ANALYSIS OF NATURALLY OCCURRING PENOLIC COMPOUNDS OF THE GENERA CLINOPODIUM L. , HYMENOCRATER FISH & C. A. MEY. AND MELISSA L

Six taxa of Lamiaceae family including three taxa of the genus Clinopodium L. (C. vulgare subsp. vulgareL., C. umbrosum (M. B.) C. Koch. and C. congstum Boiss. & Hausskn ex. Boiss. ) and two of the genus Hymenocrater Fish. & C. A. Mey. (H,longiflora Benth. H. bituminosus Fish & C. A. Mey) and one of the genus Melissa (M.officinalis subsp.fficinalis L.) were studied in order to evaluate their phenolic profile ,as these taxa were studied in such detail for the first time in Kurdistan region of Iraq . The examined phenolic substances which studied in these taxa include: Caffeine, Coumarin, Eugenol Estragole(4Allyl anisole), 2-6 Dimethyl phenol, Salicylic acid and P-Cresol, by using high performance liquid chromatography (HPLC) technique. The results showed that the most abundant phenolic compound were: Caffeine and 2-6Dimethyl phenol which found in all the studied taxa, followed by Eugenol and Salicylic acid which found in five of the six studied taxa, whereas the less prevalent phenolic compounds were )Coumarin and Estragole(4Allyl anisole),which found in just two of the studied taxa. The deference in distribution of the phenolic compound in the studied taxa can be utilized to differentiate between them in studies of genetic diversity and chemotaxonomy as it can be possible to distinguish between the genera Clinopodium, Hymenocrater and Melissa beside the distinguish between the taxa of the same genus ( Clinopodium ), using phenolic profile.


INTRODUCTION
The mint family (Lamiaceae) is the sixth largest family of flowering plants in the world and economically important ,with about seven subfamilies, 258 genera (1). Some species are traditionally used as medicinal plants, herbal teas and as raw material in cosmetic Industry. The Lamiaceae family is a rich source of polyphenolic compounds (2 ) however The level of Phenolic compounds are vary greatly within species which can be utilized as comparative data for understanding relationships, and one of main tools of chemotaxonomy ( 3). Which is one of the more modern and rapidly expanding areas of plant taxonomy as the taste and smell of plants belonging to chemical continents of plant mostly play important role in distinguishing some species, (4). Phenols are a class of chemical compound consisting of OH ( hydroxyl) group bonded directly to anaromatic hydrocarbon group.They are classified as simple phenols or polyphenols based on the number of phenol units in the molecule( 5) they are also classified by Jeffrey Harborne and Simmonds based on the number of carbons (6). Phenolic compound considered as secondary metabolite in the plants, these are less distributed from the primary metabolite, but with higher taxonomical value. A wide range of phenolic compounds have been reported from the members of this family (7). Besides, interest in phenols has increased greatly because of the antioxidant and free radical-scavenging abilities associated with some phenolics and their potential effects on human health (8). In plant materials it was difficult for detection of the phenols, however a number of methods have been proposed for the separation and determination of phenolic compounds mainly based on a high performance liquid chromatography(HPLC) technique (9and 10) and recently modern method of HPLC was conducted for analysis of naturally occurring phenolic compounds in aromatic plants such as the study of Proestos and Komaitis ( 11) and also applied that on Lamiaceae family such as the study of Verma and Trehan(11) however, because there is no phenolic profile available on the chemical composition of the Lamiaceae species growing in Kurdistan region of Iraq this study was conducted in order to use it for taxonomical relationship between these species.

MATERIALS AND METHODS Sample collection and Preparation
Plant samples of each species were obtained from deferent locations in Kurdistan region of Iraq. Leaves and stems of the samples were dried at 25 °C in darkness and analyzed after grinding in a household blender. All samples were analyzed within 3 months of collection. The extraction method used for dried samples had as follows: 50 ml of%70 methyl alcohol was added to 5 gm of dried sample, and left at room temperature for 48 hrs. The extraction mixture was then filtered, and then the extract was concentrated to adequate volume in order to get rid of alcohol by using air conditioner ,then as much as volume of Petroleum Ether (80-100 boiling point) was added to the product, mixed and shacked gently, placed in separating funnel and left for some time to separated clearly into two layer. Thereby the major part of chlorophyll dissolved in petroleum ether, and float because of its lesser density than water extraction of phenolic compounds that dissolve in water and make the lower layer, which draw from lower of funnel and injected to HPLC apparatus.

