In Vivo and In Silico Analgesic Activity of Ficus populifolia Extract Containing 2-O-β-D-(3′,4′,6′-Tri-acetyl)-glucopyranosyl-3-methyl Pentanoic Acid

Natural product-based structural templates have immensely shaped small molecule drug discovery, and new biogenic natural products have randomly provided the leads and molecular targets in anti-analgesic activity spheres. Pain relief achieved through opiates and non-steroidal anti-inflammatory drugs (NSAIDs) has been under constant scrutiny owing to their tolerance, dependency, and other organs toxicities and tissue damage, including harm to the gastrointestinal tract (GIT) and renal tissues. A new, 3′,4′,6′-triacetylated-glucoside, 2-O-β-D-(3′,4′,6′-tri-acetyl)-glucopyranosyl-3-methyl pentanoic acid was obtained from Ficus populifolia, and characterized through a detailed NMR spectroscopic analysis, i.e., 1H-NMR, 13C-DEPT-135, and the 2D nuclear magnetic resonance (NMR) correlations. The product was in silico investigated for its analgesic prowess, COX-2 binding feasibility and scores, drug likeliness, ADMET (absorption, distribution, metabolism, excretion, and toxicity) properties, possible biosystem’s toxicity using the Discovery Studio®, and other molecular studies computational software programs. The glycosidic product showed strong potential as an analgesic agent. However, an in vivo evaluation, though at strong levels of pain-relieving action, was estimated on the compound’s extract owing to the quantity and yield issues of the glycosidic product. Nonetheless, the F. populifolia extract showed the analgesic potency in eight-week-old male mice on day seven of the administration of the extract’s dose in acetic acid-induced writhing and hot-plate methods. Acetic acid-induced abdominal writhing for all the treated groups decreased significantly (p < 0.0001), as compared to the control group (n = 6) by 62.9%, 67.9%, and 70.9% of a dose of 100 mg/kg (n = 6), 200 mg/kg (n = 6), and 400 mg/kg (n = 6), respectively. Similarly, using the analgesia meter, the reaction time to pain sensation increased significantly (p < 0.0001), as compared to the control (n = 6). The findings indicated peripheral and central-nervous-system-mediated analgesic action of the product obtained from the corresponding extract.


Introduction
Small-molecule drug discovery has given credence to the natural products reservoir of diverse structures, and random and designed synthetic templates obtained through various methods and approaches in drug discovery, including metabolomics, and at best, A current literature survey of Ficus populifolia revealed that no chemical constituents are reported from this species. Herein, we report three known natural products, stigmasterol (1), β-sitosterol (2), β-amyrin (3), and a new compound (4) identified as a 3 ,4 ,6 -triacetylated glucoside derivative of 2(R)-hydroxy-3(S)-pentanoic acid, obtained for the first time from this Ficus species, Ficus populifolia. These natural products are also being reported for the first time from this Mediterranean species, F. populifolia. However, notwithstanding several different types of biological activities exhibited by the plant extract, the hereto-unknown compound (4) was pursued for its in silico activity predictions, together with the biological activity evaluation of the F. populifolia extract for its analgesic effects.
The sterols and triterpenes have been reported as potent analgesics, and extracts rich in these classes of natural products have been known for their analgesic activity [23][24][25]. Furthermore, the isolated steroids, compound 1, stigmasterol, and compound 2, β-sitosterol, as well as the triterpenoid, compound 3, β-amyrin have also been reported for their analgesic activity. For example, the stigmasterol exhibited biological properties to reduce the pain in both the early and late phases of the formalin test in a mice model [26]. Furthermore, stigmasterol and β-sitosterol have been shown to inhibit intraperitoneal acetic acid-induced chemical nociception [26,27]. Moreover, in a mouse model, β-amyrin was shown to possess peripheral analgesia and anti-inflammatory membrane stabilizing activity [28]. These compounds also showed a high binding affinity, and in silico inhibitory activity against cyclooxygenase 2 (COX-2) receptor-substrate [29][30][31]. Among the plethora of pharmacological activities, the compound (4) response, as part of the extract, to pain sensation and its relief, was taken up, as the pain relief has also been the most common biological activity, which had been exhibited by several other plant species of this genus, Ficus [7][8][9]11,32,33]. Pain, being an unpleasant sensation with multidimensional facets and involving multiple origins [34], has been treated with the widespread use of NSAIDs (non-steroidal, anti-inflammatory drugs), as well as opiates, which causes dependency, GI irritation, impairment of renal functions, inhibition of platelet aggregation, patient-compliance related troubles, together with drug tolerance issues. This has made it further pertinent to pursue the analgesic activity in more detail, including receptor binding studies, and studies on the drug-likeness of the structure (4).
Cyclooxygenase-2 (COX-2), for the majority of tissues, is the inducible form of the cyclooxygenases in pain, and its overexpression in dynamics and origins of pain together with the pain's severity connection is well-known [35,36]. Toward the in silico analysis of compound (4), COX-2 bindings, ADME (absorption, distribution, metabolism, and excretion), and toxicity studies were pursued in detail. However, owing to the quantity issues, in vivo evaluations of the primal extract of the plant material, containing compound (4), were undertaken.

