Antioxidant and Antivenom Potential of an Essential Oil, 4-(2-Oxo-propyl)-cyclopentane-1,3-dione, and Allantoin Derived from the Polyherbal Combination of Aristolochia indica L. and Piper nigrum L.

The goal of this study was to identify new compounds from a methanol extract of a polyherbal combination of Aristolochia indica L. and Piper nigrum L. (MECAIPN), two traditional medicinal plants used to cure envenomation, as well as to assess their antioxidant and antivenom properties. MECAIPN yielded EA1 (an essential oil), AA2 (4-(2-oxo-propyl)-cyclopentane-1,3-dione), and W3 ((2,5-dioxo-imidazolidin-4-yl)-urea) (Allantoin). Although EA1 had stronger radical scavenging activity, AA2 had higher DPPH and ferric ion radical scavenging activity, and W3 had higher molybdenum ion radical scavenging activity due to being a single molecule, the binding investigation revealed that EA1 has a greater Stern–Volmer quenching constant (Ksv) than AA2 and W3. Synchronous measurements indicated that EA1, AA2, and W3 bind to tryptophan and tyrosine residues in venom, causing denaturation of the secondary structure of the residue. Finally, the current study concludes that EA1 has more therapeutic antivenom potential, which could be related to the synergism of chemicals found in it. When it came to single compounds, AA2 had stronger antioxidant and antivenom capabilities than W3. To understand the mechanism of action and manufacture the green antivenom medication, more testing of the EA1 and compounds remains required.


Introduction
e genus "Scolopendra" is a venomous terrestrial arthropod whose envenomation has a negative impact on human health due to hypersensitivity. Pain, giddiness, sudden myocardial infarction, and skin-related disorders such as itching, erythema, blemishes, necrosis, aureole, and wound development are all symptoms of hypersensitive reactions. Metalloproteases, hyaluronidase, and phospholipase A2 are among the enzymes in the venom, as are CAP protein, ion channel modulators, cardiotoxin, myotoxin, and cytotoxin, among others. Envenomation also induces oxidative stress by promoting the production of damaging free radicals and damaging macromolecules (lipids, proteins, and nucleic acids). Many illnesses, including as inflammation, cancer, metabolic problems, and aging, are caused by changes in macromolecule structure and function [1]. e medicinal plant is used in the Indian traditional medical systems "Ayurveda and Siddha," which has the capacity to heal lifethreatening disorders. Both systems favor "polyherbal formulation" over uniherbal medication to gain additional therapeutic efficiency through the synergism process [2,3]. Oraon tribes of Latehar district, Jharkhand, employ the medicinal plants A. indica (root) and P. nigrum (seed) to cure envenomation [1,4]. e Malayali tribes of the Yercaud hills ingest A. indica root orally for 1 week to cure centipede envenomation [5]. Chittoor district (Andhra Pradesh) uses P. nigrum seeds to cure toxic bites [6]. In this regard, a previous study (published in Journal of Biomedicine and Pharmacotherapy) found that a methanol extract of a combination of A. indica and P. nigrum (MECAIPN) was more effective than individual plant extracts and the standard drug "Medrol" against Scolopendra morsitans L. envenomation effects [1]. As a result, the current study aims to isolate new compounds from MECAIPN and assess their antioxidant and antivenom properties.

Collection of Venom.
Scolopendra morsitans was collected in July, Dharapuram district, Tamil Nadu, India, and recognized as S. morsitans L. by the Zoological Survey of India (ZSI), Kerala (Reg. no. ZSI/WGRC/IR/INV/ 4152). e centipedes were kept in containers with air holes, soil, and litter items to keep them alive. Spraying water on the soil kept the moisture content up. e termites were offered as a source of nutrition. Venom was collected by activating the venom glands with an electrical device that had a frequency of 128 Hz, a voltage of 10 to 20 V, and a pulse width of 2 to 4 ms. Each milking occurred 2 weeks after the previous milking. e collected venom was stored at −20°C [7].

