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Pure Appl. Biol., 7(1):282-291 , March, 2018 http://dx.doi.org/10.19045/bspab.2018.70034

Research Article

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

Rahman Qadir1, Farooq Anwar1*, Tahir Mehmood1, Muhammad Shahid2 and Sadaf Zahoor1 1. Department of Chemistry, University of Sargodha, Sargodha-40100-Pakistan 2. Department of Biochemistry, University of Agriculture Faisalabad-38040-Pakistan *Corresponding author’s email: [email protected] Citation Rahman Qadir, Farooq Anwar, Tahir Mehmood, Muhammad Shahid and Sadaf Zahoor. Variations in chemical composition, antimicrobial and haemolytic activities of peel essential oils from three local Citrus cultivars. Pure and Applied Biology. Vol. 7, Issue 1, pp282-291. http://dx.doi.org/10.19045/bspab.2018.70034 Received: 11/01/2018 Revised: 13/02/2018 Accepted: 15/02/2018 Online First: 16/02/2018 Abstract 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 (), Citrus sinensis (Mussammi), and Citrus x sinensis (Red blood ). The 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 (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. Keywords: Antimicrobial agents; Cell; Citrus Peel essential oil; GC-FID; Hydrodistillation Introduction antimicrobial and bio-pesticidal properties Essential oils are odorous volatile [4, 5]. components isolated from different parts of The genus Citrus, from family, aromatic [1, 2]. essential oils comprises 1,300 and 140 genera. C. are often employed to impart to sinensis (Orange), C. paradise (), drinks and foods as well as are used as C. reticulata (), C. grandis ingredients in the formulations of (Shaddock), C. limon (), C. pharmaceuticals, and aurantium (Sour orange) and C. medica products [3]. Besides multiple industrial () are some of the important fruit applications, the essential oils are recently species of genus Citrus. Citrus are grown gaining greater recognition due to their for their fruits in several countries with potential biological such as antioxidant, tropical climates such as Egypt, Pakistan,

Published by Bolan Society for Pure and Applied Biology 282 Qadir et al.

China, Brazil, The United States, Turkey, In perspectives of value-addition, currently, India, Nigeria and [6]. there is increasing interest on the Citrus fruits are valuable food commodity exploration of under-utilized fruit which is popular across the world due to processing agro-wastes (such as peels) for high nutritional value and medicinal isolation of high-value bioactives and benefits. Several in-vitro and in-vivo volatile oils [12, 13]. In Pakistan, studies have indicated Citrus fruits to be consumption of Citrus is made on large useful against chronic diseases such as scale both in terms of whole fruit intake and cancers and cardiovascular diseases due to fruit processing by-products. As results of the presence of high amount of wide scale Citrus fruit consumption, a and phenolic bioactives with antioxidant substantial quantity of Citrus peels is potential [7]. produced annually that can be utilized for As results of large scale Citrus fruit production of essential oil. Rarely efforts consumption, huge amount of peels are have been made towards studying the generated annually, which are often comprehensive compositional analysis and discarded as agro-waste instead of biological principles of peel essential oils of revalorized into value-added products. different Citrus cultivars in Pakistan. In the According to estimates, processing of present research variations in the yield, Citrus fruit generates peels and membrane chemical composition and biological residues containing about 40-45 % of fruit activities (antifungal, antibacterial and mass as a waste fraction [8]. Interestingly, haemolytic activities) of peel essential oils Citrus peels can be explored as a valuable from three widely cultivated and consumed source of essential oil [9]. Citrus peel Citrus cultivars (Citrus reticulata, Citrus essential oils have been investigated for sinensis, Citrus×sinensis) were appraised. their potential biological roles such as Materials and methods antioxidant, antimicrobial and anti- Collection of samples inflammatory properties [10]. The volatile Fresh fruits of three Citrus cultivars such as chemical compounds such as α-terpineol, Kinnow (C. reticulata), Musammi (C. linalool and α-pinene, present in Citrus peel sinensis) and Red essential oils, can be linked to antifungal (C.×sinensis) were collected through local and antibacterial activities [11]. Recently, market of Sargodha, Pakistan. The fruits due to growing microbial drug resistance were peeled off using a sharp steel knife. there is increasing interest in the Small pieces were made from recovered investigation of essential oils as natural peels with a knife and dried under shade in antimicrobial agents [4, 5]. Nevertheless, a laboratory. The shade-dried peel material the oil yield and biochemical composition was crushed using a domestic grinder and of plant essentials oils not only vary within packed in polyethylene zipped bags. the species and its varieties but also among Isolation of the essential oil different agro-climatic and geographical The essential oil was isolated via regions [4, 5]. Various studies reveal the Clevenger- type apparatus using hydro- chemical composition and potential distillation technique [14]. The ground biological activities of Citrus peel essential Citrus peels material was hydro-distilled oils [3, 6, 8, 9], however rarely data is for 3 h. Distillates of the essential oil were available on the inter- variations in collected and dehydrated over anhydrous the volatiles composition and biological sodium sulphate (Na2SO4), filtered and then properties of Citrus peel essential oils. This kept at fridge temperature (4°C) until prompts the need to appraise the variations analysed [15]. in the chemicals profile and biological Chemical analysis of the essential oil attributes of peel essential oils from Analysis of Citrus peel essential oils was different local cultivars of Citrus. carried out using a Gas Chromatographic

