 Chemical Structure.png)
Dioscin (CCRIS 4123; Collettiside III) is a naturally occuring saponin isolated from Polygonatum Zanlanscianense Pamp, showing antitumor activities. It has antiproliferative and apoptotic agent. It can increase caspase-3 and Bax expression. Decreases Bcl-2 expression. Shows membrane disruptive and antifungal effects in vivo.
Physicochemical Properties
| Molecular Formula | C45H72O16 | |
| Molecular Weight | 869.05 | |
| Exact Mass | 868.482 | |
| Elemental Analysis | C, 62.19; H, 8.35; O, 29.46 | |
| CAS # | 19057-60-4 | |
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| PubChem CID | 119245 | |
| Appearance | White to off-white solid powder | |
| Density | 1.4±0.1 g/cm3 | |
| Melting Point | 294-296 ℃ | |
| Index of Refraction | 1.614 | |
| LogP | 7.24 | |
| Hydrogen Bond Donor Count | 8 | |
| Hydrogen Bond Acceptor Count | 16 | |
| Rotatable Bond Count | 7 | |
| Heavy Atom Count | 61 | |
| Complexity | 1600 | |
| Defined Atom Stereocenter Count | 26 | |
| SMILES | C[C@@H]1CC[C@@]2([C@H]([C@H]3[C@@H](O2)C[C@@H]4[C@@]3(CC[C@H]5[C@H]4CC=C6[C@@]5(CC[C@@H](C6)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O[C@H]8[C@@H]([C@@H]([C@H]([C@@H](O8)C)O)O)O)O)O[C@H]9[C@@H]([C@@H]([C@H]([C@@H](O9)C)O)O)O)C)C)C)OC1 |
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| InChi Key | VNONINPVFQTJOC-ZGXDEBHDSA-N | |
| InChi Code | InChI=1S/C45H72O16/c1-19-9-14-45(54-18-19)20(2)30-28(61-45)16-27-25-8-7-23-15-24(10-12-43(23,5)26(25)11-13-44(27,30)6)57-42-39(60-41-36(52)34(50)32(48)22(4)56-41)37(53)38(29(17-46)58-42)59-40-35(51)33(49)31(47)21(3)55-40/h7,19-22,24-42,46-53H,8-18H2,1-6H3/t19-,20+,21+,22+,24+,25-,26+,27+,28+,29-,30+,31+,32+,33-,34-,35-,36-,37+,38-,39-,40+,41+,42-,43+,44+,45-/m1/s1 | |
| Chemical Name | (2S,3R,4R,5R,6S)-2-[(2R,3S,4S,5R,6R)-4-hydroxy-2-(hydroxymethyl)-6-[(1S,2S,4S,5'R,6R,7S,8R,9S,12S,13R,16S)-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-ene-6,2'-oxane]-16-yl]oxy-5-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-3-yl]oxy-6-methyloxane-3,4,5-triol | |
| Synonyms |
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| HS Tariff Code | 2934.99.9001 | |
| Storage |
Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition | Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs) |
Biological Activity
| Targets | DNA; apoptosis |
| ln Vitro |
In HeLa and SiHa cells, dioscin (1.25–5 μg/mL; 6–24 h) raises intracellular calcium levels and triggers apoptosis[4]. Dioscin (1.25–5 μg/mL; 6–24 h): Dioscin down-regulates Bcl-2 and Bcl-xl level proteins and up-regulates Bak, Bax, Bid, p53, caspase-3, and caspase-9 proteins[4]. Dioscin is a natural steroid saponin derived from several plants, showing potent anti-cancer effect against a variety of tumor cell lines. In the present study, we investigated the anti-cancer activity of dioscin against human LNCaP cells, and evaluated the possible mechanism involved in its antineoplastic action. It was found that dioscin (1, 2 and 4 μmol/L) could significantly inhibit the viability of LNCaP cells in a time- and concentration-dependent manner. Flow cytometry revealed that the apoptosis rate was increased after treatment of LNCaP cells with dioscin for 24 h, indicating that apoptosis was an important mechanism by which dioscin inhibited cancer. Western blotting was employed to detect the expression of caspase-3, Bcl-2 and Bax in LNCaP cells. The expression of cleaved caspase-3 was significantly increased, and meanwhile procaspase-3 was markedly decreased. The expression of anti-apoptotic protein Bcl-2 was down-regulated, whereas the pro-apoptotic protein Bax was up-regulated. Moreover, the Bcl-2/Bax ratio was drastically decreased. These results suggested that dioscin possessed potential anti-tumor activity in human LNCaP cells through the apoptosis pathway, which might be associated with caspase-3 and Bcl-2 protein family.