Isolation and Structure Elucidation of the Constituents
The aq.-ethanolic extract of the F. populifolia plants' aerial parts were subjected to column chromatographic purification yielding three known and one unknown compound, stigmasterol (1), β-sitosterol (2), β-amyrin (3), and 2-O-β-D-(3 ,4 ,6 -tri-acetyl) glucopyranosyl-3-methyl pentanoic acid (4). All the isolated products were identified by 1 H and 13 C-NMR, and 2D-NMR spectral analyses. The 1 H -NMR spectrum of compound 4 exhibited distinguishable signals for the five methyls in the compound. Two of these methyls were resonated as the most subfield signals in the H-NMR spectrum of the compound at δ H 0.87 (triplet) and 0.93 (doublet), assigned to the methyls at the positions C-5 and C-6. In addition, another three downfield singlet methyls (δ H 1.97, single, 2.01, s, 2.06, s) were pointing to the nature of the methyls of which one was a terminal. These methyls were assigned as part of three acetoxyl functions for which three carbonyl peaks appeared in the 13 C-spectrum substantiated by the DEPT observations (Supplementary Materials, Figure S2). These methyls were resonated at δ C 20.65, 20.55, and 20.75, and were assigned the positions 3b, 4b, and 6b, respectively, by the long-range coupling correlation ( Table 1). The presence of one methy-lene at δ C 25.55 and another methylene at δ C 61.56, which were clearly observed in 13 C and DEPT-135 NMR spectra, were assigned to the C-4 and C-6' and indicated the aglycone and glycone natures of the methylenes, respectively. Five carbons of the methine nature at δ C 68. 11, 71.27, 73.20, 74.60, and δ C 95.33 were assigned to the 5-methines of the sugar part of the compound, based on their chemical shift and their long coupling with their protons and the C-6' methylene protons in the HMBC-2D NMR spectrum (Table 1). Moreover, two other methine signals appearing at δ C 76.80 and δ C 36.92 were found attached to protons that resonated at 4.53 (d, J 2.5 Hz) and 2.07 (m) in the HMQC spectrum, respectively. The positions of these two methines were assigned to be C-2 and C-3 in the aglycone part of the compound, based on their protons long coupling correlation to the carboxylic acid carbon, C-1. Furthermore, the 13 C NMR showed four most downfield quaternary signals assigned to four carbonyl groups in the compound. Three carbonyls' signals appearing at δ C 169.67, 169.97, and δ C 170.60 were attributed to the glycone, and another carbonyl appearing at δ C 168.16 was elucidated to be attached to the aglycone unit. The three carbonyls for the glycone unit were shown to be acetyloxy carbonyls. The downfield shifts of methyl and methylene as substantiated by the correlation spectroscopies of compound (4) ( Table 1 and Figure 1), confirmed the contention. The anomeric carbon, which was identified at δ C 95.33 downfield shift, indicated the presence of a sugar moiety that was identified as the tri acetyloxy glucosyl unit while the aglycone unit was constructed to structure as 2-hydroxy-3-methyl pentanoic acid based on 1 H and 13 C NMR spectra, DEPT and HSQC, and HMBC correlation peaks. The 1 H-NMR spectrum in conjunction with 13 C-DEPT, and 2D correlations of the quaternary carbon resonances, exhibited peak at δ C 168.16 ppm (COOH). Five other carbons resonating at δ C 76.80 (C-OH, C-2), δ C 36.92 (CH, C-3), δ C 25.55 (CH 2 , C-4), δ C 11.58 (CH 3 , C-5), and δ C 11.96 ppm (CH 3 , C-6), along with their corresponding hydrogens chemical shift values and coupling constants for the stereo orientations of the C-2 methine (d, 2.5 Hz, showing connectivity to C-1 and C-3 carbons), and C-3 methine groups (2.07, m, showing connectivity to C-1, and C-2 carbons) were exhibited, which confirmed the 2(S)-hydroxy-3(R)-methyl pentanoic acid-derived substructure. The 1 H-NMR spectrum also showed two methyl peaks at δ H 0.87, t. J = 7.5 and δ H 0.93, d, j = 7 for H-5 and H-6 protons, respectively. Additionally, the 1 H-NMR spectrum showed one proton doublet at δ H 4.53 (H-2), along with three other protons multiplets at δ H 1.38, δ H 1.47, and δ H 2.07 for H-4b, H-4a, and H-3 protons, respectively ( Table 1). The sugar moiety of compound (4) was assigned as 3 ,4 ,6 -tri-acetyl glucose by the presence of anomeric carbon at δ C 95.33 (C-1') and one methylene carbon at δ C 61.56 (C-6'), with other four methines carbons resonating between δ C 68.11 and δ C 74.60. The presence of one proton doublet at δ H 4.76 (H-1') and six protons resonating between δ H 3.80 and δ H 5.28 also led to elucidating the sugar moiety as acetylated glucose. Additionally, three acetylated methyl singlets resonating at δ H 1.97, δ H 2.01, and δ H 2.06 appeared in correlation with the three carbonyls at δ C 169.97, δ C 169.67, and δ C 170.60, as shown in the HMBC spectrum (Supplementary Materials, Figures S1-S6), were found attached to the C-3 , C-4 , and C-6 carbons, respectively.  