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

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BIOSCIENCES BIOTECHNOLOGY RESEARCH ASIA, March 2017. Vol. 14(1), 285-305
* To whom all correspondence should be addressed.
A Review of Genetic Taxonomy, Biomolecules
Chemistry and Bioactivities of Citrus hystrix DC
Farid Agouillal1,2 , Zarani M. Taher3, Houria Moghrani2,
Noureddine Nasrallah2 and Hesham El Enshasy3,4,5*
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.
http://dx.doi.org/10.13005/bbra/2446
(Received: 19 March 2017; accepted: 25 March 2017)
Citrus hystrix DC. with common name makrut lime or kafir lemon, is a very
popular traditional medicinal plant as well as an important spice in Asiatic countries.
The plant is native of the Indonesian island Sumbawa, then, it is cultivated in Indonesia,
Thailand, Malaysia and the tropical region of Asia. It mainly contains essential oil and
phenolic compounds. The most intense odor compounds of kafir lemon are Citronellal,
L-Linalool, 1,8-Cineole , á-Terpeneol and ä-Cadinene. Such as Citrusosides-A and
furanocoumarines, Makrut lime content also non-volatile compounds like alkaloids
and glyceroglycolipids. Citrus hystrix DC has many biological activities due to its volatile
and nonvolatile compounds, and it has been used in traditional medicine for treating
various illnesses, particularly cold pain and stomach disorder. It is also used as a juice
for its fruit or as spice for its aromatic leaves. This review covers data on the chemistry
and biological effects of Citrus hystrix DC biomolecules, and aims to lay the foundation
for further study on the extraction enhancement of these biomolecules and more useful
formulations.
Keywords: Biomolecules chemistry, Biological activities,
Citrus taxonomy, Citrus hystrix, Essential oils, Phenolic compounds.
Herbs and spices are extensively used in
Asiatic countries for culinary purposes and for
traditional medicine. Then, several organs such
as the leaves and fruits of Citrus species are widely
used to flavor foods as perfumes in ceremonial
celebrations. Among these Citrus spices commonly
planted in small garden plots of trees, C. hystrix
D.C., C. aurantijolia Swingle, C. microcarpa
Bunge, C. limn Burm. and C. mim Merr., are by far
the most economically important plant. It is believed
to have originated in south-eastern Asia, in an area
that includes China, India and the Indochinese
peninsula and nearby archipelagos1.

28 6 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
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 subfamilies2, 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 fibers5.
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-genus6.
Genus Citrus is native to tropical south-
east Asia, southern China and Malaysia. It has
been introduced and cultivated elsewhere in the
tropics and sub-tropics including in northern
Australia7, 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 non-
alcoholic drinks. In addition, the leaves can be used
fresh or dried, and can be stored frozen8. 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 world7, 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) factors9, 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. microcarpa5. 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 small6. 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 invertebrates6,13. 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 headache8,
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 shampoo8; 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 odor15. In
traditional Medicine, C. hystrix is also used to treat
flu, fever, hypertension, abdominal pains, and
diarrhea in infants16.
The fruits are used as a digestive
stimulant, blood purifier, and reduce high blood
pressure17, 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 aromatherapy19. This review aims to
summarize the main studies reporting the chemical
composition of essential oils and other extracts

287AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
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 notched6, 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, ovoid-
oblong, 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 well-
drained, neutral to slightly acid soil and direct
sunlight with ample moisture during the growing
season4. 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 genotypes6.
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. combara21. 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.
However, Liu et al. (2015)24 reported the
successful development of DArT microarrays and
their applications in phylogenetic analysis of
Citrus species, then, genetic relationships based
on neighbor-joining method show that C. hystrix
DC, C. macroptera Montr, and C. celebica Koord
are grouped into subcluster C1 which is sister to
another subcluster C2 including grapefruit (C.
paradisi Macf.), pummelo (C. grandis (L.) Osbeck),
lime (C. aurantifolia), indian wild orange (C. indica
Tan.), citron (C. medica L.), and mandarin (C.
reticulate) as shown in Fig. 4.
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 distillation8.
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 processes25, 26. Also,
essential oils are used as raw materials in many
fields, such as pharmaceutical, agronomic, food,

