GC-MS Compositional Analysis of Essential Oil of Leaf and Fruit Rind of Citrus maxima ( Burm . ) Merr . from Coastal Karnataka , India

1 Department of Prosthodontics, Crown and Bridge, AB Shetty Memorial Institute of Dental Sciences, Nitte University, Deralakatte, Mangalore, D.K, India. 2 Department of Oral and Maxillogical Surgery, AB Shetty Memorial Institute of Dental Sciences, Nitte University, Deralakatte, Mangalore, D.K, India. 3 Department of Pharmaceutical Chemistry, SDM Centre for Research in Ayurveda and Allied Sciences, Laxminarayana Nagar, Kuthpady, Udupi, India. 4 Department of Pharmacognosy and Phytochemistry, SDM Centre for Research in Ayurveda and Allied Sciences, Laxminarayana Nagar, Kuthpady, Udupi, India.


INTRODUCTION
Due to the resistance developed by microbes to the commercially available antibiotics researchers are now concentrating on herbal remedies which have less side effects but more powerful in inhibiting the microbial colonization.The ovalshaped sacs in the flavedo or colored portion of the peel of many Citrus spp are the reservoirs of oils which are natural toxic barrier to many microorganisms.Terpenes from volatile oil of medicinal plants are reported to be powerful antimicrobial agents.Effect of DL-limonene in inhibition of cell growth, ethanol formation, and sugar utilization by S. cerevisiae has also been reported (Unal et al., 2012).Essential oil C. limonum and C. aurantium were effective in inhibition of microorganism growth in multi-species biofilms in the oral cavity, which included Candida albicans, Escherichia coli, and Enterococcus faecalis responsible for atrophic candidiasis, periradicular disease, endodontic infections and several other infections (Oliveira et al., 2014).Lemon fruit peels, seeds and juice also possess activity against bacteria like Streprococcus aureus and fungus C. albicans (Pandey et al., 2011).Citrus maxima (Burm.)Merr.syn.C. grandis (Linn.)Osbeck; C. decumana Linn. is spreading, roundtopped almost thorn less tree.The fruit is nutritive, cardiotonic and refrigerant.Leaves are useful in epilepsy, chorea and convulsive cough (Nadakrni, 1954;Chopra et al., 1956).The fruit juice is used in stomach troubles (Dagar and Dagar, 1996); puerperal sepsis and insanity (Bortahnkur, 1996).Leaves are used in stomach pain due to indigestion (Chaudhari and Pal, 1976).
With the above background information, an attempt has been made to find out chemical constituent of grapefruit leaf and fruit rind essential oil using GC-MS.

Plant Materials
Mature grapefruits and leaves were collected from Manjanady, Mangalore, Dakshina Kannada district Karnataka.

Instrumentation and Techniques
Volatile oil in the drug is estimated by distilling the drug with a mixture of water and glycerin in a Clavenger's apparatus.The distillate was collected in a graduated tube in which the aqueous portion of the distillate is automatically separated and returned to the distilling flask.Volume of the oil collected on the surface of water in the graduated tube was measured.The content of the volatile oil is expressed as a percentage v/w.GC-MS was carried out using Shimadzu gas chromatograph with a SE-30 10% Chromosorb-W packed stainless steel column (2 m x 2 mm).Oven programme: 60°C (5 min), 60°-260°C (5°C/min), 260°C (10 min); carrier gasnitrogen, flow rate 40 ml/min; injector temperature 240°C; detector temperature 240°C.Individual components were identified by database of mass spectra matching with literature available in the libraries like NIST and WILEY by comparison of their mass spectrum values.The LRI of a compound is an expression of its retention time on a gas chromatographic column relative to an homologous series nalkanes.The following equation is used to calculate the Linear retention index (LRI) from the retention time (Adam, 1995).t = retention time of component n = carbon number of preceding n-alkane n+1 = carbon number of subsequent n-alkane

RESULT AND DISCUSSION
From 1500 g of leaves of grapefruit tree around 1 ml (0.07%) of pale white light volatile oil has been obtained by hydro-distillation using Clavenger's apparatus.Similarly, 1.5 ml of pale white light volatile oil was obtained from 1250 g (0.12%) of fruit rind.The composition of the volatile oil was analyzed by GC-MS.Fourty two constituents were detected from leaf, of which, 15 could not be identified as the mass fragmentation showed low similarity.Three major compounds eluted at RT 12.127, 14.481 and 21.014 mins were identified as β-Caryophyllene (16.89%), (-)-Spathulenol (9.32%), and Citronellol (28.26%) respectively.Other high concentration constituents were τ- A major compound at RT 32.034 which accounted for 11.69% could no bet identified (Table 1).
Thirty four constituents were detected from fruit rind, of which, 10 could not be identified as the mass fragmentation showed low similarity index.Limonene was the major compound accounting for 89.04%.Three other major compounds were β-Pinene (2.25%), β-Myrcene (2.06%) and β-Copaene (1.76%).All other compounds were found to be present in traces (Table 2).
Surprisingly, none of the identified compounds were found occurring in both leaf and rind, though both are from same botanical origin.The two different parts found to have distinguished chemical composition (Table 3).Antimicrobial resistance is increasing rapidly leading for the need to explore herbal remedies which does not have side effects.Thought was given to find out the components of the essential oil to explore Pomelo/Shaddock/Chakkotha, one of the fruits belonging to genus Citrus and commonly cultivated for consumption in Southern India, as a possible source of antimicrobial chemicals.
Composition of volatile oil of plants growing at different localities largely depends on the geographical conditions, and there are no studies in report on the composition of oil from this plant growing in Western Ghats.Leaf and fruit rind was considered for this study as volatile oil of Citrus species is mainly concentrated in these parts.
Citrus peels are rich sources of biologically active antimicrobial compounds like flavanones and polymethoxylated flavones, which are not found in other trees.The difference in the constitution may be due to the genetic and ecological variation of the plants.They are mostly used in food industries and also used as flavoring agents.It is also known to possess anti-inflammatory and anti-cancer properties.Constituents, such as simple sugars, carotenoids, vitamin C, flavonoids, limonoids, folic acid, fibres and potassium are good for human health.Major portion of citrus fruits produced are processed into juices, jams and concentrates (Francis and Hoang, 2015).

CONCLUSION
Citrus fruits are the most widely available and maximum consumed fruits.It has so many medicinal properties and very less toxic effect.The detection of compounds may be helpful in the study and association of various medicinal properties in the extract of leaves and fruit rind of Citrus maxima (Burm.)Merr.from Dakshina Kannada.

Table 1 :
The composition of the volatile oil from leaf.

Table 2 :
The composition of the volatile oil from rind.

Table 3 :
Linear retention index of the compounds identified from leaf and rind.