Column Chromatography and Characterization.
Based on earlier research, MECAIPN was chosen for chemical isolation. MECAIPN (50 g) was adsorbed on activated silica (60-120 mesh) while the methanol was continuously stirred. e column (59 × 4.5 cm) was packed with activated silica gel (60-120 mesh) in petroleum ether (stationary phase) using the wet packing method, and it was eluted with 600 mL of mobile phase containing various solvents such as petroleum ether (100%), petroleum ether (A): chloroform (B) (5-100%), chloroform (A): ethyl acetate (B) (5-100%), ethyl acetate (A): methanol (B) (5-100%) [in order to improve the polarity of the mobile phase, the concentration of "A" was reduced from 95, 90, 85, and 80 to 0%, and the concentration of "B" was raised by 5, 10, 15, 20, 25 up to 100%], acetic acid (1 and 2% in distilled water), and distilled water (100%). e elution volume was 10 mL and the flow rate was 3 mL/min. On a precoated silica gel 60 F254 TLC ( in layer chromatography) plate, TLC profiling was performed (Merck, Germany). In a saturated chromatographic chamber, the chromatogram was created. e produced plate was examined in an ultraviolet (UV) and iodine chamber. e formula (R f � distance travelled by the solute/distance travelled by the solvent) was used to get the R f value of the location. e elution fractions with comparable chemical signatures were grouped together based on TLC findings. From the 4 : 6 (ethyl acetate : methanol) elution, the oil EA1 (4 mL) was recovered. From 1% acetic acid in water and 100% water, the colorless crystals AA2 (4.65 g) (water : methanol : acetic acid in 8 : 1 : 1; R f : 0.85) and W3 (1.32 g) (water : acetic acid in 9.9 : 0.1; R f : 0.11) were produced [8].

Determination of Free Fatty Acid Content and an Acid
Value of EA1. One milliliter of EA1 was combined with a neutral solvent (25 mL ether, 25 mL 95% ethanol, and 1% phenolphthalein solution) and titrated against 0.1 N potassium hydroxide for 15 sec until the pink color remained persistent. Using equation (1) mL N/10 KOH (potassium hydroxide) � 0.028 g oleic acid, the free fatty acid was estimated as oleic acid [9 e acid value is calculated using the formula, Acid value (mg KOH/mL) � T × N × MW/Volume of EA1 (mL) Where, T � Titre value, N � Normality of KOH, and MW � Molecular weight of KOH.

Determination of Saponification Value of EA1.
In a flask, 2 mL of EA1 was weighed. e alcoholic KOH (25 mL) was pipetted into the mixture and allowed to drain for approximately 1 min. A blank determination was made and measured at the same time as the sample. For full saponification, a condenser was attached to the flask, and the combined sample was allowed to boil gently and continuously for 45 min. ereafter the flask and condenser were cooled, but not enough to form a gel. After disconnecting the condenser, 1 mL of phenolphthalein indicator was added to the flask's contents. With 0.5 N HCL (hydrogen chloride), the solution was titrated until the pink color faded completely [10]. Saponification value was determined using the formula saponification value � (B -S) × 56.1 × N/weight of EA1 (g), where B is the blank titre value, S is the sample titre value, and N is the HCL normality.

Determination of Molecular
Weight of EA1. From saponification and an acid value, the molecular weight [11] of EA1 was determined as MW � 168300/SV AV, where MW represents the oil's molecular weight, SV represents the saponification value, and AV represents the acid value.

Gas Chromatography-Mass Spectrum (GC-MS) Analysis of EA1.
Shimadzu GC-MS, QP2010S, was used for the GC-MS analysis. Compound separations were carried out on an Rxi-5Sil MS column with a length of 30 m, a diameter of 0.25 mm, and a thickness of 0.25 µm. e chromatographic and mass spectrometric data were processed using GC-MS Solutions software. e temperature in the GC oven was kept at 700°C for 5 min, then steadily increased to 2900°C min −1 at a rate of 50°C min −1 for another 5 min. e oil sample was divided at a 1 : 16 splitting ratio and injected in the split mode. As a carrier gas, helium was employed at a constant flow rate of 1 mL min −1 . e electron impact mode of the mass selective detector was used, with an electron energy of 70 eV. e obtained GC-MS profile was examined by comparing the chromatogram to commercially available standards using the NIST (National Institute of Standards and Technology) 11 and WILEY 8 libraries [12].

