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Abstract: A new method, namely negative-pressure cavitation extraction (NPCE), followed by liquid chromatography–tandem mass spectrometry (LC–MS/MS) is presented for the extraction and quantification of flavonoids in pigeon pea leaves. This method combines the high efficiency of NPCE and the sensitivity and accuracy of MS/MS. The influential parameters of the NPCE procedure including liquid/solid ratio, extraction time, nitrogen flow and number of extraction cycles were optimized. Under optimized conditions, the efficiency of NPCE for extracting five flavonoids was compared to microwave-assisted extraction (MAE), ultrasonic extraction (USE) and heating reflux extraction (HRE). Additionally, structural disruption to pigeon pea leaves samples with different extraction methods was investigated by scanning electron microscopy. The relative recovery with NPCE was equivalent to or higher than that with USE and obviously higher than those with MAE and HRE which are usually conducted in higher temperatures. Furthermore, because NPCE was performed with nitrogen at room temperature, the degradation and oxidation of analytes were avoided. In addition, the NPCE method was validated in terms of repeatability and reproducibility, relative standard deviation for relative recovery was lower than 5.84 and 8.83%, respectively. The method was also successfully applied for the quantification of five flavonoids in pigeon pea leaves. All these results suggest that the developed NPCE-LC–MS/MS method represents an excellent alternative for the extraction and quantification of flavonoids in other plant materials. [Copyright &y& Elsevier]

Flavonoids are important secondary metabolites in the plant growth and development process. As a medicinal plant, pigeon pea is rich in secondary metabolites. As a flavonoid, there are few studies on the regulation mechanism of naringenin in plant stress resistance. In our study, we found that naringenin can increase the pigeon pea's ability to tolerate salt and influence the changes that occur in flavonoids including naringenin, genistein and biochanin A. We analyzed the transcriptome data after 1 mM naringenin treatment, and identified a total of 13083 differentially expressed genes. By analyzing the metabolic pathways of these differentially expressed genes, we found that these differentially expressed genes were enriched in the metabolic pathways of phenylpropanoid biosynthesis, starch and sucrose metabolism and so on. We focused on the analysis of flavonoid biosynthesis related pathways. Among them, the expression levels of enzyme genes CcIFS , CcCHI and CcCHS in the flavonoid biosynthesis pathway had considerably higher expression levels. By counting the number of transcription factors and the binding sites on the promoter of the enzyme gene, we screened the transcription factors CcMYB62 and CcbHLH35 related to flavonoid metabolism. Among them, CcMYB6 2 has a higher expression level than the others. The hairy root transgene showed that CcMYB62 could induce the upregulation of CcCHI , and promote the accumulation of naringenin, genistein and biochanin A. Our study revealed the molecular mechanism of naringenin regulating flavonoid biosynthesis under salt stress in pigeon pea, and provided an idea for the role of flavonoids in plant resistance to abiotic stresses. • Naringenin enhanced the salt tolerance of pigeon pea. • Naringenin can induce the accumulation of downstream secondary metabolites. • Naringenin induced significant upregulation of CcCHS , CcCHI and CcIFS. • Revealing the potential regulation between CcMYB62 and flavonoid biosynthesis. [ABSTRACT FROM AUTHOR]

Melatonin improves plant resistance to multiple stresses by participating in the biosynthesis of metabolites. Flavonoids are an important family of plant secondary metabolites and are widely recognized to be involved in resistance; however, the crosstalk between melatonin and flavonoid is largely unknown. We found that the resistance of pigeon pea (Cajanus cajan) to salt, drought, and heat stresses were significantly enhanced by pre-treatment with melatonin. Combined transcriptome and LC-ESI-MS/MS metabolomics analyses showed that melatonin significantly induced the enrichment of flavonoids and mediated the reprogramming of biosynthetic pathway genes. The highest fold-increase in expression in response to melatonin treatment was observed for the CcF3´H family, which encodes an enzyme that catalyses the biosynthesis of luteolin, and the transcription factor CcPCL1 directly bonded to the CcF3´H-5 promoter to enhance its expression. In addition, salt stress also induced the expression of CcPCL1 and CcF3´H-5 , and their overexpression in transgenic plants greatly enhanced salt tolerance by promoting the biosynthesis of luteolin. Overall, our results indicated that pre-treatment of pigeon pea with melatonin promoted luteolin biosynthesis through the CcPCL1 and CcF3´H-5 pathways, resulting in salt tolerance. Our study shows that melatonin enhances plant tolerance to multiple stresses by mediating flavonoid biosynthesis, providing new avenues for studying the crosstalk between melatonin and flavonoids. [ABSTRACT FROM AUTHOR]

