Enzymatic assays and molecular modeling studies of Schisandra chinensis lignans and phenolics from fruit and leaf extracts

Abstract

Considerable interest has been shown in natural sources and their compounds in developing new therapeutically agents for different diseases. In this framework, investigations performed on this topic play a central role for human health and drug development process. Schisandra chinensis (Turcz.) Baill is a medicinal and edible plant showing highly advantageous bioactivity and nutritional value. The main bioactive compounds from its fruits are lignans, derivatives of dibenzocyclooctadiene whereas concerning its leaves, phenolic acids, and flavonoids are dominant. The purpose of this study was to investigate the enzyme inhibitory potential on selected carbohydrate hydrolases, cholinesterases, and tyrosinase of extracts from fruits and leaves of Schisandra in relation with their main bioactive compounds. Furthermore, the interactions between dominant compounds (schisandrol A, schisandrol B, schisandrin B, and cinnamic acid) from extracts and selected enzymes were investigated by molecular modeling and molecular dynamic studies in order to explain at a molecular level our findings.

Keywords:

• Enzyme inhibition
• molecular modeling
• lignans
• phenolics
• Schisandra chinensis

Introduction

Over the past four decades, several thousands of phytochemicals have been identified in plants. Phytochemicals are broadly found in foods, drugs, or remedies in traditional medicine. Many herbal products have health-promoting effects and, therefore, are marketed as such¹. Moreover, medicinal plants and natural products are a continuing source of inspiration in discovering novel therapeutic agents with high efficiency and less side effects². Consequently, much attention is given to phytochemicals with different biological properties such as antioxidant, antimicrobial, antiviral, antimutagenic, anticancer activity, or their use in pathologies such as Alzheimer’s disease (AD) and cardiometabolic disorders, in recent years^2–4. Among phytochemicals, phenolic compounds attracted much attention being probably the most investigated molecules of medicinal and nutritional interest^4–6. Besides, they exhibit various bioactivities such as antimicrobial, antiviral, anti-allergic, anti-inflammatory, and protective effects against cell and cutaneous aging^7,8. Their antioxidant properties as well as their beneficial effects against AD, diabetes mellitus (DM), and cardiovascular diseases are nowadays highly emphasized^6,9. After a period of being underestimated, in which synthetic compounds were favored, in recent years increasing interest of research community has been observed in the study of the biological activity of plants and the active compounds responsible for their therapeutic properties^10,11.

Schisandra chinensis (Turcz.) Baill. is a widely consumed edible and medicinal plant providing several health beneficial effects. Its fruits are used as tonic, sedative, adaptogenic, and in the treatment of several diseases such as chronic coughs, spontaneous sweating, palpitations, and fatigue^12,13. Modern research highlighted that S. chinensis has antitumor¹⁴, anti-hepatotoxic and hepatoprotective^15,16, anti-cancer¹⁷, neuroprotective¹⁸, anti-diabetic¹⁹, and antioxidant^17,20 properties. So far, its most important bioactive compounds are thought to be lignans, derivatives of dibenzocyclooctadiene, especially schisandrin (schisandrol A), schisandrol B, schisandrin A and schisandrin B^12,21 which are mainly present in the fruits. However, besides lignans, recently phenolic compounds and triterpenoids were identified in leaves and stems of S. chinensis showing promising bioactivities^22,23.

DM is considered one of the most common chronic metabolic diseases in nearly all countries. Especially the prevalence of type 2 DM, which accounts for around 90% of all diabetes cases worldwide, continues to increase due to the changing lifestyles that involve reduced physical activity and increased incidence of obesity²⁴. Increases in postprandial blood glucose levels have been linked to the absorption of carbohydrates in the small intestine. The intestinal absorption of dietary carbohydrates such as maltose and sucrose is carried out by a group of α-glucosidases and amylases. Therefore, inhibition of carbohydrate-hydrolyzing enzymes can significantly reduce postprandial hyperglycemia after a mixed carbohydrate diet and may be utilized to control DM²⁵. AD is a neurodegenerative disorder affecting generally elder people but also adults. In spite its multifactorial nature, currently just the cholinergic hypothesis is used as a therapeutic approach. Accordingly, at least some of the cognitive decline experienced by patients with AD results from a deficiency in neurotransmitter acetylcholine (ACh) and thus in cholinergic neurotransmission in brain cortical or hippocampal regions, which seems to play a fundamental role in memory²⁶. Therefore, the search for effective and safe cholinesterase (ChE) and carbohydrate enzyme inhibitors from natural sources remains an aimed objective.

