Kv2.1 channel (IC50 = 31 µM in MIN6 cells; IC50 = 43 µM in Kv2.1-overexpressing CHO cells) [1]

In MIN6 cells, vindoline (50 µM) alone had no effect on insulin secretion at 2.8 mM glucose but significantly augmented insulin secretion at 16.8 mM glucose, showing dose- and glucose-dependent enhancement of glucose-stimulated insulin secretion (GSIS) with an EC50 of 50 µM. It reduced voltage-dependent outward potassium currents through Kv2.1 inhibition. Vindoline (20 or 50 µM) did not affect insulin promoter activity (RIP-luc assay) or intracellular cAMP levels in MIN6 cells. The enhancement of GSIS by vindoline was significantly reduced by nifedipine (10 µM, an L-VDCC blocker), diazoxide (200 µM, a KATP channel opener), and linoleic acid methyl ester (LAME, 20 µM, a specific Kv2.1 channel opener). In Kv2.1N over-expressing MIN6 cells, the GSIS-enhancing ability of vindoline was lost. Vindoline (50 µM) significantly decreased the percentage of apoptotic MIN6 cells induced by cytokines (5 ng/mL IL-1β, 100 ng/mL IFN-γ, 10 ng/mL TNF-α) from 26.36% to 16.45% and reversed the cytokine-promoted cleaved caspase-3 level. This anti-apoptotic effect was lost in Kv2.1N over-expressing MIN6 cells. [1]
In primary mouse pancreatic islets, vindoline (10, 20, 50 µM) increased insulin secretion in dose- and glucose-dependent manners. [1]
In bone marrow macrophages (BMMs), vindoline (1.25, 2.5, 5, 10 µM) dose-dependently decreased the number and size (area) of TRAP-positive multinucleated osteoclasts induced by RANKL (100 ng/mL). It had no cytotoxic effect on BMMs up to 40 µM. Vindoline (5, 10 µM) dose-dependently inhibited intracellular ROS production in BMMs stimulated with RANKL. Vindoline (10 µM) treatment resulted in a drastic reduction in the size of the podosomal F-actin belt and reduced multinucleation in mature osteoclasts. Vindoline (5, 10 µM) significantly inhibited the resorptive activity of mature osteoclasts cultured on hydroxyapatite-coated plates. Vindoline (10 µM) significantly blocked the RANKL-induced phosphorylation of MAPK members (ERK, JNK, and p38) but did not affect IκBα degradation or p65 phosphorylation in the NF-κB pathway. It repressed RANKL-induced expression of c-Fos and NFATc1. Vindoline (5, 10 µM) downregulated the expression of NFATc1, CTSK, MMP9, and TRAP genes. [2]
In rat renal tissues, vindoline (20 mg/kg) significantly reduced CP-induced increases in TNF-α, IL-1β, IL-6, and NF-κB levels. It also downregulated CP-induced protein expression of Bax, cleaved caspase-3, and cleaved caspase-9, while upregulating Bcl-2 protein expression. Vindoline significantly attenuated CP-induced upregulation of JNK, ERK1/2, and p38 gene expression. [3]

In db/db mice, oral administration of vindoline (40 mg/kg/day) for 4 weeks significantly reduced fasting plasma glucose, improved glucose tolerance (OGTT), slightly decreased HbA1c level, decreased plasma triglyceride (TG) levels, and increased fasting plasma insulin levels. Immunohistochemistry showed more insulin-positive islets were preserved. [1]
In STZ/HFD-induced type 2 diabetic rats, oral administration of vindoline (20 mg/kg/day) for 4 weeks significantly reduced fasting plasma glucose, improved glucose tolerance (OGTT), largely reduced HbA1c level, decreased plasma TG levels, and increased fasting plasma insulin levels. Vindoline treatment obviously reduced STZ/HFD-induced islets disruption and preserved more insulin-positive islets. [1]
In normal C57 mice, oral administration of vindoline (50 mg/kg) after 12h fasting exerted little effect on fasting glucose levels over 8 hours, unlike tolbutamide which significantly decreased glucose levels. [1]
In ovariectomy (OVX)-induced bone loss mouse model, intraperitoneal injection of vindoline (5 or 10 mg/kg body weight) every other day for 6 weeks dose-dependently lessened the deleterious effect of OVX. Micro-CT analysis showed that high-dose (10 mg/kg) vindoline treatment considerably protected against bone loss and trabecular bone deterioration, improving BV/TV, Tb. N, and Tb. Sp. Histological assessment (TRAP staining) showed that vindoline dose-dependently reduced the OVX-induced elevation in osteoclast number and activity (N.Oc/BS and Oc.S/BS). [2]
In a rat model of cisplatin (CP)-induced nephrotoxicity, oral administration of vindoline (20 mg/kg/day) for 10 days (with CP injection on day 7) significantly reduced CP-induced increases in relative kidney weight, serum creatinine, urea, and BUN levels. It also inhibited CP-induced oxidative stress by decreasing MDA levels and increasing GSH, SOD, and CAT activities. Histopathological examination showed vindoline treatment markedly reduced CP-induced tubular necrosis, inflammatory cell infiltration, hemorrhage, and hyaline casts formation. [3]

