Chemical Constituents and Biological Activities of Zanthoxylum limonella (Rutaceae): A Review

Zanthoxylum limonella belongs to the family of aromatic deciduous trees and shrubs, Rutaceae. In traditional medicine practice, various parts of Z. limonella are used for the treatment of dental caries, febrifugal, sudorific, rheumatism, diuretic, stomach ache and diarrhea. Secondary metabolites have been isolated the stems, stem barks, and fruits. The plant contains alkaloid, amide, lignin, coumarin and terpenoid compounds. The extracts of the various parts, essential oil from the fruits and some pure compounds of Z. limonella have been found to have biological activities, for example, mosquito repellent and mosquito larvicidal, antimicrobial, antioxidant, and antitumour properties. This review compiles some scientific information on botanical description, traditional uses, phytochemical constituents and biological activities of Z. limonella. Antioxidant, Antitumour Tropical Journal of Pharmaceutical Research is indexed by Science Citation Index (SciSearch), Scopus, International Pharmaceutical Abstract, Chemical Abstracts, Embase, Index Copernicus, EBSCO, African Index Medicus, JournalSeek, Journal Citation Reports/Science Edition, Directory of Open Access Journals (DOAJ), African Journal Online, Bioline International, Open-J-Gate and Pharmacy Abstracts


INTRODUCTION
The Rutaceae family comprises almost 150 genera and 1,600 species of trees, shrubs, and climbers distributed throughout the temperate and tropical regions of the world [1].The chief genera of this family are Citrus, Zanthoxylum, Ruta, Ptelea, Murraya and Fortunella [2].Most of the Rutaceae are aromatic plants whose leaves, fruits or cotyledon in seeds contain a complex mixture of volatile aroma compounds [3].The members of Rutaceae family have been used in perfumery, gastronomy, and traditional medicine.In addition, several publications have reported the presence of secondary chemical constituents of Rutaceae.Phytochemical survey of this family reveals alkaloids, coumarins, flavonoids, limonoids, and volatile oils [4].Many of these compounds have been associated to different biological activities, for example, antimicrobial [3,5], antidiarrhoeal [6], anticholinesterasic [7], antileishmanial [8], antiprotozoal [9], larvicidal [10,11], and antioxidant activities [12,13].In family Rutaceae, the genus Zanthoxylum has been able to provide a variety of secondary metabolites with interesting phytochemical and biological activities [14].
The genus Zanthoxylum comprises over 200 species distributed worldwide in tropical and temperate regions of North America, South America, Africa, Asia, and Australia [15].The species of this genus are aromatic deciduous trees and shrubs [16].Members of the Zathoxylum have been used in traditional medicines, perfumery and pharmaceutical industry [14].Based on the used of these plants in traditional folk medicine reveals a source of valuable bioactive compounds suitable for ethnopharmaceutical applications.
Z. limonella is used in flavouring food and traditional medicine.The different parts of this species are used to treat various diseases, including dental caries, cardiac, respiratory diseases, stomach infections, and rheumatism [17,18].The present review aims to compile up to date documentations of various scientific papers related phytochemical compositions and biological properties of Z. limonella.

BOTANICAL DESCRIPTION
Z. limonella is found in India, Sri Lanka, Thailand, Myanmar, Indo-China, Peninsular Malaysia, Java, the Lesser Sunda Island, Moluccas (Wetar), Sulawesi, the Philippines and southern Papua New Guinea [19].It is a deciduous, aromatic, medium-sized tree reaching a height of 35 meters (Fig 1a).The green young bark is covered with spines while mature bark is grey with straight or ascending prickles of 2 -3 cm (Fig 1b).Small prickles occur on the twigs, and all parts of tree have a characteristic lemon-like smell.The leaves are paripinnate or imparipinnate, 30 -40 cm long.The leaflets are opposite to sub opposite, ovate to elliptical, 7 -13 cm long, 3 -5 cm wide, pellucid dots, the margins entire to glandular crenate.Inflorescense panicles have a terminal or axillary, 8 -14 cm long.The flowers are white or pale yellow, 2 -3 mm long, 4 sepals and 4 petals.The male flowers have 4 stamina and 1 rudimentary carpel while female flowers with ovary 1 carpellate.The fruit is a follicle, subglobose, 6 -7 mm in diameter, with 1 seed per carpel, green turning red when ripe.Seeds are hard and black in colour, 5 mm in diameter (Fig 2) [19].

