Phytochemical Insights into Ficus sur Extracts and Their Biological Activity

This study focused on the biological evaluation and chemical characterisation of Ficus sur Forssk. (F. sur) (Family: Moraceae). The methanolic and aqueous extracts’ phytochemical profile, antioxidant, and enzyme inhibitory properties were investigated. The aqueous stem bark extract yielded the highest phenolic content (115.51 ± 1.60 mg gallic acid equivalent/g extract), while the methanolic leaves extract possessed the highest flavonoid content (27.47 ± 0.28 mg Rutin equivalent/g extract). In total, 118 compounds were identified in the tested extracts. The methanolic stem bark extract exhibited the most potent radical scavenging potential against 2,2-diphenyl-1 picrylhydrazyl and 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (475.79 ± 6.83 and 804.31 ± 4.52 mg Trolox equivalent/g extract, respectively) and the highest reducing Cu2+ capacity (937.86 ± 14.44 mg Trolox equivalent/g extract). The methanolic stem bark extract substantially depressed tyrosinase (69.84 ± 0.35 mg kojic acid equivalent/g extract), α-amylase (0.77 ± 0.01 mmol acarbose equivalent/g extract), acetylcholinesterase and butyrylcholinesterase (2.91 ± 0.07 and 6.56 ± 0.34 mg galantamine equivalent/g extract, respectively) enzymes. F. sur extracts were tested for anticancer properties and antiviral activity towards human herpes virus type 1 (HHV-1). Stem bark infusion and methanolic extract showed antineoplastic activity against cervical adenocarcinoma and colon cancer cell lines, whereas leaf methanolic extract exerted moderate antiviral activity towards HHV-1. This investigation yielded important scientific data on F. sur which might be used to generate innovative phytopharmaceuticals.


Introduction
For millennia, humans have centred their lives on plants in an effort to maintain good health and treat common ailments. Even though the usage of plants was based simply on people's intuitive understanding, owing to a lack of suitable techniques to show plants' therapeutic potential, humans have accepted the use of many medicinal plants and included them in contemporary pharmacotherapy [1]. The Royal Botanic Gardens at Kew's Bob Allkin recognised around 28,000 plant species as medicinal plants [2]. Since the eureka moment of the discovery of Taxol, the blockbuster anti-cancer medicine produced from the Pacific yew tree, plants have demonstrated their healing ability [3]. Since then, medicinal

Antioxidant Effects
The role of oxidative stress in the initiation and progression of human diseases supports the systemic antioxidant assessment of plant extracts under investigation. Antioxidants can perform various functions, including hydrogen atom transfer, single electron transfer, and transition metal chelation [38]. In this study, a battery of antioxidant assays was used to obtain a comprehensive understanding of the antioxidant activities of the prepared extracts of F. sur. The assays were: 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric ion reducing antioxidant power (FRAP), cupric reducing antioxidant capacity (CUPRAC), metal-chelating and total antioxidant capacity (phosphomolybdenum). As previously discussed, each test has its own set of advantages and disadvantages [38]. The results are given in Table 3.
Overall, irrespective of the type of extraction solvents used, stem bark extracts demonstrated substantially higher antioxidant activities with DPPH, ABTS, CUPRAC, FRAP, and phosphomolybdenum. For instance, methanolic stem bark extract exhibited the highest DPPH radical scavenging activities (475.79 ± 6.83 mg TE/g). The ABTS assay showed that both methanolic (804.31 ± 4.52mg TE/g) and aqueous stem bark (804.91 ± 5.45 mg TE/g) extracts demonstrated remarkably high activities. ABTS can function with lipophilic and hydrophilic molecules, but DPPH can only be solubilized in organic environments [39]. Our findings confirm the findings of Kim et al. [39]. For example, the DPPH test identified the methanolic extract as the most active, while ABTS identified both methanolic and aqueous extracts as effective ABTS scavengers.
The antioxidant capacity of the extracts was further evaluated in terms of power reduction using the CUPRAC and FRAP tests. Several variables influence antioxidants' reducing potential, including their ionization potentials, the spin distribution of radical cations, and the bond dissociation energy of the phenolic O-H bond [40]. From Table 3, it can be seen that the methanolic stem bark extract possessed the most potent Cu 2+ reducing potential (937.86 ± 14.44 mg TE/g) while the aqueous stem bark extract (614.33 ± 2.79 mg TE/g) was the most robust Fe 3+ reducer.
Secondary metabolites are known to have powerful antioxidant properties due to their ability to provide electrons and because they chelate transition metals [41]. Data shown in Table 3 show that the aqueous leaves extract exhibited the highest chelating abilities (22.95 ± 0.20 mg EDTAE/g) while the methanolic stem bark extract displayed the lowest activity (4.62 ± 0.64 mg EDTAE/g). The prepared samples were also tested for their total antioxidant capacity (phosphomolybdenum assay). The latter test is based on antioxidants reducing Mo (VI) to Mo (V), resulting in the formation of a green complex in acidic conditions [42]. The aqueous stem bark extract showed the highest capacity (5.05 ± 0.05 mmol TE/g). It is noteworthy that the stem bark extracts showed stronger antioxidant ability than the leaf extracts for all assays, except the metal-chelating assay. Consequently, it can be said that the antioxidant activity of the active samples could be associated with the presence of bioactive compounds. Values are reported as mean ± SD of three parallel measurements. TE: Trolox equivalents; EDTAE: EDTA equivalents. Different letters in the same column indicate significant differences in the tested extracts (p < 0.05).

