Enhancement of vincristine under in vitro culture of Catharanthus roseus supplemented with Alternaria sesami endophytic fungal extract as a biotic elicitor.

Vincristine, one of the major vinca alkaloid of Catharanthus roseus (L.) G. Don. (Apocynaceae), was enhanced under in vitro callus culture of C. roseus using fungal extract of an endophyte Alternaria sesami isolated from the surface-sterilized root cuttings of C. roseus. Vindoline, a precursor molecule for vincristine production, was detected for the first time in the fungal endophyte A. sesami which was used as a biotic elicitor in this study to enhance vincristine content in the C. roseus callus. It was identified using high-performance liquid chromatography and mass spectroscopy techniques by matching retention time and mass data with reference molecule. Supplementing the heat sterilized A. sesami endophytic fungal culture extract into the callus culture medium of C. roseus resulted in the enhancement of vincristine content in C. roseus callus by 21.717% after 105-day culture.

Vindoline Attenuates Osteoarthritis Progression Through Suppressing the NF-κB and ERK Pathways in Both Chondrocytes and Subchondral Osteoclasts.

Disruption of extracellular matrix (ECM) homeostasis and subchondral bone remodeling play significant roles in osteoarthritis (OA) pathogenesis. Vindoline (Vin), an indole alkaloid extracted from the medicinal plant Catharanthus roseus, possesses anti-inflammatory properties. According to previous studies, inflammation is closely associated with osteoclast differentiation and the disorders of the homeostasis between ECM. Although Vin has demonstrated effective anti-inflammatory properties, its effects on the progression of OA remain unclear. We hypothesized that Vin may suppress the progress of OA by suppressing osteoclastogenesis and stabilizing ECM of articular cartilage. Therefore, we investigated the effects and molecular mechanisms of Vin as a treatment for OA in vitro and in vivo. In the present study, we found that Vin significantly suppressed RANKL-induced osteoclast formation and obviously stabilized the disorders of the ECM homeostasis stimulated by IL-1ß in a dose-dependent manner. The mRNA expressions of osteoclast-specific genes were inhibited by Vin treatment. Vin also suppressed IL-1ß-induced mRNA expressions of catabolism and protected the mRNA expressions of anabolism. Moreover, Vin notably inhibited the activation of RANKL-induced and IL-1ß-induced NF-κB and ERK pathways. In vivo, Vin played a protective role by inhibiting osteoclast formation and stabilizing cartilage ECM in destabilization of the medial meniscus (DMM)-induced OA mice. Collectively, our observations provide a molecular-level basis for Vin's potential in the treatment of OA.

Vindoline Inhibits RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss in Mice.

Osteolytic bone diseases, for example postmenopausal osteoporosis, arise from the imbalances between osteoclasts and osteoblasts in the bone remodeling process, whereby osteoclastic bone resorption greatly exceeds osteoblastic bone formation resulting in severe bone loss and deterioration in bone structure and microarchitecture. Therefore, the identification of agents that can inhibit osteoclast formation and/or function for the treatment of osteolytic bone disease has been the focus of bone and orthopedic research. Vindoline (Vin), an indole alkaloid extracted from the medicinal plant Catharanthus roseus, has been shown to possess extensive biological and pharmacological benefits, but its effects on bone metabolism remains to be documented. Our study demonstrated for the first time, that Vin could inhibit osteoclast differentiation from bone marrow macrophages (BMMs) precursor cells as well as mature osteoclastic bone resorption. We further determined that the underlying molecular mechanism of action of Vin is in part due to its inhibitory effect against the activation of MAPK including p38, JNK, and ERK and intracellular reactive oxygen species (ROS) production. This effect ultimately suppressed the induction of c-Fos and NFATc1, which consequently downregulated the expression of the genes required for osteoclast formation and bone resorption. Consistent with our in vitro findings, in vivo administration of Vin protected mice against ovariectomy (OVX)-induced bone loss and trabecular bone deterioration. These results provided promising evidence for the potential therapeutic application of Vin as a novel treatment option against osteolytic diseases.

Expression of tabersonine 16-hydroxylase and 16-hydroxytabersonine-O-methyltransferase in Catharanthus roseus hairy roots.

