INTRODUCTION:

India is abundantly supplied with a diverse range of medicinal plants. Clitoria ternatea commonly known as butterfly blue pea belonging to the family Fabaceae. It is a perennial herb widely seen in India and distributed in humid low and tropical region, east and west indies¹.The plant have variety of ethnical medicinal significance and traditionally used in the treatment of various ailments including Jaundice, migraine, eye infection and other disorders². Clitoriaternatea is a great valuable medicinal plant possess many bioactive principles and also acts as a source of therapeutic agent³. The extracts of Clitoria ternatea have been utilised as an ingredient in Medhyarasayana, a rejuvenating herbal combination intended to treat a variety of neurological illnesses and improve cognitive capacity^4. It has recently sparked a lot of interest because of its potential applications in modern health and agriculture, as well as a natural source of food colorants and antioxidants.

Clitoria ternatea has also been widely used in traditional medicine, particularly as a supplement to improve cognition and relieve the symptoms of a numerous of diseases, including fever, inflammation, pain, and diabetes^5. Moreover, the medicinal properties of this plant have been scientifically validated, particularly at an international level, and have been reported to have a variety of biological and pharmacological activities, including antioxidant, anti-inflammatory, anti-microbial, anti-diabetic, and hepatoprotective properties⁶.

Figure 1. Clitoria ternatea Linn

Gas chromatography–Mass spectroscopy (GC–MS) is a method for identifying different chemicals within a test sample that combines the features of gas liquid chromatography and mass spectroscopy⁷. GC-MS is a powerful and versatile analytical technique with a wide range of scientific applications⁸. The presence of bioactive compounds is established by GC-MS analysis of the bioactive fraction, which is further confirmed by library data. The molecular mass of a substance and its elemental composition are determined using mass spectroscopy⁷. The present study shows the ethylacetate of leaf of Clitoria ternatea subjected to GC-MS analysis to detect the presence of various phytoconstituents in this plant².

MATERIALS AND METHODS:

Plant material collection and authentification:

The plant material (leaves) was collected from Orchards botanical garden, Pattambi, Kerala, India. The specimens collected from garden were identified using standard literature and authenticated with legitimate voucher specimens. The plant materials, leaves were taxonomically identified by the Botanist, Dr. Ranjusha A.P, Department of Botany, NSS College, Ottapalam. The specimen of Clitoria ternatea Linn (accession no: 18365) has been deposited at Kerala Forest Research Institute (KFRI).

Extraction:

The plant matter was dried in the shade and roughly pulverised. 20grams of coarsely powdered leaves were placed in a Soxhlet apparatus and extracted with 250 millilitres of ethyl acetate solvent (approx.2days). The extract has been gathered. The extract was then concentrated after being filtered through Whatman No. 1 filter paper. After that, the extract was subjected to GC-MS analysis.⁹

GC-MS analysis:

Gas chromatography Mass spectroscopy analysis of ethyl acetate extract was performed using Shimadzu GC-MS Model number: QP2010S equipped with Column - ELITE-5MS (30 meter length, 0.25mm ID, and 0.25µm thicknesses). Electron ionization system was used; details of GC programme were given in the table 1. The oven temperature was programmed from 70.000C which is given in table 2.

Table 1. GC programme [GC-2010]

GC MS Parameters	Programme
Column oven Temp	60.0°C
Injection Temp	260.00 °C
Injection Mode	Split
Flow control Mode	Linear Velocity
Pressure	57.5 KPa
Total Flow	24.0 ml/min
Column Flow	1.00 ml/min
Linear Velocity	36.5 cm/sec
Purge Flow	3.0 ml/min
Split Ratio	20.0
Splitter Hold	OFF

Table 2. Oven temperature programme

Rate	Temperature	Hold time
-	60.0	0.00
5.00	280.0	5.00

Table 3. GC-MS Programme [GCMS-QP2010]

GC-MS Parameters	Programme
Ion source temperature	200.00⁰C
Interface temperature	280.00 ⁰C
Solvent cut time	5.00 min
Detector gain mode	Relative
Detector gain	0.98 kV +0.20 kV
Threshold	500

Table 4. MS Table

Mass spectroscopy parameters	Programme
Start time	5.10min
End time	49.00min
ACQ Mode	Scan
Event time	0.50sec
Scan speed	1250
Start m/z	50.00
End m/z	650.00
Sample inlet unit	GC

Helium gas was used as the carrier gas. Details of GC-MS programme was given in table 3. Programme specifications regarding Mass Spectra were depicted in table 4. GCMS Software: GCMS Solutions, Libraries used: NIST 11& WILEY 8.¹⁰

Identification of compounds:

The constituents in the extract were identified by comparing their relative retention times, and the mass spectra were confirmed by comparing them to databases from the Library of NIST 11 and Wiley8, as shown in the GC-MS Chromatogram in Figure 2.

RESULTS AND DISCUSSION:

GC MS is a hyphenated analytical technique that is used in the separation and analysis of multi component mixtures such as essential oils, hydrocarbons and solvents. GC MS analysis of ethylacetate of extract Clitoria ternatea were carried out 16 compounds were identified which are depicted in table 4. GC –MS result showed the presence of steroidal and phenolic derivatives and other biologically and pharmacologically active compounds. The compound identified are Benzene 1,3–Bis(1,1-Dimethylethyl) (3.08%), Tetradecane(1.68%), Eicosane (2.87%), 2,4-Ditert-butylphenol-2.74%), Neophytadiene(6.27%), Eicosane (3.06%), Phytol(7.14%), Pentatriacontane (2.05%), Lupeol(5.99%), Tricosane(2.51%), Eicosane(6.53%), Farnesyl bromide(29.77%), Docosane (10.16%), Squalene(5.93%), Tetratetracontane (6.97%). Each of these constituents is responsible for various biological and pharmacological activities.

