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.     

Schisandra extract ameliorates arthritis pathogenesis by suppressing the NF-κB and MAPK signalling pathways

Schisandra chinensis is a medicinal plant used to treat various diseases. Extracts from the leaves or fruits of S. chinensis and their components are used in osteoarthritis (OA). The OA inhibitory effect of schisandrol A, one of its components, has been previously confirmed. We aimed to confirm the OA inhibitory effect of Schisandra (including components like schisandrol A) to identify why the inhibitory effect of the Schisandra extract is better. First, we investigated the effects of the Schisandra extract on OA as a potential therapeutic. Experimental OA was induced in a mouse model via destabilized medial meniscus surgery. The animals were orally administered the Schisandra extract; the inhibition of cartilage destruction was confirmed using histological analysis. In vitro analysis showed that the Schisandra extract attenuated osteoarthritic cartilage destruction by regulating IL-1β-induced MMP3 and COX-2 levels. The Schisandra extract inhibited IL-1β-induced degradation of IκB (NF-κB pathway) and IL-1β-induced phosphorylation of p38 and JNK (mitogen-activated protein kinase (MAPK) pathway). RNA-sequencing analysis showed that the Schisandra extract decreased the expression of IL-1β-induced MAPK and NF-κB signalling pathway-related genes more than schisandrol A alone. Therefore, Schisandra extract may be more effective than schisandrol A in preventing OA progression by regulating MAPK and NF-κB signalling.     

Effect of Tacrolimus on the pharmacokinetics of bioactive lignans of Wuzhi tablet (Schisandra sphenanthera extract) and the potential roles of CYP3A and P-gp

We recently reported that Wuzhi tablet (WZ), a preparation of the ethanol extract of Wuweizi (Schisandra sphenanthera), had significant effects on blood concentrations of Tacrolimus (FK506) in renal transplant recipients and rats. The active lignans in WZ are schisandrin A, schisandrin B, schisandrin C, schisandrol A, schisandrol B, schisantherin A, and schisantherin B. Until now, whether the pharmacokinetics of these lignans in WZ would be affected by FK506 remained unknown. Therefore, this study aimed to investigate whether and how FK506 affected pharmacokinetics of lignans in WZ in rats and the potential roles of CYP3A and P-gp. After a single oral co-administration of FK506 and WZ, the blood concentration of lignans in WZ was decreased by FK506; furthermore, the AUC of schisantherin A, schisandrin A, schisandrol A and schisandrol B was only 64.5%, 47.2%, 55.1% and 57.4% of that of WZ alone group, respectively. Transport study in Caco-2 cells showed that these lignans were not substrates of P-gp, suggesting decreased blood concentration of lignans by FK506 was not via P-gp pathway. Metabolism study in the human recombinant CYP 3A showed that these lignans had higher affinity to CYP3A than that of FK506, and thus had a stronger CYP3A-mediated metabolism. It was concluded that the blood concentrations of these lignans were decreased and their CYP3A-mediated metabolisms were increased in the presence of FK506 since these lignans had higher affinity to CYP3A.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Lignans from the fruit of Schisandra sphenanthera,and their inhibition of HSV-2 and adenovirus

The systematic isolation of the EtOAc extract from Schisandra sphenanthera fruit was performed during a search for HSV-2 and adenovirus inhibitors. Sixteen lignans were obtained, with compound 1 representing a new and rare type of lignan in the genus Schisandra. Their structures were elucidated by spectroscopy and comparison with literature data. Among all the lignans tested for their antiviral activities, compound 14 was the most active against HSV-2 with a selectivity index value up to 29.83. Moreover, the new compound 1, and the known ones (4, 6, 7, 10 and 14) also exhibited moderate inhibition of HSV-2 and adenovirus. To the best of our knowledge, this is the first report that these lignans from Schisandra genus were shown to have modest activity against HSV-2 and adenovirus. Meanwhile, structure–activity relationships of some lignans for the inhibitory activity against HSV-2 and adenovirus were discussed in this study.     

