Essential Oils from the Malaysian Citrus (Rutaceae) Medicinal Plants

This review article appraises the extraction methods, compositions, and bioactivities of the essential oils from the Citrus species (family: Rutaceae) endemic to Malaysia including C. aurantifolia, C. grandis, C. hystrix, and C. microcarpa. Generally, the fresh peels and leaves of the Citrus species were extracted using different methods such as steam and water distillation, Likens-Nikerson extraction, solvent extraction, and headspace solid-phase micro-extraction (HS-SPME). Most of the Citrus oils were found to be rich in monoterpene hydrocarbons with limonene (1) as the major component identified in the peels of C. aurantifolia (39.3%), C. grandis (81.6%–96.9%), and C. microcarpa (94.0%), while sabinene (19) was the major component in the peels of C. hystrix (36.4%–48.5%). In addition, citronellal (20) (61.7%–72.5%), linalool (18) (56.5%), and hedycaryol (23) (19.0%) were identified as the major components in the oil of C. hystrix leaves, C. grandis blossom and C. microcarpa leaves, respectively. The C. hystrix essential oil has been experimentally shown to have antimicrobial and antifeedant activities, while no bioactivity study has been reported on the essential oils of other Malaysian Citrus species.


Introduction
The Citrus species of the family Rutaceae provide several popular edible fruits in the world. The species are widely distributed in the Indo-Malaysia region, South-East Asia, India and China, but cultivated worldwide [1]. The fruits and the leaves of the Citrus species contain a variety of essential oils with various distinct flavors, and biologically-active compounds, which are important to human nutrition and diet, which include vitamin C, folic acid, potassium, flavonoids, coumarins, pectin, and dietary fibers. In Malaysia, the oils from the fruits and the leaves are commercially used as flavors and fragrances, as well as in cooking, perfumery and medical treatments, especially in aromatherapy [2]. Recent studies on the Malaysian Citrus plants have reported the identification and composition of essential oils of several Citrus species including C. aurantifolia, C. grandis, C. hystrix, and C. microcarpa. A monoterpene hydrocarbon, limonene (1), is the major component in the essential oils from the peels of these Malaysian Citrus species [3][4][5]. This review focuses on the details of extraction methods, identification and composition of essential oils from the Malaysian Citrus species and also on their biological properties.

Citrus hystrix
Citrus hystrix (synonym: kaffir lime) ( Figure 4) is known as 'limau purut' or 'wild lime'. C. hystrix leaves and fruits are widely used as spices in preparation of 'tomyam', either white or red, and it is famous dish in Malaysia and Thailand [22]. The height of this plant is about 3-5 m and the fragrant green leaves are 7.5-10 cm long. It has white flowers with 4-6 petals. The diameter of pear-shaped fruits is about 5.0-7.5 cm with wrinkle on the surface of fruit. The fruit is dark green, and yellow when ripe [23]. The essential oil of C. hystrix is used in aromatherapy and an essential ingredient of various cosmetic and beauty products [20].
In traditional medicine, C. hystrix is used to treat flu, fever, hypertension, abdominal pains, and diarrhea in infants [24]. The fruits are used as a digestive stimulant, blood purifier, and reduce high blood pressure [25,26]. Additionally, the fruits are used in cooking for flavoring and also in the production of shampoo as an insecticide for washing the head [27]. In addition, the fruit juice is used in softening the skin and the mixture of the fruit juice with bath water can be used to eliminate body

