A Novel Trypsin Kunitz-Type Inhibitor from Cajanus cajan Leaves and Its Inhibitory Activity on New Cancer Serine Proteases and Its Effect on Tumor Cell Growth.

A novel trypsin inhibitor from Cajanus cajan (TIC) fresh leaves was partially purified by affinity chromatography. SDS-PAGE revealed one band with about 15 kDa with expressive trypsin inhibitor activity by zymography. TIC showed high affinity for trypsin (Ki = 1.617 µM) and was a competitive inhibitor for this serine protease. TIC activity was maintained after 24 h of treatment at 70 °C, after 1 h treatments with different pH values, and ß-mercaptoethanol increasing concentrations, and demonstrated expressive structural stability. However, the activity of TIC was affected in the presence of oxidizing agents. In order to study the effect of TIC on secreted serine proteases, as well as on the cell culture growth curve, SK-MEL-28 metastatic human melanoma cell line and CaCo-2 colon adenocarcinoma was grown in supplemented DMEM, and the extracellular fractions were submitted salting out and affinity chromatography to obtain new secreted serine proteases. TIC inhibited almost completely, 96 to 89%, the activity of these serine proteases and reduced the melanoma and colon adenocarcinoma cells growth of 48 and 77% respectively. Besides, it is the first time that a trypsin inhibitor was isolated and characterized from C. cajan leaves and cancer serine proteases were isolated and partial characterized from SK-MEL-28 and CaCo-2 cancer cell lines. Furthermore, TIC shown to be potent inhibitor of tumor protease affecting cell growth, and can be one potential drug candidate to be employed in chemotherapy of melanoma and colon adenocarcinoma.

Methylglyoxal metabolism is altered during defence response in pigeonpea (Cajanus cajan (L.) Millsp.) against the spotted pod borer (Maruca vitrata).

Pigeonpea (Cajanus cajan ) production can be affected by the spotted pod borer (Maruca vitrata ). Here, we identified biochemical changes in plant parts of pigeonpea after M. vitrata infestation. Two pigeonpea genotypes (AL 1747, moderately resistant; and MN 1, susceptible) were compared for glyoxalase and non-glyoxalase enzyme systems responsible for methylglyoxal (MG) detoxification, γ-glutamylcysteine synthetase (γ-GCS), glutathione-S-transferase (GST) and glutathione content in leaves, flowers and pods under control and insect-infested conditions. MN 1 had major damage due to M. vitrata infestation compared to AL 1747. Lower accumulation of MG in AL 1747 was due to higher activities of enzymes of GSH-dependent (glyoxylase I, glyoxylase II), GSH-independent (glyoxalase III) pathway, and enzyme of non-glyoxalase pathway (methylglyoxal reductase, MGR), which convert MG to lactate. Decreased glyoxylase enzymes and MGR activities in MN 1 resulted in higher accumulation of MG. Higher lactate dehydrogenase (LDH) activity in AL 1747 indicates utilisation of MG detoxification pathway. Higher glutathione content in AL 1747 genotype might be responsible for efficient working of MG detoxification pathway under insect infestation. Higher activity of γ-GCS in AL 1747 maintains the glutathione pool, necessary for the functioning of glyoxylase pathway to carry out the detoxification of MG. Higher activities of GST and GPX in AL 1747 might be responsible for detoxification of toxic products that accumulates following insect infestation, and elevated activities of glyoxylase and non-glyoxylase enzyme systems in AL 1747 after infestation might be responsible for reducing reactive cabanoyl stress. Our investigation will help the future development of resistant cultivars.

Safety Assessment and Hepatic-Renal Protection of Cajanus cajan (L.) Millsp. Root and Its Soy Isoflavone Contents.

Cajanus cajan (L.) Millsp., also known as pigeon pea, has roots that have exhibited much pharmacological potential. The present study was conducted to assess the safe dose of the ethanolic extract of C. cajan roots (EECR95) and to analyze the main soy isoflavones contents. In vitro, we investigated the mutagenicity and cytotoxic effect of EECR95 on Salmonella typhimurium-TA98 and TA100 (by Ames tests) and RAW 264.7, L-929, and HGF-1 cell lines (by MTT tests) for 24 h of incubation. We found no mutagenic or cytotoxic effects of EECR95. After administration of 0.2 or 1.0 g/kg bw of EECR95 to both male and female Wistar rats for 90 days, there were no significant adverse effects on the behaviors (body weight, water intake, and food intake), organ/tissue weights, or immunohistochemical staining, and the urine and hematological examinations of the rats were within normal ranges. EECR95 potentially decreases renal function markers in serum (serum uric acid, BUN, CRE, and GLU) or liver function markers (cholesterol, triglyceride, and glutamic-pyruvate-transaminase (GPT)). We also found that EECR95 contained five soy isoflavones (genistein, biochanin A, daidzein, genistin, and cajanol), which may be related to its hepatorenal protection. Based on the high dose (1.0 g/kg bw) of EECR95, a safe daily intake of EECR95 for human adults is estimated to be 972 mg/60 kg person/day.

