A Review of Genetic Taxonomy, Biomolecules Chemistry and Bioactivities of Citrus hystrix DC

1Research Unit on Analysis and Technological Development in Environment (URADTE), Centre de Recherche Scientifique et Technique en Analyses Physico-Chimiques (CRAPC), Tipaza, Algeria. 2Laboratory of Reaction Engineering (LGR), Faculty of Mechanical Engineering and Process Engineering (FGMGP), Houari Boumediene University of Sciences and Technology (USTHB), Algiers, Algeria. 3Institute of Bioproduct Development (IBD), Universiti Teknologi Malaysia (UTM), Johor Bahru, Malaysia. 4Faculty of Chemical Engineering and Energy, Universiti Teknologi Malaysia (UTM), Johor Bahru, Malaysia. 5Genetic Engineering and Biotechnology Research Institute, City of Scientific Research and Technology Applications (CSAT), New Burg Al Arab Alexandria, Egypt.

The genus Citrus belongs to the subfamily Aurantioideae and order of Sapindales of the Rutaceae family which comprises of about 140 genera and 1,300 species distributed between 7 subfamilies 2, 3, 4 .The fruits and the leaves of the Citrus species contain a variety biologically-active compounds including essential oils with various distinct flavors which are important to human nutrition and diet, vitamin C, folic acid, potassium, flavonoids, coumarins, pectin, and dietary fibers 5 .
The name Mauritius papeda, or Kaffir lime, is widely used in the Netherlands and Germany while in France, Italy and Spain, the lime is often called 'combava'. Later in 1824, the name "Mauritius papeda" was introduced to Kaffir lime by De Candolle (DC), who brought the seeds from Mauritius to his botanical garden in Montpellier in southern France. Before that time, De Candolle had studied and classified C. hystrix as the first species of the Papeda sub-genus 6 .
Genus Citrus is native to tropical southeast Asia, southern China and Malaysia. It has been introduced and cultivated elsewhere in the tropics and sub-tropics including in northern Australia 7,8 . However, due to its aromatic, strong, unique and spicy flavor, both fruit and leaves of C. hystrix are popular used ingredient in Asian cooking. They are frequently used for instance in soups, curries, or to add flavour to rice. It can also be used as an infusion for both alcoholic and nonalcoholic drinks. In addition, the leaves can be used fresh or dried, and can be stored frozen 8 . In Thailand, the fruit is used for seasoning and to prepare drinks teas such as Citrus hystrix flavonoid-rich sachet, which has been promoted to have great potential as a natural antioxidant health product. The essential oil is normally produced from fresh leaves by steam distillation and serves as a source of kaffir lime leaf flavours and essences in a large variety of internationally marketed products. The main producers of kaffir lime leaves are Thailand, Indonesia, Malaysia and India. Recently, Thai growers have developed and started growing a kaffir lime without wrinkles that is easier to pack and ship around the world 7,8 .
In aromatic plants, the composition of essential oils usually varies considerably because of intrinsic (sexual, seasonal, ontogenetic, and genetic variations) and extrinsic (ecological and environmental aspects) factors 9,10 .
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 5 . Due to its high content of phenolic and flavonoid compounds, studies showed that citrus peel could be, also, potential source for natural antioxidant. In general, three types of flavonoid compounds are found on citrus peel; flavanone (e.g. hesperidin, naringin, and hesperitin), polymethoxylated flavone (e.g.nobiletin and tangeretin), and flavonol (e.g. rutin) 11,12 .
In Europe, North America, Asia and Australia, dried kaffir lime leaves are available in most of the marts. Usually, a bag of dried leaves can be stored in a sealed airtight container for a couple of years with little physical change.
However, the quality of the dried kaffir lime leaf product depends mostly on the drying methods that are industrially employed. The leaves can be harvested all year round, especially when the trees are small 6 . In addition to these uses, the hesperidium is still used to wash hair (as was noted in the seventeenth century by Rumphius) and, in Sri Lanka, to keep off leeches, apparently reflecting its insecticidal compounds having a rather broad range of effects on invertebrates 6,13 . The leaves can be used to treat stomach ache caused by dyspepsia and insect bites; also, the rind has been prescribed for treatment for worms and headache 8,14 . In Peninsular Malaysia, the fruit and leaves have been also used for washing hair; the fruit is halved and the grated rind is rubbed on the head or the whole fruit is boiled and used as shampoo 8 ; 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 15 . In traditional Medicine, C. hystrix is also used to treat flu, fever, hypertension, abdominal pains, and diarrhea in infants 16 .
The fruits are used as a digestive stimulant, blood purifier, and reduce high blood pressure 17,18 . In Malaysia, the oils from the fruits and the leaves of C. hystrix DC are commercially used as flavors and fragrances, as well as in cooking, perfumery and medical treatments, especially in aromatherapy 19 . This review aims to summarize the main studies reporting the chemical composition of essential oils and other extracts from the kaffir lime leaf , the used extraction processes, and discus their main biological activities. Finally, the formulation and safety level are considered.

