Responses to lead stress in Scrophularia striata: insights into antioxidative defence mechanisms and changes in flavonoids profile.

Lead (Pb) induces oxidative stress in plants, which results in different responses, including the production of antioxidants and changes in the profile of secondary metabolites. In this study, the responses of Scrophularia striata exposed to 250mgL-1 Pb (NO3 )2 in a hydroponic environment were determined. Growth parameters, oxidative and antioxidative responses, redox status, and the concentration of Pb were analysed in roots and shoots. Malondialdehyde and hydrogen peroxide (H2 O2 ) levels in the roots were significantly increased and reached their highest value at 72h after Pb treatment. Superoxide dismutase, catalase, and peroxidase, as an enzymatic antioxidant system, were responsible for reactive oxygen species scavenging, where their activities were increased in the shoot and root of Pb-treated plants. Enzymatic antioxidant activities were probably not enough to remove a significant H2 O2 content in response to Pb treatment. Therefore, other defence responses were activated. The results stated that the flavonoid components of S. striata progressed towards the increase of isoflavone, flavanol, and stilbenoid contents under Pb treatment. In general, S. striata stimulates the enzymatic defence system and activates the non-enzymatic system by modulating the profile of flavonoids toward the production of flavonoids with high antioxidant activity, such as quercetin and myricetin in response to Pb stress.

A metabolism-based study of the mechanism of action of Scrophularia ningpoensis Hemsl. on nephrogenic edema.

Nephrogenic edema (NE) is a type of edema with hypoproteinemia and water and sodium retention as a result of renal injury. Traditional Chinese medicine has proved that Scrophularia ningpoensis Hemsl. has an effect on NE, but its mechanism is not clear. In this study, the main components and blood components of S. ningpoensis were identified using ultra-high-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS). Pathological section and blood biochemical analysis were used to estimate the therapeutic effect of S. ningpoensis on NE. Network pharmacology was used to predict the potential pathways of S. ningpoensis. The metabolomics method was used to study the changes in small-molecule metabolites in the body. The results showed that S. ningpoensis could relieve NE by regulating relative to renal function and body edema, and its mechanism may be related to the regulation of energy metabolism, recovery of renal injury, and reduction in inflammation. The active component harpagoside may be one of the important compounds of S. ningpoensis in the treatment of NE. We confirmed that S. ningpoensis has a therapeutic effect on NE, which provides a solid scientific research basis for the clinical application of S. ningpoensis.

Hydroalcoholic extract of Scrophularia striata has a significant therapeutic effect on thioacetamide-induced liver cirrhosis in rats.

OBJECTIVES: Liver cirrhosis is one of the most important causes of death from liver diseases. Nowadays, the use of herbal medicines has increased due to its availability, less side effects and cheapness for the treatment of liver diseases. The present study was conducted to examine therapeutic effects of hydroalcoholic extract of Scrophularia striata (S. striata) on thioacetamide-induced liver cirrhosis in rats through evaluate its effects on oxidative stress markers and the expression of metalloproteinase 1 (TIMP 1), toll-like receptor-4 (TLR-4), and Mitofusin (MFN2) genes. METHODS: 24 male rats were selected by simple random sampling. Rats were randomly assigned to four groups: group I: healthy rats, group II: thioacetamide (TAA) injected rats, group III: TAA injected rats+100 mg/kg bw of S. striata and group IV: TAA injected rats+200 mg/kg bw of S. striata. Liver cirrhosis was induced in rats by a 300 mg/kg bw TAA administration twice with an interval of 24 h. After 8 weeks of treatment by S. striata at doses of 100 and 200 mg/kg bw, biochemical factors and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) were measured using spectrophotometric methods. Also, gene expression of TIMP 1, TLR-4, and MFN2 were analyzed using real-time PCR. ANOVA and Bonferroni post hoc test analysis were applied to evaluate the data. RESULTS: The results showed the S. striata extract significantly improve the serum ALT, AST and ALP levels, TIMP 1, TLR-4, and MFN2 genes and oxidative stress markers (SOD, TAC, GPX, CAT and MDA) in the liver tissues when compared to control group (p<0.05). Also, it was found that the beneficial effects of the S. striata were dose-dependent. CONCLUSIONS: Based on the results obtained S. striata by reducing the expression of TIMP 1, TLR-4, and MFN2 genes and improving oxidative stress might be used as adjuvant treatment for liver cirrhosis.