To determine the effect on Kv2.1 and KATP channels, whole-cell patch-clamp assays were performed using an Axon 200B amplifier. MIN6 cells or channel-overexpressing CHO cells were held at -70 mV, and outward currents were recorded with voltage steps to +130 mV in 10 mV increments. For MIN6 cells, the extracellular solution contained nifedipine to block L-type Ca2+ currents. Vindoline was applied, and its inhibition of sustained outward K+ current was measured. The IC50 for vindoline on current blockage in MIN6 cells was estimated by fitting a dose-response curve. [1]
To evaluate the effect on MAPK and NF-κB pathways, BMMs were serum-starved, pretreated with or without vindoline (10 µM) for 1 hour, and then stimulated with RANKL (100 ng/mL) for 0, 5, 10, 20, 30, or 60 minutes. Total cellular proteins were extracted, separated by SDS-PAGE, transferred to nitrocellulose membranes, and immunoblotted with specific antibodies against total and phosphorylated forms of ERK, JNK, p38, IκBα, and p65. Protein bands were visualized and quantified. [2]
For apoptosis pathway analysis in kidney tissues, proteins were extracted from homogenized renal tissues, separated using 12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked and then incubated with primary antibodies for Bax (1:2000), Bcl-2 (1:1500), caspase-3 (1:1300), and caspase-9 (1:1000) overnight at 4°C, followed by HRP-labeled secondary antibodies. Protein bands were detected using chemiluminescence and normalized to β-actin. [3]

For insulin secretion assay (GSIS), MIN6 cells were plated in 24-well plates (5 x 10^5 cells/well). After 2h starvation in glucose-free KRB buffer, cells were incubated with vindoline and different glucose concentrations (2.8, 5.6, 11.2, 16.8, 25 mM) for 2h. Insulin in the supernatant was measured by Elisa kit. For primary islets, isolated mouse islets were starved in glucose-free KRB buffer for 2h and then stimulated with vindoline (10, 20, 50 µM) and glucose for 2h. [1]
For cell viability (cytotoxicity) assay, BMMs were seeded in 96-well plates and treated with different concentrations of vindoline (1.25, 2.5, 5, 10, 20, 40 µM) for 48h. Cell viability was assessed using the CCK-8 assay by measuring absorbance at 450 nm. [2]
For osteoclast differentiation assay, BMMs were seeded in a 96-well plate (6 x 10^3 cells/well) and stimulated with RANKL (100 ng/mL) and M-CSF (50 ng/mL) in the absence or presence of vindoline (1.25, 2.5, 5, 10 µM) for 5 days. Cells were then stained for TRAP activity, and TRAP-positive multinucleated cells (≥3 nuclei) were counted and area quantified using ImageJ. [2]
For intracellular ROS measurement, BMMs stimulated with RANKL without or with vindoline (5, 10 µM) for 48h were incubated with DCFH-DA for 40 min. Fluorescence intensity (DCF) was detected under a fluorescence microscope and analyzed by ImageJ. [2]
For podosomal F-actin belt staining, BMM-derived osteoclasts stimulated with RANKL without or with vindoline (5, 10 µM) for 5 days were fixed, permeabilized, stained with Rhodamine-conjugated phalloidin (for F-actin) and DAPI (for nuclei), and visualized by fluorescence microscopy. [2]
For hydroxyapatite resorption assay, pre-osteoclasts (stimulated with RANKL for 3 days) were reseeded on hydroxyapatite-coated OsteoAssay plates and treated without or with vindoline (5, 10 µM) for 48h. Cells were removed, and the resorption pit area was quantified. [2]
For quantitative real-time PCR (qPCR), total RNA was extracted from cells using TRIzol reagent, reverse transcribed to cDNA, and amplified using SYBR Green PCR Master Mix with specific primers for CTSK, MMP9, NFATc1, and TRAP. Gene expression was normalized to GAPDH using the 2-ΔΔCT method. [2]
For gene expression analysis in rat kidney tissue, total RNA was extracted, and RT-PCR was performed for JNK, p38, and ERK1/2 using SYBR green One-step Quantitative RT-PCR ReadyMix Kit. GAPDH was used as a housekeeping gene, and relative expression was calculated using the 2-ΔΔCT method. [3]

