Metabolomics of potential contenders conferring antioxidant property to varied polar and non-polar solvent extracts of

Background: The Himalaya hotspot, habitat to numerous indigenous species, harbours an endemic and endangered, cucurbit climber Herpetospermum darjeelingense (C.B.Clarke) H.Schaef. & S.S.Renner [Edgaria darjeelingensis C.B.Clarke]. Seeds of this plant have traditionally been used for ethno-veterinary treatment. The present probe aims to provide an insight into the antioxidant-related biochemical attributes of this phytochemically unexplored plant. Results: DPPH scavenging activity of solvent extracts showed more potential towards the polar end with its maximum activity in methanolic extract (84.64%). Acetone extract showed more ferric reducing power than the ethanol fractions. Maximum flavonoid quantification was recorded in ethanol extract. GC–MS of methanolic extract detected 39 compounds of which 22 have preventive and curing properties for several ailments. Thirteen compounds were reported with antioxidant activity and cover an area of 63.92% among the total detected compounds by GC–MS. 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid; Hexadecanoic acid, methyl ester; ß sitosterol and Phytol are present in significant quantity and are the prime candidates conferring antioxidant potential. Chromatographically fractionalized methanol extract with non-polar to polar solvents and their subsequent DPPH scavenging analysis exhibited a major peak in ethanol fraction due to the presence of Phytol in considerable high quantity and some other compounds. Further sub-fractionation of ethanolic fraction provides a supportive evidence of ethanol solubility of the compounds. Biosynthetic lineage reveals fatty acid synthesis, intermediates of Shikimate pathway and terpenoid metabologenesis schemes lead to the several antioxidant molecules detected in our extract. Conclusion: Metabolomics of antioxidants will provide a better understanding in its intracellular free radical quenching activity and isolation of these bioactive compounds can boost pharmaceutical industry.


Background
India is a mega-diversity nation owing to its rich floral and faunal variations. It harbours several hotspots of which the Himalaya Hotspot is habitat to numerous endemic and indigenous species. The Eastern Himalaya including Darjeeling hills (average 6700 ft) shares boundaries with Eastern Nepal in the west and Bhutan and Sikkim in the North East and thus can be considered as a typical geographical terrain assembling and uniting the various endemic as well as exotic flora of India, Bhutan and Nepal. Out of the various endemic species and among the 11.61% indigenous climbers of the Darjeeling Eastern Himalayas (https ://shodh ganga .infli bnet.ac.in/bitst ream/10603 /14712 3/14/14_chapt er_09); a very phytochemically active but biochemically under investigated endemic Cucurbit climber is Edgaria darjeelingensis C.B.Clarke [Herpetospermum darjeelingense (C.B.Clarke) H. Schaef. & S.S. Renner]. The plant is not only endemic but is also enlisted under the endangered category confronting a very high risk of extinction in the wild in the near future as per scientific records. The angiospermic family Cucurbitaceae ranks second (only next to Papilionaceae) among the climber dominating families in the Darjeeling hills of Eastern Himalaya with about twenty-seven reported species (https ://shodh ganga .infli bnet.ac.in/bitst ream/10603 /14712 3/14/14_chapt er_09).
The referred plant occurs at an altitude of 1450-3000 m and is a climber characterized by axillary shoot tendril where axillary buds are modified into tendrils for climbing purposes. E. darjeelingensis is prevalently known as Cathil in the Chitwan district of Nepal and its fruits are economically useful as a source of vegetable to the local people specifically the ancient tribal ethnic group 'Tharu' (Mueller-Boeker 1993). The 'Tamang' community in Central Nepal and Darjeeling Eastern Himalayas also employs E. darjeelingensis in herbal veterinary medications where it is commonly known as 'Tangsarkato' among the Tamang's and 'Jangali Karela' among the local Nepali inhabitants. The seeds of the plant are mentioned to be used for bovine treatment as per the ethno medicinal records of the 'Tamang' community. The methodology involves the mixing of the pounded seeds with corn flour and is fed to the cattle for relief from fever (Shrestha and Khadgi 2019). Thus, E. darjeelingensis is an endemic, endangered and ethno-medicinally important climber Cucurbit of Darjeeling Eastern Himalaya hotspot that has remained neglected from the biochemical and molecular characterizations till date. The present probe aims to explore and unveil the various antioxidant-related phytochemical attributes of the under explored Cucurbit climber.

Collection of plant material
Plant materials [leaves of E.darjeelingensis (ED)] were collected from the junction of Lebong Cart Road and Happy Valley Tea Estate, Darjeeling (27.050904°N and 88.260809°E). The plant was field identified by Senior Taxonomist of Darjeeling Government College, Darjeeling. Since this plant is an endangered species, utmost care was taken during sample collection so as to cause minimal injury to the plant body. Vegetative leaves were detached from mature shoots, filled in zipper bags and preserved in insulated ice packed boxes for transference to laboratory.

