INTRODUCTION- Plants play an important role in the prevention and treatment of diseases and can minimize the side effects of conventional treatments^[1]. They can be a source of active components of biological and medicinal importance. They have traditionally occupied an important position in the socio-cultural, spiritual and medicinal arena of rural and tribal living in India ^[2]. Plants have been the sources of successful drugs from ancient times and will continuously be important for investigations of new medicinal compounds^[3]. For proper standardization of herbals and its formulations the identification and quantification of active principal compounds is the need of the hour^[4]. Thousands of these chemicals have been reported but still, there are many important components which have not been reported yet. Metabolic profiling has very wide range of applicability. Currently, the most effective technique for metabolic profiling is the use of Gas chromatography united with electron impact (EI) quadrupole or time of flight (TOF) mass spectroscopy (GC-MS). It has the capacity of identification and rapid quantification of few hundreds of metabolites within single extract ^[5]. Volatile compounds are directly separated and quantified by using Gas Chromatography and Mass Spectroscopy approach ^[6]. Another advantage of this technique is the rapid identification of metabolites using databases of mass spectra^[7]. GC-MS studies have been used on a large scale for the analysis of medicinal plants as this technique has proved very useful for the investigation of several chemical compounds.

R. graveolens L. commonly known as Rue is a small evergreen sub-shrub or semi-woody perennial 0.6 to 0.9 m tall and almost as wide, originally native to the Mediterranean region. It belongs to Rutaceae family in the order of Sapindales that contains about 160 genera and more than 1600 species. Due to its cultural and medicinal value, rue has been introduced in various countries of North, Central and South America, China, India, Middle East and South Africa. The bioactive compounds of Ruta species are of great interest in medicinal chemistry, as they show a broad range of biological activity ^[8]. Hence the objective of the present study is to identify the phytochemical constituents in different extracts of R. graveolens with the aid of GC-MS technique.

MATERIALS AND METHOD

Plant material- The plants of R. graveolens L. were bought from Melghat region of Amravati District of Maharashtra State and were identified by Dr. S. R. Manik, Former Head Department of Botany, Sant Gadge Baba Amravati, University, Amravati, India.

Plant sample extraction- The collected R. graveolens leaves, stem and roots were air-dried at room temperature until constant weights and coarsely powdered with an electric grinder. Dried and powdered samples (3 gram each) were subjected to extraction with petroleum ether and chloroform separately for 24 hours using Soxhlet apparatus and concentrated using water bath. The extracts were then filtered through Whatman filter paper No. 42 to obtained the free and clear extract. This extract was then concentrated to 5 ml and stored in refrigerator for further use.

Gas Chromatography-Mass Spectrometry (GC-MS) analysis- GC-MS analysis of the samples was carried out using Shimadzu Make QP-2010 with nonpolar 60 M RTX 5MS Column. Helium gas was used as a carrier gas and the temperature programming was set with initial oven temperature at 40ºC and held for 3 min and the final temperature of the oven was 480^ºC with rate at 10ºC/min. Total 2 μL sample was injected with splitless mode. Mass spectra was recorded over 35–650 amu range with electron impact ionization energy 70 eV. The total running time for a sample was 45 min ^[9].

Identification of chemical constituents- The Interpretation of mass spectra obtained by the GC-MS method was conducted using the database of National Institute of Standards and Technology (NIST) having more than 62,000 patterns. The spectrums of the unknown components from the samples were compared with the spectrum of the known components stored in the NIST library. Name, molecular weight and structure of the components of the tested materials were ascertained.

RESULTS- In the present study, the in vivo plant samples (Leaves, Stem and Roots) were investigated for biologically active compounds by GC-MS analysis. The extracts showed the presence of many secondary metabolites belonging to different groups. It was observed that the R. graveolens L. on which scientific studies has been carried out are validated in their uses in various parts of the world. The GC-MS analysis revealed the presence of 1, 4 and 6 chemical compounds in chloroform extracts of leaves, stem and roots respectively. (Table 1, Fig. 1, 2 and 3). In chloroform extract of leaf only one compound Tricosane (RT 35.72) with 100% peak area was found whereas in stem extract the highest peak area 57.49% and the lowest peak area 2.34% in Chloroform extract was of Benzene, 1-(chloromethyl)-2-fluoro-(RT. 15.13) and phytol (RT. 28.16) respectively. The other compounds identified in stem extracts were 14.94% Ethane hexachloro (RT. 11.50) and 25.22% 1, 2-Benzenedicarboxylic acid, diisononyl ester (RT. 23.90). Similarly in chloroform root extracts, the highest peak area (%) of 52.86 and the lowest peak area (%) of 2.50 was of Adamantane, 1,3-dichloro (RT 32.73) and Phosphonic acid, dioctadecyl ester (RT 30.48) respectively. The other compounds revealed in GC-MS analysis of Chloroform extract of roots were 6.17% 1-Octadecanol (RT.29.69), 11.42% 1-Bromodocosane (RT. 32.35), 16.03% Cyclotetracosane (RT.33.14) and 11.03% Di-n-octyl phthalate (RT. 33.56).

