Hydroxyl-rich ferrofluid for efficient liquid phase microextraction of cinnamic acid derivatives in traditional Chinese medicine.

In this study, a hydroxyl-rich ferrofluid was prepared by dispersing silica-coated magnetic nanoparticles into a methyltrioctylammonium chloride-glycerol deep eutectic solvent and then employed in the preconcentration of trace-level of cinnamic acid derivatives (caffeic acid, p-hydroxycinnamic acid, ferulic acid, and cinnamic acid) in traditional Chinese medicine prior to high-performance liquid chromatography analysis. The structures of the synthesized materials were characterized by X-ray diffraction and infrared spectroscopy. The experimental parameters affecting the extraction performance, such as deep eutectic solvent composition, dosage of ferrofluid, pH of aqueous sample solution, salt concentration, extraction time, type, and volume of desorption solvent, were studied and optimized. Under the optimum conditions, the enrichment factors of four cinnamic acid derivatives were in the range of 107-114. Low detection limits (0.2-0.9 ng/mL), good precisions (relative standard deviations 1.2%-9.5%), and satisfactory recoveries (96.0%-104.7%) were achieved. Subsequently, the possible microextraction mechanism of the proposed method was explored and elucidated. It showed that the prepared ferrofluid is easily dispersed in the aqueous sample and achieved recovery after the extraction. The developed approach is a simple, convenient, and efficient method for preconcentration and determination of cinnamic acid derivatives in complex matrices.

Regioisomers Salviprolin A and B, Unprecedented Rosmarinic Acid Conjugated Dinorditerpenoids from Salvia przewalskii Maxim.

Salvia przewalskii Maxim is a perennial plant from the genus Salvia (family Lamiaceae). The roots of S. przewalskii were long used as a traditional herb to treat blood circulation related illnesses in China. As part of our continuing interest in polycyclic natural products from medicinal plants, two unprecedented adducts comprised of a dinor-diterpenoid and a 9'-nor-rosmarinic acid derivative, linked by a 1,4-benzodioxane motif (1 and 2), were isolated from the roots of S. przewalskii. Their structures were established by extensive spectroscopic approaches including 1D, 2D NMR, and HRFABMS. Their cytotoxic activities against five human tumor cell lines were evaluated.

Comparative Analysis of Phenolic Composition of Six Commercially Available Chamomile (Matricaria chamomilla L.) Extracts: Potential Biological Implications.

Several phytochemical-containing herbal extracts are increasingly marketed as health-promoting products. In particular, chamomile (Matricaria recutita L.) is well known for its anti-inflammatory, analgesic, and antitumor properties. Here, we evaluated differences in chemical composition among six commercially available products and their potential impact on biological activity in human immortalized colonocytes. Our investigation encompassed: (i) preparation of dry extracts and yield evaluation; (ii) qualitative and quantitative analysis of phenol content; (iii) modulation of redox state; and (iv) bioavailability of main bioactive compounds. We demonstrated that apparently identical products showed huge heterogeneity, in terms of yield extraction, chemical composition, and antioxidant effects. All samples contained high amounts of flavonoids and cinnamic acid derivatives, but differentially concentrated in the six extracts. Depending on polyphenol content, chamomile samples possessed variable antioxidant potential, in terms of decreased radical generation and increased reduced glutathione levels. The observed effects might be ascribed to flavones (apigenin, luteolin, and their glycones) highly represented in the six extracts. Nonetheless, chamomile extracts exerted cytotoxic effects at high concentrations, suggesting that a herbal medicine is not always safe. In conclusion, due to the complexity and variability of plant matrices, studies evaluating effectiveness of chamomile should always be accompanied by preliminary characterization of phytochemical composition.

Effect of Elicitation with (+)-Usnic Acid on Accumulation of Phenolic Acids and Flavonoids in Agitated Microshoots of Eryngium alpinum L.

