Chemo-Divergence in Essential Oil Composition among Thirty One Core Collections of Ocimum sanctum L . Grown under Sub-Tropical Region of Jammu , India

Evaluation of thirty one core collections of Ocimum sanctum L. synonyms O. tenuiflorum L. collected from different ecological regions representing contrasting environment of India was carried out. All the collections were grown under sub-tropical region of Jammu, India. Study revealed wide range of variability in quantitive and qualitative attributes of oil. Essential oil content ranged between 0.16% ± 0.01% 0.55% ± 0.08% showing the presence of fifteen constituents. Methyl eugenol (1.54% 93.16%) and Eugenol (0.06% 70.41%), were the major constituent. The other major constituent of the oil was β-Caryophyllene (4.60% 33.77%) which was detected in almost all the collections. Borneol, Copane, α Caryophyllene were other constituents detected in almost all the accessions. α selinene was detected in traces in only three accessions (OS-01, OS-03, OS-50) and β-selinene was detected in four accessions (OS-01, OS-03, OS-50, OS-72. Accession OS-70 collected from Patna, showed distinct chemical profile having β-Elemene (32.81%), β-Caryophyllene (16.37%), Germacrene-D (18.05%), β-Ocimene (17.69%) and Copane (5.738%). Being distinct in oil profiling, Patna collection was designated as distinct chemotype. Collections OS-50 from Gwalior from Central India and OS-59 from Rajkot Western India have been identified as methyl eugenol (93.16%) and eugenol (70.41%) rich genotypes. The data collected provided useful information with respect to composition of essential oil among core collection evaluated representing various agro-climatic zones.


Introduction
Genus Ocimum belonging to family Lamiaceae, consists of about 160 species [1] represents versatile group of aromatics.It is well discussed in Ayurveda as healing system.Ocimum sanctum (Tulsi) leaves have potent medicinal properties, acting as an expectorant and antiseptic, in addition to having use as insect-repellent [2].Leaves are diaphoretic, anti-periodic; they are also used in bronchitis, gastric and hepatic disorders.Decoction of leaves is recommended for cough, malaise and in colds.It is a good mosquito repellant as well.Oil extracted from flowers is used in skin diseases and ring worm infection.Various studies have been performed with Ocimum sanctum for its antibacterial, antioxidant, antiulceric, antimalarial, antidiabetic, anti-inflammatory, antilipidemic, anticancer and immunomodulatory properties [3].The oils from the leaves of O. basilicum and O. sanctum contain phenols as aldehydes [4].
The therapeutic value of this is ascribed to its essential oils contents [5,6].The perfume, pharmacy and food industries use aromatic essential oil extracted from the leaves and flowers of basil in variety of products [6].The European genotype or a sweet basil is considered to have the highest quality aroma, containing linalool and methyl chavicol as major constituents [7].Seasonal variation in the content of essential oil and its major constituents eugenol, methyl eugenol, and caryophyllene, in leaves of O. sanctum has been reported [8].Eugenol-rich (54% of essential oil) genotype of sacred basil O. sanctum has been identified [9].Use of sensitive analytical tools, GC-MS of the essential oil of O. sanctum leaves allowed the separation of 46 compounds of which 31 were completely or partially identified [10].
The therapeutic potential is distributed variably in offtypes of Ocimum sanctum L. of different geographical and agro-climatic zones which implies that there is morpho-chemical and molecular diversity in it [11,12].The natural variability needs to be studied and tapped for Seeds of all the core collections were subjected to seed viability test by triphenyl tetrazolium chloride (TTC) solution before sowing for raising nursery in well prepared pots in the second week of April.Six week old seedlings of all the accessions were planted in Farm Yard Manure (FYM) treated experimental plots during 2009 and 2011 at Experimental Farm of the Institute (32˚44'N Latitude and 74˚55'E Longitude and 400 masl) with a temperature ranging from 5˚C -45˚C and total rain fall 500 mm with plant distance of 50 cms.The soil of experimental plots was sandy loam with pH 6.8, organic carbon 0.27%, available nitrogen, phosphorous and potash (200 kg/ha, 14 kg/ha and 136 kg/ha) respectively.