Enzymatic Inhibitory Activities
In the present study, the ability of F. sur extracts to modulate the activity of enzymes related to Alzheimer's disease [acetylcholinesterase (AChE) and butyrylcholinesterase (BChE)], diabetes type 2 (α-amylase and α-glucosidase), and skin hyperpigmentation (tyrosinase) was investigated. The results are presented in Table 4.
Because enzymes in the human body contribute to the genesis of disease, inhibiting these enzymes can be advantageous in health care. Cholinesterase inhibitors, for example, are drugs that prevent the breakdown of acetylcholine, a neurotransmitter in the central nervous system that, when present in excessive concentrations, can cause neurodegenerative diseases, such as Alzheimer's and Parkinson's disease [43]. Our study explored the anti-cholinesterase activity in various extracts of F. sur. High anti-AChE and anti-BChE activities were recorded with the methanolic stem bark extract (2.91 ± 0.07 and 6.56 ± 0.34 mg GALAE/g, respectively). However, the aqueous leaves extract was inactive against AChE and BChE.
Inhibitors of α-amylase and α-glucosidase diminish carbohydrate digestion in the small intestine and, as a result, lower postprandial blood glucose levels, making them an essential therapy option for type II diabetes patients [44]. The methanolic stem bark extract of F. sur was observed to substantially depress α-amylase (0.77 ± 0.01 mmol ACAE/g) but was found to be inactive against α-glucosidase. Instead, the methanolic leaves extract showed high anti-glucosidase activity (3.98 ± 0.03 mmol ACAE/g).
Tyrosinase inhibitors help to protect the skin and prevent hyperpigmentation. They are strongly promoted by the pharmaceutical and cosmetics industries [45]. The methanolic stem bark extract displayed the strongest anti-tyrosinase activity (69.84 ± 0.35 mg KAE/g), while the aqueous leaves extract showed the lowest activity (0.35 ± 0.08 mg KAE/g). It is noteworthy that the methanolic stem bark extract showed the highest activity against four enzymes, namely AChE, BChE, tyrosinase, and α-amylase, although, the extract did not show the highest TFC and TPC.

Cytotoxicity Evaluation
Cytotoxicity evaluation revealed that the infusion and methanolic extract from Ficus sur leaves exerted low toxicity on normal kidney fibroblasts (VERO); the exact CC 50 values could not be evaluated because they were above the tested concentration range (Table 5).

