Essential oil of Citrus hystrix DC.: A mini - review on chemical composition, extraction method, bioactivities, and potential applications in food and pharmaceuticals

Citrus hystrix DC. is a common herb in tropical regions. Its essential oils are now widely researched and applied because of their high economic value and safety for humans and are interesting materials for future trends. This review provides an extensive overview of the biological activities of C. hystrix essential oil, characterized predominantly by citronellal, β - pinene, sabinene, limonene


Introduction
The scientific name of this plant is Citrus hystrix DC., and it is part of the genus Citrus belonging to the Rutaceae family.It grows well in the typical warm and humid climate of tropical and subtropical regions and is native to Southeast Asia and South China.Being known and commonly used in daily life by indigenous peoples, it is called by different names, including Kaffir lime, Thai Lime, and Chúc.C. hystrix is a small tree that is about 3-6 m high and 2.5-3 m wide with crooked spiny branches.Its leaves are unifoliolate, alternate, 7.5-10 cm long, 5 cm wide.It has small and white flowers.Fruits are large, warty, or bumpy, verrucose, globose to ovoid, green turning yellowish-green when ripe (1).In addition to its trunk and branches, its fruit, which is unpopular and generally harvested with its leaves in June and July, is a by-product of the exploitation process and is used to make essential oil (EO) through distillation (2).
In traditional medicine, C. hystrix is also used to treat colds, fever, hypertension, colic, and diarrhea in infants.In addition, the EO from its fruit and leaves is commercially used as a flavouring, and in cooking, perfume, insecticides, food technology, and medical treatments (1).In Malaysia, fruit and leaves are used to wash hair, and fruit juice is applied to the skin to soften and control body odors.Besides, the fruits are also used to separate leeches from the skin, while fresh leaves are frequently used to brush teeth and gum to improve oral health (3).Moreover, Thai people use fresh leaves to make Tom Yum Gai and Tom Yum Kung, as well as Kroeung in Cambodia and Laos, and Sayur asem in Indonesia (1).Those are the special dishes and spices of these countries.

Chemical composition of essential oil from the leaves and fruit peels of C. hystrix
According to the study of Sato et al., 58 chemical compounds were found in C. hystrix EO.Citronellal, a major component (81%) of leaf oil, has the highest antioxidant activity, making it a feature of C. hystrix leaf oil.In contrast, the oil from the fruit peel is rich in β-pinene (25.93%) and sabinene (20.36%) (4).There is also regional variation in the composition of leaf oil.For example, citronellal concentration in leaf oil from Long Xuyen province (Vietnam) is significantly high at 86.89% (5).

Antimicrobial activity
The chemical composition of C. hystrix EO was found to have antimicrobial activity.In the study of Chanthaphon et al., peel oil showed higher antimicrobial activity than other citrus fruit oils.It acted against Saccharomyces cerevisiae, Bacillus cereus, Staphylococcus aureus, and Escherichia coli due to antibacterial components, such as βpinene (30.6%), limonene (29.2%) and sapinene (22.6%) (7).Additionally, Sreepian et al. (8) indicated that grampositive bacteria were more sensitive to C. hystrix oil than gram-negative bacteria.Overall, the peel oil of C. hystrix could be considered a potent ingredient with high antimicrobial properties and natural origin, suitable for use as an alternative to synthetic substances in the pharmaceutical and food industries.

Antioxidant activity
Similarly, C. hystrix EO has antioxidant properties, and oils extracted from different plant parts show differing antioxidant activity.According to Warsito et al. (9), leaf oil had an antioxidant activity that was twice as high as peel oil and oil from branches.Citronellal, which is abundant in leaf oil at 85.07%, typically demonstrates the antioxidant characteristics of leaf oil (9).Note that, the extraction method also influences antioxidant activity and this property can be attributed to bioactive compounds obtained (10).In addition, Paramitha and Nabila evaluated the leaf oil produced by steam distillation and the extract collected by macerating C. hystrix leaves in ethanol.The results showed that the extract had higher antioxidant activity than the leaf oil because the oil was impaired by the high temperature in the steam distillation process (11).
These studies also indicate that the extraction method affects antioxidant properties because antioxidant constituents are lost during extraction.In addition, the C. hystrix EO from different parts also showed differing antioxidant activity, and the leaf oil was considered to have higher antioxidant activity, which explains its more common use.

Insect repellent activity
Another exciting feature that local people have long used daily is repelling insects with fresh leaves and fruit, which are toxic to insects but safe for humans.Experimental findings showed that all fly larvae were killed at a 50 percent lethal concentration (LC50) of 71 g/L, and mature flies were killed at a 50% lethal dose (LD50) of 408.63 μg/fly (12).The study has also indicated that compounds, such as citronellol, limonene, pinene, and terpinene-4-ol, in EO, positively impact mosquito repellent, and an addition of 5% vanillin prolongs the protection time to three hours because pure oil is volatile at room temperature (13).This shows that this material has a very high potential for controlling insects that cause infectious diseases.

Anti-inflammatory and anti-tumor activity
C. hystrix is also used as an herbal medicine to treat common diseases, such as cold, fever, cough, and abdominal pain.However, recent studies on the action of this material at the molecular level have shown that it directly or indirectly inhibits protein and enzyme molecules, which in turn induces anti-inflammatory and anti-tumor effects.For example, Lertsatitthanakorn et al. reported the antiinflammatory property of C. hystrix EO when it could act against Propionibacterium acnes and suppress 5lipoxygenase, which is an inflammatory enzyme, with the contribution of D-Limonene (14).In addition, Othman et al. also revealed that citrus EO had effective antiproliferative activity against the HeLa cervical cancer cell line with a half maximum inhibitory concentration (IC50) of 25.91 µg/mL (15).
Other reports also showed that C. hystrix peel oil with a 220-300 μg/mL concentration had a potential antimelanoma effect on three human cell lines, WM793, A375, and HTB140, by inhibiting hyaluronidase and tyrosinase.Furthermore, it is safe and non-toxic to human skin cells (16).

