|Origanum vulgare L.; Hydrodistillation; Essential oil;
Hydrophilic fraction; Composition
|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 . 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 .
|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 . The
genus, Origanum is known for its huge morphological and chemical
diversity . The chemotypic and ontogenic variations occurring in the essential oil composition of Indian oregano (Origanum vulgare L.)
have been explored [15-18].
|Aqueous distillate volatiles of some Indian aromatic plants have
been subjected to phytochemical and antimicrobial studies [1,2,19-24]. 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.
|Materials and Methods
|The fresh samples of O. vulgare chemotypes (I & II) were collected
from experimental field of Central Institute of Medicinal and Aromatic
Plants, Research Centre, Purara, Uttarakhand on 29th June, 2009 when
the plants were in flowering stage. Climatologically, the site falls in
temperate region (1250 m) of western Himalaya where the monsoon
usually breaking in June and continuing up to September.
|Isolation of essential oil
|Freshly harvested plant materials were hydro-distilled for 3 hrs in a
slightly modified all glass Clevenger’s apparatus in which the recycling of
distillate was stopped and diverted to a collection flask so that essential oil and hydrosol can be collected simultaneously (like field distillation
without cohobation) . 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°C at 3°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.
|Identification of components
|Constituents were identified on the basis of a Retention Index (RI,
determined with reference to homologous series of n-alkanes, C8-C30,
under identical experimental conditions), co-injection with standards
(Aldrich and Fluka) or known essential oil constituents, MS Library
search (NIST/EPA/NIH version 2.1 and WILEY registry of MS data 7th
edition), by comparing with the MS literature data . The relative
amounts of individual components were calculated based on the GC peak area (FID response) without using a correction factor.
|Results and discussion
|Primary and secondary volatile oils obtained from the
hydrodistillation of fresh herbs of O. vulgare chemotypes (I & II)
were analysed by GC/FID and GC/MS. Altogether, 55 constituents
representing 95.2-98.3% of the total oil compositions were identified
(Table 1). Chemotypes ‘I’ was rich in thymol, p-cymene, and γ-terpinene.
While the chemotype II contained carvacrol, p-cymene, and γ-terpinene
as major constituents. The amount of thymol and carvacrol was found to
be very low in primary oil (30.8% and 1.0%, respectively) as compared
to secondary oil (83.4% and 5.9%, respectively) of chemotype ‘I’. On
the other hand, primary oil possessed higher amounts of γ-terpinene
(21.4%), p-cymene (19.0%), myrcene (3.2%), carvacrol methyl ether
(3.2%), α-terpinene (2.4%), (E)-caryophyllene (2.1%), (E)-β-ocimene
(2.0%), and (Z)-β-ocimene (1.3%) as compared to secondary oil of this
chemotype. Furthermore, the primary oil of chemotype ‘II’ contained
lesser amount of carvacrol (42.3%) in comparison to secondary oil
(94.7%). However, the amounts of γ-terpinene (23.4%), p-cymene
(20.9%), myrcene (2.0%), and α-terpinene (2.0%) were noticed to be
higher in primary oil than secondary oil of chemotype II.
|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.
|We are thankful to Director, CSIR-Central Institute of Medicinal and Aromatic
Plants, Lucknow, India for continuous support.
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