Citrus hystrix
Citrus hystrix (synonym: kaffir lime) ( Figure 4) is known as 'limau purut' or 'wild lime'. C. hystrix leaves and fruits are widely used as spices in preparation of 'tomyam', either white or red, and it is famous dish in Malaysia and Thailand [22]. The height of this plant is about 3-5 m and the fragrant green leaves are 7.5-10 cm long. It has white flowers with 4-6 petals. The diameter of pear-shaped fruits is about 5.0-7.5 cm with wrinkle on the surface of fruit. The fruit is dark green, and yellow when ripe [23]. The essential oil of C. hystrix is used in aromatherapy and an essential ingredient of various cosmetic and beauty products [20].
In traditional medicine, C. hystrix is used to treat flu, fever, hypertension, abdominal pains, and diarrhea in infants [24]. The fruits are used as a digestive stimulant, blood purifier, and reduce high blood pressure [25,26]. Additionally, the fruits are used in cooking for flavoring and also in the production of shampoo as an insecticide for washing the head [27]. In addition, the fruit juice is used in softening the skin and the mixture of the fruit juice with bath water can be used to eliminate body odor [28]. Furthermore, the essential oil of C. hystrix has been reported to have various bioactivities such as antioxidant, antibacterial, antileukimic, and antitussive [26].
The antibacterial susceptibility of the essential oils and oil emulsions of Malaysian C. hystrix was evaluated against Escherichia coli, Bacillus subtilis, and Staphylococcus aureus using the disc diffusion method. Pure essential oil with a percentage of 2% by weight exhibited a strong inhibitory effect against E. coli and B. subtilis with the zones of inhibition of 16.0 and 15.0 mm, respectively. Meanwhile, the formulated emulsions with surfactant mixture of Tween 80 and Span 80 (90:10) with 2% (by weight) essential oil displayed the most potential antibacterial activity against E. coli with the zones of inhibition ranging between 11.0 to 18.0 mm [2].
The topical application bioassay on uniform weighted second instar larvae in the laboratory was carried out to determine the insecticidal properties of the essential oil from the leaves of the Malaysian C. hystrix against Spodoptera litura (tobacco army worm). The study demonstrated considerable repellant activity of C. hystrix essential oil against the S. litura larvae after 24 and 48 h of treatment with LD 50 values of 29.25 and 26.75 µg/mL, respectively [29].

Citrus microcarpa (Bunge) Wijnands
Citrus microcarpa (synonym: C. madurensis) ( Figure 5), common name: 'limau kasturi' in Malaysia, is used in the preparation of beverages. C. microcarpa is 3-5 m tall with abundant of long spine on the stem, branches and twigs. The dark green leaves of C. microcarpa are between 2.5-6.8 cm long and 2-3 cm thick. The round or oblong-shaped green leaves of this plant are 2.5-3.8 cm in diameter. This plant is used to treat fever, cough, and pharyngitis [30]. The juice is traditionally used to prevent respiratory diseases, strengthen the bones and act as growth stimulant for children. The juice is also commonly used in cooking as flavoring ingredients and additives. The leaves of this plant can be used in the treatment of skin diseases, relieve headache and also act as a mouth wash to treat sore throat [31]. Essential oil from C. microcarpa is used commercially in perfumes, food, cosmetics and detergents. It is one of the ingredients in pharmaceutical, aromatherapy and antiseptic products [32].

Conclusions
Extraction and identification of the essential oils from the Malaysian Citrus species showed that limonene (1) (96.9%) and sabinene (19) (48.5%) were the major components in C. grandis and C. hystrix, respectively. Sample collections from different locations, and differences in extraction methods resulted in different composition and percentage of yields. Moreover, extraction of essential oils from different parts of Citrus plants also gave different major components. The bioactivity studies on the C. hystrix essential oil revealed strong antimicrobial activity against E. coli and good antifeedant properties against S. litura. More bioactivity studies on the essential oils of the Malaysian Citrus plants need to be carried out to acquire better bioactivity profiles of these oils.

Conclusions
Extraction and identification of the essential oils from the Malaysian Citrus species showed that limonene (1) (96.9%) and sabinene (19) (48.5%) were the major components in C. grandis and C. hystrix, respectively. Sample collections from different locations, and differences in extraction methods resulted in different composition and percentage of yields. Moreover, extraction of essential oils from different parts of Citrus plants also gave different major components. The bioactivity studies on the C. hystrix essential oil revealed strong antimicrobial activity against E. coli and good antifeedant properties against S. litura. More bioactivity studies on the essential oils of the Malaysian Citrus plants need to be carried out to acquire better bioactivity profiles of these oils.