Pigeon pea penta- and hexapeptides with antioxidant properties also inhibit renin and angiotensin-I-converting enzyme activities.

Pigeon pea protein was sequentially digested with pepsin followed by pancreatin and the hydrolysate separated into 18 fractions using reversed-phase high-performance liquid chromatography. Fractions were analyzed for in vitro antioxidant properties (radical scavenging, metal chelation, and ferric iron reducing ability) in addition to inhibition of renin and angiotensin-converting enzyme (ACE). The most active fractions were analyzed by mass spectroscopy followed by identification of 10 peptide sequences (7 pentapeptides and 3 hexapeptides). All the peptides showed a wide range of multifunctional activity by scavenging hydroxyl (31.9-66.8%) and superoxide (25.6-100.0%) radicals in addition to ACE inhibition (7.4-100%) with significant (p < .05) differences between the peptides. AGVTVS, TKDIG, TSRLG, GRIST, and SGEKI were the most active; however, AGVTVS had the highest hydrophobic residue and exhibited the strongest activity against ACE, renin as well as superoxide and hydroxyl radicals. PRACTICAL APPLICATIONS: There is an increasing attraction of researchers to food peptides especially from legume proteins. Enzymatic digestion as well as high performance liquid chromatography (HPLC) purification has become an important process used to separate peptides with significant biological activities and health-promoting effects. There is useful information regarding the bioactive and functional (in vitro antioxidant, antidiabetic, in vitro/in vivo antihypertensive) properties of hydrolyzed and ultra-filtered pigeon pea fractions but scant research output still exists for purified peptides from pigeon pea establishing their therapeutic potential. The present study aimed to separate peptide fractions from pigeon pea hydrolysate and identify available amino acid sequences from the parent protein. Therefore, peptide sequences generated from the most bioactive fractions showed prospects for the expanded industrial utilization of pigeon pea. Further promoting its application as functional ingredient or additive for alleviating angiotensin-converting enzyme-related diseases.

Health-Promoting Phenolic Compound Accumulation, Antioxidant Response, Endogenous Salicylic Acid Generation, and Biosynthesis Gene Expression in Germinated Pigeon Pea Seeds Treated with UV-B Radiation.

Germinated pigeon pea seeds (GPPSs) are good dietary supplements with satisfactory nutritional and medicinal values. In this study, UV-B treatment was used to promote the accumulation of health-promoting phenolic compounds (10 flavonoids and 1 stilbene) in GPPS. The total yield of 11 phenolic compounds (235 839.76 ± 17 118.24 ng/g DW) significantly improved (2.53-fold increase) in GPPSs exposed to UV-B radiation (3 W/m2) for 8 h, whereas free amino acid and reducing sugar contents exhibited a decreasing tendency during UV-B exposure. Meanwhile, the positive response in the antioxidant activities of enzymes and nonenzymatic extracts was noticed in UV-B-treated GPPSs. Moreover, UV-B radiation could cause tissue damages in hypocotyls and cotyledons of the GPPSs and enhance the generation of endogenous salicylic acid, thus activating the expression of biosynthesis genes (especially CHS and STS1). Overall, the simple UV-B supplementation strategy makes GPPSs more attractive as functional foods/nutraceuticals in diet for promoting human health.

Kinome-Wide Profiling Identifies Human WNK3 as a Target of Cajanin Stilbene Acid from Cajanus cajan (L.) Millsp.