[1] In this study, we for the first time demonstrated that Dioscin suppressed RANKL-mediated osteoclast differentiation and bone resorption in vitro in a dose-dependent manner. The suppressive effect of dioscin is supported by the reduced expression of osteoclast-specific markers. Further molecular analysis revealed that dioscin abrogated AKT phosphorylation, which subsequently impaired RANKL-induced nuclear factor-kappaB (NF-κB) signaling pathway and inhibited NFATc1 transcriptional activity. [2] To determine if Dioscin protects cardiac cells from ischemia/reperfusion (I/R) injury by preventing apoptosis.Cardiac H9c2 cells were subjected to simulated I/R. Cell viability was evaluated by the methyl thiazolyl tetrazolium (MTT) colorimetry assay. Reactive oxygen species (ROS) were detected with dichlorodihydrofluorescein (DCF). Apoptosis was evaluated with flow cytometric assay. Rhodamine 123 (Rho123) was used to measure mitochondrial membrane potential (ΔΨm). ELISA was used to detect cytochrome c (Cyt-c) release from mitochondria to the cytosol. Bax and Bcl-2 mRNA expressions were measured with RT-PCR.Dioscin reduced cell death and lactate dehydrogenase (LDH) release in cells subjected to I/R. I/R induced apoptosis and cytochrome c release from mitochondria to the cytosol and this was prevented by dioscin. In support, dioscin decreased Bax but increased Bcl-2 mRNA expression. Dioscin prevented I/R induced dissipation of ΔΨm. Finally, dioscin increased superoxide dismutase (SOD) expression but reduced intracellular ROS and malondialdehyde (MDA) levels.Dioscin protects H9c2 cells from H/R injury by modulating the mitochondrial apoptotic pathway through attenuation of oxidative stress.[3] Dioscin, a natural product, has activity against glioblastoma multiforme, lung cancer and colon cancer. In this study, the effects of dioscin against human cervical carcinoma HeLa and SiHa cells were further confirmed, and the possible mechanism(s) were investigated. A transmission electron microscopy (TEM) assay and DAPI staining were used to detect the cellular morphology. Flow cytometry was used to assay cell apoptosis, ROS and Ca(2+) levels. Single cell gel electrophoresis and immunofluorescence assays were used to test DNA damage and cytochrome C release. The results showed that dioscin significantly inhibited cell proliferation and caused DNA damage in HeLa and SiHa cells. The mechanistic investigation showed that dioscin caused the release of cytochrome C from mitochondria into the cytosol. In addition, dioscin significantly up-regulated the protein levels of Bak, Bax, Bid, p53, caspase-3, caspase-9, and down-regulated the protein levels of Bcl-2 and Bcl-xl. Our work thus demonstrated that dioscin notably induces apoptosis in HeLa and SiHa cells through adjusting ROS-mediated DNA damage and the mitochondrial signaling pathway [4]. |
| ln Vivo |
There is no subchronic toxicity in female rats and mild subchronic toxicity in male rats after dosing with Dioscin (75-300 mg/kg, 10 mL/kg; oral gavage; 90 days). In male rats, dolicin produced modest dilation of the gastrointestinal tract and showed hemolytic anemia upon hematological examination [5]. Rat liver ischemia-reperfusion injury can be lessened by dioscin through the inhibition of oxidative-nitrative stress, apoptosis, and inflammation[6]. Dioscin is the major active compound in many traditional Chinese medicines (TCMs), while safety evaluation of this natural product has not yet been investigated. Therefore, the aim of this study was to evaluate the 90-day subchronic toxicity of Dioscin in rats. The rats were divided into four groups and Dioscin was administered orally at doses of 0, 75, 150 and 300 mg/kg/day, respectively. The toxicity of dioscin was evaluated based on clinical observations, ophthalmic examination, body weight, food and water consumption, urinalysis, hematology, clinical biochemistry and pathology. The results showed that dioscin had no subchronic toxicity in female rats and had slight subchronic toxicity in male rats. However, male rats in the 300 mg/kg/day group showed slight gastro-intestinal tract distension during the treatment period and hemolytic anemia in the hematology assessment. Compared with the control group, body weight gain was significantly decreased in male rats. Other significant changes were not associated with dioscin in the male and female groups. In conclusion, the no-observed-adverse-effect level (NOAEL) and the lowest-observed-adverse-effect level (LOAEL) of dioscin are estimated to be 300 mg/kg/day for female and male rats, respectively. Our work provides useful data for further research and new drug exploration of dioscin.