283 Pure Appl. Biol., 7(1):282-291 , March, 2018 http://dx.doi.org/10.19045/bspab.2018.70034 system (Schimadzu) attached to an FID Sample-less discs were employed as a (flame ionization detector). Compounds negative control. For bacteria and fungi, were separated on DB-5 capillary column. Amoxycillin (30 µg/dish) (Oxoid, UK) and Nitrogen (mobile phase) was flushed at a Flumequine (30 µg/disk] (Oxoid, UK) were flow rate of 5 mL/min. Initial column set as positive reference to compare temperature was held at 80 °C for 2 min and sensitivities of strain/isolate. After placing auto increased to 240 °C at the rate of 10 °C for 2 h at 4oC, plates were incubated at 37oC /min. The percent composition of the for about 18 h for bacteria and at 28oC for compounds was reported relative to total 24 h for fungal strains. By calculating the peak areas. diameters of the growth inhibition zones Antimicrobial activity (zone reader) of the organisms, Microbial strains antimicrobial activity was evaluated and Two Gram-positive bacteria such as S. compared with the controls [16, 17]. aureus (Staphylococcus aureus) API Staph Haemolytic activity TAC 6736152, and B. subtilis (Bacillus Haemolytic activity of the tested essential subtilis) JS 2004 and two Gram-negative oils was evaluated by a prescribed bacteria such as E. coli (Escherichia coli) procedure. [18, 19]. Freshly heparinized ATCC 25922, and P. multocida human blood (3mL) was taken from healthy (Pasteurella multocida) (local isolate) were volunteers. The bold was centrifuged for 5 employed to test the isolated essential oils. min at 1000 x g; plasma was poured off and Four pathogenic forms of fungi including cells were washed three times with 5 mL of C. albican (Candida albicans), M. canis chilled (4oC) sterile isotonicphosphate- (Microsporum canis), A. flavus buffered saline (Aspergillus flavus) and F.solani (PBS) of pH7.4. In each assay,erythrocytes (Fusarium solani) were used to test the were preserved at 108 cells per mL. Each antifungal potential of the essential oils. cultivar’s essential oil (100μL) was taken The above mentioned pure microbial strains and agitated with RBC (108cells/mL) were acquired from the Clinical Medicine independently. Incubation of samples was and Surgery Department, University of done for 35 min at 37oC. Instantaneously, Agriculture, Faisalabad, Pakistan. Purity after incubating, the samples were kept in and identity were verified and authenticated ice for 5 min and then centrifuged for 5 min by the Department of Microbiology at at 1000 x g. A 100- μL supernatant was University of Agriculture, Faisalabad, collected from each tube and diluted 10 Pakistan. The bacterial strains were time with chilled (4oC) PBS. The positive cultured in Nutrient agar (NA, Oxoid) and negative controls employed were overnight at 37 ºC while fungal strains Triton X-100 and phosphate buffer saline were cultured in Potato dextrose agar (PBS), respectively. Using μQuant (PDA, Oxoid) overnight at 28 ºC. (Bioteck, USA) the absorbance was Disc diffusion method observed at 576 nm and % RBCs lysis for The antimicrobial activity of the essential each sample was computed. oils was evaluated by disc diffusion method Results and discussion [4, 5]. Briefly, a 100 µL tested Peel portion and essential oils yield microorganism’s suspension, having 107 The results regarding % peel portion and % colony-forming units (CFU)/mL of bacteria oil yield of essential oils obtained for three and 106 spores/mL of fungi, were spread on cultivars of Citrus including Citrus NA and PDA medium, respectively. The reticulata (Musammi), Citrus sinensis compound’s solution was added in filter (Kinnow), Citrus x sinensis (Red blood discs (6 mm in diameter) and employed on orange orange) are presented in (Table 1). the agar plates which had formerly been The content of % peel portion from fruits of inoculated with the tested microorganisms. Red blood orange was higher (36.0%)

