Research into Microscopic Structure and Essential Oils of Endemic Medicinal Plant Species Satureja Subspicata Bartl

In this study we looked into the cells and histological organization of leaves (Saturejae folium) as well as a phyto-chemical composition of overground parts (Saturejae herba) of endemic species Satureja subspicata Bartl. ex Vis. (Lamiaceae) collected during year  on south slopes of mountain Velez in Herzegovina. Microscopic organization was analyzed in wet slides using light microscope. Estimation of stomata index was done according to Ph. Yug. IV. Chemical composition of overground material extracts was determined by thin layer chromatography (TLC) using thymol as a reference. In our research we found the following: Leaf structure of the analyzed species Satureja subspicata points at numerous specifi cities in anatomical and histological sense. In histological sense, leaf is of ventral type, with diff erentiated upper and lower epidermis and palisade and spongy tissue in between. Stoma index assigned according to Ph. Yug. IV leads to a conclusion that it is the case of diastitic stomata, which is common feature of most species from Lamiaceae family. Comparative qualitative analysis of essential oils in species Satureja subspicata showed similarities with other species from Lamiaceae family such as Th ymus L. (thymol). In fact, we found more common substances that are part of the species Satureja montana L. extract, but in diff erent concentrations.


Introduction
Th e application of medicinal plants with essential oils is continuously rising.Aromatic plants and essential oils are used in pharmaceutical and chemical industries but also in pharmacies for mixing of various sanative preparations.One of the advantages of essential oils therapy is the simplicity of their application.It can be part of regular care (bath, face care etc.) without changes in everyday routine (, , , ).Identifi cation of pharmaco-dynamic characteristics and antimicrobial activity of isolated plant components are set up as main tasks, which imply successful phytochemical research.Today, those are undertaken in order to fi nd new cures from nature, especially from chemically low examined or unknown plants (, , , , , ).Essential oils are mixtures of aliphatic, aromatic and hydro-aromatic substances as well as various carbohydrates, alcohols, aldehydes, ketones, esters, phenols, acids etc. (, ) (Figure ).Two very important phenols, thymol and carvacrol, are known to possess antiseptic properties for a long time.Th ey can be found in Th ymus serpyllum L. s. lat., Origanum vulgare L., Ocimum basilicum L. and Satureja hortensis L (, , , , , ).Certain extent of antimicrobial activity is experimentally demonstrated for citral, citronellal, cinnamon and oil-aldehyde, cineole, some gumerzines and other ingredients.Genus Satureja L. is represented with several species in the fl ora of B&H.Many of those are endemic and relic elements.Th ey are widely applied in ethno-medicine and ethnobotany of old.Signifi cant proportion are offi cinal plants that have important role in the contemporary pharmaceutical industry.Due to their content of various essential oils and antimicrobial eff ects they are widely used in various types of phyto-therapies and cosmetics.However, many species are not thoroughly studied.One of them is Satureja subspicata.It is an endemic Balkan species, which, in our country, is found in the area of sub-Mediterranean rocks ().Therefore, we designed this study in order to conduct basic biological and phytochemical research using applicable methods.The amount of reference data in this area demonstrates an increasing interest in exploration of biological, phyto-chemical, pharmaceutical and pharmaceutical/technological properties of the most common species of this type (, , ).

BOTANICAL AND SYSTEMATIC CHARAC-TERISTICS OF THE STUDIED PLANT SPECIES
Description of plant: Vertical, half-laid or half-risen halfbush - cm high (Figure ).Th e plant is four-partite, smooth, and almost completely bare.The leaves are thick, leathery, with glands at the edges or very rough.Th e fl owers are composed into levels and make a false ear.The calyx is wide, bell-like, dark green or dirty pink, containing numerous oil glands and  distinguishable nerves.It blooms from August till October.

THE FIELD STUDY Th e process of collecting and drying herbs
Plant material of species Satureja subspicata was collected at two locations in sub-Mediterranean area of mountains Prenj and Velez, where this species grows on geological foundation and lower grounds, mostly of neutral or alkali reaction.Th e material was collected from several populations in various phenological phases.Part of the material was deposited in herbarium, and part preserved for microscopic analysis.After the drug was dried an extract was made, which was used for other chemical and microbiological analysis.

LABORATORY RESEARCH Methods of microscopy
Standard light microscopy was used for the analysis of leaf cross section (Saturejae folium) as well as for the analysis of leaf vertical section (Saturejae folium), which was necessary for the purpose of stoma index determination in accordance with Ph.Yug.IV ().