Botany and plant taxonomy
Citrus hystrix is generally a small tree of 3-6 m high and a width of 2.5-3 m, often not straight, crooked with glabrous and spiny branches (Fig. 1). Leaves of kaffir lime are unique among the citrus varieties, they are alternate, unifoliolate, broadly ovate to ovate-oblong, 7.5-10 cm long, dark green on top, lighter on the bottom, very fragrant with long petiole expanded into prominent wings, 15 cm long by 5 cm wide, then, each leaf comes in two parts, seemingly a double leaf ( Fig.  1.e). Leaf and expanded petiole appear to be a single "pinched" leaf. Leaf base is cuneate, or rounded, apex obtuse or slightly acuminate or notched 6,8 . Flowers ( Fig. 1.d) are small, fragrant, white; calyx cuspidate 4-lobed, white with violet fringe; petals 4-5, ovate-oblong, yellowish white tinged with pink; stamens 24-30 free. Fruit is large, verrucose, warty or bumpy, globose, ovoid to elliptic, green turning yellowish-green when ripe, approximately 5-7 cm diameter, rind thick, pulp yellowish, very acid and bitter with wrinkle on the surface of fruit ( Fig. 1.b) . Seeds are numerous, ridged, ovoidoblong, 1.5-1.8 by 1-1.2 cm, monoembryonic with white cotyledons (Fig. 1.c) 6,8 .
Kaffir lime grows well in a warm subtropical or tropical climate and prefers welldrained, neutral to slightly acid soil and direct sunlight with ample moisture during the growing season 4 . Citrus taxonomy is still controversial due to the large degree of morphological diversity found in the group, sexual compatibility between the species and apomixes in many genotypes 6 . Based on morphological and phenotypic data, the two major classification systems currently used are those of Swingle and Reece (1967) 6 and Tanaka (1977) 20 . According to Tanaka system, it is hypothesized that citrus is originated in Asia about 30 million years ago from C. hystrix, C. latipes, C. macroptera and C. combara 21 . Based on both bootstrap analysis of RAPD, SCAR data and cpDNA markers, Nicolosi et al. (2000) 21 have separated eight clusters as C. hystrix belong to Micrantha cluster with C. micrantha, C. macroptera and C. latipes, all belonging to subgenus Papeda. These results place C. hystrix in a cluster genetically distinct from the 'citron' cluster as showed in Fig. 2. 21,22 Studying the genetic relationships among members of the Aurantioideae, especially of the genus Citrus and based on the chloroplast matK gene sequences analysis, Penjor et al. (2013) 23 confirmed the results of Nicolosi et al. (2001) 21 study that C. hystrix belongs to the Pummelo cluster which differs and stands out between the Mandarin cluster and the Citron Cluster as shown in Fig. 3.

Chemical composition
Based on chemical analysis, C. hystrix DC, is riche on bioactive molecules such as essential oils, phenolic compounds, glycerolipides and others; The main compounds of C. hystrix DC, will be described in the following paragraphs.