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

Table 1. Peel portion and yield of peel essential oil from different Citrus cultivars Cultivar Common Name % Peel portion % Oil yield Citrus sinensis (Musammi) 34.50 ± 0.50 1.76 ± 0.10 Citrus x sinensis (Red blood orange) 36.00 ± 1.00 1.06 ± 0.10 Citrus reticulata (Kinnow) 31.67 ± 1.15 0.86 ± 0.05 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 C. x sinensis were limonene, linalool and essential oils citranellal, respectively. Nerol, eugenol, The percent composition of major chemical geraniol and ethyl benzoate β-myrcene, and components identified in the essential oils linalool oxide were also present in from peels of three different Citrus considerable amounts. cultivars is depicted in (Table 2). The data, The present study values are in close derived from GC-FID analysis, revealed a agreement with the results of Geraci et al. significant qualitative and quantitative [27] who described that the key variation in the chemical profile of the oils components of these oils were myrcene, from selected Citrus cultivars. Based upon limonene,nerol, β-pinene and geraniol, . matching the relative retention time with Lota et al. [28] reported two major those of pure standards, major chemical monoterpenes: limonene and γ-terpinene in constituents (>1%) detected in essential oils essential oils from peels of Citrus reticulata of peels from C. reticulata, C. sinensis and [mandarin]. According to Feger et al. [29], the chief component in peel oils of

285 Pure Appl. Biol., 7(1):282-291 , March, 2018 http://dx.doi.org/10.19045/bspab.2018.70034

Brazillian Murcot was noted to quite comparable chemical composition be limonene. [30, 31]. The differences in the composition A comparative study, reported on the of citrus peel oils from different cultivars chemical composition of peel essential oils and regions can be attributed to the agro- isolated from Italian Sweet as well as climatic and genetic factors [32, 33]. Bergamot, revealed that both the oils have

Table 2. GC-FID percent chemical composition of peel essential oil from different Citrus cultivars Volatile Compound Retention Musammi Red Blood Kinnow Time Orange Ethyl acetate 1.152 0.89 0.15 0.15 Camphene 1.463 0.32 ---- 0.18 Acetaldehyde 1.620 3.56 3.56 3.56 Geraniol 1.960 10.02 24.0 12.67 Limonine 2.823 48.9 46.30 54.57 Myrcene 3.780 4.67 ---- 2.89 ᵞ-undecalactone 3.948 5.00 ------Citraniol 5.545 10.05 10.40 14.00 Eugenol 6.013 7.5 12.90 8.90 Others (Unidentified) 1.48 2.70 3.08 The data are mean ±S.D. of peel essential oil from three different samples of each Citrus cultivar analysed in triplicate Antimicrobial activities microbial strains i.e. B. subtilis, (26.16 mm) The results related to antibacterial activity S. aureus (25.50 mm), E.coli (37.42 mm) of peel essential oils from different Citrus and P.multocida (35.50 mm) as compared cultivars against two Gram +ve bacteria to peel essential oil of such as S. aureus (Staphylococcus aureus, Musammi (C. sinensis) 17.33,15.42,30.33, API Staph TAC 6736152) and Bacillus 21.00 mm and Red blood orange subtilis (B. subtilis JS 2004) and two Gram- (C.×sinensis) 25.55, 23.0,31.83,30.58 mm, ve bacteria: E. coli (Escherichia coli ATCC respectively. These values revealed that C. 25922), and P. multocida (Pasteurella reticulata peel essential oil is more active multocida, local isolate) are presented in against all the bacterial strains as compared (Table 3). In the present study Kinnow (C. to essential oils of C.sinensis and C. x reticulata) peel essential oil revealed sinensis [34]. maximum zone of inhibitions against