28 8 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
sanitary, cosmetic, and perfume industries27.
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
antitussive28,29. Limonene, a monoterpene
hydrocarbon, is the major component in the
essential oils from the peels of the Malaysian
Citrus species30, 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 oil32,33,34.
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%), â-
citronellol (10.0%–14.0%), and limonene (5.0%–
7.0%) as major components.
In addition, citronellal (72.4%), â-
citronellol (6.7%), and citronellyl acetate (4.1%)
were reported to be the major components in kaffir
lime leaves, followed by â-pinene (1.9%) and
limonene (0.1%) as minor components5. Studying
the chemical composition and antimicrobial activity
of the essential oils from New Caledonian C.
macroptera and C. hystrix from leaves, Waikedre
et al. (2010)35, reported the presence of 38
constituents. The obtained essential oils were
characterized by high contents of terpinen-4-ol
(13.0%), â-pinene (10.9%), á-terpineol (7.6%), 1,8-
cineole (6.4%), citronellol (6.0%) and p-cimene
(5.6%), but poor in limonene (4.7%).
However, from the kaffir lime peels (from
Masjid Tanah, Melaka in Malaysia), â-pinene
(39.3%), limonene (14.2%), citronellal (11.7%), and
terpinen-4-ol (8.9%) were identified as the principal
components. Then, â-pinene (23.5%) and sabinene
(20.1%) appeared as the major components
followed by citronellal (12.6%), limonene (11.8%),
and â-citronellol (3.3%) found in C. hystrix peel
and reported by Nor (1999)36.
Other study on essential oils extraction
using automated steam distillation process with
uncontrolled temperature carried out by Nurhani
et al. (2013)34 reported that the oil composition was
as follows: sabinene (31.224%), â-pinene (32.967%),
limonene (20.687%), á-pinene (3.338%), camphene
(0.135%) , myrcene (1.735%), á-terpineol (0.938%)
and citronellal (7.531 %). Other compounds were
identified using the same process but in the
controlled temperature like terpinolene, linalool,
terpinen-4-ol and citronellol. It was also reported
that the essential oil isolated from Malaysian
variety of kaffir lime peel contained sabinene (36.0%
- 49.0%), limonene (17.0%–33.0%), citronellal
(3.0%–11.0%), and â-pinene (8.0%–14.0%) as major
components37. However, citronellal (66.9%) and â-
citronellol (6.6%) were the major components
essential oil in kaffir lime peel (from Selangor),
obtained using the hydro-distillation method. Other
research also reported that the essential oil of fresh
fruit-peel is mainly consisted of monoterpene
hydrocarbons, with limonene (30.73%) and â-
pinene (18.76%) as the principal components with
other minor components such as terpinene-4-ol
(10.63%), á-terpineol (8.35%), ã-terpinene (6.18%),
á-terpinene (5.09%) and terpinolene (4.33%)38. In
other study, citronellal was found to be the major
component (80.04%) in C. hystrix leaf oil; In
contrast, C. hystrix fruit peel essential oil consisted
of other components: limonene (40.65%),
terpinene-4-ol (13.71%) and á-terpineol (13.20%)39.
Recent research of Aumeeruddy-Elalfi et al.
(2016)40 found that the main compounds of
essential oils from C. hystrix leaves as á-pinene
(3.02%), limonene (83.89%), â-pinene (0.78%) and
â-myrcene (0.89%) with traces of Methyl-eugenol
(0.21%). From the study of Juraithip et al. (2010)41,
we can draw another conclusion, that C. hystrix
peel and leaf showed similar patterns of essential
oils chemical compositions. The major constituents
of C. hystrix peel and leaf were citronellal (about
23.85-23.41%) and trace components were elemol
(6.59-4.17%), ä-cadinene (5.96-4.74), geranylacetate
(5.12-4.45%), á-terpineol (5.15-5.40%), L-linalool
(4.22-4.36%), â-pinene (1.82%), limonene (1.13%)
and á-humulene (1.09-0.94%). The chemical
structure of theses essential oils compounds are
given in Fig. 5.
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 vegetables42. In terms of chemical
structure, phenolic compounds have at least one
aromatic ring to which one or more hydroxyl

289AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
(a)
(b) (c) (d) (e)
Fig.1. Tree (a), Fruit (b), Seeds (c), Flowers (d) and Leaves (e) of Citrus hystrix.
*
*
Fig. 2. Analysis of RAPD and SCAR data (Left) and cpDNA markers (Right); Evidence of C. hystrix cluster
distinction from the “Citron” cluster21

29 0 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Fig. 3. Chloroplast matK gene sequences analysis of genus Citrus; Maximum likelihood tree (Left) and neighbor-
joining tree (Right) showing the three clusters: Citron, Pummelo and Mandarin. Penjor et al. (2013)23

291AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
groups are bonded to aromatic or aliphatic
structures43.
Phenolic compounds range from simple
phenolic molecules to highly polymerized
compounds. However, these phenolic compounds
were obtained mainly in ethanolic extracts44.
Besides their antioxidant activities,
flavonoids have been demonstrated to have a wide
range of biochemical and pharmacological effects
including anti-inflammatory, anti-viral, anti-
allergenic, anti-carcinogenic, anti-ageing activity,
anti-oxidant and anti-allergic effects45,46. 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
tannins47.
The group of non-flavonoids is mainly
represented by benzoic and cinnamic acid known
as phenolic acids44. 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 flavonoid–
aluminium 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 activity48,49.
Interestingly, the peels of C. hystrix have been
reported to contain a variety of phenolic
compounds, mainly flavanone, flavone and
flavonol50. In C. hystrix, hesperidin is reported as
component, which is responsible for radical
scavenging activity51,52.
Using supercritical carbon dioxide
extraction, vanillic acid, p-coumaric acid, sinapic
acid, m-coumaric acid, benzoic acid and cinnamic
acid were isolated from the plant leaves53. Three
known coumarins, bergamottin, oxypeucedanin
and 5-[(6’,7’-dihydroxy-3’, 7’-dimethyl-2-
octenyl)oxy] psoralen were exhibited inhibitory
activities against both lipopolysaccharide (LPS)
and interferon- ã (IFN-ã )-induced nitric oxide (NO)
generation in RAW 264.7 cells54. The chemical
structures of these main phenolic compounds
found in C. hystrix are showed in Fig. 6.55
Other extracts
Two glyceroglycolipids were isolated by
Murakami et al. (1995)56, from the leaves of C.
hystrix, and identified as l,2-di-O-a-linolenoyl-3-
O-beta-galactopyranosyl-sn-glycerol (DLGG) and
a mixture of two compounds, l-O-a-linolenoyl-2-O-
palmitoyl-3-O-beta-galactopyranosyl-sn-glycerol
and its counterpart (LPGG). These compounds
inhibit the tumor-promoting activity of 12-O-
Tetradecanoylphorbol 13-Acetate in Mouse skin.
However, in addition of two coumarins (hystrixarin
and hopeyhopin, an benzenoid derivatives
(hystroxene-I), and an quinolinone alkaloid
(hystrolinone), as shown in Fig. 6, were isolated
from the crude acetone extract of root of C. hystrix57.
Fig. 4. Neighbor-joining method based on DArT microarrays study of 23 Citrus species24

29 2 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Citronellal β-Citronellol Limonene Citronellyl acetate
β-Pinene Terpinen-4-ol α-Terpineol 1,8-Cineole
p-Cimene Sabinene Camphene β-Myrcene
Terpinolene Linalool Elemol δ –Cadinene
Geranyl acetate γ-Terpinene α-Terpinene α-Copaene
β -Cubebene β -Caryophyllene d-Germacrene Citronellic acid
Nerolidol α-Pinene Methyl eugenol α-Humulene
Fig. 5. Structures of essential oils obtained from leaves and peels of C. hystrix
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, have tested the
leaves essential oil against three Gram positive
bacteria (Staphylococcus aureus, Staphylococcus
epidermidis and Bacillus subtilis), two Gram
negative bacteria (Klebsiella pneumonia and
Escherichia coli), and five fungal strains
(Aspergillus fumigates, Candida albicans,
Cryptococcus neoformans, Saccharomyces
cerevisiae and Trichophyton mentagrophytes).
The tested essential oil was inactive against

293AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Table 1. Summary of main essential oils compounds from C. hystrix DC.
leaves and peels as reported by previous studies
Location and Main % Part of references
years of study components plant
Malaysia, 1996 β-Pinene 39.3 Peels 30
Limonene 14.2
Citronellal 72.4 Leaves
Malaysia, 1999 L-Citronellal 61.73-72.45 Leaves 36
12.56 Peels
L-Limonene 5.90-6.78 Leaves
11.78 Peels
Sabinene 1.60-2.03 Leaves
20.13 Peels
Linalool 0.96-1.56 Leaves
1.82 Peels
β-Citronellol 10.34-13.43 Leaves
3.34 Peels
Citronellyl acetate 1.22-2.02 Leaves
1.67 Peels
Thailand, 2007 Limonene 30.73 Peels 38
β-Pinene 18.76
Terpinene-4-ol 10.63
α-Terpineol 8.35
γ-Terpinene 6.18
α-Terpinene 5.09
Terpinolene 4.33
New Caledonia, 2010 Terpinen-4-ol 13.0 Leaves 28
β-Pinene 10.9
Limonene 4.7
α-Terpineol 7.6
1,8-Cineole 6.4
Citronellol 6.0
Thailand, 2010 Sabinene 2.21 Leaves 41
1.55 Peels
β-Pinene 1.67 Leaves
1.82 Peels
Limonene 1.18 Leaves
1.13 Peels
L-Linalool 4.36 Leaves
4.22 Peels
Citronellal 23.41 Leaves
23.85 Peels
α-Terpineol 5.40 Leaves
5.15 Peels
Citronellol 1.40 Leaves
1.48 Peels
Citronellyl acetate 3.75 Leaves
3.82 Peels
α-Copaene 2.35 Leaves
2.16 Peels
Geranyl acetate 4.45 Leaves
5.12 Peels
β-Cubebene 2.46 Leaves
2.34 Peels

29 4 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
β-Caryophyllene 3.51 Leaves
3.73 Peels
d-Germacrene 1.82 Leaves
2.01 Peels
δ-Cadinene 4.74 Leaves
5.69 Peels
Elemol 4.17 Leaves
6.59 Peels
Malaysi, 2011 β-Citronellal 66.85 Leaves 37
β-Citronellol 6.59
Linalool 3.90
Citronellol 1.76
Thailand, 2012 Limonene 40.65 Leaves 39
Terpinene-4-ol 13.71
α-Terpineol 13.20
Thailand, 2013 Citronellic acid 4.5 Leaves 29
Nerolidol 2.14
δ-Cadinene 1.49
Citronellal 1.41
Citronellol 1.39
Malaysia, 2013 Sabinene 27.498 - 45.594 Peels 32
Limonene 28.649 - 32.455
Citronellal 8.293 - 17.487
â-Pinene 7.147 - 8.974
á-Pinene 1.255 - 2.515
Malaysia, 2013 Sabinene, 46.165 - 48.5 Peels 23
Limonene, 25.742 - 27.714
β-Pinene 8.759 - 10.088
Citronellal 3.247 - 7.146
α-Pinene, 2.959 - 3.223
Myrcene 1.338 - 1.469
Terpinen-4-ol 0.457 - 1.054
Malaysia, 2013 Sabinene 31.224 - 46.573 Peels 34
β-Pinene 13.509 - 32.967
Limonene 17.232 - 20.687
Citronellal 4.616 - 7.829
α-Pinene 3.047 - 3.546
Myrcene 1.804 - 1.985
Terpinen-4-ol 1.823 - 2.822
γ-Terpinene 0.735 - 1.775
Linalool 0.633 - 1.156
α-Terpineol 0.438 - 0.907
Mauritius, 2016 Limonene 83.89 Leaves 40
α-Pinene 3.02
α-Myrcene 0.89
β-Pinene 0.78
bacteria but showed moderate activity against
Cryptococcus neoformans and Saccharomyces
cerevisiae with MIC of (50 mg/ml). This value is
about tenfold lower than the used antifungal agent
standard (ketoconazole :5 mg/ml). The GC-MS of
the tested essential oil was characterized by high
contents of terpinen-4-ol (13.0%), a-terpineol
(7.6%), 1,8-cineole (6.4%), and citronellol (6.0%).
Testing the antibacterial activities of the two
essential oils of makrut leaf and makrut fruit peel
against 411 isolates of groups A, B, C, F, G
Streptococci, Streptococcus pneumoniae,

295AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Hesperidin Vanillic acid p-Coumaric acid
Sinapic acid m–Coumaric acid Benzoic acid
Cinnamic acid Bergamottin, Oxypeucedanin
5-[(6’,7’-dihydroxy-3’, 7’-dimethyl-2-octenyl)oxy]psoralen Hystrixarin
Hopeyhopin Hystroxene-I Hystrolinone
Fig. 6. Structure of phenolic compounds, coumarins and a quinolinone alkaloid from C. hystrix DC
Haemophilus influenzae, Staphylococcus aureus
(Methicillin-Resistant and -Sensitive S. aureus) and
Acinetobacter baumannii, obtained from patients
with respiratory tract infections, Vimol et al.
(2012)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