The monoterpene indole alkaloids vindoline and catharanthine, which are exclusively synthesized in the medicinal plant Catharanthus roseus, are the two important precursors for the production of pharmaceutically important anti-cancer medicines vinblastine and vincristine. Hairy root culture is an ideal platform for alkaloids production due to its industrial scalability, genetic and chemical stability, and availability of genetic engineering tools. However, C. roseus hairy roots do not produce vindoline due to the lack of expression of the seven-step pathway from tabersonine to vindoline [Murata & De Luca (2015) Plant Journal, 44, 581-594]. The present study describes the genetic engineering of the first two genes tabersonine 16-hydroxylase (T16H) and 16-O-methyl transferase (16OMT) in the missing vindoline pathway under the control of a glucocorticoid-inducible promoter to direct tabersonine toward vindoline biosynthesis in C. roseus hairy roots. In two transgenic hairy roots, the induced overexpression of T16H and 16OMT resulted in the accumulation of vindoline pathway metabolites 16-hydroxytabersonine and 16-methoxytabersonine. The levels of root-specific alkaloids, including lochnericine, 19-hydroxytabersonine and hörhammericine, significantly decreased in the induced hairy roots in comparison to the uninduced control lines. This suggests tabersonine was successfully channeled to the vindoline pathway away from the roots competing pathway based on the overexpression. Interestingly, another two new metabolites were detected in the induced hairy roots and proposed to be the epoxidized-16-hydroxytabersonine and lochnerinine. Thus, the introduction of vindoline pathway genes in hairy roots can cause unexpected terpenoid indole alkaloids (TIA) profile alterations. Furthermore, we observed complex transcriptional changes in TIA genes and regulators detected by RT-qPCR which highlight the tight regulation of the TIA pathway in response to T16H and 16OMT engineering in C. roseus hairy roots.

Medicinal plants used in the traditional management of diabetes and its sequelae in Central America: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Globally 387 million people currently have diabetes and it is projected that this condition will be the 7th leading cause of death worldwide by 2030. As of 2012, its total prevalence in Central America (8.5%) was greater than the prevalence in most Latin American countries and the population of this region widely use herbal medicine. The aim of this study is to review the medicinal plants used to treat diabetes and its sequelae in seven Central American countries: Belize, Costa Rica, El Salvador, Guatemala, Honduras, Nicaragua and Panama. MATERIALS AND METHODS: We conducted a literature review and extracted from primary sources the plant use reports in traditional remedies that matched one of the following disease categories: diabetes mellitus, kidney disease, urinary problems, skin diseases and infections, cardiovascular disease, sexual dysfunctions, visual loss, and nerve damage. Use reports were entered in a database and data were analysed in terms of the highest number of use reports for diabetes management and for the different sequelae. We also examined the scientific evidence that might support the local uses of the most reported species. RESULTS: Out of 535 identified species used to manage diabetes and its sequelae, 104 species are used to manage diabetes and we found in vitro and in vivo preclinical experimental evidence of hypoglycaemic effect for 16 of the 20 species reported by at least two sources. However, only seven of these species are reported in more than 3 studies: Momordica charantia L., Neurolaena lobata (L.) R. Br. ex Cass., Tecoma stans (L.) Juss. ex Kunth, Persea americana Mill., Psidium guajava L., Anacardium occidentale L. and Hamelia patens Jacq. Several of the species that are used to manage diabetes in Central America are also used to treat conditions that may arise as its consequence such as kidney disease, urinary problems and skin conditions. CONCLUSION: This review provides an overview of the medicinal plants used to manage diabetes and its sequelae in Central America and of the current scientific knowledge that might explain their traditional use. In Central America a large number of medicinal plants are used to treat this condition and its sequelae, although relatively few species are widely used across the region. For the species used to manage diabetes, there is variation in the availability and quality of pharmacological, chemical and clinical studies to explain traditional use.

Completion of the seven-step pathway from tabersonine to the anticancer drug precursor vindoline and its assembly in yeast.

Antitumor substances related to vinblastine and vincristine are exclusively found in the Catharanthus roseus (Madagascar periwinkle), a member of the Apocynaceae plant family, and continue to be extensively used in cancer chemotherapy. Although in high demand, these valuable compounds only accumulate in trace amounts in C. roseus leaves. Vinblastine and vincristine are condensed from the monoterpenoid indole alkaloid (MIA) precursors catharanthine and vindoline. Although catharanthine biosynthesis remains poorly characterized, the biosynthesis of vindoline from the MIA precursor tabersonine is well understood at the molecular and biochemical levels. This study uses virus-induced gene silencing (VIGS) to identify a cytochrome P450 [CYP71D1V2; tabersonine 3-oxygenase (T3O)] and an alcohol dehydrogenase [ADHL1; tabersonine 3-reductase (T3R)] as candidate genes involved in the conversion of tabersonine or 16-methoxytabersonine to 3-hydroxy-2,3-dihydrotabersonine or 3-hydroxy-16-methoxy-2,3-dihydrotabersonine, which are intermediates in the vindorosine and vindoline pathways, respectively. Biochemical assays with recombinant enzymes confirm that product formation is only possible by the coupled action of T3O and T3R, as the reaction product of T3O is an epoxide that is not used as a substrate by T3R. The T3O and T3R transcripts were identified in a C. roseus database representing genes preferentially expressed in leaf epidermis and suggest that the subsequent reaction products are transported from the leaf epidermis to specialized leaf mesophyll idioblast and laticifer cells to complete the biosynthesis of these MIAs. With these two genes, the complete seven-gene pathway was engineered in yeast to produce vindoline from tabersonine.