TRADITIONAL USE
The different parts of Z. limonella have been used in Thai folk medicine.The bark contains febrifugal, sudorific, and diuretic properties, while the essential oil of fruit is used for treatment of dental caries [20,21].
In India traditional medicine, the bark has been used to treat cardiac, respiratory diseases, tooth infection, stomach infection and rheumatism [17,18].The fruits are used as spice and the essential oil extracted from the fruits is known as "Mullilam oil" used as anti-inflammatory, antiseptic, anticholera, diarrhoea, hypocholesterolemic, mosquito repellent and soothing agent for dental caries [22,23].The Kanikkars tribe prepare a paste of hard spines prepared by rubbing them against rock with water and apply the extract to the breast of a nursing mother to relief pain and also to increase milk supply [24].In the Phillippines, the pounded bark mixed with oil is a good formula to treat stomach ache.In addition, the bark decoction is also taken to treat chest pain and chewed bark applied as antidote for snake bites [19].

PHYTOCHEMICAL CONSTITUENTS
Previous phytochemical investigations on the different parts of Z. limonella showed quite wide variety of chemical substances.A number of secondary metabolites, alkaloids, aromatic and aliphatic amides, terpenes, sterols, and phynylpropanoid-lignans and coumarins have been isolated from this plant [25,26].A variety of compounds found in Z. limonella are compiled in Table 1.
Phytochemical studies on the ethyl acetate extract from the stem bark of Z. limonella initiated by Somanabandhu et al [28] have resulted in the isolation and structural elucidation of main component triterpenoid: lupeol (7) and the minor compounds such as three coumarins: xanthoxyletin (8), osthol (9), scopoletin (10) and one alkaloid: rutaecarpine (11) (Fig 4).Further work done by Charoenying et al [29] on the ethyl acetate extract from the fruit of Z. limonella have resulted in the isolation of xanthoxyline (2hydroxy-4,6 dimethoxy acetophenone) (12) as phenolic compound (Fig 5).The structure of this compound was determined by 1 H, 13 C NMR spectroscopy, and by the comparison of the spectral data with the data reported in the literature.
Thus, variation of environmental, ecological, geographical conditions as well as culture conditions and extraction techniques results in a diversity of volatile oil chemical components.Such conditions are likely to affect the biosynthetic pathway of the plant resulting in diverse essential oils.

BIOLOGICAL ACTIVITIES
The biological and pharmacological activities, mosquito repellent and larvicidal activity, antimicrobial activity, antitumour activity, have been frequently reported and detailed in Table 2.

Mosquito repellent and larvicidal activity
Essential oil and petroleum ether extract of the fruits of Z. limonella have been evaluated as repellent against Aedes albopictus mosquito in both a mustard and coconut oil.All repellents have been tested at three different concentrations (10, 20 and 30 %).The fruit oil and extract in mustard oil provide better protection than in coconut oil.The fruit oil offered the longest duration of protection effective against mosquitoes.Repellent efficacy of 30 % concentration of fruit oil in mustard oil mixture gave the longest protection time of repellency (296 -304 min) against A. albopictus [23].In another study, Trongtokit et al [21] investigated the repellency of essential oil from the fruit of Z. limonella using an arm-in-cage test.The undiluted fruit volatile oil gave complete protection time for 2 h against A. aegypti, Culex quinquefasciatus, and Anopheles dirus.Moreover, Mostel, the repellent product by the Insecticide Research Unit, Mahidol University, Thailand, containing binary mixture of 10 % clove oil (essential oil from Syzygium aromaticum) plus 10 % makaen oil (essential oil from the fruit of Z. limonella) in a gel form gave 100 % protection for 4-5 h against A. aegypti, C. quinquefasciatus, and A. dirus by arm in cage methods [36].Mostel provides protection against A. stephensi for 4.5-5 h by the cage test and gave protection from free flying mosquitoes in a mosquito proof room for 7-8 h [21].
The essential oil from the fruit of Z. limonella was effective against fourth instar larvae of A. aegypti in 24 h with the lethal concentration of 50 % and 95 % mortality (LC 50 and LC 95 ) values were 24.61 and 55.81 ppm, respectively [37].Rabha et al [38]