Scrophularia buergeriana Extract (Brainon) Attenuates Neuroinflammation in BV-2 Microglia Cells and Promotes Neuroprotection in SH-SY5Y Neuroblastoma Cells.

Microglia-induced neuroinflammation is one of the causative factors in cognitive dysfunction and neurodegenerative disorders. Our previous studies have revealed several benefits of Scrophularia buergeriana extract (Brainon®) in the central nervous system, but the underlying mechanism of action has not been elucidated. This study is purposed to investigate the anti-inflammatory and neuroprotective mechanisms of Brainon in the BV-2 condition SH-SY5Y model. Lipopolysaccharide (LPS)-induced BV-2 conditioned media (CM) were used to treat SH-SY5Y cells to investigate neuroprotective effects of the extract against microglial cytotoxicity. Results demonstrated that pretreated Brainon decreased nitric oxide release, the inducible nitric oxide synthase expression level, and expression of cytokines like interleukin-6, interleukin-1ß, and tumor necrosis factor-α by blocking expression of TLR4/MyD88 and NLRP3 and suppressing nuclear factor κB/AP-1 and p38/JNK signaling pathways in LPS-induced BV-2 cells. In addition, when SH-SY5Y cells were treated with CM, pretreatment with Brainon increased neuronal viability by upregulating expression of antioxidant proteins like as SODs and Gpx-1. Increased autophagy and mitophagy-associated proteins also provide important clues for SH-SY5Y to prevent apoptosis by Brainon. Brainon also modulated mTOR/AMPK signaling to clear misfolded proteins or damaged mitochondria via auto/mitophagy to protect SH-SY5Y cells from CM. Taken together, these results indicate that Brainon could reduce inflammatory mediators secreted from BV-2 cells and prevent apoptosis by increasing antioxidant and auto/mitophagy mechanisms by regulating mTOR/AMPK signaling in SH-SY5Y cells. Therefore, Brainon has the potential to be developed as a natural product in a brain health functional food to inhibit cognitive decline and neuronal death.

Antidiabetic effects of Scrophularia striata ethanolic extract via suppression of Pdx1 and Ins1 expression in pancreatic tissues of diabetic rats.

One of the factors that causes severe metabolic imbalance and abnormal changes in many tissues, especially in the pancreas, is the pathological disease of diabetes mellitus. Therefore, in this study, the therapeutic effects of Scrophularia striata were investigated using an animal model in the control of diabetic injury and pancreatic complications caused by diabetes. A total of 66 rats (weight 220-250 g) were randomly divided into: Healthy Control group (rats without diabetes receiving Propylene glycol as solvent); Diabetic control group; 3 experimental healthy groups (receiving the extract with doses of 100, 200 and 400 mg/kg bw/day); 3 treatment groups; and3 pretreatment groups. Diabetes was induced in rats by intraperitoneal STZ (60 mg/kg bw). FBS, HbA1c and insulin were measured after 4 weeks. Pdx1 and Ins1 gene expression was assessed by RT-PCR. The histological evaluation was also performed with H&E staining. The data were analyzed by SPSS ver20 using ANOVA and Tukey tests. By treatment with S. striata ethanolic extract, these factors were close to the normal range. The expression of the Pdx1 and Ins1 genes increased in the treated rats with S. striata extract. Analysis of the obtained data indicates the effect of S. striata in improving the complications of diabetes in rats and can be considered for therapeutic purposes.

The isolation and expression analysis of cinnamate 4-hydroxylase and chalcone synthase genes of Scrophularia striata under different abiotic elicitors.