Stem bark extracts showed a similar effect on VERO cells. Selective toxicity towards HeLa cancer cells was observed for Ficus sur leaves methanolic extract (FLM) and infusion (FLI)
with SI of >3.62 and >2. 36. In contrast, in the case of RKO, only FLM showed selective toxicity (SI > 3.13). Significant antineoplastic activity towards both cancer cell lines was observed ( Figure 2) for Ficus sur stem bark methanolic extract (FSBM) and infusion (FSBI) with CC 50 values ranging from 36.8 to 56.12 µg/mL. The anticancer selectivity of FSBM and FSBI towards HeLa cells was 7.1 and 9.24, respectively, whereas against RKO, it was found to be 5.37 and 7.01, respectively. Multiple studies describe the anticancer potential of Ficus spp, ex. Ficus carica [46,47], Ficus salicifolia [46], Ficus religiosa [48], Ficus beecheyana [49], Ficus pandurata H [50] and Ficus exasperata (Vahl) [51], against various cancer cell lines, however, to the best of our knowledge, this is the first report showing Ficus sur stem bark extracts as a possible source of antineoplastic molecules. however, to the best of our knowledge, this is the first report showing Ficus sur stem bark extracts as a possible source of antineoplastic molecules.

Antiviral Potential
The Ficus sur extracts were incubated with an HHV-1 infected VERO cell line to evaluate the antiviral potential. After CPE was found in the virus control cells, the influence on CPE was observed in extract-treated infected cells. It was found that only one extract, namely FLM at 250 µ g/mL, decreased, but did not abolish altogether, CPE formation, as can be seen in Figure 3. The collected samples were further subjected to an end-point dilution assay to evaluate the infectious titer of HHV-1. The data on HHV-1 titer reduction contained in Table 6 confirmed that FLM 250 µ g/mL exerted antiviral activity, decreasing the infectious titer by 2.86 log. However, since it is generally agreed that the tested sample should reduce the infectious titer by at least 3 log to show significant antiviral potential, FLM cannot be regarded as such. However, plant extracts are complex mixtures of compounds belonging to various groups of secondary metabolites, and the biological activity

Antiviral Potential
The Ficus sur extracts were incubated with an HHV-1 infected VERO cell line to evaluate the antiviral potential. After CPE was found in the virus control cells, the influence on CPE was observed in extract-treated infected cells. It was found that only one extract, namely FLM at 250 µg/mL, decreased, but did not abolish altogether, CPE formation, as can be seen in Figure 3. The collected samples were further subjected to an end-point dilution assay to evaluate the infectious titer of HHV-1. The data on HHV-1 titer reduction contained in Table 6 confirmed that FLM 250 µg/mL exerted antiviral activity, decreasing the infectious titer by 2.86 log. However, since it is generally agreed that the tested sample should reduce the infectious titer by at least 3 log to show significant antiviral potential, FLM cannot be regarded as such. However, plant extracts are complex mixtures of compounds belonging to various groups of secondary metabolites, and the biological activity of such extracts depends on their composition, and the relative amount of particular substances and possible biological interactions (ex. synergism or antagonism). One of the end-point dilution assays performed for virus-infected cells treated is presented in Figure 4; in this particular experiment, the reduction of HHV-1 titer was 3.1 log. Considering this, the reported results can be regarded as interesting, and the observed antiviral activity will be further evaluated to elucidate the compounds responsible. We have previously reported that Oenanthe aquatica and Oenanthe silaifolia extracts possess significant antiviral activity, and the observed effect may be related to the presence of caffeic acid and its derivatives (caffeic acid glucoside, chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid) present in those extracts [52]. Interestingly, caffeic acid derivatives were identified in the FLM, which showed the highest anti-HHV-1 activity, and in FSBI, which exerted a noticeable, though much lower, influence on the tested herpes virus, reducing the infectious titer only by 0.92 log. Furthermore, methyl gallate, detected exclusively in the FLM, was proven to be a potent and specific inhibitor of HHV-2 [53]. Additionally, FLM was the only extract showing the presence of 5,8-dihydroxy-7-methoxyflavone-Oglucoside-rhamnoside; there are reports of antiviral activity of some flavone compounds ex. 5,7-dihydroxy-3,4 -dimethoxyflavone (ermanin) and 5,7,4 -trihydroxy-3-methoxyflavone (isokaempferide) against polio [54] or 5,7,4 -trihydroxy-8-methoxyflavone against influenza virus [55], while 5-hydroxy-7-methoxyflavone and 5,7-dimethoxyflavone were found to be protease inhibitors active against HIV-1, HCV, and HCMV (Human cytomegalovirus, HHV-5, CMV) at micromolar concentrations [56]. The kaempferol-O-glucoside present in FLM was also isolated from Securigera securidaca and reported to inhibit HHV-1 attachment to the cell membrane, virus entry and viral polymerase [57], and showed potent anti-HIV-1 reverse transcriptase activity [58]. Flavone glycosides, namely quercetin-3-Orutinoside, kaempferol-3-O-rutinoside and kaempferol-3-O-robinobioside, were reported by Yarmolinsky et al. [59] as being responsible for the antiviral potential of Ficus benjamina. Interestingly, isolated glycosides exerted significant antiviral activity against HHV-1 and HHV-2, especially when added to infected cells during and after infection, but no activity was found against HHV-3 (varicella-zoster virus, VZV). Flavone aglycones, kaempferol and quercetin, obtained as standards, showed significantly lower activity [59]. Finally, FLM was the only extract that showed the presence of (epi)-afzelechin-7-O-glucoside, and of note, ent-epi-afzelechin-(4-8)-epiafzelechin was reported to inhibit HHV-2 by disrupting virus penetration and interfering with late stages of the viral replication cycle [60].