Sample extracts preparation
ED leaves were washed thoroughly in running tap water to remove any adherent dust particles followed by final cleansing with distilled water. Wet leaves were then surface-dried using tissue paper and mechanically grinded to obtain a powdery mass. 3 g of powdered leaf sample was measured in a digital balance and poured in each of the reaction tubes containing a range of non-polar to polar solvents (hexane, toluene, chloroform, diethyl ether, ethyl acetate, acetone, ethanol, methanol and water). Reaction mixtures were allowed to stand for 48 h at room temperature after which the extracts were filtered through ash-less filter paper. Next the respective solvents in the reaction tubes were made to completely dry out at a mild temperature and finally the remnants were dissolved in methanol adjusting the concentration to 0.5 g/ml. For GC-MS analysis, the methanolic extract was adjusted at a concentration of 25 mg/ml.

Assay of antioxidant activity DPPH induced free radical scavenging assay
The protocol of Bhattacharya et al. (2009) was followed for analysing free radical scavenging activity through DPPH assay technique. 0.3 ml ED leaf extract was added to 2 ml methanolic solution of 0.2 mM DPPH (2,2-diphenyl-1-picrylhydrazyl). The reaction mixture was vortexed vigorously and incubated at room temperature for 30 minutes in dark. Absorbance was measured at 517 nm using UV-Vis spectrophotometer and ascorbic acid (µg/ ml methanol) was taken as standard. DPPH scavenging percentage was calculated as follows: where A Control = absorbance value of Control and A Sample = Absorbance value of Sample. Ascorbic acid equivalent of potential DPPH scavenger was calculated from the standard curve.
The study of interaction and DPPH inhibition kinetics of ED leaf extracts in acetone, ethanol and methanol were conducted at 517 nm. 0.3 ml ED leaf extract was added to 2 ml methanolic solution of 0.2 mM DPPH in a quartz cuvette and placed in UV-Vis spectrophotometer for 30 minutes. Absorbance kinetics obtained from the graph was used to determine the time required for 50% scavenging activity.

Flavonoid quantification
To 250 µl of ED leaf extract in a test tube, 1.25 ml double distilled water and 75 µl 5%NaNO 2 (sodium nitrite) were added and allowed to stand at room temperature for 5 minutes. Then, 150 µl 10%AlCl 3 (Aluminium chloride) was added to the reaction mixture and kept stationary for 6 minutes at room temperature. After that 500 µl of 1 mM sodium hydroxide (NaOH) and 275 µl of double distilled water were subsequently added and the solution was incubated for 30 minutes. Finally, the absorbance was measured at 510 nm utilizing UV-Vis spectrophotometer (Zou et al. 2004).

Gas chromatography-mass spectrometry analysis
GC-MS analysis was conducted following the protocol of Majumder et al. (2020). Methanol extract of ED (25 mg/ ml) was used for GC-MS. 1 µl of extract was injected in split mode in the instrument (GCMS-QP2010 Plus). Injection temperature (260 °C) and interface temperature (270 °C) were maintained. Ion Source temperature was adjusted to 230 °C. Helium was used as carrier gas. Total flow rate was 16.3 ml min −1 and column flow rate was 1.21 ml min −1 . Mass spectra were recorded at 5 scan sec −1 with a scanning range of 40-650 m/z. Quantification of compounds was done on the basis of their peak areas. The data obtained from GCMS analysis were further analysed through the available literature.

Chromatographic separation and DPPH scavenging assay
Bioactive molecules conferring antioxidant potential differ by their preference for solvent. So, the extract in methanol was fractionized by solvents like toluene, chloroform, diethyl ether, ethanol, ethanol/methanol (1:1), methanol, methanol/water (1:1) and water to obtain their respective fractions. A part of the ethanol fraction was further fractionated by ethanol in a silica column and were collected as 10 ml fractions. DPPH scavenging ability for each fraction was conducted and calculations were made in accordance with previously described protocol (Bhattacharya et al. 2009).

Statistical analysis
Statistical analysis, viz. mean and standard deviation, was calculated on the basis of results obtained from three replications. Ascorbic acid (µg AE/ml) and quercetin (mg QE/g) equivalent were calculated based on the mean value. Standard error bars are provided in graphical representation of data.