The phenylpropanoid pathway serves as a rich source of metabolites in plants, and it is considered as a starting point for the production of many other important compounds such as the flavonoids, flavonols, coumarins, and lignans. Scrophularia striata is a member of the Lamiaceae family with some biological activities similar to flavonoid compounds such as antioxidant, antibacterial, anti-inflammatory and analgesic activities. Cinnamate 4-hydroxylase (C4H) and Chalcone synthase (CHS) are key enzymes of the phenylpropanoid pathway, leading to the biosynthesis of several secondary metabolites. In this study, two S. striata CHS and C4H were isolated and then analyzed. The investigation of the expression of these genes was performed under the effects of three salicylic acid (SA), jasmonic acid (JA), and gibberellic acid (GA) at concentrations of 100 and 300 ppm with a completely randomized design at the transcript level using Real Time PCR method. These have different expression patterns at developmental stages. Moreover, these genes present different sensitivities to hormonal treatment. Considering the total results, it was found that the amount of expression of these genes during the reproductive phase is higher than that of the vegetative phase. Additionally, the treatment of 300 ppm SA in the reproductive phase is the most effective treatment on increasing the corresponding phenylpropanoid compounds. A correlation analysis was performed between the phenylpropanoid compounds content and both CHS and C4H expression values at different phenological development stages. The results indicate that the expression variations of both CHS and C4H are significantly related to the changes in total phenolic content. We believe that the isolation of CHS and C4H can be helpful in better understanding phenylpropanoid metabolis.

Anti-inflammatory secondary metabolites from Scrophularia kotschyana.

The species belonging to Scrophularia genus grow mainly in Irano-Turanian and Mediterranean regions and have been used as folk remedy for inflammatory-related diseases since ancient times. The present study was aimed to evaluate the anti-inflammatory activity of the extracts of Scrophularia kotschyana as well as the isolated compounds. The aerial parts and the roots of the plant were separately extracted with methanol. Anti-inflammatory activities of both extracts were evaluated with formalin test in mice. As the methanolic extract of the aerial parts significantly (p < .05) inhibited inflammation, it was then submitted to successive solvent extractions with n-hexane, dichloromethane, ethyl acetate and n-butanol to yield subextracts. Anti-inflammatory activities of the subextracts were evaluated within the same test system. Among the subextracts tested, the n-butanol subextract produced a significant (p < .05) anti-inflammatory activity at all doses (5, 10, and 30 mg/kg, ip.). Sequential chromatographic separation of the n-butanol subextract yielded 8-O-acetyl-4'-O-(E)-p-coumaroylharpagide, 8-O-acetyl-4'-O-(Z)-p-coumaroylharpagide, ß-sitosterol 3-O-ß-glucopyranoside, apigenin 7-O-ß-glucopyranoside, apigenin 7-O-rutinoside, luteolin 7-O-ß-glucopyranoside and luteolin 7-O-rutinoside. The anti-inflammatory activities of the isolates were evaluated at 5 mg/kg dose. Luteolin 7-O-ß-glucopyranoside and apigenin 7-O-rutinoside caused a significant (p < .05) inhibition of oedema formation.

Daily dynamics of intermediate metabolite profiles lead to time-dependent phenylethanoid glycosides production in Scrophularia striata during the day/night cycle.

Phenylethanoid glycosides (PhGs) are important medicinal compounds found in Scrophularia striata, one of the plant species native to Iran. Since almost all aspects of plant life are controlled by night/light cycle, studying its relationship to valuable plant metabolites production will help us to determine the right time for their extraction. Therefore, the aim of this investigation is to figure out whether the diel light oscillations control PhGs production and how it relates to daily changes in upstream metabolic reactions and circadian clock in S. striata. For this, daily rhythms of metabolic pathways were examined every 4 h during a day/night cycle in 3 groups of control (16 h light/8 h dark), continuous light and darkness. The results showed that acteoside and echinacoside levels in each group peaked during the night and day, respectively. Thus, the PhGs production follows a rhythmic behavior in S. striata, which is probably controlled by circadian clock. Also, the levels of photosynthetic pigments, carbohydrates, amino acids, phenolic acids, phytohormones and phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) enzyme activities varied diel in a similar or different way among study groups. The observations revealed that light/dark cycle controls the carbon and energy flow from light reception to the production and consumption of starch, biosynthesis of phenylalanine, tyrosine, cinnamic acid and coumaric acid, activation of hormonal signaling pathways and enzymes involved in phenylpropanoid pathway. Overall, it can be concluded that PhGs accumulation time-dependent patterns is likely due to daily fluctuations in upstream metabolic reactions induced by light/dark cycle.