Antimicrobial activity
Z. limonella has been used medicinally for the treatment of infectious diseases.It has been reported to contain antibacterial and antifungal activities, which has been investigated in vitro.The crude chloroform extract from the fruits of Z. limonella showed antituberculous activity against Mycobacterium tuberculosis H37Rv with minimum inhibitory concentration (MIC) of 200 µg/mL [39].Wannissorn et al [40] has evaluated antibacterial activity of essential oil from the fruits of Z. limonella against 5 enteropathogens, Salmonella typhimurium TISTR 292, S. enteritidis DMST 17368, Escherichia coli TISTR 292, Clostridium perfringens DMST 15191 and Campylobacter jejuni DMST 15190.The results from disk diffusion assay indicated that the essential oil had antibacterial activity against all the tested bacterial strains.Maximum activity was observed against C. perfringens (27.0 mm), followed by S. typhimurium (20.5 mm), C. jejuni (18 mm), S. enteritidis (16.3 mm) and E. coli exhibited weak inhibitory activity (13.5 mm).
Nanasombat and Wimuttigosol [46]  Their results showed that the fruits essential oil had inhibitory activity against all of tested strains.This oil was found to be more effective to tested yeasts and fungi as compared to the bacterial strains.The essential oil possessed strong inhibitory activity against tested yeasts such as Rhodotorula glutinis, Schizosaccharomyces pombe, Hanseniaspora uvarum with the large zone of inhibition (42.3, 34.0 and 33.3 mm) and the MIC values of 1, 2 and 1 mg/mL, respectively.In this study, the essential oil had inhibitory activity against all tested fungi, especially Aspergillus ochraceus and Fusarium moniliforme with inhibition zones of 25.8 and 23.5 mm and the MIC values of these tested strains were equivalent at 1 mg/mL.The antibacterial activity of essential oil was also observed on the bacteria Bacillus cereus and Staphylococcus aureus with the wide zone of inhibition (20.5 and 21.5 mm) and the MIC values for these bacteria were the same (6 mg/mL).
In another study, Tangjitjaroenkun et al [24] reported that the antibacterial activities of crude essential oil, the distilled fractions (fraction I, fraction II, and fraction III) from Z. limonella fruits and pure major compounds (sabinene, limonene and terpinen-4-ol) were tested against various bacteria (B.subtilis ATCC 6633, S. aureus (ATCC 25923; methicillin -sensitive S. aureus (MSSA)), E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853)) by paper disk diffusion method with a final concentration of each test sample of 5 mg per disk.The crude oil, fraction I, fraction II and sabinene showed a potent antibacterial activity against B. subtilis, S. aureus and E. coli (> 10 mm inhibition diameter) but weak against P. aeruginosa (< 10 mm inhibition diameter).The crude oil exhibited higher antibacterial activity than distilled fractions and sabinene, suggesting that the untested minor components in the crude oil might have synergistic or additive effects against the tested bacteria.In addition, the crude oil and major compounds, sabinene, were further examined the antibacterial activity against the drugsensitive strains (MSSA, E.coli, P. aeruginosa) and multi-drug resistant (MDR) bacteria of S. aureus ATCC 43300 (methicillin-resistant S. aureus (MRSA)), extend spectrum β-lactamase (ESBL)-producing E. coli and the clinical isolated MDR P. aeruginosa using the quantitative microdilution susceptibility assay.The essential   In this study, the bactericidal activities of essential oil and sabinene were determined by using an in vitro time killing assay at dose equivalent to MBC, 2 × MBC and 4 × MBC, respectively.The trend of time-kill curves of crude oil and sabinene appears to be both timeand concentration-dependent.At higher concentration (2 × MBC and 4 × MBC) and longer contact time of interaction, more bacteria were killed.The essential oil and sabinene at the concentration of 4 × MBC (8 and 67.6 g/L, respectively) killed MSSA, E. coli, ESBLproducing E. coli and MRSA within 3, 6, 15 and 60 min, respectively.The effect of crude oil and sabinene were rapidly bactericidal at 2 × MBC (4 g/L and 33.8 g/L) achieving a complete elimination of MSSA and E.coli within 10 min and MRSA and ESBL-producing E.coli within 90 min exposure time.Considering that the essential oil had a higher bactericidal acitivity than sabinene, and the MBC values of the sabinene were eight times higher than those of crude oil.These results indicated that the essential oil is a complex mixture of wide variety compounds and their active components might have a synergistic or additive effect against tested organisms.