Table 1: GC-MS analysis of chloroform extracts of R. graveolens L.

Plant part used	Peak No	RT	Name of Compounds	Peak area (%)	MW	MF
Leaf	1	35.72	Tricosane	100	324	C₂₃H₄₈
Stem	1	11.50	Ethane, hexachloro	14.94	234	C₂Cl₆
	2	15.13	Benzene, 1-(chloromethyl)-2-fluoro	57.49	144	C₇H₆ClF
	3	23.90	1,2-Benzenedicarboxylic acid, diisononyl ester	25.22	418	C₂₆H₄₂O₄
	4	28.16	Phytol	2.34	296	C₂₀H₄₀O
Root	1	29.69	1-Octadecanol	6.17	270	C₁₈H₃₈O
	2	30.48	Phosphonic acid, dioctadecyl ester	2.50	586	C₃₆H₇₅O₃P
	3	32.35	1-Bromodocosane	11.42	388	C₂₂H₄₅Br
	4	32.73	Adamantane, 1,3-dichloro	52.86	204	C₁₀H₁₄Cl₂
	5	33.14	Cyclotetracosane	16.03	336	C₂₄H₄₈
	6	33.56	Di-n-octyl phthalate	11.03	390	C₂₄H₃₈O₄

RT= Retention time; MW= Molecular weight; MF= Molecular Formula

Fig. 3: GC-MS Chromatogram of Chloroform Extract of Root of R. graveolens L.

The GC-MS analysis of petroleum ether extracts of leaves, stem and root extracts showed 1, 5 and 3 chemical constituents respectively (Table 2; Fig. 4, 5 and 6).which contribute to the pharmacological activity of the plant. The major compound found in leaf extract was Ergosta-5, 7-dien-3-ol, 3.beta. (Peak area 100%). The highest peak area of 52.07% and the lowest peak area 1.62% in petroleum ether extract of the stem was of tetratriacontane (RT 35.89) and DOP; 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester (RT 33.59) respectively. The other compounds identified were 36.53% Toluene (RT. 6.26), 7.96% eicosanoic acid (RT.23.14) and 1.82% octadecane (RT. 35.51). Similarly, the highest peak area 49.41% and the lowest peak area 24.05% in petroleum ether extract of root was of 1,3,5-Cycloheptatriene (RT 6.60) and hexacosane (RT 30.95) respectively. The other compound identified was 26.53% nonacosane (RT. 39.69).

Table 2: GC-MS Analysis of Petroleum ether extracts of R. graveolens L.

Plant part used	Peak No	RT	Name of Compounds	Peak area (%)	MW	MF
Leaf	1	30.81	Ergosta-5,7-dien-3-ol, (3.beta.)-	100	398	C28H46O
Stem	1	6.26	Toluene	36.53	92	C7H8
	2	23.14	Eicosanoic acid	7.96	312	C20H40O2
	3	33.59	DOP ; 1,2-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester	1.62	390	C24H38O4
	4	35.51	Octadecane	1.82	254	C18H38
	5	35.89	Tetratriacontane	52.07	478	C34H70
Root	1	6.60	1,3,5-Cycloheptatriene	49.41	92	C7H8
	2	30.95	Hexacosane	24.05	366	C26H54
	3	39.69	Nonacosane	26.53	408	C29H60

RT= Retention time; MW= Molecular weight; MF= Molecular Formula

Fig. 6: GC-MS Chromatogram of Petroleum Ether Root Extract of R. graveolens L.

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Fig. 7: Molecular Structure of Compounds identified in different Extracts of R. graveolens L.

