Identification and characterization of phytoconstituents of ethanolic root extract of Clitoria ternatea L. utilizing HR-LCMS analysis

Medicinal plants act as a vital source in improving health and overcoming the side effects of modernday medicine. Many evidence-based reports are present in the literature about the benefits of medicinal plants. Clitoria ternatea L. belongs to the family Fabaceae and is known to be one of the important Ayurvedic medicinal plant whose uses are specified mainly for the modification of nervous system activities. ‘Medhyarasayana’ is one of the Ayurvedic formulations which is used to promote the intellectual capacity, revive the body and nervous tissue, Clitoria ternatea serves as a major constituent of ‘Medhyarasayana.’ Identification and characterization of active metabolites of C. ternatea will help to isolate the important phytoconstituents responsible for the central nervous system effects, isolated components can be utilized in future for the formulation of new medicine for various neurodegenerative disorders. In the present study, the phytochemical evaluation of the ethanolic root extract of C. ternatea (EECT) was performed using the HR-LCMS technique. Preliminary qualitative phytoconstituents analysis showed the presence of tannins, alkaloids, saponins, steroids, carbohydrate, protein, flavonoids and triterpenoids in the ethanolic root extract. Almost 42 compounds were identified when the EECT subjected to HR-LCMS analysis.


Introduction
Medicinal plants are considered as amusing resources of ingredients that can be used in drug discovery and development as they are a very vital source to improve health and to overcome adverse effects of allopathic medicine. Many evidence-based reports are present in the literature about the benefits of medicinal plants and their biochemical and molecular effects (1). Worldwide a huge percentage of the population utilize medicinal plants and herbs for their health purpose. Therefore, scientific scrutiny of their phytoconstituents, therapeutic potential, biological properties and safety will be valuable in making wise decisions about their use. (2,3) Ayurveda is one of the most popular Indian traditional health care systems which labels several herbal preparations which are well-known to uphold health and endurance. 'Rasayana' is the common term representing one of such herbal preparations which is ultimate for the progress of tissue functions in addition to their role as micronutrients (4). 'Medhyarasayana' is an Ayurvedic preparation made from the selected plant extracts to revitalize the brain by acting on the nervous system (5).
Clitoria ternatea L. belongs to the family Fabaceae, is a perennial twining herb with terete steam. It possess two varieties-white-flower and blue flower varieties (6). The local name is 'Shankhpushpi' and this is one of the 'Medhyarasayana' ingredients and is reported to promote intellectual capability, revive the body and nervous tissue and because of all these properties it has been widely used as a brain tonic (6). Scientific studies also reported other medicinal properties including antidepressant and anticonvulsant (7), anti-inflammatory, analgesic and antipyretic (8), local anesthetic (9), purgative (10) and anti-diabetic (11) activity. It is also used for the treatment of snakebite and scorpion sting in India (12). In the present study, the phytochemical evaluation of the ethanolic root extract of C. ternatea (EECT) was performed using the HR-LCMS technique.

Collection and Preparation of Clitoria ternatea root extract
Fresh roots of the white variety of wild C. ternatea were collected from Kerala, India. Authentic identification was carried by taxonomist Prof.P.Jayaraman, Director, Plant Anatomy and Research Centre, West Tambaram, Chennai. India. A voucher specimen (SES.CLBM.NO. 1458) has been deposited at the Herbarium of Department of Pharmacognosy, C.L.Baid Metha College of Pharmacy, Chennai, India. The collected materials were shade dried at room temperature to remove moisture, then coarsely powdered by using an electric grinder. The powdered materials were stored in an air-tight container and used for further extraction.

Extraction procedure
Extraction of roots was carried out using ethanol by continuous hot extraction method using Soxhlet apparatus. The obtained extract was concentrated by gentle heating followed by using rotarat vacuum evaporator. The concentrated extract was then weighed, calculated the percentage yield and stored. The extract was subjected to various preliminary phytochemical tests and HR-LCMS analysis (13). The qualitative phytochemical tests were performed for alkaloids, flavonoids, glycosides, phenolics, terpenoids, saponins, carbohydrate, protein, amino acids and triterpenoids (14,15).