Table 3. Antibacterial activity of peel essential oils from different Citrus cultivars Inhibition zone diameters (mm)

Gram-positive bacteria Gram-negative bacteria Cultivar B. subtilis S. aureous E. coli P. multocida a a a a Citrus reticulata 26.16 ± 0.28 A 25.50± 0.50 A 37.42± 0.38 B 35.50 ± 0.50 C b b b b Citrus sinensis 17.33 ± 0.58 A 15.42± 0.58 B 30.33 ± 0.58 C 21.00 ± 1.00 D c c b c Citrus x sinensis 25.55 ± 1.00 A 23 ± 1.00 B 31.83 ± 1.04 C 30.58± 0.52 C Standard drug 43.00 ± 1.00d 38.41± 0.38d 42.50± 1.32d 43.00± 1.00d (Amoxycillin) A B A A Values 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

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The considerable antibacterial activity of Antifungal activity peel essential oil (EO) of Kinnow (C. The results of antifungal activity of the reticulata) is in line with the results of tested Citrus peel essential oils against C. Sultana et al. [35], and Javaid et al. [36], albican (Candida albicans), M. canis who reported that among Citrus cultivars (Microsporum canis), A. flavus tested, Citrus reticulata was highly (Aspergillus flavus) and F.solani effective against all microbial strains. Peel (Fusarium solani) are shown in (Table 4). EO’s of Citrus species exhibited Peel EO of Red blood orange (Citrus x appreciable equal antibacterial activity on sinensis) presented maximum zone of both gram +ve and gram -ve bacteria [37, inhibitions against A. flavus (29.0 mm) and 38]. The variable antibacterial activity of F.solani (32.0 mm). But in case of M.canis Citrus EOs can be mainly attributed to the (23.30 mm) and C.albicans (22.50 mm) it variation in the composition of constituents gave less zone of inhibitions than Kinnow such as oxygenated monoterpenes and (C. reticulata) A.flavus (26.00 mm), phenolics in due part to their genetic make- F.solani (31.0 mm), M.canis (24.17 mm), up. [39, 41]. C.albicans (22.83mm).

Table 4. Antifungal activity of peel eessential oils from different Citrus cultivars Inhibition zone diamtere (mm) Cultivar C. albican M. canis A. flavus F.solani a a a a Citrus reticulate 22.83 ± 0.76 A 24.17 ± 0.29 B 26.00 ± 1.00 C 31.00 ± 1.00 D b b b b Citrus sinensis 13.67 ± 0.58 A 17.5 ± 0.50 B 25.67 ± 0.58 C 23.50 ± 0.50 D A a c c Citrus x sinensis 22.50 ± 0.50 A 23.30 ± 0.26 B 29 ± 1.00 C 32.00 ± 1.00 D c c d d Flumequine 37.33 ± 1.1 A 38.00 ± 1.00 B 39.00 ± 0.50 C 42.00 ± 1.00 D Values 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

