Phytochemical investigation of seeds of Trachyspermum ammi Linn. by GC-MS

The present study embraces phytochemical investigation of the essential oil extracted from the mature seeds of Trachyspermum ammi Linn for different constituents by subjecting the oil to gas chromatography-mass spectrometry (GC-MS) analysis. The identification of the constituents is based upon retention indices and by comparison of their mass spectral fragmentation patterns against the commercial library mass spectra (Wiley, Nist etc.). Ellagic acid (EA), which is a natural phenol antioxidant, has been isolated from methanol extract from the mature seeds of Trachyspermum ammi Linn. Also, Thymol (Thl), a naturally occurring phenolic compound, has been crystallized by the reported standard procedure from oil extracted from these mature seeds. Both these compounds have been evaluated for their possible anti-cancer effect against a selected panel of human cancer cell lines by means of sulforhodamine B assay.


INTRODUCTION
Trachyspermum ammi Linn Synonym Carum copticum Linn (Family: Umbelliferae or Apiaceae) is widely recognized as ajowan or ajwain in India. Cultivation of this plant species was instigated in Egypt. It nurtures widely around Mediterranean sea and in South-West Asia encompassing Iraq to India, particularly in North Indian parts including Punjab, Haryana, Uttar Pradesh, Maharashtra, Bihar, Madhya Pradesh, Rajasthan, Gujarat, and West Bengal and is a well-regarded medicinal herb from the earlier times. Trachyspermum ammi Linn (Omum) is a herbaceous winter annual that reaches up to height of 90 cm, stem is profusely branched, nearly 7-9 mm thick near bottom and is striated all over. The leaves are pinnately divided, 24×14 cm with clasping leaf bases bearing tiny white-petaled flowers in umbels that ultimately develop into small, oval-shaped, compressed, about 2 mm long grayish brown seeds marked with vertical stripes on their outer surface Joy et al. (2001); Asif et al. (2014).
Ajwain has been extensively employed in the traditional ayurvedic and unani medicines for different ailments and as enhancer of body's resistance Asif et al. (2014). Ajwain seeds, with their distinguishing aroma and sharp taste are extensively used as spice in cooking foods, for preservation and for getting oil for ultimate use in perfumery Ranjan et al. (2011). In traditional Indian medicinal system, paste of crushed seeds has been applied externally as a poultice for relieving colic pains, decoction made from the seeds has been used as remedy for diarrhea, amoebiasis, febrile conditions and stomach disorders including flatulence and indigestion as their active ingredients help to boost the digestive function of the intestinal tract by assisting release of the gut juices Rao et al. (2003). Hot dry fomentation of the ajwain fruits applied on chest has been commonly used as a asthma therapy Singh et al. (2003). T. ammi has been reported by Siripornvisal  Hussein et al. (2000). Being motivated by the extensive pharmacological activities and medicinal applications of Trachyspermum ammi Linn, we have analyzed the essential oil extracted from mature seeds for different constituents by subjecting the oil to hyphenated GC-MS technique. Ellagic acid has been isolated from methanol extract of mature seeds of this plant species. Thymol has been crystallized by the reported standard procedure of storing the oil Phytochemical investigation of seeds of Trachyspermum ammi Linn. by GC-MS extracted from mature seeds under low temperature condition for overnight Guenther (1950). These isolated compounds have been evaluated for their possible anticancer potential against a panel of selected human cancer cell lines by Sulforhodamine B assay Monks et al. (1991).

Experimental
Thin Mass spectra were recorded on Varian GC-MS/MS 4000 instrument with workstation using electron impact method, FID detector, injector temperature 230ºC, column oven at100ºC, held for 5 minutes to 250ºC at the rate of 10 deg/min, held for 10 minutes, helium carrier gas with flow rate of 1 ml/min and Varian CP-SIL 8 CB MS column (30m ×0.32mm, 1μm film thickness), temperature 250ºC.

Extraction of oil from seeds of Trachyspermum ammi Linn
The air dried seeds of T. ammi Linn (500gm) were crushed, powdered and hydro-distilled in Clevenger-like apparatus for 12 hours to yield 20 ml of essential oil, which was brownish in color and dried over anhydrous sodium sulphate. The essential oil was subjected to gas chromatography-mass spectrometry analysis. When this essential oil was stored at 0 o C for overnight, colorless needles of thymol got separated.

Isolation of EA
The air dried material from previous step, after extraction of essential oil, was exhaustively extracted with petroleum ether at 60-80°C and then successively extracted with chloroform and then methanol by using Soxhlet apparatus. Both extracts were evaporated under vacuum to dryness. The methanol extract in the form of slurry was chromatographed on a silica gel column eluting with chloroform-methanol gradient. Fractions were collected (100 ml each) and monitored by TLC. Fractions of similar composition as determined by TLC analysis were combined and again chromatographed on silica gel column using n-hexane-ethyl acetate gradient as eluent in the order of increasing proportion of ethyl acetate, that yielded pure ellagic acid (EA).

