β -Sitosterol Mediates Gastrointestinal Smooth Muscle Relaxation Induced by Coccoloba uvifera via Muscarinic Acetylcholine Receptor Subtype 3

: Coccoloba uvifera is a Mayan medicinal plant, and these leaves are used as antidiarrheal and diuretic agents. In the present work, we develop in-vitro, ex-vivo, in-vivo, and in-silico strategies to evaluate several aqueous extracts of C. uvifera leaves. In vitro tests showed that decoction extract (CuDe) presented the best yield and chlorophyll, phenol, and flavonoid content; however, CuDe showed low antioxidant activity (DPPH model). All aqueous extracts exert spasmolytic and vasore-laxant activity in a concentration-dependent manner (ex vivo), and in vivo tests showed that CuDe exerts the best antiperistaltic and diuretic effects. The in-silico analysis suggests that C. uvifera triter-penes act as a ligand of GPCR, and β -sitosterol could act as an antagonist of muscarinic acetylcholine receptor subtype 3 (m3AChR). In the context of aqueous extracts of C. uvifera , β -sitosterol and their heterosides were identified by FTIR and 1 H-NMR spectroscopy. The concerted binding of β -sitosterol and other triterpenes within the m3AChR binding site may be relevant for the induction of relaxant effects at the gastrointestinal smooth muscle level. In this context, C. uvifera is a high-value plant species that requires analytical and pharmacological studies to confirm traditional medicinal use.


Introduction
As noted in the literature, there has been an increase in information related to the involvement of chemical entities in the pharmacological effects induced by medicinal plants [1].Currently, many medicinal plants are used in cardiovascular, respiratory, gastrointestinal, and urinary diseases, among others [2].The therapeutic potential of medicinal plants increases with regional idiosyncrasy, as well as with a shortage of allopathic medicines [3].In a health system, herbal preparations represent a relevant strategy for therapeutic diseases.In addition, it is the historical strategy used for the identification of new chemical entities with pharmacological applications.
Historically, the study of natural products has been interested in the isolation of chemical entities, physicochemical characterization [4], and their pharmacological evaluation.Technological development has allowed for the application of bio-guided strategies and the identification of numerous chemical entities with therapeutic applications [5].The evolution in the study of natural products is directed toward the use of mathematical and statistical methods in the chemical context [6], allowing for the application of computational approaches in the discovery of drugs from natural products [7].Nowadays, more and more research simultaneously uses computational models, chemical or cellular experiments, and well-characterized and controlled animal models [8].
Despite extensive studies being conducted, few of them report on the functional properties at the gastrointestinal smooth muscle level.In this context, C. uvifera is a highvalue plant species that requires pharmacological and phytochemical studies to confirm traditional medicinal use.Then, the present work focuses on the phytochemical and pharmacological evaluation (in silico, in vitro, ex vivo, and in vivo) of several aqueous extracts from the leaves of C. uvifera.

Plant Material
Leaves of C. uvifera (LCu) were collected in the urban coastal area of San Francisco de Campeche, Campeche, Mexico, in April 2021.The specimens collected were identified and deposited in UAC-Herbarium (V: P-7570).

Preparation of the Extracts
After drying at room temperature (seven days), LCu were crushed and grounded to storage in sealed plastic bags (Ziploc ® ).All aqueous extraction methods, a fixed mass of C. uvifera, and a volume of distilled water (ratio: 1/28; g/mL) were employed.
For maceration (CuMa), LCu was kept in contact with water for 24 h at room temperature.Percolation (CuPe): A column (i.d. 2 cm; 25 mL) was packed by LCu, and separation was started by eluting distilled water (24 • C) at 1 mL/min flux (±25 min).Infusion (CuIn): Water at 100 • C was transferred to the LCu flask, homogenized, and removed from the heat source; the extraction process was carried out until the extract reached room temperature (±35 min).Decoction (CuDe): The temperature of the LCu extraction system was raised from room temperature to boiling point (±97 • C) and kept for 15 min, and then it was removed from the heat source.Digestion (CuDi): The temperature of the LCu extraction system was raised to 45 • C and kept for 15 min.The extraction process for CuDe and CuDi was carried out until reaching 23 • C. Finally, all the extracts were filtered and dried using forced air at 60 • C and stored at 5 • C for further analysis.

