Comparison of Chemical Profile and Antibacterial Activity of Cinnamomum cassia Bark Essential Oil from Three different Vietnamese Provinces 

Cinnamomum cassia (C. cassia), commonly known as Chinese cinnamon or cassia, is a tropical aromatic evergreen plant belonging to the Lauraceae family. The species is natively cultivated for its barks in Southern China, India, Vietnam and other Southeast Asian countries. Dried cinnamon bark is commonly used as a flavor for a large variety of food and beverages. Cinnamon bark is highly rich in essential oil (EO) which is traditionally used as aromatherapy and is now widely incorporated into many healthcare products like medicated oils, soaps and transdermal patches. C. cassia has long been traditionally used as a medicinal herb for the treatment of various illnesses including fevers, coughs, digestive symptoms, arthritis, muscle pain and menstrual discomfort. Contemporary research data have shown that cinnamon has a wide range of significant pharmacological properties, including anti-carcinogenic, anti-inflammatory, anti-diabetic, antimicrobial, cardiovascular protective and neuroprotective effects. Phytochemical analysis of C. cassia essential oil revealed the presence of starch, glycosides, mucilage, tannins and essential oil. The predominant component in essential oil is cinnamaldehyde (65-85%), a bioactive aromatic aldehyde compound (Kosari et al., 2020), along with other components mainly belonging to classes of terpenoids and phenylpropanoids (Barceloux, 2009), has demonstrated notable antioxidant, anticancer, anti-inflammatory and antimicrobial properties.
       
In general, the quality and quantity of essential oil and its molecules might be extremely variable depending on various factors including plant species, genotypes, geographical origin, climatic conditions, cultivation practices, harvesting time and experimental conditions. Vietnam is well known as one of the best sources of high-quality cinnamon, partly owing to the soil and climate considerably contributing to favorable conditions for growing premium quality organic cinnamon. The cinnamon growing area in Vietnam is approximately estimated to be 150,000 hectares, accounting for 17% of the global cinnamon growing area (Center for WTO and International Trade Vietnam Chamber of Commerce and Industry, 2023). In the first half of 2023, Vietnam exported 43,186 tons of cinnamon to fastidious markets like the EU, USA, Japan, Bangladesh and India. In 2022, Vietnam was the largest supplier of cinnamon to India, accounting for 32,650 tons of cinnamon (85%) of India’s total imported cinnamon output. Vietnamese cinnamon is commonly cultivated on large scales in several provinces ranging from the North to the Central of Vietnam; however, three provinces, namely Yen Bai, Quang Ngai and Kon Tum, are best known as the most cinnamon growing regions of Vietnam where cinnamon is cultivated commercially for domestic and international market (Fig 1).


       
In this regard, this present research aimed to investigate the variation of essential oil composition from the bark of cinnamon cultivated in three different provinces as mentioned above. The physicochemical properties and antibacterial activity of obtained essential oils were also subjected to the investigation.

Selection and preparation of materials
 
Fresh cinnamon barks (Fig 2) were collected in March 2024 from various farms located in three selected provinces (Yen Bai- 21°41'35"N, 104°52'22"E, QN- 15°072'263"N, 108°482'423"E and KT- 14°212'423"N, 108°002'303"E) and then washed several times with running water to remove any impurities. The barks were immediately air-dried, cut into small pieces and then ground into smaller particles. The extraction of essential oil was carried out at the International University- Vietnam National University by hydro-distillation for 5-6 h using a Clevenger circulatory hydro-distillation apparatus (Bicchi and Maffei, 2012), (Naik et al., 2017). Once the distillation had been complete, the obtained essential oil was dehydrated using anhydrous sodium sulfate and then stored in sealed opaque brown bottles at 4°C for further analysis.

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Gas chromatography - Mass spectroscopy analysis
 
Gas chromatography - Mass spectroscopy (GC-MS) was employed to analyze obtained EO (Son L.Hoang and Nhi H.M.Nguyen, 2023). GC-MS analyses were performed using Nexis GC-2030. Helium was used as carrier gas at a constant flow rate of 1 mL/min. The oven temperature was initially programmed at 50°C for 1 min and then increased to 80°C at 30°C/min. Shortly afterward, it was increased to 230°C at 5°C/min and finally to 280°C at 25°C/min where it was thermally held for 3 min. The injector temperature was set at 250°C and the split ratio was set at 1:30. Fragmentation was done by electron impact under a field of 70 eV. The mass spectra were recorded over the mass range of 50-500 amu with the full-scale mode at a rate of 1s/scan.