Recent studies have shown S. chinensis stems extracts and sub-fractions as possessing important antidiabetic potential via the inhibition of tyrosine phosphatase 1B (PTP1B) and α-glucosidase²⁷. Moreover, previous research highlighted the acetylcholinesterase inhibitory effects of schisandrol B and gomisin A isolated from Schisandra fruits. However, up-to-date little is known about the enzyme inhibitory potential of Schisandra leaves. In a previous study, we reported that S. chinensis fruits are an important source of lignans in comparison with leaves and stems which contain significant amounts of phenolic compounds and cinnamic acid (CA), exhibiting high antioxidant activity²³. In the present work, the antidiabetic and neuroprotective activities of S. chinensis methanolic extracts (fruits and leaves) were evaluated through enzymatic assays using selected carbohydrate hydrolases (α-amylase and α-glucosidase), ChE, and tyrosinase. To better understand the inhibition mechanisms, docking experiments and molecular dynamics (MD) were performed on selected major compounds previously found in the extracts.

Materials and methods

Plant material and extraction procedure

Dried fruits and leaves of S. chinensis (Turcz.) Baill. were harvested from a local cultivator from Cluj-Napoca, Romania. The herbal material was obtained from an ecological culture and a voucher specimen was deposited in the Herbarium of the Department of Pharmaceutical Botany, Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy. Extracts were prepared according to the procedure previously described by Mocan et al.²³ Each sample (1 g) was extracted with 30 mL of methanol at room temperature in a sonication bath for 1 h. The extract was centrifuged and filtered and the residue was re-extracted twice, each time with an additional portion of 30 mL of methanol. Then the extracts were combined and evaporated under reduced pressure (t < 40 °C). Afterwards, the residue was re-dissolved in 10 mL of methanol. Prior to LC-DAD/ESI-ToF-MS analyses, the extracts were syringe filtered. For the bioassay, extracts obtained were evaporated under reduced pressure and further kept in a vacuum desiccator to fully remove traces of solvents. For each analysis, three different samples were used and the assays were performed in triplicate.

Chemicals and reagents

Lignans chemical standards (all >96%) were purchased from from ChromaDex (Irvine, CA). Phenolics standards (all >95%) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, Germany). Methanol and acetonitrile were purchased from VWR International GmbH (Darmstadt, Germany). All solvents were of LC grade and water was of Milli-Q-quality.

Enzyme inhibitory activity

Cholinesterase inhibition

ChE inhibitory activity was measured using Ellman’s method, as previously reported²⁸. Sample solution (50 μL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid) (125 μL) and AChE (acetylcholinesterase (Electric ell acetylcholinesterase, Type-VI-S, EC 3.1.1.7, Sigma-Aldrich)), or BChE (butyrylcholinesterase (horse serum butyrylcholinesterase, EC 3.1.1.8, Sigma-Aldrich)) solution (25 μL) in Tris-HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI) or butyrylthiocholine chloride (BTCl) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the ChE inhibitory activity was expressed as milligrams of galantamine equivalents (mg GALAE/g extract).

α-Amylase inhibition

α-Amylase inhibitory activity was performed using Caraway-Somogyi iodine/potassium iodide (IKI) method²⁸. Sample solution (25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma-Aldrich) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37 °C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37 °C, then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the IKI solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as millimoles of acarbose equivalents (mmol ACE/g extract).

α-Glucosidase inhibition

α-Glucosidase inhibitory activity was performed by the previous method²⁸. Sample solution (50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma-Aldrich) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside) (50 μL) in a 96-well microplate and incubated for 15 min at 37 °C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as millimoles of acarbose equivalents (mmol ACE/g extract).

Tyrosinase inhibition

Tyrosinase inhibitory activity was measured using the modified dopachrome method with L-DOPA as substrate, as previously reported²⁸ with slight modifications. Sample solution (25 μL) was mixed with tyrosinase solution (40 μL) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25 °C. The reaction was then initiated with the addition of L-DOPA (40 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after 10-min incubation at 25 °C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as equivalents of kojic acid (mg KAE/g extract).

