Analysis of the Hydrosol Aroma of Indian Oregano

The essential oils or volatile oils are complex mixture of terpenoids (generally C10 and C15) and some other classes of components, isolated from aromatic plants by physical means, e.g. steam-distillation, hydro-distillation, hydro-cum-steam distillation, and hydro-diffusion processes. These oils find extensive application in flavour, perfumery, cosmetic, and pharmaceutical industries. The process of distillation of aromatic plant material yields essential oil as a main product (primary oil or prime oil) and distillate water, also known as hydrosol as a byproduct. Typically, in commercial production, the essential oils are skimmed off and the hydrosol is discarded as waste or cohobated back to the source solution. The slightly hydrophilic part of essential oil, passes in to the aqueous phase during process of distillation, gives pleasant aroma to the resulted hydrosol. Typically, the essential oil fraction present in hydrosol is highly aromatic and its composition is often different from the primary essential oil [1,2]. However, the major components are generally the same of those present in oxygenated fraction of corresponding essential oils [3]. Biological and organoleptic properties of the hydrosols make them useful for food and cosmetic industries [2,4-9]. In addition, hydrosols also find application in biological agriculture against mushrooms, mildew, insects, and for fertilization of soils [10].


Introduction
The essential oils or volatile oils are complex mixture of terpenoids (generally C 10 and C 15 ) and some other classes of components, isolated from aromatic plants by physical means, e.g. steam-distillation, hydro-distillation, hydro-cum-steam distillation, and hydro-diffusion processes. These oils find extensive application in flavour, perfumery, cosmetic, and pharmaceutical industries. The process of distillation of aromatic plant material yields essential oil as a main product (primary oil or prime oil) and distillate water, also known as hydrosol as a byproduct. Typically, in commercial production, the essential oils are skimmed off and the hydrosol is discarded as waste or cohobated back to the source solution. The slightly hydrophilic part of essential oil, passes in to the aqueous phase during process of distillation, gives pleasant aroma to the resulted hydrosol. Typically, the essential oil fraction present in hydrosol is highly aromatic and its composition is often different from the primary essential oil [1,2]. However, the major components are generally the same of those present in oxygenated fraction of corresponding essential oils [3]. Biological and organoleptic properties of the hydrosols make them useful for food and cosmetic industries [2,[4][5][6][7][8][9]. In addition, hydrosols also find application in biological agriculture against mushrooms, mildew, insects, and for fertilization of soils [10].
In Indian subcontinent, Origanum vulgare L. is locally known as 'Jungali Tulsi' or 'Oregano' or 'Himalayan marjoram' . The plant is widely used as a very popular spice. Oregano is of great economic value owing to its various traditional and modern applications. It is used as a traditional remedy to treat various ailments such as whooping and convulsive coughs, digestive disorders, and menstrual problems [11,12]. Dried Origanum leaves and essential oils are used by the flavouring industry in various liqueur formulations, tomato sauces, condiments, in baked goods such as pizzas and salad dressings [13]. The genus, Origanum is known for its huge morphological and chemical diversity [14]. The chemotypic and ontogenic variations occurring in the essential oil composition of Indian oregano (Origanum vulgare L.) have been explored [15][16][17][18].
Aqueous distillate volatiles of some Indian aromatic plants have been subjected to phytochemical and antimicrobial studies [1,2,[19][20][21][22][23][24]. However, literature survey revealed that there were no such reports available on emerging aromatic crop, Indian oregano; hence a comparison of the composition of primary (main essential oil) and secondary (dissolved fraction of essential oil) volatile oils of two chemotypes of O. vulgare have been conducted in this research. oil and hydrosol can be collected simultaneously (like field distillation without cohobation) [2]. The oil collected directly in extraction burette was separated and dehydrated by anhydrous sodium sulphate and designated as 'primary oil' . The hydrosol collected simultaneously in a separate flask was used for isolation of 'dissolved' or 'secondary oil' .

Isolation of dissolved essential oil
The hydrosol collected in a separate flask was shaken vigorously with hexane (10:1×2) using separatory funnel for 30 minutes to recover the dissolved oil. The mixture was then allowed to settle and the organic layer was separated. The organic layer was then dried over anhydrous sodium sulphate, filtered and the solvent evaporated (35°C) under reduced pressure to get the 'secondary oil' . The primary and secondary oils were kept in a cool and dark place prior to analysis.

Gas chromatography (GC)
The GC analysis of the oil sample was carried out on a Nucon gas chromatograph model 5765 equipped with FID and DB-5 (30 m×0.32 mm; 0.25 µm film coating) fused silica capillary column. Oven temperature programming was done from 60-230°C at 3°C/min. Hydrogen was the carrier gas at 1.0 ml/min. The injector and detector temperatures were 220°C and 230°C, respectively. The injection volume was 0.02 µl neat (syringe: Hamilton 1.0 µl capacity, Alltech USA) and the split ratio was 1:30.

Gas chromatography-mass spectrometry (GC/MS)
GC/MS analysis of the essential oil sample was carried out on a PerkinElmer AutoSystem XL GC interfaced with a Turbomass Quadrupole Mass Spectrometer fitted with an Equity-5 fused silica capillary column (60 m × 0.32 mm i.d., film thickness 0.25 µm). The oven temperature was programmed from 60-210 ο C at 3 ο C/min using helium as the carrier gas at 1.0 mL/min. The injector temperature was 210°C, injection volume 0.1 µl prepared in n-hexane (dilution 10%), split ratio 1:40. MS were taken at 70 eV with a mass scan range of 40-450 amu and scan rate 1 sec with interscan delay 0.5 sec.
Further, the class compositions of the primary and secondary volatile oils of O. vulgare chemotypes also showed clear and considerable differences in their nature. The primary oils of O. vulgare chemotypes were dominated by hydrocarbons (chemotype I: 57.6%; chemotype II: 53.4%), followed by oxygenated compounds (chemotype I: 38.1%; chemotype II: 44.9%). However, secondary oils were dominated only by oxygenated compounds (chemotype I: 92.0%; chemotype II: 96.2%). These variations are observed due to relatively higher solubility of oxygenated compounds in water over the solubility of hydrocarbons.
Thus, on the basis of present study, it can be said that the hydrosol of O. vulgare populations should not be discarded as usually done with commercial distillation of aromatic crops. It could be redistilled by introducing cohobation system in field distillation unit to improve the organoleptic property of the primary oil or reused for distillation of fresh herb to minimize the loss of valuable components of the essential oil. Alternatively, hydrosols could also be used for isolation of pure compounds (thymol, and carvacrol) or it can be used as such for disinfection and cosmetic applications.