INTRODUCTION:

The composition of active principles in herbs has been the subject of many research studies. They are responsible for the wide aroma and therapeutic herb effects [1]. The curative efficiency of herbs depends on their quality, the time of harvest, the drying and storage procedure and on the climate [2-4]. A simple and rapid method was used in for the isolation and detection of selected bioactive compounds such as terpenes, flavonoids, tannins, flavanoids, etc. found in some well-known medicinal plant, like Delonix elata (L.) Gamble (Caesalpiniaceae), Clerodendrum phlomidis.L, (Family: Verbenaceae)

In the development of medicinal plant industry, plant medicines are classified into three groups, herbs (jamu), standardized extracts and phytopharmaceuticals (5). There are some strict requirements for standardizing the extracts .Some of them are correctness and restorative power proven, uniformity of active constituents , its efficacy, safety and its assurance, both in quality and quantity(6,7).

Delonix elata (L.) Gamble (Caesalpiniaceae) family is found in some part of south India and it is a fast growing tree in the footing of rivers and channel banks(8,9,10) The height of the tree is 6-9m height, tolerably smooth and ash color. In the Indian system of medicine the leaves of plant is used for rheumatism, anti-microbial (11,12), flatulence (13), antiulcerogenic, antioxidant, and membrane stabilizing.

In addition we reported that the plant having analgesic, anti pyretic and anti-inflammatory activity (Kilimozhi et al), unpublished data. Clerodendrum phlomidis.L, (Family: Verbenaceae) is found in some part of south India and widely distributed waste lands. It is a large bush (or) small tree, reaching 9m height with more or less pubescent leaves and branches. They are growing in mesic habitats with moderate rainfall and mild temperature.

The leaves of plant are used in inflammation. The decoction of the root and leaves of the herb is used in rheumatism, nervous diseases, convalescence of measles, piles, chronic bronchitis, etc., (14) Consequently, the present study has been undertaken to illustrate the beneficial effect of the leaf extract of DE and CP in GC-MS instrumentation (gaschromatography - mass spectrometry) was used for the separation, identification and quantification of the extract compounds. The aim of our paper is to validate a rapid method for the quantitative determination of organic compounds in herbs using the rapid fingerprint procedure.

MATERIALS AND METHODS:

Plant material:

Taxonomic identification of the plants Delonix elata (L.) Gamble and Clerodendrum phlomidis(L.) made from Rapinat Herbarium, St. Joseph’s college of arts and sciences, Trichy, Tamilnadu, India.The fresh leaves of the plants were collected from Jeyankondam, Perambalur (Dist), Tamilnadu, India. The leaves were dried under shade, segregated, pulverized by a mechanical grinder and passed through 40 mesh sieves.

GC-MS chromatogram of ethanolic extract of Delonix elata

kilimozhi etal. Fig.1.

GC-MS chromatogram of ethanolic extract of Clerodendrum phlomidis

Kilimozhi etal. Fig.2.

Phytocomponents identified in the extract of the Plant Delonix elata [GC- MS study]