Structural investigation and characterization of lignans and phenolic compounds by HPLC-DAD/ESI-ToF-MS

The structural characterization of lignans and phenolic compounds from fruits and leaves was carried out as described by Mocan et al.²³ Therefore, the analysis was carried out on a LC-DAD/ESI-ToF-MS system consisting of an HPLC Agilent 1200 Series with Agilent Mass Hunter B.05.01 software (Agilent Inc., Santa Clara, CA), and an ESI-TOF Agilent 6224 mass spectrometer with software Agilent Mass Hunter B.05.01 (Agilent Inc.) and MestReNova 7 (Mestrelab Research SL, Santiago de Compostela, Spain). The HPLC system consisted of a binary pump, an autosampler, a column compartment, and a diode array detector. The LC-DAD method was similar to the LC-DAD method as described above with the exception of a reduced flow rate of 0.5 mL/min. ESI-ToF experiments were recorded in the positive mode for lignans and negative mode for phenolics and a scan range between 110 and 1500 m/z in both cases. Identified compounds and their amounts in the samples are listed in Table 1. For further details on characterization, identification, and fragmentation patterns of the compounds, please refer to the study of Mocan et al.²³.

Table 1. Bioactive compounds in Schisandra chinensis fruits and leaves: identification and quantification (mg/g d.w. material) – mean ± SD.

Compound	MW(g/mol)	Leaves(mg/g dw)	Fruits(mg/g dw)
Schisandrol A*	432	1.29 ± 0.02	4.24 ± 0.21
Schisandrol B*	416	0.63 ± 0.01	1.45 ± 0.14
Schisandrin A*	416	0.23 ± 0.00	0.89 ± 0.09
γ-Schisandrin*	400	1.01 ± 0.01	2.27 ± 0.09
Cinnamic acid*	148	3.54 ± 0.06	ND
Protocatechuic acid*	154	0.53 ± 0.01	0.34 ± 0.01
3-O-Caffeoylquinic acid (neo-chlorogenic acid)	354	2.95 ± 0.02	ND
4-O-p-Coumaroylquinic acid	338	0.26 ± 0.01	ND
3-O-p-Coumaroylquinic acid	338	0.87 ± 0.04	ND
5-O-Caffeoylquinic acid* (chlorogenic acid)	354	4.99 ± 0.22	ND
3-O-Ferruloylquinic acid	368	0.19 ± 0.01	ND
4-O-Caffeoylquinic acid (crypto-chlorogenic acid)	354	0.15 ± 0.01	ND
trans-5-O-p-Coumaroylquinic acid	338	1.05 ± 0.05	ND
cis-5-O-p-Coumaroylquinic acid	338	0.41 ± 0.02	ND
Rutin*	610	0.38 ± 0.01	ND
Quercetin-3-O-glucoside-7-O-rhamnoside	610	1.70 ± 0.12	ND
Quercetin-3-O-galactoside	464	2.49 ± 0.05	ND
Quercetin-3-O-glucoside*	464	3.68 ± 0.01	ND
Quercetin-3-O-dirhamnoside	594	0.42 ± 0.01	ND
Quercetin-3-O-xyloside	434	0.49 ± 0.02	ND
Kaempferol-3-O-glucoside-7-O-rhamnoside	594	5.68 ± 0.09	ND
Kaempferol-3-O-glucoside	448	5.12 ± 0.07	ND
Kaempferol-3-O-arabinose	418	0.48 ± 0.01	ND
Cinnamic acid*	148	3.54 ± 0.06	ND
Quercetin*	302	0.29 ± 0.01	ND
Kaempferol*	286	0.63 ± 0.02	ND

Compounds with* were identified with references accurately. Data according to Mocan et al.²³

Molecular modeling studies

Receptors preparation

All the x-ray enzyme structures have been downloaded from the Protein Databank RCSB PDB²⁹: acetylcholinesterase (pdb:4X3C)³⁰ in complex with tacrine-nicotinamide hybrid inhibitor, butyrylcholinesterase (pdb:4BDS)³¹ in complex with tacrine, amylase (pdb:1VAH)³² in complex with r-nitrophenyl-α-D-maltoside, glucosidase (pdb:3AXI)³³ in complex with maltose, and tyrosinase (pdb:2Y9X)³⁴ in complex with tropolone. The enzymes were separated from their crystallographic inhibitors, also water molecules, ions, and other small molecules were removed by using UCSF Chimera software³⁵. Then the proteins were neutralized at pH 7.4 by Epik implemented in Maestro suite, seleno-cysteines and seleno-methionines when present, were converted respectively to cysteine and methionine, all the missing fragments and other errors present in the crystal structures were automatically solved by the Wizard Protein Preparation implemented in Maestro suite³⁶.