The present work was aimed at studying the potential of elicitation on the accumulation of phenolic compounds in in vitro shoot cultures of Eryngium alpinum L., a protected plant from the Apiaceae family. The study examined the influence of (+)-usnic acid on the biomass growth as well as on the biosynthesis of the desired flavonoids and phenolic acids in the cultured microshoots. The phenolic compound content was determined by HPLC-DAD. The flavonoid of the highest concentration was isoquercetin, and the phenolic acids of the highest amount were rosmarinic acid, caffeic acid and 3,4-dihydroxyphenylacetic acid, both in the non-elicited and elicited biomass. Isoquercetin accumulation was efficiently increased by a longer elicitation with a lower concentration of lichenic compound (107.17 ± 4.67 mg/100 g DW) or a shorter elicitation with a higher concentration of acid (127.54 ± 11.34 and 108.37 ± 12.1 mg/100 g DW). Rosmarinic acid production generally remained high in all elicited and non-elicited microshoots. The highest content of this acid was recorded at 24 h of elicitation with 3.125 µM usnic acid (512.69 ± 4.89 mg/100 g DW). The process of elicitation with (+)-usnic acid, a well-known lichenic compound with allelopathic nature, may therefore be an effective technique of enhancing phenolic compound accumulation in alpine eryngo microshoot biomass.

The new rapid and accurate analytical HPLC-ECD method for the determination of rosmarinic acid in meat products.

A new rapid method has been developed for the determination of low levels of rosmarinic acid extracted from rosemary (Rosmarinus officinalis L.) and has been used as an antioxidant in meat and meat products after cold storage at 4°C. The method is a high performance liquid chromatography using a coulometric electrochemical detector. It provides a significant improvement on the limit of detection, which was 0.33 ppb, while the limit of quantification was 1 ppb of rosmarinic acid. The advantage of the method also lies in the simpler and faster sample preparation, which can quantify a very low concentration of rosmarinic acid (60 ppb), and is more than 40 ppb below the limits of previously existing methods. A coulometric method is well suited for determining low analyte concentrations and is one of the most sensitive analytical approaches available today, in addition to being time efficient and cost effective. PRACTICAL APPLICATION: A new method for determining low concentrations (60 ppb) of rosmarinic acid in meat and meat products is presented. The method is user-friendly, as it does not require complex sample preparation. It is a selective, precise, and accurate method that makes it useful for routine applications in the meat and other food industries.

Synthesis, antitumor activity and in silico analyses of amino acid derivatives of artepillin C, drupanin and baccharin from green propolis.

Breast cancer has the highest incidence and mortality in females, while prostate cancer has the second-highest incidence in males. Studies have shown that compounds from Brazilian green propolis have antitumor activities and can selectively inhibit the AKR1C3 enzyme, overexpressed in hormone-dependent prostate and breast tumors. Thus, in an attempt to develop new cytotoxic inhibitors against these cancers, three prenylated compounds, artepillin C, drupanin and baccharin, were isolated from green propolis to synthesize new derivatives via coupling reactions with different amino acids. All obtained derivatives were submitted to antiproliferative assays against four cancer cells (MCF-7, MDA MB-231, PC-3, and DU145) and two normal cell lines (MCF-10A and PNT-2) to evaluate their cytotoxicity. In general, the best activity was observed for compound6e, derived from drupanin, which exhibited half-maximal inhibitory concentration (IC50) of 9.6 ± 3 µM and selectivity index (SI) of 5.5 against MCF-7 cells.In silicostudies demonstrated that these derivatives present coherent docking interactions and binding modes against AKR1C3, which might represent a possible mechanism of inhibition in MCF-7 cells.

Production of trans-cinnamic acid by whole-cell bioconversion from L-phenylalanine in engineered Corynebacterium glutamicum.