No	RT	Name of the compound	Molecular Formula	MW	Peak Area %	Compound Nature
1	3.61	Butane, 1,1-diethoxy-2-methyl-	C₉H₂₀O₂	160	0.59	Ether compound
2	4.81	2-Undecenal	C₁₁H₂₀O	168	3.24	Aldehyde
3	5.29	1-Butanol, 3-methyl-, formate	C₆H₁₂O₂	116	12.83	Butanol compound
4	6.91	Benzoic acid, 2-butoxy-, methyl ester	C₁₂H₁₆O₃	208	1.01	Aromatic compound
5	7.09	Hydroxylamine, O-decyl-	C₁₀H₂₃NO	173	0.29	Nitrogen compound
6	8.39	3-tert-Butyl-5-chloro-2- hydroxybenzophenone	C ₁₇H₁₇ClO₂	288	0.18	Chloro compound
7	8.56	2-Tridecenal, (E)-	C₁₃H₂₄O	196	0.50	Aldehyde
8	8.77	Octane, 1-chloro-	C₈H₁₇Cl	148	0.25	Chloro compound
9	9.33	4-Tridecene, (Z)-	C₁₃H₂₆	182	0.37	Alkene compound
10	9.47	1-Tridecene	C₁₃H₂₆	182	1.11	Alkene compound
11	9.62	Dodecanal	C₁₂H₂₄O	184	0.60	Aldehyde
12	10.03	3-Pentanol, 2,4-dimethyl-	C₇H₁₆O	116	0.47	Alcoholic compound
13	10.89	3-Hexadecyloxycarbonyl-5-(2-hydroxyethyl)-4-methylimidazolium ion	C₂₄H₄₅N₂O₃	409	0.25	Alkaloid
14	11.23	-Undecanethiol, 2-methyl-	C₁₂H₂₆S	202	0.34	Sulfur compound
15	11.50	Pentadecanal-	C₁₅H₃₀O	226	1.22	Aldehyde
16	11.67	cis-9,10-Epoxyoctadecan-1-ol	C₁₈H₃₆O₂	284	0.57	Alcoholic compound
17	12.70	1-Hexadecyne	C₁₆H₃₀	222	0.44	Alkene
18	12.96	2-Tetradecanone	C₁₄H₂₈O	212	2.30	Ketone
19	13.21	Oxirane, tetradecyl-	C₁₆H₃₂O	240	4.38	Oxirane compound
20	14.13	Undecanoic acid	C₁₁H₂₂O₂	186	0.91	Fatty acid
21	14.52	9,9-Dimethoxybicyclo[3.3.1]nona-2,4-dione	C₁₁H₁₆O₄	212	1.66	Ketone
22	15.69	3,7,11,15-Tetramethyl-2-hexadecen-1-ol	C₂₀H₄₀O	296	2.05	Terpene alcohol
23	16.20	9-Octadecenal	C₁₈H₃₄O	266	0.82	Aldehyde
24	16.71	Pentadecanoic acid, 14-methyl-, methyl ester	C₁₇H₃₄O₂	270	1.07	Ester compound
25	17.54	n-Hexadecanoic acid	C₁₆H₃₂O₂	256	16.20	Palmitic acid
26	17.94	Tetradecanoic acid, ethyl ester	C₁₆H₃₂O₂	256	1.69	Myristic acid ester
27	20.08	Phytol	C₂₀H₄₀O	296	6.82	Diterpene
28	20.60	Hexadecenoic acid, Z-11-	C₁₆H₃₀O₂	254	22.37	Unsaturated fatty acid
29	20.90	Octadecanoic acid	C₁₈H₃₆O₂	284	6.56	Stearic acid
30	22.62	-Hexadecenal, (Z)-	C₁₆H₃₀O	238	1.66	Aldehyde
31	24.43	1-Cyclohexylnonene	C₁₅H₂₈	208	0.40	Alkene compound
32	32.63	2,6,10-Dodecatrien-1-ol, 3,7,11-trimethyl-, (E,E)-	C₁₅H₂₆O	222	3.87	Terpene alcohol

Kilimozhi et al Fig 3 Mass spectrum and structure of n-Hexadecanoic acid

Kilimozhi et al Fig 4 Mass spectrum and structure of 1-butanol, 3-methyl-, formate

Preparation of extract:

The powdered leaves (1000g) were successively extracted with ethanol (70-80ºC) for 24 hrs by continuous hot percolation method using soxhlet apparatus. The fraction was separated from the solvent by distillation under reduced pressure to yield (9 .2% w/w) solid mass was obtained, it was stored in refrigerator and used for further studies

GC –MS tecniques:

GC-MS analysis was carried out on a GC clarus 500 Perkin Elmer system comprising a AOC-20i auto sampler and gas chromatograph interfaced to a mass spectrometer (GC-MS) instrument employing the following conditions: column Elite-1 fused silica capillary column ((30mm×0.25mm ID ×1µM df, composed of 100% Dimethyl poly siloxane), operating in electron impact mode at 70 eV; helium (99.999%) was used as carrier gas at a constant flow of 1ml/min and an injection volume of 0.5 µl was employed (split ratio of10:1) injector temperature 250 °C; ion-source temperature 280 °C. The oven temperature was programmed from 110 °C (isothermal for 2 min), with an increase of 10°C/min, to 200 °C, then 5 °C/min to 280 °C, ending with a 9 min isothermal at 280 °C. Mass spectra were taken at 70 eV; a scan interval of 0.5 s and fragments from 40 to 550 Da.

Kilimozhi et al Fig 5 Mass spectrum and structure of 3,7,11,15-Tetramethyl-2-hexadecen-1-ol

Kilimozhi et al Fig 6 Mass spectrum and structure of n-Hexadecanoic acid

Identification of Components:

Interpretation on mass spectrum GC-MS was conducted using database of National Institute Standard and Technology (NIST) 12, 62 having more than 62000 patterns. The spectrum of the unknown component is compared with the spectrum of the known components stored in the NIST library .The name, molecular weight and structure of the components of the test materials is ascertained.

RESULTS:

The active principles content in the etanolic extract of Delonix elata are presented in table.1 shows high quantities of bioactive compounds such as Hexadecenoic acid (22.37 %), Z-11-,1-Butanol, 3-methyl-, formate (12.83 %) but 3,7,11,15-Tetramethyl-2-hexadecen-1-ol, one of the active principles responsible for anti-inflammatory activity. Fig.3,4,5 shows mass spectrum and structure of this compound which is suggested to be a plasticizer compound and used as an antimicrobial, arthritic and, anti-inflammatory activity.