The attachments of the acetyl carbons to the sugar were assigned according to the HMBC long-range correlations and are depicted in Figure 1, which clearly shows a correlation between δH 5.28 (H-3') and δC 169.97 for the C-3′ (acetoxy C=O) and δH 5.04 (H-4') with δC 169.67 for the C-4′ (acetoxy C=O), in addition to long-range coupling between The attachments of the acetyl carbons to the sugar were assigned according to the HMBC long-range correlations and are depicted in Figure 1, which clearly shows a correlation between δ H 5.28 (H-3') and δ C 169.97 for the C-3 (acetoxy C=O) and δ H 5.04 (H-4') with δ C 169.67 for the C-4 (acetoxy C=O), in addition to long-range coupling between δ H 4.10 and δ H 4.26 of H-6' and δ C 170.60 for the C-6 (acetoxy C=O). Although partially acetylated glucose/glycone is rarely found in nature, nonetheless there are few reports of acetylated sugars, e.g., mono-acetylated allose as part of the flavonoid-glycoside structure isolated from Stachys anisochila [37]. However, partially acetylated glucose in structures ( Figure 1) was also confirmed by the presence of three methyl singlets appearing at δ H 1.97 (3 -acetoxy methyl), δ H 2.01 (4 -acetoxy methyl), and δ H 2.06 (6 -acetoxy methyl), in connectivity with the 13 C-NMR spectrum showing carbons at δ C 20.65, δ C 20.55, and δ C 20.75. Additionally, the 13 C-DEPT (Table 1) confirmed the presence of four quaternary carbons for the previous three acetyls along with one carboxylic acid carbon resonating at δ C 168.16 for the C-1 (aglycone part) carbon, which was found coupled with the proton doublet resonating at δ H 4.53 (H-2) in the HMBC spectrum. The chemical shift values of the acetylated methyls revealed that these acetyl groups must be attached to the sugar moiety of compound (4) and not to the aglycone part [38]. The distinct downfield shift of the sugar protons H-3' and H-4', as compared to the reported values of the fastigitin-A showed protons, peaks additionally downfield by δ +1.1, and +0.86 ppm, respectively [20]. Moreover, the long-range coupling appearing in 2D analyses confirmed that the quaternary carbon at δc 169.97, 169.67, and δ C 170.60 is the nodal carbon for the three acetyl groups, which are attached to the sugar carbons at C-3', C-4', and C-6', respectively. The attachment of the triacetylated glucose to the aglycone part was assigned through the long-range coupling in the HMBC spectrum between the doublet proton resonates at δ H 4.76 of the anomeric proton (H-1') with the δ C 76.80 for the C-2. In addition, the glycone was assigned as β-D-triacetylated glucose based on the J-coupling of the anomeric proton, which was found as 8.5 Hz that showed the β-linkage of the glucose to the aglycone part in parallel to the fastigitin-A, which has been reported from Rhodiola fastigiata [39], the significant difference being the presence of tri-acetoxy functions in the glucose unit. Therefore, the 1 H and 13 C-NMR correlations and HMBC led to elucidating the structure of compound (4) as a (3S)-2-hydroxy-3-methyl pentanoic acid derivative with a 3 ,4 ,6triacetoxy-β-D-glucosyl unit ( Figure 1).
Compound (1), stigmasterol, isolated as a white amorphous powder with a melting point of 170 • C, was identified based on spectral data, which were similar to β-sitosterol, compound (2); except for the presence of two multiple olefinic protons at δ H 5.03 and δ H 5.20, in addition to the presence of two olefinic carbons at δ C 129.25 and δ C 138.34 ppm. The structure was also confirmed through 2D-NMR spectral analyses, and literature comparisons [40,41].
Compound (2) was isolated as a white amorphous powder with a melting point of 141 • C. The IR spectrum showed absorption peaks at 1050 cm −1 (C-O), in addition to stretching at 3428 cm −1 (O-H) and 1640 cm −1 (C=C). The 1 H-and 13 C-NMR spectra showed a similar pattern to the published data of β-sitosterol. The structure was confirmed through the presence of a multiplet peak at δ H 3.51 ppm (H-3), along with a double doublet at δH 2.26 for the methylene protons (H-7) and with the multiple peaks at δ H 5.33 ppm for the olefinic proton (H-6). Additionally, the 13 C-NMR showed peaks at δ C 140.76 (C) and 121.85 ppm (CH) for the C-5 and C-6 carbons, respectively. The carbon signal at δ C 71.81 ppm for C-3 along with another 26 aliphatic carbon signals that strongly supported the β-sitosterol structure for compound (2), which was also supported by the 2D-NMR spectral data [42,43].
Compound (3) was isolated as a pale amorphous mass with a melting point of 189 • C and identified as β-amyrin based on the NMR spectral data and in comparison with the reported values. The presence of δ H 3.21 (H-3) as a double doublet and the peak at δ H 5.17 (H-12) as a triplet in addition to the carbon's signals resonating at δ C 79.90 (C-3) and δ C 121.750 ppm (C-12) with the quaternary carbon at δ C 145.30 ppm, confirmed the β-amyrin structure. The spectral data from 1 H and 13 C-NMR, DEPT, HMBC, and HMQC spectra conclusively identified the β-amyrin structure [44].

