SREBP1c (transcription inhibition) [2]
SREBP2 (transcription inhibition) [2]
miR-33a (level reduction) [2]
miR-33b (level reduction) [2]

Methyl protodioscin inhibited proliferation of A549 human lung cancer cells in a dose- and time-dependent manner. MTT assay after 48 h treatment showed dose-dependent cytotoxicity with IC50 not explicitly stated but significant effect at 20 μM. [1]
Treatment with 20 μM Methyl protodioscin for 48 h induced typical apoptotic morphological changes including cell shrinkage, nuclear fragmentation, and apoptotic body formation as observed by Hoechst 33258 staining. [1]
Flow cytometry analysis revealed that Methyl protodioscin (20 μM for 48 h) induced significant G2/M phase cell cycle arrest and apoptosis in A549 cells in a time-dependent manner. [1]
Western blot analysis showed that Methyl protodioscin treatment resulted in marked decrease of Bcl-2 protein expression and drastic increase in the expression of Bax and Bak proteins. [1]
Methyl protodioscin induced a time-dependent mitochondrial transmembrane depolarization (decrease of mitochondrial membrane potential) and concomitant time-dependent cytochrome c release from mitochondria into cytosol in A549 cells. [1]
Caspase-3 activity was increased upon Methyl protodioscin treatment (20 μM for 48 h), and pretreatment with caspase-3 inhibitor z-DEVD-fmk (20 μM for 2 h) significantly inhibited MPD-induced cell death and caspase-3 activation. [1]

MTT assay for cell viability: A549 cells were seeded in 24-well plates at 4.0 × 10^4 cells/well, incubated for 24 h, then treated with various concentrations of Methyl protodioscin (0, 5, 10, 15, 25, 35 μM) for 48 h. MTT dye (5 mg/ml) was added, then DMSO (150 μl) was added to dissolve crystals. Absorbance was measured and IC50 calculated using Bliss software. [1]
Hoechst 33258 fluorescent staining for apoptosis morphology: A549 cells (2 × 10^5) were seeded in 6-well plates and treated with 20 μM Methyl protodioscin for 48 h. Cells were stained with Hoechst 33258 and observed for chromatin condensation and fragmentation. [1]
Cell cycle analysis by flow cytometry: A549 cells were treated with 20 μM Methyl protodioscin for 48 h, washed with PBS, fixed with 70% ice-cold ethanol overnight at 4°C, then resuspended in staining solution containing 1% Triton X-100, 0.1 mg/ml RNase, and 6 μg/ml propidium iodide (PI). After 30 min incubation at 37°C in the dark, samples were analyzed on a flow cytometer. [1]
Mitochondrial membrane potential assay: A549 cells (1 × 10^6 cells/ml) were incubated with 10 μM rhodamine 123 for 10 min at 37°C, washed twice with PBS, then resuspended in PBS. Fluorescence intensity was measured at 530 nm using a fluorescence spectrophotometer. Cells were treated with 20 μM Methyl protodioscin for various times. [1]
Caspase-3 activity assay: A549 cells were treated with 20 μM Methyl protodioscin for 48 h, harvested, washed, and lysed in lysis buffer on ice for 30 min. Lysates were centrifuged at 10,000 × g for 10 min, and supernatant (20 μl) was collected for caspase-3 activity assay using DEVD-pNA as a specific substrate. pNA concentration was measured at 405 nm. For inhibition studies, cells were pretreated with 20 μM z-DEVD-fmk for 2 h prior to MPD treatment. [1]
Mitochondrial cytochrome c release assay: A549 cells (1 × 10^6 cells/ml) were incubated with 20 μM Methyl protodioscin for up to 4 h. Cells were suspended in lysis buffer (195 mM mannitol, 65 mM sucrose, 2 mM HEPES pH 7.4, 0.05 mM EGTA, 0.01 mM MgCl2, 0.5 mg/ml BSA) and lysed with 0.01% digitonin. Cytosolic fraction was obtained by centrifugation at 10,000 × g for 10 min and collected for cytochrome c assay using a commercial immunoassay kit. [1]
Western blot analysis: Cells were lysed and protein concentrations determined by Bradford assay. Protein extracts (20 μg per sample) were separated by SDS-PAGE, transferred to PVDF membranes, incubated with primary antibodies (against Bax, Bak, Bcl-2) for 12 h at 4°C, washed, then incubated with HRP-conjugated secondary antibody for 1 h at room temperature. Protein bands were visualized using chemiluminescence detection. [1]

[1]. Methyl protodioscin induces G2/M cell cycle arrest and apoptosis in A549 human lung cancer cells. Pharmacogn Mag. 2014 Jul;10(39):318-24.

