Phytochemical Composition and Bioactivity Assessment of Essential Oil of Cinnamomum bejolghota (Buch.-Ham.) Sweet from Xuan Son National Park, Vietnam

MATERIALS AND METHODS

Study area: This work was carried out from July, 2021 to July, 2022 at the Faculty of Natural Sciences, Hung Vuong University and the Institute of Natural Products Chemistry (INPC), Vietnam Academy of Science and Technology (VAST).

Plant materials: Stem bark of C. bejolghota (Buch.-Ham.) Sweet was collected from Xuan Son National Park, Phu Tho Province, Vietnam (21°9'N, 104°56'E) in July, 2021.

Research procedure
Isolation of essential oil and chemical component investigation: A total of 500 g dried bark of C. bejolghota (Buch.-Ham.) Sweet was used for hydrodistillation for 3 hrs by a Clevenger-type apparatus. Essential oils were separated, dehydrated and investigated as described by Nguyen et al.¹¹.

Microbial strains: Seven microorganism strains from American Type Culture Collection (ATCC, Manassas, VA, USA) (Bacillus subtilis subsp. spizizenii (ATCC 6633), Staphylococcus aureus subsp. aureus (ATCC 25923) Escherichia coli (ATCC 25922), Pseudomonas aeruginosa (ATCC 10145), Aspergillus niger (ATCC 6275), Fusarium oxysporum (ATCC 7601) and Candida albicans (ATCC 10231)) and two strains, Saccharomyces cerevisiae (VTCC-Y-62) and Salmonella enterica (VTCC 12277), from the Vietnam Type Culture Collection (Institute of Microbiology and Biotechnology, Vietnam National University, Ha Noi, Vietnam) were used for antimicrobial activity of C. bejolghota bark oils.

The antibacterial property of essential oil was tested using the agar disk diffusion technique^12-14. Prior to testing, microorganisms kept at -80°C were switched on by culture medium to a concentration of 1.0×10⁶ CFU mL^–1. A total of 100 μL of inoculum solution was brought and applied uniformly to the agar surface. The positive (B. subtilis subsp., spizizenii (ATCC 6633) and S. aureus subsp., aureus (ATCC 25923)) and negative Gram bacteria (E. coli (ATCC 25922), Salmonella enterica (VTCC 12277)) were cultured in the Trypcase Soy Broth medium (TSB-Sigma). Using an aseptic approach, three holes (approximately 6 mm in diameter each) were created on agar plates. A pipette was used to place 50 μL of essential oil into each hole. The Petri dishes were kept at 37°C for 18-24 hrs after being incubated at room temperature for 2-4 hrs. On each plate culture, the presence or absence of growth surrounding each hole carrying an antibacterial agent was noted. The ruler with millimeter graduations was used to measure the diameters of the inhibiting growth zones.

The standard liquid dilution method¹⁵ was performed to determine the in vitro antimicrobial activity experiments on a 96-well microtiter plate as previous description¹⁶. The fungi were grown in the Sabouraud Dextrose Broth medium (SDB- Sigma). After that, the agar plates were kept at 30°C or 37°C for 24 or 48 hrs depending to the fungal species. The MIC (Minimum inhibitory concentration) value was calculated. For respective Gram (+) and Gram (-) bacteria, streptomycin and tetracycline were used as positive controls whereas nystatin was used for fungi and yeasts.

Statistical analysis: Statistical investigation of all data was carried out using the ANOVA and means sorted out by Duncan’s Multiple Range Test at the 5% level of significance (p = 0.05).

RESULTS AND DISCUSSION

Composition analysis of essential oil of C. bejolghota (Buch.-Ham.) Sweet: The essential oil obtained by hydrodistillation of C. bejolghota bark with a refractive index of 1.4752 (20°C), was a pale-yellow color liquid with a pleasant odor.

Fig. 1:

GC/MS total ion chromatogram of the bark essential oil of C. bejolghota

The extraction yield was 1.1% (v/w), quantified on a dry weight basis.

The chromatogram of the essential oil from C. bejolghota bark was presented in Fig. 1. A total of 53 compounds, accounting for 99.45 % of the essential oil composition were identified (Table 1). The main classes of compounds found in the essential oil were oxygenated monoterpenes (68.43%), followed by monoterpene hydrocarbons (14.78%), sesquiterpene hydrocarbons (11.32%), oxygenated sesquiterpene (2.42%) and non-terpenes (2.54%). Major components of the essential oil were geraniol (26.2%), 1,8-cineole (24.73%), followed by α-terpineol (9.30%), terpinene-4-ol (3.90%), α-pinene (3.34%), β-pinene (2.40), β-caryophyllene (2.69%), α-copaene (2.11%), respectively.