Fourier Transform Infrared Spectroscopy (FT-IR)
Analysis. To make transparent sample discs, EA1 (0.5 mL) and 10 mg of AA2 and W3 crystal powder were encapsulated in 100 mg of KBr pellet. To identify the distinctive peaks and their functional groups, the powdered sample was put into an FT-IR (Fourier transform infrared) spectroscopy (Perkin Elmer Spectrum GX) in the (4000-400) cm −1 range. e FTpeak IR's values were recorded. For spectrum confirmation, the procedure was done twice [13].

Nuclear Magnetic Resonance Spectroscopy (NMR).
e signal processing information base was used to determine the 1 H-NMR and 13 C-NMR spectra. At room temperature, 1 H (−2-15 ppm) and 13 C (0-220 ppm) NMR were recorded on a Bruker DPX 400 spectrometer (ppm, J in Hz) running at 400 MHz 1 H (−2-15 ppm) and 13 C (0-220 ppm). Chemical changes were measured in parts per million (ppm). e internal standard was tetramethylsilane (TMS). Ten milligrams of compounds were dissolved in a maximum of 2 mL of DMSO, which was chosen as the solvent of choice due to the compounds' solubility. Clean Pasteur pipettes were used to pipette the impurity-free solutions into NMR tubes for examination.

Energy Dispersive X-Ray Analysis (EDX).
e EDX was used to determine the elemental makeup of the AA2 and W3 (Brukers). Gold sputtering was used to cover the compounds prior to analysis. Low electron energies of 1 keV to 10 keV were used to capture secondary electron pictures.

Single-Crystal X-Ray Diffraction Analysis (Sc-XRD).
e X-ray crystallographic investigation was performed on a clear vivid white columnar-like specimen with approximate dimensions of 0.165 mm × 0.201 mm × 0.365 mm (AA2). e X-ray intensity measurements were collected in a Bruker D8 Quest over a 3:04-h room temperature exposure. A narrowframe method was used to merge the frames using the Bruker Saint Software package. e multi-scan approach was used to adjust for absorption effects in the data (SADABS). e solvent Apex II program was used to solve the structure. Table 1 shows the data gathering and refining procedure for AA2. For the X-ray crystallographic study, a clear bright white columnar-like specimen with approximate dimensions of 0.558 × 0.168 × 0.030 mm 3 (W3) was placed on a Mitgencryoloop in random orientation (Bruker AXS SMART APEX CCD). Table 2 shows the specifics of W3's data collecting and refining procedures.

In Vitro Antioxidant Assay
2.13.1. DPPH • Scavenging Activity. Approximately 3 mL of 0.1 mM methanolic DPPH solution was added to the aliquots of the samples and standards (BHT) and vigorously shaken. e negative control was made by mixing 100 µL methanol with 3 mL of 0.1 mM DPPH methanol solution. At 27°C, the tubes were left to stand for 30 min. e sample's absorbance was measured at 517 nm and represented as an IC 50 , which was the sample concentration necessary to inhibit 50% of DPPH concentration [8].

Ferric Reducing Antioxidant Power Assay.
A quantity of 2.5 mL of 10 mM TPTZ in 40 mM HCL, 2.5 mL of 20 mM FeCl 3 .6H 2 O, and 25 mL of 0.3 M acetate buffer were combined to make the FRAP reagent (pH 3.6). e FRAP reagent (900 µL) was combined with 270 µL of distilled water and 100 µL of test material after being newly produced and incubated at 37°C. e test sample was diluted to 1/34 in the reaction mixture at the end. In a water bath, the test samples and reagent blank were incubated for 30 min at 37°C. A spectrophotometer was used to measure the absorbance at 593 nm against the reagent blank at the conclusion of the incubation. As a baseline, BHT was employed. For the calculations, a methanolic solution with known Fe(II) concentrations ranging from 500 to 4000 mM (FeSO 4 ·7H 2 O) was used.
e equivalent concentration was estimated as the antioxidant concentration that increased the absorbance in the FRAP experiment to the predicted absorbance value of 1 mM Fe(II) solution [8].