BACKGROUND: trans-cinnamic acid (t-CA) is a phenylpropanoid with a broad spectrum of biological activities including antioxidant and antibacterial activities, and it also has high potential in food and cosmetic applications. Although significant progress has been made in the production of t-CA using microorganisms, its relatively low product titers still need to be improved. In this study, we engineered Corynebacterium glutamicum as a whole-cell catalyst for the bioconversion of L-phenylalanine (L-Phe) into t-CA and developed a repeated bioconversion process. RESULTS: An expression module based on a phenylalanine ammonia lyase-encoding gene from Streptomyces maritimus (SmPAL), which mediates the conversion of L-Phe into t-CA, was constructed in C. glutamicum. Using the strong promoter PH36 and ribosome binding site (RBS) (in front of gene 10 of the T7 phage), and a high-copy number plasmid, SmPAL could be expressed to levels as high as 39.1% of the total proteins in C. glutamicum. Next, to improve t-CA production at an industrial scale, reaction conditions including temperature and pH were optimized; t-CA production reached up to 6.7 mM/h in a bioreactor under optimal conditions (50 °C and pH 8.5, using NaOH as base solution). Finally, a recycling system was developed by coupling membrane filtration with the bioreactor, and the engineered C. glutamicum successfully produced 13.7 mM of t-CA (24.3 g) from 18.2 mM of L-Phe (36 g) and thus with a yield of 75% (0.75 mol/mol) through repetitive supplementation. CONCLUSIONS: We developed a highly efficient bioconversion process using C. glutamicum as a biocatalyst and a micromembrane-based cell recycling system. To the best of our knowledge, this is the first report on t-CA production in C. glutamicum, and this robust platform will contribute to the development of an industrially relevant platform for the production of t-CA using microorganisms.

Development and validation of ultra-high performance supercritical fluid chromatography method for quantitative determination of six compounds in Guizhi Fuling capsule and tablet samples.

A fast and simple ultra-high performance supercritical fluid chromatography method has been developed for the determination of six analytes, namely (paeonol, coumarin, cinnamic alcohol, cinnamic acid, paeoniflorin, and amygdalin) in Guizhi Fuling capsule and tablet samples. The influence of the key chromatographic parameters for the separation purposes was evaluated. The optimal column was Trefoil CEL1 column. The optimal mobile phase was a gradient mixture of carbon dioxide and methanol at flow rate of 1.0 mL/min. The back pressure of the system was set to 1.38 × 107  Pa and the temperature to 45°C. The six compounds were separated within 11 min by the proposed ultra-high performance supercritical fluid chromatography method with satisfactory resolution. Method validation confirmed that the procedure is accurate with the recovery rates from 87.04 to 104.30%, intraday precision values less than 4.81% and interday precision less than 5.22%, and linear with R2 higher than 0.9967. Therefore, this work provides a simple and novel method for the simultaneous analysis of six compounds in Guizhi Fuling capsule and tablet samples.

The Contents of Polyphenols in Perilla frutescens (L.) Britton var. frutescens (Egoma) Leaves are Determined by Vegetative Stage, Spatial Leaf Position, and Timing of Harvesting during the Day.

The leaf of Perilla frutescens (L.) Britton var. frutescens (egoma) is a rich source of polyphenolic compounds, including rosmarinic acid. However, there is still a lack of detailed information concerning the content of phenolic compounds in these leaves. Since some flavonoids were found as a conjugated form, leaves were used untreated or hydrolyzed using ß-glucuronidase for analysis. Enzymatic hydrolysis method successfully identified some polyphenols, which have not been reported before. Scutellarin, a flavone glucuronide with a molecular mass similar to that of luteolin 7-O-glucuronide, was present in egoma leaves. Scutellarin was the second most abundant polyphenolic compound, after rosmarinic acid. Egoma leaves at the top of the plant contained a higher amount of rosmarinic acid and scutellarin compared to that in the leaves below. The difference in plant growth stage also influenced the rosmarinic acid and scutellarin contents, while the time of harvesting during the day did rosmarinic acid contents only. This is the first time that scutellarin, a traditional Chinese medicine, widely used for the treatment of cerebrovascular disease, was quantitatively determined in egoma leaves. The present study may help adding value to egoma leaves, developing dietary supplements, functional foods, and cosmetics.

Honey Phenolic Compound Profiling and Authenticity Assessment Using HRMS Targeted and Untargeted Metabolomics.

