Variations in chemical composition , antimicrobial and haemolytic activities of peel essential oils from three local Citrus cultivars

The present research work was carried out to appraise the variations in the chemical composition, antimicrobial and haemolytic activities of hydro-distilled essential oils from peels of three Citrus cultivars including Citrus reticulata (Kinnow), Citrus sinensis (Mussammi), and Citrus x sinensis (Red blood orange). The essential oil yield from the peels of these cultivars was found to be 0.86, 1.70 and 1.07 %, respectively. Overall, the major chemical constituents (GC-FID analysis) in the peel essential oils of C. reticulata, C. sinensis and C. x sinensis were identified to be limonene (46.30-54.57%), geraniol (10.02-24.00 %) and citraniol (10.05-14.00%). Among the oils tested, peel essential oil of C. reticulata exhibited maximum zone of inhibition against bacterial strains whereas that of C. x sinensis against fungal strains. The tested peel essential oils exhibited small extent of haemolytic activity (0.29 to 1.09%) indicating negligible cytotoxicity. The antibacterial, antifungal and haemolytic activities of the tested Citrus peel essential oils varied considerably (p<0.05) among cultivars depending upon the variable composition of the oils.

Citrus fruits are valuable food commodity which is popular across the world due to high nutritional value and medicinal benefits.Several in-vitro and in-vivo studies have indicated Citrus fruits to be useful against chronic diseases such as cancers and cardiovascular diseases due to the presence of high amount of vitamin C and phenolic bioactives with antioxidant potential [7].As results of large scale Citrus fruit consumption, huge amount of peels are generated annually, which are often discarded as agro-waste instead of revalorized into value-added products.According to estimates, processing of Citrus fruit generates peels and membrane residues containing about 40-45 % of fruit mass as a waste fraction [8].Interestingly, Citrus peels can be explored as a valuable source of essential oil In perspectives of value-addition, currently, there is increasing interest on the exploration of under-utilized fruit processing agro-wastes (such as peels) for isolation of high-value bioactives and volatile oils [12, 13].In Pakistan, consumption of Citrus is made on large scale both in terms of whole fruit intake and fruit processing by-products.As results of wide scale Citrus fruit consumption, a substantial quantity of Citrus peels is produced annually that can be utilized for production of essential oil.Rarely efforts have been made towards studying the comprehensive compositional analysis and biological principles of peel essential oils of different Citrus cultivars in Pakistan.In the present research variations in the yield, chemical composition and biological activities (antifungal, antibacterial and haemolytic activities) of peel essential oils from three widely cultivated and consumed Citrus cultivars (Citrus reticulata, Citrus sinensis, Citrus×sinensis) were appraised.

Collection of samples
Fresh fruits of three Citrus cultivars such as Kinnow (C.reticulata), Musammi (C.sinensis) and Red blood orange (C.×sinensis) were collected through local market of Sargodha, Pakistan.The fruits were peeled off using a sharp steel knife.Small pieces were made from recovered peels with a knife and dried under shade in a laboratory.The shade-dried peel material was crushed using a domestic grinder and packed in polyethylene zipped bags.

Isolation of the essential oil
The essential oil was isolated via Clevenger-type apparatus using hydrodistillation technique [14].The ground Citrus peels material was hydro-distilled for 3 h.Distillates of the essential oil were collected and dehydrated over anhydrous sodium sulphate (Na2SO4), filtered and then kept at fridge temperature (4°C) until analysed [15].Chemical analysis of the essential oil Analysis of Citrus peel essential oils was carried out using a Gas Chromatographic system (Schimadzu) attached to an FID (flame ionization detector).Compounds were separated on DB-5 capillary column.Nitrogen (mobile phase) was flushed at a flow rate of 5 mL/min.Initial column temperature was held at 80 °C for 2 min and auto increased to 240 °C at the rate of 10 °C /min.The percent composition of the compounds was reported relative to total peak areas.