Essential oils
According to the International Standard Organization on Essential Oils (ISO 9235: 2013) and the European Pharmacopoeia, the term "essential oil" is reserved for a product obtained from vegetable raw material, either by distillation with water or steam, or from the epicarp of citrus fruits by a mechanical process, or by dry distillation 8 . Essential oils are complex mixtures of volatile to semi-volatile compounds, usually with a strong odor, rarely colored, soluble in organic solvents, and insoluble in water. They comprise volatile compounds of terpenoid and non-terpenoid origin, synthetized through different biosynthetic routes and with distinct primary metabolic precursors. In nature, essential oils can be found in various plant organs (flowers, fruits, seeds, leaves, stems, and roots), and they play very important roles in plant defense and signaling processes 25,26 . Also, essential oils are used as raw materials in many fields, such as pharmaceutical, agronomic, food, sanitary, cosmetic, and perfume industries 27 .
The essential oil of C. hystrix is used in aromatherapy and as essential ingredient of various cosmetic and beauty products; furthermore, the essential oil of C. hystrix has been reported to have various bioactivities such as antioxidant, antibacterial, antileukimic, and antitussive 28,29 . Limonene, a monoterpene hydrocarbon, is the major component in the essential oils from the peels of the Malaysian Citrus species 30,31 . In general, hydro diffusion steam distillation system, steam distillation with induction heating system, and automated steam distillation process with optimized temperature were applied to extract kaffir lime peel essential oil 32,33,34 . From the fresh leaves of C. hystrix, the essential oil extracted by the steam distillation and the Likens-Nikerson extraction methods, was found to be dominated by citronellal (61.0%-73.0%), âcitronellol (10.0%-14.0%), and limonene (5.0%-7.0%) as major components.

Phenolic and Flavonoid compounds
C. hystrix also function to scavenge radical activities, due to their phenolic compounds which has beneficial implications in human health, phenolic compounds (PC) are widely distributed in fruits and vegetables 42 . In terms of chemical structure, phenolic compounds have at least one aromatic ring to which one or more hydroxyl groups are bonded to aromatic or aliphatic structures 43 . Phenolic compounds range from simple phenolic molecules to highly polymerized compounds. However, these phenolic compounds were obtained mainly in ethanolic extracts 44 .
Besides their antioxidant activities, flavonoids have been demonstrated to have a wide range of biochemical and pharmacological effects including anti-inflammatory, anti-viral, antiallergenic, anti-carcinogenic, anti-ageing activity, anti-oxidant and anti-allergic effects 45, 46 . Flavonoids represent the widely distributed group of plant phenolics, including the anthocyanin pigments, flavonols, flavones, flavanols, and isoflavones. The flavanols tend to polymerize to condensed tannins 47 .
The group of non-flavonoids is mainly represented by benzoic and cinnamic acid known as phenolic acids 44 . Flavonoids are the most common and widely distributed group of plant phenolic compounds that are characterized by a benzopyrene structure, which is ubiquitous in fruits and vegetables; and can be analyzed using colorimetric method by reaction with sodium nitrite and the development of coloured flavonoidaluminium complex formation using aluminium chloride. The presence of polyphenolic compounds like gallic acid, hesperidin, and naringin in citrus fruits have been suggested to be responsible for the anti-diabetic activity 48,49 . Interestingly, the peels of C. hystrix have been reported to contain a variety of phenolic compounds, mainly flavanone, flavone and flavonol 50 . In C. hystrix, hesperidin is reported as component, which is responsible for radical scavenging activity 51,52 .

Other extracts
Two glyceroglycolipids were isolated by Murakami et al. (1995) 56

Biological activities
According to the diversity of chemical compounds extract from C. hystrix, several works have undertaken for the assessment of some biological activities both in vitro or in vivo systems. Antimicrobial: antibacterial and antifungal activities Waikedre et al. (2010) 35 39 , report that both essential oils were effective against all tested pathogens with minimal inhibitory concentration (MIC) ranges of 0.06-68 mg/ml and 0.03-17.40 mg/ml, respectively for leaves and fruit essential oils from C. hystrix. The GC-MS analysis of the used essential oils revealed that citronellal was found to be the major component (80.04%) in the leaf essential oil and had the lowest . 7. Structure of two glyceroglycolipids from C. hystrix with anti-tumor promoting activity MIC. In contrast, fruits peel essential oil consisted of mixture of components (limonene 40.65%, terpinene-4-ol 13.71%, á-terpineol (13.20%), and the most active fraction was á-terpineol, followed by terpinene-4-ol, and limonene 39 41 .
The volatile oil of C. hystrix leaf, did not show any inhibitory activity against tested organisms, but interestingly, growth of Mycobacterium phlei was inhibited by the volatiles of C. hystrix peel with MIC of 3.5 mg/ml, with the similar patterns of essential oils in both C. hystrix peels and leaves. This bioactive fraction composed of citronellal (about 23%) and similar trace components as L-linalool (4.22%), â-pinene (1.82%) and limonene (1.13%) 41 .
It have been also reported hydrodistillation and ethyl acetate extract of C. hystrix peels showed broad spectrum of inhibition against three Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus and Listeria monocytogenes), one yeast (Saccharomyces cerevisiae var. sake) and one mold (Aspergillus fumigatus TISTR 3180) 58 . The tested ethyl acetate extracted essential oils of C. hystrix peel had stronger antibacterial activity than the volatile obtained from hydrodistillation.
It exhibited minimum inhibitory concentration (MIC) values of 0.28 and 0.56 mg/ml against the tested yeast and B. cereus, respectively while the minimum bactericidal concentration (MBC) values against both microbes were 0.56 mg/ ml. The MIC values of the ethyl acetate extracted essential oils against L. monocytogenes, S. aureus and the mold were 1.13 mg/ml while the MBC values against L. monocytogenes as well as the mold A. fumigatus TISTR 3180 and S. aureus were 2.25 and 1.13 mg/ml, respectively. The GC-MS analyses revealed that the major components of the ethyl acetate extracted essential oil were limonene (31.64 %), citronellal (25.96 %) and â-pinene (6.83 %) whereas â-pinene (30.48 %), sabinene (22.75 %) and citronellal (15.66 %) appeared to be major compounds of the essential oil obtained by hydrodistillation 58 .