Honey consumption is attributed to potentially advantageous effects on human health due to its antioxidant capacity as well as anti-inflammatory and antimicrobial activity, which are mainly related to phenolic compound content. Phenolic compounds are secondary metabolites of plants, and their content in honey is primarily affected by the botanical and geographical origin. In this study, a high-resolution mass spectrometry (HRMS) method was applied to determine the phenolic profile of various honey matrices and investigate authenticity markers. A fruitful sample set was collected, including honey from 10 different botanical sources (n = 51) originating from Greece and Poland. Generic liquid-liquid extraction using ethyl acetate as the extractant was used to apply targeted and non-targeted workflows simultaneously. The method was fully validated according to the Eurachem guidelines, and it demonstrated high accuracy, precision, and sensitivity resulting in the detection of 11 target analytes in the samples. Suspect screening identified 16 bioactive compounds in at least one sample, with abscisic acid isomers being the most abundant in arbutus honey. Importantly, 10 markers related to honey geographical origin were revealed through non-targeted screening and the application of advanced chemometric tools. In conclusion, authenticity markers and discrimination patterns were emerged using targeted and non-targeted workflows, indicating the impact of this study on food authenticity and metabolomic fields.

Preparative separation of six compounds from Salvia yunnanensis Radix using pH-zone-refining counter-current chromatography.

Phenolic acids are the main active components in Salvia yunnanensis Radix, which have significant effects such as cardiovascular protection, anti-thrombosis, anti-oxidant, and anti-inflammation. In this study, pH-zone-refining counter-current chromatography (pH-ZRCCC) was successfully applied to the preparative separation of phenolic acids from S. yunnanensis Radix. First, a two-phase solvent system composed of Pet-EtOAc-ACN-H2 O (1.5:2.5:1:5, v/v) [TFA (10 mM) was added in the upper phase and NH3 ·H2 O (30 mM) was added in the lower phase] was used for the separation of 4.0 g of the crude sample to obtain 55.6 mg of rosmarinic acid (1), 69.0 mg of caffeic acid (2), 18.9 mg of protocatechualdehyde (3), 14.6 mg of 8-epiblechnic acid 9-methyl ester (4), and a mixture containing two compounds. After the recovery, 1.3 g of the mixture was obtained and separated using the MtBE-H2 O (1:1, v/v) solvent system containing TFA (5 mM) and NH3 ·H2 O (60 mM) to obtain 259.9 mg of salvianolic acid B (5) and 28.75 mg of lithospermic acid (6). Moreover, a systematic separation pattern for separating the relatively low-polarity phenolic acids from natural products by pH-ZRCCC was summarized for the first time. This study provided technical support for the pharmacological activity and quality control research of S. yunnanensis Radix.

Development and validation of a rapid high-performance thin-layer chromatographic method for quantification of gallic acid, cinnamic acid, piperine, eugenol, and glycyrrhizin in Divya-Swasari-Vati, an ayurvedic medicine for respiratory ailments.

Divya-Swasari-Vati is a calcium containing polyherbal ayurvedic medicine prescribed for the lung-related ailments observed in the current pandemic of Severe Acute Respiratory Syndrome Coronavirus 2 infections. The formulation is a unique quintessential blend of nine herbs cited in Ayurvedic texts for chronic cough and lung infection. Analytical standardization of herbal medicines is the pressing need of the hour to ascertain the quality compliance. This persuaded us to develop a simple, rapid, and selective high-performance thin-layer chromatographic method for Divya-Swasari-Vati quality standardization. The developed method was validated for the quantification of marker components, gallic acid, cinnamic acid, piperine, eugenol and glycyrrhizin, against reference standards in five different batches of Divya-Swasari-Vati. The analytes were identified by visualization at 254 nm, and by matching their retention factor with authentic standards. The developed method was validated as per the guidelines recommended by the International Council for Harmonization for parameters like, linearity, limit of detection, limit of quantification, accuracy, and precision. Therefore, the developed novel high-performance thin-layer chromatographic process could be employed for rapid standardization of Divya-Swasari-Vati and other related herbal formulation, which would aid in quality manufacturing and product development.

Protective effect of Zataria multiflora Boiss. and its main compound, rosmarinic acid, against malathion induced oxidative stress and apoptosis in HepG2 cells.