Plant Materials and Preparation of Extracts
Ficus sur samples were collected in the village of Prikro (city of Brobo, Côte d'Ivoire), in January 2020. The species was identified by a plant taxonomist at the National Floristic

Plant Materials and Preparation of Extracts
Ficus sur samples were collected in the village of Prikro (city of Brobo, Côte d'Ivoire), in January 2020. The species was identified by a plant taxonomist at the National Floristic

Plant Materials and Preparation of Extracts
Ficus sur samples were collected in the village of Prikro (city of Brobo, Côte d'Ivoire), in January 2020. The species was identified by a plant taxonomist at the National Floristic Center (Universite Felix Houphouet Boigny, Abidjan, Côte d'Ivoire). Voucher specimens were deposited at the herbarium of the above-mentioned center. The leaves and stem barks of the plant samples were dried in shade conditions at room temperature for about one week. Then, the samples were powdered with a mill and stored in dark conditions. Different solvents (methanol and water) were used to obtain the extracts in this study. Maceration was used as the extraction method for methanol extracts. In addition, the infusion was prepared. For the maceration, the plant materials (10 g) were macerated with 200 mL methanol at room temperature overnight. After that, the mixtures were filtered, and the solvents were evaporated. In preparing the water extracts, the plant materials (10 g) were kept with 200 mL boiled water for 15 min and then filtered. Water extracts were lyophilized, and all extracts were stored at 4 • C until analysis.

Chromatographic Conditions
The separation was performed on a C18 Gemini ® column (3 µm i.d. with TMS endcapping, 110 Å, 100 × 2 mm) supported with a guard column (Phenomenex Inc, Torrance, CA, USA), at a flow rate of 0.2 mL/min under a gradient program operated by Agilent 1200 Infinity HPLC (Agilent Technologies, Santa Clara, CA, USA). Solvent A was water with 0.1% formic acid (v/v), whereas solvent B was 0.1% formic acid in acetonitrile (v/v). Both solvents were mixed according to the following program: 0-60% B for 45 min., next 60-95% B for 1 min., and 95% B for 4 min. The stop time was at 50 min. 10 µL of the sample was injected into a thermostated (20 • C) chromatographic column.

Detection Conditions
Mass spectra were acquired by the Agilent 6530B QTOF Accurate-Mass QTOF system equipped with Dual Agilent Jet Stream spray source (ESI) (Agilent Technologies, Santa Clara, CA, USA) connected with N 2 generator (Parker Hannifin Corporation, Haverhill, MA; generating N 2 at purities >99%). Negative ion mode was applied for MS and MS/MS acquisition with drying gas temp: 275 • C, drying gas flow: 10 L/min, sheath gas temp: 325 • C, sheath gas flow: 12 L/min; nebulizer pressure: 35 psig, capillary V (+): 4000 V, skimmer 65 V, fragmentor 140 V. Two spectra per sec were recorded in a range between 100 and 1000 m/z with a collision energy of 10 and 40 eV. The identification of compounds was based on fragmentation patterns and supported by a comparison of obtained mass spectra with those available in databases and the scientific literature.