[5] It was found that Dioscin significantly decreased serum alanine aminotransferase and aspartate aminotransferase activities, increased survival rate of rats, and improved I/R-induced hepatocyte abnormality. In addition, Dioscin obviously increased the levels of SOD, CAT, GSH-Px, GSH, decreased the levels of MDA, TNOS, iNOS, NO, and prevented DNA fragmentation caused by I/R injury. Further research indicated that Dioscin markedly decreased the gene expressions of interleukin-1β, interleukin-6, tumor necrosis factor-α, intercellular adhesion molecule-1, MIP-1α, MIP-2, Fas, FasL, decreased the protein expressions of NF-κB, AP-1, COX-2, HMGB-1, CYP2E1, Bak, caspase-3, p53, PARP, Caspase-9, decreased the levels of JNK, ERK and p38 MAPKs phosphorylation, and upregulated the levels of Bcl-2 and Bcl-x. Conclusion: The above results suggest that Dioscin has potent actions against hepatic I/R injury through suppression of inflammation, oxidative-nitrative stress, and apoptosis, which should be developed as a new drug to treat hepatic I/R injury in the future[6]. |
| Enzyme Assay |
Transmission Electron Microscope (TEM) Assay [4] The HeLa and SiHa cells (2 × 105 cells/mL) were plated in 6-well plates, treated with Dioscin, harvested, and fixed overnight at 4 °C in 2% glutaraldehyde. The samples were implemented as previously described The obtained sections were then stained and observed using a transmission electron microscope. DAPI Staining [4] HeLa and SiHa cells were seeded in six-well plates and cultured overnight, then treated separately with Dioscin (1.25, 2.5 and 5.0 μg/mL) for 12 h and 24 h. For DAPI staining, the cells were treated as mentioned above, and then stained with DAPI (1.0 μg/mL) solution. Finally, the images were photographed with a fluorescence microscope. Detection of Intracellular ROS Accumulation [4] The HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated by Dioscin(1.25, 2.5 and 5.0 μg/mL). The cells were collected and re-suspended in 500 μL DCFH-DA (10.0 μM), which were all analyzed by flow cytometry. Detection of Cell Apoptosis and Intracellular Ca2+Release [4] HeLa and SiHa cells were plated in 6-well plates at the density of 1 × 105 cells/well and treated with Dioscin (1.25, 2.5 and 5.0 μg/mL), then collected and resuspended in 500 μL of Fluo-3/AM (2.5 μM), which were all analyzed by flow cytometry. Single Cell Gel Electrophoresis Assay [4] After the cancer cells being treated with Dioscin (1.25, 2.5 and 5.0 μg/mL), single cell gel electrophoresis (SCGE) method was used to detect Dioscin-induced DNA damage. The images of the cells were obtained by a fluorescence microscope (Olympus) according to the manufacturer’s instructions. Eventually, over 50 cells were randomly selected from each of the three repeated wells and analyzed by the Comet Assay Software Project (CASP) 1.2.2. Detection of Cytochrome C Release [4] HeLa and SiHa cells were seeded in six-well plates, treated with Dioscin (1.25, 2.5 and 5.0 μg/mL), and then incubated with the primary antibody overnight. After that, the plates were incubated with the secondary antibody for 1 h at 37 °C, and dyed with DAPI (5.0 μg/mL) for 5 min. The images of the cells were obtained by a laser scanning confocal microscope. |
| Cell Assay |