Pigeon Pea (Cajanus cajan (L.) Millsp.) is a common food crop used in many parts of the world for nutritional purposes. One of its chemical constituents is cajanin stilbene acid (CSA), which exerts anticancer activity in vitro and in vivo. In an effort to identify molecular targets of CSA, we performed a kinome-wide approach based on the measurement of the enzymatic activities of 252 human kinases. The serine-threonine kinase WNK3 (also known as protein kinase lysine-deficient 3) was identified as the most promising target of CSA with the strongest enzymatic activity inhibition in vitro and the highest binding affinity in molecular docking in silico. The lowest binding affinity and the predicted binding constant pKi of CSA (-9.65 kcal/mol and 0.084 µM) were comparable or even better than those of the known WNK3 inhibitor PP-121 (-9.42 kcal/mol and 0.123 µM). The statistically significant association between WNK3 mRNA expression and cellular responsiveness to several clinically established anticancer drugs in a panel of 60 tumor cell lines and the prognostic value of WNK3 mRNA expression in sarcoma biopsies for the survival time of 230 patients can be taken as clues that CSA-based inhibition of WNK3 may improve treatment outcomes of cancer patients and that CSA may serve as a valuable supplement to the currently used combination therapy protocols in oncology.

The Dietary Use of Pigeon Pea for Human and Animal Diets.

Pigeon pea (PP) [Cajanus cajan (L.) Huth] plays an important role in preserving poor smallholders' major source of income in the tropics and subtropics by improving food and feed security, particularly protein intake. In the meantime, protein deficiency is frequent in tropical and subtropical regions due to rapidly increasing human populations and the high cost of animal-origin proteins. As a result, pulse crops should be their primary source of protein. Among these, PP is the most important pulse crop utilized as a food component in rain-fed agricultural conditions with the lowest costs, and it is the best source of protein supplements in typical cereal-based diets to fill the nutritional deficit. Despite this, it is the world's least-used pulse crop. Therefore, the primary goal of this review was to provide and synthesize scientifically confirmed and up-to-date information on the dietary usage of pigeon pea for food and feed. Protein, carbohydrates, minerals, vitamins, and essential amino acids are all present in reasonable amounts in both mature and immature PP seeds. PP has the most potential for usage as food and feed, and its nutrients are comparable to those of soybeans and maize. PP's green leaves, roots, seeds, and pods are high in phenolic compounds, which have anti-inflammation, antibacterial, antioxidant, anticarcinogenic, and antidiabetic properties, as well as the ability to cure diseases like measles, smallpox, chicken pox, sickle cell anemia, fever, dysentery, hepatitis, and antimalarial medications for the body. Furthermore, the addition of pigeon pea and its by-products improves ruminant and nonruminant animal feeding performance significantly. In general, PP products such as dried grain, fresh (aerial portion), and green pods are used as a low-cost (low-cost) source of high-quality and quantity of protein food and feed for tropical and subtropical populations' livelihoods.

GC-MS and LC-MS: an integrated approach towards the phytochemical evaluation of methanolic extract of Pigeon Pea [Cajanus cajan (L.) Millsp] leaves.

Pigeon Pea is a well-received traditional nutritious and medicinal plant in India since ancient times. The plant is identified for its impressive pharmacological activities like antimicrobial, anti-inflammatory, antitubercular, antioxidant, neuroprotective, antihypertensive, antihyperglycemic and anticancer properties. The metabolites from Pigeon Pea leaves methanolic extract were identified using hyphenated instruments namely Gas Chromatography-Mass Spectroscopy (GC-MS) and Liquid Chromatography-Mass Spectroscopic (LC-MS) method. GC and LC chromatogram showed proper separation and presence of several bioactive compounds such as flavonoids, terpenoids, essential oils, alkaloids, and coumarins. LC-MS investigation reported the presence of Selidin (coumarin) and Tamarixetin (monomethoxyflavone) in the Pigeon Pea Leaves. The present investigations have demonstrated that methanolic extract of Pigeon Pea leaves exhibited impressive antibacterial activity against E. coli and antifungal activity against C. albicans with MIC values 50 µg/ml 250 µg/ml respectively. The extracts studied here have demonstrated to be a new remedial source used against diseases caused by these microorganisms.

Green Extraction of Antioxidant Flavonoids from Pigeon Pea (Cajanus cajan (L.) Millsp.) Seeds and Its Antioxidant Potentials Using Ultrasound-Assisted Methodology.

