Mitochondrial pathway (Bax, Bcl-2, caspase-3, caspase-8, caspase-9, AIF, Apaf-1, cytochrome C) - no IC50/Ki/EC50/DC50 values reported
ROS-mediated pathway - no quantitative binding data reported
No specific receptor or enzyme targets identified in these studies. [1][2]

α-Hederin is cytotoxic and, at comparatively low concentrations, prevents both cell lines from proliferating. Treated cells exhibit less mitotic activity when exposed to α-Hederin [2].

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Proliferation inhibition: alpha-Hederin significantly inhibited proliferation of HGC-27 and SGC-7901 gastric cancer cells in a dose- and time-dependent manner with IC50 values of 20.86 µM and 25.91 µM respectively at 24 hours (CCK-8 assay). EdU retention assay showed concentration-dependent inhibition of DNA synthesis. Colony formation assay revealed reduced colony numbers. [1]
Cell cycle arrest: Flow cytometry showed G1 phase arrest with percentages of 46.29% (control), 62.02% (low dose), 65.44% (middle dose), and 69.62% (high dose). Western blot showed increased P16, P21, P53, and CDK2 protein levels and decreased Cyclin D1. [1]
Apoptosis induction: Hoechst 33258 staining revealed apoptotic nuclei with cell shrinkage, membrane blebbing, chromatin condensation, and nuclear fragmentation. Apoptotic rate increased dose-dependently. Western blot showed increased Bax, cleaved caspase-3, caspase-8, caspase-9 and decreased Bcl-2. [1]
GSH depletion: Intracellular GSH was depleted gradually with increasing alpha-Hederin concentration (0-40 µM). Pretreatment with BSO (GSH inhibitor) augmented the anti-cancer effects, while NAC (GSH precursor) attenuated them. [1]
ROS accumulation and ATP depletion: alpha-Hederin increased intracellular ROS and decreased ATP levels; BSO amplified these changes, NAC reversed them. JC-1 staining showed loss of mitochondrial membrane potential, augmented by BSO and attenuated by NAC. [1]
Mitochondrial pathway activation: Western blot of mitochondrial and cytosolic fractions showed decreased mitochondrial AIF, Apaf-1, and cytochrome C levels with corresponding increases in cytosolic fractions upon alpha-Hederin treatment; BSO amplified, NAC attenuated these changes. [1]
In vitro cytotoxicity in B16 melanoma and 3T3 fibroblasts: alpha-Hederin was cytotoxic at concentrations above 5 µg/mL after 8 hours in serum-free medium; at 10 µg/mL caused significant cell death; effects were dose- and time-dependent. Cytotoxicity decreased in presence of FCS or BSA. [2]
Morphological changes: Phase microscopy revealed cytoplasmic vacuoles within hours of treatment with 5 µg/mL alpha-Hederin; vacuoles were not stained by hematoxylin-eosin or fat red. Scanning electron microscopy showed crater-like depressions on cell surfaces and blebs. Transmission electron microscopy revealed empty vacuoles, lamellar bodies inside phagolysosomes, and membrane breaks. [2]
Biochemical changes in B16 and 3T3 cells: alpha-Hederin (3-5 µg/mL, 72h) decreased cell numbers; increased melanin and protein content in B16 cells at 5 µg/mL; increased DNA and protein in 3T3 fibroblasts. Mitotic index decreased and necrotic index increased in both cell lines. [2]

Similar to thymoquinone, α-hederin also acts as a preventative measure in sensitized rats. It might suppress the inflammatory response by interfering with the expression of miRNA-126, which would disrupt the IL-13 secretion pathway [3].
Xenograft tumor model: Male BALB/c nude mice with subcutaneous gastric cancer xenografts were treated with alpha-Hederin at 2.5, 5, and 10 mg/kg via intraperitoneal injection every other day. Tumor growth was significantly inhibited in a dose-dependent manner; final mean tumor weights were 1.85 g (control), 1.59 g (2.5 mg/kg), 1.18 g (5 mg/kg), and 0.87 g (10 mg/kg). [1]
Histological analysis: H&E staining showed tumor cells arranged closely with larger nuclei; some mitotic figures observed. TUNEL staining revealed significantly more apoptotic cells in alpha-Hederin treated groups in a concentration-dependent manner. No metastases were found in lung, liver, or brain of any group. [1]
No significant differences in liver or kidney function biomarkers (ALT, AST, urea, creatinine) were observed among groups, indicating favorable safety profile. [1]