MTT Assay for Cell Viability [1] The cytotoxic effect of Dioscin on LNCaP cells was determined by MTT assay according to the documented method. Briefly, 8×103 cells were seeded into and incubated in 96-well flat-bottom plates with 200 μL complete growth medium each well. After 24 h, LNCaP cells were treated with various concentrations of Dioscin (0–10 μmol/L) for 12, 24 and 48 h. Thereafter, 10 μL of 5 mg/mL MTT was added to each well and the plates were incubated for another 4 h. At the end of the experiment, the medium was removed from each well and 150 μL DMSO was added to terminate the reaction. The absorbance (A) at 570 nm wavelength was measured using an automated ELISA microplate reader. Inhibition rate (%) was calculated based on the following equation: Inhibition rate (%)=[(Acontrol–Atreated)/Acontrol] ×100%, where Acontrol was A value of the vehicle-treated wells, and Atreated the A value of the dioscin-treated wells. The cytotoxicity of dioscin on LNCaP cells was expressed as IC50 values (50% inhibition of cell viabilities for cells treated with the tested drug with respect to the 0.1% DMSO-treated cells, and calculated by Probit normal method) Flow Cytometrical Detection of Cell Apoptosis [1] Cell apoptosis was flow cytometrically determined by using an Annexin Ⅴ-FITC/PI apoptosis detection kit by following instructions. In brief, 2×105 cells were harvested and washed with ice-cold PBS (pH 7.4) two times. Afterward, cells were suspended in 500 μL binding buffer and incubated in the dark at room temperature for 15 min after dual labeling with 5 μL Annexin Ⅴ-FITC and 5 μL propidium iodide (PI). Then, about 1×104 of the stained apoptotic cells were flow cytometrically counted, and the results were defined as follows: the lower-left quadrant represents the living cells (Annexin V– /PI– ); the lower-right quadrant represents the early apoptotic cells (Annexin V+ /PI– ); the upper-right quadrant represents the late apoptotic cells (Annexin V+ /PI+ ); the upper-left quadrant represents the primary necrotic cells (Annexin V– /PI+ ). Western Blotting [1] The expression of caspase-3, Bcl-2 and Bax was determined by Western blotting. In brief, after treated with Dioscin for 24 h, cells were harvested, placed on ice, washed with ice-cold PBS, and then lysed on ice for 30 min in lysis buffer. After the buffer was centrifuged at 15 000 g for 10 min at 4°C, the resulting supernatant was collected as total cellular protein. Protein concentrations were detected by using the BCA protein assay kit. Protein extracts (20 μg in 20 μL) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinyldifluoride (PVDF) membrane by electroblotting. The transferred membranes were blocked with 5% non-fat dried milk in TBST (1 mol/L Tris-buffered saline, pH 7.4; 0.1% Tween-20) for 1 h at room temperature, and then cultured overnight at 4°C with primary antibodies (cas-pase-3, Bcl-2 and Bax, 1:1000 dilution). The membranes were washed three times in TBST buffer, and then incubated at room temperature for 90 min with the appropriate secondary antibodies linked with horseradish peroxidase (1:2000 dilution). After washing the membranes 4 times with TBST, the proteins in the blots were developed on films by using the enhanced chemiluminescence reagent. The expression level of the aforementioned proteins was densitometrically evaluated by using Bio-Rad Quantity One V4.62. β-actin was detected in the same way as described above and used as an internal loading reference. Cell viability assay [2] The anti-proliferative effect of Dioscin on BMMs cells was assessed with a cell counting kit-8. Briefly, after treatment, 10 μl CCK-8 solution was added to each well; after 4 h incubation, absorbance was measured at 450 nm using a microplate reader. The effect of Dioscin on cell viability was expressed as percent cell viability with vehicle-treated control cells set at 100%. In vitro osteoclastogenesis assay [2] In vitro osteoclastogenesis assays were preformed to examine the effects of Dioscin on osteoclast differentiation. Bone marrow macrophages (BMM) cells were prepared as previously described. Briefly, cells extracted from the femur and tibiae of a 6-week-old C57/BL6 mouse were incubated in complete cell culture media and 30 ng/mL M-CSF in a T-75 cm2 flask for proliferation. When changing the medium, the cells were washed in order to deplete residual stromal cells. After reaching 90% confluence, cells were washed