The pharmacological activities of these compounds present in the leaf can be used to develop a novel drug. Each of these constituents are responsible for various biological and pharmacological activities. The retention time shows high in tetratetracontane (44.122) and low in Benzene 1,3-Bis(1,1 –Dimethylethyl) (12.915).The GC –MS spectrum confirmed the presence of various components with different retention times as illustrated in table :5

Figure 2. GC-MS chromatogram of ethylacetate extract of Clitoria ternatea Linn

Table 5: GC-MS analysis of ethylacetate extract of Clitoria ternatea Linn

Peak #	R.Time	Area	Area%	Height	Height %	Name	Base m/z
1	12.915	615455	3.08	260105	4.63	Benzene 1,3-Bis(1,1 –Dimethylethyl)	175.05
2	13.510	335059	1.68	147173	2.62	Tetradecane	57.05
3	19.002	574419	2.87	233741	4.16	Eicosane	57.05
4	19.575	547726	2.47	202509	3.60	2,4-Ditert-butylphenyl	191.00
5	23.930	651984	3.26	276004	4.91	Eicosane	57.05
6	26.681	1253424	6.27	379258	6.75	Neophytadiene	68.05
7	28.354	611185	3.06	226361	4.03	Eicosane	57.05
8	32.005	1427723	7.14	529348	9.41	Phytol	71.00
9	35.742	409324	2.05	46653	0.83	pentatriacontane	57.05
10	37.848	1196507	5.99	117033	2.08	Lupeol	57.00
11	38.375	501188	2.51	197240	3.51	Tricosane	204.10
12	41.352	1305795	6.53	490654	8.73	Eicosane	57.05
13	42.677	5951551	29.77	840006	14.94	Farnesyl bromide	69.05
14	42.756	2030176	10.16	713793	12.70	Docosane	57.05
15	43.258	1186369	5.93	434447	7.73	Squalene	69.05
16	44.122	1393640	6.97	528157	9.39	Tetratetracontane	57.05

Table 6: Pharmacological activity of phytocomponents in ethylacetate leaf extract of Clitoria ternatea Linn. leaves

Peak No	Retention time	Compound detected	Compound nature	Activity
1	12.915	Benzene 1,Bis(1,1 –Dimethylethyl)	Phenyl propanes	Anti-oxidant Lipid oxidation¹¹
2	13.510	Tetradecane	Alkane hydrocarbon	Anti-fungal activity Biomedical uses¹²
3	19.002	Eicosane	Alkanes hydrocarbon	Anti-fungal activity¹⁷
4	19.575	2,4-Ditert-butylphenyl	Organo-phosphorus compound	Anti-oxidant activity Anti-fungal activity¹²
5	26.681	Neophytadiene	Alkene Diterpene	Anti-microbial activity Anti-ulcerative activity Anti-inflammatory activity Anti-pyretic¹⁴
6	32.005	Phytol	Diterpene alcohol	Anti-microbial Anti-oxidant Anti-inflammatory Anti-nociceptive Anti-asthmatics Anti-malarial activity¹³
7	35.742	pentatriacontane	Long chain hydrocarbon	Anti-bacterial Anti-viral¹⁵
8	37.848	Lupeol	Triterpene	Anti-cancer Anti-tumor Chemopreventive¹³
9	38.375	tricosane	Alkane hydrocarbon	Anti-bacterial¹⁵
10	42.677	Farnesyl bromide	Fattyacids	Anti-oxidant Anti-microbial¹²
11	42.756	docosane	Alkane	Anti-microbial activity¹³
12	43.258	squalene	Triterpene	Anti-inflammatory Anti-cancer Anti-tumor Chemopreventive¹¹
13	44.122	Tetratetracontane	Alkane	Antioxidant Cytoprotective activities¹⁶

CONCLUSION:

In the present study, sixteen chemical constituents have been identified from the ethyl acetate extract of Clitoria ternatea Linn. leaves. The present study revealed that the ethylacetate extract of Clitoria ternatea of GC-MS analysis proves the presence of numerous active phytoconstituents responsible for various pharmacological activities and justifies the medicinal use of this plant in Folklore medicine. The GC-MS analysis showed the presence of steroids, flavonoid and terpenoids etc. The obtained bioactive compounds were identified as potentially active and can be used for the treatment of various diseases and also for the formulation of an anti-inflammatory Gel. The presence of various phytoconstituents helps in future research to explore full pharmacological potential of the plant.

CONFLICTS OF INTEREST:

The authors have no conflict of interest.

ACKNOWLEDGEMENT:

We thank Department of Pharmacognosy and Phytochemistry, Nehru College of Pharmacy, Thiruvilwamala, Thrissur, Kerala and Kerala Forest Research Institute, Peechi, Kerala for helping us to carry out this research.

ABBREVIATION USED:

GC-MS: Gas Chromatography- Mass Spectroscopy, NIST 11: National Institute of Standard and Technology 11, RT: Retention time.

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Received on 23.11.2021 Modified on 10.01.2022

Asian J. Pharm. Ana. 2022; 12(1):49-52.

DOI: 10.52711/2231-5675.2022.00009