Physicochemical analysis
 
Determination of specific gravity (SG)
 
The specific gravity of EO was determined using a 50-mL density bottle (pycnometer) at 20°C according to the procedure described in the AOAC Official Method (AOAC 920.212, 1920). The density bottle must be clean and dry prior to the assay. The empty dry bottle with a stopper was initially weighed on the analytical balance and documented as W0. The bottle was then filled with distilled water and the stopper was inserted, followed by gently tapping the sides of the bottle to remove the air bubbles. Once the bottle had been carefully wiped off, it was weighed and recorded as W₁. The same process was repeated but using EO instead of distilled water. The bottle was then weighed and noted as W₂.
 

 

 
Determination of optical rotation
 
A 10-mL polarimeter tube containing oil was placed at 20! in the trough of the instrument between the polarizer and analyzer. This test was done by a polarimeter of Kruss equipment (AOAC 920.142, 1920).
 
Determination of total acid number (TAN)
 
One gram of EO was accurately weighed into a conical flask containing 25 mL of ethanol mixed with 25 mL of diethyl ether. Two drops of phenolphthalein were added and then titrated with 0.1 N KOH solution until the color of the endpoint turned pale pink and must persist for at least 30 sec. The AV was then calculated using the following formula according to the standard procedure (AOAC 969.17, 1995).
 

 

 
V = Volume of potassium hydroxide used
N = Normality of potassium hydroxide
W = Weight in g of the sample
 
Determination of saponification value (SV)
 
Two grams of EO were accurately weighed into a conical flask containing 30 mL of 0.2 N ethanolic KOH solution. The mixture was then refluxed with continuous agitation for 2 h, followed by the addition of two drops of phenolphthalein. Shortly thereafter, the mixture was titrated with 0.5 N HCl until the pink color disappeared completely. Titration was simultaneously conducted for blank determination (AOAC 920.160, 1920).
 

Saponification value = (T-B) × N × 56.1

 
B = mL of HCl required by blank
T = mL of HCl required by the oil sample
N = Normality of HCl
W = Weight of oil in gram

Determination of ester value (EV)
 
The ester value was determined based on the acid and saponification values (ISO 709:2001, 2001) using the following formula:
 

Ester Value = Saponification Value - Acid Value

 
Determination of iodine value (IV)
 
0.2 g of respective oil was weighed into an Erlenmeyer flask containing a mixture of 10 mL of 0.1 N iodine in ethanol and the solution was kept in the dark for 30 min at room temperature. The resulting solution was titrated against 0.1 M sodium thiosulphate (Na₂S₂O₃) until the color was dark yellow. Starch was used as an indicator; if it was green, adjusted until a straw yellow color appeared. A blank sample was simultaneously prepared under the same condition. The iodine value (ISO 3961:2018, 2018) was then calculated by the following formula:
 

 
V₁ = Volume of Na₂S₂O₃ in blank (mL)
V₂ = Volume of Na₂S₂O₃ in EO (mL)
M = Weight of used EO(g)
 
Determination of peroxide value (PV)
 
Two grams of EO were accurately weighed into the 250-mL glass-stoppered Erlenmeyer flask containing 30 mL of a mixture of three volumes of glacial acetic acid and two volumes of chloroform. The flask was promptly closed with the glass stopper and then swirled until completely dissolved. Shortly afterward, 1 mL of freshly prepared saturated KOH solution was added to the mixture. The flask was then closed and agitated for 60 sec in the dark and subsequently, 30 mL of distilled water was added and titrated against a standard solution of sodium thiosulfate (0.01 N) till the yellow color was almost gone. Thereafter, 0.5 mL of starch solution (1%) was added to the mixture with continuous agitation, followed by titration with sodium thiosulfate until the blue color disappeared. A blank titration was simultaneously carried out under the same condition (ISO 3960:2017, 2017). The volume of sodium thiosulfate was recorded. The peroxide value (milliequivalent peroxide/ kg of sample) was calculated using the following formula:
 

 

 
Where:
S = mL of sodium thiosulfate.
N = Normality of sodium thiosulfate.
 