Chromatographic and Biological Screening of Chosen Species of Schisandraceae Family: Schisandra chinensis,S. rubriflora,S. sphenanthera,S. henryi and Kadsura japonica

HPLC and TLC profiling was carried out for leaf and fruit extracts of five Schisandraceae species: Schisandra chinensis, S. rubriflora, S. spehenanthera, S. henryi and Kadsura japonica. HPLC measurements confirmed presence of lignans and phenolic compounds in fruits and leaves of all tested species. The most abundant in lignans was S. chinensis fruit extract in which 15 compounds were detected (e. g.: schisandrol A, schisanhenol, γ-schisandrin, gomisin N). The effect-directed detection, i. e., TLC-direct bioautography against Bacillus subtilis, showed exceptionally high activity for S. chinensis and S. rubriflora fruit extracts. On the other hand, TLC-DB enzyme tests (α-glucosidase, lipase, tyrosinase and acetylcholinesterase (AChE) inhibition assays) showed that all fruit and leaf extracts have ability to inhibit the above-mentioned enzymes (except for the K. japonica fruit). The leaf extracts showed much stronger antioxidant activity than the fruit ones, which were assessed and compared using both TLC-direct bioautography and spectrophotometric measurements based on ABTS, DPPH and FRAP tests.     

Genetic diversity and population structure of two important medicinal plant species <Emphasis Type="Italic">Schisandra chinensis</Emphasis> and <Emphasis Type="Italic">Schisandra sphenanthera</Emphasis> revealed by nuclear microsatellites

Seven polymorphic and transferable nuclear microsatellites were used to investigate the population structure of genetic diversity of Schisandra chinensis and Schisandra sphenanthera for facilitating their conservation and sustainable utilization. High levels of gene diversity were revealed in these two medicinal species, the majority of genetic diversity was harbored within populations, and population structure was might due to restricted gene flow among populations. Isolation by distance was close to significance in S. chinensis but not in S. sphenanthera. In S. chinensis, null alleles were identified as a cause for excess of homozygotes at loci G24 and WGA60, but inbreeding might also be partly responsible for the positive F _IS values in this species. In contrast, null allele frequencies were high at all the seven loci in S. sphenanthera and resulted in overestimation of fixation index. The strategy for ex situ conservation of these two medicinal species is discussed based on the genetic results.     

Lignans and Terpenoids from the Leaves of Schisandra chinensis

Fifteen constituents, including one new lignan (schisandroside E) and one new terpenoid (schisandenoid A) as well as nine known lignans and four known terpenoids, were isolated from Schisandra chinensis leaves. The structures of schisandroside E and schisandenoid A were established by entirely meticulous spectroscopic analysis (NMR, MS, CD, IR and UV). All compounds were tested for cytotoxicity against MGC‐803, Caco‐2 and Ishikawa cell lines. Some compounds showed strong cytotoxicity against these three cancer cell lines with IC₅₀<1 μm .     

Rapid discrimination of Schisandra sphenanthera and Schisandra chinensis using electronic tongue and ultra-performance liquid chromatography coupled with chemometrics

Both Schisandra sphenanthera (S. sphenanthera) and Schisandra chinensis (S. chinensis) are used as traditional Chinese medicines, but they have different medicinal properties. Because S. sphenanthera is cheaper, it is often used as a counterfeit product for S. chinensis. In the present study, an electronic tongue (e-tongue) was used for discrimination of the two Schisandraceae species. In addition, the contents of schisandrin, schizandrol B, schisantherin A, deoxyschizandrin, and schisandrin B were determined simultaneously by ultra-performance liquid chromatography. Principal component analysis (PCA) and discriminant factor analysis (DFA) were used to establish the mathematical models for species identification, and the classification rates for both methods reached 100%. The e-tongue coupled with multivariate analysis exhibited the excellent performance and classification accuracy, and this was validated by the ultra-performance liquid chromatography results. This simple e-tongue technique could be useful for rapid and accurate identification of S. sphenanthera and S. chinensis.     

New Thymoquinol Glycosides and Neuroprotective Dibenzocyclooctane Lignans from the Rattan Stems of Schisandra chinensis

Three new lignans ( 1 – 3 ), together with four new thymoquinol glycosides ( 4 – 7 ), were isolated from 70%‐EtOH extract of the rattan stems of Schisandra chinensis. The structures of 1 – 7 were elucidated by detailed spectroscopic analyses, and these new compounds were identified as pinobatol‐9‐O‐β‐d ‐glucopyranoside ( 1 ), 1,2,13,14‐tetramethoxydibenzocyclooctadiene 3,12‐O‐β‐d ‐diglucopyranoside ( 2 ), 3,7‐dihydroxy‐1,2,13,14‐tetramethoxydibenzocyclooctadiene 12‐O‐β‐d ‐glucopyranoside ( 3 ), thymoquinol 2‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 4 ), thymoquinol 2‐O‐α‐d ‐arabinofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 5 ), thymoquinol 5‐O‐β‐d ‐apiofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 6 ), and thymoquinol 5‐O‐α‐d ‐arabinofuranosyl‐(1→6)‐β‐d ‐glucopyranoside ( 7 ). The neuroprotective activity of 1 – 7 was evaluated on PC12 cells with neurotoxicity induced by amyloid‐beta 1 – 42 (Aβ_{1 – 42}). Compounds 2 and 3 showed protecting activity against Aβ‐induced toxicity in PC12 cells.     

