Phytochemistry, GC-MS Analysis, Antioxidant and Antimicrobial Potential of Essential Oil From Five Citrus Species

Citrus essential oils were extraction from hydro distillation technique yielding Citrus oil with reasonable yield. Phytochemical screening of all five Citrus oils showed that alkaloids, tannins, sterols, terpenoids, saponoins, flavonoids were present (50-80%). GC/MS analysis showed highest percentage of limonene (58-89%) in Citrus oils. Antioxidant study revealed that Citrus peel oils have strong scavenging activity (83%-91%). Antimicrobial activity was evaluated by agar well method against eight common pathogens depicted marked antimicrobial potential especially tangerine (4.9-1.9 cm inhibition zones) and grapefruit oil (4.5-1.2 cm) inhibition zones. The studies emphasized the therapeutic and commercial utilization of Citrus peel essential oils as food preservatives, phytomedicine and antioxidant agent.


Introduction
All over the world Citrus is one of the widespread genus due to its prominent production. Citrus essential oils are naturally occurring, volatile and odoriferous oils synthesized by non woody parts of aromatic plants such as seeds, buds, leaves, flowers, stems, fruits, twigs and roots etc. and accumulated in secretory or epidermis cells and also sometimes in cavities (Ahmad, 2006). Essential oil from Citrus fruit peel is the fundamental product of genus Citrus and typically isolated by distillation or solvent extraction (Mondello et al., 2005). These are the complex mixtures of about 400 compounds of which 1-15% are non-volatile whereas 85-99% is the volatile constituents (Nannapaneni et al., 2009). Other organic compounds present in Citrus essential oils are aliphatic hydrocarbons, alcohols (linalool), aldehydes (citral), acids, esters and some aromatic compounds (Sharma & Tripathi, 2006). Svoboda & Greenaway (2003) reported the chief chemical constituent of Citrus essential oils is limonene and have a range of 32 to 98%. Citrus essential oils act as natural antioxidants because flavanone glycosides namely naringin, narirutin, hesperidin and neohesperidin are valuable phenolic compounds found in Citrus peel oil which make them liable to avert rancidity of food (Anagnostopoulou et al., 2006). Essential oils of Citrus peels are medicinally very important and show variety of biological effects because they are rich in flavonoids (flavone, flavonol and flavanone), terpenes, carotenes and coumarines which are responsible for antimicrobial activity (Tepe et al., 2005). Consequently Citrus essential oils are extensively used in pharmaceutics as an antimicrobial, anti-diabetic, antioxidant, insect repellent, carminative, larvicidal, antiviral, antihepatotoxic and antimutagenic agent (Kanaze et al., 2008).
The rapidly growing importance of Citrus based essential oils in food, pharmaceuticals, perfumes, flavor and fragrance has forced Pakistan to import increasing amounts of Citrus oils despite its rich variegated acreage of Citrus fruits and one of the largest Citrus fruits producing country of the world. These factors provide the opportunity for the production of highest grade essential oils from Citrus fruit peel. So there was an urgent need to focus on the extraction of essential oils as solid waste management and to improve our economy. This study was aimed for the assessment of phytochemical constituents, antioxidant as well as antimicrobial activities of five Citrus species, Citrus sinensis (L.) var. Malta, C. sinensis (L.) var. Mousami, C. reticulate (L.) var. Tangerine, C. reticulate (L.) var. Mandarin and C. paradisi (L.) Grapefruit.

Extraction of Citrus Essential Oil
Essential oils of selected Citrus species was extracted by hydro distillation unit for 3-4 hours extraction. Mixture of Citrus oils and water was incorporated which was separated, in two liquid layers which was isolated. . Hydro-distilled pure oil obtained was stored in dark brown sealed vials at 4°C until analysis.

Physicochemical and Phytochemical Investigation of Citrus EO
Physicochemical characteristics of Citrus essential oils including refractive index, optical rotation, specific gravity, color, odor and solubility were analyzed by the method of AOAC (2005). The chemical tests were carried out for screening of bioactive compounds present in Citrus essential oils using standard methods (Sofowora, 1993;Trease & Evans, 1989).

GC-MS Analysis of Citrus EO
Citrus essential oils were analyzed for their chemical composition by GC-MS analysis. GC/MS JOEL model JMS-A × 5050 H mass spectrometer (JOEL, Japan) Hewlett Packard 5890 Gas Chromatograph (JOEL, Japan). Helium as carrier gas, split ratio 1:100, electrical energy 70 eV, ionization current 200 µA, ionization temperature 250°C, column temperature with 6°C/min rise to 230°C. The chemical constituents were identified by their retention time and compared with known spectrum deposited in the National Institute Standard and Technology (NIST) library (NIST147.LIB).

DPPH Assay
Antioxidant potential was assessed by evaluating scavenging effect of each of five varieties of Citrus peel oils on DPPH. 500 µL of each essential oil was added in 3 ml of 0.002% methanolic solution of DPPH and shaken well. Absorbance was noted at 517 nm for all sample solutions and blank (contain only DPPH) after a stay time of 30 min in dark (Amin et al., 2006). All measurements were performed in triplicates. Scavenging potential of Citrus peel oils was determined in terms of percentage inhibition (I %) of DPPH by given formula:

Percent inhibition = (A blank -A sample / A blank ) x 100
Where A blank represents absorbance of DPPH only at 517 nm and A sample represents absorbance of sample under investigation at 517 nm.

Test Organisms
Antimicrobial activity of Citrus peel essential oils were studied against the two Gram positive bacteria Listeria monocytogenesn and Corynebacterium minutissimum and three Gram negative bacteria Escherichia coli, Yersinia sp. and Klebsiella planticola whereas three fungal strains named as Aspergillus flavus, A. fumigates and A. niger were used. All microorganisms were obtained from First Fungal Culture Bank of Pakistan (FCBP), Institute of Agricultural Sciences, University of the Punjab, Lahore.

