1. Introduction
Kaffir lime, also known as Thai lime, is a tropical fruit of the genus Citrus in the Rutaceae family (Kaffir lime is the more common name). Its scientific name is Citrus hystrix DC. (Srifuengfung et al., 2020). The kaffir lime tree is about 3-5 m high and its fruit is round to egg-shaped and often has a distinct nipple-like structure near the apex. They have thick, wrinkled green skin that turns yellow after ripening (Abirami et al., 2014). Essential oils (EO) exist in the leaves, flowers, and fruit; the oils exist in the highest concentrations in the green skin of the fruit. The EO content of the kaffir lime fruit is 3 to 5 times higher than other citrus fruits in the same family (Giao et al., 2022).
People use almost every part of the plant, such as the leaf, fruit, seed, and root, to treat many diseases. Phytochemicals isolated from different parts of C. hystrix have been found to have various pharmacological activities, such as antibacterial, antifungal, antioxidant, anti-inflammatory, and anticancer activities (Abirami et al., 2014). The EO of C. hystrix was reported to have antibacterial and anti-inflammatory activity against some typical bacteria, such as Propionibacterium acnes. D-limonene in EO could also inhibit P. acnes and reduce inflammation, which in turn reduced post-acne scar formation and faded acne dark spots (Lertsatitthanakorn et al., 2006). On the other hand, C. hystrix EO extracted from fruit peel showed anti-proliferative effects on human skin fibroblasts and melanoma cells A375 and WM793 (Borusiewicz et al., 2017). It also inhibited the growth of dandruff-causing Candida albicans on the scalp (Foo-trakul and Watchiradatsatiean, 2005).
The bioactive compounds in C. hystrix peel EO (ChpEO) are pretty diverse such as D-limonene, β-pinene, sabinene, citronellal, terpinen-4-ol, etc. (Sreepian et al., 2019). Their chemical composition and content differ when collected in different regions, harvest times, and extraction methods. The obtained EOs have usually been isolated using steam or hydro distillation. In addition to its medical and pharmaceutical technology application, ChpEO is also noticed in the agricultural field. It can be considered an antifungal agent, for instance, Chit-aree et al. (2021) proved that C. hystrix EO could be applied to protect against mango fungal contamination (Colletotrichum gloeosporioides). For the food industry, the EO of C. hystrix peel is of little interest and is mainly used for culinary purposes.
In fact, there have been many studies on C. hystrix EO; however, the EO from different locations has different chemical compositions and properties. Meanwhile, C. hystrix is quite commonly cultivated in Tinh Bien, An Giang province (Vietnam), mainly for its fruit. The EO in the fruit peel of C. hystrix is considered a by-product, so its value has not been fully exploited yet. This study aims to determine the chemical composition and some physicochemical and biological properties of C. hystrix EO to further understand the value of this EO and its potential application in food technology and pharmacology.
2. Materials and methods
The EO was extracted from the peel of C. hystrix harvested in the Tinh Bien district, An Giang province (Vietnam) (Coordinates: 10°37'55.6"N 104°59'34.8"E). The fruits had reached the desired maturity (3-4 months old) and the weight of each fruit was 46.84±5 g. The peel was used to extract the EO by steam distillation. The strong point of steam distillation is that it produces clean and pure oils. The obtained EOs can contain many temperature-sensitive compounds with specific biological properties that may be damaged or changed by other extraction methods. The fruits were washed and dried naturally at room temperature, then peeled and cut into small pieces. On average, about 2.5 kg of whole fruit produced 1 kg of peel; the per-batch yield was 50-60 kg peel/batch. The distillation time was 3 h at 100℃. The yield of the distillation process was about 2.8%. The obtained EO was stored in dark glass jars at room temperature.
The bacteria used in this study included 4 strains: 2 gram-positive bacteria, Staphylococcus aureus (ATCC 25923) and Bacillus cereus (ATCC 11778), and 2 gram-negative bacteria, Pseudomonas aeruginosa (ATCC 27853) and Salmonella typhimurium (ATCC 13311). They were provided by the Institute of Biotechnology and Food Technology, Industrial University of Ho Chi Minh City.