Anti-inflamatory and Antioxidant activities
On studying anti-Inflammatory response with a model based on lipopolysaccharideactivated RAW 264.7 Murine Macrophages, Tuntipopipat et al. (2009) 59 , found that among 13 plants, the extract (with 70% ethanol ) of the freezedried fresh leaf C. hystrix, with extract from seven other plant, inhibited NO and TNF-a production in a dose-dependent manner without exerting cytotoxicity. Kaffer lime extract should be used in a concentration of 29.2+2.1 μg/mL and 35.4+1.5 μg/ mL to reach the IC 50 , respectively for the inhibition of NO Production and for TNF-a Secretion by LPS-Activated RAW 264.7 Cells. For antioxidant activity evaluation, based on DPPH radical scavenging capacity method, the methanolic extracts from leaf and peel of C. hystrix, have promising a potent antioxidant activity with IC 50 of 24.6 and 66.3 microg/ml respectively for leaves and peel 41 . The antioxidant activity of fresh juice of C. hystrix was evaluated by employing different in vitro assays covering applied for the contents of total phenolics, tannins, and total flavonoids ranged that tanged respectively 836. Swiss albino mice model. Leaf extracts were administrated at the dose of 200 mg/kg body weight for 7 days and toxicity was induced by paracetamol (2 g/kg) on day 5, Liver function markers (ALT, AST, ALP), total bilirubin and total protein in blood serums and hepatic antioxidants (SOD, CAT, GSH and GPx) in liver homogenate were estimated after that animals were sacrificed on the 7 th. day.
However, the recent study conducted by Abirami et al. (2015) 61 , shows that methanolic extracts of C. hystrix leaf possess hepatoprotective action against murine paracetamol induced hepatotoxicity; The level of enzyme markers (alanine transaminase, aspartate transaminase and alkaline phosphatase) in experimental rats were significantely restorated of by the interventions C. hystrix leaves extract to the comparable level of normal control.
Pretreatment with C. hystrix extracts brought back the oxidative stress markers (superoxide dismutase, catalase and glutathione peroxidase) in the range of normal control rats. In the same study, the histopathological examination have confirmed that pretreatment with methanolic extracts of C. hystrix leaf in paracetamol intoxicated rats showed recovery of the hepatocytes from necrosis indicating that sample extracts preserved the structural integrity of the hepatocellular membrane and liver cell architecture damaged by paracetamol action.