Dynamic analysis of secondary metabolites in various parts of Scrophularia ningpoensis by liquid chromatography tandem mass spectrometry.

The roots of Scrophularia ningpoensis are used as traditional medicines for thousands of years in China, nevertheless the stems and leaves were discarded as non-medicinal parts. Modern research have indicated the chemical constituents in the stems and leaves are similar to the identified in the roots, and the therapeutic effects of stems and leaves are superior to roots for some disease. In the study, the chemical constituents in roots, stems and leaves of S. ningpoensis were analyzed qualitatively by HPLC-Q-TOF-MS/MS. 40 compounds including 17 iridoid glycosides, 15 phenylpropanoids and 8 flavonoids were identified. Meantime, the dynamic accumulations of six index constituents in various parts were measured by HPLC-DAD. The results indicated the S. ningpoensis stems contained high content of aucubin (30.09 mg/g) and harpagide (28.4 mg/g) in August, and the leaves contained high content of harpagoside (12.02 mg/g) in July. The study provides the basis for the full development and utilization of the resource of stems and leaves from S. ningpoensis.

Time-dependent behavior of phenylpropanoid pathway in response to methyl jasmonate in Scrophularia striata cell cultures.

KEY MESSAGE: MeJA triggers a time-dependent behavior of the phenylpropanoid compounds. Plant cells produce a large number of metabolites in response to environmental factors. The cellular responses to environmental changes are orchestrated by signaling molecules, such as methyl jasmonate (MeJA). To understand how the MeJA changes the behavior of amino acids, carbohydrates, and phenylpropanoid compounds such as phenolic acids, phenylethanoid-glycosides, and flavonoids in Scrophularia striata cells; we monitored the metabolic responses for different times of exposure. In this study, we performed a time course analysis of metabolites and enzymes in S. striata cells exposed to MeJA (100 µM) and evaluated the metabolic flux towards carbon-rich secondary metabolites production. Moreover, we calculated the biosynthetic energy cost for free amino acids. Our results indicated that MeJA accelerates the sucrose degradation and directs the metabolic fluxes towards a pool of flavonoids and phenylethanoid glycosides through a change in enzyme behavior in the entry point and center of the phenylpropanoid pathway. MeJA also decreased and then raised the amino acid biosynthesis cost in S. striata cells in a time-dependent manner, indicating the cells evolve to utilize amino acids more economically by reducing cell growth. Finally, we classified the marked changes in the metabolites level and enzyme activities into three groups including early-, late-, and oscillatory-response groups to MeJA and summarized our findings as a model depicting pathway interactions during MeJA elicitation. Determination of metabolic levels in response to MeJA suggests that the changes in metabolic responses are time-dependent.

Effects of ecological factors on the antioxidant potential and total phenol content of Scrophularia striata Boiss.

Scrophularia striata, commonly known as figwort, is one of the most important medicinal plants that mainly grows in cold regions of the Zagros Mountains (West of Iran). Although the chemical composition of this plant species has not yet been explored, people living in Ilam province (W Iran) have used it for many years to treat different illnesses. The present study aims to analyze the effect of some ecological factors on the antioxidant potential and the amount of phenol present in this plant species, using a random factorial design with two factors (elevation and region) and three replicates. The fruits of the plant were gathered from three different elevations. They were collected from three regions of the Ilam province (Badreh, Dareshahr, and Dehloran) in June 2016, when the fruits appear. Moreover, to analyze different soil chemical and physical features, soil samples were gathered from a depth of 0.5 m under the shrubs. The antioxidant action of the methanol extract from the plant samples and the total amount of phenol compounds were measured using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and the Folin-Ciocalteu method, respectively. The results showed that the effects of site and elevation, and the interaction between these factors, on the antioxidant potential and total phenol amount were significant with a probability of error of 1%. The maximum extract efficiency (19.37 ± 3.07%), antioxidant potential (126.5656 ± 0.96 µg/mL), and total amount of phenol (55.7689 ± 3.17 µg/mL) were obtained from Dareshahr at an elevation of 600 m above mean sea level. The minimum amount of total phenol (24.6544 ± 3.21 µg/ml) was recorded at the lowest elevation of Badreh, at which phosphorus, potassium, organic carbon, organic material, nitrogen, acidity, lime, and silt were present at the lowest amount. However, the antioxidant activity and total amount of phenol had a strong direct correlation in the two districts of Dareshahr and Badreh, but were reversely and strongly correlated in Dehloran. Therefore, it can be stated that Scrophularia striata has the potential for antioxidant activity, however, the complexity of the effect of ecological factors on one hand, and the emergence of different chemical processes in the plant under such effects on the other hand, has led to the synthesis of different compounds with antioxidant potential in the plant in different regions.