In comparison to the essential oil components of C. bejolghota reported in the literature, there was a slight difference between the essential oil compositions of C. bejolghota growing in northern of Vietnam and in other regions, including India^8,9 and Thailand¹⁰ (Table 2). The content of monoterpene hydrocarbons of Vietnamese essential oil samples was lower than in other samples. However, the content oxygenated monoterpene of Vietnamese essential oil samples was quite high, almost equivalent to the Thailand oil sample and was significantly superior to the Indian samples. There were a few interesting points such as α-Terpineol, the chemical composition was detected in lower quantity in this study in comparison with the previous studies^8,9. The content of cineole-1,8 (eucalyptol) of C. bejolghota grown in Phu Tho, Vietnam, was significantly higher than C. bejolghota grown in India, but lower than sample collected in Thailand. Especially, the high content of Geraniol was discovered for the first time in our study. Similarities and differences between the study results could be explicated by a variety of factors including the nature and age of the plants as well as depending on growth area and other conditions¹⁷.

Bioactivity analysis of essential oil of Cinnamomum bejolghota (Buch.-Ham.) Sweet: The biological activities of C. bejolghota bark essential oil were determined (Fig. 2 and Table 3) with regard to inhibitory zone diameter and MIC. The essential oil from the bark of C. bejolghota inhibited E. coli and S. enterica growth, with inhibitory zone diameters of 6.55±1.10 mm and 4.75±1.20 mm, respectively. The MIC value of the essential oil from the bark of C. bejolghota was 500 μg/mL for S. cerevisiae and F. oxysporum. These MIC values for both fungi were higher for six other examined microorganisms (Table 3).

Although the antibacterial effects of essential oils from Cinnamomum spp., have been extensively documented, the efficiency of C. bejolghota bark oil against pathogenic species has been poorly investigated. Atiphasaworn et al.¹⁰, found that the essential oil of C. bejolghota bark from Thailand was efficient against bacterial and fungal infections.

Fig. 2:

Antibacterial property of essential oils from barks of C. bejolghota (mean±standard deviation and n = 3)

The antibacterial and antifungal effects of C. bejolghota bark essential oil might be attributed to the range of its bioactive compounds. Geraniol (26.20%) and 1,8-cineole (24.73%), the two principal components of C. bejolghota bark essential oils, exhibit many biological activities, including antibacteria¹⁸, fungicidal^18,19, anti-inflammatory, anticarcinogenic and antioxidant²⁰. The antimicrobial activity (lipophilic character) of Geraniol may be explained by its capacity to cling to the microorganism's cell membrane lipids, join with its components, making it more permeable and adhering important intracellular spots, thereby destroying their structures.

CONCLUSION

This work surveyed the components and biological activities of the essential oil from the bark of C. bejolghota grown in Xuan Son National Park in Vietnam. The main chemical classes of components were oxygenated monoterpene (68.43%), monoterpene hydrocarbons (14.78%), sesquiterpene hydrocarbons (11.32%), oxygenated sesquiterpene (2.42%) and non-terpenes (2.54%), respectively. The dominant compounds of the essential oil were geraniol (26.2%) and 1,8-cineole (24.73%). Biological activity analysis demonstrated that C. bejolghota bark essential oil has potent antibacterial and antifungal properties. From the foregoing, the essential oil of C. bejolghota might be a source of potential antimicrobial agents.

SIGNIFICANCE STATEMENT

The phytochemical compositions and antibacterial activity of the essential oil from the bark of C. bejolghota grown in Northern Vietnam were first investigated in this work. Some components, such as geraniol and other chemicals in trace amounts, were discovered in C. bejolghota essential oil for the first time. As a result, the essential oil of C. bejolghota might be considered a bioactive natural product which had many potential applications in aesthetic or postharvest conservation. This work helps to reveal the components and biological activities of the essential oil of C. bejolghota grown in northern Vietnam that many other researchers did not explore.

ACKNOWLEDGMENT

This work was supported by Hung Vuong University (Project grant No. 04/2021/HĐKH).