Docking Studies for Analgesic Activity
For the study, the human cyclooxygenase-2 (COX-2) protein was selected. COX-2 converts arachidonic acid to prostaglandin. Prostaglandin is subsequently metabolized by the downstream tissue-specific synthases into potent signaling molecules that play fundamental roles in both the regulation of physiological homeostasis as well as in disease states such as nociceptive inflammation and cancer [45]. The compounds 1-3 are known for their potential analgesic activity [26][27][28] and have shown a high binding affinity and in silico inhibitory activity against COX-2 [29][30][31]. In this study, the binding efficiency of the 3 ,4 ,6 -triacetylated pentanoic acid glucoside (compound 4) was calculated. Lower binding energy, resulting from the association of the compound with the targeted protein, is an indication of a higher binding efficiency. NAG was used in this study as a COX-2 inhibitor by comparing the binding affinity of compound 4 (∆G of −8.1) with NAG (∆G of −7.6). It was found that compound (4) showed a very good binding affinity. These results of the in silico protein-screened metabolites interactions showed that the following amino acid in the protein target participated actively in the interactions (VAL 349, TYR 355, LEU 359, VAL 116, SER 530, ALA 527, and GLY 526) through the number of hydrogens, and followed hydrophobic interactions, Table 2

Physiochemical and ADMET Profiling
The drug-likeness behavior of compound (4) was estimated using the Swiss ADME program [46]. The listed data in Tables 3 and 4 show that, compound 4, 3 ,4 ,6 -triacetylated pentanoic acid glucoside, has a molecular weight <500, which increased the transport and absorption and improved the transmissibility of the compound to the membranes. The topological polar surface area (TPSA) of this compound found in the correct published range is less than 140 Å 2 (Tables 3 and 4) [47,48]. Lipophilicity (Log P) values ranged within 1.5-4.81, which is considered compatible by Lipinski's rule of five. The lipophilicity, which is also related to toxicity generation at a certain scale, has demonstrated that the toxicity is significantly higher for derivatives with log Po/w > 5 and TPSA < 75 Å 2 [49]. The obtained results showed that the newly investigated compound (4) has physicochemical characteristics that are within the appropriate range, as also demonstrated by the bioavailability radar ( Figure 5). Thus, the study indicated that compound (4) is non-toxic in nature and has the suitable physico-chemical and ADMET properties for being fit to be a lead compound for future development.