Ligands preparation

Lignans cp1, cp4, cp24 were selected from the pool of bioactive compounds found previously in our extracts, with the aim to study their docking properties on our enzymatic set. These compounds were prevalent in fruit and leaf extracts of S. chinensis, also the scarce literature on their activity have stimulated our interest. CA was present in the leaf extract in relevant amount, thus it has been selected for docking studies being a very well-known bioactive compound. All the chemical structures were downloaded from Zinc databases and in the case of the lignans the CAS number was also checked by SciFinder database³⁷ in order to confirm the chemical structures. The molecules and their alternative names used for molecular modeling experiments are reported in Scheme 1. The ligands were prepared by the ligand preparation tool embedded in Maestro, neutralized at pH 7.4 by Epik and minimized³⁶.

Scheme 1. Chemical structures of compounds cp1, cp4, cp24, and cinnamic acid (CA) employed for docking and molecular dynamic experiments.

Molecular docking

Dockings of compounds cp1, cp4, cp24 and CA have been performed for each enzyme used in this paper for the in vitro biological evaluation. The software Gold 6.0³⁸ has been employed for the docking calculations by using all the available scoring function: GoldScore (GS), ChemScore (CS), ASP, and PLP. The binding pocket was determined automatically by centering the grid on the crystallographic inhibitor and by extending the grid area in a radius of 10 Angstroms from the center. The best pose for each enzyme was selected among the four obtained with the application of the four scoring functions implemented in the software, by qualitative manual analysis with preferences with the presence in the docking complex of hydrogen bonds, coordinative bonds, pi-pi interactions over hydrophobic interactions. Figures 1, 3, 4A and B, and 5 reported the best and representative obtained enzyme–ligand complexes. These complexes have also been subjected to MD calculation (see Figures 2, 4C, and 6).

Figure 1. (A and C) best pose of compound cp1 in complex to acetylcholinesterase (GoldScore); (B and D) best pose of compound cp4 in complex to acetylcholinesterase (PLP).

Figure 2. RMSD in Angstrom of 26 ns MD simulation of compounds cp1 (GoldScore) (yellow), cp4 (PLP) (blue) in complex to acetylcholinesterase compared to the simulation of the crystallographic ligand tacrine-nicotinamide (light red).

Figure 3. (A and C) best pose of cp1 (PLP) in complex to glucosidase; (B and D) best pose of cp4 (PLP) in complex to glucosidase.

Figure 4. (A and B) best pose of cp1 (PLP) in complex to amylase; (C) RMSD in Angstrom of 26 ns MD simulation of compound cp1 (PLP) in complex to amylase.

Figure 5. (A and B) best pose of cp4 (GoldScore) in complex to tyrosinase; (C and D) best pose of CA (GoldScore) in complex to tyrosinase.

Figure 6. RMSD in Angstrom of 26 ns MD simulation of compounds cp4 (GoldScore), cp24 (ChemScore) and CA (GoldScore) in complex to tyrosinase.

Molecular dynamics

In order to gain further information about time-dependent behavior and stability of the compounds cp1, cp4, cp24 and CA docked in the binding pocket of the selected enzymes, MD experiments were carried out by Amber 14³⁹. The partial charges of the ligands were determined by using the AM1-BCC method of Antechamber suite. The complexes of the docking compounds and the relative enzyme were prepared by tleap suite, General Amber force field (GAFF) parameters were assigned to the ligands and ff14SB to the proteins. The complexes were hydrated in a cubic water box of 20 Angstroms, and the net charge of the system has been neutralized by adding Na⁺or Cl⁻ ions to the complex. Then the complexes were minimized for 10 000 steps of steepest descent followed by conjugate gradient or until a convergence of 0.05 kcal/A mols was reached. In a second stage, the MD trajectories were run on the minimized complexes were subjected to MD simulation. MD trajectory was run by using the cuda version of PMEMD implemented in Amber 14, and the simulations were analyzed for 26 ns, by recording 1 frames every 0.01 ns. The MD trajectories were analyzed by using UCSF Chimera³⁵. The plotting of RMSD versus time (ns) of the selected compounds is reported in Figures 2, 4(C), and 6.

