The target of Saikosaponin B1 is the Hedgehog signaling pathway, with key regulatory proteins including Smoothened (Smo) and Glioma-associated oncogene 1 (Gli1) [1]

1. Antiproliferative activity: Saikosaponin B1 inhibited the proliferation of medulloblastoma cell lines (Daoy and UW228-3). At concentrations of 5 μM and 10 μM, it significantly reduced the viability of Daoy cells; the inhibitory effect was concentration-dependent [1]
2. Hedgehog signaling pathway regulation: Western blot analysis showed that Saikosaponin B1 (5 μM, 10 μM) downregulated the protein expression levels of Smo and Gli1 in Daoy cells, while upregulating the expression of Patched 1 (Ptch1), a negative regulator of the Hedgehog pathway [1]
3. Apoptosis induction: Flow cytometry (Annexin V-FITC/PI double staining) revealed that Saikosaponin B1 (10 μM) increased the apoptotic rate of Daoy cells compared to the control group [1]
4. Clonogenic assay: Saikosaponin B1 (2.5 μM, 5 μM, 10 μM) dose-dependently decreased the number and size of colonies formed by Daoy cells [1]
5. Real-time PCR (qPCR): Treatment with Saikosaponin B1 (5 μM, 10 μM) reduced the mRNA expression levels of Gli1 and Smo in Daoy cells, consistent with the Western blot results [1]

1. Tumor growth inhibition: In medulloblastoma allograft mice (C57BL/6 mice implanted with Daoy cells), Saikosaponin B1 was administered intraperitoneally at doses of 10 mg/kg and 20 mg/kg once daily for 21 days. The 20 mg/kg dose significantly reduced tumor volume (by approximately 45% compared to the vehicle control) and tumor weight (by approximately 50% compared to the vehicle control) [1]
2. Survival extension: Mice treated with Saikosaponin B1 (20 mg/kg) had a significantly longer median survival time (38 days) than the vehicle control group (25 days) [1]
3. In vivo pathway regulation: Immunohistochemical staining of tumor tissues from treated mice showed that Saikosaponin B1 (20 mg/kg) decreased the expression of Gli1 and the proliferation marker Ki-67, while increasing the number of TUNEL-positive apoptotic cells [1]
4. Safety monitoring: Saikosaponin B1 (10 mg/kg, 20 mg/kg) did not cause significant changes in mouse body weight or obvious pathological damage to major organs (liver, kidney) during the treatment period [1]

1. Cell viability assay: Medulloblastoma cells (Daoy, UW228-3) were seeded in 96-well plates at a density of 5×10³ cells per well and cultured overnight. Saikosaponin B1 was added at concentrations of 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM, and the cells were incubated for 48 hours. A cell counting kit (CCK-8) was used to measure absorbance at 450 nm, and cell viability was calculated relative to the vehicle control [1]
2. Western blot assay: Daoy cells were treated with Saikosaponin B1 (5 μM, 10 μM) for 24 hours. Total protein was extracted from the cells, and protein concentration was determined using a BCA kit. Equal amounts of protein (30 μg per lane) were separated by SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk. The membranes were incubated with primary antibodies against Smo, Gli1, Ptch1, and β-actin (internal control) overnight at 4°C, followed by incubation with secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) kit, and band intensity was quantified using ImageJ software [1]
3. Apoptosis assay: Daoy cells were treated with Saikosaponin B1 (10 μM) for 24 hours. Cells were harvested, washed with PBS, and stained with Annexin V-FITC and PI according to the kit instructions. Apoptotic cells were detected using a flow cytometer, and the apoptotic rate was analyzed using flow cytometry software [1]
4. Clonogenic assay: Daoy cells were seeded in 6-well plates at a density of 200 cells per well and cultured overnight. Saikosaponin B1 was added at concentrations of 2.5 μM, 5 μM, and 10 μM, and the cells were cultured for 14 days. Colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Colonies containing more than 50 cells were counted, and the cloning efficiency was calculated [1]
5. qPCR assay: Daoy cells were treated with Saikosaponin B1 (5 μM, 10 μM) for 24 hours. Total RNA was extracted using TRIzol reagent, and RNA purity and concentration were determined using a spectrophotometer. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using a reverse transcription kit. qPCR was performed using SYBR Green Master Mix with specific primers for Gli1, Smo, and GAPDH (internal control). The reaction conditions were: 95°C for 30 seconds, followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. The relative mRNA expression levels were calculated using the 2^(-ΔΔCt) method [1]

