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.
GC–MS analysis revealed the presence of thirteen compounds with reported antioxidant activity which must have contributed towards scavenging DPPH. Of these compounds 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid; Hexadecanoic acid, methyl ester; Phytol and Stigmast-5-en-3-ol, (3.beta.)-or ß-sitosterol are present in high quantity. 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; 9,12-Octadecadienoic acid (Z,Z)-, methyl ester; Squalene; and Stigmasta-5,24(28)-dien-3-ol, (3.beta.)- or Fucosterol have moderate quantitative presence while 3,7,11,15-Tetramethyl-2-hexadecen-1-ol; Hexadecanoic acid,2-hydroxy-1-(hydroxymethyl) ethyl ester; Dioctyl phthalate and Ergost-5-en-3-ol, (3.beta.,24R)- are much lower in quantity.
We tried to assess the contribution of detected antioxidant molecules in scavenging DPPH. For this, the antioxidant molecules present in extracts was grouped on the basis of their solubility. In our DPPH scavenging experiment with non-polar to polar solvent extract we had moderate activity in acetone (69.23%) and ethanol (54.22%) but high activity in methanol (84.64%) extract. Compounds like 9,12-Octadecadienoic acid (Z,Z), methyl ester and Hexadecanoic acid, methyl ester are soluble in acetone; 9,12-Octadecadienoic acid (Z,Z)-, methyl ester and Phytol are soluble in ethanol while 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic-acid; Hexadecanoic-acid, methyl-ester; Stigmast-5-en-3-ol, (3.Beta.)-or ß-Sitosterol; Stigmasta-5, 24(28)-dien-3-ol,(3.Beta.)- or Fucosterol; Squalene and Ergost-5-en-3-ol, (3.Beta.,24R)- are soluble in methanol. High DPPH activity of these three organic solvents must be due to synergistic contribution of these compounds in their respective solvents. Significantly compounds like 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid (soluble in ethanol and methanol), Hexadecanoic acid, methyl ester (soluble in acetone and methanol) and Stigmast-5-en-3-ol, (3.beta.)-or sitosterol,.beta (soluble in ethanol and methanol) are present in high quantity, and thus, these three solvent extracts exhibit higher degree of DPPH scavenging activity. So, we may infer that these three compounds—1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid, Hexadecanoic acid, methyl ester and Stigmast-5-en-3-ol, (3.beta.)-or ß-sitosterol, are the prime candidates conferring antioxidant potential to ED. However, we acknowledge the contribution of other antioxidant molecules present in it for providing an overall impact on antioxidant potential.
Antioxidant flavonoids and biomolecules extractable in flavonoid fraction (https://sg.inflibnet.ac.in/jspui/bitstream/10603/148741/11/11_tables) like 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-; 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid; 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (or Phytol); Hexadecanoic acid, methyl ester; 9,12-Octadecadienoic acid (Z,Z)-, methyl ester and Stigmast-5-en-3-ol, (3.beta.)- or ß-sitosterol, while other bioactive flavonoids like 1,2-Benzenedicarboxylic acid, diethyl ester and 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- took main role in quantifying the flavonoids in extract. Diethyl ether extract was quantified to have 4.064 mg QE/g flavonoid. This quantity is due to the presence of Hexadecanoic acid, methyl ester in high amount and 9,12-Octadecadienoic acid (Z,Z)-, methyl ester. Acetone (10.192 mg QE/g) showed higher flavonoid content due to the presence of 1,2-Benzenedicarboxylic acid, diethyl ester over Hexadecanoic acid, methyl ester and 9,12-Octadecadienoic acid (Z,Z)-, methyl ester. Ethanol (18.528 mg QE/g) with the highest quantity of flavonoid dissolves 3,7,11,15-Tetramethyl-2-hexadecen-1-ol or Phytol and 9,12,15-Octadecatrienoic acid, methyl ester, (Z,Z,Z)- in high quantity and 9,12-Octadecadienoic acid (Z,Z)-, methyl ester in moderate amount. Methanol (2.488 mg QE/g) reported the lowest amount of flavonoid despite containing molecules like 4H-Pyran-4-one,2,3-dihydro-3,5-dihydroxy-6-methyl-; Stigmast-5-en-3-ol,(3.beta.)- or ß-sitosterol and Hexadecanoic acid, methyl ester. So, we can infer that besides these antioxidant and other major bioactive molecules, there must be other flavonoids that contribute towards summative effect in respect of its quantity.