Phytocomponents identified in the extract of the plant of Clerodendrum phlomidis [GC MS study]

No	RT	Name of the compound	Molecular Formula	MW	Peak Area %	Compound Nature
1	2.72	Propane, 1,1-diethoxy-2-methyl-	C₈H₁₈O₂	146	1.13	Ether compound
2	3.65	Butane, 1,1-diethoxy-2-methyl-	C₉H₂₀O₂	160	0.68	Ether compound
3	4.54	Oxirane, (butoxymethyl)-	C₇H₁₄O₂	130	0.29	Oxirane compound
4	5.05	Propane, 1,1,3-triethoxy-	C₉H₂₀O₃	176	1.02	Ether compound
5	9.69	2-Decen-1-ol	C₁₀H₂₀O	156	1.02	Alkene compound
6	10.83	2-Undecanethiol, 2-methyl-	C₁₂H₂₆S	202	4.47	Sulphur compound
7	12.04	1-Cyclohexylnonene	C₁₅H₂₈	208	2.94	Hydrocarbon
8	13.11	2-Nonen-1-ol	C₉H₁₈O	142	0.63	Alkene compound
9	16.20	1-Octadecyne	C₁₈H₃₄	250	3.23	Alkene compound
10	16.66	Cyclopentaneundecanoic acid, methyl ester	C₁₇H₃₂O₂	268	1.13	Fatty acid ester
11	17.30	n-Hexadecanoic acid	C₁₆H₃₂O₂	256	28.14	Palmitic acid
12	17.70	Pentadecanoic acid, 2,6,10,14-tetramethyl-, methyl ester	C₂₀H₄₀O₂	312	3.05	Fatty acid ester
13	19.41	Z-10-Tetradecen-1-ol acetate	C₁₆H₃₀O₂	254	4.41	Alkene compound
14	19.74	Phytol	C₂₀H₄₀O	296	5.15	Diterpene
15	20.10	Z-10-Pentadecen-1-ol	C₁₅H₃₀O	226	11.59	Alkene compound
16	20.43	9,9-Dimethoxybicyclo[3.3.1]nona-2,4-dione	C₁₁H₁₆O₄	212	3.45	Ketone compound
17	20.61	3-Hexadecanol	C₁₆H₃₄O	242	0.63	Alcoholic compound
18	23.72	d-Mannitol, 1-decylsulfonyl-	C₁₆H₃₄O₇S	370	2.15	Sugar alcohol compound
20	25.93	2-[3-(4-tert-Butyl-phenoxy)-2-hydroxy-propylsulfanyl]-4,6-dimethyl-nicotinonitrile	C₂₁H₂₆N₂O₂S	370	4.86	Nitrogen, sulfur- compound
21	31.12	6,10-Dodecadien-3-ol, 3,7,11-trimethyl-	C₁₅H₂₈O	224	6.61	Alkene compound

Kilimozhi et al Fig 7 Mass spectrum and structure of Vitamin E acetate

Kilimozhi et al Fig 8 mass spectrum and structure of 2 (3-(4-tert-Butyl-phenoxy)-2-hydroxy-propylsulfanyl)-4,6-dimethyl-nicotinnonitrile.

The active principles content in the ethanolic extract of 3 Clerodendrum phlomidis are presented in table.2 shows high quantities of bioactive compounds such as n- Hexadecenoic acid (28.37 %), but Vitamin E acetate (13.45 %) and 2-[3-(4-tert-Butyl-phenoxy)-2-hydroxy-propylsulfanyl]-4,6-dimethyl-nicotinonitrile, one of the active principles responsible for anti-inflammatory activity. Fig.6, 7, 8 shows mass spectrum and structure of this compound which is suggested to be a compound and used as an antimicrobial, arthritic and anti-inflammatory activity.

CONCLUSION:

The utilization of mass spectrometry was effective for the identification of the minor active components from Delonix elata and Clerodendrum phlomidis and most of these compounds are reported for first time in these plants. Their low concentration in plant material and the similarity of skeletons justify the fact that these compounds were not previously reported in conventional phytochemicalstudies of Delonix elata and Clerodendrum phlomidis. The importance of the study was to identify the active components and the biological activity of some of these compounds (13). In the case of Delonix elata and Clerodendrum phlomidis to be related to activity of anti-inflammatory, analgesic, antioxidant, antimicobial and anti- arthritic activity. The present study suggests that the relative contribution of the active components on the pharmacological activity should be evaluated (14).

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Received on 26.06.2009 Modified on 10.08.2009

Asian J. Research Chem. 2(3): July-Sept., 2009, page 344-348