Natural product vindoline stimulates insulin secretion and efficiently ameliorates glucose homeostasis in diabetic murine models.

ETHNOPHARMACOLOGICAL RELEVANCE: Catharanthus roseus (L). Don (Catharanthus roseus) is a traditional anti-diabetic herb widely used in many countries, and the alkaloids of Catharanthus roseus are considered to possess hypoglycemic ability. AIM OF THE STUDY: To systematically investigate the potential anti-diabetic effects and the underlying anti-diabetic mechanisms of vindoline, one of the alkaloids in Catharanthus roseus. MATERIALS AND METHODS: The regulation of vindoline against the glucose-stimulated insulin secretion (GSIS) was examined in insulinoma MIN6 cells and primary pancreatic islets. Insulin concentration was detected by Elisa assay. Diabetic models of db/db mice and type 2 diabetic rats induced by high-fat diet combining with streptozotocin (STZ/HFD-induced type 2 diabetic rats) were used to evaluate the anti-diabetic effect of vindoline in vivo. Daily oral treatment with vindoline (20mg/kg) to diabetic mice/rats for 4 weeks, body weight and blood glucose were determined every week, oral glucose tolerance test (OGTT) was performed after 4 weeks. RESULTS: Vindoline enhanced GSIS in both glucose- and dose-dependent manners (EC50 = 50 µM). It was determined that vindoline acted as a Kv2.1 inhibitor able to reduce the voltage-dependent outward potassium currents finally enhancing insulin secretion. It protected ß-cells from the cytokines-induced apoptosis following its inhibitory role in Kv2.1. Moreover, vindoline (20mg/kg) treatment significantly improved glucose homeostasis in db/db mice and STZ/HFD-induced type 2 diabetic rats, as reflected by its functions in increasing plasma insulin concentration, protecting the pancreatic ß-cells from damage, decreasing fasting blood glucose and glycated hemoglobin (HbA1c), improving OGTT and reducing plasma triglyceride (TG). CONCLUSION: Our findings suggested that vindoline might contribute to the anti-diabetic effects of Catharanthus roseus, and this natural product may find its more applications in the improvement of ß-cell dysfunction and further the potential treatment of type 2 diabetes.

Simultaneous determination of secondary metabolites from Vinca rosea plant extractives by reverse phase high performance liquid chromatography.

BACKGROUND: Vinca rosea (Apocynaceae) is one of the most important and high value medicinal plants known for its anticancer alkaloids. It is the iota of the isolated secondary metabolites used in chemotherapy to treat diverse cancers. Several high performance liquid chromatography (HPLC) methods have been developed to quantify the active alkaloids in the plant. However, this method may serve the purpose in quantification of V. rosea plant extracts in totality. OBJECTIVE: To develop and validate the reverse phase (RP)-HPLC method for simultaneous determination of secondary metabolites, namely alkaloids from V. rosea plant extracts. MATERIALS AND METHODS: The quantitative determination was conducted by RP-HPLC equipped with ultraviolet detector. Optimal separation was achieved by isocratic elution with mobile phase consisting of methanol:acetonitrile:ammonium acetate buffer (25 mM) with 0.1% triethylamine (15:45:40 v/v) on a column (Zorbax Eclipse plus C(18), 250 mm % 4.6 mm; 5 µm). The standard markers (vindoline, vincristine, catharanthine, and vinblastine) were identified by retention time and co-injected with reference standard and quantified by external standard method at 297 nm. RESULTS: The precision of the method was confirmed by the relative standard deviation (R.S.D.), which was lower than 2.68%. The recoveries were in the range of 98.09%-108%. The limits of detection (LOD) for each marker alkaloids were lower than 0.20 µg. Different parts of the V. rosea extracts shows different concentrations of markers, flower samples were high in vinblastine content, while methanol extract from the leaves contains all the four alkaloids in good yield, and there is no significant presence of markers in water extracts. CONCLUSION: HPLC method established is appropriate for the standardization and quality assurance of V. rosea plant extracts.