Pigeon pea is an important pea species in the Fabaceae family that has long been used for food, cosmetic, and other phytopharmaceutical applications. Its seed is reported as a rich source of antioxidants and anti-inflammatory flavonoids, especially isoflavones, i.e., cajanin, cajanol, daidzein, and genistein. In today's era of green chemistry and green cosmetic development, the development and optimization of extraction techniques is increasing employed by the industrial sectors to provide environmentally friendly products for their customers. Surprisingly, there is no research report on improving the extraction of these isoflavonoids from pigeon pea seeds. In this present study, ultrasound-assisted extraction (USAE) methodology, which is a green extraction that provides a shorter extraction time and consumes less solvent, was optimized and compared with the conventional methods. The multivariate strategy, the Behnken-Box design (BBD) combined with response surface methodology, was employed to determine the best extraction conditions for this USAE utilizing ethanol as green solvent. Not only in vitro but also cellular antioxidant activities were evaluated using different assays and approaches. The results indicated that USAE provided a substantial gain of ca 70% in the (iso)flavonoids extracted and the biological antioxidant activities were preserved, compared to the conventional method. The best extraction conditions were 39.19 min with a frequency of 29.96 kHz and 63.81% (v/v) aqueous ethanol. Both the antioxidant and anti-aging potentials of the extract were obtained under optimal USAE at a cellular level using yeast as a model, resulting in lower levels of malondialdehyde. These results demonstrated that the extract can act as an effective activator of the cell longevity protein (SIR2/SIRT1) and cell membrane protector against oxidative stress. This finding supports the potential of pigeon pea seeds and USAE methodology to gain potential antioxidant and anti-aging (iso)flavonoids-rich sources for the cosmetic and phytopharmaceutical sectors.

Effects and mechanism of Pigeonpea (Cajanus cajan (L.) Millsp.) leaves on proliferation, migration, and tube formation of hypoxic human umbilical vein endothelial cells in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Pigeonpea (Cajanus cajan (L.) Millsp) leaves (PL) are widely used for treating avascular necrosis of the femoral head. PL has an ideal effect on bone angiogenesis in patients with hormone-induced avascular necrosis of the femoral head and could promote the repair of blood vessels in the necrotic femoral head. Angiogenesis is beneficial to the treatment of myocardial ischemia. PL can be used to treat ischemic heart disease; however, no studies have examined whether it could protect the myocardium against ischemia injury via promoting angiogenesis. AIM: The present study aimed to investigate whether PL could encourage angiogenesis on hypoxic human umbilical vein endothelial cells (HUVECs) and whether estrogen receptor (ER-α), protein kinase B (Akt), and vascular endothelial growth factor (VEGF) (the ischemia injury salvage kinase pathway, phosphoinositide-3 kinase (PI3K)) are involved in this effect. METHODS: A hypoxic HUVEC model was established by culture in the hypoxia incubator. The proliferation ability of HUVECs was determined by the 2,3-Bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) method, the migration rate of HUVECs was inspected by the Transwell method, the tube formation was evaluated by the Matrigel method, and the expression of PI3K, phosphorylated Akt (p-Akt), and VEGF was detected by Western blotting. RESULTS: The proliferation, migration, and tube formation were promoted by the PL extract on hypoxic HUVECs, and the hypoxia-induced downstream signaling was counteracted, leading to increased expression of PI3K, p-Akt, and VEGF in HUVECs. CONCLUSIONS: The current findings showed that the PL extracts encourage angiogenesis. In addition, the above effects could be mediated via ER-α and PI3K/Akt/VEGF pathways.

Simultaneous determination of 79 pesticides in pigeonpea grains using GC-MS/MS and LC-MS/MS.

Pigeonpea grains are important sources of vegetarian proteins. It is the paramount importance to check the pesticide residues due to their frequent use during production. The LC-MS/MS and GC-MS/MS analytical method was developed and validated for the simultaneous determination of 79 pesticide residues in pigeonpea. The LOD and LOQ of the analytical method were in the range of 0.53 to 3.97 and 1.60 to 10.05 µg kg-1, respectively, with a correlation coefficient of more than 0.997. Average recoveries were in the range of 80 to 118.8%, with the RSD of less than 15%. Measurement uncertainty (Ux) for pesticides was in the range of 3.42 to 12.76 µg kg-1 evaluated at 50 µg kg-1. The method was applied to analyze the sample collected from the farmer's field. This method could be useful for routine analysis of selected pesticide residue for monitoring purposes.

Longistylin A, a natural stilbene isolated from the leaves of Cajanus cajan, exhibits significant anti-MRSA activity.