Spectroscopic Analysis
In total, 5 mg of CuMa, CuPe, CuIn, CuDe, and CuDi and 195 mg of KBr were mixed in an agate mortar, and FTIR analyses were carried out.For this, pellets were loaded in Thermo Nicolet Nexus 670: 500-4000 cm −1 at 4 cm −1 of resolution.analyses were carried out using the Varian-Agilent AR Premium Compact spectrometer (Santa Clara, CA, USA).The spectra 1 H were acquired with 64 and 50,000 scans, and the spectral width was 16 and 250 ppm, respectively.

Ex Vivo Evaluation
All experimental procedures and protocols with animals were developed according to NOM-062- ZOO-1999 [19], and they comply with the international guidelines on the care and use of laboratory animals (12 h light/dark cycle; 25 • C; food and water ad libitum).Adult male rats, Wistar strain (±275 g), were used after a three-day adaptation period.After abdominal dissection, the ileum and thoracic aorta were obtained free of connective tissue and fat.All tissues were maintained in a Ringer-Krebs-Henseleit solution (RKHS).
For spasmolytic evaluation [20], segments of the ileum (±0.5 cm) were used; tissues were stabilized at one gram of tension for 30 min.The activity exerted by aqueous extracts of C. uvifera (0.03 → 300 µg/mL) and papaverine (0.001 → 3 µg/mL) solutions was compared to the basal state of the spasmodic activity of the ileum.For vasorelaxant evaluation [21], aortic rings (±0.3 cm) were stabilized at three grams of tension for 30 min.Then, all the aorta ring tissues were sensitized with noradrenaline (NA, 1 × 10 −7 M) for 15 min, washed with RKHS, and re-sensitized with NA after 30 min.The presence of endothelium was corroborated by the response to carbachol (1 × 10 −6 M).The vasorelaxant activity induced by aqueous extracts of C. uvifera (0.1 → 300 µg/mL) and carbachol (0.00056 → 2 µg/mL) solutions was compared with the maximal contraction induced by NA.Acqknowledge software (MP150, Biopac Instruments, Co., Goleta, CA, USA) was used, and six independent experiments were performed (n = 6) for each extract.

In Vitro Tests
The chlorophyll content [22] in CuIn, CuMa, CuPer, CuDe, and CuDi was estimated by measuring absorbance at λ = 645 and 663 nm, as well as the use of Chl TOT (µg/mL) 20.2 (A 645 ) + 8.02 (A 663 ) formula.Neutral and acidified (HCl 0.1 N; t = 2 h) aqueous extracts of C. uvifera (10 mg/mL) were evaluated using the Lieberman-Burchard test; the change in color to green was considered positive [23].
For simple phenols [24], 0.4 mL from GA (1.04-8.2µg/mL) or extract stock solution (10 µg/mL) and 0.4 mL of Folin-Ciocalteu reagent were transferred to 1 mL of distilled water and incubated for 30 min in darkness.The reaction was stopped with Na 2 CO 3 10% (1.6 mL), and the absorbance was recorded at 765 nm.The content of simple phenols was expressed as equivalents (mg/g) of gallic acid (GA).For flavonoids [25], 0.3 mL of quercetin (1-10 µg/mL) or extract stock solution (10 µg/mL) was added to 0.9 mL of MeOH, 0.15 mL of AlCl 3 10% solution, 0.15 mL of CH 3 CO 2 K 1 M, and 1.8 mL of distilled water.All samples were incubated for 30 min at room temperature in the dark, and the absorbance was recorded at 415 nm.The content of flavonoids in aqueous extracts was expressed as equivalents (mg/g) of quercetin.
For antioxidant activity [26], 0.2 mL of C. uvifera extracts (100 µg/mL), C. sinensis (50 µg/mL) extract, or quercetin (10 µg/mL) solution was added to 1.8 mL of DPPH 0.2 M.After mixing, all the samples were incubated at room temperature in the dark for 30 min.Then, absorbance was recorded at 517 nm, and antioxidant activity was calculated.The percentage of the discoloration (%) of DPPH was calculated using: % = 1 − Amuesta ADPPH * 100.All samples were subjected to three independent experiments with three replicates each.