Total Phenolic and Flavonoid Content
Total levels of phenolics and flavonoids were assessed based on previously reported methods [61,62]. Total phenolic levels were expressed as mg gallic acid equivalents (GAE)/g dry extract, and mg rutin equivalents (RE)/g dry extract was used to evaluate total flavonoids. All experimental details are given in the Supplementary Materials. The experiments were performed in triplicate, and the results were assessed by ANOVA assays (Tukey's test).

Antioxidant and Enzyme Inhibitory Assays
In the current investigation, the antioxidant effects of the tested extracts were detected by different assays [61]. The assays were: [1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2 -azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid (ABTS) radical scavenging, cupric ion reducing antioxidant capacity (CUPRAC), ferric ion reducing antioxidant power (FRAP), metal chelating ability (MCA) and phosphomolybdenum assay (PDA)]. For DPPH, ABTS, CUPRAC and FRAP assays, data were expressed as mg Trolox equivalents (TE)/g extract, whereas in MCA and PDA, mg EDTA equivalents (EDTAE)/g extract and mmol TE/g extract, respectively, were used. The experimental details for acetylcholinesterase, butyrylcholinesterase, tyrosinase, amylase and glucosidase assays were previously provided. Galanthamine was used as a positive control in cholinesterase assays, and data were evaluated as mg galanthamine equivalents (GALAE)/g extract. Kojic acid was used as a standard inhibitor in tyrosinase inhibitory assay, and the results were expressed as mg kojic acid equivalents (KAE)/g extract [61,62]. Acarbose was selected as an inhibitor of both amylase and glucosidase in the antidiabetic assays, and the results are given as mmol acarbose equivalents (ACAE)/g extract. All experimental details are given in the Supplementary Materials. The assays were performed in triplicates, and the differences in the extracts were evaluated by ANOVA (Tukey's test).

Cytotoxicity Testing
The evaluation of cytotoxicity was performed against normal kidney fibroblasts (VERO) and cancer cell lines derived from cervical adenocarcinoma (HeLa) and colon cancer (RKO) using microculture tetrazolium assay (MTT) as previously described [52]. Briefly, the cell monolayers were incubated with serial dilutions of the tested extracts for 72 h, and then cellular viability was assessed using the MTT protocol. Details can be found in the Supplementary Materials. The collected data were analyzed using GraphPad Prism to calculate the CC 50 values (50% cytotoxic concentration). Additionally, selectivity indexes (SI) were calculated by comparing CC 50 values obtained for VERO with those observed for cancer cells (SI = CC 50 VERO/CC 50 Cancer, SI > 1 indicates selectivity towards cancer cells).

Evaluation of Antiviral Potential
The extracts in non-toxic concentrations were tested for their influence on HHV-1 replication in the virus-infected VERO cells after 72 h incubation as previously described [52]. Briefly, the monolayer of VERO cells was treated with HHV-1 (100-fold CCID 50 , CCID 50 -50% cell culture infections dose) for 1 h, followed by washing with PBS (phosphate-buffered saline) and further incubated until a cytopathic effect (CPE) was recorded in the virus control (VC). Subsequently, after three cycles of freezing (−72 • C) and thawing, the HHV-1 infectious titer in the collected samples was measured using an end-point titration assay. Finally, the HHV-1 titer (∆log) difference was calculated (∆log = logCCID 50 VC-logCCID 50 FE, FE-Ficus extract). The difference of ≥3 log is regarded as significant.

Conclusions
In conclusion, the F. sur methanolic stem bark extract demonstrated substantial in vitro antioxidant potential with DPPH, ABTS, and CUPRAC assays, but not with FRAP, metalchelating and phosphomolybdenum assays. The methanolic stem bark extract significantly depressed tyrosinase, α-amylase, AChE and BChE activity. To date, no evidence of enzyme inhibitory actions of Ficus members has been discovered. In this regard, the presented work is the first scientific demonstration of the enzyme inhibitory effects of F. sur extracts, and it may offer a substantial contribution to the scientific platform. Herein, we would like to report that the F. sur leaves methanolic extract exerted noticeable, but limited, antiviral activity against HHV-1, diminishing CPE development and reducing the virus titer by 2.86 log. Furthermore, antineoplastic activity against cervical adenocarcinoma and colon cancer cell lines was observed for stem bark infusion and methanolic extract. However, more study, including in vivo and clinical investigations, is needed to further examine these aforementioned properties to incorporate this traditional herb as a possible therapeutic element.