Research Journal of Phytochemistry

Year: 2017 | Volume: 11 | Issue: 3 | Page No.: 150-169
DOI: 10.17311/rjphyto.2017.150.169

Metabolite Profiling and Antioxidant Potency of Couroupita guianensis Aubl. Using LC-QTOF-MS Based Metabolomics

Mital Kaneria , Kalpna Rakholiya , Ruchi Jakasania, Rajesh Dave and Sumitra Chanda

Abstract: Background and Objective: Couroupita guianensis Aubl. is found throughout India in plains, is native to South India and Malaysia, which is used in traditional medicine to treat colds, stomach aches, skin diseases, malaria and disinfect wounds. Optimization of the best extraction method for the extraction of antioxidant bioactive metabolite and identification of these metabolites by LC-QToF-MS technique has been carried out from this species. Present study was designed to find out the best method for the extraction of antioxidant compounds from Couroupita guianensis (C. guianensis) and identification of those metabolites. Materials and Methods: The extraction was done by six different methods: Decoction extraction, ethanolic maceration extraction, methanolic maceration extraction, cold percolation extraction, microwave assisted extraction and infusion extraction method. Antioxidant activity and total phenol content were determined in all different extracts of various extraction methods of C. guianensis leaf, stem and flowers. Antioxidant activity was tested by 2, 2-Diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity and ferric reducing antioxidant power. Metabolite profile was carried out by applying non-targeted LC-QToF-MS. Results: The results showed that the extracting solvent significantly altered the antioxidant property estimations of C. guianensis leaf, stem and flowers. The aqueous extract of leaf obtained by cold percolation method had maximum phenol and showed best DPPH free radical scavenging activity and ferric reducing antioxidant power, therefore, it was selected for the further characterization of active metabolites and total 39 compounds were identified. Conclusion: High correlations between phenolic compositions and antioxidant activities of various extracts were observed. Cold percolation extraction method proved to be the best extraction method for the extraction of antioxidant from C. guianensis. Total 39 compounds belongs to different groups were detected and identified from the most potent extract of C. guianensis.

Keywords: extraction optimization, characterization, metabolite profiling, metabolomics, Couroupita guianensis, compounds identification, structure elucidation and LC-QToF-MS

INTRODUCTION

Medicinal plants have been widely used by both ancient and modern man of all cultures for treating different illnesses and for other purposes as well. Plants are a good source of biologically active natural products that are all biodegradable and, more importantly, they are renewable. In recent years, the use of plants based bioactive compounds as a replacement for synthetic drugs has increased. Bioactive plant compounds are preferred over synthetic compounds due to their safety^1-3. Recently, several extraction techniques have been developed to maximize the extraction of such compounds from different plant sources⁴. During the extraction process some variables need to be evaluated including temperature, time and solvent concentration, therefore, an optimization of the technique is essential in order to reach the maximum potential of extraction.

Antioxidant and other health promoting activities of plants are due to the presence of various biologically active compounds. Antioxidants play an important role in preventing the oxidation of food, which is a deterioration process that involves reactions among lipids, vitamins, proteins and sugars with reactive nitrogen and oxygen species^5-7. The ROS are constituted by a large amount of reactive molecules derived from molecular oxygen and free radicals formed in organisms by oxygen consumption, water reduction, lipid oxidation, glycosylation and environmental causes such as smoking and exposition to irradiation and air pollutants⁸. Antioxidant activity of phytoconstituents is mainly due to their redox properties, which play an important role in adsorbing and scavenging free-radicals, quenching oxygen and decomposing peroxides⁹.

Couroupita guianensis Aubl., commonly known as Cannon ball tree, locally known as ‘Kailashpati’ and/or ‘Shivalingi’ is found throughout India in plains, is native to South India and Malaysia¹⁰. The pharmacological functions of Kailashpati include antibacterial^11-13, antimicrobial^14-17, antibiofilm¹⁸, antioxidant^19-22, ovicidal^23,24, larvicidal^25,26, antiulcer^27,28, anti-arthritic and anti-platelet²⁹, antioxidant and antimicrobial^30-32, antidiarrhoeal³³, analgestics³⁴, anti-inflammatory³⁵, antifertility³⁶, anticancer^37-39, neuropharmacological⁴⁰, anxiolytic⁴¹, antiplasmodial⁴², antidepressant^43,44, antinociceptive⁴⁵, immunomodulatory⁴⁶, anti-quorum sensing⁴⁷, antimalarial⁴⁸, wound healing⁴⁹, antioxidant and anticancer⁵⁰, repellency and toxicity⁵¹ and cytotoxicity⁵².