The chemicals used in this study included 2,2-diphenyl-1-picrylhydrazyl (DPPH, Sigma, St. Louis, Missouri, USA) and dimethyl sulphoxide (DMSO, Nanjing, China). The culture and antibacterial media included Mueller Hinton Agar (HiMedia, Thane, India), Nutrient Broth (HiMedia, Thane, India), and other chemicals meeting analytical standards.
The physicochemical properties of the EO, such as the freezing point, acid value (AV), saponification value (SV), ester value (EV), specific gravity, and density, were determined respectively according to following standards: ISO 1041 (1973), ISO 1242 (2023), ISO 3657 (2013), and ISO 279 (1998).
The chemical composition of the EO was analysed by gas chromatography-mass spectrometry analysis method (GC-MS) according to the method described by Van et al. (2022) with some minor changes. First of all, 20 μL of sample was dissolved in methanol and made up 1 mL with methanol. Then, 1 mL of EO was injected into an Agilent DB-5MS gas chromatograph with a capillary column (30 m×0.25 mm, 0.25 μm) at an injection temperature of approximately 220°C. The temperature was set up to 70°C in 1 min, then increased by 12°C/min to reach 280°C and then held for 10 min. The mass range was 29-650 amu. Helium was used as the carrier gas at a constant flow rate of 1.2 mL/min.
The antioxidant capacity (AC) of ChpEO was assessed in terms of the radical-scavenging activity (RSA) against DPPH according to the procedure described by Quoc (2020) with some slight modifications. The EO was dissolved in ethanol (96%) to obtain different concentrations (1,040, 520, 260, 130, 65, and 32.5 mg/mL). Then, 0.3 mL of the obtained EO was mixed with 2.7 mL of 0.1 mM DPPH. The mixture was kept in the dark at room temperature for 30 min. The decolourisation of DPPH was determined by measuring the absorbance at a wavelength of 517 nm using a spectrophotometer (Genesys 20, Thermo Scientific, Waltham, Massachusetts, USA). The AC of the EO was compared with ascorbic acid, as a control. The AC was calculated using the formula below:
Acontrol: Absorbance of a solution containing only DPPH solution.
Asample: Absorbance of the EO sample in the presence of DPPH.
The AC of the EO was estimated by the IC50 value (concentration of the sample required to inhibit 50% of DPPH free radical scavenging).
The antibacterial activity (AA) of the EO was determined by the paper disc diffusion method according to the procedure of Quoc (2021). First, 100 μL of bacterial suspension (0.5 McFarland, approximately 1.5×108 CFU/mL) was spread on a Petri dish containing Mueller Hinton Agar medium. Then, sterile paper plates (6 mm diameter) impregnated with EO (5 μL/disc) were placed on the plate with gentamicin (10 μg/disc) and DMSO solution (5%), which were used as the positive and negative controls, respectively. The plates were then incubated at 37°C for 24 h. The AA was assessed by measuring the diameter of the bacterial inhibition zone in millimetres (mm).
Three independent experiments were performed, and the results were expressed as mean±standard deviation (SD). The Statgraphics Centurion XV software was used for the statistical analysis of the results, using one-way analysis of variance (ANOVA) and Fisher’s least significant difference (LSD) procedure with a significance level of 5%.
3. Results and discussion
The obtained EO is an insoluble liquid in water, has a pale yellow colour, a characteristic aroma, and is very volatile. Its smell is similar to grapefruit EO and lemon EO.
The physical and physicochemical properties of the EO are expressed in Table 1 through parameters such as the freezing point, density, specific gravity, AV, SV, and EV. The freezing point of ChpEO was measured at −40°C, but there was no crystallisation phenomenon at this condition. This experiment has shown that the ChpEO has a very low freezing point when compared to Ageratum conyzoides L. EO, which had a freezing point of −10.33°C in the study by Quoc (2020). Thus, the freezing point of ChpEO is much lower than that of A. conyzoides L. The freezing point of ChpEO has not been mentioned in previous studies, so it is difficult to compare our results.