These values of inhibition zones revealed transfer complex with fungal cells [43]. that peel EO of Red blood orange (C. x Furthermore, the variable susceptibilities of sinensis) is more active against A.flavus and the tested organisms to citrus peel essential F.solani while that of Kinnow oils might be ascribed to the variations in (C.reticulata) is more potent against the rate of essential oil constituent’s C.albicans and M.canis. Meanwhile, penetration via the cell wall and cell Musammi (C. sinensis) peel EO exhibited membrane components of organisms [44- weaker activity against A.flavus (25.67 46]. This indicates that antimicrobial mm), F.solani (23.50 mm), M.canis (17.50 activity of the tested Citrus EO’s are not mm), C.albicans (13.67mm) than peel EO only cultivars dependent nevertheless this of Red blood orange (C. x sinensis) and potential is also dependant on the genetic Kinnow (C. reticulata). Citrus essential oils make of the microbial strains [47]. possess considerable anti-fungal activities. Cytotoxic /haemolytic activity The presence of chemical compounds such Essential oils from peels of different Citrus as α-pinene, linalool and α-terpineol can be cultivars exhibited negligible cytotoxicity linked to antifungal potential of the oils (0.29-1.09%) as compared to positive [42]. Citral is one of the main components control, Triton X-100 which offered 100% of essential oils that acts as a fungicidal haemolytic activity as since it is capable of forming a charge expressed in (Table 5).

287 Pure Appl. Biol., 7(1):282-291 , March, 2018 http://dx.doi.org/10.19045/bspab.2018.70034

Table 5. Cytotoxic activity of peel essential oils from different Citrus cultivars Cultivar Cytotoxic/Haemolytic Activity (%) Citrus reticulate 0.29 ± 0.02 Citrus sinensis 1.09 ± 0.34 Citrus x sinensis 0.62 ± 0.12 PBS 0.00 Triton-x-100 100

Obtained results clearly depicted that Citrus be utilized as natural antioxidant and peel essential oils have very low cytotoxic antimicrobial agents leading to value- activity and can be safe to use for nutra- addition in Citrus fruit processing industry. pharmaceutical applications [48]. This Authors’ contributions cytotoxic is interesting property of many Conceived and designed the experiments: F saponins to bring about haemolysis i.e. the Anwar & T Mehmood, Performed the release of haemoglobin from erythrocytes experiments: R Qadir, S Zahoor & M as result of change in membrane Shahid, Analyzed the data: F Anwar, T permeability [49]. Mehmood & M Shahid, Some other plants have also been studied Contributed reagents/ materials/ analysis for the haemolytic activity towards humans tools: F Anwar & T Mehmood, Wrote the or animal erythrocytes [50]. Although the paper: F Anwar & R Qadir. cytotoxicity of essential oils of Citrus References reticulata and Citrus sinensis have been 1. Ahmadi L, Mirza M, & Shahmir F checked against pests, but no data is (2002). The volatile constituents of available on their haemolytic activities Artemisia marschaliana Sprengel and its against human red blood cells. secretory elements. Flav Fragr J 17: Conclusion 141-143. Among the Citrus cultivars selected, 2. Bezic N, Samanic I, Dunkic V, Musammi (C. sinensis) peels exhibited Besendorfer V & Puizina J (2009). greater yield of essential oil as compared to Essential oil composition and internal Kinnow and then Red blood orange peels. transcribed spacer (ITS) sequence Limonene was found as a major component variability of four South-Croatian in three Citrus cultivars. All Citrus peel oils Satureja species (). Molecules revealed variable volatile composition 14: 925-938. owing to differences in genetic makeup of 3. Kamaliroosta L, Zolfaghari M, Shafiee the cultivars. The tested Citrus peel S, Larijani K & Zojaji M (2016). essential oils exhibited appreciable Chemical identifications of Citrus peels antibacterial, antifungal activities. Kinnow essential oils. J Food Bio Tech 6 (2): 69- (Citrus reticulata) peel essential oil 76. exhibited maximum antibacterial and 4. Hussain AI, Anwar F, Sherazi STH antifungal activity, while other Citrus peel & Przybylski R (2008). Chemical oils revealed relatively lesser biological composition, antioxidant and activities. The tested essential oils also antimicrobial activities of basil gave negligible level of cytotoxicity and (Ocimum basilicum) essential oils thus can be safe to use as depend on seasonal variations. Food valuable ingredients in nutra- Chem 108: 986-995. pharmaceutical products. Moreover, it can 5. Hussain AI , Anwar F, Chathta SAS be depicted that hydrodistillation is an , Latif S, Sherazi STH , Ahmad A, appropriate choice for isolation of essential Worthington J & Sarker SD (2013). oils from under-utilized Citrus peels. The Chemical composition and bioactive hydrodistilled Citrus peel essential oils can studies of the essential oils from two

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