mode, with an HP-5MS UI column (30 m × 0.25 mm id. × 0.25 μm film thickness, Agilent Technologies). System installation: carrier gas was Helium (flow rate of 1 mL/min); injection volume: 1 μL, split ratio 1:50; the temperature was programmed from 60 °C (kept for 1 min) to 240 °C (kept for 4 min) at a rate of 4 °C/min; injector temperature: 300 °C, MS Quad temperature: 150 °C, transfer line temperature: 300 °C, MS source: 230 °C; ionization energy: 70 eV, and mass range: 50–550 amu (2.0 scan/s). Identification of the oil components was performed based on comparing their mass spectra value to that of the NIST14 library and then confirmed by comparing retention indices with reference to a homologous series of n-alkanes. The percentage of the relative peak area was used for quantification.
The anti-microbial bioassays were performed on Gram-positive bacterial strains (Enterococcus faecalis ATCC 299212, Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 14579), and Gram-negative bacterial strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella enterica ATCC 13076), and a pathogenic yeast (Candida albicans ATCC 10231). All strains were provided by National Institute for Food Control (No. 65 Pham Than Duat Street, Mai Dich Ward, Cau Giay District, Ha Noi, Vietnam). The antimicrobial activity of the essential oil prepared from the leaves and rhizomes of C. vietnamense was assessed by microdilution broth susceptibility assay method, which strictly followed the standards issued by the Clinical and Laboratory Standards Institute (CLSI supplement M100, 2020) (
The microbial inhibition rate expressed as a percentage of inhibition was calculated using the following formula (
Where: Ao: absorbance of blank sample. Aoc: absorbance of the culture medium without cells. At: absorbance of the test sample. Hc and Lc: high and low concentration (%) of test agent, respectively. Hi and Li: inhibition percentage at high and low concentrations, respectively.
The yields of the hydrodistilled essential oils from the leaves and rhizomes of Conamomum vietnamense were 0.43% and 0.37% (w/w, fresh weight), respectively. The chemical composition of essential oils was analyzed and presented in Table
Chemical compositions of essential oil distilled from C. vietnamense rhizomes and leaves.
No. | Compounds | RT (min) | RI (calc) | RI (db) | Concentration (%) | |
---|---|---|---|---|---|---|
Rhizomes | Leaves | |||||
1 | α-Pinene | 5.548 | 939 | 937 | 3.60 | 4.91 |
2 | Camphene | 5.908 | 955 | 952 | 1.42 | 0.66 |
3 | β-Pinene | 6.612 | 981 | 979 | 0.42 | 1.27 |
4 | β-Myrcene | 6.938 | 993 | 991 | 1.25 | 2.36 |
5 | α-Phellandrene | 7.333 | 1007 | 1005 | 3.30 | 3.77 |
6 | 3-Carene | 7.499 | 1013 | 1011 | 0.34 | 0.09 |
7 | α-Terpinene | 7.676 | 1020 | 1017 | 1.70 | 1.47 |
8 | p-Cymene | 7.905 | 1029 | 1025 | 1.33 | 1.29 |
9 | Limonene | 8.048 | 1034 | 1030 | 18.74 | 26.20 |
10 | Eucalyptol | 8.134 | 1037 | 1032 | 40.47 | 49.49 |
11 | cis-β-Ocimene | 8.271 | 1042 | 1038 | 0.11 | 0.07 |
12 | trans-β-Ocimene | 8.58 | 1052 | 1049 | 1.50 | 0.34 |
13 | γ-Terpinene | 8.924 | 1063 | 1060 | 0.64 | 1.37 |
14 | Terpinolene | 9.845 | 1091 | 1088 | 0.74 | 0.71 |
15 | 2-Nonanone | 9.937 | 1094 | 1092 | 0.05 | 0.16 |
16 | Linalool | 10.177 | 1100 | 1099 | 3.23 | 0.87 |
17 | exo-Fenchol | 10.635 | 1116 | 1115 | 0.40 | 0.18 |
18 | Camphor | 11.659 | 1149 | 1145 | 0.10 | - |
19 | Camphene hydrate | 11.779 | 1153 | 1148 | 0.07 | - |
20 | endo-Borneol | 12.334 | 1170 | 1167 | 2.19 | 0.49 |
21 | Terpinen-4-ol | 12.723 | 1181 | 1177 | 0.58 | 0.46 |
22 | α-Terpineol | 13.158 | 1193 | 1189 | 2.43 | 1.84 |
23 | Fenchyl acetate | 14.142 | 1224 | 1123 | 0.17 | - |
24 | Bornyl acetate | 16.317 | 1289 | 1285 | 0.74 | - |
25 | Dihydroedulan | 16.408 | 1291 | 1293 | - | 0.06 |
26 | 2-Undecanone | 16.545 | 1295 | 1294 | - | 0.15 |
27 | α-Copaene | 19.229 | 1382 | 1376 | 0.87 | - |
28 | Methyl cinnamate | 19.418 | 1385 | 1380 | - | 0.08 |
29 | Caryophyllene | 20.608 | 1423 | 1419 | 0.57 | 0.21 |
30 | Humulene | 21.661 | 1458 | 1454 | 0.14 | - |
31 | Alloaromadendrene | 21.884 | 1465 | 1461 | 0.12 | - |
32 | Cadina-1(6),4-diene | 22.268 | 1478 | 1481 | 0.33 | - |