Antibacterial activity
 
Microbial strains
 
The antibacterial effectiveness of EOs from C. cassia was tested against ten different microbial strains. Gram-positive species were Staphylococcus aureus ATCC 25923 (S. aureus 25923), Streptococcus pneumoniae ATCC 49619 (S. pneumoniae 49619), Streptococcus pyogenes ATCC 19615 (S. pyogenes 19615), Enterococcus faecalis ATCC 29212 (E. faecalis 29212) and Bacillus subtilis ATCC 15245 (B. subtilis 15245). Gram-negative strains included Escherichia coli ATCC 25922 (E. coli 25922), Pseudomonas aeruginosa ATCC 27853 (P. aeruginosa 27853), Klebsiella pneumoniae ATCC 700603 (K. pneumoniae 700603), Acinetobacter baumanni ATCC 19606 (A. baumanni 19606) and Proteus mirabilis ATCC 12453 (P. mirabilis 12453). The identity of the microorganisms assayed in this research was confirmed by morphological studies and standard biochemical tests (Franco-Duarte  et al., 2019), (Gundappa et al., 2021).
 
Determination of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)
 
The MIC (Djahida and Houcine, 2021) and MBC were determined using the broth macro-dilution method (Ivan G. Ivanov  et al., 2019) . In this assay, 1.76 mL of the EO was diluted with 20.24 mL of Muller-Hinton broth (MHB) and DMSO (1 %). Two-fold serial dilutions of EOs were then prepared to achieve a series of decreasing concentrations ranging from 8 to 0.0156 % (8, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, 0.0313 and 0.0156 %). Each concentration was divided into 11 tubes consisting of 100 µL each. 50 µL of the bacterial suspension (prepared in a saline solution) was added to each tube in a final concentration of McFarland 0.5. The EO was not added to the positive control tube while microorganisms were not included in the negative control sample. All tubes were incubated at 37°C for 24 h.
       
After 24 h of incubation, the turbidity of each test tube was compared against the corresponding control, followed by the selection of two adjacent tubes having turbidity equal to and greater than the standard, respectively. Thereafter, 50 µL of each selected tube was taken and spread on a petri dish with TSA medium by sterile cotton swabs. After 24 h of incubation, the growth of microorganisms was evaluated by observing the appearance of colonies growing on plates. The MIC value was defined as the lowest EO concentration that did not show any growth of bacteria. The MBC value was determined as the lowest concentration of the EO showing negative subculture after incubation at 37°C for 24 h.
 
Statistical analysis
 
All experiments were conducted in triplicate and the results were expressed in terms of Mean ± Standard Error of Mean (SEM). Statistical analysis was performed by SPSS and analysis of variance (ANOVA) with a level of significance ñ < 0.05.

Essential oil yield
 
The yield of the YBO, QNO and KTO were 1.315 ± 0.136, 2.600 ± 0.037 and 1.505 ± 0.045 %, respectively. The yield of QNO (2.600 ± 0.037 %) was approximately equal to that of C. cassia bark essential oil from Guangdong and Guangxi (2.55%) which was conducted by (Abdurahman Nour  et al., 2016).
 
Chemical compositions of essential oils
 
Fig 3 and Table 1 show the identified components present in EOs of YB, QN and KT, respectively. The EO from C. cassia bark of YB consists of 19 compounds accounting for 99.91% of the total oil. Meanwhile, the EO from C. cassia bark of QN contains 19 constituents representing 99.98% of the total oil. EO from C. cassia bark of KT, on the other hand, includes 21 compounds accounting for 99.88% of the total oil. As expected, trans-cinnamaldehyde was the most abundant in three EOs which was accounted to compose more than 94% of the constituents of total oil. Noticeably, the KT C, cassia bark EO provided the highest trans-cinnamaldehyde concentration at 96.07% of 99.91% total, followed by YB (94.72%) and QN (94.69%). The amount of trans-cinnamaldehyde present in YBO, QNO and KTO was extremely greater than that of the C. cassia bark EOs from the Guangdong and Guangxi provinces (66.28 - 71.22% and 73.56 - 77.21%, respectively) which was conducted by (Li et al., 2013). Notably, GC-MS analysis revealed the presence of nine other compounds in all three EOs (Fig 4 -A, B). However, four components were only detected in YBO (Fig 4 -C1), seven in QNO (Fig 4 -C2) and nine in KTO only (Fig 4 -C3).

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Organoleptic and physicochemical properties
 
Table 2 details the physicochemical and organoleptic characteristics of the C. cassia bark essential oils from YB, QN and KT. All three essential oils had the same color (light yellow), odor (cinnamon-like note) and taste (hot and spicy). There were generally no significant differences in physicochemical parameters among three C. cassia bark essential oils, namely total acid number, saponification value, ester value and peroxide value, but specific gravity, optical rotation and iodine value.


