Composition of Volatile Oil and Methanolic Extract of Jordanian Melissa Officinalis L . and actions against Human Cancer Cell Lines SALEM

The essential oil of Jordanian Melissa officinalis L. were obtained by hydro-distillation and analyzed by Gas Chromatography – Mass Spectrometry. Components representing 96.40% of the total oil were identified. The methanolic extract and the volatile oil of Melissa officinalis L, were tested and showed anti-proliferation activities against 3 cancer cell lines. keywords: Melissa officinalis L, Jordan Flora, LC-Ms/Ms analysis, Cancer Cell line Methanolic Extract.


INTROdUCTION
Most people living in less developed countries rely almost exclusively on traditional medicines for their healthcare needs.In Jordan also many people use herbal medicines as alternative, additional or complementary medicine [1][2] .However, most of the plants used in traditional medicine in Jordan lack detailed phytochemical study and biological evaluation 3 .
One of the most interesting medicinal plant species in Jordan is Melissa, a genus of the madder family Labiatae 4 .It is widely cultivated in Europe and the United States 5 .There has been considerable interest in the biological effects of essential oils from a variety of plants 6 and in their antimicrobial 7 and antioxidant properties [7][8][9] .The content and composition of the oil Melissa officinalis vary with its origin within a given country 10 , from country to country [11][12][13] , and under the influence of nitrogenous fertilizers 14 and growth regulators 15 .This variability increases the importance of the study of a wide range of Melissa samples.In addition to the melissa essential oil composition, the aromatic and polyphenolic composition of herbal tea made from lemon balm (Melissa) has been reported 16 .
Malissa has traditionally been used to treat a wide range of conditions such as fever, flatulence, headache, influenza and toothache 2,5 .Numerous specific biochemical activities have been reported 17 such as acetylcholinesterase inhibition and antioxidant activity 18 and its use for the treatment of cancer 19,20 and of diabetes 21 have been evaluated.
Milessa occurs rarely in Jordan but is found in restricted regions at Wadi Rajeb.It is a neglected, underutilized plant and threatened by wild herbs.Preliminary work was carried out by Syouf with collections from 3, not previously, studied sites in Jordan 22 the locations are shown in Table 1 The essential oil composition of Melissa from Jordan and its anti-cancer proliferative activity have not been studied previously.Herein the analysis of the essential oil from these Melissa species is described for the first time.

Plant materials
A large sample of wild Melissa officinalis SP were collected in Jordan in 2011.The plants were identified by Dr. Maha Al-Syuof.(Biodiversity department, NCARE [22]) .The aerial parts of the plants were dried at room temperature and then coarsely powdered.

Extraction, isolation and identification of the essential oil
Dried leaves of M. officinalis were subjected to hydrodistillation for 3 hours using a Clevenger Caryophellene Oxide 30.5 type apparatus 23 .The oil was collected, dried over anhydrous sodium sulphate and stored in the dark in a refrigerator until analyzed.

Gas Chromatographic -Mass Spectral (GC-MS) Analysis
About 1 µl aliquot of each oil sample, diluted in n-hexane, was subjected to GC-MS analysis.GC-MS analysis was performed using a Varian Chrompack CP-3800 GC/MS/MS-200 (Saturn, Netherlands) equipped with split-splitless injector and DB-5 (5% diphenyl, 95% dimethylpolysiloxane) capillary GC column (30m x 0.25mm ID, 0.25 µl film thickness).The carrier (ultra-pure helium) flow rate was 1ml/min.The column temperature was kept at 100°C for 3 min and then programmed at rate of 10°C/min up to 250°C, and then held at 250°C for 60 min.The total run time was 56.98 min the mass detector was set to scan ion between 35-500 m/z.A mixture of n-alkanes (C 8 -C 20 ) was analyzed separately under the same conditions using the same DB-5 column.The compounds in the volatile oil s were identified, using built in libraries (NIST Co and Wiley Co, USa)

Identification of the compounds
The compounds were identified by comparing the retention time, retention index and mass spectrum of the chromatographic peaks with that of the standards available .
The identification of other components was by computer matching with the Wiley, NIST and ADAMS libraries 24 based on their retention indices 25 determined by reference to a homologous series of n-alkanes, (C8-C20) and by comparison of their mass spectral fragmentation patterns with those reported in the literature 24 and stored on the MS library data system.Using the van den Dool equation 26

RESULTS
In the Table (1-2).whichfollow the major oil components (component %>3) are indicated in bold.The total amount content was 0.1 ml.
The Chemical composition of Melissa officinalis from Wadi Rujb /Ajlun is shown in Table 1-4.The amount of oil collected was 0.2 ml.

Antiproliferative Activity Against Human Cancer Cell Lines
The methanolic extract of Melissa officinalis SP was tested on three cancer cell lines namely : two types of colorectal (SW480), (HCT116), and prostate (PC3).As were the volatile oils of the plants.
The cells growth were evaluated using MTT assay as illustrated.

CONCLUSIONS
Here in is reported the chemical composition of the volatile oils obtained by hydrodistillation from the two Melissa species, The plant also evaluated for its anti-proliferative activities using (SW480), (HCT116), and (PC3), cancer cell lines.Results revealed that the methanolic extract of Melissa officinals has an effect in cell viability.Further studies are needed for determination of the mode of action(s) of these plants antiproliferative activities.
The present study strengthen evidence that the search for new anti cancer agent should emphasize to the screening of natural flora of the different countries.
It can be noticed generally that the chemical composition of Jordanian Melissa officinlis sp ,and Melissa officinalis in other countries have not identical components .
The composition of the oil from M. officinalis harvested in Algeria was dominated by neral, geranial and citronellal.This composition was qualitatively the same that the oils from Serbia (Dukic et al., 2004), Slovak 22 , Egypt (Shalaby El-Gengaihi and Khattab, 1995), France (Carnatet al., 1998)   (2005) reported that caryophyllene oxide (12.6 %) and b-pinene (18.2 %) were also the most abundant constituents in the oil of M. officinalis from Greece but neral and geranial were not detected in the oil.Oils from Cuba 13 ) and Brazil 15 were dominated by neral (29.9 % and39.3%) and geranial (41.0 % and 47.3 %) respectively.
Which clearly leads to conclusion that the chemical composition of the plant and volatile oil composition may vary according to location.
, to get RI values helps by predicting the closet component from the top ten component summary which are given from the data system.tx = retention time of unknown component.tn = retention time of preceding n-alkane.t(n+1) = retention time of following n-alkane.n = carbon number of preceding n-alkane.RI: arithmetic index as reported in literature.KI: Kováts index as reported in literature.