with phosphate-buffered saline (PBS) three times and trypsinised for 30 min to harvest BMMs. Adherent cells on dish bottoms were classified as BMMs; these BMMs were plated in the 96-well plates at a density of 8 × 103 cells/well in triplicate and incubated in a humidified incubator containing 5% CO2 at 37 °C for 24 h. The cells were then treated with various concentrations of Dioscin (0, 1, or 4 μM) plus M-CSF (30 ng/mL) and RANKL (50 ng/mL). After five days, cells were fixed and stained for TRAP activity. TRAP+ multinucleated cells with more than five nuclei were counted as osteoclasts. Resorption pit assay [2] For the bone resorption assay were carried out as previously described, BMMs were seeded on bone slices in 96-well plates at a density of 8 × 103 cells/well with three replicates and stimulated with M-CSF (30 ng/mL) plus RANKL (50 ng/mL). Three days later, cells were treated with the indicated concentrations of Dioscin for 48 h post-culture. Cells were then fixed with 2.5% glutaraldehyde. Bone slices were imaged using a scanning electron microscope (SEM; FEI Quanta 250) with 200× magnification and at 10 kV. Three view fields were randomly selected for each bone slice for further analysis. Pit areas were quantified using Image J software. Similar independent experiments were repeated for at least three times. Western blot analysis [2] BMMS cells were seeded at 5 × 105 cells/well into 6-well plates and pretreated with or without Dioscin (4 μM) for 4 h prior to RANKL stimulation (50 ng/mL) for the indicated times (0, 5, 15 or 30 min). BMMs were seeded at 5 × 105 cells/well into 6-well plates and treated with or without Dioscin (4 μM) and RANKL (50 ng/mL) for the indicated times. Cells were lysed in RIPA lysis buffer containing 50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 1 mM sodium fluoride, 1 mM sodium vanadate, 1% deoxycholate, and protease inhibitor cocktail. The lysate was centrifuged at 12,000 rcf for 10 min, and the protein in the supernatant was collected. Protein concentrations were measured though BCA assay. Thirty micrograms of each protein lysate was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using 8–10% gels, and proteins were then transferred to polyvinylidene difluoride membranes. Nonspecific interactions were blocked with 5% skim milk for 1 h, and membranes were then probed with the indicated primary antibodies overnight at 4 °C as indicated. Membranes were incubated with the appropriate secondary antibodies conjugated with IRDye 800CW (molecular weight, 1166 Da), and the antibody reactivity was detected by exposure in an Odyssey infrared imaging system (Li-COR). Luciferase reporter gene activity assay [2] The effects of Dioscin on RANKL-induced NF-κB activation were measured using RAW264.7 cells that had been stably transfected with an NF-κB luciferase reporter construct, as previously described. Briefly, cells were seeded into 48-well plates and maintained in cell culture media for 24 h. Cells were then pretreated with or without the indicated concentrations of Dioscin for 1 h, followed by addition of RANKL (50 ng/mL) for 8 h. Luciferase activity was measured using the Promega Luciferase Assay System and normalised to that of the vehicle control. Similarly, the effect of dioscin on RANKL-induced AP-1- or NFATc1-dependent luciferase reporter assays was determined as described previously |
| Animal Protocol |
Based on a previously published study on the acute toxic effects of Dioscin (Liang et al., 2010), the rats received Dioscin at doses of 0 (control), 75, 150, and 300 mg/kg/day, respectively. Dioscin was mixed with a solution of 0.5% carboxymethylcellulose sodium in distilled water. An appropriate amount of Dioscin or control (vehicle) was administered daily (7 days/week) to each rat for 90 consecutive days. Each animal was dosed by oral intubation using a ball-tipped gavage needle attached to an appropriate syringe. Dosing was at the same time each day ±1 h. Individual doses were calculated based on body weight and were adjusted each day to maintain the