29 6 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
l,2-di-O-a-linolenoyl-
3-O-beta-galactopyranosyl-sn-glycerol (DLGG)
l-O-a-linolenoyl-2-O-palmitoyl-
3-O-beta-galactopyranosyl-sn-glycerol (LPGG)
Fig. 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 limonene39. The antimicrobial
activities of volatile oils and extracts of eight
Thailand species, C. hystrix essential oil were
investigated against eight bacteria (3 Gram positive
bacteria, Bacillus subtilis (ATCC 6051),
Staphylococcus epidermidis (ATCC 12228), S.
aureus (ATCC25923); 5 Gram negative bacteria:
Escherichia coli (ATCC 25922), Enterococcus
faecalis (ATCC 1406), Proteus mirabilis (ATCC
14153), Pseudomonas aeruginosa (ATCC 27853);
Mycobacterium: Mycobacterium phlei (ATCC
11758) ) and three fungi (Candida albicans (ATCC
10231), C. parasilosis (ATCC 90018) and C.
tropicalis (ATCC 13803)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
hydrodistillation58.
Anti-inflamatory and Antioxidant activities
On studying anti-Inflammatory response
with a model based on lipopolysaccharide-
activated RAW 264.7 Murine Macrophages,
Tuntipopipat et al. (2009)59, found that among 13
plants, the extract (with 70% ethanol ) of the freeze-
dried 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 IC50, respectively for the inhibition
of NO Production and for TNF-a Secretion by LPS-
Activated RAW 264.7 Cells.

297AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Citrusosides A Citrusosides B
Citrusosides C
Citrusosides D
Eudesmane-4b,11-diol;
R
1
=OH, R
2
=Me or R
1
= Me, R
2
= OH
1-O-isopropyl-beta-D-Glucopyranoside
6’-hydroxy-7’-methoxybergamottin 6’, 7’-dihydroxybergamottin Isoimperatorin
Fig. 8. Chemical structure of citrusosides A-D; 6’-hydroxy-7’-methoxybergamottin; 6’, 7’-dihydroxybergamottin
and isoimperatorin and other molecules responsible of cholinesterase inhibition activity58
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
IC50 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 836.90
mg gallic acid equivalent (GAE)/L, 507.61 mg gallic
acid equivalent(GAE)/L and 224.88 mg rutin
equivalent/L. Antioxidant potential based on FRAP
assay show a value of 30504.40 mmol of ferrous
equivalents/L juice, DPPH• and ABTS•+ scavaging
are respectively about 10903,28 mmol of trolox
equivalents/L juice, and 33830.69 mg of EDTA
equivalents/L juice. Both superoxide radical and
hydroxyl radical scavenging activity are in the
range of 19.89 % and 42.91 %, respectively. Also,
metal chelating activity reaches 7.73 mg of EDTA
equivalents/L juice. These results indicated that
fresh juice of C. hystrix could be used as a source
of antioxidant agents 60.
Hepatoprotective activity
Abirami et al. (2015)61 have evaluated the
hepatoprotective effects of C. hystrix methanolic
leaf extracts on paracetamol induced toxicity in a

29 8 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Fig. 9. Chemical structure of flavonol compound
possessing a 3-O-beta-D-glucopyranosyl- 3,5,7,4’-
tetrahydroxy-6,8,3’-trimethoxyflavonol nucleus in the
prenylfuranocoumarin–HMGA conjugate70
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 7th. 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 reported the plant
anti-tumor properties on mouse skin in a two-stage
induced by dimethylbenz[a]anthracene (DMBA)
and 12-O-tetradecanoylphorbol 13-acetate (TPA).
The results showed that the IC50 values of two
compounds were strikingly lower than those of
representative used cancer preventive agents such
as a-linolenic acid, beta-carotene, or (-)
epigallocatechin gallate. Also, one compound
exhibited anti-tumor-promoting activity was
identified as l,2-di-O-a-linolenoyl-3-O-beta-
galactopyranosyl-sn-glycerol (DLGG). The second
compound was identified as a mixture of l-O-a-
linolenoyl-2-O-palmitoyl-3-O-beta-
galactopyranosyl-sn-glycerol and its counterpart
(LPGG) as shown in Fig 7.
However, its well known that several
medicinal plant exhibit anti-proliferative activity62,
Manosroi et al.(2006)63, have investigated the anti-
proliferative 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 assay64.
The cytotoxicity bioassays showed that
the ethyl acetate fraction exhibited the highest
cytotoxicity, with IC50 values of 19.0±0.6, 35.3±1.4,
21.8±0.4, and 19.8±1.0 ìg/ml, in response to the 4
leukemic cell lines (HL60, K562, Molt4, and U937),
respectively.
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 activity64.
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

299AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
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 IC50 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 lC50 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.
The cytotoxic effect of kaffir lime leaf
extracts on cervical cancer and neuroblastoma cell
lines based on the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay was
carried out by Woro et al. (2014)66. They showed
that both ethyl acetate and chloroform extracts
have an IC50 for HeLa cells, UKF-NB3, IMR-5 and
SK-N-AS parental cells of 40.7-17.6; 28.4-18.9; 14.1-
6.4 and 25.2-9.4 (ìg/mL) respectively. Then, kaffir
lime extract reduces the viability of cervical and
neuroblastoma cell lines and may have potential
as anti-cancer compounds.
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 acetylcholine67. The
juices of C. hystrix possess strong anti-
cholinesterase 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 have isolated 4 new citrusosides A-
D, six furanocoumarins, a sesquiterpene
(eudesmane-4b,11-diol), 5 monoterpenes, and 1-
O-isopropyl-beta-D-glucopyranoside. The
Butyryl-cholinesterase inhibitory activity of the
isolated fractions was investigated and 62 -
hydroxy-72 -methoxybergamottin was found as a
compound to possess the highest potency,
showing an IC50 value of 11.2+/-0.1 ìM against
3.2+0.2 ìM of galanthamine (a positive control).
However, 62 ,72 -dihydroxybergamottin and
isoimperatorin showed IC50 values of 15.4+/-0.3 and
23+/-0.2 ìM, respectively. These molecules are
represented in 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 butyryl-
cholinesterase (BChE) inhibitory activities. The
tested essential oil exhibited inhibitory activity on
BChE higher than on AChE.
Among sixteen compounds isolated by
Chonticha et al.(2016)70, from the ethyl acetate
extract of the fruit peels of C. hystrix, only one
flavonol compound, (3-O-beta-D-glucopyranosyl-
3,5,7,4’-tetrahydroxy-6,8,3’-trimethoxyflavonol
nucleus in the prenylfuranocoumarin–HMGA
conjugate, Fig. 9) showed very potent butyryl-
cholinesterase inhibitory activity with IC50 value
of 10.12 ± 0.22 μM, against galanthamine, a positive
control compound with IC50 value of 11.2 ± 0.09
μM.
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.
GCMS analyses of the tested essential
oil revealed the presence of 29 compounds with
dominance of beta-citronellal as major compound
(66.85%) of total essential oil followed by beta-
citronellol (6.59%), linalool (3.90%) and citronellol
(1.76%)37.
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 dengue72; Result suggests
that high concentrations of Citrus hystrix leaves
ethanol extract can be used for the eradication of
A. aegypti; then, concentrations of 2.4-2.1-1.8-1.5
and 1.2% of the tested leaves ethanol extract
caused 99.5-85.5-62.5-26.5 and 2% mortality of

30 0 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
Aedes larvae in 24 hrs, respectively.
Ansori et al. (2015)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 LC90 = 2,885 ppm
compared with polar extract fraction which has an
LC90 = 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%.
Against Anopheles minimus Theobald,
essential oil from C. hystrix leaf had strong
combined irritant and repellent activity responses
at 1–5% concentrations (90.0–96.4% escape) and
the strongest spatial repellent activity at 1% and
2% (85.9% and 87.2% escape), respectively. The
peel essential oil exhibited good excitation with
repellency at concentrations of 2.5% (89.8%
escape) and 5% (96.28% escape), while
concentrations 1–5% showed more moderate
repellent activity.
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 mosquitoes74.
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 extract75.
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 melanomas76. Abirami et al. (2014)60 ,
reported that C. hystrix juice exhibited excellent
tyrosinase inhibitory activity of 80.79%, against
90.87% of the used reference compound (Kojic
acid).
A recent study conducted by
Aumeeruddy-Elalfi et al. (2016)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 IC50 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)

301AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
activity, and observed by light microscope the
cardio-hepato-histopathology of a doxorubicin-
induced 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 Safety79.
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, â-
pinene, 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-beta-
galactopyranosyl-sn-glycerol (DLGG) and a
mixture of l-O-a-linolenoyl-2-O-palmitoyl-3-O-beta-
galactopyranosyl-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.

30 2 AGOUILLAL et al., Biosci., Biotech. Res. Asia, Vol. 14(1), 285-305 (2017)
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-a-
linolenoyl-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-O-
beta-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.
REFERENCES
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