Anti-Cancer Effect
The early study to assess the anticancer properties of methanolic extract of C. hytrix fresh leaves by Murakami et al. (1995) 55  However, its well known that several medicinal plant exhibit anti-proliferative activity 62 , Manosroi et al.(2006) 63 , have investigated the antiproliferative activity of essential oil extracted from 17 Thai medicinal plants on human mouth epidermal carcinoma (KB) and murine leukemia (P388) cell lines using MTT assay. The IC50 value of both C. hytrix fruit and leaf, was, respectively, 0.0997 -1.1479 mg/ml in KB cell line and 0.0746-0.3977 in P388 cell line. Then, these two C. hytrix essential oils have an anti-proliferative activity on cervical cancer (KB cell) and mouse leukemia (P388 cell).
Five fractions of crude extract (hexane, ethanol, ethyl acetate, butanol and methanol) from the leaves of C. hystrix were investigated in vitro for their potential cytotoxic activity on 4 leukemic cell lines (HL60, K562, Molt4, U937), and normal human peripheral blood mononuclear cells (PBMCs) using the MTT assay 64 .
These were higher than those of fractions from hexane, ethanol, and butanol. However, none of the five fractions had cytotoxic effects on PBMCs; Also, the methanol fraction did not exhibit any cytotoxic activity 64 .
The cytotoxicity effects and apoptosis induced by three different kaffir lime leaves extract (ethanol, ethyl acetate, and hexane) to cervical cancer cell line (HeLa cells) were studied by Nastiti et al. (2013) 65 . They used cytotoxicity assay via MTT assay, and apoptosis test with double staining method (ethidium bromide-acrydine orange). The three kaffir lime crude extract exhibited dose dependently HeLa cells proliferation inhibition. The IC 50 of ethanolic and ethyl acetate extract was 82,034 and 57,845 ìg/mL, respectively, these two extract were able to induce apoptosis of HeLa cells by increasing the number of apoptotic cells. On the other hand hexane extract was not cytotoxic with lC 50 of 203, 992 ìg/mL. In addition, the results showed that ethyl acetate extract of kaffir lime was the most potential to induce apoptosis in HeLa cells.

Cholinesterase inhibition activity
Increasing communication between nerve cells that use acetyl-choline as a chemical messenger produce a therapeutic effect in patients with Alzheimer's disease, glaucoma, myasthenia gravis, and for the recovery of neuromuscular block in surgery, then, acetylcholine breakdown in the brain can be prevented by the inhibition of acetyl cholinesterase activity, which subsequently increases the concentration of acetylcholine 67 . The juices of C. hystrix possess strong anticholinesterase activity of 79.74% against 86.89% of the used reference compound (Eserine) 60 .
From the hexanes and dichloromethane extracts of the peels of C. hystrix fruits, Youkwan et al. (2010) 68 Fig. 8.
In the study undertaken by Wantida et al. (2010) 69 , essential oils of C. hystrix were tested for their acetyl-cholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitory activities. The tested essential oil exhibited inhibitory activity on BChE higher than on AChE.

Insecticidal and larvicidal activities
The study of insecticidal properties of essential oil from Citrus hystrix DC fresh leaves against tobacco armyworm Spodoptera litura fabricius, using topical application bioassay on uniform weighted second instar larvae, demonstrated considerable repellant activity against the armyworm larvae after 24 and 48 h of treatment with LD50 values of 29.25 and 26.75 μg/ mL, respectively. Also, the growth and development study in the antifeedant test showed that weight gained of larvae treated with C. hystrix essential oil were lower as compared to control treatment.
In another study, Mya et al. (2015) 71 , used ethanol extract of Citrus hystrix leaves to assess their larvicidal effects against Aedes aegypti which is the primary vector of dengue 72 73 , tested (methanol) and non-polar (n-hexane) extract fractions of C. hystrix leaves, with concentrations of 500 ppm, 1375 ppm, 2250 ppm, 3125 ppm, and 4000 ppm against the 3rd instar larvae of A. aegypti; The number of mosquito larvae mortality was calculated after 24 hours of treatment. The results reported that non-polar extract fraction is more toxic and is an effective biolarvicide with LC 90 = 2,885 ppm compared with polar extract fraction which has an LC 90 = 3,180 ppm.
Using an excito-repellency test system, Nararak et al. (2016) 74 , studied the effect of essential oils of the leaf and peel of kaffir lime at four different concentrations (0.5, 1.0, 2.5, and 5.0% v/v) for their repellency, excitation, and knockdown properties against laboratory strains of A. aegypti (L.) and Anopheles minimus Theobald at the 3-5 day aged old mosquito starved 24 h before testing.
For repellency against A. aegypti, leaf volatile oil produced the greatest response for both contact (56.1% escape) and non-contact trials with 63.3% escape at 2.5%, while peel volatile oil produced the strongest response with 46.5% escape at 2.5%.
However, knockdown responses above 50% were only observed in A. aegypti exposed to 5% leaf essential oil. Then, the tested Kaffir lime essential oils were more active against Anopheles minimus Theobald than A. aegypti mosquitoes 74 . Leaf significantly appear more active then peel essential oils at each concentration against Anopheles minimus in contact and non-contact trials, except at the highest (5%) concentration.