Longistylin A (LLA) is an abundant stilbene isolated from the leaves of Cajanus cajan (L.) Millsp. However, the antibacterial effect of LLA is not yet understood. Therefore, in this study, a detailed investigation of the antibacterial effect of LLA, particularly against methicillin-resistant Staphylococcus aureus (MRSA), was conducted. In vitro, LLA exhibited strong antibacterial activity against MRSA with a minimum inhibitory concentration (MIC) of 1.56 µg/mL and displayed much more rapid bactericidal activity (3-log decrease in MRSA survival within 8 h) than vancomycin. A membrane-targeting experiment suggested that the antibacterial activity of LLA is associated with perturbation of the bacterial membrane potential and increased membrane permeability. Notably, LLA had relatively weak cytotoxicity to murine macrophages [50% cytotoxic concentration (CC50) = 8.61 ± 0.57 µg/mL]. In vivo, topical treatment of a skin injury with LLA improved wound healing and closure in an MRSA-infected wound healing mouse model. After 3 days treatment, LLA decreased MRSA bacterial counts in the wounded region, reduced the accumulation of immune cells at the injury site, and alleviated induction of the inflammatory cytokines tumour necrosis factor-alpha (180.74 ± 10.78 pg/mL vs. 606.57 ± 68.99 pg/mL) and interleukin-6 (87.25 ± 10.19 pg/mL vs. 280.58 ± 42.27 pg/mL) in serum.

Cajanonic acid A regulates the ratio of Th17/Treg via inhibition of expression of IL-6 and TGF-ß in insulin-resistant HepG2 cells.

BACKGROUND: The objectives of the present study are to investigate whether cajanonic acid A (CAA) can reduce insulin resistance (IR) in HepG2 cells and to gain a preliminary understanding of the mechanisms underlying this effect. METHODS: Following induction of IR in HepG2 cells, we tested the regulatory effect of CAA on glucose consumption and evaluated hepatocyte production of IL-6, TGF-ß, and key molecules in the insulin transduction pathway. A transwell co-culturing system was used to assess the effect of CAA on IR in HepG2 cells during the differentiation of CD4+ T cells by calculating the ratio of (Th17)/regulatory T cell (Treg). We evaluated the effect of CAA on the expression of IL-17RC cells and HepG2 cell apoptosis by immunofluorescence and flow cytometry assay. RESULTS: CAA improved dexamethasone-induced reduction in glucose consumption in HepG2 cells, inhibited hepatocyte production of IL-6 and TGF-ß, increased the expression of IL-17RC cell, and increased cellular apoptosis in insulin-resistant HepG2 cells. When co-cultured with CD4+ T cells, insulin-resistant HepG2 cells induced a decrease in the ratio of Th17/Treg, but CAA dampened the effect. Application of IL-6 and TGF-ß, together with CAA, reversed the effect of CAA on insulin-resistant HepG2 cells. Overexpression of IL17R, however, counteracted the effect of IL-6 neutralizing antibody within the culture system. CONCLUSION: CAA can regulate the ratio of Th17/Treg by mediating the expression of IL-6 and TGF-ß in insulin-resistant HepG2 cells.

Characterization of in vitro antioxidant activity, bioactive components, and nutrient digestibility in pigeon pea (Cajanus cajan) as influenced by germination time and temperature.

Germination of legumes is potential bioprocessing technique to improve the nutrient digestibility and enhancement of bioactive components. The present investigation studies the effects of different germination conditions on the bioactive components, antioxidant activity, and in vitro nutrient digestibility of pigeon pea. Results obtained indicated that increase in germination time and temperature modifies the bioactive components and nutritional digestibility of the pigeon pea. Studies have shown that increase in germination time from 12 to 48 hr and temperature from 25 to 35°C, results in significant increase in accumulation of total pheolics and flavonoid content as a result of cell wall degrading enzymes. Germination for prolonged time at higher temperature also significantly increases the antioxidant potential and reducing power of the germinated pigeon pea. Increased activity of hydrolytic enzymes alters the structure of starch and proteins and thus enhanced in vitro starch and protein digestibility and also lowers down the hot paste viscosity of germinated pigeon pea. PRACTICAL APPLICATIONS: Currently, consumers are increasingly interested in high-quality natural health foods with high biological value and better nutrient digestibility. Germinated grains legumes become popular among people engrossed in improving and maintaining their health by changing dietary habits. Germinated pigeon pea exhibited better nutrient digestibility with improved texture and flavor, higher antioxidant activity and more bioactive components with lower pasting viscosity due to enzymatic modification of starch and protein in the grain. For this reason, use of germinated pigeon pea flour can provide an excellent example of functional food with high dietary protein value. Germinated pigeon pea flour can be utilized as a functional ingredient in the preparation of novel function foods, and it would intensify metabolism, strengthen immunity, reimburse deficiencies of vitamins and mineral, lower the risk of various diseases and exert health-promoting effects.