Results and discussions

In vitro enzyme inhibitory properties of S. chinensis extracts by microtiter assays

The worldwide prevalence of some diseases such as AD and DM is increasing significantly. For example, 420 million people (65+ years) were affected by AD in 2000 and there are estimated over 1 billion cases by 2030⁴⁰. Regarding DM, more than 350 million people have DM in 2013 and the number of cases is expected at 592 million in next 25 years⁴¹. In this sense, new strategies or alternative therapeutic approaches are crucial to control these health problems⁴². Amongst these strategies, key enzyme inhibition theory is one of the most accepted approaches. For example, acetylcholinesterase, which hydrolyzes ACh in the synaptic gap, is a key enzyme and its inhibition is considered an effective tool for managing AD. This approach is specifically called as “cholinergic hypothesis”⁴³. Likewise, α-amylase and α-glucosidase are main enzymes in the carbohydrate metabolism (in the hydrolysis of starch) and therefore play a major role in controlling blood glucose level⁴⁴. Tyrosinase is a copper-containing enzyme and catalyzes the synthesis of melanin. Thus, tyrosinase is considered as a main target for treatment of skin disorders (SD)⁴⁵.

From this point, many synthetic drugs (galanthamine and tacrin for AD; acarbose and viglibose for DM; kojic acid and corticosteroids for SD) are produced as inhibitors, but the usages of these inhibitors have several concerns due to their undesirable side effects^46–48. All in all, novel inhibitors from natural sources have gained recent interest as safe and effective agents^49,50.

According to previous studies of the authors, S. chinensis fruit and leaf methanolic extracts are rich in lignans and phenolic compounds, respectively. As seen in Table 1, the dominant compounds in the fruits were schisandrol A, schisandrol B, schisandrin A and schisandrin B, and for the leaves one of the dominant compounds was CA²³.

The enzyme inhibitory effects of S. chinensis leaves and fruits were tested against ChE (AChE and BChE), tyrosinase, α-amylase, and α-glucosidase. The results are presented in Table 2. The fruits and leaves exhibited close AChE inhibitory effect (0.97 mg GALAE/g extract for leaves and 0.98 mg GALAE/g extract for fruits), but leaves presented no inhibitory effects on BChE (1.78 mg GALAE/g extract for fruits). The observed ChE inhibitory effects might be explained with the high concentration of lignans from the fruit extracts. Several researchers have reported that some lignans possess remarkable cholinesterease inhibitory effects, for example, study of Hung et al.⁵¹ reported for S. chinensis lignans; study of Azhar-Ul-Haq et al.⁵² reported for Vitex negundo lignans; and study of Wang et al.⁵³ reported for Valeriana officinalis lignans. Most ChE inhibitors affect the catalytic site of the ChE and this activity is associated with their aromatic rings⁵⁴. Similarly, some lignans (gomisin A, gomisin C, gomisin G, gomisin D, and schisandrol B) from S. chinensis have both aromatic ring and hydroxyl groups on their structure and they exhibited remarkable AChE inhibitory activity⁵¹.

Table 2. Enzyme inhibitory effects of Schisandra chinensis leave and fruit extracts.

Samples	AChE inhibition (mg GALAE/g extract)	BChE inhibition (mg GALAE/g extract)	Amylase inhibition (mmol ACAE/g extract)	Glucosidase inhibition (mmol ACAE/g extract)	Tyrosinase inhibition (mg KAE/g extract)
Leaves	0.97 ± 0.01*	NA	0.36 ± 0.02	8.54 ± 0.02	15.53 ± 0.12
Fruits	0.98 ± 0.01	1.78 ± 0.01	0.46 ± 0.01	0.31 ± 0.04	10.24 ± 0.34

*Three parallel experiments ± SD.

GALAE: galanthamine equivalents; ACAE: acarbose equivalents; KAE: kojic acid equivalents; NA: not active.