Physiochemical and ADMET Profiling
The drug-likeness behavior of compound (4) was estimated using the Swiss ADME program [46]. The listed data in Tables 3 and 4 show that, compound 4, 3′,4′,6′triacetylated pentanoic acid glucoside, has a molecular weight < 500, which increased the transport and absorption and improved the transmissibility of the compound to the membranes. The topological polar surface area (TPSA) of this compound found in the correct published range is less than 140 Å 2 (Tables 3 and 4) [47,48]. Lipophilicity (Log P) values ranged within 1.5-4.81, which is considered compatible by Lipinski's rule of five. The lipophilicity, which is also related to toxicity generation at a certain scale, has demonstrated that the toxicity is significantly higher for derivatives with log Po/w> 5 and TPSA < 75 Å 2 [49]. The obtained results showed that the newly investigated compound (4) has physicochemical characteristics that are within the appropriate range, as also demonstrated by the bioavailability radar ( Figure 5). Thus, the study indicated that compound (4) is non-toxic in nature and has the suitable physico-chemical and ADMET properties for being fit to be a lead compound for future development.      Table 5 depicts the toxicity data of compound (4), 3 ,4 ,6 -triacetylated pentanoic acid glucoside. The ADMET lab 2.0 software used the AMES toxicity test that described the potential carcinogenic effect of the compound [50] and according to the human ether-a-gogo-related gene (hERG) toxicity test, the blockage of the potassium channel of that gene leads to cardiac toxicity [51]. Furthermore, the software estimated the oral rat chronic toxicity (LOAEL), hepatotoxicity, and skin sensitization.   Table 5 depicts the toxicity data of compound (4), 3′,4′,6′-triacetylated pentanoic acid glucoside. The ADMET lab 2.0 software used the AMES toxicity test that described the potential carcinogenic effect of the compound [50] and according to the human ether-ago-go-related gene (hERG) toxicity test, the blockage of the potassium channel of that gene leads to cardiac toxicity [51]. Furthermore, the software estimated the oral rat chronic toxicity (LOAEL), hepatotoxicity, and skin sensitization. In addition to the toxicity model, the toxicophoric rules were also estimated for identifying the ability of compound (4) to induce cancer by mutations (genotoxic carcinogenicity rule), or by any other mechanism rather than the mutation (non-genotoxic carcinogenicity rule) [52]. These results revealed that the tested compound (4) had a nontoxicity impact in any toxic model (Table 5), and thus, proved to be of interest for further development in the drug discovery program. The predictions of the pharmacokinetics properties, with one violation involving the N or O atoms abundance which is part of the molecular framework and can be improved through use of bioisosteric group replacement in subsequent design(s), further supported the notion of the drug-likeliness of the product compound (4).