Disc diffusion method
The antimicrobial activity of the essential oils was evaluated by disc diffusion method [4, 5].Briefly, a 100 µL tested microorganism's suspension, having 10 7 colony-forming units (CFU)/mL of bacteria and 10 6 spores/mL of fungi, were spread on NA and PDA medium, respectively.The compound's solution was added in filter discs (6 mm in diameter) and employed on the agar plates which had formerly been inoculated with the tested microorganisms.
Sample-less discs were employed as a negative control.For bacteria and fungi, Amoxycillin (30 µg/dish) (Oxoid, UK) and Flumequine (30 µg/disk] (Oxoid, UK) were set as positive reference to compare sensitivities of strain/isolate.After placing for 2 h at 4 o C, plates were incubated at 37 o C for about 18 h for bacteria and at 28 o C for 24 h for fungal strains.By calculating the diameters of the growth inhibition zones (zone reader) of the organisms, antimicrobial activity was evaluated and compared with the controls [16, 17].

Haemolytic activity
Haemolytic activity of the tested essential oils was evaluated by a prescribed procedure.[18, 19].Freshly heparinized human blood (3mL) was taken from healthy volunteers.The bold was centrifuged for 5 min at 1000 x g; plasma was poured off and cells were washed three times with 5 mL of chilled (4 o C) sterile isotonicphosphatebuffered saline (PBS) of pH7.4.In each assay,erythrocytes were preserved at 10 8 cells per mL.Each cultivar's essential oil (100μL) was taken and agitated with RBC (10 8 cells/mL) independently.Incubation of samples was done for 35 min at 37 o C. Instantaneously, after incubating, the samples were kept in ice for 5 min and then centrifuged for 5 min at 1000 x g.A 100-μL supernatant was collected from each tube and diluted 10 time with chilled (4 o C) PBS.The positive and negative controls employed were Triton X-100 and phosphate buffer saline (PBS), respectively.Using μQuant (Bioteck, USA) the absorbance was observed at 576 nm and % RBCs lysis for each sample was computed.

Peel portion and essential oils yield
The results regarding % peel portion and % oil yield of essential oils obtained for three cultivars of Citrus including Citrus reticulata (Musammi), Citrus sinensis (Kinnow), Citrus x sinensis (Red blood orange orange) are presented in (Table 1).The content of % peel portion from fruits of Red blood orange was higher (36.0%) followed by Musammi (34.50%) and then Kinnow (31.67%).These outcomes are in accordance with the findings of other scientists.Weiss [20], observed that peel portions of mandarin, sweet orange and lemon were 28.0, 25.0 and 40.0%, respectively.In another work of Manthey & Grohmann [21], peel portions from citrus fruits such as orange, grape fruit and lemon generated 25.6 -33.0, 21.5 -38.1, 33.7 -36.4 and 32.0 -46.6% of peel mass, respectively.Such variations in the peel mass of selected species from Sargodha region can be attributed to changing environmental factors of area as well as the genotype.These variable morphological and genetical traits of fruits play effective role in altering the composition of peels [22].Among the Citrus species, extracted through Clevenger apparatus using hydrodistillation method, Musammi (C.sinensis) peels exhibited maximum oil yield (1.76%) followed by Red blood orange (C.x sinensis) (1.06%) and Kinnow (C.reticulata) (0.86%).The results obtained were compatible with the findings of Weiss [20] who reported the peel essential oil yield from sweet orange, lemon as well as mandarin to be 0.80, 0.90 and 0.80 % respectively, while bergamot orange had 0.45-0.65%peel oil yield.Notable variations in the oil yield from different Citrus cultivars can be mainly linked to the fruit genotype.In another study, cold pressed peel oil yield from oranges, and bergamot petitgrain was 0.5% each whereas, mandarin oil had 0.2% yield, Anon [23].According to Ahmad et al. [24], the highest oil yield (1.21%) was obtained from Malta peel followed by Eureka,s lemon (1.12%), Musammi (0.98%) and Grapefruit (0.73%).Kamal et al. [25], reported that highest oil yield was exhibited by C. sinensis (0.23-1.08%), followed by C. reticulata (0.31-0.52%) and then C. paradise (0.21-0.43%).Tue et al. [26] reported that yield of peel essential oils differed from 0.2-2.0 % among different citrus species.