The objective of this study was to develop a rapid, reproducible and simple extraction technique, considering significant bioactive metabolites from the C. guianensis, also determined as complement to the phytochemical characterization, regardless the effect of extraction technique and antioxidant activity by liquid chromatography coupled to quadrupole-time of flight-mass spectrometry (LC-QToF-MS). However, no studies have so far been reported on effects of different extraction techniques on antioxidant activity of C. guianensis with metabolite profiling of potent extract.

MATERIALS AND METHODS

Chemicals and reagents: Petroleum ether, methanol, hydrochloric acid (HCl), Folin-Ciocalteu’s reagent, sodium carbonate, ferric chloride (FeCl₃), ferrous sulfate (FeSO₄), 2, 2-Diphenyl-1-picrylhydrazyl (DPPH), 2,4,6-Tri-(2-pyridyl)-5-triazine (TPTZ), gallic acid and ascorbic acid were obtained from Sigma, Hi-media, Merck and SRL. Water was purified with a Milli-Q system (Millipore, Bedford, USA). All solvents and chemical used were of analytical grade.

Plant collection: The flower, leaf and stem of Couroupita guianensis were collected in August, 2015 from Motibaug, Junagadh, Gujarat, India. The plant parts were washed thoroughly with tap water, shade dried and homogenized to fine powder and stored in air tight bottles.

Extraction procedures: Six different extraction processes were employed in this study, i.e., decoction, ethanolic maceration, methanolic maceration, cold percolation, microwave assisted and infusion extraction method. After extraction, the extract was filtered with eight layers of muslin cloth and centrifuged at 5,000 rpm (Remi Centrifuge, India) for 10 min. The supernatant was collected and the solvent was evaporated using a rotary vacuum evaporator (Equitron, India) to dryness. The extract was stored at 4°C in an airtight bottle.

Decoction extraction method: For the decoction, method was followed as previously used by Li et al.⁵³, 5 g of dried powder was extracted with 100 mL of deionized water at 100°C for 30 min in a water bath.

Ethanolic maceration extraction method: For the ethanolic maceration, method was followed as previously used by An⁵⁴, 5 g of dried powder was extracted with 100 mL of 50% aqueous ethanol at 25°C for 42 h in static condition.

Methanolic maceration extraction method: For methanolic maceration, method was followed as previously used by Cai et al.⁵⁵, 5 g of dried powder was extracted with 100 mL of 80% aqueous methanol at 35°C for 24 h in an incubator.

Cold percolation extraction method: For cold percolation extraction, method was followed as previously used by Parekh and Chanda⁵⁶, 10 g of dried powder was taken in 150 mL petroleum ether in a conical flask, plugged with cotton wool and then kept on a rotary shaker at 120 rpm for 24 h. After 24 h, it was filtrated through eight layers of muslin cloth and the solvent was evaporated from the powder. This dry powder was then taken in 150 mL of deionized water and was kept on a shaker at 120 rpm for 24 h.

Microwave assisted extraction method: For microwave assisted extraction, method was followed as previously used by Jaitak et al.⁵⁷, 1 g of dried powder was extracted with 200 mL of deionized water in a conical flask in a microwave (Magicook 20S (Galaxy), India) at different power levels ranging from 20-160 W with extraction time range between 30 sec to 5 min with a temperature range of 10-90°C.

Infusion extraction method: For infusion extraction, method was followed as previously used by Martins et al.⁵⁸, 2 g of dried powder was extracted with 400 mL of boiling deionized water and were left to stand at room temperature for 5 min.

Quantitative phytochemical analysis by total phenolic content (TPC) estimation: Quantitative phytochemical analysis of the different extracts obtained by different extraction techniques from flower, leaf and stem of C. guianensis, was carried out by the estimation of the TPC by modified Folin-Ciocalteu’s reagent method^59-61. The extract (0.5 mL) and 0.1 mL of Folin-Ciocalteu’s reagent (0.5 N) were mixed and the mixture was incubated at room temperature for 15 min. Then, 2.5 mL of sodium carbonate (2 M) solution was added and further incubated for 30 min at room temperature and the absorbance was measured at 760 nm using a digital spectrophotometer (Systronic 1823, India), against a blank sample. The calibration curve was made by preparing gallic acid (10-100 μg mL^–1) solution in distilled water^62,63. The TPC is expressed in terms of gallic acid equivalent (mg g^–1 of extracted compound).