For the db/db mouse model, 8-week old male db/db mice (approx. 30g) were orally administered vehicle or vindoline (40 mg/kg/day) daily for 4 weeks. Body weight and fasting plasma glucose were measured weekly. After 4 weeks, an oral glucose tolerance test (OGTT) was performed after 6h fasting by oral administration of 2g/kg glucose, and blood glucose was measured at 0, 15, 30, 60, 90, 120 min. At termination, plasma insulin, HbA1c, and TG were measured. Pancreas was removed for immunohistochemistry. [1]
For the STZ/HFD-induced type 2 diabetic rat model, male SD rats fed with HFD for 2 weeks were injected with STZ (25 mg/kg, i.p.). Diabetic rats were orally administered vehicle or vindoline (20 mg/kg/day) daily for 4 weeks. OGTT was performed after overnight fasting by oral glucose (2g/kg) administration. Blood glucose, plasma insulin, HbA1c, and TG were measured. Pancreas was removed for histology and immunohistochemistry. [1]
For the acute hypoglycemia assay in normal mice, 6-week old normal C57/BL6 mice were fasted for 12h and then orally administered vindoline (50 mg/kg) or tolbutamide (50 mg/kg). Glucose levels were measured from tail blood at 0, 1, 2, 4, 6, 8h. [1]
For the OVX-induced bone loss mouse model, 11-week old female C57BL/6J mice underwent bilateral OVX or sham operation. Seven days post-operation, mice were intraperitoneally injected every other day with normal saline (Sham and OVX groups) or with vindoline (5 or 10 mg/kg body weight) for 6 weeks. After sacrifice, tibias were resected for micro-CT analysis and histological assessment (TRAP staining). [2]
For the cisplatin-induced nephrotoxicity rat model, male Wistar rats (170-180g, 6-8 weeks old) were divided into four groups (n=6). Vindoline (20 mg/kg/day) was administered orally for 10 days. On the 7th day, a single intraperitoneal injection of cisplatin (5 mg/kg) was given. At the end of the 10-day period, rats were sacrificed. Blood and kidney tissues were collected for biochemical analysis, histopathology (H&E staining), and Western blotting. [3]

The difference between the in vitro IC50 of vindoline against Kv2.1 channel (31 µM) and its in vivo efficacy dose (20-40 mg/kg) may be caused by its low oral bioavailability (data not shown). [1]

In db/db mice and STZ/HFD-induced type 2 diabetic rats treated with vindoline for 4 weeks, few adverse effects on body and tissue weights were observed. [1]
Unlike tolbutamide, vindoline (50 mg/kg, oral) exerted little effect on fasting glucose levels in normal mice, suggesting a lower potential side-effect of hypoglycemia. [1]
Vindoline (up to 40 µM) showed no cytotoxic effect on BMM cell viability in vitro. [2]
In the rat model of CP-induced nephrotoxicity, vindoline (20 mg/kg) did not cause any significant changes in nephrotic marker levels or tissue histology compared to the control group, indicating a lack of toxicity at this dose. [3]

[1]. Natural product vindoline stimulates insulin secretion and efficiently ameliorates glucose homeostasis in diabetic murine models. J Ethnopharmacol. 2013 Oct 28;150(1):285-97.