Crosstalk among polyamines, phytohormones, hydrogen peroxide, and phenylethanoid glycosides responses in Scrophularia striata to Cd stress.

Plants respond to Cadmium (Cd) as a hazardous heavy metal through various mechanisms depending on their available metabolite resources. In this research, the physiological and signaling pathways mediating the responses to Cd stress in Scrophularia striata seedlings were characterized after they were exposed to different Cd concentrations at different time periods. The results showed that the polyamines (PAs), Abscisic acid (ABA) and hydrogen peroxide (H2O2) contents were significantly enhanced at 48 h. Moreover, the enzyme activity of phenylalanine ammonia-lyase (PAL) and tyrosine ammonia-lyase (TAL) as regulator enzymes in the phenylpropanoid pathway was increased, related to the reinforcement of phenolic compounds such as phenylethanoid glycosides (as a special compound of this plant). This metabolic profiling indicates that the signal transduction of Cd stress increased the activity of different enzymes (PAL and TAL) by regulating the PAs metabolism, the modulation of ABA, and the H2O2 content. As a result, it caused the accumulation of phenolic compounds, especially echinacoside and acteoside, both of which are required to improve the response of Cd stress in S. striata.

A nutritional and rumen ecological evaluation of the biorefinery by-product alfalfa silage cake supplemented with Scrophularia striata extract using the rumen simulation technique.

BACKGROUND: By-products of the food production chain are gaining importance as feedstuffs for ruminants. Alfalfa silage cake (AC) is an unexploited biorefinery by-product rich in fiber. The aim of this study was to test AC, using an in vitro rumen simulation technique (Rusitec), for its suitability as a fiber source for cattle. Three diets with similar crude protein (CP) content were formulated; they contained the biorefinery by-product AC, the original alfalfa silage (OA), or a fiber-rich hay. As fibrous feedstuffs are known to promote ruminal methanogenesis, we additionally tested a plant extract of Scrophularia striata (60 mg g-1 dry matter) for its methane mitigation and antimicrobial properties. RESULTS: Diets containing AC displayed lower nutrient degradability, with the largest difference in CP degradation (P < 0.001). Sequencing of microbial DNA revealed several effects of the diet and of the addition of S. striata extract, but no inhibitory effect on methanogens. Likewise, methane production, which, in general, is lower with AC and OA diets, was not inhibited by S. striata extract, while the short chain fatty acid (SCFA) profiles were unaffected. CONCLUSION: Although CP degradation of the AC diet was lower, degradation of the fiber fractions was similar among diets. According to the present results, AC can be used as fibrous feedstuff for ruminants. Supplementation with S. striata extract did not inhibit methane formation. © 2019 Society of Chemical Industry.

Water stress alleviation by polyamines and phenolic compounds in Scrophularia striata is mediated by NO and H2O2.