Antioxidant activity assay DPPH induced free radical scavenging assay
Non-polar to polar solvent extracts of ED showed variable ability to scavenge DPPH exhibiting an ample range of free radical scavenging activity (Fig. 1a). Ascorbic acid equivalent (AE) calculated from the standard curve, y = 0.876x + 6.591 (R 2 = 0.993) for DPPH scavenging activity of methanol extract, was found to be 89.005 µg AE/ml. Water extract was divided into two parts-methanol soluble (Water-M) and water soluble (Water-W) parts. Part that was insoluble in methanol but soluble in water (Water-W) exhibited more DPPH scavenging property. Solvent extracts of hexane, toluene, chloroform, diethyl ether and ethyl acetate showed DPPH scavenging activity below 40%. Extracts with acetone and ethanol exhibited moderate activity while methanol showed maximum potential in scavenging DPPH. Interaction and DPPH inhibition kinetics of ED leaf extracts in acetone, ethanol and methanol are depicted in Fig. 1b-d. Graphical representation of absorbance kinetics in acetone extract portrayed consecutive rise and fall in DPPH scavenging activity while a steady increase in DPPH quenching activity was recorded in ethanol and methanol extracts, respectively. The time taken for 50% scavenging activity was minimum in acetone extract (2.9minute) followed by ethanol (5.2minute) and methanol (7.5minute).

200
mg/ml-4.2604 ± 0.539261 and 300 mg/ ml-3.3941 ± 0.274685) and then gradually declined in activity (Fig. 2). However, in all the concentrations acetone extract exhibited more FRP than the ethanolic fractions. The ferric reducing power portrayed was almost at par with the ferric reducing power of ascorbic acid used as standard.

Quantification of flavonoids
Flavonoids belong to a group of natural biomolecules with variable phenolic structures available in almost  all plant parts (Middleton 1998). They confer antioxidant property, so flavonoids present in diethyl ether, acetone, ethanol and methanol extracts were quantified and expressed as quercetin equivalent (mg QE/g). Variable quantities of flavonoids were detected in the solvent extracts ( Fig. 3) with mean maximum values recorded in the ethanolic extract (18.528 ± 0.486124 mg QE/g).

Chromatographic separation for confirmation
Preference of solvents for molecules is not same. So, initiative was made to chromatographically fractionalize the methanol extract with solvents like toluene, chloroform, diethyl ether, ethanol, ethanol/methanol (1:1), methanol, methanol/water (1:1) and water to execute DPPH scavenging ability for each fraction. The DPPH scavenging activity of solvent fractions showed variable results and is represented in Fig. 7a. The graph showed a major peak in DPPH scavenging activity with ethanol fraction (84.99%). Further sub-fractionation of ethanol fraction and collecting the sub-fractions in recurring 10 ml volume was conducted to confirm ethanol solubility of the antioxidant molecules described previously. The results of DPPH scavenging activity of the fractions collected as recurring 10 ml volume are depicted in Fig. 7b. The graph is a clear indicative of the fact that the molecules described earlier as ethanol soluble are confirmed as truly and highly soluble in ethanol as they were extracted in the first 10 ml volume of the chromatographic fraction.

Biosynthesis pathway of potential antioxidant compounds
We consider four compounds namely-1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid (10.8%); Hexadecanoic acid, methyl ester (9.47%); Phytol (19.24%); and Stigmast-5-en-3-ol,(3.beta.)-or ß-sitosterol (12.96%), present in considerably higher quantities as the chief candidates for conferring antioxidant property to the plant extract, though rest compounds also have their role in enhancing the free radical quenching activity, and altogether a cumulative effect may be concluded in respect of the remarkable antioxidant potential of ED. The pathway trends of all these potent radical quenching compounds would decipher important information regarding their site of cellular origin and molecular chemistry.
Lastly, Dioctyl phthalate [a phthalate ester(1%)], though have been described as an antioxidant (Gunalan et al. 2014), has been excluded from any further analysis due to its uncertain origin and environmental effects (Reid et al. 2007).