PRODUCTION OF HERBAL EXTRACT
Procedure of hydro-distillation:  g of chopped up overground part of drug (Satureja subspicata) was mixed with  ml of water in a distilla- tion fl ask.Th e fl ask was heated over asbestos net to the boiling point and distilled.Th e distillate was collected in Erlenmeyer flask during that time.Before distillation, apparatus was washed with water, followed with dichrome-sulfuric acid and again with water.Th e harvested distillate was transferred into separation funnel.Th en, this solution was extracted with dichloromethane four times.Th e layer of etheric oil was collected in the glass until there was no layer between oil and watery phase.Anhydrous Na  SO  was added to etheric oil extract in order to remove residual water.Subsequently it was removed by filtration.Extracted and purified oil was then left to absorb steam (Figures ,  For the purpose of detection it was exposed to iodine vapor.In every sample yellow zones (stains) appeared or, to be more precise, one stain for thymol standard and four stains in Satureja montana etheric oil standard.For the purpose of detection, chromatogram was, before being exposed to iodine vapor, examined under UV lamp and extinguishing of fl uorescence appeared at  nm in zones.Four zones, where extinguishing of fl uorescence appeared, were detected for sample.One zone was detected for thymol standard, and eight zones, where extinguishing of fl uorescence appeared, for standard of essential oil of species Satureja montana.Later, chromatogram was sprayed with specifi c reagents vanillin-sulfuric acid (solution +solution ) for the purpose of visualization.x  is violet, x  is pink), and x  showed extinguishing of fl uorescence at  nm, too.Th ymol standard showed one pink stain, and extinguishing of fluorescence appeared at the same place, too.The second standard containing essential oil of species Satureja montana resulted in fi ve colored stains (x  is brown, x  is light green, x  is blue, x  is dark green, x  is violet), and extinguishing of fl uorescence appeared in all of them, except x  .
Rf values (Table ) were counted for all zones visible in chromatogram according to the formula (): Rf=d/d d−distance of chromatographic substance in cm d−distance of eluant in cm d=,  cm

Discussion
PLANT MICROSCOPY Anatomical-histological picture of leaf species Satureja subspicata points at numerous specifi cities in comparison with other dicotyledonous plants, even the species from the same family, Lamiaceae.According to its cy-tological diff erentiation, it would be a form of dorsalventral equalfacial leaf.Th e existence of two layers of palisade tissue (epipalisade and hypo-palisade tissue), which is a result of plant adaptation to sunlight utilization leads us to that conclusion.Namely, increase in palisade tissue compensates for a quite small leaf area.Such palisade tissue organization is an important feature of this plant's microscopic organization and can be successfully used for identifi cation.Also, the leaf has some elements of sclerophilly.Not only palisade, but also spongy tissue illustrate that.Namely, there is no intercellular space, and that is clear characteristic of this histological product inside of spongy tissue.Structure of epidermis hairs and cytological organization of gland apparatus can be used as important characteristics for this plant's microscopic identifi cation.In comparison with histological diff erentiation of close species Rosmarini folium and Salviae folium () numerous diff erences were noticed.Primarily, the organization of the tested leaves is more sclerophyllic.Th e analysis of stoma index according to Ph. Yug.IV () Satureja subspicata has diacitical stomas, like most of the species from La-  ().Etheric oils of related species Satureja and Satureja montana are very complex.Very complex structure of essential oil was determined by gas chromatography -spectrometric analysis in the samples of Satureja montana from diff erent regions of Serbia, B&H and Macedonia.Th ere are more than  diff erent components () in its structure.Among those are thymol and carvacrol and their quantity varies depending on the region (, , , , ).Evaluation of antimicrobial activity of Satureja subspicata oil was evaluated using agar diffusion and broth microdillution methods.Testing of antimicrobial activity showed that the oils had a great potential against all  bacterial and  fungal strains.Gram-positive bacteria are more sensitive to the examined oil, with a range of . to . μl/ml comparing to a signifi cantly higher range of . to . μl/ml for Gram-negative bacteria.
Results presented here suggest that the essential oil of S. subspicata possesses antimicrobial properties, and is therefore a potential source of antimicrobial ingredients for the food and pharmaceutical industry ().