Malathion (MT) is one of the most widely used organophosphorus insecticides which induces toxicity through oxidative stress induction, free radical production and acetylcholinesterase inhibition. In this work, HepG2 cells were used to determine the effect of Zataria multiflora methanolic extract (MEZM) and rosmarinic acid (RA) on MT-induced cytotoxicity, oxidative stress, and apoptosis. Total phenolic content (TPC) and total flavonoid content (TFC) were determined and plant was further standardized based on RA content using HPLC method. The cultured HepG2 cells were pretreated with MEZM (1 µg/ml) and RA (0.1 µg/ml) for 4 h and exposed to MT (100 µM). Cell viability, oxidative stress biomarkers, ROS production, and cell death were examined after 24 h. The amount of RA was determined 73.48 mg/g dried extract. IC50 values of MEZM and MT were 368.56 µg/ml and 99.43 µM, respectively. Pretreatment with MEZM and RA decreased the cytotoxicity, oxidative stress, and cell percentage in the late apoptosis and necrosis stages induced by MT. There was no significant difference between MEZM and RA effects. The present study showed the significant protective effects of MEZM against toxicity induced by MT in hepatocytes which can be attributed to the plant antioxidant constituents including RA.

Application of smartphone in detection of thin-layer chromatography: Case of salvia miltiorrhiza.

In this work, a smartphone-based device was constructed for thin-layer chromatography (TLC) detection and semi-quantitative analysis of the components of Salvia miltiorrhiza. The key construction and shooting parameters were investigated by the relative peak area and signal-to-noise ratio. The best conditions were as follows: shooting height, 17 cm; angle between the UV lamp and TLC plate, 58°; exposure compensation, 0~0.2 EV; and shutter speed under daylight and UV 365 nm, 1/50 s and 1/5 s, respectively. These ideal conditions could be replicated by smartphones from different brands with different versions of software. With good precision, repeatability and stability, the developed device was used for the semi-quantitative analysis of salvianolic acid B, rosmarinic acid, cryptotanshinone, tanshinone I, tanshinone IIA, and miltirone in the TLC analysis of 10 batches of S. miltiorrhiza. The results were compared with those obtained by a TLC densitometric scanner and two common types of image processing software, i.e., Gelanalyzer and ImageJ. Except for salvianolic acid B in the TLC densitometric scanner, all results were not significantly different among these methods, which suggested that smartphones might be a useful tool for the quality control of traditional Chinese medicines.

Protective effect of rosmarinic acid-rich trichodesma khasianum clarke leaves against ethanol-induced gastric mucosal injury in vitro and in vivo.

BACKGROUND: Although gastroprotective drugs have been used for peptic ulcer disease prevention and treatment, side effects have been observed. Finding a safe and effective treatment strategy is important. PURPOSE: Edible Trichodesma khasianum (T. khasianum) Clarke leaves are considered to protect against peptic ulcers. However, scientific evidence of this effect of T. khasianum Clarke leaves remains limited. STUDY DESIGN/METHODS: In this study, we aimed to evaluate the effect of T. khasianum Clarke leaves on ethanol-induced gastric injury and gut microbiota using RAW 264.7 cells, RGM-1 cells, and BALB/c mice, respectively. RESULT: The rosmarinic acid was identified as the major component of T. khasianum Clarke leaves extracted by 80% ethanol (80EETC). The results showed that 80EETC suppressed inflammatory mediator protein levels in LPS-induced RAW 264.7 cells. Additionally, heat shock protein expression, antiapoptotic ability, and wound healing migration capability were increased by 80EETC pretreatment in RGM-1 cells with the ethanol-induced injury. Remarkably, pretreatment with 80EETC (150 mg/kg b.w.) promoted gastric mucosal healing by decreasing oxidative stress, inflammatory response, proapoptotic protein expression, and gastric mucosa damage in ethanol-induced gastric injury in mice. Crucially, no liver or kidney toxicities were observed by 80EETC oral gavage. Moreover, 80EETC increased gut microbiota diversity and short-chain fatty acid production. CONCLUSION: Our results illustrated the remarkable gastroprotective effect by 80EETC treatment in vitro and in vivo. These findings are the first to demonstrate the powerful protective effect of T. khasianum Clarke leaves against gastric mucosal injury development.