In Silico Approach
Petra/osiris/molinspiration analysis (POM) is one approach that has been used in Medicinal Chemistry to identify the pharmacophore site that affects the pharmacological activity.In this sense, POM analysis predicts biological activities of chemical entities through steric/electrostatic properties with ligand-receptor interaction [27], is validated with ±7000 drug molecules available in databases, and has been used in the evaluation of natural products with applications in hypertension [28].
On the other hand, to determine the interaction with m3AChR, molecular docking studies with m3AChR were performed.In this sense, the active site on m3AChR was validated using scopolamine (muscarinic receptor antagonist and natural product isolated from Solanaceae species) as the native ligand (PDB: 5ZHP).RMSD was set to less than 2.5 Å to determine the best docking position between m3AChR and ligands using Autodock Vina [29].The validation was performed with 1000 poses, each in ten replicates, selecting the lowest energy value.Protein visualization and overlap were carried out using Pymol 3.1 (Schrödinger, San Diego; http://www.pymol.org/).
The docking of each ligand (β-sitosterol, lupeol, α-amyrin, β-amyrin, and royleanone) was simulated using the program AutoDock Vina, which has been used to estimate the conformation of protein-ligand complexes and significantly improves the average accuracy of the binding mode predictions.All calculations for protein-fixed ligand-flexible docking were analyzed using the Lamarckian Genetic Algorithm (LGA) method.The docking site on m3AChR was defined by establishing a grid box using Pymol 3.1.The grid box size for the coordinates x, y, and z was 25 Å, with a grid spacing of 0.375 Å, centered on x = −2.265,y = 7.164, and z = 17.568Å.The best conformation was chosen based on the lowest binding energy after the docking search was completed.Autodock Vina was set for 1000 modes, and in terms of exhaustiveness (exhaustiveness of the global search, roughly proportional to time) for each ligand structure, and for each run, the best pose was saved.The average binding energy for the best poses was used as the final binding energy value; this process was repeated ten times [29].

Data Analysis
Data were stored in a database and processed through Origin Microcal 8.0.The results are expressed as the mean ± standard error of the mean.Finally, an analysis of variance was performed followed by Tukey's test; values of p < 0.05 were considered significant.

Phytochemical In Vitro Test
The use of different extraction methods in the C. uvifera leaves allowed for recording variations in the extraction yield as well as other phytochemical parameters.Table 1 shows the extraction yields, chlorophyll, phenols, flavonoid content, and antioxidant activity for different C. uvifera leaf extracts.Colorimetric analysis through the Lieberman-Burchard test indicates the presence of sterols; likewise, the color intensity increased in the aqueous extracts subjected to acid hydrolysis.
The chlorophyll, simple phenols, and flavonoid content improve with an increase in temperature (CuDe > CuMa; p < 0.05) and time (CuMa > CuPer; p < 0.05) of extraction.In the DPPH model, all extracts were evaluated at 100 mg/mL, the methanol extract of C. sinensis was evaluated at 50 mg/mL, and quercetin was evaluated at 10 mg/mL.The antioxidant activity induced by CuPer (E max : 64.3 ± 1.1%) was significantly greater than CuMa, CuDi, CuIn, and CuDe; however, it was lower than C. sinensis (E max : 53.28 ± 0.2%) and quercetin (E max : 91.62 ± 0.13%).The antioxidant activity exerted by C. uvifera decreases with increasing temperature and extraction time (Table 1).