Others bioactivities: Antifertility, Tyrosinase inhibitory activity and Cardioprotective effect
Pawinee et al. (1995) 75 investigated the effect of oral adminstration of both alcohol and chloroform extract of C. hystrix DC fruit peel for antifertility activity in pregnant adult female rats (Wistar) by oral administration at different periods of gestation.
They showed an enhancement of the uterotrophic effect of estradiol when both extract were simultaneously given; additionally, the extract stimulated uterine contractions. These two effects may be responsible for the interruption of pregnancy associated with the extract. Then, alcohol and chloroform extract of C. hystrix were found to effectively inhibit implantation, produce abortion and slightly hasten labor time when it was given from day 2 to 5, day 8 to 12 and day 15 until labor, respectively.
Administration of the chloroform extract in a dose of 1 g/kg produces a 62.2 f 14.5% inhibition of implantation. However, administration of the chloroform extract at a dose of 1 g/kg twice a day from day 8 to 12 interupted pregnancy by 91.9+5.5% while the same amount of the alcohol extract produced the effect by 86.3+ 9.6%.
According to the anti-implantation effect, they founded that chloroform extract also possesses a higher abortifacient activity than the alcohol extract 75 .
Tyrosinase is responsible for the formation of melanin in the human body; however, surplus expression of tyrosinase is a major problem which can lead to several skin hyperpigmentation disorders such as, seborrheic keratoses, melasma, diabetic dermopathy, tinea versicolor, melasmas and malignant melanomas 76 77 showed the potency of 19 essential oils from exotic and endemic medicinal plants from Mauritius. The results tend to show that essential oils extracted from these medicinal plants can exhibit anti-tyrosinase activities and may be potential candidates for the cosmetic, food and pharmaceutical industries. Results showed that C. hystrix essential oils exhibit an IC 50 of 2.08 ± 0.253ìg/ml. Putri et al. (2013) 78 , analyzed the effects of C. hystrix peel ethanolic extract on blood serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activity, and observed by light microscope the cardio-hepato-histopathology of a doxorubicininduced cardiac and hepatic toxicity animal model (female Sprague Dawley rats). In the animal groups receiving 500 mg/kg to 1000 mg/kg C. hystrix peel ethanolic extract, cardiohistopathology profile of doxorubicin induced cardiotoxicity and hepatotoxicity rats was repaired, but neither hepatohistopathology profile was did repaired nor serum activity of aminotransferase (ALT) and aspartate aminotransferase (AST) was reduced; Thus, Putri et al. (2013) 78 , conclude that the ethanolic extract of C. hystrix, can be developed as cardioprotector agent.

Safety issues
Regarding the chemical structures and the biological activities of biomolecules from C. hystrix, and also, the several domestic uses of this plant and their extracts, it appears that a bio-safety issue for this plant may be highlighted.
No information was founded on toxicity profile of C. hystrix biomolecules in the second edition of a Guide for Health Care Professionals of Essential Oil Safety 79 .
From, the released draft tentative report of the Cosmetic Ingredient Review Expert Panel (May and October, 2016), entitled ''Safety Assessment of Citrus Flower-and Leaf-Derived Ingredients as Used in Cosmetics''. Thus, it appear that C. hystrix leaf extract produced by extracting dried leaves with 80% ethanolic solution is reported as safe and to be non-irritating and non-sensitizing.