 
Minimum inhibitory concentration and minimum bactericidal concentration
 
There were significant differences in the susceptibilities of C. cassia bark EOs against ten studied microbial strains (Table 3). Indeed, KTO generally exerted powerful effectiveness against ten selected microbial strains as both MIC and MBC were recorded as low as 0.0156-0.125% and 0.0313-0.250%, respectively. Differences in the susceptibility of tested microorganisms to KTO were quantitatively identified. Accordingly, KTO was able to powerfully inhibit the growth of S. pyogenes 19615 at the lowest dose of 0.0156% whereas five microbial strains, namely S. aureus 25923, E. faecalis 29212, B. subtilis 15245, E. coli 25922 and A. baumanni 19606, displayed the least sensitivity to KTO with the same highest MIC value of 0.125%. Notably, the same trend had been identified in MBC, but at different levels.


       
QNO also exhibited potent antibacterial capacity against ten selected microbial strains as MIC and MBC were documented to be 0.0313-0.250% and 0.0625-0.500%, respectively. P. mirabilis 12453, in contrast to KTO, was found the most susceptible to QNO as both MIC and MBC were recorded to the lowest dose of 0.0313% and 0.0625%, respectively. Three microbial strains, namely P. aeruginosa 27853, K. pneumoniae 700603 and A. baumanni 19606, displayed the same MIC and MBC values of 0.0625% and 0.125%, respectively. However, the differences in susceptibility of six other tested microbial strains to QNO could be noted as MIC of three microbial strains, namely S. aureus 25923, S. pneumoniae 49619 and S. pyogenes 19615, was valued at the same dose of 0.125%, whereas E. faecalis 29212, B. subtilis 15245 and E. coli 25922 had the same dose of 0.250%. The same pattern had been observed in MBC but at different levels.
       
On the other hand, YBO of the lowest concentration (MIC value of 0.0625% and MBC value of 0.125%) was able to inhibit six studied microbial strains (S. aureus 25923, S. pneumoniae 49619, E. coli 25922, P. aeruginosa 27853, A. baumanni 19606, P. mirabilis 12453). B. subtilis 15245, on the contrary, exerted the least sensitivity to YBO with MIC and MBC values of 0.250% and 0.500%, respectively.
       
Although trans-cinnamaldehyde was predominant in all three EOs, representing more than 94% of the total oil, significant differences in antibacterial activity against ten studied microbial strains were documented, reflecting the effects of other components in C. cassia bark essential oils. EO is a complex mixture of naturally occurring volatile compounds, primarily terpenoids, phenylpropanoids and phenolics. Certain components present in EO might interact with others, resulting in variations in binding affinity to different lipophilic molecular structures of lipids, protein, glycolipids, lipoprotein and glycoprotein present in the bacterial cell wall and cytoplasmic membrane. In addition to the diversified and complicated nature of phytocomponents present in EOs, the differences in susceptibility of Gram-negative and Gram-positive to a variety of EOs considerably attribute to the effects of antibacterial activity against microorganisms. Nevertheless, the present results would hopefully encourage more studies to elucidate the mechanisms behind which C. cassia bark essential oils exhibited antibacterial effects, for the purpose of exploiting the potential of this valuable EO to the fullest.

In conclusion, trans-cinnamaldehyde was found to be the major constituent in all three essential oils claiming more than 94% of the total composition of essential oils. There were generally no significant differences in essential oil compositions and physiochemical parameters but varying in antibacterial activities against ten studied microbial strains. This present research scientifically contributes to the herbal database system and promotes the significant potential of high-quality C. cassia bark essential oils from three selected regions of Vietnam that might be exploited to develop in the food and pharmaceutical industry, bringing huge benefits to human health, which encourages increased planting of crops for export and domestic industry. The extraction of C. cassia bark essential oils was conducted using an eco-friendly method, minimizing environmental impact and ensuring sustainability. Nevertheless, further studies are necessary to establish the international standard for essential oils from the cinnamon bark of C. cassia planted in three selected geographic regions of Vietnam, particularly the regulation of specific quantity ranges of major active components present in C. cassia bark essential oils. Perhaps, the design of sampling on certain geographic regions is the limitation of this study. Future research should expand more studies on geographical scope, investigating molecular mechanisms, assessing health benefits, developing novel pharmaceutical products and considering other varieties within this species based on geographical and environmental factors.

The authors are grateful to the authorities at the Department of Applied Biochemistry, Ho Chi Minh City International University- Vietnam National University for providing the facilities to conduct this study.

The authors declare to have no conflict of interest.