Agar Well Diffusion Method
Antimicrobial potential of Citrus peel essential oils was assessed using agar well method (Kim et al., 1995). Each microbial concentration was made 10 6 CFU/ml. Wells (8 mm) were prepared in plates (single well in case of fungi whereas four wells in case of bacteria). About 60 µL of the essential oil was dripped into the wells. Water was used as control. The inoculated plates were incubated for 24 hours at 37°C for bacterial isolates and for 72 h at 27°C for fungi isolates. Study was conducted in triplicates. Biostatic efficacy against test organisms was investigated by measuring the inhibition zones in comparison to a control.

Yield of Citrus EO by Hydro Distillation
For Citrus oil extraction by the hydro distillation, five batches of Citrus species were carried out to determine average productivity of Citrus oils (Table 1). Citrus oils yield were in the range of (0.28-0.45%) for 3-4 hours extraction, comparable and better in some varieties as compared to other extraction techniques reported in literature (Minh Tu et al., 2002;Lota et al., 2000). The highest yield among all Citrus essential oil was calculated for C. paradise Grapefruit 0.45% followed by C. sinensis var. Malta 0.37%, C. reticulate var. Mandarin 0.33%, C. sinensis var. Mousami 0.30%, C. reticulate var. Tangerine 0.28% (Table 1). According to the previous work of Minh Tu et al. (2002) the yield of orange essential oil was 0.13% and tangerine essential oil was 0.25%. The essential oil yielded from various cultivars of mandarin was reported, 0.1% to 0.45% (Lota et al., 2000). Varying yields of essential oil are due to different extraction methods, units, soil and climatic conditions (Huet, 1991).

Physicochemical Characterization
Specific gravity of Citrus essential oils in present work was ranged from 0.842-0.858, refractive indices between 1.465-1.476 and all essential oils were found optically active (Table 2). These results were in line with preceding work on essential oils of Citrus species (Guenther, 1948).

Chemical Composition by GC-MS Analysis
Limonene was identified as the key element of Citrus peel oils. GC-MS analysis of Citrus peel oils revealed that among five Citrus essential oils, Grapefruit essential oil displayed highest concentration of limonene (89.84%) followed by essential oils of Malta (88.57%), Mousami (87.84%), Mandarin (87.45%) and Tangerine (58.50%).
www.ccsen   (Figure 2A). The essential oils had various degrees of inhibition potential against the five bacterial strains. Tangerine oil depicted maximum bacteriostatic activity against all the test bacteria i.e., E. coli, Y. sp. K. planticola, L. monocytogenesn with inhibition zones (4.9 cm, 4.8 cm and 4.6 cm and 1.9 cm, respectively) except C. minutissimum var. Mandarin essential oil revealed second highest inhibitory potential against E. coli and C. minutissimum (4.7 cm and 2.8 cm) while Mousami oil against both L. monocytogenesn (1.3 cm) and Y. sp. (4.7 cm). Grapefruit essential oil displayed marked bactericidal activity against K. planticola depicting the inhibition zone of 4.4cm. Least antibacterial effect was shown by Mousami in case of C. minutissimum, E. coli and K. planticola (1.6 cm, 3.7 cm and 3.2 cm, respectively), whereas Grapefruit and Mandarin against Y. sp. (4.5 cm each). The growth of L. monocytogenesn was least affected by Malta oil (1.1 cm). C. minutissimum and L. monocytogenesn were least susceptible to growth inhibition by Citrus essential oils.
The antimicrobial activities of Citrus species are strongly related with chemical constituents like flavonoids and phenols (Viuda-Martos et al., 2008). The active ingredients responsible for antimicrobial potential of Citrus peel oils are monoterpene components (Pavithra et al., 2009). D-limonene, linalool or citral are major attributors for the antimicrobial capacity of Citrus peel oils. Previous work revealed that the inhibitory influence of Citrus peel essential oils is owed to the presence of linalool rather than limonene (Fisher & Phillips, 2006 been found that antimicrobial activity is not only produced by one particular major component but also due to the antagonistic and synergistic effects of variety of compounds (Deba et al., 2008).
The essential oils of Grapefruit, Mousami, Malta, Mandarin and Tangerine showed the tendency to impede the growth of molds A. flavus, A. fumigates and A. niger ( Figure 2B). In all molds, Tangerine and Grapefruit oils revealed great antifungal potentials. In case of A. flavus and A. fumigatus, Tangerine peel oil was the best growth inhibitor (3.4 cm-4.0 cm). Followed by Grapefruit and Malta peel oils, which showed almost equal reduction in growths for these two molds. Mandarin and Mousami showed the lowest growth reductions of A. flavus and A. fumigatus. The mycelium growth of A. niger was most susceptible to grapefruit essential oil with inhibition zone (2.8 cm) while least affected by mousami peel oil with inhibition zone (0.7 cm). Viuda-Martos et al. (2008) studied inhibitory influence of mandarin, orange and grapefruit peel essential oils on four fungal pathogens. A. flavus growth was best prevented by Mandarin essential oil whereas A. niger growth was most susceptible to Orange peel oil. Grapefruit essential oil was the most efficient against P. verrucosum and P. chrysogenum.

Conclusion
The results of present investigation are the basis for extraction of essential oil of Citrus by cheaper methods to design pilot plant to extract EO for industrial production. Some major restraints in viable industrial exploitation of medicinal plants are due to the poor agricultural practices, quality control trials, strain in marketing and dearth of research on process and product development. Coordination among various institutes and organizations of the country can lead for sustainable commercial utilization.