Conclusion
Based on the fi ndings of microscopic study and the analysis of essential oils of endemic Dinaric medicinal plant Satureja subspicata it is possible to reach the following conclusions: -Th e leaf of the analyzed species in anatomical-histological sense pints at numerous specifi cities.In histological sense, leaf is of ventral type, with diff erentiated upper and lower epidermis and palisade and spongy tissue in between.It is unique type of sclerophillic leaf and important for microscopic identifi cation of this species; -Stoma index assigned according to Ph. Yug.IV. leads to the conclusion that the stoma is diactitic, which is common feature of most species from family Lamiaceae; -Comparative qualitative analysis of essential oils in species Satureja subspicata showed similarities with other species from Lamiaceae family such as Th ymus L. (thymol).In fact, we found more common substances that are part of the species Satureja montana L. extract, but in diff erent concentrations.-Results of this study indicate that the essential oils and other components of S. subspicata may be a great resource in pharmaceutical industry and modern aromatherapy.
and ).THIN-LAYER CHROMATOGRAPHY OF ESSENTIAL OILS (TLC)Procedure: On a fi ctitious start line drawn on previously activated chromatography panel (absorbance: silica gel  F  ; Merck, Germany;  minutes at °C) we applied etheric oil extract of over ground part of herb Satureja subspicata in two different concentrations, solution of thymol in ethanol (standard ) (Thymol, C  H  O, Kemika Zagreb) and essential oil extract of herb Satureja montana (standard ).After the stains were dried, the panel was placed vertically in a chromatography chamber coated with filter paper and saturated with solvent (eluant) vapor (rising chromatography).Nevertheless, we ensured that the eluant level was - cm below the start line.Developing solution (eluant) was a mixture of toluene-ethyl-acetate (:).When the analyzed solution reached the desired level, the panel was removed from the chamber and dried.After that, the position of separated substances was detected by illuminating the panel with UV lamp at  and  nm (UV lamp Spektroline (model CM- ENF- C/F producer: Spectronics corporation, Westbury, New York, USA).The substances were visualized by spraying the panel with universal and specifi c reagents, which develop characteristic color with the analyzed substances.Iodine vapor was used as a universal reagent for the detection of organic chemical compounds.Th e panel was exposed to iodine vapor in ex-SULEJMAN REDŽIĆ ET AL.: RESEARCH INTO MICROSCOPIC STRUCTURE AND ESSENTIAL OILS OF ENDEMIC MEDICINAL PLANT SPECIES SATUREJA SUBSPICATA BARTL.EX VIS.(LAMIACEAE) icer and as a result, characteristic brown stains appeared.Reagents specifi c for components of essential oils are: a)  solution of vanillin in ethanol (solution ) b)  solution of sulfuric acid in ethanol (solution ) The panel was sprayed first with solution , then with solution  and first, he board was sprayed with solution , and then with solution  and subsequently heated at  o C for about  minutes in a dryer.The result was characteristic coloring.Results PLANT MICROSCOPIC ANALYSIS Th e leaf of analyzed species Satureja subspicata has differentiated upper and lower epidermis with palisade and spongy tissue between them (Figure ).Mesophyll consists of two series of palisade at both upper and lower side, with regularly lined cells.Central part consists of spongy tissue with isodiametrically shaped cells (there is no stressed intercellular area).Collateral vascular bundles with approximately balanced proportion of phloem and xylem cells are also located in the central part.Epidermis is built of thick compressed cells with no intercellular area and chloroplasts and with stressed external inner side.Th e lower epidermis contains stomas of Helleborus type.Epidermis also contains numerous glands with etheric oils and many multicellular hairs.Vertical section of leaf of the studied plant species (Saturejae folium) was analyzed under microscope in order to estimate stoma index, which was defined on the basis of the number of stomas and number of epidermis cells according to formula (): Stoma index=xS/E+S S−number of stomas per leaf area unit =  E−number of epidermis cells per leaf area unit =  Stoma index (SI) =,  ESSENTIAL OILS Figure  shows chromatogram where a mixture of toluene ethyl-acetate (:) was used a developing agent.
SULEJMAN REDŽIĆ ET AL.: RESEARCH INTO MICROSCOPIC STRUCTURE AND ESSENTIAL OILS OF ENDEMIC MEDICINAL PLANT SPECIES SATUREJA SUBSPICATA BARTL.EX VIS.(LAMIACEAE) miaceae family.Stomas are surrounded by two accompanist cells that make right angle with adductor cells.ESSENTIAL OILS Similarities with other species from Lamiaceae family are underlined through our study of qualitative structure of essential oils of species Satureja subspicata (, , ).Th at was enabled by comparison between Rf values of etheric oil of Satureja subspicata and Rf values of thymol and etheric oil of species Satureja montana, which were used as standards.Based on the results we can ascertain that the sample was not heterogeneous.Component with the highest contents was thymol, and that was verifi ed by comparing thymol as a standard on the basis of Rf value and characteristic stains.Comparison with etheric oils of species Satureja montana revealed - shared components, but their concentrations are diff erent (, , , , , ).Th e latest study () describes phytochemical profi le and antimicrobial activity of Satureja subspicata Vis.essential oils, collected in Dalmatia (Croatia).Th ree samples of essential oils were obtained from the aerial parts of the plant by hydrodistillation and analyzed by GC-MS.From the  compounds representing . of the oils, carvacrol (.), α-pinene (.), p-cymene (.), γ-terpinene (.) and thymol methyl ether (.) appear as the main components.Th e oils also contained smaller percentages of myrcene, linalool, β-caryophyllene, limonene, geranyl acetate, -Octen--ol, nerol, thymol and borneol