Antioxidant activity
Oxidants/free radicals relevant to human physiology are mainly derived from oxygen (reactive oxygen species/ ROS) and nitrogen (reactive nitrogen species/ RNS).They are produced intracellulary in the body, by endogenous sources, and also from the environment, man-made sources, and exogenous sources [47,48].Free radicals can damage the cellular components such as DNA, cell membrane lipids and modify several biochemical compounds [49].Free radicals has been linked to the various diseases including cancer, cardiovascular disease, stroke, atherosclerosis, arthritis, Alzheimer's disease, Parkinson's disease, diabetes and the ageing process [50].Antioxidants are substances that neutralize free radicals and protect cell constituents from oxidative damage caused by unstable molecules.These antioxidants may be synthesized in the body or obtained from plants such as fruits, vegetable, spices and herbs [51].Plants produce a wide variety of substances that possess antioxidant activity [52].
Tangjitjareonkun et al [53] reported that free radical scavenging activity of Z. limonella essential oil from the fruits and crude dichloromethane extracts of leaves (LD) and stems (SD); as well as methanol extracts of leaves (LM) and stems (SM) in both cell free and cell-based systems.The quantitative screening of antioxidant activity of extract and essential oil were evaluated by determining its effect on diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and trolox equivalent antioxidant capacity (TEAC).In DPPH antioxidant assay, IC 50 values of SD, SM and essential oil were 54.63 ± 2.89, 117.47 ± 4.66 and 5,764.67 ± 6.45 µg/mL, respectively, which was comparable to the standard antioxidant butylated hydroxyltoluene (BHT) (IC 50 value = 19.70 ± 0.2 µg/mL).TEAC values of SM, SD and essential oil were 15.47 ± 0.34, 14.34 ± 0.31 and 7.05 ± 0.34 µM, respectively.These data showed the ranking order of free radical scavenging activity, SM > SD > essential oil.However, the LD and LM were not determined the free radical scavenging activity due to the low antioxidant activity.Furthermore, the antioxidant capacity of Z. limonella crude extracts (SD, SM) and essential oil were investigated on prostate adenomacarcinoma cell lines, PC-3 and DU-145.All extract (SD, SM) and essential oil at 0.5, 5 and 10 µg/mL exhibited weak activity to reduce the malondialdehyde (MDA) level in both normal (untreated) cell lysates [53].
These results indicated that the extracts and essential oil cannot provide direct protection against free radical to protect the cell membrane from the damage caused by lipid peroxidation.On the other hand, the pretreated PC-3 and DU-145 with various concentrations of SD, SM, and essential oil at 0, 0.5 and 5 µg/mL for 24 h significantly decreased the MDA level of cell lysate.The intracellular antioxidant system, glutathione (GSH) and catalase (CAT) levels of cell lysates from PC-3 and DU-145 pretreated with SD and SM seemed to be significantly increased when compared to the control group, while the pretreated PC-3 and DU-145 with essential oil at all concentrations expressed high CAT level in dose-dependent manner more than GSH level in both cell lines.The SD and SM might have effect on levels of GSH and CAT, while essential oil may play a role in the regulation of CAT activity.These data suggested that the crude extracts, SM and SD, and essential oil may also regulated by increasing expression of the gene encoding the endogenous antioxidants, GSH and CAT.
In another study by Nanasombat and Wimuttigosol [46] the essential oil from the fruits of Z. limonella was determined by four different methods and exhibited strong antioxidant activity with IC 50 value of 5.66 mg/mL (DPPH assay), 66.16 % antioxidant activity (β-carotene bleaching test), 0.26 mM/mg reducing capacity (ferric reducing antioxidant power assay), and 79.07 % (superoxide anion scavenging activity assay).In addition, the free radical scavenging activity of the aqueous seed extract of this plant was measured by TEAC assay and the TEAC value was 5.059 mM trolox/gdw [54].

Antitumour activity
In vitro assessment of the antitumour promoting activity of methanolic fruit extract of Z. limonella was measured by the in vitro 12-Otetradecanoylphorbol-13-acetate (HPA, 40 ng/mL) induced Epstein-Barr virus early antigen (EBV-EA) activation assay in lymphoid cell line, Raji cells.The fruit extract showed strongly inhibitory effect (IE ≥ 70 %) at 200 µg/mL.The result indicated that this extract might contain effective antitumour promoters or cancer chemopreventive agents [55].

CONCLUSION
Z. limonella is used as folk medicine and flavouring food.The present review describes the botanical description, traditional uses, phytochemical constituents and biological activities.Available scientific literature shows that diverse secondary metabolites have been isolated from bark, stem, root and fruit of Z. limonella such as alkaloids, amides, terpenoids, coumarins, sterols, volatile oil and others.Various reports indicate that many of these phytoconstituents exhibit biological activities such as mosquito repellent and larvicidal, antimicrobial, antioxidant, and antitumour properties.Thus, there is little doubt about the potential of Z. limonella as an important resource for novel therapeutic agents.Therefore, this literature review may provide assistance to researchers who wish to further investigate the pharmacological properties of the plant.

Fig 1 :Fig 2 :
Fig 1: Zanthoxylum limonella (a) Appearance of the overall plant and (b) appearance of stem

Table 1 : Chemical constituents found in Z. limonella Plant part Compound Chemical category Reference Stem
also found that the essential oil hydrolate of Z. limonella exhibited strong activities against A. albopictus and C. quinquefasciatus larvae after 24 h.The LC 50 and LC 90 values of Z. limonella oil were 11 and 19.4 (% v/v), respectively against A. albopictus larvae and

Table 2 :
Reported biological activities of Z. limonella

Table 2 :
Reported biological activities of Z. limonella (continued