CCK-8 cytotoxicity assay: Cells seeded in 96-well plates at 5×10^3 cells/well were treated with alpha-Hederin (0-80 µM) for 0-36 hours, or pretreated with 2 mM BSO or 5 mM NAC for 12 hours before alpha-Hederin treatment. CCK-8 solution (10 µL in 100 µL medium) was added, incubated for 2 hours, and absorbance measured at 450 nm. Cell viability calculated as (experimental absorbance/control absorbance) × 100%. [1]
EdU proliferation assay: Cells (2×10^4/well in 24-well plates) treated with alpha-Hederin (0-20 µM) for 24 hours, incubated with 50 µM EdU for 2 hours, fixed in 4% paraformaldehyde, permeabilized with 0.5% Triton X-100, stained with Apollo reaction cocktail and Hoechst 33342, and imaged under fluorescence microscope. [1]
Colony formation assay: Cells (1-5×10^3/well in 6-well plates) with or without BSO/NAC pretreatment were treated with 10 µM alpha-Hederin, medium replaced every 2-3 days for ~14 days, fixed with 4% paraformaldehyde, stained with Wright-Giemsa, colonies (≥50 cells) counted. [1]
Hoechst 33258 apoptosis staining: Cells (5×10^4/well on cover glasses in 6-well plates) treated with alpha-Hederin (0-20 µM) for 24 hours or with BSO/NAC pretreatment, fixed, stained with Hoechst 33258, and examined under fluorescence microscope for apoptotic nuclear morphology. [1]
Flow cytometry cell cycle and apoptosis: Cells digested with trypsin, fixed in 70% ethanol at 4°C overnight. For cell cycle: stained with propidium iodide (1 mg/mL) with RNase (100 µg/mL) for 30 min at 4°C. For apoptosis: stained with Annexin V-FITC and propidium iodide for 15 min at room temperature. DNA content analyzed by flow cytometry. [1]
GSH content measurement: Cells treated with alpha-Hederin (0-40 µM) for 24 hours, lysed with protein removal reagent M, frozen/thawed in liquid nitrogen, centrifuged at 10,000×g for 10 min. GSH/GSSG measured at 412 nm using GSH/GSSG Assay Kit. [1]
ROS measurement: Cells treated with 10 µM alpha-Hederin for 24 hours with/without BSO/NAC, loaded with 10 µM DCFH-DA for 20 min at 37°C, fluorescence measured by microplate reader or fluorescence microscope. [1]
ATP measurement: Cells treated with 10 µM alpha-Hederin for 24 hours with/without BSO/NAC, lysed with ATP lysis buffer, ATP detection solution added, luminescence measured within 30 min using microplate reader. [1]
Mitochondrial membrane potential (JC-1): Cells treated with 10 µM alpha-Hederin for 24 hours with/without BSO/NAC, incubated with JC-1 staining solution for 20 min at 37°C, washed, and analyzed by laser confocal fluorescence microscopy. [1]
Mitochondrial/cytosolic protein fractionation: Proteins extracted using Cell Mitochondria Isolation Kit, separated by SDS-PAGE, transferred to PVDF membranes, immunoblotted with antibodies against Bax, Bcl-2, cleaved caspase-3/8/9, AIF, Apaf-1, cytochrome C, Cox-IV, and GAPDH, detected using two-color infrared imaging. [1]
MTT cytotoxicity assay (Danloy et al.): Cells seeded in 96-well plates (5,000-50,000 cells/well depending on treatment duration), treated with alpha-Hederin (1-15 µg/mL) in serum-free DMEM-F12-N2 for 8-72 hours, cell survival measured by MTT reduction assay. [2]
Serum effect on cytotoxicity: B16 cells treated with 10 µg/mL alpha-Hederin in DMEM-F12-N2 with increasing BSA or FCS concentrations for 72 hours, cell survival measured by MTT. [2]
Cell proliferation/necrosis analysis: Cells (150,000/Petri dish) treated with alpha-Hederin (3 or 5 µg/mL) for 24 or 48 hours, fixed in Bouin's solution, stained with hematoxylin-eosin, mitotic and necrotic cells counted (2,000 cells/slide). [2]
DNA/protein/melanin quantification: Cells (350,000 B16 or 1,050,000 3T3) treated with alpha-Hederin (3 or 5 µg/mL) for 72 hours, cell counts performed, melanin measured by Prasad method, proteins by Bradford, DNA by Labarca method. [2]
Electron microscopy: Cells treated with alpha-Hederin, fixed with glutaraldehyde and osmium tetroxide, embedded in Epon 812 for TEM, or dehydrated and gold-palladium coated for SEM. [2]