target dose level in all rats. All doses were administered volumetrically after correcting for dilution. Doses were administered to all groups at a constant dose volume of 10 mL/kg. The control group received vehicle only at the same volume as the test animals. The present study was conducted based on the US Food and Drug Administration principles (FDA, 2000) and OECD Test Guideline 408 for ‘Repeated dose 90-day oral toxicity study in rodents’. [5] Ophthalmological examination : An ophthalmological examination was conducted on all rats (40 males; 40 females) on the first day of the experiment prior to the administration of Dioscin and on the 90th day at the end of the study. The peripheral and internal structure of both eyes in each rat was examined by the naked eye and indirect ophthalmoscopy, respectively. The animals were allowed to adapt to the environment for 1 week before the experiments. As shown in Figure 5(B), in the prophylactic test, Dioscin was administered intragastrically to the animals at the doses of 20, 40, and 60 mg/kg once daily for seven consecutive days. The rats in the sham operation and model groups were administered with vehicle. On the eighth day, the murine model of 70% partial hepatic ischemia was established as described previously. Briefly, the rats were anesthetized, and the livers were exposed by midline laparotomy, then the inflow of the left lateral and median lobes of the livers was choked by placing a bulldog clamp, whereas the right lobes were remainedly perfused to prevent intestinal congestion occlusion. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the liver was reperfused for 6 hr. At the end of the surgery, the animals were anesthetized for collecting the blood and then killed to collect the left lateral and middle lobes of the livers. As shown in Figure 5(C), in the therapeutic test, the rats were randomized into three groups, of which the animals in sham and model groups were given vehicle, and the rats in Dioscin group received Dioscin intragastrically at a dose 60 mg/kg once 2 hr before I/R. After 60 min of hepatic ischemic, the bulldog clamp was removed, and the orbital blood samples were collected at 0.5, 1, 2, 4, 6, 12, 24, 48, and 96 hr after reperfusion.. In addition, the survival rate of the animals was assayed. [6] Background: Dioscin shows potent effects against liver damage in our previous studies; however, the action of it on hepatic ischemia-reperfusion (I/R) injury is still unknown. In the present article, the effects and possible mechanisms of dioscin against hepatic I/R injury were investigated. Methods: Seventy percent partial hepatic warm ischemia was induced in Wistar rats for 60 min followed by succedent reperfusion. In the prophylactic test, dioscin was administered intragastrically to the rats at doses of 20, 40, and 60 mg/kg once daily for seven consecutive days before I/R. In the therapeutic test, the rats received dioscin intragastrically at a dose of 60 mg/kg once 2 hr before I/R. [6] |
| Toxicity/Toxicokinetics | mouse LD50 subcutaneous >300 mg/kg Japanese Kokai Tokyo Koho Patents., #91-271224 |
| References |
[1]. Dioscin-induced apoptosis of human LNCaP prostate carcinoma cells through activation of caspase-3 and modulation of Bcl-2 protein family. J Huazhong Univ Sci Technolog Med Sci. 2014 Feb;34(1):125-30. [2]. Dioscin inhibits osteoclast differentiation and bone resorption though down-regulating the Akt signaling cascades. Biochem Biophys Res Commun. 2014 Jan 10;443(2):658-65. [3]. Dioscin prevents the mitochondrial apoptosis and attenuates oxidative stress in cardiac H9c2 cells. Drug Res (Stuttg). 2014 Jan;64(1):47-52. [4]. Dioscin Induces Apoptosis in Human Cervical Carcinoma HeLa and SiHa Cells through ROS-Mediated DNA Damage and the Mitochondrial Signaling Pathway. Molecules. 2016 Jun 4;21(6):730. [5]. A 90-day subchronic toxicological assessment of dioscin, a natural steroid saponin, in Sprague-Dawley rats. Food Chem Toxicol. 2012 May;50(5):1279-87. [6]. Dioscin attenuates hepatic ischemia-reperfusion injury in rats through inhibition of oxidative-nitrative stress, inflammation and apoptosis. Transplantation. 2014 Sep 27;98(6):604-11. |