Ex Vivo and In Vivo Evaluation
CuInf, CuMa, CuDe, and CuDi extracts induce partial spasmolytic and vasorelaxant (endothelium +) effects in a concentration-dependent manner; decreased vasorelaxant activity is observed in CuPer.The potency and efficacy of spasmolytic and vasorelaxant effects were lower than papaverine (Figure 4A) and carbachol (Figure 4B, Table 2).

Ex Vivo and In Vivo Evaluation
CuInf, CuMa, CuDe, and CuDi extracts induce partial spasmolytic and vasorelaxant (endothelium +) effects in a concentration-dependent manner; decreased vasorelaxant activity is observed in CuPer.The potency and efficacy of spasmolytic and vasorelaxant effects were lower than papaverine (Figure 4A) and carbachol (Figure 4B, Table 2).

In Silico Approaches
Fourteen metabolites have been described in C. uvifera leaves, and each one has different theoretical physicochemical properties that determine the extraction in aqueous systems (Table 3).

In Silico Approaches
Fourteen metabolites have been described in C. uvifera leaves, and each one has different theoretical physicochemical properties that determine the extraction in aqueous systems (Table 3).Molinspiration is a virtual tool that allows for the calculation of molecular properties that influence the bioavailability of chemical entities that are pharmaceutical candidates [33].Sterols and pentacyclic triterpenes registered low TPSA values, which suggests low aqueous solubility, unlike flavonoid glycosides.Royleanone, gallic acid, and anthraquinones (emodin, chrysophanol, physcion, rhein) registered cLogP values appropriate for absorption at the membrane level and did not break Lipinski's rule, so they could cross the enterocyte membrane, among others.The bioavailability of metabolites with one or more violations of the Lipinski rule could be justified using active transport mechanisms.
β-sitosterol, lupeol, α-amyrin, and β-amyrin described in C. uvifera leaves show the best biological (Table 4) by acting as a nuclear receptors ligand (NRL), enzyme inhibitors (Eis), or a ligand of the G protein-coupled receptor (GPCR), and, to a lesser extent, as protease inhibitors (PIs) and ion channel modulators (ICMs).The aqueous nature of traditional preparations and their oral use allow us to infer that the induction of biological effects will be primarily in the gastrointestinal tract (stomach, duodenum, ileum, jejunum, and colon).In the heat map, the green color is applied to metabolites with potential biological activities and low toxicity (Table 4).Emodin, gallic acid, chrysophanol, physcion, rhein, and royleanone present in the leaves of C. uvifera are secondary metabolites with toxicological

Molecular Docking m3AChR
m3AChR participates in physiological and pathophysiological conditions of gastrointestinal motility [34].To identify interactions of diterpenes (royleanone) and triterpenes (β-sitosterol, lupeol, αamyrin, β-amyrin) from C. uvifera with m3AChR, we carried out molecular docking simulation between terpenoids and m3AChR.First, the active site of the m3AChR chain was validated with a co-crystallized native ligand: scopolamine (a muscarinic antagonist).Comparison of the poses obtained by the AutoDock Vina program against those of the crystallized protein yielded a root mean square deviation (RMSD) = 2.48 Å, indicating an appropriate optimization score.These values are small and support binding at the simulation site with the original orientation of the co-crystallized molecule (Figure 6A,B).
I: irritant; R: reproductive; S: solubility; DL: drug-likeness; DS: drug score.Positive (+); negative The green color is applied to metabolites with potential biological activities and low toxicity.T red color indicates low biological activity and high toxicity.