Table 1. Peel portion and yield of peel essential oil from different Citrus cultivars
The data are mean ±S.D. of peel essential oil from three different samples of each Citrus cultivar analysed in triplicate

Chemical composition of citrus peel essential oils
The percent composition of major chemical components identified in the essential oils from peels of three different Citrus cultivars is depicted in ( The presence of chemical compounds such as α-pinene, linalool and α-terpineol can be linked to antifungal potential of the oils [42].Citral is one of the main components of essential oils that acts as a fungicidal since it is capable of forming a charge transfer complex with fungal cells [43].Furthermore, the variable susceptibilities of the tested organisms to citrus peel essential oils might be ascribed to the variations in the rate of essential oil constituent's penetration via the cell wall and cell membrane components of organisms [44][45][46].This indicates that antimicrobial activity of the tested Citrus EO's are not only cultivars dependent nevertheless this potential is also dependant on the genetic make of the microbial strains [47].Cytotoxic /haemolytic activity Essential oils from peels of different Citrus cultivars exhibited negligible cytotoxicity (0.29-1.09%) as compared to positive control, Triton X-100 which offered 100% haemolytic activity as expressed in (Table 5).
Introduction Essential oils are odorous volatile components isolated from different parts of aromatic plants [1, 2].Plant essential oils are often employed to impart flavor to drinks and foods as well as are used as ingredients in the formulations of pharmaceuticals, perfumes and cosmetics products [3].Besides multiple industrial applications, the essential oils are recently gaining greater recognition due to their potential biological such as antioxidant, antimicrobial and bio-pesticidal properties [4, 5].The genus Citrus, from Rutaceae family, comprises 1,300 species and 140 genera.C. sinensis (Orange), C. paradise (Grapefruit), C. reticulata (Tangerine), C. grandis (Shaddock), C. limon (Lemon), C. aurantium (Sour orange) and C. medica (Citron) are some of the important fruit species of genus Citrus.Citrus are grown for their fruits in several countries with tropical climates such as Egypt, Pakistan, China, Brazil, The United States, Turkey, India, Nigeria and Spain [6].
[9].Citrus peel essential oils have been investigated for their potential biological roles such as antioxidant, antimicrobial and antiinflammatory properties [10].The volatile chemical compounds such as α-terpineol, linalool and α-pinene, present in Citrus peel essential oils, can be linked to antifungal and antibacterial activities [11].Recently, due to growing microbial drug resistance there is increasing interest in the investigation of essential oils as natural antimicrobial agents [4, 5].Nevertheless, the oil yield and biochemical composition of plant essentials oils not only vary within the species and its varieties but also among different agro-climatic and geographical regions [4, 5].Various studies reveal the chemical composition and potential biological activities of Citrus peel essential oils [3, 6, 8, 9], however rarely data is available on the inter-cultivar variations in the volatiles composition and biological properties of Citrus peel essential oils.This prompts the need to appraise the variations in the chemicals profile and biological attributes of peel essential oils from different local cultivars of Citrus.

Table 2
The differences in the composition of citrus peel oils from different cultivars and regions can be attributed to the agroclimatic and genetic factors [32, 33] .

Table 2 . GC-FID percent chemical composition of peel essential oil from different Citrus cultivars Volatile Compound Retention Time
The data are mean ±S.D. of peel essential oil from three different samples of each Citrus cultivar analysed in triplicateAntimicrobial activitiesThe results related to antibacterial activity of peel essential oils from different Citrus cultivars against two Gram +ve bacteria such as S. aureus (Staphylococcus aureus, API Staph TAC 6736152) and Bacillus subtilis (B.subtilis JS 2004) and two Gramve bacteria: E. coli (Escherichia coli ATCC 25922), and P. multocida (Pasteurella multocida, local isolate) are presented in (Table3).In the present study Kinnow (C.reticulata) peel essential oil revealed maximum zone of inhibitions against microbial strains i.e.B. subtilis, (26.16 mm) S. aureus (25.50 mm), E.coli (37.

Table 3 . Antibacterial activity of peel essential oils from different Citrus cultivars Cultivar Inhibition zone diameters (mm)
AValues expressed as means ± SD of three separate experiments performed (n=3 ×3).Different caps letters in subscript within the same row express significant (p<0.05)variations of means among bacterial strains.Difference of superscript letters within the same column express significant (p<0.05)differences of means among the Citrus cultivars

Table 4 . Antifungal activity of peel eessential oils from different Citrus cultivars
DValues expressed as means ± SD of three separate experiments (n=3 ×3).Various caps letters in subscript within the same row express significant (p<0.05)differences of means among different fungal strains.Difference superscript letters within the same column express significant (p<0.05)differences of means among Citrus species