| Additional Infomation |
Dioscin is a spirostanyl glycoside that consists of the trisaccharide alpha-L-Rha-(1->4)-[alpha-L-Rha-(1->2)]-beta-D-Glc attached to position 3 of diosgenin via a glycosidic linkage. It has a role as a metabolite, an antifungal agent, an antiviral agent, an antineoplastic agent, an anti-inflammatory agent, a hepatoprotective agent, an apoptosis inducer and an EC 1.14.18.1 (tyrosinase) inhibitor. It is a spirostanyl glycoside, a spiroketal, a hexacyclic triterpenoid and a trisaccharide derivative. It is functionally related to a diosgenin. It derives from a hydride of a spirostan. Dioscin has been reported in Dioscorea collettii, Dioscorea deltoidea, and other organisms with data available. See also: Dioscorea polystachya tuber (part of). In this study, we have verified for the first time that natural compound Dioscin inhibited osteoclast differentiation and bone resorption, suggesting an additional protective effect of dioscin on osteoclast-related diseases. In addition, we revealed the molecular mechanisms of dioscin on osteoclasts are through suppressing Akt/NF-κB and Akt/NFATc1 signaling pathways. In osteoclasts, the Akt signaling cascades is a critical downstream of three osteoclast surface receptors including c-fms, αvβ3 integrin and RANK. Previous studies demonstrated Akt phosphorylation is activated upon both M-CSF and RANKL stimulation and play critical roles in osteoclastogenesis by affecting both NF-κB and NFATc1 activation. Moon et al. demonstrated that overexpression of Akt in BMMs strongly induced NFATc1 expression and lead to enhanced osteoclastogenesis. Besides, activation of NF-κB can be initiated by several different kinases such as Akt and NF-κB inducing kinase. Gingery et al. demonstrated that AKT/NF-κB axis is critical in osteoclastogenesis and maintaining mature osteoclast survival. In consistent with these studies, we demonstrated that dioscin inhibited Akt phosphorylation and thus suppressed the RANKL-induced NF-κB activity and NFATc1 activity, both of which are critical for osteoclast differentiation. Interestingly, in the process of detecting the function of dioscin on MAPKs pathway, no significantly inhibitory impact was witnessed. In summary, dioscin is capable of inhibiting osteoclast formation and function, indicating additional therapeutic benefits of dioscin for osteoclast-related diseases. In addition, this study also clearly revealed the molecular mechanisms of dioscin on osteoclasts are via impairing Akt/NF-κB and Akt/NFATc1 signaling pathways in vitro. In addition, our in vitro results further verified the bone protective role of dioscin on LPS-induced osteolysis model. However, further investigation of dioscin on other cells within bone is still required. [1] Dioscin induced apoptosis of human cervical cancer HeLa and SiHa cells through inducing ROS-mediated DNA damage and activating the mitochondrial signaling pathway. However, based on the in vitro results, the molecular mechanism of action and the drug targets of dioscin need further investigation.[4] Due to the medicinal value and widespread use of Dioscin, this natural compound has great potential in future application and research in the pharmaceutical field. Thus, it is critically important to evaluate the toxicity of dioscin. The results from our 90-day subchronic toxicity study showed changing trends in dose dependency on individual body weight. Compared with the control group, body weight was significantly decreased in all male groups, especially in the male 300 mg/kg treatment group. This change was considered to be related to the properties of saponins such as reduced feeding caused by gastro-intestinal tract distension (Shen et al., 2008), decreased intestinal motility (Klita et al., 1996), and protein digestibility (Potter et al., 1993). Due to reduced feeding, food consumption was decreased and therefore body weight was lost. In the urinalysis, changes