In Vivo Analgesic Activity Evaluations
Analgesic activity of the compound containing extract was undertaken since the material quantity was in adequate. Furthermore, the plant extract contains significant In addition to the toxicity model, the toxicophoric rules were also estimated for identifying the ability of compound (4) to induce cancer by mutations (genotoxic carcinogenicity rule), or by any other mechanism rather than the mutation (non-genotoxic carcinogenicity rule) [52]. These results revealed that the tested compound (4) had a non-toxicity impact in any toxic model (Table 5), and thus, proved to be of interest for further development in the drug discovery program. The predictions of the pharmacokinetics properties, with one violation involving the N or O atoms abundance which is part of the molecular framework and can be improved through use of bioisosteric group replacement in subsequent design(s), further supported the notion of the drug-likeliness of the product compound (4).

In Vivo Analgesic Activity Evaluations
Analgesic activity of the compound containing extract was undertaken since the material quantity was in adequate. Furthermore, the plant extract contains significant amounts of steroids and tetraterpenoids, which are well-known for their analgesic and anti-inflammatory properties [53,54]. As observed, the number and percentage of acetic acid induced abdominal writhing were decreased significantly, (p < 0.0001). It was observed for all the treated groups in comparison to the control group (27.6 ± 1.53) by 10.00 ± 1.37, 8.67 ± 0.95, 7.83 ± 1.25, 6.33 ± 0.84, and 62.9%, 67.9%, 70.9%, and 76.5% for the extract groups at 100 mg/kg, 200 mg/kg, and 400 mg/kg, and the diclofenac 5 mg/kg group, respectively ( Figure 6). The reaction time to pain, measured using an analgesia meter, increased significantly (p < 0.0001) as compared to the control group (7.53 ± 0.95) by 10.95 ± 0.95, 10.32 ± 1.05, and 11.80 ± 1.20, and 13.00 ± 1.03 for all the extract groups 100 mg/kg, 200 mg/kg, and 400 mg/kg, and the diclofenac 5 mg/kg group, respectively (Figure 7).
10.00 ± 1.37, 8.67 ± 0.95, 7.83 ± 1.25, 6.33 ± 0.84, and 62.9%, 67.9%, 70.9%, and 76.5% for the extract groups at 100 mg/kg, 200 mg/kg, and 400 mg/kg, and the diclofenac 5 mg/kg group, respectively ( Figure 6). The reaction time to pain, measured using an analgesia meter, increased significantly (p < 0.0001) as compared to the control group (7.53 ± 0.95) by 10.95 ± 0.95, 10.32 ± 1.05, and 11.80 ± 1.20, and 13.00 ± 1.03 for all the extract groups 100 mg/kg, 200 mg/kg, and 400 mg/kg, and the diclofenac 5 mg/kg group, respectively (Figure 7).  The extract of the plant F. populifolia showed peripherally mediated analgesic activity using an acetic acid-induced writhing model. Researchers have previously described acetic acid-induced writhing [55] and the hot plate method [56] using an analgesia meter as the model for the evaluation of analgesic activity mediated at the peripheral and central levels, respectively. The peripheral level mediation is hypothesized to act through the inhibition of cyclooxygenases, especially and primarily through COX-2. Data from our current study confirmed the analgesic activity in an acetic acid-induced writhing model for the first time using the extract of the F. populifolia plant. The data are also in confirmation with other species of Ficus in their analgesic activity [57][58][59][60][61].
Pain induced by the thermal stimulus of the hot plate is specific for centrally mediated nociception [32]. The ability of the F. populifolia extract to increase the latency of pain reaction induced by a hot plate, described here for the first time, indicates the central analgesic potential of the extract. However, other constituents, e.g., flavonoids, steroids, and tannins are also known to have analgesic activities [59].