Similar to ChE, different results were obtained in terms of amylase and glucosidase inhibitory effects. Particularly, the glucosidase inhibitory effects for leaves (8.54 mmol ACAE/g extract) were considerable higher than for fruits (0.31 mmol ACAE/g extract). This might probably be related to the high concentrations of phenolics from the leaves, compared to fruits as kaempferol and quercetin glucosides were the main phenolics in the leaves²³. These findings are agreement with previous studies^55–57, which indicate stronger glucosidase inhibitory effects for phenolics such as kaempferol, quercetin, CA, and their glucosides.

The anti-tyrosinase effect of leaves (15.53 mg KAE/g extract) was about 1.5-fold higher than that of fruits (10.24 mg KAE/g extract). This activity might be attributed to the high level of phenolics in addition to lignans which are distributed in the fruits as well as in the leaves, but in significantly lower amounts. Similar results are reported by Hu et al.⁵⁸ and Azhar-Ul-Haq et al.⁵⁹ who found that phenolics and lignans have strong anti-tyrosinase effects. Hydroxyl groups in the phenolics may directly interact with active sites of these enzymes. This case was observed in several studies^60,61. Also, some phenolics (kaempferol, quercetin, etc.) have the ability to penetrate into the active site of the enzymes⁶². Moreover, schisandrin B from S. chinensis was reported by Yan et al.⁶³ as a strong tyrosinase inhibitor. They suggested that ionic state of schisandrin B might interfere with the substrates or tyrosinase, so it could inhibit the enzyme activity. In this direction, molecular docking is considered as a valuable tool to predict the inhibition mechanism.

Molecular modeling and molecular dynamic studies

In the nutraceutical field, computational techniques have been successfully used for the prediction of ligand–target binding affinity and to better understand the molecular basis of the biological responses. In silico studies also provide additional insights into the possible mechanism of action and binding mode of active compounds against metabolic key enzymes⁶⁴.

From a detailed literature analysis, it has been found that cp1 and cp24 should not possess any inhibition activity on AchE⁵¹ whereas cp4 showed a strong inhibition activity (EC50 = 12.5 μM), which was also confirmed by Kim et al.⁶⁵ (EC50 = 15.5 μM). For CA, based on the data reported by Szwajgier⁶⁶, it should not be an inhibitor of AchE, neither of BChE. The best docking pose found for cp1 and cp4 on AchE is reported in Figure 1. In its best pose (found by GoldScore scoring function) cp1 interacts to the receptor with the residues Ser122, Tyr121, and Phe330, whereas, the docking complex of cp4–AchE obtained by using the PLP scoring function is stabilized by two pi-pi interactions respectively with Phe330, Trp84, and a hydrogen bond with Gly117. These poses were further studied by MD simulation. The RMSD graphic of a 26 ns simulation reported in Figure 2 shows a high time-dependent stability of cp4 in the complex with AchE, with a RMSD oscillating within 0.2 A, reflecting a high stability of the found pose; on the other hand, compound cp1 changed its conformation after 6 ns, demonstrating a certain degree of instability of the found pose, this being also compatible with the literature data which reported cp4 to be an effective inhibitor of AchE in the opposite of cp1.