Xenograft study: Male BALB/c nude mice (4-6 weeks) were subcutaneously inoculated with gastric cancer cells. When tumors reached ~150 mm³, mice were randomly allocated to 4 groups (n=6): control (saline), low-dose (2.5 mg/kg), middle-dose (5 mg/kg), and high-dose (10 mg/kg) alpha-Hederin. Drug or saline was administered via intraperitoneal injection every other day. Tumor size measured every 3 days using calipers; tumor volume calculated as (width² × length)/2. At experiment end, tumors harvested, weighed, and analyzed by H&E and TUNEL staining. Body weight and liver/kidney function biomarkers (ALT, AST, urea, creatinine) were monitored. [1]
In vitro cell culture experiments (Danloy et al.): B16 melanoma and 3T3 fibroblasts cultured in MEM or DMEM with 10% FCS and penicillin. After 24 hours incubation, medium removed, cells rinsed with PBS, and treated with DMEM-F12-N2 containing alpha-Hederin (1-15 µg/mL) for 8-72 hours at 37°C in 5% CO2. For serum effect experiments, medium contained 10 µg/mL saponin with increasing BSA or FCS. [2]

In vitro cytotoxicity: In serum-free medium, alpha-Hederin was cytotoxic at concentrations above 5 µg/mL in B16 melanoma and 3T3 fibroblasts after only 8 hours of treatment. At 10-15 µg/mL, significant cell death occurred across all time points (8-72 hours). Cytotoxicity was reduced in the presence of FCS or BSA, indicating binding to serum proteins. [2]
Cell death induction: alpha-Hederin treatment (3-5 µg/mL, 24-48 hours) increased necrotic index and decreased mitotic index in both B16 and 3T3 cells. Vacuolization, membrane alterations, and cell retraction were observed. [2]
In vivo toxicity: In xenografted mice, alpha-Hederin treatment at 2.5, 5, and 10 mg/kg (i.p., every other day) showed no significant differences in liver function (ALT, AST) or kidney function (urea, creatinine) compared to control group, indicating favorable safety profile. No metastases were observed in lung, liver, or brain. [1]
No LD50, plasma protein binding, or drug-drug interaction data were reported in these studies. [1][2]

[1]. Wang J, et al. α-Hederin induces the apoptosis of gastric cancer cells accompanied by glutathione decrement and reactive oxygen species generation via activating mitochondrial dependent pathway. Phytother Res. 2020;34(3):601-611.
[2]. Danloy S et al. Effects of alpha-hederin, a saponin extracted from Hedera helix, on cells cultured in vitro. Planta Med, 1994 Feb, 60(1):45-9.
[3]. Maryam Fallahi et al. Effect of Alpha-Hederin, the active constituent of Nigella sativa, on miRNA-126, IL-13 mRNA levels and inflammation of lungs in ovalbumin-sensitized male rats. Planta Med, 1994 Feb, 60(1):45-9.

Kalopanax saponin A is a triterpenoid saponin composed of hederogenin linked by a glycosidic bond to a 2-O-(6-deoxy-α-L-mannopyranosyl)-α-L-arabinopyranosyl residue at position 3. It has been isolated from the stem bark of Kalopanax pictus. It possesses anti-inflammatory activity and is a plant metabolite. It is a pentacyclic triterpenoid compound, a triterpenoid saponin, a disaccharide derivative, and a hydroxy monocarboxylic acid. Its function is related to hederogenin. α-Hederogenin has been reported in honeysuckle (Lonicera macrantha), anemone taipaiensis, and several other organisms with relevant data.