The present study was designed to evaluate the chemical extraction, chemical composition, and antioxidant and antibacterial properties of the total flavonoids in Willow Buds (TFW). We investigated the optimal extraction of TFW using response surface methodology (RSM). Chemical compounds were analyzed using Q-Orbitrap LC–MS/MS. The DPPH radical scavenging capacity, hydroxy radical inhibitory ability, and superoxide anion radical inhibitory ability were explored to determine the antioxidant properties of flavonoid extractions. The antibacterial effect was assessed via minimal inhibitory concentration. The results demonstrated that the optimal extraction conditions were an ethanol concentration of 50%, a time of 35 min, and a liquid/material ratio of 70:1 mL/g. Under these conditions, the yield of TFW was 7.57%. Eight flavonoids, a phenolic glycoside, and an alkaloid were enriched in the Willow Buds. The TFW exhibited significant antioxidant activity, with IC50 values of 0.18-0.24 mg/mL and antimicrobial activity against Escherichia coli, Salmonella enterica, Staphylococcus aureus, and Streptococcus pneumoniae. TFW may be explored as potential and natural compounds in food and pharmacological applications. [ABSTRACT FROM AUTHOR]

In this study, different types of natural deep eutectic solvents (NADES) were used to extract flavonoids from sweet tea. A total of 7 flavonoids, primarily dihydrochalcones, were successfully extracted. Among the NADESs tested, a ternary deep eutectic solvent Bet-PG-Ur (betaine: propanediol: urea = 1: 2: 1, molar ratio) demonstrated the highest extraction efficiency. The chemical and morphological changes of both the NADES and extracted compounds were analyzed using Fourier transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). To optimize the extraction process and evaluate the total flavonoid yield, response surface methodology (RSM), artificial neural network (ANN), and random forest (RF) techniques were employed. The NADES demonstrated consistent extraction performance even after three cycles of recovery using the SPE method. The results revealed that all three models successfully predicted the TFC in the green extraction ultrasonic process. The ANN model exhibited superior result consistency compared to the others, which can be attributed to the enhanced statistical parameters obtained. The highest total flavonoid content (TFC) of 167.561 mg/g was predicted by ANN and achieved under the following conditions: liquid/solid ratio of 19, water content of 50 %, temperature of 45 °C, and ultrasonic power of 400 W. [Display omitted] • A ternary DES-ultrasonic assisted was used for extraction of 7 flavonoids from sweet tea. • For the first time, machine learning was used to forecast the parameters for the TFC in sweet tea. • In terms of process modeling and prediction, ANN is better than RF and RSM. • The extraction efficiency of DES can reach 80% after 3 cycles of recovery by SPE. • Ultrasound-assisted extraction of 7 flavonoids from sweet tea using NADESs. [ABSTRACT FROM AUTHOR]