Phosphomolybdenum Activity.
In a test tube, triplicates of 100 µL of samples were mixed with 3 mL of reagent solution (0.6 M sulphuric acid, 28 mM sodium phosphate, Evidence-Based Complementary and Alternative Medicine 3 and 4 mM ammonium molybdate) and varied quantities of standard (ascorbic acid in 1 mM dimethyl sulphoxide). e test tubes were incubated for 90 min in a water bath at 95°C. e absorbance of the combination was measured at 695 nm against the reagent blank after the samples had cooled to room temperature. A typical blank solution had 3 mL of reagent solution and water in the place of the sample, and it was incubated at the same temperature as the other samples. As a control, the synthetic antioxidant BHT was utilized. e findings were given as mean values in mg of ascorbic acid equivalents (AAE) per gram of the sample [8].

Venom Protein Binding Studies.
e venom was dissolved in phosphate buffer (µL/mL) (pH 7.2). For the venom protein binding investigation, the excitation wavelength of venom was measured at 280 nm and the emission wavelength was measured at 346 nm. For all of the trials, the excitation and emission slit widths and scan speeds were kept constant. For all of the studies, a concentrated stock solution of the samples was generated by dissolving the relevant materials in DMSO, phosphate buffer, and diluting appropriately with deionized water to the needed concentrations (percentage of DMSO (v/v) in the final solution). Titrations were carried out manually with the addition of the samples (10-100 µM) using a micropipette. e same concentration of venom and samples was utilized for synchronous fluorescence spectra, and the spectra were taken at two distinct Δλ values (difference between the excitation and emission wavelengths of venom), such as 15 and 60 nm. A 1 cm quartz cell on a JASCO FP 6600 spectrofluorimeter [14] was used for fluorescence and synchronous measurements. e quenching ability was evaluated by the Stern-Volmer equation: where I o is the emission intensity in the absence of samples, I corr is the corrected emission intensity in the presence of the   In order to correct the inner filter effect, the following equation is used [15]: where I corr is the corrected fluorescence value, I obs is the measured fluorescence value, A exc is the absorption value at the excitation wavelength, and A em is the absorption value at the emission wavelength. e binding constant (K b ) and the number of binding sites (n) may be calculated using the static quenching interaction, assuming comparable and independent binding sites are considered in the biomolecule [16]. e Scatchard equation may be used to solve this problem, which is as follows: e number of binding sites per molecule (venom) can be determined by the slope and intercept of a double log- where K b is the binding constant for the compound-venom protein interaction and "n" is the number of binding sites per molecule (venom). At room temperature, the values of "n" are roughly equal to 1, indicating that there was just one binding site in the venom for the samples.

Statistical Analysis.
Data expressed as mean ± standard deviation were statistically analyzed using SPSS 20.0 by means of one-way ANOVA followed by Duncan's test, significant when p < 0.05.  (Figure 1(a)). e existence of 63 compounds was discovered using GC-MS (Table 3), and their GC chromatogram is shown in Figure 1 (Figure 2(b)). Peaks were found at chemical shifts of 174.05 ppm (CH and CH 2 ); 157.41 and 157.21 ppm (C�C); and 62.03 ppm (Figure 2(c)) (CH 2 ). In the compound AA2, EDX examination revealed the existence of components such as carbon (C) and oxygen (O) (Figure 2(d)). Tables 4 and 5 exhibit Sc-XRD data for the specified bond lengths and orientations of the compound AA2. is chemical crystallized in the P 1 21/c 1 monoclinic space group. Table 6 shows the atomic coordinates and corresponding isotropic atomic displacement parameters (Å 2 ) of compound AA2. Table 7 lists the torsion angles (°), whereas Tables 8 and 9 provide the anisotropic atomic displacement parameters (Å 2 ) and hydrogen atomic coordinates and isotropic atomic displacement parameters (Å 2 ). e molecular structure of the chemical AA2 was determined using these data. e molecule was identified as 4-(2oxo-propyl)-cyclopentane-1,3-dione using the Chem Draw Ultra program 7.0.1 version and its molecular formula is C 8 H 6 O 3 (Molecular weight: 150.13 g/mol). Its chemical structure is depicted in Figure 2(e). e compound AA2 was assigned the number 1574042 by the Cambridge Crystallographic Data Centre (CCDC).