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Background: alpha-Hederin is a monodesmosidic triterpenoid saponin extracted from Hedera helix L. (Araliaceae) and present in other plants such as Nigella species. It has been used in Chinese traditional medicine and possesses various biological activities including antispasmodic, anti-inflammatory, antioxidant, antileishmanial, antifungal (in vitro and in vivo), molluscicidal, anthelmintic, antiamoebic, and trichomonicidal properties. It has no mutagenic effect according to the Ames test and decreases the mutagenicity of benzo[a]pyrene and smoker urine. [1][2]
Chemical properties: alpha-Hederin is a monodesmosidic saponin with one sugar moiety at C-3. It has hemolytic activity. Purity used in studies was ≥90% (Wang et al.) and >98% (Danloy et al.). Stock solutions prepared in DMSO (1 mM) or distilled water (200 µg/mL). [1][2]
Mechanism of action: alpha-Hederin induces apoptosis via mitochondrial pathway: (1) intracellular GSH depletion leading to oxidative stress; (2) ROS accumulation; (3) loss of mitochondrial membrane potential; (4) ATP depletion; (5) release of AIF, Apaf-1, and cytochrome C from mitochondria to cytosol; (6) activation of caspase-9, caspase-3, and caspase-8; (7) increased Bax and decreased Bcl-2 expression. It also induces cell cycle arrest at G1 phase via P16, P21, P53 upregulation and Cyclin D1 downregulation. In addition, alpha-Hederin causes membrane alterations, cytoplasmic vacuolization, and formation of phagolysosomes containing lamellar bodies. [1][2]
Efficacy: alpha-Hederin shows promising antitumor activity against gastric cancer cells (HGC-27, SGC-7901) with IC50 values of 20.86 µM and 25.91 µM respectively, and inhibits xenografted tumor growth in vivo. It also shows cytotoxic effects in B16 melanoma, 3T3 fibroblasts, and HeLa cells. It potentiates the efficacy of 5-fluorouracil in HT-29 colon cancer cells. [1][2]
Potential therapeutic applications: alpha-Hederin has potential as a natural anticancer agent for gastric cancer treatment with favorable safety profile, though further molecular mechanisms need elucidation. [1]

C41H66O12

750.9556

750.455

27013-91-8

73296

White to off-white solid powder

1.3±0.1 g/cm3

849.6±65.0 °C at 760 mmHg

215ºC (dec.)

250.7±27.8 °C

0.0±0.6 mmHg at 25°C

1.601

8.37

7

12

6

53

1440

18

O(C1([H])C([H])(C([H])(C([H])(C([H])([H])O1)O[H])O[H])OC1([H])C([H])(C([H])(C([H])(C([H])(C([H])([H])[H])O1)O[H])O[H])O[H])C1([H])C([H])([H])C([H])([H])C2(C([H])([H])[H])C([H])(C([H])([H])C([H])([H])C3(C([H])([H])[H])C4(C([H])([H])[H])C([H])([H])C([H])([H])C5(C(=O)O[H])C([H])([H])C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C5([H])C4=C([H])C([H])([H])C32[H])C1(C([H])([H])[H])C([H])([H])O[H]

KEOITPILCOILGM-LLJOFIFVSA-N

InChI=1S/C41H66O12/c1-21-28(44)30(46)31(47)33(51-21)53-32-29(45)24(43)19-50-34(32)52-27-11-12-37(4)25(38(27,5)20-42)10-13-40(7)26(37)9-8-22-23-18-36(2,3)14-16-41(23,35(48)49)17-15-39(22,40)6/h8,21,23-34,42-47H,9-20H2,1-7H3,(H,48,49)/t21-,23-,24-,25+,26+,27-,28-,29-,30+,31+,32+,33-,34-,37-,38-,39+,40+,41-/m0/s1

(4aS,6aR,6aS,6bR,8aR,9R,10S,12aR,14bS)-10-[(2S,3R,4S,5S)-4,5-dihydroxy-3-[(2S,3R,4R,5R,6S)-3,4,5-trihydroxy-6-methyloxan-2-yl]oxyoxan-2-yl]oxy-9-(hydroxymethyl)-2,2,6a,6b,9,12a-hexamethyl-1,3,4,5,6,6a,7,8,8a,10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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 (~133.16 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.33 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 25.0 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (3.33 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 25.0 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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (3.33 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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