Prenylated stilbenes and flavonoids from the leaves of Cajanus cajan.

Three new prenylated stilbenes, named as cajanusins A-C (1-3), and one new natural product cajanusin D (4), along with six known derivatives (5-10) were isolated from the leaves of Cajanus cajan. Their structures were fully elucidated by means of extensive spectroscopic methods and comparison with data in the reported literatures. The new compounds of 1 and 2 were evaluated for in vitro cytotoxic activities against a panel of human cancer cell lines.

Nutraceutical protein isolate from pigeon pea (Cajanus cajan) milling waste by-product: functional aspects and digestibility.

The aim of the present study was to utilize the protein from pigeon pea milling waste by-product for the preparation of protein isolate (PPI). The PPI was exposed to pepsin hydrolysis to reveal functional aspects like increased antioxidant capacity along with gelling ability. Complexation study demonstrated the use of PPI as an encapsulating material for water insoluble bioactive compounds (curcumin). Fluorescence spectroscopy revealed the formation of a complex through hydrophobic interaction. Ex vivo digestibility of the major proteins of PPI resulted in many peptides after digestion with human gastrointestinal enzymes. The peptide sequences identified may have potential biological activities, as revealed by bioinformatic data analysis. The presence of bioactive peptides and essential amino acids in the ex vivo digest suggests the role of PPI as a nutraceutical protein. The study provides a new value addition to pigeon pea milling waste by-product, a novel nutraceutical protein that can be used in functional food formulations.

The Cholesterol-Modulating Effect of Methanol Extract of Pigeon Pea (Cajanus cajan (L.) Millsp.) Leaves on Regulating LDLR and PCSK9 Expression in HepG2 Cells.

Pigeon pea (Cajanus cajan (L.) Millsp.) is a legume crop consumed as an indigenous vegetable in the human diet and a traditional medicinal plant with therapeutic properties. The current study highlights the cholesterol-modulating effect and underlying mechanisms of the methanol extract of Cajanus cajan L. leaves (MECC) in HepG2 cells. We found that MECC increased the LDLR expression, the cell-surface LDLR levels and the LDL uptake activity in HepG2 cells. We further demonstrated that MECC suppressed the proprotein convertase subtilisin/kexin type 9 (PCSK9) mRNA and protein expression, but not affected the expression of other cholesterol or lipid metabolism-related genes including inducible degrader of LDLR (IDOL), HMG-CoA reductase (HMGCR), fatty acid synthase (FASN), acetyl-CoA carboxylase (ACC1), and liver X receptor-α (LXR-α) in HepG2 cells. Furthermore, we demonstrated that MECC down-regulated the PCSK9 gene expression through reducing the amount of nuclear hepatocyte nuclear factor-1α (HNF-1α), a major transcriptional regulator for activation of PCSK9 promoter, but not that of nuclear sterol-responsive element binding protein-2 (SREBP-2) in HepG2 cells. Finally, we identified the cajaninstilbene acid, a main bioactive stilbene component in MECC, which significantly modulated the LDLR and PCSK9 expression in HepG2 cells. Our current data suggest that the cajaninstilbene acid may contribute to the hypocholesterolemic activity of Cajanus cajan L. leaves. Our findings support that the extract of Cajanus cajan L. leaves may serve as a cholesterol-lowering agent.

Cajanolactone A from Cajanus cajan Promoted Osteoblast Differentiation in Human Bone Marrow Mesenchymal Stem Cells via Stimulating Wnt/LRP5/ß-Catenin Signaling.