Abstract: The present study aims to extract active components from pigeon pea leaves by ionic liquid-based microwave-assisted extraction (ILMAE) for quantitative analysis. Compared with ionic liquid-based heating extraction (ILHE) and ultrasonic-assisted extraction (ILUAE), ILMAE exhibited higher extraction efficiency. A series of 1-alkyl-3-methylimidazolium ILs with different anions and alkyl chain lengths of cations were investigated, and 1-butyl-3-methylimidazolium bromide ([C4MIM]Br) solution was selected as extractants. Subsequently, ILMAE parameters were optimized, and the optimized conditions were [C4MIM]Br concentration 0.75M, extraction temperature 60°C, solvent to solid ratio 12:1mL/g, and irradiation time 7min. Under the optimal conditions, the extraction efficiency increased by 11.0–38.9% compared with ILHR and ILUAE, and the recovery achieved more than 95.3%. Furthermore, a rapid analysis method, HPLC-DAD with an UPLC column, was established and validated. This method exhibited satisfactory linearity (r 2 >0.999), precision (RSD<4.6%) and recovery (97.14–103.38%), and was successfully applied for the quantification of nine components in pigeon pea leaves within 25min. The results obtained provide a fast and safe method for determination of active components in pigeon pea leaves. [Copyright &y& Elsevier]

In this study, the aqueous and ethanolic extracts (leaves, stems and fruits) from Morus alba L., a traditional Chinese medicine, were evaluated for their antioxidant and antimicrobial properties. Ethanolic extracts showed higher contents of both total phenolics and flavonoids than aqueous extracts. The total phenolic content was in the order of: leaf extracts > fruit extracts > stem extracts, whereas the total flavonoids was: leaf extracts > stem extracts > fruit extracts. Using DPPH assays, the concentrations providing 50% inhibition (IC50) values of aqueous extracts from leaves, stems and fruits were 7.11 ± 1.45 mg/ml, 86.78 ± 3.21 mg/ml and 14.38 ± 2.83 mg/ml, respectively, whereas the IC50 values of ethanolic extracts were 3.11 ± 0.86 mg/ml, 14.62 ± 2.45 mg/ml and 12.42 ± 2.76 mg/ml, respectively. In sum, the antioxidant activities of ethanolic extracts from M. alba L. were stronger than the aqueous extracts, and in the order of: leaf extracts > fruit extracts > stem extracts. The ethanolic extracts exhibited moderate antimicrobial activities, whereas the aqueous extracts showed poor antimicrobial properties in our test system. This study validated the medicinal potential of M. alba L. [ABSTRACT FROM AUTHOR]

SUMMARY: Calcineurin B‐like (CBL)‐interacting protein kinases (CIPKs) play a central role in Ca2+ signalling and promote drought tolerance in plants. The CIPK gene family in pigeon pea (Cajanus cajan L.), a major food crop affected by drought, has not previously been characterised. Here, we identified 28 CIPK genes in the pigeon pea genome. Five CcCIPK genes were strongly upregulated in roots upon drought treatment and were selected for further characterisation. Overexpression of CcCIPK13 and CcCIPK14 increased survival rates by two‐ to three‐fold relative to controls after 14 days of drought. Furthermore, the three major flavonoids, genistin, genistein and apigenin, were significantly upregulated in the same transgenic plants. Using CcCIPK14 as bait, we performed a yeast two‐hybrid screen and identified six interactors, including CcCBL1. CcCIPK14 exhibited autophosphorylation and phosphorylation of CcCBL1 in vitro. CcCBL1‐overexpressed plants displayed higher survival rates upon drought stress as well as higher expression of flavonoid biosynthetic genes and flavonoid content. CcCIPK14‐overexpressed plants in which CcCBL1 transcript levels were reduced by RNA interference had lower survival rates, which indicated CcCBL1 in the same pathway as CcCIPK14. Together, our results demonstrate a role for the CcCIPK14‐CcCBL1 complex in drought stress tolerance through the regulation of flavonoid biosynthesis in pigeon pea. Significance Statement: This study systematically elucidates the mechanism by which the CIPK‐CBL complex regulates flavonoid accumulation and drought resistance in pigeon pea, which lays a foundation for in‐depth exploration of relevant molecular mechanisms in plants. [ABSTRACT FROM AUTHOR]