Chitosan stimulates secondary metabolite production and nutrient uptake in medicinal plant Dracocephalum kotschyi.

BACKGROUND: A wide variety of secondary metabolites are synthesized from primary metabolites by plants which have a vast range in pharmaceutical, food additive and industrial applications. In recent years, the use of elicitors has opened a novel approach for the production of secondary metabolite compounds. Dracocephalum kotschyi is a valuable herb due to pharmaceutical compounds like rosmarinic acid, quercetin and apigenin. In the current study, foliar application of chitosan (0, 100, 400 mg L-1 ) as an elicitor was used. RESULTS: After chitosan treatment, the amounts of hydrogen peroxide (H2 O2 ) increased and the plant was able to increase the activities of enzymatic (guaiacol peroxidase, catalase and phenylalanine ammonium lyase) and non-enzymatic (total phenols and flavonoids) defensive metabolites. Also, foliar spray of chitosan promoted nutrient absorption which led to the accumulation of macroelements in the plant. CONCLUSIONS: Chitosan was found to be a very effective elicitor for improving rosmarinic acid and quercetin content (up to 13-fold). Also, the content of apigenin (anticancer flavonoid) showed 16-fold enhancement compared to the control. Therefore, the treatment of D. kotschyi leaves with chitosan caused a very large increase in the induction and production of important pharmaceutical compounds such as rosmarinic acid and quercetin. © 2020 Society of Chemical Industry.

Supercritical Fluid Chromatography development of a predictive analytical tool to selectively extract bioactive compounds by supercritical fluid extraction and pressurised liquid extraction.

Selective extraction is a great concern in the field of natural products. The interest is to apply specific conditions favouring the solubility of targeted secondary metabolites and avoiding the simultaneous extraction of unwanted ones. Different ways exist to reach selective extractions with suited conditions. These conditions can be determined from experimental studies through experimental design, but a full experimental design takes time, energy, and uses plant samples. Prediction from varied solubility models can also be applied allowing a better understanding of the final selected conditions and eventually less experiments. The aim of this work was to develop and use a chromatographic model to determine optimal extraction conditions without the need for numerous extraction experiments. This model would be applied on the selective extraction of the desired antioxidant compounds in rosemary leaves (rosmarinic and carnosic acids) vs chlorophyll pigments to limit the green colour in extracts. This model was achieved with Supercritical Fluid Chromatography (SFC) and then applied to Supercritical Fluid Extraction (SFE) and Pressurised Liquid Extraction (PLE) assays. SFC models predicted low solubility of chlorophylls for low (5%) and high (100%) percentage of solvent in carbon dioxide. Also, low solubility was predicted with acetonitrile solvent compared to methanol or ethanol. This was confirmed with different extractions performed using SFE with different percentages of solvent (5, 30, and 70%) and with the three solvents used in the SFC models (acetonitrile, methanol and ethanol). Also extractions using PLE were carried out using the same neat solvents in order to confirm the SFC models obtained for 100% of solvent. Globally, extractions validated the SFC models. Only some differences were observed between ethanol and methanol showing the complexity of plant extraction due to matrix effect. For all these extracts, the content of carnosic acid and rosmarinic acid was also monitored and selective extraction conditions of bioactive compounds could be determined.

Rindera graeca (Boraginaceae) Phytochemical Profile and Biological Activities.