Results
W3 absorption bands in the FT-IR spectrum are shown in Figure 3 (Figure 3(d)). Tables 10 and 11 provide Sc-XRD data for the specified bond length and bond angles of compound W3. e chemical W3 formed monoclinic space group P 21/ c crystals. Table 12 shows the distance between hydrogen acceptor and donor, whereas Figure 3(e) shows hydrogen bonding. e atomic coordinates (×10 4 ) and comparable isotropic displacement parameters (Å 2 × 10 3 ) described in Tables 13-15 show anisotropic displacement parameters (Å 2 × 10 3 ) and hydrogen coordinates (×10 4 ), as well as isotropic displacement parameters (Å 2 × 10 3 ). Table 16 shows the torsional angles (°) of W3. W3's chemical formula was predicted using these data as C 4 H 6 N 4 O 3 (molecular weight: 158.13 g/mol). Figure 3(f ) depicts the chemical structure of W3 as predicted by Chem Draw Ultra software 7.0.1 version as (2,5-dioxo-imidazolidin-4-yl)-urea. Compound W3 has a CCDC number of 1574043. e radical scavenging activity of EA1 was greater than that of AA2 and W3 for DPPH (IC 50 � 23.17 µg/mL), ferric (23303.57 ± 68.53 Fe(II) E/g of the sample), and molybdenum (709.17 ± 1.51 mg AAE/g of the sample) (Table 17). When compared to EA1, the synthetic antioxidant BHT had stronger radical scavenging activity. However, when it came to single compounds, AA2 was better at scavenging DPPH and ferric ions radicals, whereas W3 was better at scavenging molybdenum radicals.  Table 18. e Scatchard plot ( Figure 6) was used to calculate the binding constant (K bin ) and the number of binding sites (n), and the findings are provided in Table 18. EA1 had a higher binding constant (9.52 ± 0.08 10 6 ) and a larger number of binding site factor (1.479) than both AA2 and W3. ere was a drop in the emission intensity of the venom corresponding to tyrosine and tryptophan residues with a red shift after adding the samples (Figures 7(a)-7(c) and 8(a)-8(c)). 4-(2-Oxo-propyl)-cyclopentane-1,3-dione, on the other hand, had higher potential in reducing the intensity of venom emission than (2,5-Dioxo-imidazolidin-4-yl)-urea as a single chemical.