Discussion
Reactive oxygen species and antioxidants have multifold roles in the life of an organism. Most of the diseases and disorders are attributed to the asymmetry in pro-oxidation and anti-oxidation reactions occurring in an organism (Bhattacharya and Chakraborty 2015). DPPH assay primarily explores the capability of plant extracts to scavenge or neutralize free radicals and thus, is considered a major experimental indicator of antioxidant activity of the extract. The percentage of recorded DPPH activity in solvent extracts showed an increase towards the polar end as the antioxidant molecules are more soluble to polar solvents. Ferric reducing power assay of acetone and ethanol reveals acetone extract to be more capable over ethanol extract. A similar type of result was observed by Labar et al. (2019) in extracts made from fresh green leaves of Camellia sinensis. Interaction and DPPH inhibition kinetics of ED extracts of acetone, ethanol and methanol further confirm DPPH scavenging potential of methanol extract. The contributor for antioxidant activity of ED was unearthed by GC-MS analysis.
The antioxidant pathway analysis deduced three separate metabologenesis schemes to explain the origin of these varied antioxidant molecules in ED. Figure 8 represents the summative pathway diagram representing the biosynthesis of these antioxidant compounds. Terpenoid molecules originate via the MEP/DOXP or MVA pathway; FA and their derivatives are anabolized through the FA biosynthesis pathway and Phenolic counterparts are synthesized by the SP.
HA-methyl ester (C 17 H 34 O 2 ) commonly known as methyl palmitate belongs to the class of organic compounds called fatty acid methyl ester. This saturated fatty acid is commonly found in plants, animals and microorganisms. Synthesis of HA initiates in the CP with A-CoA as raw material being imported from the Tri-Carboxylic acid (TCA) cycle occurring in MT and proceeds through the FA biosynthesis pathway with Malonyl-CoA (M-CoA) as the first synthesized metabolic intermediate; by the enzyme A-CoA carboxylase (EC 6.4.1.2) attributed to be main enzyme controlling the fatty acid metabologenesis scheme (Ohlrogge and Jaworski 1997). The M-CoA produced is transformed to  ] to generate Acetoacetyl-ACP (Ac-Ac-ACP). The Ac-Ac-ACP is subsequently converted to D-3-Hydroxybutyryl-ACP (D-3-H-ACP) by the enzyme β-Ketoacyl-ACP reductase (EC 1.1.1.100). This reaction is followed by the successive conversion of D-3-H-ACP to Crotonyl-ACP in the presence of biocatalyst 3-Hydroxyacyl-ACP dehydratase (EC 4.2.1.59) which is finally transformed to terminal metabolite Butyryl-ACP by enzyme Enoyl-ACP reductase [EC 1.3.1.10]. This reaction cycle is being repeated with M-ACP adding 2-Carbon units in every subsequent round until the formation of P-ACP (16-Carbon compound) followed by a terminal hydrolysis reaction aided by an enzyme Thioesterase (EC 3.1.2.14) to ultimately yield HA. 2-hydroxy-1-(hydroxymethyl)-ethyl-ester commonly regarded as 2-palmitoyl glycerol (C 19 H 38 O 4 ), too is biosynthesized/derived from HA. It is a β-monoacyl glycerol and a 2-monoglyceride where the acyl group is hexadecanoyl (palmitoyl). Further, addition of 2-Carbon M-CoA to HA produces Octadecanoyl-CoA/Stearic acid in SER, which post twice successive rounds of desaturation synthesize Linoleic acid, an essential FA, identified as a methyl esterified derivative in ED owing to methanolic leaf extract being used for GC-MS analysis. This metabolite Methyl linolenate or 9,12-Octadecadienoic acid(Z,Z)-, methyl ester (C 19 H 34 O 2 ), is an ester of omega-6 polyunsaturated fatty acid (PUFA), attributed to be a powerful antioxidant molecule. 1,3,4,5-Tetrahydroxy-cyclohexane-carboxylic acid (C 7 H 12 O 6 ), commonly known as Quinic acid or Kinic acid, is a conjugate acid of quinate. Chemically, it is a cyclitol, a cyclic polyol, and a cyclohexanecarboxylic acid synthesized by the SP occurring in the CP. Even 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-or Pyranone (C 6 H 8 O 4 ) too follow the footsteps of the same SP and is synthesized as a subsidiary product in the synthesis of simple phenolics and flavone moieties in ED. The initial precursors involved in SP, i.e. PEP and E-4-P, reacts to generate 3-Deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) aided by the enzyme DAHP synthase. DAHP synthase (EC 2.5.1.54), a transferase class enzyme, may be attributed to be the chief initial biocatalyst controlling the metabolic outputs of the SP, as it regulates the gateway leading to synthesis of the first Shikimate scheme intermediate DAHP (Tzin et al. 2012). This DAHP is further converted to 3-Dehydroquinate (3-DHQ) in the presence of EC 4.2.3.4 (3-DHQ synthase) which post a dehydrogenation reaction (probable enzyme EC 4.2.1.10/3-DHQ dehydratase) yields Quinic acid.
The above-described independent pathways operate parallelly and cumulatively affecting the metabologenesis of these varied classes of antioxidant metabolites which confers this remarkable free radical scavenging potential to ED plant extract.

Conclusion
Antioxidants have diverse role to play in the life of an organism as major diseases and disorders are mainly attributed to the imbalance between pro-oxidation and antioxidation reactions occurring in the living system. Edgaria darjeelingensis is a treasure of antioxidant molecules of which some of them are in significant quantities. Isolation of these molecules for pharmaceutical industry can be a boon for mankind.