HPLC Analysis
All standards were prepared as stock solutions in methanol. Working standards were made by diluting stock solutions in 62.5% aqueous methanol containing BHT 1 g/L, and 6 M HCL to yield concentrations ranging between 0.5-25 mg/L. Stock working solutions of the standards were stored in darkness at −18 °C (11). The analytical of high performance liquid chromatography apparatus system was employed .The separation was achieved on Analytical column: Eurospher 100, C18, 5µm, 250 x 4.6 mm at ambient temperature. The mobile phase consisted of water-acetonitrile water: concentrated phosphoric acid (400:600: 3± 0.05). The flow rate was 0.8 mL/min and the injection volume was 20 μL. The monitoring wavelength was 254. The identification of each compound was based on a combination of retention time and spectral matching.

RESULTS AND DISCUSSION
The kinds of phenolic compounds which detected in the studied species are presented in Table 1 ,with the Retention times of each of them and the standard curves of them using HPLC technique was illustrated in Figure  1,with their structure (figure 2) . The results of the kinds of phenolic compounds which obtained by methanolic extracts of the plant material from the six samples, show that there is a increased variability in the analysed taxa and found that the most abundant phenolic compounds were: Caffeine and2-6Dimethyl phenol which found in all the studied taxa (Table2), followed by Eugenol and Salicylic acid which found in five of the six studied species ,whereas the less abundant phenolic compounds were Coumarin and Estragole which found in just two of the studied taxa.  phenol, ,Salicylic acid and P-Cresol The identification of each phenolic compound was based on a combination of retention time and spectral matching, since polyphenols absorb in the ultraviolet (UV) region and using aqueous methanol for performing the extraction due Methanol has a protective role. It can prevent phenolic compounds from being oxidized by enzymes, such as phenoloxidases this is in agreement with (13). The deference in distribution of these phenolic compound in the studied species can be utilized to differentiate between them in studies of chememotaxonomy. from these results it can be possible to distinguish between the species of the genus Clinopodium by their phenolic profile (Figure 3,4,5) , as all the three species of this genus shared the samee phenolic compounds (Caffeine, 2-6Dimethyl phenol, P-Cresol) however C. congstum distinguished from the other species by containing another phenolic compound, (Coumarin and Eugenol), beside C. umbrosum species can be distinguish by having the phenolic compound Eugenol in addition to the shared ones, whereas C. congstum was the richest of phenolic compounds, it have all the studied phenolic compounds except Coumarin. The two studied species of the genus Hymenocrater have its unique profile compared with the other genera as they shared the same phenolic compounds (Caffeine, Coumarin, Eugenol, 2-6Dimethyl phenol, Salicylic acid), ( Figure 6,7) and finally the species Melissa officinalis was found rich of phenolic compounds, it have all the studied phenolic compounds except Coumarin (figure 8). So there were differences in the phenolic profile of the studied species because of the deference in their genomic structure and this is in agreement with (11) who found that the presence of polyphenols in any plant is largely influenced by genetic factors. At last determination of phenolic content in any plant using HPLC was very necessary as in the event of complex plant matrix selection of appropriate chromatographic condition for HPLC is a matter of great importance as well as a potentials analytical problem and extremely reduce time and efforts compared with other chromatographic method .And this study has it s importance that it was done for the first time on the new conformed taxa in Kurdistan region of Iraq.

Conclusions
The presence of phenolic compounds, usually called polyphenols, in aromatic plants proved by employing high performance liquid chromatography ,which was found in the studied species in deferent profile ,not only between the genera, but also within the species of the same genus ,which add information about the chemical structure of these species and can be utilized for determining taxonomic relationship between the species belong to these genera.