in urine pH, urine specific gravity and urine protein were noted in the male 75 mg/kg treatment group. Similar changes in the 300 mg/kg treatment group were not found. These changes were incidental without apparent dose dependence. In the hematology assessment, changes in RBC and HCT were recorded in the male 300 mg/kg treatment group, suggesting hemolytic anemia. The bioactive compound of steroidal saponin has hemolytic activity (Santos et al., 1997, Zhang et al., 1999) and saponin is known to have a lytic action on erythrocyte membranes (Howard and Wallace, 1953), which was reported to result from the affinity of aglycone moieties for membrane sterols, particularly cholesterol, forming insoluble complexes in open pores in the membranes (Bangham et al., 1962, Cho et al., 2009). Membrane rupture of erythrocytes could release into the blood and cause slight nephrotoxicity. There were significant changes in BUN of males in the clinical biochemistry assessment, but no histopathological lesions were observed in the kidneys. Other changes in hematology were incidental as a dose–response relationship was not observed. In the clinical biochemistry assessment, increases in ALT observed in the female 300 and male 300 mg/kg treatment groups were judged to be related to Dioscin treatment, however, the changes in AST, AKP and T-BIL in the groups showed no apparent dose dependence. Although the changes in ALT in the groups were suggestive of hepatic damage, no changes in liver weight and no abnormalities on histopathological examination were observed. The significant changes in BUN in male and female rats indicated mild nephrotoxic effects, however, no histopathological lesions were recorded in the kidneys. While both males and females showed significant fluctuations in Cr, TG, γ-GT and glucose, the changes were incidental without apparent dose dependence or toxicological significance. In the evaluation of organ weight and histopathology, although significant changes in absolute and relative weights were observed in organs such as the liver, kidneys, heart, and brain, no histopathological changes were observed and these changes could be considered to be due to decreased body weight. Body weight loss and relative organ weight increases are shown in Table 5, and these changes are considered to be incidental due to additional fat. Conclusion: The development potential of the medicinal constituent, Dioscin, is significant. However, it is also necessary to focus on the toxicity of Dioscin based on the results obtained in this study. Our findings provide some evidence on the safety of dioscin for potential clinical application and the widespread use of dioscin, however, prior to the clinical application of dioscin further evaluation is required. In conclusion, dioscin at a dose of 300 mg/kg/day in female rats was identified as the NOAEL (no-observed-adverse-effect-level) and a dose of 300 mg/kg/day in male rats was identified as the LOAEL (lowest-observed-adverse-effect level) in this study. [5] In conclusion, dDioscin has a good protective effect against hepatic I/R injury in rats through attenuating oxidative-nitrative stress, inflammation, and apoptosis, which should be developed as a new and potent candidate for treatment of I/R-induced liver injury in the future. Of course, mechanisms, drug-target, and clinical applications of Dioscin are needed further investigations. [6] |
Solubility Data
| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.25 mg/mL (2.59 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 22.5 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. Solubility in Formulation 2: ≥ 2.25 mg/mL (2.59 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 22.5 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. Solubility in Formulation 3: ≥ 2.25 mg/mL (2.59 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 22.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.1507 mL | 5.7534 mL | 11.5068 mL | |
| 5 mM | 0.2301 mL | 1.1507 mL | 2.3014 mL | |
| 10 mM | 0.1151 mL | 0.5753 mL | 1.1507 mL |