Molecular Docking m3AChR
m3AChR participates in physiological and pathophysiological conditions of gast intestinal motility [34].To identify interactions of diterpenes (royleanone) and triterpen (β-sitosterol, lupeol, α-amyrin, β-amyrin) from C. uvifera with m3AChR, we carried o molecular docking simulation between terpenoids and m3AChR.First, the active site the m3AChR chain was validated with a co-crystallized native ligand: scopolamine muscarinic antagonist).Comparison of the poses obtained by the AutoDock Vina progra against those of the crystallized protein yielded a root mean square deviation (RMSD 2.48 Å, indicating an appropriate optimization score.These values are small and supp binding at the simulation site with the original orientation of the co-crystallized molecu (Figure 6A,B).β-sitosterol, lupeol, α-amyrin, β-amyrin, and royleanone exhibited docking pos with high binding affinities (in terms of coupling energy).The binding energies and ami acid interactions for each triterpene with m3AChR are given in Table 5.The binding int actions of β-sitosterol (Figure 6) with the active site of m3AChR are stabilized throu hydrophobic interactions.From docking results, binding energies for m3AChR with sitosterol and royleanone are ±10 and 16.6% less than scopolamine.β-sitosterol, lupeol, α-amyrin, β-amyrin, and royleanone exhibited docking poses with high binding affinities (in terms of coupling energy).The binding energies and amino acid interactions for each triterpene with m3AChR are given in Table 5.The binding interactions of β-sitosterol (Figure 6) with the active site of m3AChR are stabilized through hydrophobic interactions.From docking results, binding energies for m3AChR with β-sitosterol and royleanone are ±10 and 16.6% less than scopolamine.The Ki value for the binding of β-sitosterol with m3AChR was lower than scopolamine (Ki = 0.21 vs. 0.03 µM; ±10 times less); royleanone and β-amyrin registered ±16 and ±1700 times less affinity to m3AChR, respectively.Docking analysis showed that β-sitosterol with hydrophobic interactions has a higher binding affinity for m3AChR concerning β-amyrin and royleanone.The alkyl chain in C17 of β-sitosterol forms alkyl-alkyl interactions with Leu144, Tyr148, Leu225, and Tyr529, and pi-alkyl and pi-sigma interactions were observed with Phe221 and Trp525.H-bonds in β-amyrin (Asn526) seem not to be relevant in the interaction with Acho-M3r, pi-sigma interactions were observed with Trp525, and alkyl interactions were prevalent with ring A. All details of the bonding are given in Table 5.