Essential Oil Extraction
Freshly harvested foliage (100gm) was used for extraction of essential oil by hydro-distillation method using Clevenger-type apparatus at 60˚C for 3 h.The oil was further analysed by the combination of GC and GC-MS.Essential oil content (percentage) was determined on air dried weight basis as an average of three samples.Mean (X): The mean value of the character was worked out by dividing the totals by corresponding number of observations.
Mean: Sum of X values/N (Number of values).Range: Lowest and highest values of each of character were recorded.Standard error: Standard error of difference of two means was calculated with the help of error mean square from the analysis of variance.
GC analysis: Column oven temperature was programmed from 50˚C -240˚C at 5˚C per min.Injector and detector temperatures were optimized at 250˚ and 270˚C respectively.Nitrogen gas at a flow rate of 1mL/min was used as the mobile phase.Injector split ratio was 1:80.
Gas chromatograph-mass spectrometer: GC-MS analysis was carried out on a Varian GC-MS 4000 fitted with a Varian Factor Four VF-5 ms fused silica capillary column (30 m × 0.25 mm id, film thickness 0.25 um).Temperature programming of oven was from 50˚C to 240˚C at 5˚C/min rising rate.Helium was used as carrier gas at flow rate of 1 ml/min.Mass spectra were recorded over 50 -300 amu range at 1 scan per sec with E.I. at 70 eV.
Identification of peaks was carried out by comparing their retention times with authentic samples injected under similar chromatographic conditions.Comparison of Kovat indices with literature values was carried out.The mass spectra were compared with those reported in the NIST and WILEY computer libraries and those published in literature [13] The relative amount (%) of individual components of the oil is expressed as percent peak area relative to total peak area from the GC/FID analyses of the whole extracts.

Results and Discussion
The chemical constituents of oil obtained from various samples were examined by the combination of GC, GC-MS and Kovat indices and data recorded is presented in Table 2.
β-Caryophyllene was identified as major constituent which was detected in almost all the analysed collections.Its content ranged between 4.60% -33.77%, with highest value (33.77%) recorded in accession OS-01 collected from Jammu.The presence of α-Caryophyllene in high proportion (19.42%) was only detected in (OS-01) whereas α-Caryophyllene Borneol, Copane were detected in almost all the accessions.α-selinene was present in OS-01, OS-03 and OS-50 in traces and β-selinene was detected in four accessions (viz.OS-01, OS-03, OS-50 and OS-72).Δ Cadinene, Elemol, Linalool and Caryophyllen oxide were present in traces in some accessions.The collection OS-70 from Patna, Bihar showed β-Ele-  from different eco-zones representing quite contrasting environments.Diversity among collected accessions which depicts divergence in chemical spectrum of essential oil could possibly be attributed due to soil type, pH, extractable nutrients, temperature in various ecozones located in different parts of India.Secondly, relatively less conservative nature of the synthesis or accumulation of these constituents at harvest could possibly be another reason for variation in composition of oil.The collected data further validate and supports the earlier findings where the role of environmental conditions on basil productivity, oil content, and composition [16,33] have been documented.The present study and the earlier reports clearly point towards importance of germplasm evaluation in Ocimum sanctum, which needs to be carried out to identify high yielding chemotype rich in molecules of therapeutic importance.This will further help to exploit particular genotype for commercial purpose.The study lead to identify OS-50 and OS-59 collected from Gwalior from central India and Rajkot from western India, respectively as methyl eugenol (93.16%) and eugenol (70.41% ) rich cultivars.On the basis of data OS-70 collected from Patna (Bihar) was quite distinct which suggest that this collection could be characterized as distinct chemotype having β-Elemene (32.81%), β-Caryophyllene (16.37%),Germacrene-D (18.05%) and β-Ocimene (17.69%) as major constituents among the thirty one evaluated core collections.

Conclusion
The present investigation has broaden knowledge base with respect to existing diversity with respect to essential oil array in thirty one core collections of Ocimum sanctum from different eco-geographical conditions having contrasting environment.This has helped to identify elite clone rich in particular components which could be exploited further for bioprospection of biomolecules.