Discussion
An essential oil, and two colorless crystal compounds such as AA2-4-(2-oxo-propyl)-cyclopentane-1,3-dione-and W3-(2,5-dioxo-imidazolidin-4-yl)-urea (Allantoin)were extracted using column chromatography in this study. e existence of electronegative elements "oxygen, carbon, and hydrogen," which is a defining property of an essential oil, was verified by FT-IR analysis of EA1. e volatile oil is also known as an essential oil, and the GC-MS data showed the existence of a volatile antioxidant molecule termed heptadecane, which reduces pro-inflammatory gene expression [17]. Similarly, "ledol," a sesquiterpene, has anti-inflammatory and NF-κB inhibitory characteristics [18], and phenol, 2,4-bis(1,1, dimethylethyl), is an anti-inflammatory, analgesic, and antioxidant molecule [19]. EA1 also included anti-inflammatory compounds such as globulol [20] and hexadecanoic acid [21]. Analgesic and antipyretic properties of neophytadiene have been observed [22].    e crystal AA2 is a new compound that resembles the structure of 2-methyl-1,3-cyclopentanedione, which was patented (US2016168190) for its composition and to treat traumatic brain injury (TBI); dermatological disorders such as wounds, scars, itching, and ulcers (A61P17/02); joint diseases such as arthritis and arthrosis (A61P19/08); and bone diseases such as rachitis. e crystal W3 is an Allantoin, which is a well-known chemical (PubChem CID: 204) possessing anti-inflammatory, anti-healing, and anti-irritating characteristics. Because of its capacity to heal tiny wounds and promote healthy skin, Allantoin is a common e anisotropic atomic displacement factor exponent takes the form −2π2[h2a * 2U11 + · · · + 2hka * b * U12]. 10 Evidence-Based Complementary and Alternative Medicine      Evidence-Based Complementary and Alternative Medicine ingredient in anti-acne treatments, sun care products, and clarifying lotions. It is also found in mouthwash, toothpaste, shampoos, lipsticks, other cosmetic lotions, creams, and medicinal items [23]. e capacity of a medication to scavenge free radicals is seen as crucial. Many studies have shown a significant link between antioxidant and antivenom action of substances [24]. EA1 had higher antioxidant activity because it included  heptadecanol. ese chemicals may act together to stabilize free radicals. In the case of single chemical therapeutic activity, the mechanism of the synergism was not investigated. As a result, the antioxidant potential of AA2 was shown to be much higher than that of W3. As a consequence, the findings imply that EA1 and AA2 may have therapeutic potential in combating the oxidative stress and pathology caused by S. morsitans envenomation.
In addition, fluorescence detection is a key method for determining the pharmacological action of (antivenom) substances [25]. e potential of the samples to quench the emission intensity of S. morsitans venom was shown by the linearity of the emission spectra [26]. e observed quenching might be ascribed to changes in venom protein secondary structure or denaturation, suggesting sample binding to S. morsitans venom protein [27,28]. e type and amount of the drug-venom protein interaction have a significant impact on the drug's pharmacological activity. e binding characteristics are helpful in determining a drug's pharmacological response and designing a dosing range [29,30].
Scolopendra venom contains the aromatic amino acids tyrosine and tryptophan [31]. Tyrosine is found in venom at 15 nm and tryptophan at 60 nm, according to the research.
e anisotropic displacement factor exponent takes the form: −2π2[h2a * 2U11 + · · · + 2hka * b * U12].    e blue shift occurs when the AA2 and W3 are added, indicating an increase in hydrophobicity surrounding the fluorophore moiety. With a red shift in synchronous spectra, the EA1 revealed a reduction in emission intensity corresponding to tyrosine and tryptophan residues. In S. morsitans venom, red shift indicates an increase in hydrophilicity near the fluorophore [32][33][34]. e molecular environment in the region of the fluorophore moieties of S. morsitans venom was studied using synchronized fluorescence [35]. When comparing the quenching and binding constants, the antivenom activity of EA1, AA2, and W3 was shown to be much higher than that of S. morsitans venom. Allantoin, an alkaloid derived from the A. radix, was shown to be effective against the haemorrhagic components found in the venoms of Naja naja atra and Bungarus multicinctus [36]. Allantoin was included to the national library of medicine and toxicology data network because it can be utilized as an antivenom [37]. Due to their greater quenching and binding constants, 4-(2-oxo-propyl)-cyclopentane-1,3-dione (AA2) and an essential oil (EA1) may be more potent antivenom agents than Allantoin (W3).

Conclusion
Overall, the findings of this investigation backed with the conventional claim linked with A. indica and P. nigrum literature. It also aids in the promotion of medication usage in the present competitive pharmaceutical industry, which is rife with ineffective medicines. Further research into the interactions of isolated chemicals 4-(2oxo-propyl)-cyclopentane-1,3-dione, Allantoin, and EA1 with S. morsitans venom will aid in a better understanding of the venom neutralization process and the development of a green antivenom medication for human benefit.

Data Availability
e data used to support the findings of this study are included in the article.

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