CONCLUSION
C. hystrix has been used as food and medicine with long history mainly in the Asian region. It is also a flavor food with health value. The present study reported phylogenetic taxonomy based on bootstrap analysis of RAPD, SCAR data, cpDNA markers and the chloroplast matK gene sequences analysis showed that C. hystrix belong to Pummelo cluster which is genetically distinct from the Citron cluster and the Mandarin cluster. The chemical structures of bioactives molecules explain its traditional uses and his potential to be used in cosmeticeucal and pharmaceuticals.
C. hystrix essential oils are the mostly studied biomoleculs and they have potential beneficial therapeutic actions in the management of bacterial and fungal infections. The chemical composition revealed that essential oil of leaves and fruit peel of C.hystrix have generally a different profile; then, amoung reported studies, the C.hystrix leaves are characterized by citronellal, â-citronellol and terpinen-4-ol as major components. However, citronellyl acetate, âpinene, limonene, alpha-terpineol, 1,8-cineole, citronellol, p-cimene, and limonene were identified as minor components. Whereas, kaffir lime peels content respectively limonene, â-pinene, sabinene and citronellal as major components with other minor components like terpinene-4-ol, a-terpineol, g-terpinene, a-terpinene and terpinolene. Other essential oils compounds of C. hystrix were detected in little amount in both leaves and peels such as elemol, delta-cadinene, geranylacetate and L-linalool. The reported studies show that some of tested essential oils were inactive against bacteria. Mainly those content terpinen-4-ol as major compounds. However, essential oils with citronellal as major component were more effective against bacterial strains.
In the future, more deep analysis and profiling of the volatile oils are needed to allow further elaboration of a chemotype of C. hystrix based on essential oils profile. Interestingly, phenolic compounds of the peels of C. hystrix contain a variety of flavanone, flavone and flavonol; Vanillic acid, p -coumaric acid, sinapic acid, m -coumaric acid, benzoic acid and cinnamic acid were isolated from C. hystrix leaves. In addition, flavonoids such as cyanidin, myricetin, peonidin, quercetin, luteolin, hesperetin, apigenin and isorhamnetin, as flavanone compounds, didymin and hespiridine were isolated from both leaves and fruit juice and as flavone compounds, rutin and diosmin were isolated just from the leaves. Glyceroglycolipids, l,2-di-O-a-linolenoyl-3-O-betagalactopyranosyl-sn-glycerol (DLGG) and a mixture of l-O-a-linolenoyl-2-O-palmitoyl-3-O-betagalactopyranosyl-sn-glycerol and its counterpart (LPGG) were identified in the C. hystrix leaves. Benzenoid derivatives, (hystroxene-I), quinolinone alkaloid (hystrolinone) were isolated from the crude acetone extract of root of Citrus hystrix.
With diversity of contents in the total phenolics, both ethanolic and methanolic extract have promising a potent antiinflamatory, antioxidant activity and hepatoprotective effects. Fresh leaves methanolic extract content l,2-di-O-alinolenoyl-3-O-beta-galactopyranosyl-sn-glycerol (DLGG) which appear an anti-tumor-promoting agent. Also, some essential oils extracted from fruit and leaves C.hytrix have an anti-proliferative activity on cervical cancer (KB cell) and mouse leukemia (P388 cell). Also, Ethyl acetate fraction of leaves of kaffir lime exhibited the highest cytotoxicity activity on leukemic cell lines inducing, also, highest apoptosis in HeLa cells and reducing the viability of cervical and neuroblastoma cell lines. Cholineserase inhibitory activity was manifested by juices, hexane and dichloromethane extracts of C. hystrix, the implicated molecules are citrusosides compounds and 6'-hydroxy-7'methoxybergamottin and a compound with 3-Obeta-D-glucopyranosyl-3,5,7,4'-tetrahydroxy-6,8,3'-trimethoxyflavonol nucleus in the prenylfuranocoumarin-HMGA conjugate.
Essential oil from C. hystrix DC fresh leaves showed larvicidal effects against tobacco armyworm Spodoptera litura fabricius; and a repellant effect, with the peel essential oils, against adult mosquito of Aedes aegypti (L.) and Anopheles minimus Theobald, However, larvicidal effects against Aedes aegypti is reported with the ethanolic extract of Citrus hystrix leaves. Others bioactivities such as antifertility effect, tyrosinase inhibitory activity, cardioprotective and hepatoprotective effects were reported. Alcohol and chloroform extract of C. hystrix were found to effectively inhibit implantation and produce abortion, therefore, the antifertility effect were proposed. Both essential oils and juice of C. hystrix exhibited excellent tyrosinase inhibitory activity and the peel ethanolic extract, showed cardiotoxic and hepatotoxic protective effects on rat model. Both emulsions and microcapsules have been described for the formulation of C. hystrix essential oil. Finally, we think that the safety issues in terms of toxicity profile related to the daily use of these biomolecules should be further investigated. In addition, further studies on non-conventional extraction processes involving less solvent and energy use are also needed.