The absolute density and specific gravity of the ChpEO was 0.8478 g/mL and 0.8533, respectively. In previous research, a study performed by Wulandari et al. (2019) showed that C. hystrix leaf oil had a specific gravity of 0.843, while Husni et al. (2021) indicated an absolute density of 0.87 g/mL for C. hystrix peel oil, the materials for both of these studies were collected in Indonesia. It can be clearly seen that there is a negligible difference between these results and the results of our study. In addition, the refractive index (RI) of ChpEO reached 1.4699, compared with a value of 1.5 for Thymus zygis EO and 1.33 for Selaginella willdenowii EO (Radi et al., 2022). This shows that the RI is completely discrepant with different materials. The RI of EO also indicates the quality of the EO.
The AV and EV are criteria used to evaluate the quality of EOs. In this study, the AV and EV of ChpEO were 0.561 mg KOH/g and 10.66 mg KOH/g, respectively. In comparison, patchouli EO harvested in Indonesia had an AV and EV of 23.9 mg KOH/g and 8.48 mg KOH/g, respectively (Lubis et al., 2022), indicating that the AV of ChpEO was 42.5 times lower than that of patchouli EO, while the EV was not significantly higher. The above results demonstrate the special physicochemical properties of ChpEO and highlight its high potential EO in the food and pharmaceutical industry in the future, with a low acid index, and a higher ester index compared with other EOs.
The difference in physicochemical indexes with other studies is due to the difference in raw material sources, the different parts of the plant used to extract the EO, such as the leaves, roots, bark, fruits, etc., as well as the difference in harvest time, which leads to difference in the properties and chemical compositions of the EOs.
Table 2 shows that ChpEO has 22 volatile compounds identified, which account for 99.98% of the total number of compounds identified. The number of compounds in this study was significantly different from C. hystrix EO obtained from other locations. For instance, leaf oil originating in Malaysia had 29 compounds accounting for 99.75% (Loh et al., 2011), while peel oil from Thailand had 27 compounds accounting for approximately 89.98% (Sreepian et al., 2019). In general, the chemical composition of C. hystrix EO fluctuates quite considerably, which might be due to climate, plant variety and soil characteristics in each area, as well as differences in conditions and extraction methods. The main compounds of ChpEO were also present in juniper EO, as in the study by Zheljazkov et al. (2017), including α-pinene, β-pinene, β-myrcene, sabinene, and limonene.
The major compounds of ChpEO identified in this study were β-pinene (30.19%), D-limonene (22.15%), and sabinene (21.37%), and these compounds were also present in ChpEO from Malaysia and Thailand. However, there was an insignificant variation in the content of the main chemical compositions; for instance, β-pinene, limonene, and sabinene accounted for 21.1%, 25.28% and 14.99% respectively in ChpEO from Thailand (Sreepian et al., 2019). Chanthaphon et al. (2008) also reported β-pinene as having the highest concentration in ChpEO in Thailand. In addition, sabinene might contribute to the anti-microbial activity (Chanthaphon et al., 2008) and D-limonene has been shown to have a powerful antioxidant activity against cancer (Miller et al., 2011). Thereby, it can be seen that ChpEO had many precious compounds with different biological activities. The combination of these substances with different ratios in the EO can also create special effects due to their ability to interact synergistically. Some precious compounds with high biological activity but low content were also identified in the oil, such as germacrene D (0.15%), camphene (0.17%), caryophyllene (0.22%), etc. This finding demonstrates that ChpEO is a potential material worthy of future research for application in the food and medical fields.
Based on the obtained results, the chemical composition of ChpEO includes: Monoterpene (monoterpene hydrocarbon and monoterpenoids) and Sesquiterpene. There were 19 of the most predominant substances belonging to the Monoterpene group (99.3%), of which Monoterpene hydrocarbons accounted for 83.52% and Monoterpenoids accounted for 15.78%. Three compounds belonged to the Sesquiterpene group, with 0.68% Sesquiterpene hydrocarbon including copaene, caryophyllene, and germacrene D. Therefore, it can be seen that the characteristic composition of ChpEO is Monoterpenes (99.3%), which is also the most common group present in most EOs.