Antioxidant activity-DPPH free radical scavenging assay: The antioxidant activity of the different extracts obtained by different extraction techniques from flower, leaf and stem of C. guianensis, was measured by using DPPH^. radical scavenging capacity by the modified method of McCune and Johns⁶⁴ and Rakholiya et al.⁶⁵. The reaction mixture (3.0 mL), consisted of 1.0 mL DPPH in methanol (0.3 mM), 1.0 mL methanol and 1.0 mL (100 μg mL^–1) of different extracts diluted by methanol, was incubated for 10 min, in dark, after which the absorbance was measured at 517 nm using a digital spectrophotometer (Systronic 1823, India), against a blank sample. Ascorbic acid (2-16 μg mL^–1) was used as positive control^66,67. Percentage of inhibition was calculated using the following formula⁶⁸:

Ferric reducing antioxidant power: The reducing ability of the different extracts obtained by different extraction techniques from flower, leaf and stem of C. guianensis, was determined by ferric reducing antioxidant power (FRAP) assay of Benzie and Strain⁶⁹ and Kaneria et al.⁷⁰. The FRAP assay is based on the ability of antioxidants to reduce Fe³⁺ to Fe²⁺ in the presence of TPTZ, forming an intense blue Fe²⁺-TPTZ complex with an absorption maximum at 593 nm. This reaction was pH-dependent (optimum pH 3.6). About 0.1 mL of the different solvent extract was added to 3.0 mL FRAP reagent [10 parts 300 mM sodium acetate buffer at pH 3.6, 1 part 10 mM TPTZ in 40 mM HCl and 1 part 20 mM FeCl₃] and the reaction mixture was incubated at 37°C for 10 min. And then, the absorbance was measured at 593 nm using a UV-VIS Spectrophotometer (Shimadzu, Japan), against a blank sample. The calibration curve was made by preparing a FeSO₄ (100-1000 μM mL^–1) solution in distilled water^71,72. The antioxidant capacity based on the ability to reduce ferric ions of sample was calculated from the linear calibration curve and expressed as M FeSO₄ equivalents per gram of extracted compounds⁷³.

Metabolite profiling by LC-QToF-MS technique: Metabolite profiling by liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (LC-QToF-MS) technique was done from Food Testing Laboratory, Department of Biotechnology, Junagadh Agricultural University, Junagadh. Metabolite analysis of C. guianensis leaf aqueous extract obtained by cold percolation method was carried out using an Agilent 6540 LC-QToF-MS system consisting of an Agilent 1290 LC with a 6540 UHD accurate-mass QToF mass spectrometer. Separation of metabolites was performed using (4.6 mm×100 mm, 3.5 μm) Agilent ZORBAX Eclipse XDB-C18 column at 25°C. The mobile phase consisted of 0.1% of formic acid in water (phase A) and acetonitrile (phase B). Gradient elution was as follows: 5% B for 7 min then increased to 95% B up to 12 min, held for 6 min, followed by decrease to 5% B and maintained at 5% B for 7 min. Total run time was 30 min. The applied flow rate was 0.7 mL min^–1 and injection volume was 10.0 μL. MS analysis were carried out using a 6540 Agilent Ultra-High-Definition Accurate-Mass QToF-MS coupled to the LC, equipped with an Agilent Dual Jet Stream electrospray ionization (Dual AJS ESI) interface in negative ionization mode at the following conditions: Drying gas flow (Nitrogen): 8.0 L min^–1, nebulizer pressure: 45 psi, gas drying temperature: 325°C, capillary voltage: 4000 V, mass scan range: 100-1700 m/z and fragmentor voltage: 120 V. Integration and data elaboration were performed using Mass Hunter software (Agilent Technologies, Santa Clara, CA, USA)⁷⁴. Agilent Technologies has provided the METLIN Personal Compound Database with accurate mass MS/MS Library (PCDL). The METLIN PCDL includes all compounds and additionally accurate mass Q-TOF MS/MS library reference spectra.

Statistical analysis: All the experiments were performed in triplicate and results are presented as Mean±SEM (Standard Error of Mean).