[2]. Methyl protodioscin increases ABCA1 expression and cholesterol efflux while inhibiting gene expressions for synthesis of cholesterol and triglycerides by suppressing SREBP transcription and microRNA 33a/b levels. Atherosclerosis. 2015 Apr;239.

Methylprodiosgenin is a steroidal saponin. It has been reported to exist in Smilax china, Polygonatum sibiricum, and other organisms with relevant data.

---

Methyl protodioscin (MPD) is a furostanol bisglycoside purified from Dioscorea collettii var. hypoglauca with purity >97% by HPLC. It has been previously tested by the National Cancer Institute's (NCI) anti-cancer drug discovery screen against a panel of 60 human cancer cell lines. The mechanism of action in A549 cells involves G2/M cell cycle arrest and mitochondria-mediated apoptosis through downregulation of Bcl-2, upregulation of Bax and Bak, loss of mitochondrial membrane potential, release of cytochrome c into cytosol, and activation of caspase-3. Potential therapeutic application: lung cancer (non-small cell lung cancer, adenocarcinoma). [1]

C52H86O22

1063.2261

1062.561

54522-52-0

11263254

White to off-white solid powder

1.4±0.1 g/cm3

1.620

4.17

12

22

15

74

1920

31

C[C@H]1[C@H]2[C@H](C[C@@H]3[C@@]2(CC[C@H]4[C@H]3CC=C5[C@@]4(CC[C@@H](C5)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O[C@H]7[C@@H]([C@@H]([C@H]([C@@H](O7)C)O)O)O)O)O[C@H]8[C@@H]([C@@H]([C@H]([C@@H](O8)C)O)O)O)C)C)O[C@@]1(CC[C@@H](C)CO[C@H]9[C@@H]([C@H]([C@@H]([C@H](O9)CO)O)O)O)OC

HSSJYSJXBOCKQM-GVTGEURHSA-N

InChI=1S/C52H86O22/c1-21(20-66-46-40(61)39(60)36(57)31(18-53)70-46)10-15-52(65-7)22(2)33-30(74-52)17-29-27-9-8-25-16-26(11-13-50(25,5)28(27)12-14-51(29,33)6)69-49-45(73-48-42(63)38(59)35(56)24(4)68-48)43(64)44(32(19-54)71-49)72-47-41(62)37(58)34(55)23(3)67-47/h8,21-24,26-49,53-64H,9-20H2,1-7H3/t21-,22+,23+,24+,26+,27-,28+,29+,30+,31-,32-,33+,34+,35+,36-,37-,38-,39+,40-,41-,42-,43+,44-,45-,46-,47+,48+,49-,50+,51+,52-/m1/s1

(2S,3R,4R,5R,6S)-2-[(2R,3S,4S,5R,6R)-4-hydroxy-2-(hydroxymethyl)-6-[[(1S,2S,4S,6R,7S,8R,9S,12S,13R,16S)-6-methoxy-7,9,13-trimethyl-6-[(3R)-3-methyl-4-[(2R,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxybutyl]-5-oxapentacyclo[10.8.0.02,9.04,8.013,18]icos-18-en-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

NSC-698790; Smilax saponin B; NSC 698790; NSC698790

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 : ~100 mg/mL (~94.05 mM)
H2O : ~25 mg/mL (~23.51 mM)

Solubility in Formulation 1: ≥ 3.5 mg/mL (3.29 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 35.0 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: 3.5 mg/mL (3.29 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 35.0 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.

---

View More

Solubility in Formulation 3: ≥ 3.5 mg/mL (3.29 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 35.0 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.)