Seven New Sesquiterpenoids from the Fruits of Schisandra sphenanthera

Fractionation of the EtOH extract from the fruits of Schisandra sphenanthera resulted in the isolation of seven new sesquiterpenoids, 1 – 7 , in addition to the known metabolites 8 – 23 . Among them, schiscupatetralin A ( 1 ) possesses an unprecedented structure with a C? C bond between cuparenol and tetralin. The isolated new compounds were evaluated for their anti‐HSV‐1 and anti‐inflammatory activities. The results revealed that compound 4 exhibited anti‐HSV‐1 activity, while compound 6 showed a significant anti‐inflammatory activity.     

Anti-liver fibrotic lignans from the fruits of Schisandra arisanensis and Schisandra sphenanthera

Three new polyoxygenated C₁₈-dibenzocyclooctadiene lignans, arisanschinins M and N (1 and 2) and schisphenin A (3), together with eight related metabolites (4–11), were isolated from the fruits of Schisandra arisanensis and Schisandra sphenanthera, respectively. The structures of 1–3 were elucidated on the basis of extensive spectroscopic and 2D NMR (HSQC, HMBC, and NOESY) analyses. The configuration of the biphenyl moiety in the octadiene ring was determined by circular dichroism (CD). Compound 1 possessed an unprecedented 3-(1-hydroxypropan-2-yl)-3-methyl-1,4-dioxo-2-one lactonide ring system attaching at C-6/C-14. Pharmacological studies revealed that compounds 3, 4, 6, 7, and 10 exhibited significant anti-hepatic fibrosis activity, while 9 and 11 showed cytotoxicity against HSC-T6 cells. The biogenetic pathway for compound 1 was also proposed.     

Floral ontogeny of <Emphasis Type="Italic">Schisandra chinensis</Emphasis> (Schisandraceae): implications for androecial evolution within <Emphasis Type="Italic">Schisandra</Emphasis> and <Emphasis Type="Italic">Kadsura</Emphasis>

The organogenesis of staminate and carpellate flowers of Schisandra chinensis (Schisandraceae) was investigated with scanning electron microscopy, with observations on the development of tepals reported for the first time. The results showed that there is no interval between the initiation of the last tepal and that of the first stamen or carpel, and that the shapes of tepal, stamen, and carpel primordia are similar. The tepals and stamens of staminate flowers are initiated acropetally in a continuous spiral Fibonacci phyllotaxis, with no carpel structures observed; the filaments are not connate. The organogenesis of the carpellate flowers is similar to that of the staminate flowers, but with no evidence of stamen development. The carpels are ascidiate without postgenital fusion. Three androecial characters of Schisandra and Kadsura are discussed in a phylogenetic context. The subglobose or obovoid androecium of Schisandra propinqua and Schisandra plena may be homologous with that in sections Kadsura and Sarcocarpon. The plesiomorphic form of the androecium within the two genera is likely to be elongate with more than ten free stamens.     

Isolation and characterization of polymorphic microsatellite loci in a traditional Chinese medicinal plant, Schisandra sphenanthera

Schisandra sphenanthera (Schisandraceae) is a traditional Chinese medicinal plant. In this study, nine polymorphic microsatellite loci were developed and characterized for the species. A genomic DNA enrichment protocol was used to isolate microsatellite loci and polymorphism was explored using 36 individuals sampled from a natural population. The observed number of alleles ranged from 2 to 10 with an average of 5.2. Expected and observed heterozygosity ranged from 0.30 to 0.82 and 0.33 to 0.97, respectively. These microsatellites have been directly applied to both population and conservation genetic studies of S. sphenanthera.     

A Cytotoxic Neolignan from Schisandra propinqua （Wall.） Baill.