RESULTS AND DISCUSSION

Metabolomic approaches allow a comprehensive profiling of the cell metabolome or "Library of metabolites" that provides chemical signatures of cell dynamics and metabolic activity. Various analytical approaches are used to identify metabolites. Metabolic profiling is often referred to as targeted or nontargeted. In the targeted approach, specific metabolites of known identity are profiled. Nontargeted profiling involves the use of NMR or MS for simultaneous measurement of as many metabolites as possible in a biological specimen. The major approaches in metabolomics studies include MS-based techniques. Modern MS platforms such as those that incorporate time of flight mass analyzers offer very high mass resolution and mass accuracy. Coupling such MS instrumentation with high-resolution chromatographic technologies has made it possible to resolve literally thousands of individual small molecules. The high mass accuracy of these methods facilitates peak identification through databases such as METLIN, HMDB and KEGG.

Polyphenols have many favorable effects on human health like inhibiting the oxidation of low-density proteins, thereby decreasing the risk of heart diseases^75,76. They have anti-inflammatory and anti-carcinogenic properties. Flavonoids and many other phenolic compounds of plant origin have also been reported as scavenger of reactive oxygen species (ROS) and are viewed as promising therapeutic drugs for free radical pathogens⁷⁷. Thus, measurements of polyphenols and antioxidant activity in herbs have become important tools to understand the reactive values of plant species⁷⁸ from a health point of view. The total phenol content of different extracts of all extraction methods of flower, leaf and stem of C. guianensis are shown in Table 1. Highest amount of total phenol content was in cold percolation method in leaf, while lowest amount of total phenol content was in microwave assisted method in leaf. Different extracts of flower and stem had almost similar total phenol content (Table 1).

Several methods have been used to measure free radical scavenging capacities of plant. The DPPH radical scavenging activity has been widely used as a model system to investigate the scavenging activity of natural compounds^79-82. Among the various methods to evaluate the radical scavenging activity of natural compounds, DPPH method received more attention due to its fast, reliable results, relatively simple, stable and the DPPH was available commercially in high purity^83,84. The DPPH is a commercial oxidizing radical, which can be reduced/scavenged by antioxidants through the donation of a proton forming the reduced DPPH. The color changes from purple to yellow after reduction, which can be quantified by its decrease of absorbance at wavelength 517 nm^85,86. Radical scavenging activity increases with increasing percentage of the free radical inhibition. The DPPH free radical scavenging activity of different extracts at 100 μg mL^–1 of all extraction methods of flower, leaf and stem of C. guianensis are shown in Table 1. All the different extracts showed varied level of percentage inhibition at a fix concentration of 100 μg mL^–1. Ascorbic acid was used as a positive control and it shows 43.9% inhibition at 10 μg mL^–1 concentration. The highest DPPH free radical scavenging activity was showed by aqueous extract obtained by cold percolation method in leaf followed by maceration ethanol method in flower (Table 1).

The antioxidant activity was also determined on the basis of the ability of antioxidant in the plant extracts to reduce ferric (III) iron to ferrous (II) iron in FRAP reagent⁸⁷. Generally, FRAP assay is used due to its reproducibility. The ferric reducing antioxidant power (FRAP) of different extracts of all extraction methods of flower, leaf and stem of C. guianensis are shown in Table 1. All the different extracts showed varied level of antioxidant potential. Amongst all the extracts, maximum FRAP was in aqueous extract obtained by cold percolation method in leaf followed by aqueous extracts obtained by infusion and decoction methods in flower (Table 1). Meanwhile, FRAP assay was used to determine the antioxidant ability by utilizing the electron-donating capacity of the antioxidant to reduce Fe³⁺ to Fe^{2+ 88}.

In the present study, it was observed that the greatest antioxidant activity, i.e., DPPH and FRAP had a direct correlation with quantities of total phenols (Fig. 1). Phenolic compounds are the main class of natural antioxidants and there is a close relationship between the phenolic content and antioxidant activity of plant extracts^89-92. Several studies have shown that higher antioxidant activity associated with medicinal plants is attributed to their total phenolic compounds^93-95.

The Leaf Cold Percolation Aqueous Extract (LeCpAq) showed significant activity, therefore, it was selected for the further characterization of active metabolites. Metabolite profile of C. guianensis LeCpAq assessed by applying non-targeted LC-QToF-MS using ESI in negative ionization mode is shown in Table 2. Name of the detected and identified compounds, IUPAC name of the compounds, molecular formula, PubChem compound identification number, retention time (t_R), experimental mass (m/z), height of the peak and area covered by the individual peak of about the identified compounds is given in Table 2. Molecular structures of the detected and identified metabolites of C. guianensis LeCpAq by applying non-targeted LC-QToF-MS in negative ionization mode is shown in Fig. 2a-d. The total ion current (TIC) base peak chromatogram (BPC) of C. guianensis leaf aqueous extract obtained in extracted ion chromatogram in negative ionization mode is shown in Fig. 3. Total 39 metabolites were detected in LC-QToF-MS and identified as shown in Table 2 by using Mass Hunter software of Agilent Technologies. From the detected and identified compounds, 14 were belongs to lipid group, 5 flavonoids, 4 glycosides, 3 alkaloids, 2 triterpenes, 2 tripeptides, 2 vitamins, remaining were polycyclic and aromatic compounds.