Plants respond to water stress through a variety of mechanisms, depending on metabolites preferences and their available resources. This work was performed to elucidate the cross-talk between signaling molecules (polyamines (PAs), hydrogen peroxide (H2O2) and nitric oxide (NO)), phenolic compounds and osmolytes (phenylethanoid glycosides (PhGs), phenolic acids, flavonoids, soluble sugars and amino acids) under water stress in Scrophularia striata plants. The results revealed that PAs, NO levels were enhanced in the plants, earlier in response to polyethylene glycol-induced water stress. The antioxidative mechanisms with increased activity of catalase (CAT), guaiacol peroxidase (GPX) and superoxide dismutase (SOD) and also phenylalanine ammonia-lyase (PAL), tyrosine ammonia-lyase (TAL), as key enzymes in phenolic pathway were deployed in response to the stress. Mannose, glucose, xylose/rhamnose which are involved in PhGs biosynthesis as well as in serving osmotic adjustment were modulated. The elevated content of arginine and methionine as PAs precursors and tyrosine and phenylalanine as PhGs precursors was enhanced by water stress and was significantly associated with PAs and PhGs accumulations. Metabolic profiling revealed new information about relationship between stress signal molecules; PAs, NO and H2O2, osmolytes (sugers, PhGs) and phenolic compounds which involved in the improvement of water stress tolerance in S. striata.

New Iridoids from Scrophularia ningpoensis.

Five new compounds including five iridoids (1-5) and six known compounds were isolated from the rhizomes of Scrophularia ningpoensis. Their structures were determined by extensive NMR and IR, MS spectroscopic data analyses. The anti-inflammatory, antibacterial, antifungal, and cytotoxic activities of the isolated compounds were evaluated. Compound 11 exhibited significant inhibitory effects on lipopolysaccharide-induced nitric oxide production in RAW264.7 macrophage cells.

Extracts of Scrophularia frigida Boiss display potent antitumor effects in human breast cancer cells by inducing apoptosis and inhibition of expression of the human epidermal growth factor receptor 2.

Some species of the Scrophularia genus have been extensively used as a natural remedy for treatment of various medical conditions. The objective of this study was to evaluate the growth inhibitory activity of Scrophularia frigida Boiss extracts as well as to study the effect of the potent extracts on the induction of apoptosis and cell cycle arrest on human breast cancer cells. S. frigida Boiss extracts exhibited obvious inhibitory effects on the growth of cancer cells and induced apoptosis. It is suggested that the extracts exert their anti-proliferative effect through multiple implications such as suppressing growth, arresting the cell cycle, increased DNA fragmentation, downregulation of the expression of human epidermal growth factor receptor 2 and myeloid cell Leukemia-1, and upregulation of pro-apoptotic messenger RNAs like caspase-3 and caspase-9. Taken together, the results obtained indicate that S. frigida Boiss extracts may contain effective compounds that can be used as a therapeutic anticancer agent.

Induction of aromatic amino acids and phenylpropanoid compounds in Scrophularia striata Boiss. cell culture in response to chitosan-induced oxidative stress.

Manipulation of cell culture media by elicitors is one of most important strategies to inducing secondary metabolism for the production of valuable metabolites. In this investigation, inducing effect of chitosan on physiological, biochemical, and molecular parameters were investigated in cell suspension cultures of Scrophularia striata Boiss. The results showed that chitosan concentration and time of elicitation are determinants of the effectiveness of the elicitor. Accumulation of aromatic amino acids (phenylalanine [Phe] and tyrosine [Tyr]), phenylpropanoid compounds (phenolic acids [PAs] and echinacoside [ECH]), hydrogen peroxide (H2O2) production, phenylalanine ammonia-lyase (PAL) activity and gene expression, and antioxidant enzymes (superoxide dismutase [SOD], peroxidase [POX], catalase [CAT]) activities were altered by changing the exposure time of elicitation. Results showed that, upon elicitation with chitosan, oxidative events were induced, antioxidant responses of S. striata cells were boosted through enhanced activity of an effective series of scavenging enzymes (SOD, CAT, and POX), and biosynthesis of non-enzymatic antioxidants (ECH and PAs [cinnamic, p-coumaric and, caffeic acids]). The increase in amino acid content and PAL activity at early days of exposure to chitosan was related with rises in phenolic compounds. These results provide evidence that chitosan by up-regulation of PAL gene differentially improves the production of phenylpropanoid compounds, which are of medical commercial value with good biotechnological prospects.

Profiling of acidic (amino and phenolic acids) and phenylpropanoids production in response to methyl jasmonate-induced oxidative stress in Scrophularia striata suspension cells.