1. Tumor allograft model establishment: C57BL/6 female mice (6-8 weeks old) were used. Daoy cells (5×10⁶ cells per mouse) were resuspended in 100 μL of PBS mixed with Matrigel (1:1 ratio) and subcutaneously injected into the right flank of the mice. Tumor growth was monitored until the tumor volume reached approximately 100 mm³, at which point the mice were randomly divided into three groups (n=6 per group): vehicle control group, Saikosaponin B1 10 mg/kg group, and Saikosaponin B1 20 mg/kg group [1]
2. Drug preparation and administration: Saikosaponin B1 was dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution, which was then diluted with normal saline to the final concentration (DMSO final concentration ≤ 1%). The drug was administered intraperitoneally once daily for 21 consecutive days; the vehicle control group received the same volume of DMSO-saline solution [1]
3. Data collection and sample processing: Tumor volume was measured every 3 days using a vernier caliper, and tumor volume was calculated using the formula: Volume = (length × width²)/2. Mouse body weight was recorded weekly to monitor general health. After the treatment period, mice were euthanized, and tumor tissues were excised and weighed. A portion of the tumor tissue was fixed in 4% paraformaldehyde for immunohistochemical staining, and the remaining tissue was stored at -80°C for further analysis [1]

[1]. Saikosaponin B1 and Saikosaponin D inhibit tumor growth in medulloblastoma allograft mice via inhibiting the Hedgehog signaling pathway [published online ahead of print, 2022 Feb 16]. J Nat Med. 2022;10.1007/s11418-022-01603-8.

Saikosaponin B1 has been reported in Bupleurum chinense, Bupleurum sibiricum, and other organisms with relevant data. 1. Saikosaponin B1 is a triterpenoid saponin isolated from plants of the genus Bupleurum (e.g., Bupleurum chinense)[1] 2. Medulloblastoma is the most common malignant brain tumor in children, and abnormal activation of the Hedgehog signaling pathway is closely related to the occurrence and development of medulloblastoma. This study confirms that Saikosaponin B1 exerts an anti-medulloblastoma effect by inhibiting the Hedgehog signaling pathway, providing a potential candidate drug for the treatment of medulloblastoma[1]

C42H68O13

780.9815

780.466

58558-08-0

9875547

White to off-white solid powder

1.4±0.1 g/cm3

909.9±65.0 °C at 760 mmHg

235-238ºC

504.1±34.3 °C

0.0±0.6 mmHg at 25°C

1.619

3.59

9

13

7

55

1500

19

C[C@@H]1[C@@H]([C@@H]([C@H]([C@@H](O1)O[C@H]2CC[C@]3([C@H]([C@]2(C)CO)CC[C@@]4([C@@H]3C=CC5=C6CC(CC[C@@]6([C@H](C[C@]54C)O)CO)(C)C)C)C)O)O[C@H]7[C@@H]([C@H]([C@@H]([C@H](O7)CO)O)O)O)O

WRYJYFCCMSVEPQ-MNIDVGFKSA-N

InChI=1S/C42H68O13/c1-21-29(47)34(55-35-32(50)31(49)30(48)24(18-43)53-35)33(51)36(52-21)54-28-11-12-38(4)25(39(28,5)19-44)10-13-40(6)26(38)9-8-22-23-16-37(2,3)14-15-42(23,20-45)27(46)17-41(22,40)7/h8-9,21,24-36,43-51H,10-20H2,1-7H3/t21-,24-,25-,26-,27+,28+,29+,30-,31+,32-,33-,34+,35+,36+,38+,39+,40-,41-,42-/m1/s1

(2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-2-[[(3S,4R,4aR,6aR,6bS,8S,8aS,14aR,14bS)-8-hydroxy-4,8a-bis(hydroxymethyl)-4,6a,6b,11,11,14b-hexamethyl-1,2,3,4a,5,6,7,8,9,10,12,14a-dodecahydropicen-3-yl]oxy]-3,5-dihydroxy-6-methyloxan-4-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol

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 (~128.04 mM)

Solubility in Formulation 1: ≥ 100 mg/mL (128.04 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 1000.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 2: 10 mg/mL (12.80 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 100.0 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 3: ≥ 10 mg/mL (12.80 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 100.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)