Cajanolactone A (CLA) is a stilbenoid discovered by us from Cajanus cajan (L.) Millsp. In our study, CLA was found to promote osteoblast differentiation in human bone marrow mesenchymal stem cells (hBMSCs), as judged by increased cellular alkaline phosphatase activity and extracellular calcium deposits, and elevated protein expression of Runx2, collagen-1, bone morphogenetic protein-2, and osteopontin. Mechanistic studies revealed that hBMSCs undergoing osteoblast differentiation expressed upregulated mRNA levels of Wnt3a, Wnt10b, LRP5/6, Frizzled 4, ß-catenin, Runx2, and Osterix from the early stage of differentiation, indicating the role of activated Wnt/ß-catenin signaling pathway in osteoblast differentiation. Addition of CLA to the differentiation medium further increased the mRNA level of Wnt3a, Wnt10b, Frizzled 4, LRP5, and ß-catenin, inferring that CLA worked by stimulating Wnt/LRP5/ß-catenin signaling. Wnt inhibitor dickkopf-1 antagonized CLA-promoted osteoblastogenesis, indicating that CLA did not target the downstream of canonical Wnt signaling pathway. Treatment with CLA caused no changes in mRNA expression level, as well as protein secretion of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-B ligand (RANKL), indicating that CLA did not affect the OPG/RANKL axis. Our results showed that CLA, which promoted osteoblast differentiation in hBMSCs, through activating Wnt/LRP5/ß-catenin signaling transduction, is a promising anti-osteoporotic drug candidate.

Cajanine promotes osteogenic differentiation and proliferation of human bone marrow mesenchymal stem cells.

BACKGROUND: Seed cells - mesenchymal stem cells (MSCs) - appear to be an attractive tool in the context of tissue engineering. Bone marrow represents the main source of MSCs for both experimental and clinical studies. However, the number limitation of bone marrow MSCs (BMSCs) and decreased function caused by proliferation make the search for adequate alternative sources of these cells for autologous and allogenic transplant necessary. OBJECTIVES: This study was aimed to investigate the roles of cajanine isolated from the extracts of Cajanus cajan L. Millsp. in the proliferation and differentiation of BMSCs, and to discover the mechanism of proliferation of BMSCs promoted by cajanine. MATERIAL AND METHODS: Bone marrow mesenchymal stem cells were cultured in high-glucose Dulbecco's Modified Eagle's Medium (DMEM) and osteogenic differentiation was induced by adding dexamethasone, ascorbic acid and ß-glycerophosphate supplements. Bone marrow MSCs were cultured in medium without cajanine or supplemented with cajanine. The information about the proliferation and osteogenic differentiation of BMSCs was collated. The osteogenic differentiation potential of BMSCs was also assessed at the 3rd passage by Von Kossa staining. To observe cell signal transduction changes of BMSCs after culturing them with cajanine for 24 h, the western blot analysis was performed to detect phosphorylated cell cycle proteins and activated cyclins. RESULTS: After osteogenic induction, the differentiation of BMSCs was accelerated by cajanine treatment. Osteogenesis markers were upregulated by cajanine treatment at both protein and mRNA levels. Cajanine obviously promoted the proliferation of BMSCs. After BMSCs were cultured with cajanine for 24 h, the cell cycle regulator proteins were phosphorylated or upregulated. CONCLUSIONS: Cajanine can promote the expansion efficiency of BMSCs, at the same time keeping their multi-differentiation potential. Cajanine can activate the cell cycle signal transduction pathway, thus inducing cells to enter the G1/S phase and accelerating cells entering the G2/M phase. This study can contribute to the development of cajanine-based drugs in tissue engineering.

Polyphenol enriched ethanolic extract of Cajanus scarabaeoides (L.) Thouars exerts potential antifilarial activity by inducing oxidative stress and programmed cell death.

Development of antifilarial drug from the natural sources is considered as one of the most efficacious, safe, and affordable approaches. In this study, we report the antifilarial activity of a leguminous plant Cajanus scarabaeoides (L.) Thouars. The polyphenol-rich ethanolic extract obtained from the stem part of the plant C. scarabaeoides (EECs) was found to be efficient in killing the filarial nematode Setaria cervi in all the three developmental stages viz. oocytes, microfilariae (Mf) and adults with LD50 values of 2.5, 10 and 35 µg/ml, respectively. While studying the molecular mechanism of action, we found that induction of oxidative stress plays the key role in inducing the mortality in S. cervi. The redox imbalance finally results in activation of the nematode CED pathway that executes the death of the parasite. Intriguingly, EECs was found to be selectively active against the worm and absolutely non-toxic to the mammalian cells and tissues. Taken together, our experimental data demonstrate that C. scarabaeoides can be chosen as an affordable natural therapeutic for treating filarial infection in the future with high efficacy and less toxicity.