MAIN CONCLUSION: A metabolic profiling including calculation of energy cost of amino acids biosynthesis in cultured cells of Scrophularia striata showed that methyl jasmonate-inducible oxidative stress elicited secondary metabolites formation derived from phenylalanine and tyrosine and increased energy cost for these amino acids biosynthesis. Understanding of the metabolic pathways in cell culture of Scrophularia striata, an aromatic plant species, facilitates means of production of pharmaceutical metabolites under oxidative stress. In this study, we evaluated the effects of MeJA on the S. striata metabolic pathway and the responses to oxidative stress. Exposure to methyl jasmonate (MeJA) affects plant growth, effectively induces production of reactive oxygen species (ROS) and inserts oxidative stress at the cellular level which results in alteration of primary metabolites and production of phenylepropanoid compounds. Cells treated with MeJA indicated increase in the activities of three antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (GPx) as well as intracellular H2O2 and MDA contents compared with mock-treated cells. High performance liquid chromatography (HPLC)-based metabolome analysis revealed dynamic metabolic changes in oxidatively stressed S. striata cells, e.g., general phenylpropanoid pathway, phenylethanoid-glycosides, lignans, and increased energy cost of biosynthesis and accumulation of amino acids. Furthermore, principal component analysis (PCA)-derived score plots demonstrated that MeJA affects cellular metabolism in S. striata cells and significantly alters metabolite composition under MeJA-inducible oxidative stress. These observations suggest that MeJA-elicited cell suspension cultures of S. striata balanced the production of primary and secondary metabolites in coordination with ROS-scavenging system.

Chemical Elicitor-Induced Modulation of Antioxidant Metabolism and Enhancement of Secondary Metabolite Accumulation in Cell Suspension Cultures of Scrophularia kakudensis Franch.

Scrophularia kakudensis is an important medicinal plant with pharmaceutically valuable secondary metabolites. To develop a sustainable source of naturaceuticals with vital therapeutic importance, a cell suspension culture was established in S. kakudensis for the first time. Friable calli were induced from the leaf explants cultured on a Murashige and Skoog (MS) medium containing 3.0 mg·L(-1) 6-benzyladenine (BA) in a combination with 2 mg·L(-1) 2,4-dichlorophenoxy acetic acid (2,4-D). From the callus cultures, a cell suspension culture was initiated and the cellular differentiation was investigated. In addition, the effect of biotic elicitors such as methyl jasmonate (MeJa), salicylic acid (SA), and sodium nitroprusside (SNP) on the accumulation of secondary metabolites and antioxidant properties was demonstrated. Among the elicitors, the MeJa elicited the accumulation of total phenols, flavonoids, and acacetin, a flavonoid compound with multiple pharmaceutical values. Similarly, the higher concentrations of the MeJa significantly modulated the activities of antioxidant enzymes and enhanced the scavenging potentials of free radicals of cell suspension extracts. Overall, the outcomes of this study can be utilized for the large scale production of pharmaceutically important secondary metabolites from S. kakudensis through cell suspension cultures.

[LC-LTQ-Orbitrap analysis on chemical constituents in Scrophulariae Radix extract and their metabolites in rat plasma].

Chemical constituents in extract of Scrophulariae Radix and their metabolites in rat plasma after oral administration were identified by HPLC-LTQ-Orbitrap. Samples were separated by a Venusil MP C18 column using a binary gradient elution. The information on the total ion chromatogram, the extraction chromatogram and the mass spectrogram in a negative mode were synthetically analyzed by comparing the retention time, MS and MS/MS spectra with literature data and some of reference standards to conduct a qualitative study on constituents of Radix Scrophulariae extract in vivo and in vitro. Totally 37 compounds from Scrophularia ningpoensis extract were detected including 12 iridoid glycosides, 20 phenylpropanoids and 5 unknown compounds. In vivo, harpagide, harpagoside and angoroside C were confirmed to enter into the blood in prototype forms. Besides, another 2 prototype compounds and 2 metabolites were detected in rat plasma after oral administration of S. ningpoensis extract. The results are beneficial for the determination of bioactive substances of S. ningpoensis and significant for further studies on S. ningpoensis.