[2]. Vindoline Inhibits RANKL-Induced Osteoclastogenesis and Prevents Ovariectomy-Induced Bone Loss in Mice. Front Pharmacol. 2020 Jan 22;10:1587.

[3]. Vindoline mitigates cisplatin-mediated kidney damage by alleviating redox imbalance, apoptosis and inflammation through extracellular signal-regulated kinase pathway modulation. J Physiol Pharmacol. 2024 Dec;75(6).

[4]. Vindoline Attenuates Osteoarthritis Progression Through Suppressing the NF-κB and ERK Pathways in Both Chondrocytes and Subchondral Osteoclasts. Front Pharmacol. 2022 Jan 12;12:764598.

[5]. Mechanism of interaction of vinca alkaloids with tubulin: catharanthine and vindoline. Biochemistry. 1991 Jan 22;30(3):873-80.

Vindoline is a periwinic alkaloid belonging to the alkaloid ester, organic heteropentane compound, methyl ester, acetate ester, tertiary amine compound, and tertiary alcohol classes. It is the conjugate base of vincainium(1+). Vindoline has been reported in periwinic flowers (Catharanthus trichophyllus), rose periwinic flowers (Catharanthus roseus), and other organisms with relevant data. Vindoline is an indole alkaloid that exerts antimitotic activity by inhibiting microtubule assembly. (NCI)

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Vindoline is a natural product derived from Catharanthus roseus, a plant traditionally used as an antidiabetic folk medicine. The study suggests that vindoline contributes to the anti-diabetic effects of this plant. [1]
Vindoline functions as a Kv2.1 inhibitor, which enhances glucose-stimulated insulin secretion (GSIS) and protects pancreatic β-cells from apoptosis. Its glucose-dependent stimulation feature may allow it to avoid the hypoglycemia side-effect common with sulfonylureas. The study highlights the potential of this natural product in anti-diabetic drug lead compound development. [1]
Vindoline possesses anti-osteoclastogenic and anti-resorptive properties, making it a potential therapeutic agent for osteoclast-mediated osteolytic diseases such as postmenopausal osteoporosis. It inhibits osteoclast differentiation and function by suppressing the RANKL-induced MAPK pathway and intracellular ROS production, which consequently represses the induction of c-Fos and NFATc1. [2]
Vindoline mitigates cisplatin-induced nephrotoxicity in rats through a multifaceted mechanism that includes antioxidant, anti-inflammatory, and anti-apoptotic properties, potentially via modulation of the ERK pathway. [3]

C25H32N2O6

456.5314

456.226

2182-14-1

260535

White to off-white solid

1.3±0.1 g/cm3

569.8±50.0 °C at 760 mmHg

163-165ºC

298.4±30.1 °C

0.0±1.6 mmHg at 25°C

1.626

2.2

1

8

6

33

864

6

O(C(C([H])([H])[H])=O)C1([H])[C@](C(=O)OC([H])([H])[H])([C@]2([H])[C@@]3(C4C([H])=C([H])C(=C([H])C=4N2C([H])([H])[H])OC([H])([H])[H])C([H])([H])C([H])([H])N2C([H])([H])C([H])=C([H])[C@]1(C([H])([H])C([H])([H])[H])[C@@]23[H])O[H]

CXBGOBGJHGGWIE-ACSXSLCXSA-N

InChI=1S/C25H32N2O6/c1-6-23-10-7-12-27-13-11-24(19(23)27)17-9-8-16(31-4)14-18(17)26(3)20(24)25(30,22(29)32-5)21(23)33-15(2)28/h7-10,14,19-21,30H,6,11-13H2,1-5H3/t19-,20+,21+,23+,24+,25-/m0/s1

methyl (1R,9R,10S,11R,12R,19R)-11-acetyloxy-12-ethyl-10-hydroxy-5-methoxy-8-methyl-8,16-diazapentacyclo[10.6.1.01,9.02,7.016,19]nonadeca-2(7),3,5,13-tetraene-10-carboxylate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~250 mg/mL (~547.61 mM)

Solubility in Formulation 1: ≥ 6.25 mg/mL (13.69 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 6.25 mg/mL (13.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 6.25 mg/mL (13.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 62.5 mg/mL clear DMSO stock solution to 900 μL corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)