High-Resolution Liquid Chromatography and Mass Spectrometry (HR-LCMS) analysis
The HR-LCMS analysis of the extract was carried out in Sophisticated Analytical Instrument Facility (SAIF), IIT Bombay, Mumbai. Methanol was used as the solvent for the preparation of extract and this process was done before subjecting the extract for analysis. Agilent high-resolution liquid chromatography and mass spectrometry model-G6550A (0.01% mass resolution) was used to prepare the chemical fingerprints of the subjected extract. The acquisition method was set to be Mass-minimum range 50 dalton (M/Z) and maximum 1000 Dalton (M/Z). The scanning was done with a rate of each spectrum per second (16).

Identification of components
Interpretation on mass spectrum HR-LCMS was carried out by comparing the spectrum of unknown components with known components spectrum. For comparison, we have utilized the SAIF -IIT Bombay database, where they have been stored more than 62000 patterns of the spectrum. The name, molecular weight and structure of the components of the trial materials were determined.

Results and Discussion
The percentage yield of (EECT) was found to be 10.4%w/w. Preliminary phytochemical evaluation of EECT showed the presence of tannins, alkaloids, saponins, steroids, carbohydrate, protein, flavonoids and triterpenoids (Table 1).
HR-LCMS analysis of EECT showed different major peaks indicating the presence of various phytochemical constituents. The characterization and identification of constituents were done by performing a comparison with the HRLC-MS spectrum of SAIF library compounds. The HR-LCMS study was performed for both positive and negative mode of ionization, the respective chromatogram is represented in Fig. 1 and Fig. 2. The fingerprint obtained was interpreted and mentioned ( Table 2, 3). Positive ionization ESI of EECT showed 24 compounds and negative ionization ESI of EECT showed 18 compounds. The MS zoom spectrum of few important compounds identified by both positive and negative ionization ESI are also represented (Fig. 3,  Fig. 4).
Neuropharmacologic effects of various crude root extract of C. ternatea were reported by different researchers. It was reported that the oral intubation of CT aqueous root extract had shown a significant increase in learning and memory of postnatal and young adult Wistar rats (17). In another study, there are reports the in vitro effects of 200 mg/ml of C. ternatea aqueous root extract on proliferation, differentiation and growth of anterior subventricular zone neural stem cells derived from prenatal and postnatal rat pups (18). Acetylcholine (ACh) and Acetylcholinesterase (AChE) activity modification in connection with memory and cognitive enhancement of laboratory rodents upon administration of various root extracts of C. ternatea was reported by various researchers (19,20). Anti-depressant and anti-anxiety effects of different root extracts have been studied and reported by different scientists (21,22).
Even though the preclinical trial on rodents with various crude root extract of C. ternatea reported promising results on nervous system, a detailed study on isolated compounds from the root was not done so far. The present study imparts light on various constituents in root ethanolic extract. As per the results of the present study, the identified compounds like Chelidonine, Gibberellin, Elephantopin, Deoxy sapponone B 7,3'-dimethoxy ether acetate, 3 hydroxy-3'4'-dimethoxy flavone, Tubernoic acid, Pectolinarin, Isotectorigenin 7-methyl ether, Mucronulatol, Biochanin A dimethyl ether and different amino acids may be responsible for the reported effects produced by the root. For the confirmation, a detailed fractionation and constituent's isolation research study have to be performed on its roots. A welldesigned constituent isolation and preclinical studies with those isolated compounds will confirm the safety and efficacy of C. ternatea against different neurological disorders.

Conclusion
The present study investigated and specified the various active metabolites found in the ethanolic root extract of Clitoria ternatea by carrying out different qualitative phytochemical screening and HR-LCMS analysis. The results serve as a potential resource to explore the isolation, purification and pharmacological screening of       various secondary active metabolites from this traditionally well-known medicinal plant.