Abstract: This study evaluated the antioxidant activity of the main active compound of an endophytic fungus MD-R-1 from pigeon pea. This fungus was identified as Aspergillus fumigates by ITS sequence. The antioxidant capacity was evaluated by DPPH, hydroxyl radical scavenging, reducing power, lipid peroxidation and XOD inhibitory effect assays. Among the four fractions, the EtOAc fraction showed the best antioxidant activity (IC50 values were 16.38, 35.20, 19.69, 22.64 and 193.24μg/mL, respectively) and the highest phenolic and flavonoid content. Furthermore, it possessed good protective activity from oxidative DNA damage. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione reductase (GR) in HepG2 cells treated with MD-R-1 EtOAc extracts were also determined. The results showed that EtOAc extracts significantly increased the activities of SOD, CAT and GR in HepG2 cells in a dose-dependent manner. The LC–MS/MS was used to analyze EtOAc fraction and the result pointed to a notable antioxidant compound, luteolin. The present investigation suggests that MD-R-1 represents a valuable natural antioxidant source and may be applicable in health food industry potentially. [Copyright &y& Elsevier]

Abstract: An enzyme incubation–water extraction (EI–WE) method was developed and optimised for the extraction of the natural antioxidant taxifolin and of the total flavonoids from wood sawdust of Larix gmelini (Rupr.) Rupr. A factorial design and a central composite design approach were used for method optimisation. Optimal conditions were 0.5mg/ml cellulase and 0.5mg/ml pectinase, a pH of 5.0, a temperature of 32°C and 18h incubation time. The flavonoids and taxifolin were extracted in hot water at 50°C for 30min, with a solid to liquid ratio of 1:20. Under optimised conditions, the yields of taxifolin and total flavonoids increased from 1.06±0.08 to 1.35±0.04mg/g and 4.13±0.17 to 4.96±0.29mg/g, respectively. DPPH and BHT assays revealed that the EI–WE samples had 1.8- and 1.68-fold higher antioxidant activities than the controls. SEM results revealed the structural disruption of wood sawdust with enzyme incubation. [ABSTRACT FROM AUTHOR]

A novel approach for synchronous extraction and enrichment of hydrophilic component and hydrophobic component with CO 2 -based switchable-solvent (SHS) from pigeon pea leaves was developed in the present study. After screening of SHS, N, N-dimethyl-cyclohexylamine (DMCHA)/water system was selected as the properest SHS for the extraction and enrichment process. To optimize this process, we conducted single factor experiments with the Box-Behnken design. The optimal parameters were as follows: solid-liquid ratio R (S/L) 40:1 (mg/mL), ultrasound time 15 min and bubbling CO 2 time 79 min (0.5 mL/s). Under the circumstances, the extraction yields of representative flavonoid vitexin and stilbene cajaninstilbenes acid (CSA) reached 1.80 ± 0.12 mg/g and 9.32 ± 0.08 mg/g, respectively, which were 1.06 and 1.49 folds to those by the traditional methods. And the contents of target compounds vitexin and CSA in the enriched extracts were 2.3% and 12.1%, which increased above 6.76 and 4.58 folds to that in extracts using 65% ethanol-UAE. And the results suggested that the N, N-dimethyl-cyclohexylamine (DMCHA)/water system can be guaranteed for at least 5 cycles with relatively stable extraction yields of vitexin and CSA. Furthermore, a theoretical study using density functional theory was put into effect to explore the properties of SHS and their relationship with the vitexin and CSA. The mechanism study showed that SHS had higher biocompatibility with target compounds due to the H-bonds. The present method was an eco-friendly, promising, and sustainable alternative to extract and enrich simultaneously hydrophilic and hydrophobic phytochemicals from plant materials. Image 1 • This paper developed a novel approach for efficient and recyclable extraction and enrichment of phytochemicals from the disused pigeon pea leaves in one solvent system. • The mechanism study showed that SHS had higher biocompatibility with target compounds due to the H-bonds using the density functional theory. • The switchable solvents have usually been used in the extraction of oil. Moreover, the exploit of SHS considering bioactive compounds from plants material has hardly been reported yet. [ABSTRACT FROM AUTHOR]