In vitro cytotoxicity of the isolated compounds
In vitro cytotoxicity of the isolated compounds was assessed against the selected human cancer cell lines according to the standard procedure via protein-binding sulforhodamine B dye to estimate cell growth Skehan et al. (1990). The human cancer cell lines used in present study were acquired from National Cancer Institute, Frederick, U.S.A.

GC-MS analysis of the oil extracted from mature seeds of Trachyspermum ammi Linn
Different constituents were identified from the essential oil from mature seeds of Trachyspermum ammi Linn on the basis of evaluation of their retention indices, EI-MS, MS/MS spectra and GC data with mass spectral databases of reference mass spectral libraries (Wiley and NIST) ( Figure 1). The different chemical constituents identified along with their retention time (Rt) and percentage composition in the essential oil from mature seeds are listed in table1.

β-Pinene
The GC separated constituent of oil exhibited molecular ion peak at 136 m/z in EI-MS/MS spectrum, analyzed as C 10 H 16 . The other fragments ion peaks were obtained at m/z 121, 107, 93, 77, 69, 63 and 43.

α-Pinene
The GC separated constituent of oil exhibited molecular ion peak at 136 m/z in EI-MS/MS spectrum, analyzed as C 10

Limonene
The GC separated constituent of oil exhibited molecular ion peak at 136 m/z in EI-MS/MS spectrum, analyzed as C 10 H 16 . The other fragments ion peaks were at m/z 119, 107, 93, 77, 66, 63 and 43.

Thymol
The GC separated constituent of oil exhibited molecular ion peak at 150 m/z in EI-MS/MS spectrum, analyzed for C 10 H 14 O. The other fragments ion peaks were at m/z 135, 115, 107, 91, 77 and 65.

γ-Terpinene
The GC separated constituent of oil exhibited molecular ion peak at 136 m/z in EI-MS/MS spectrum, analyzed as C 10 H 16 . The other fragments ion peaks were at m/z 107 and 121.

∆-3-Carene
The GC separated constituent of oil exhibited molecular ion peak at 136 m/z in EI-MS/MS spectrum, analyzed as C 10 H 16 . The other fragments ion peaks were at m/z 121, 105, 107, 93, 77 and 45.

p-Cymene
The GC separated constituent of oil exhibited molecular ion peak at 134 m/z in EI-MS/MS spectrum, analyzed as C 10 H 14 . The other fragments ion peaks were at m/z 119, 105, 91, 77 and 65.

Carvacrol
The GC separated constituent of oil exhibited molecular ion peak at 150 m/z /MS spectrum, analyzed as C 10 H 14 O. The other fragments ion peaks were at m/z 135, 115, 107, 91, 77 and 66.

Ellagic acid (EA) HPLC:
Under the LC conditions employed as stated in experimental section, the purified ellagic acid got eluted at retention time (Rt) of 3.3 minutes, giving a single peak.

LC-ESI-MS:
The LC peak exhibited a molecular adduct at m/z 302.1 under positive ionization mode of ESI-MS, without any fragment ions produced.
IR (CHCl 3 , cm -1 ): Ellagic acid showed a strong peak at 1617.21 cm -1 representing an aromatic system beside strong bands at 3413.73, 3474.44 cm -1 due to hydroxyl groups and a sharp peak at 1692.59 cm -1 due to lactone carbonyl.

In vitro cytotoxicity of the isolated compounds
The results in terms of percent growth inhibition for the tested compounds along with the standards have been summarized in Table 2. From the resulting cytotoxicity data, ellagic acid (EA) has been found to possess considerable cytotoxicity with more than 70% growth inhibition at 1x10 -5 M concentration for A-549 (Lung) and THP-1 (Leukemia) human cancer cell lines, which is comparable to the standard anticancer drugs used for this study. Not much significant effect of thymol (Thl) has been observed on the tested cancer cell lines.

CONCLUSION
The present work reveals analysis of the essential oil extracted from mature seeds of Trachyspermum ammi Linn for different constituents by gas chromatography-mass spectrometry (GC-MS) analysis. Also, ellagic acid (EA), a pharmacologically significant polyphenol, has been isolated from methanolic extract from the mature seeds and thymol (Thl) has been crystallized by storing the oil extracted from mature seeds of this plant species under low temperature condition. Both the isolated compounds have been evaluated for their anticancer potential against a panel of selected human cancer cell lines by sulforhodamine B assay. The activity results obtained have demonstrated that ellagic acid (EA) possesses considerable cytotoxic activity against some human cancer cell lines used for the present study.
Phytochemical investigation of seeds of Trachyspermum ammi Linn. by GC-MS