Moreover, one derivative of fluconazole antifungal drug fluconazole glucuronide was detected as well as, one diterpenoid potent anticancer compound bruceantin was also detected.

Metabolomics is the profiling of the total set of metabolites or low molecular weight biochemical intermediates, resulting from the physiological, developmental or pathological state of a cell, tissue, organ or organism.

Compound identification is a key element in untargeted metabolomics experiments. The level of confidence in the identification is directly dependent on the quality of the database used to assign compound identity. Metabolomics databases used for the accurate identification of the detected compounds were: HMDB, BiGG, PubChem Compound, SYSTOMONAS, LIPID MAPS (LMSD), MetaCyc (MetaCyc is a database of nonredundant, experimentally elucidated metabolic pathways). Encyclopedia of Metabolic Pathways; The Molecular Ancestry Network (MANET: MANET database traces evolution of protein architecture onto biomolecular networks), Metabolite and Tandem MS database (METLIN: The METLIN Metabolite Database is a repository of metabolite information as well as tandem mass spectrometry data)^96-98.

Begum et al.⁹⁹ reported three triterpenes from the n-hexane and carbon tetrachloride soluble fractions of a methanolic extract of the stem bark of the C. guianensis using NMR spectra. Identified compounds were (1) β-amyrin, (2) Betulin-3β-caffeate and (3) Lupeol-3β-caffeate, as structures are shown in Fig. 4a and b. Al-Dhabi et al.¹⁸ reported one compound, Indirubin from the chloroform extract of the fruit of C. guianensis using HPLC-DAD technique, as structure is shown in Fig. 4c. Prabhu and Ravi³⁷ reported two compounds, Stigma sterol and Quercetin from methanol extract of the fresh flowers of C. guianensis using HPTLC, IR, NMR and MS techniques, as structures are shown in Fig. 4d and e. Sukumar and Shakira¹⁰⁰ reported one compound, Quercetin-3-O-rutinoside (rutin) from 85% ethanol fraction of the fresh pinkish white flowers of C. guianensis using NMR, as structure is shown in Fig. 4f. Tayade and Adivarekar¹⁰¹ reported two pigments, namely blue pigment-Indigotin and pink pigment-Indirubin from fruits of C. guianensis using UV, FTIR and NMR techniques, as structures are shown in Fig. 4g and h. The 3D structures of some detected and identified compounds generated using freely available online structure generator-Molinspiration Galaxy 3D Structure Generator v2016.01 beta (http://www. molinspiration.com/cgi-bin/galaxy) from C. guianensis LeCpAq is shown in Fig. 5a and b. According to the results, it can be stated that cold percolation extraction method is an efficient method for the determination antioxidant activity and this method should be considered for extracting higher quality and quantity of antioxidants. Further docking studies on interaction and elucidation of mechanism of antioxidant actions is underway.

CONCLUSION

The results of the present study showed that the cold percolation extraction method was best than the other extraction methods used in the present investigation, maybe by concentrating active principles and by removing interferences to substances of the plant C. guianensis leaves. This could be due to the presence of an enormous amount of the bioactive compounds, which are responsible for the immense antioxidant property, a number of thirty nine were here identified and reported for the first time. The study also revealed the possible antioxidant mechanism of the extracts may be due to hydroxyl groups existing in almost each of the detected compounds that can scavenge the free radicals.

SIGNIFICANCE STATEMENTS

The measurement and interpretation of the endogenous metabolite profile from a biological sample have provided many opportunities to investigate the changes induced by external stimuli or enhance knowledge of inherent biological variation. This article focused on the metabolic profiling of the plant C. guianensis by using LC-QToF-MS technique. Total 39 metabolites were detected and identified belongs to various groups. This study will help the researcher to uncover the positive relation of metabolites and antioxidant activity of C. guianensis.

ACKNOWLEDGMENT

The authors thank to Prof. S.P. Singh, Head, Department of Biosciences (UGC-CAS), Saurashtra University, Rajkot, Gujarat, India for providing necessary facilities and timely supports in order to complete research work.

REFERENCES