33 | γ-Muurolene | 22.348 | 1480 | 1477 | 0.3 | - |
34 | Aristolochene | 22.599 | 1488 | 1487 | 0.21 | 0.1 |
35 | β-Eudesmene | 22.657 | 1490 | 1486 | 0.16 | - |
36 | α-Selinene | 22.914 | 1498 | 1494 | 0.35 | - |
37 | Epizonarene | 23.023 | 1501 | 1051 | 0.71 | - |
38 | β-Bisabolene | 23.292 | 1511 | 1509 | 0.10 | - |
39 | γ-Cadinene | 23.481 | 1518 | 1513 | 0.20 | 0.08 |
40 | Cadina-3,9-diene | 23.755 | 1527 | 1518 | 1.47 | - |
41 | Cadina-1(10),4-diene | 23.824 | 1530 | 1524 | 1.04 | 0.05 |
42 | Cubenene | 24.018 | 1537 | 1532 | 0.98 | - |
43 | α-Calacorene | 24.333 | 1547 | 1542 | 0.09 | - |
44 | (±)-trans-Nerolidol | 24.911 | 1567 | 1564 | 1.21 | 1.1 |
45 | Palustrol | 25.06 | 1572 | 1567 | 0.07 | - |
46 | Caryophyllenyl alcohol | 25.129 | 1574 | 1570 | 0.11 | - |
47 | (-)-Globulol | 25.529 | 1588 | 1580 | 0.19 | - |
48 | Guaiol | 25.924 | 1600 | 1596 | 0.11 | - |
49 | Epicubenol | 26.788 | 1633 | 1627 | 0.43 | - |
50 | γ-Eudesmole | 26.880 | 1636 | 1631 | 0.63 | - |
51 | α-epi-Cadinol | 27.183 | 1647 | 1640 | 0.77 | - |
52 | δ-Cadinol | 27.286 | 1651 | 1645 | 0.29 | - |
53 | β-Eudesmol | 27.400 | 1655 | 1649 | 0.68 | - |
54 | α-Eudesmol | 27.486 | 1658 | 1653 | 1.52 | - |
55 | (+)-Intermedeol | 27.646 | 1664 | 1667 | 0.18 | - |
Total | 99.35 | 99.83 |
The two oil samples were then evaluated for their antimicrobial activities against several bacterial and fungal strains. The investigated strains included Enterococcus faecalis (causing endocarditis, sepsis, urinary tract infections, and meningitis), Staphylococcus aureus (causing bacteremia, infective endocarditis), Bacillus cereus (causing food-borne illnesses with symptoms of nausea, vomiting, and diarrhea), Escherichia coli (causing diarrhea, urinary tract infections, respiratory illnesses, and pneumonia), Pseudomonas aeruginosa (causing pneumonia, urinary tract infections, and wound, ear, and bloodstream infections), Salmonella enterica (food-borne pathogen causing gastroenteritis), and Candida albicans (causing hospital-acquired infections, vaginal yeast infection, and bloodstream infection). The bioassays used streptomycin and cycloheximide as positive controls (Table
Antimicrobial activities of essential oil distilled from C. vietnamense rhizomes and leaves.
Microorganisms | MIC (µg/mL) | |||
---|---|---|---|---|
Leaf oil* | Rhizome oil* | Streptomycin | Cycloheximide | |
E. faecalis ATCC 299212 | 32 ± 2.65 | 32 ± 1.89 | 256 | NT |
S. aureus ATCC 25923 | 128 ± 1.56 | 128±2.64 | 256 | NT |
B. cereus ATCC 14579 | 128 ± 2.14 | 64±3.24 | 128 | NT |
E. coli ATCC 25922 | 256 ± 4.35 | 128±2.58 | 32 | NT |
P. aeruginosa ATCC 27853 | 128 ± 2.17 | 128±1.39 | 256 | NT |
S. enterica ATCC 13076 | 128 ± 2.13 | 64±3.25 | 128 | NT |
C. albicans ATCC 10231 | 32 ± 1.45 | 64±2.45 | NT | 32 ± 0.07 |
Eucalyptol and limonene were the two main components of C. vietnamense leaf and rhizome essential oils, which occupied 75.69 and 59.21% of the total oil content, respectively. Eucalyptol, or 1,8-cineol, is a bicyclic terpenoid, which naturally occurs in many plants, especially Eucalyptus species with high-content (60–97.32%) (
The essential oils were prepared from C. vietnamense leaves and rhizomes by hydrodistillation method with yields of 0.43% and 0.37% (w/w, fresh weight), respectively. The chemical composition of both oil samples was identified by GC-MS analysis, indicating that eucalyptol and limonene were the two dominant components (40.47–49.49% and 18.74–26.20%, respectively). The essential oils were then evaluated for their antimicrobial activities. The leaf oil was the most active against E. faecalis and C. albicans with MIC values of 32 µg/mL while the rhizome oil showed potential inhibitory effects on E. faecalis (MIC 32 µg/mL), B. cereus (MIC 64 µg/mL), S. enterica (MIC 64 µg/mL), and C. albicans (MIC 64 µg/mL). The study results showed that the essential oil of C. vietnamense can be a resource for eucalyptol and limonene and possessed potential antimicrobial effects. Remarkably, this is the first report on the chemical constituents and antimicrobial activity of the essential oil prepared from Conamomum vietnamense.