Pre-treatment methods
Pre-treatment aims to improve the efficiency of the oil extraction process by using physical or biological agents.One of the most traditional, simple, and economical methods is reducing material size with physical processes such as cutting, grinding, and crushing, which increase the surface area of the material, increase heat transfer, and weaken the cell wall.In addition, a high percentage of broken cells leads to better mass transfer, effectively transporting constituents to the outside.A size reduction of C. hystrix leaves from 600 to 400 µm increased the yield efficiency from 15.97 to 23.52% (17).
Most materials must be dehydrated to maintain quality and minimize the growth of fungi and other microbiomes during storage and transport.Dehydration helps reduce free water, and the compounds are pulled out, which makes the solvent penetration process easier before extraction.This also explains why volatile substances are lost or altered during dehydration (18).Specifically, prolonged dehydration time decreases oil yield efficiency.In one study, C. hystrix leaves were dehydrated at ambient temperature.The results showed that the yield efficiency peaked at 1.26 percent on the 4 th day and then decreased gradually (19).Therefore, choosing the appropriate method, temperature, and time during the dehydration process is crucial, which consequently influences EO quality.Dehydration temperatures between 40 and 60°C, and material size reduction, microwave, ultrasound, or shortening processing time are reasonable options.
Another pre-treatment method related to biology is using microbiome enzymes.Enzyme activity breaks down cellulose linkages, thus weakening the cell wall.The compounds are subsequently pulled out, which assures extraction efficiency and the best EO quality.The effects of enzyme pre-treatment are presented in Table 1.
Modern pre-treatment techniques, such as ultrasound, are applied to highly bioactive and thermally unstable compounds, which helps reduce the alteration and loss of essential chemical compounds.Ultrasound involves the formation and implosive rupture of cavitation bubbles, which disrupt the cell wall and facilitate solvent penetration.The extracted compounds then easily flow out without altering nature, thus enhancing oil yield and quality, shortening extraction time by up to 70%, and improving the oil's antimicrobial and antioxidant activity.However, a prolonged ultrasound pre-treatment time results in the escape of volatile substances and diminishes oil quality and output (23).

Essential oil extraction methods
Hydro-distillation is a method for distilling EOs in which water vaporizes to form a heterogeneous mixture and then liquefies by indirect cooling in a condenser that flows to a separator.One feature of hydro-distillation is that the material is completely immersed in water, which protects it from heat.Moreover, steam distillation is also a preferred and popular method on an industrial scale.It is used to extract plant materials that have a high oil content and are heat-sensitive.In the same way as hydro-distillation, however, the material does not directly contact water in this method; therefore, extraction efficiency and oil quality are higher (24).
In addition, soxhlet extraction is a standard method used in oil extraction.The material continuously contacts the new solvent, which prevents solvent saturation and optimizes the distillation procedure.Nowadays, at a higher technical level, people use pressurized liquid extraction (PLE) with liquid solvents at high temperatures and pressures to extract EOs.The extraction process takes place within a set period once the desired pressure and temperature are reached.Haiyee and Winitkitcharoen also applied this method to extract the EO from C. hystrix leaves, obtaining an excellent yield efficiency of 42.27% (dry weight basis), respectively (25).Based on the points mentioned above, depending on the technical and economic conditions, we can choose an appropriate extraction method (Table 2).

Applications in food technology and pharmaceuticals
People utilize the antibacterial and antioxidant properties of C. hystrix EO in food preservation.However, the direct use of EOs is quite limited because they are volatile compounds.Therefore, many methods are used to slow down the loss of EOs by combining them with other materials, such as proteins, lipids, and polysaccharides, which prolong the action time of EOs (31).For example, C. hystrix leaf oil can be combined with cassava starch to form an edible coating, which prolongs the shelf life of fresh beef, according to Utami et al. (32).This coating prevents the accumulation of by-products related to lipid oxidation and inhibits microbial growth.As a result, this method extends the shelf life of fresh beef (Table 3).
In addition, Pumival et  results showed an excellent ability to inhibit this fungus and hence offered worthy pharmaceutical applications (33).
So, using C. hystrix EO is a solution for future trends because citrus EOs that come from natural plants are considered to be safe, eco-friendly, antimicrobial, and antioxidant, and they can replace synthetic preservatives.

Conclusion
Concern for public health worldwide is increasing; therefore, research to discover a new direction from natural ingredients to replace synthetic substances in food and pharmaceutical products is an inevitable trend.C. hystrix EO is a potential research field because of its antimicrobial, antioxidant, anti-inflammatory, and anti-tumour properties and its abundant material resources.

Table 1 .
al. also evaluated the antifungal ability of C. hystrix leaf EO.They applied a microemulsion of C. hystrix leaf oil to treat foot fungus caused by Trichophyton mentagrophytes var.interdigitale.The Pretreatment time and microbiome species in correlation with yield efficiency of Citrus hystrix essential oil

Table 2 .
Comparison of the efficiency, advantages, and disadvantages of popular Citrus hystrix essential oil extraction methods

Table 3 .
Application of Citrus hystrix essential oil combined with other compounds in food