Chemistry: Plant Material, Extraction, and Isolation of Products
Ficus populifolia aerial parts were collected from Qassim, Saudi Arabia, in fruiting season and were authenticated by the institutional taxonomist at the College of Figure 7. Evaluation of centrally mediated analgesic activity using the analgesia meter method. Pain reaction values expressed as mean ± standard error, n = 6 mice per group. Extract groups: total extract of F. populifolia. *** Data differed significantly at p < 0.0001 when compared with the control group in the relevant column.
The extract of the plant F. populifolia showed peripherally mediated analgesic activity using an acetic acid-induced writhing model. Researchers have previously described acetic acid-induced writhing [55] and the hot plate method [56] using an analgesia meter as the model for the evaluation of analgesic activity mediated at the peripheral and central levels, respectively. The peripheral level mediation is hypothesized to act through the inhibition of cyclooxygenases, especially and primarily through COX-2. Data from our current study confirmed the analgesic activity in an acetic acid-induced writhing model for the first time using the extract of the F. populifolia plant. The data are also in confirmation with other species of Ficus in their analgesic activity [57][58][59][60][61].
Pain induced by the thermal stimulus of the hot plate is specific for centrally mediated nociception [32]. The ability of the F. populifolia extract to increase the latency of pain reaction induced by a hot plate, described here for the first time, indicates the central analgesic potential of the extract. However, other constituents, e.g., flavonoids, steroids, and tannins are also known to have analgesic activities [59].

Chemistry: Plant Material, Extraction, and Isolation of Products
Ficus populifolia aerial parts were collected from Qassim, Saudi Arabia, in fruiting season and were authenticated by the institutional taxonomist at the College of Agriculture, Qassim University. A voucher sample number QPP-125 was kept at the College of Pharmacy, Qassim University, Saudi Arabia. The freshly collected plant material (1.1 kg) was shade-dried, grinded, and extracted over a period of 6 h cycle in a Soxhlet apparatus (BLS.2307.13) with light petroleum ether, followed by 95% aq. ethanol to yield 22 gm and 64 gm of viscous masses, respectively, after concentration on vacuum rotatory evaporator under reduced temperature and pressure. The EtOH extract was examined on the TLC (Merck, Darmstadt, Germany, catalogue number, HX99037354) and subjected to silica gel (70-230 mesh, Supelco, Tokyo, Japan, Lot MKCP3826) column chromatography (CC) (CG119720) in CHCl 3 and CHCl 3 -MeOH (v/v) and was afforded fractions with impure products which were further purified by the Sephadex TM LH-20 (GE Healthcare Bio-Sciences, Uppsala, Sweden) and silica gel column chromatography in hexane-ethyl acetate varying polarities. Of the purified products, compound (1) was identified as stigmasterol, compound (2) as β-sitosterol, and compound (3) as β-amyrin, based on their reported spectral data including 2D NMR analyses. Compound (4) was isolated as a gummy substance (CHCl 3 -MeOH, 88/12: v/v, fractions 29-33, 1 × 50 mL) and was further purified by reverse-phase silica-C 18 (Sigma, Buchs, Switzerland, Lot BCC82847) CC using methanol-water mixture (1:0.5) as mobile phase; R f 0. 6

Biological Activity: Animal Groups
Thirty eight-week-old male mice were obtained from the animal facility, College of Pharmacy, Qassim University. The animals were housed in polyacrylic cages and maintained at room temperature with a relative humidity of 45-65% in controlled light and dark cycles. The Research Ethics Committee, College of Pharmacy, Qassim University, Saudi Arabia, approved all the experimental procedures (21-04-06). The care of laboratory animals was carried out as per the Guide for the Care and Use of Laboratory Animals. The extract doses (100 mg/kg, 200 mg/kg, and 400 mg/kg) were selected based on the previously published article [61].

Evaluation of Peripherally Mediated Analgesic Activity Using Acetic Acid-Induced Writhing Model
Induction of writhing using acetic acid was performed as previously described by Koster et al. [56] method. Briefly, mice were divided into 5 groups (n = 6/group). The control group received normal saline (10 mL/kg oral route (p.o.). Groups 2-4 received 100 mg/kg, 200 mg/kg, and 400 mg/kg extract p.o. daily for one week, while the positive group received Diclofenac 5 mg/kg, i.p, on the day of the test. On the seventh day, 30 min after the last dose, 0.05% acetic acid/100 g, i.p, was injected into all the 5 groups. Five minutes after the dose, abdominal constrictions were counted for each mouse for 10 min. Data were recorded, and percentage inhibition of writhing was calculated using the formula: where c = Mean number of writhing in control group and t = Mean number of writhing in treated group.