Rindera graeca is a Greek endemic plant of the Boraginaceae family which has never been studied before. Consequently, this study attempted to phytochemically examine the aerial parts of this species. Nine phenolic secondary metabolites were identified, consisting of seven caffeic acid derivatives and two flavonol glucosides, namely rutin and quercetin-3-rutinoside-7-rhamnoside. These flavonoids, together with rosmarinic acid, were isolated via column chromatography and structurally determined through spectral analysis. Quercetin-3-rutinoside-7-rhamnoside is an unusual triglycoside, which is identified for the first time in Rindera genus and among Boraginaceae plants. This metabolite was further examined with thermal analysis and its 3D structure was simulated, revealing some intriguing information on its interaction with biological membrane models, which might have potential applications in microcirculation-related conditions. R. graeca was also analyzed for its pyrrolizidine alkaloids content, and it was found to contain echinatine together with echinatine N-oxide and rinderine N-oxide. Additionally, the total phenolic and flavonoid contents of R. graeca methanol extract were determined, along with free radical inhibition assays. High total phenolic content and almost complete inhibition at experimental doses at the free radical assays indicate a potent antioxidant profile for this plant. Overall, through phytochemical analysis and biological activity assays, insight was gained on an endemic Greek species of the little-studied Rindera genus, while its potential for further applications has been assessed.

Simultaneous determination of eight bioactive components in Marsdenia tenacissima extract in rat plasma by LC-MS/MS and its application in a pharmacokinetic study.

As a traditional Chinese medicine, Marsdenia tenacissima (Roxb.) Wight et Arn. plays an indispensable role in clinical practice owing to its specific efficacy in treating malignant tumors, leukocythemia, cystitis and asthma. This study aimed to establish a novel and scientific LC-MS/MS approach to simultaneously determine tenacissoside B, H, G and I, caffeic acid, cryptochlorogenic acid, chlorogenic acid and neochlorogenic acid from M. tenacissima extract within the rat plasma samples. Digoxin was used as the internal reference. All determinations were carried out using the Eclipse Plus C18 column, and water (containing 0.1% formic acid) was used as the mobile phase A, while acetonitrile was the mobile phase B for gradient elution. The UPLC methods were validated, including calibration curves, accuracy, precision, stability and recovery of the total eight analytes, in accordance with the requirements for biopharmaceutical analysis. Moreover, the proposed approach was also used in comprehensive pharmacokinetic research on those eight analytes in rats following M. tenacissima extract gavage. According to the pharmacokinetic parameters, tenacissoside B, I, H and G are the long-acting and primary bioactive constituents in M. tenacissima extract, with long mean residence times and high concentrations. Our findings shed light on the absorption mechanism and provide significant information for the clinical application of M. tenacissima.

Influence of photoperiod on growth, bioactive compounds and antioxidant activity in callus cultures of Basella rubra L.

Basella rubra L. is an important green leafy vegetable vine and is known for its health benefits in traditional medicine. Light is a basic physical factor essential to the development and bioactive secondary metabolite production in in vitro callus cultures. The present study researched the impact of different photoperiods on biomass, bioactive compounds, and antioxidant activity in callus cultures of B. rubra. The in vitro seedling based cotyledonary leaf explants responded differently, when cultured on Murashige and Skoog (MS) medium with varying concentrations and combination of auxins and cytokinins. The best callus proliferation was found in MS medium with 0.1 mg.L-1 1-naphthaleneacetic acid (NAA) and 6 mg.L-1 6-benzylaminopurine (BAP), with greenish callus inception by about 2 weeks. The growth curve recorded for 6 weeks of culturing revealed that the photoperiod effect was found to be pivotal for acquiring biomass. At the fifth week, the continuous light supported maximum biomass (12.42 g) production followed by the 16:8 h photoperiod (9.02 g) and continuous darkness (4.28 g). The 80% ethanol extract of 1-week-old callus that grows under the 16:8 h photoperiod showed the highest total phenolic content (TPC) (74 mg.100 g-1 fresh weight, FW) when compared to all other extracts at different stages. The ferric reducing antioxidant power assay showed the highest (336.23 mg.100 g-1 FW) activity in methanol extractions of first-week callus cultures maintained in the continuous light condition. HPLC-UV identification and quantification of individual phenolics and flavonoids, such as gallic, trans-cinnamic, quercetin, protocatechuic and rutin, were highest in the callus cultures. The outcome of this study is significant to this plant, as B. rubra is familiar for its important health constituents with high-value bioactives and applications in the pharma and nutraceutical industries.