DISCUSSION- The members of Rutaceae family are used in medicine of which R. graveolens (Rue) is in the forefront and is native to Europe but not found all over the world^[10]. Rue contains various active compounds belonging to the group flavonoids, coumarins, derivatives, furoquinolines, volatile oils etc^[11]. Many of these compounds are yet to be identified. The recent scenario shows that there is again revival of interest in herbal medicines. The World Health Organisation has a strong thrust upon evaluation of these herbal drugs for some major diseases against modern drugs^[12]. Hence, traditional and herbal medicine is the need of hour ^[13]. GC-MS analysis is an interesting technique to evaluate the large number of chemical constituents used in cosmetic, pharmaceutical and food industry^[14]. The gas chromatogram shows the relative concentrations of various compounds getting eluted as a function of retention time. The heights of the peak indicate the relative concentrations of the components present in the plant. The mass spectrometer analyzes the compounds eluted at different times to identify the nature and structure of the compounds.

In the present study, the extraction of different plant parts was carried out to acquire the valuable information about secondary metabolites present in R. graveolens L. The in vivo plant parts like leaves, stem and roots were investigated for biologically active compounds by GC-MS analysis which revealed the occurrences of 11 compounds in chlorofom extracts and 9 compounds in petroleum ether extracts. The screening of plants for medicinal value has been carried out by the number of researchers^[15]. The presence of 79 metabolites from in vitro cultures of R. graveolens with a wide range of bioactivities like photobiological, anti-microbial, anti-oxidative ^[16], anti-inflammatory ^[17] among others with potential economic importance have been also reported^[18]. Metabolite screening is very much important in identifying new source of therapeutically and industrially valuable compounds having medicinal importance, to make the best and judicious use of available natural resources. The selection of plant parts, which gives maximum number of bioactive compounds, was the important step in such investigations^[19]. Several numbers of chemical constituents such as alkaloids, coumarins, terpenoids, steroids, flavonoids and furoquinolines have been isolated from different parts of the plant^[20]. Besides these, several essential oils have been reported in R. graveolens having anti-inflammatory and cytotoxic properties^[21]. The identification and isolation of these active compounds could lead to the new drug discovery at a cheaper cost which would be useful in medicine. The steroid Phytol from R. graveolens, was found to be cytotoxic against breast cancer cell lines (MCF7) ^[22,23]. R. graveolen possess diuretic, antimicrobial and anti-inflammatory activity^[24]. The level of these metabolites in plants varies with the changing seasons, age and location^[25]. The biological effects of furanocoumarins and furoquinolone alkaloids make them attractive for pharmaceutical uses, hence the considerable interest is shown in their availability and sources. In our earlier reports, we have reported the presence of 51 compounds in callus extracts of R. graveolens and 25 compounds in methanolic extracts of the same taxon with a wide range of bioactivities and potential economic importance^[26,27]. Due to the presence of various secondary metabolites in the extracts, rue plant has the potential application in various pharmaceutical industries^[28].

CONCLUSIONS- In the present study, twenty chemical constituents have been identified from chloroform and petroleum ether extracts of leaves, stem and roots of the R. graveolens L. by GC-MS analysis. The presence of these chemical constituents justifies the use of the plant for various diseases by traditional people. The study would lead to understanding the richness of bioactive components in different parts of R. graveolens L. The present study would form a basis for understanding the richness of bioactive components in different extracts of R. graveolens L.

Further study should be carried out for large scale production and isolation of these compounds in pure form. The abundance of these components and their isolation would have potential applications in drug designing as well as in the medicine and healthcare industries.

ACKNOWLEDGMENTS- The authors express sincere thanks to Coordinator, Common Facility Center (CFC), Shivaji University, Kolhapur, Maharashtra, India for supporting instrumental analysis for this research work.

CONTRIBUTION OF AUTHORS

Research concept- Dr. V. N. Nathar

Research design- Mr. Malik Aabid Hussain

Supervision- Dr. V. N. Nathar

Materials- Mr. Malik Aabid Hussain

Data collection- Dr. Malik Aabid Hussain

Data analysis and Interpretation- Mr. Malik Aabid Hussain

Literature search- Mr. Malik Aabid Hussain

Writing article- Mr. Malik Aabid Hussain

Critical review- Dr. V. N. Nathar

Article editing- Dr. V. N. Nathar

Final approval- Dr. V. N. Nathar

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