The AC is one of the most important properties for determining the quality of an EO or its potential for application in the preservation of food products. With an increasing concentration of EO from 32.5 to 1,040 mg/mL, Fig. 1 shows that the AC becomes more effective when the amount of ChpEO increases. The AC increased rapidly from a concentration of 130 to 520 mg/mL and increased slowly from a concentration of 520 to 1,040 mg/mL. The IC50 of ChpEO was determined at approximately 500 mg/mL and the AC reached 79.6% at an EO concentration of 1,040 mg/mL. In particular, the AC of the pure EO only inhibited about 92%. ChpEO showed a much lower AC than the control sample ascorbic acid. This is acceptable because ascorbic acid is a potent antioxidant and its IC50 was determined at a concentration of 36 μg/mL (Fig. 2).
According to previous studies, most EOs have antioxidant properties. When compared with ChpEO from other regions, the IC50 of this study was approximately 500 mg/mL, while ChpEO from Indonesia had an IC50 of 72.78 mg/mL according to the study of Zuhra et al. (2014), and this value (IC50 = 500 mg/mL) was >250 μg/mL for material from Thailand (Wungsintaweekul et al., 2010). According to the study by Othma et al. (2023), the IC50 of EO from C. hystrix leaves was 279.03 mg/mL, which is 1.8 times lower than the peel oil in this study, indicating that the AC of leaf oil is stronger. The difference in AC of EOs can greatly depend on their origin, growing regions, and chemical composition. The AC of ChpEO is caused by the presence of several strong antioxidant compounds, especially β-pinene (30.19%), D-limonene (22.15%), and sabinene (21.37%). These substances have been shown to have good AC (Amorati et al., 2013).
Table 3 shows that the inhibitory ability of gentamicin in ascending order is as follows: S. typhimurium / S. aureus / P. aeruginosa < B. cereus. The diameters of the inhibition zone of gentamicin range from 12.67 to 15.33 mm. The obtained results indicate that the tested bacteria, including S. aureus, S. typhimurium, and P. aeruginosa (inhibition diameter between 8 and 14 mm), are sensitive to gentamicin, while B. cereus (inhibition diameter between 14 and 20 mm) is very sensitive to gentamicin (Sebei et al., 2015). In addition, for ChpEO, the ascending order of bacterial inhibition is as follows: S. typhimurium < P. aeruginosa / B. cereus < S. aureus. The AA is strongest against S. aureus (inhibition diameter of 22.33 mm) and weakest against S. typhimurium (inhibition diameter of 19 mm). B. cereus, S. aureus, and P. aeruginosa are extremely sensitive to ChpEO (inhibition diameter ≥ 20 mm). Notably, all inhibitory zone diameters for the EO were superior to those of the positive control (gentamicin) with regard to the 4 strains of Gram-negative and Gram-positive bacteria used in the study.
The mechanism of action of EOs on microbial cells follows various pathways. For instance, EOs can penetrates into the cell membrane and increase cell membrane permeability, and denature proteins. It can also inhibit ATP synthesis through energy metabolism, or damage the cell membrane and inhibit peptidoglycan synthesis through the cell wall. Through a genetic material pathway, it can inhibit gene expression or impede DNA repair. The antibacterial mechanism of the compounds present in the EO may be different. Therefore, the antibacterial mechanism of an EO is often not a single mode of action but includes many different activities. Genetic material plays a key role in the process of growth, development, and reproduction; and can control the synthesis and metabolism of protein in the body, hence controlling life activities. Previous reports have shown that terpenoids and phenolic compounds in EOs inhibit bacterial DNA replication and repair (Ni et al., 2021).
Given the superior antibacterial properties of ChpEO above, the application of a combination of this oil with other agents is a possibility in the food industry, to increase the effectiveness of the preservation process in future.
4. Conclusions
In summary, the chemical composition of C. hystrix EO from fruit peel harvested in Tinh Bien, An Giang province (Vietnam) was analysed by GC-MS and found to contain 22 volatile compounds. The main compounds accounting for a high concentration of the oil include β-pinene (30.19%), sabinene (21.37%), and D-limonene (22.15%). The ChpEO could inhibit the growth of both specific Gram-positive and Gram-negative bacteria, including P. aeruginosa, S. aureus, S. typhimurium, and B. cereus. The oil also had specific physicochemical properties (RI, AV, SV, EV, etc.) compared to other raw materials and potentially has a wide application in food preservation technology in order to replace synthetic food preservatives.