Discussion
Aqueous preparations are typically made from polar solvents and use continuous and discontinuous processes of extraction [35]; additionally, temperature and time exert variations in the chemical content of the aqueous extracts.In this context, several aqueous extracts from dry leaves of C. uvifera were obtained and tested; CuDe favors the best extraction yield, but the content of chlorophylls, phenols, and flavonoids, however, exerts low antioxidant activity.CuMa demonstrates excellent yield; however, the aglycone extraction capacity is limited.In this sense, the yield of CuDe and CuMa could be related to the extraction of heterosides among other components of polar nature.
Time, temperature, polarity solvent, and pH are involved in the extraction procedure to obtain raw material [35,36].In CuDe, the increased temperature favors the solubility of metabolites as well as their diffusion [37]; however, it is reported that pH, temperature, and oxidizing environments modify the stability of aglycones and heterosides of flavonoid [38], temperatures above 50 • C decrease the content of polyphenols due to thermal degradation [39], phenolic alcohols and carboxylic acids could be related to the formation of artifacts [40], and aromatic alcohols are prone to the oxidation and formation of H 2 O 2 , quinones, and semiquinones [41], facts that suggest the loss of antioxidant activity.CuPer induces increased antioxidant activity and is a low thermal stress process.
The partial spasmolytic and vasorelaxant effects induced by C. uvifera are exerted in a concentration-dependent manner through actions at the smooth muscle level; on the other hand, temperature and time extraction reduce the effects at the smooth muscle level.In the context of aqueous extracts of C. uvifera, pharmacological bases are established for the search for new drugs or their use in the practice of clinical and traditional medicine [42].It is highlighted that flavonoids [43], terpenoids [44], and stilbenoids [45], among other metabolites, are identified as smooth muscle relaxants.
The organoleptic properties of aqueous extracts are related to the extraction method; in the in vivo model, ad libitum administration of the aqueous extracts of C. uvifera leaves modify oral consumption and consequently the dosage, a situation that differentially affects intestinal transit and diuresis.Emodin, chrysophanol, physcion, and rhein are reported to exert laxative effects through a potential interaction with c-kit, 5-HT4, and AQP3 [46]; however, aqueous extracts of C. uvifera reduce stool output.The absence of laxative effects in the experimental groups can be related to the low abundance of anthraquinones.More than 0.5% of anthraquinones in dry material are recommended to be considered a laxative medication [47].Quantitative studies are required to corroborate this assertion.
To recognize the metabolite(s) involved in the effects induced by C. uvifera, POM analysis was developed.In this context, the main transport mechanism of aglycones is simple diffusion.The biological activity may be related to enzymatic inhibition and interaction with G protein-coupled receptors distributed in the gastrointestinal tract [48][49][50][51][52][53].Muscarinic receptors play an important role in the control of gastrointestinal and vascular smooth muscle [54].m3AChR is coupled to the G protein (G q ) and mediates an increase in intracellular calcium (Ca 2+ ) that typically causes the constriction of smooth muscle; additionally, in vascular endothelial cells, it increases the production of nitric oxide (NO) and causes relaxation and vasodilation [55].Triterpenes such as ursolic acid [21], oleanolic acid, erythrodiol [56], and others exert relaxant effects on smooth muscle tissues.
Phytochemical reports [17] as well as chemical and spectroscopic analyses suggest the presence of β-sitosterol in C. uvifera leaves.Aglycone and β-sitosterol heterosides have been reported in various plant species [57,58], and the presence of heterosides favors solubility in aqueous extraction systems (e.g., CuIn, CuMa, CuPer, CuDe, or CuDi); the acid or enzymatic hydrolysis [59] of β-sitosterol heterosides could improve the release of β-sitosterol, as well as the spasmolytic effect.The docking of scopolamine used as the native ligand, with the modeled m3AChR protein, was simulated using the AutoDock Vina.To explore the binding affinity and the molecular basis of the interactions with m3AChR, docking analysis of di-and triterpenes generates negative values for free energy, suggesting a high affinity for the binding pocket.β-sitosterol has a higher binding affinity for m3AChR based on lower binding energies that contribute to a stable protein-ligan complex and thus induce a tone reduction in smooth muscle.The spasmolytic effects induced by aqueous extracts of C. uvifera could be exerted through actions with m3AChR and a reduction in the peristalsis of the gastrointestinal tract [48].Figure 7 explains the molecular mechanism of β-sitosterol on gastrointestinal smooth muscle relaxation.Finally, temperature favors the diuretic effect.Flavonoid heterosides hydrolysis could be related to vasorelaxant and diuretic effects.The bioavailability of flavonoid heterosides or their aglycones (myricetin and quercetin) could be related to the diuresis exerted by C. uvifera (CuDe) concerning the vehicle (±26.8%) and furosemide (±10.5%)groups.Myricetin 3-O-rhamnoside (myricitrin) exerts diuresis, natriuresis, and kaliuretic effects in a dose-dependent manner [60]; on the other hand, Quercetin-3-O-β-D-glucopy- Finally, temperature favors the diuretic effect.Flavonoid heterosides hydrolysis could be related to vasorelaxant and diuretic effects.The bioavailability of flavonoid hetero-sides or their aglycones (myricetin and quercetin) could be related to the diuresis exerted by C. uvifera (CuDe) concerning the vehicle (±26.8%) and furosemide (±10.5%)groups.Myricetin 3-O-rhamnoside (myricitrin) exerts diuresis, natriuresis, and kaliuretic effects in a dose-dependent manner [60]; on the other hand, Quercetin-3-O-β-D-glucopyranoside is related to diuretic activity in a concentration-dependent manner [61].Myricetin exerts a wide variety of biological effects, including ACE inhibition and the release of nitric oxide or prostacyclin at the endothelium level [62].Quercetin exerts a vasodilation effect by stimulating the vascular CaV1.2 channel current [63].This result suggests a potential diuretic effect of aqueous extracts from C. uvifera leaves.However, it is required to determine whether the effects are dose-dependent.