On amylase, no specific literature data have been found for our four selected compounds. Based on the manual analysis of the obtained docking poses, the cp1 docking pose on amylase was selected for further analysis on MD (Figure 4). The complex of cp1 and amylase is stabilized by two hydrogen bonds with Asp300 and Gln63 and the RMSD of the fluctuation of the ligand docked to the enzyme is 0.2 Angstroms, reflecting a good stability of the found pose. With regard to glucosidase enzyme, as reported by Fang et al.²⁷ cp1, cp4, cp24 are not strong inhibitors of this enzyme. Compounds cp1, cp4, and cp24 have been previously tested to a limit of 50 μM (IC₅₀ > 50 μM)²⁷ and no further data were reported for higher concentrations. The manual analysis of the complex cp1–glucosidase, obtained by the docking experiments by employing the scoring function PLP, revealed a series of five pi-pi interactions between the molecule cp1 and the Phe303, Arg352, Arg442, Arg315, Gln353 residues on the enzyme. For compound cp4, docked to glucosidase using the scoring function PLP, the best pose showed three hydrogen bonds between the ligand and Arg442, Gln353 (two hydrogen bonds for this residue) and one pi-pi interaction with Phe303 residues. cp4 with glucosidase formed one pi-pi interaction with Phe303 and one hydrogen bond with Gln353. All these poses are compatible with a modest to weak inhibition activity on this enzyme (Figure 3). CA has been previously reported by Adisakwattana et al.⁶⁷ to not inhibit glucosidase (>5000 μM), so that the observed inhibition activity on α-glucosidase found in our in vitro tests may be probably attributed to kaempferol, which is also present in significant amount in our extracts and its glycosides, which have been found previously to be an effective glucosidase inhibitor (11.6 μM/L)⁵⁶. Tyrosinase is well known to be targeted by lignans¹⁸. However, some data found in literature report contrasting data; in fact, following the data published by Yan et al.⁶³ cp24 was reported to inhibit tyrosinase, whereas Ye et al.⁶⁸ reported to be an activator on the same enzyme (enhancement of activity +40%). In our docking experiments, we have found that cp24 could bind to tyrosinase, but its best interaction is outside of the enzymatic pocket, so that cp24 was not able to chelate the Cu atoms present in the enzymatic pocket of tyrosinase, and these findings could be in agreement with the activating mechanism as reported by Ye et al.⁶⁸ (the best pose of cp24 to tyrosinase is reported in the Supplementary materials). On the contrary, the docking of cp4 on tyrosinase, obtained by GS scoring function, revealed a pose with several points of interaction, respectively three pi-pi interactions with His259, His85, His263 residues and the chelation of both Cu atoms, Cu400 and Cu401 (Figure 5A–C). CA has been previously reported to be a good inhibitor >350 μM⁶⁹ of tyrosinase. Indeed, in our docking experiments, it has shown the properties to chelate both the Cu atoms present in the enzymatic pocked of tyrosinase (GS scoring function) and to establish other relevant interactions with the aromatic portion (Figure 5B–D). The complexes of cp4, cp24, and CA with tyrosinase were also subjected to MD experiments. The RMSD of cp24 on tyrosinase showed a movement from the original poses after 12 ns with a RMSD of 0.7 Angstroms, so that this pose cannot be considerate very stable. Whereas the MD simulation carried out on cp4 docked to tyrosinase showed a very stable pose with a RMSD within a range of 0.3 Angstroms. Also the pose found for CA showed a RMSD in the range of 0.2 A, so that both cp4 and CA may form stable complexes with the tyrosinase by chelating the Cu atoms and establishing other relevant interactions with the amino acid residues surrounding the enzymatic pocket (Figure 6). Also these latter findings are in agreement with the literature data reported previously^68,69. All the best poses of compounds cp1, cp4, cp24, CA to AChE, BChE, amylase, glucosidase, tyrosinase are reported in the Supplementary materials.

Conclusions

Computational techniques have been successfully used for the prediction of ligand–target binding affinity and to better understand the molecular basis of the biological responses. In silico studies also provide additional insights into the possible mechanism of action and binding mode of active compounds against metabolic key enzymes. The herein study reported the enzyme inhibitory effects of extracts from fruits and leaves of S. chinensis in relation to their bioactive components (lignans and phenolics) by in vitro assays and molecular docking studies. As a peculiarity, the leaves extract showed a high inhibitory effect on glucosidase, whereas only fruit extracts were active on BChE. Moreover, both extracts exhibited a high antityrosinase effect. Following our molecular modeling experiments, we were able to show the interactions found for the exemplificative compounds cp1, cp4, cp24 (lignans) and CA, with the tested enzymes. Cp1 was found to bind strongly to the residues present in the enzymatic pocket of amylase and glucosidase; cp4 was found to have a stable and effective docking pose on AChE and moderately to glucosidase, confirming the literature data that indicated this compound as a strong inhibitor of AChE and moderate inhibitor of glucosidase. cp4 and CA have shown very good interactions to tyrosinase by chelating the Cu atoms present in the enzymatic pocket of the enzyme, and cp24 revealed a good binding pose on glucosidase. All together, these data confirm both the literature data and our findings reported in the experimental part of this work. To complete the panel of the activity, it is worth noting the presence of many other well-known bioactive substances in the leave extract contents, i.e. kaempferol and chlorogenic acids, which have been reported to be effective inhibitors of glucosidase and tyrosinase respectively. The results from the present study indicate that besides fruits, leaves of Schisandra can be regarded as an alternative source of multifunctional agents and a starting point for new bioactive formulations development.

Declaration of interest

The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Supplementary materials provided on line