Chromatographic separation of methanol extract with solvents and DPPH scavenging ability for each fraction resulted in a major peak with ethanol fraction. During the process of column chromatographic fractionation, as we expected the bioactive compounds must have been separated on the basis of their preferred solvent. Compounds like 9,12-Octadecadienoic acid (Z,Z)-, methyl ester and Phytol are soluble in ethanol. But, 9,12-Octadecadienoic acid (Z,Z)-, methyl ester is also soluble in toluene and diethyl ether, so during our process of fractionation they must have been separated by these two solvents towards the non-polar end and phytol was only left behind and accumulated in ethanol fraction. Phytol in considerable high quantity has expressed its activity as antioxidant and that has been reflected in DPPH scavenging activity. Ethanol/ methanol (1:1) contains 4H-Pyran-4-one, 2,3-dihydro-3,5-dihydroxy-6-methyl-; 1,3,4,5-Tetrahydroxy-cyclohexanecarboxylic acid; Stigmast-5-en-3-ol, (3.Beta.)-or ß-Sitosterol; Stigmasta-5,24(28)-dien-3-ol,(3.Beta.)- or Fucosterol; Squalene; Ergost-5-en-3-ol, (3.Beta.,24R)-, so its DPPH scavenging activity is due to synergistic activity of all these compounds. Hexadecanoic acid, methyl ester though soluble in methanol gets separated by diethyl ether towards the non-polar end. Finally, trace amount of bioactive compounds are left behind to show their potential in following methanol, methanol/water (1:1) and water fractions.
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.
The MVA pathway initiates in the cytosol with A-CoA being the initial metabolite channelled from the MT and successively transformed to 3-Hydroxy-3-Methylglutaryl-CoA (HMG-CoA). It is converted to MVA by the enzyme HMG-CoA reductase (1.1.1.34) [HMGR], an important enzyme regulating the synthesis of MVA and its corresponding downstream metabolites (Nagegowda 2010). MVA is modified to Isopentyl phosphate that serves as the immediate precursor in the synthesis of Isopentyl pyrophosphate (IPP). The MEP/DOXP pathway initiates with glycolytic terminal product pyruvate combining with Erythrose-4-phosphate in the CP, to synthesize 1-Deoxy-D-xylulose-5-phosphate; which through a series of reactions yields (E)-4-Hydroxy-3-methylbut-2-enyl-diphosphate (HMBPP). HMBPP serves as the common precursor in the production of IPP and Dimethylallyl pyrophosphate (DMAPP). IPP and DMAPP are inter-convertible isomers of each other and their metabolic equilibrium is maintained by the enzyme Isopentyl diphosphate isomerase (IDI), an important rate determining enzyme of the terpenoid backbone biosynthesis scheme (Nagegowda 2010). One molecule each of IPP and DMAPP combines with each other to synthesize Geranyl pyrophosphate (GPP), which on addition of another molecule of GPP produces geranyl geranyl pyrophosphate (GGPP). GPP ultimately gives to monoterpenoid metabolite Linalool (C10H18O), a tertiary alcohol which is an octa-1,6-diene substituted by methyl groups at positions 3,7 and a hydroxy group at position 3 while GGPP synthesizes 3,7,11,15-Tetramethyl-2-hexadecen-1-ol or Phytol (C20H40O), an acyclic diterpene alcohol and a constituent of chlorophyll ubiquitous in all members of Plantae in the presence of the enzyme geranyl geranyl diphosphate synthase (GGPS) [EC 2.5.1.29], another rate limiting enzyme of the terpenoid metabologenesis corridor (Nagegowda 2010). Two molecules of IPP and one molecule of DMAPP add up to generate Farnesyl pyrophosphate (FPP). Two molecules of FPP subsequently condense to synthesize Squalene (C30H50). Squalene is a triterpene and is a precursor for synthesis of all plant and animal sterols. Squalene is further converted to (S)-Squalene-2,3-epoxide by the enzyme Squalene monooxygenase (EC 1.14.14.17). This 2,3-Oxidosqualene gives rise to D:A-Friedooleanan-7-ol,(7.Alpha.)- [C30H52O] or Epiputranjivol; and Cycloartenol in two separate reactions. The conversion of (S)-Squalene-2,3-epoxide to Cycloartenol is accomplished in the presence of the biocatalyst Cycloartenol synthase (EC 5.4.99.8). Cycloartenol serves as a common metabolic precursor in the biosynthesis of the three sterolic triterpenoid antioxidants, viz. Fucosterol (C29H48O), β-Sitosterol (C29H50O) and Campesterol (C28H48O), respectively.
HA-methyl ester (C17H34O2) 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 Malonyl-ACP (M-ACP) in the presence of biocatalyst Malonyl transacylase (EC 2.3.1.39), which further reacts with Acetyl-ACP [a modified A-CoA biomolecule aided by the enzyme A-CoA transacylase (EC 2.3.1.38)] 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 (C19H38O4), 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 (C19H34O2), 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 (C7H12O6), 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 (C6H8O4) 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.