Conclusions
The temperature and extraction time modify the content of the pigments, simple phenols, and flavonoids present in C. uvifera extracts and negatively affect the antioxidant activity; however, these factors favor the extraction of metabolites with actions at the smooth muscle level.
C. uvifera exerts a spasmolytic and vasorelaxant effect in a concentration-dependent manner, suggesting interactions with specific molecular targets on the smooth muscle.POM analysis indicates that metabolites interact with gastrointestinal enzymes and GPCRs.Molecular docking indicates that β-sitosterol is an antagonist of m3AChR and affects the gastrointestinal level function; the presence of β-sitosterol and their heterosides was by FTIR and 1H-NMR.In the context of the antiperistaltic effects, it is necessary to determine the effective dosage of CuDe extract and β-sitosterol, among other participating metabolites.
Finally, the flavonoid heterosides and their aglycones (myricetin and quercetin) from C. uvifera leaves are related to diuretic effects.

Figure 2 .
Figure 2. Two-dimensional structure of β-sitosterol glucoside.Red bonds indicate the electronegativity induced by oxygen atoms.

Figure 2 .
Figure 2. Two-dimensional structure of β-sitosterol glucoside.Red bonds indicate the electronegativity induced by oxygen atoms.

Figure 3 .
Figure 3. Representative H-NMR profiles (region 0 to 14 ppm) of the aqueous extracts from C. uvifera leaves obtained by different extraction methods.

Figure 3 .
Figure 3. Representative H-NMR profiles (region 0 to 14 ppm) of the aqueous extracts from C. uvifera leaves obtained by different extraction methods.

Figure 2 .
Figure 2. Two-dimensional structure of β-sitosterol glucoside.Red bonds indicate the electronegativity induced by oxygen atoms.

Figure 3 .
Figure 3. Representative H-NMR profiles (region 0 to 14 ppm) of the aqueous extracts from C. uvifera leaves obtained by different extraction methods.

igure 5 .
Consumption of food, as well as urine and feces production induced by CuInf, CuMa, CuPer, CuDe, and CuD, furosemide, and vehicle groups.Results are expressed as the mean ± SEM of four experiments (n = 12; p < 0.05 vs. * furosemide; vs. vehicle).

Figure 6 .
Figure 6.Ligand binding site of m3AChR protein with co-crystalized native scopolamine (gre and scopolamine, as posed by the Autodock Vina program (pink; (A)).Molecular docking simu tion showing the interaction of β-sitosterol with active site residues of m3AChR (grey; (B)).

Figure 6 .
Figure 6.Ligand binding site of m3AChR protein with co-crystalized native scopolamine (green) and scopolamine, as posed by the Autodock Vina program (pink; (A)).Molecular docking simulation showing the interaction of β-sitosterol with active site residues of m3AChR (grey; (B)).

Table 1 .
Yield and chlorophyll, phenols, and flavonoid content in different aqueous extracts derived from leaves of C. uvifera.
Note: Results are expressed as a mean ± SEM of six experiments (* p < 0.05 vs. CuMa); % of dry matter.

Table 3 .
Physicochemical properties by Osiris calculations of chemical entities identified and/or isolated from leaves of C. uvifera.

Table 4 .
Molecular properties of chemical entities identified in leaves of C. uvifera.

Table 5 .
Binding affinity for the molecular coupling in m3AChR with scopolamine and triterpenes described in C. uvifera leaves.