Research Article (Open access) |
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SSR Inst. Int. J. Life Sci., 6(3):
2536-2543,
May 2020
Phytochemical
Profiling of Ruta graveolens L. a
Vital Medicinal Plant Using Gas Chromatography-Mass Spectrometry
Malik
Aabid Hussain1*,
Varsha
Nitin Nathar2
1Research Scholar, Department
of Botany, Sant Gadge Baba Amravati University, Amravati, Maharashtra, India
2Professor, Department of Botany, Sant Gadge Baba Amravati
University, Amravati, Maharashtra, India
*Address for Correspondence: Mr. Malik Aabid
Hussain, Department of Botany, Sant Gadge Baba Amravati University, Amravati,
Maharashtra, India
E-mail: maabid1990@gmail.com
ABSTRACT-
Background: Ruta graveolens is a small evergreen sub-shrub of the
family Rutaceae, 2-3 feet tall and almost as wide. It contains a mixture of
alkaloids, coumarins, flavonoids, acridone alkaloids, essential oils etc.
having various biological activities. The present study was designed to
determine the volatile compounds
present
in various extracts of R. graveolens L. (Rue) using gas chromatography-mass spectrometry (GC-MS).
Methods: The leaves, stem and roots of R. graveolens
were extracted using solvents like petroleum ether and chloroform by using
Soxhlet apparatus for 24 hours and the extracts were analyzed through GC-MS.
The mass spectra of the compounds observed were matched by the National
Institute of Standards and Technology (NIST) library.
Results: The results revealed the presence
of 1, 4 and 6 chemical compounds in chloroform extracts of leaves, stem and
roots, respectively, while as 1, 5, and 3 chemical constituents were identified
in petroleum ether extracts of leaves, stem and roots respectively.
Conclusion: The
study is a base to understand the richness of bioactive components in different
extracts of R. graveolens L. Their isolation would have potential
applications in drug designing as well as in the medical and healthcare
industries.
Key
Words: Gas Chromatography-mass spectrometry (GC-MS),
Phytoconstituents, Ruta graveolens, Rutaceae, Volatile compounds
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 480ºC 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.
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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