Evaluation of Centrally Mediated Analgesic Activity Using Analgesia Meter Method
Hot plate tests were performed as described previously [25,55]. Briefly, the temperature on the analgesia meter was set to 55 ± 1 • C. The time taken by the mice for licking or jumping was recorded with 15 s as a cutoff point. All the mice were separated into five groups (n = 6/group). The control group received normal saline (10 mL/kg, p.o.), groups 2-4 received 100 mg/kg, 200 mg/kg, and 400 mg/kg extract p. o. daily for one week, while the positive group received diclofenac 5 mg/kg, i.p on the day of the test. On the seventh day, 30 min after the last administration, the mice were placed on a hot plate, and the sensitivity to pain in seconds was documented.

In Silico Studies: COX-2 Docking
The MOE 2019.012 suite [62] was applied to carry out the docking studies for the 3 ,4 ,6 -triacetylated pentanoic acid glucoside to propose their mechanism of action as analgesic, through evaluating their binding scores and modes compared with NAG (2acetamido-2-deoxy-β-D-glucopyranose) as the co-crystallized ligand. The 3 ,4 ,6triacetylated pentanoic acid glucoside was introduced into the MOE window, subjected to partial charges addition, and its energy was minimized. Then, the prepared compound was inserted into one database with NAG and saved as an MDB file to be uploaded in the ligand icon during the docking step. The X-ray crystallography of the target human cyclooxygenase-2 (COX-2) was obtained from the Protein Data Bank (https: //www.rcsb.org/structure/5IKR, accessed on 25 October 2022). Moreover, it was prepared for the docking process following the previously described steps in detail. Notably, the downloaded protein was corrected for any errors, loaded with 3D hydrogens, and the was energy minimized as well. The 3 ,4 ,6 -triacetylated pentanoic acid glucoside was inserted into a general docking process in place of the ligand site. The docking site was chosen to be the co-crystallized ligand site and the docking process was initiated after adjusting the default program specifications described before [3]. Briefly, the dummy atoms method was used to select the docking position. Triangle matcher and London dG were selected as the placement and scoring methodologies, respectively. Both the refinement methodology and the scoring one were changed to the rigid receptor and GBVI/WSA dG, respectively, to extract the best 10 poses produced from 100 poses for the docked molecule. The best pose for the ligand with the most acceptable score, binding mode, and RMSD value was selected for further studies. It is worth clarifying that a program validation step was performed first for the applied MOE program by re-docking the co-crystallized ligand (NAG) at its binding pocket of the prepared target ( Figure 8). Valid performance scores were confirmed by obtaining a low RMSD value (1.43) between the screened compound and the re-docked, co-crystallized ligand (NAG). The output from MOE software was further visualized by Discovery Studio 4.0 software.

Statistical Analysis
Data were reported as mean ± standard error of the mean (SE). The difference between groups was analyzed using one-way ANOVA followed by post-hoc test using Dunnett's multi-group comparison on GraphPad Prism 8.0.2. The data were considered significant if p < 0.05. docked molecule. The best pose for the ligand with the most acceptable score, binding mode, and RMSD value was selected for further studies. It is worth clarifying that a program validation step was performed first for the applied MOE program by re-docking the co-crystallized ligand (NAG) at its binding pocket of the prepared target ( Figure 8). Valid performance scores were confirmed by obtaining a low RMSD value (1.43) between the screened compound and the re-docked, co-crystallized ligand (NAG). The output from MOE software was further visualized by Discovery Studio 4.0 software. Figure 8. Two-dimensional and three-dimensional molecular interactions of the re-docked cocrystalized ligand (NAG) with human cyclooxygenase-2 (COX-2). The hydrogen bonds are represented as green dotted lines.

Conclusions
In silico studies confirmed the analgesic potential of the isolated new compound (4), 2-O-β-D-(3 ,4 ,6 -tri-acetyl)-glucopyranosyl-3-methyl pentanoic acid, and the in vivo experimental data from the current study indicated the significant nociceptive potential of the F. populifolia extracts at all the evaluated doses. Further research is required on the presence and role confirmation of the predicted active constituent of the plant.