In the course of our study of bioactive natural products from Schisandra plants, we isolated a neolignan from an EtOAc extract of the stems of Schisandra propinqua （Wall.） Baill. The structure of the new com- pound was determined to be 4, 4-di （4-hydroxy-3-methoxyphenly）-2, 3-dimethylbutanol （compound 1） on the basis of 1H- and 13C-NMR spectra and 2D NMR methods. Eight known compounds, compounds 2-9, were also isolated and identified, of which compounds 3, 4, 6 and 9 were isolated for the first time from this plant. In addition, compounds 1-4 were evaluated for cytotoxicity by an 3-（4,5-dimethyl-2 thiazoyl）-2,5-diphenyl-2H- tetrazolium bromide （MTT） assay. Compound 1 showed significant potential cytotoxic ability in the bioassay.     

Nortriterpenoids and lignans from Schisandra sphenanthera

Nortriterpenoids, sphenadilactone C (1) and sphenasin A (2), together with four known lignans (3-6), were isolated from the leaves and stems of Schisandra sphenanthera. Their structures were elucidated by extensive analysis of 1D and 2D NMR spectroscopic data and compound 2 was further confirmed by single-crystal X-ray diffraction. Compound 1 features a partial enol moiety and an acetamide group in its structure. In addition, compounds 1, 3-6 showed weak anti-HIV-1 activity with EC₅₀ values in the range of 15.5-29.5 μg/mL.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Improved production of dibenzocyclooctadiene lignans in the elicited microshoot cultures of Schisandra chinensis (Chinese magnolia vine)

Dibenzocyclooctadiene lignans are a specific group of secondary metabolites that occur solely in Schisandra chinensis. The aim of the presented work was to boost the accumulation of lignans in the agitated microshoot cultures of S. chinensis, using different elicitation schemes. The experiments included testing of various concentrations and supplementation times of cadmium chloride (CdCl₂), chitosan (Ch), yeast extract (YeE), methyl jasmonate (MeJa), and permeabilizing agent—dimethylsulfoxide (DMSO). After 30 days, the microshoots were harvested and evaluated for growth parameters and lignan content by LC-DAD method. The analyses showed enhanced production of lignans in the elicited S. chinensis microshoots, whereas the respective media samples contained only trace amounts of the examined compounds (< 5 mg/l). Elicitation with CdCl₂ caused up to 2-fold increase in the total lignan content (max. ca. 730 mg/100 g DW after the addition of 1000 μM CdCl₂ on the tenth day). Experiments with chitosan resulted in up to 1.35-fold increase in lignan concentration (max. ca. 500 mg/100 g DW) after the supplementation with 50 mg/l on the first day and 200 mg/l on the tenth day. High improvement of lignan production was also recorded after YeE elicitation. After the elicitation with 5000 mg/l of YeE on the first day of the growth period, and with 1000 and 3000 mg/l on the 20th day, the lignan production increased to the same degree—about 1.8-fold. The supplementation with 1000 mg/l YeE on the 20th day of the growth cycle was chosen as the optimal elicitation scheme, for the microshoot cultures maintained in Plantform temporary immersion system—the total content of the estimated lignans was equal to 831.6 mg/100 g DW.

     

Schisandra chinensis Peptidoglycan-Assisted Transmembrane Transport of Lignans Uniquely Altered the Pharmacokinetic and Pharmacodynamic Mechanisms in Human HepG2 Cell Model

Schisandra chinensis (Turz Baill) (S. chinensis) (SC) fruit is a hepatoprotective herb containing many lignans and a large amount of polysaccharides. A novel polysaccharide (called SC-2) was isolated from SC of MW 841 kDa, which exhibited a protein-to-polysaccharide ratio of 0.4089, and showed a characteristic FTIR spectrum of a peptidoglycan. Powder X-ray diffraction revealed microcrystalline structures within SC-2. SC-2 contained 10 monosaccharides and 15 amino acids (essential amino acids of 78.12%w/w). In a HepG2 cell model, SC-2 was shown by MTT and TUNEL assay to be completely non-cytotoxic. A kinetic analysis and fluorescence-labeling technique revealed no intracellular disposition of SC-2. Combined treatment of lignans with SC-2 enhanced the intracellular transport of schisandrin B and deoxyschisandrin but decreased that of gomisin C, resulting in alteration of cell-killing bioactivity. The Second Law of Thermodynamics allows this type of unidirectional transport. Conclusively, SC-2 alters the transport and cell killing capability by a “Catcher-Pitcher Unidirectional Transport Mechanism”.