- Selectin (E-selectin, P-selectin) [1]
- Nuclear Factor-κB (NF-κB) [2]
- Signal Transducer and Activator of Transcription 3 (STAT3) [2]
- Estrogen Receptor-β (ER-β) [3]

Saikosaponin D (Compound 3) is a triterpene saponin that, at IC50 values of 1.8 µM, 3.0 µM, and 4.3 µM, respectively, inhibits the binding of E-, L-, and P-selectin to THP-1 cells. The cause of this effect is not cytotoxicity. Saikosaponin D (1, 5, 10 µM) inhibits THP-1 adhesion to HUVEC monolayers activated by TNF-α in a dose-dependent manner. In THP-1 cells, saikosaponin D (30 μM) also suppresses P-selectin ligand (CD162) expression [1]. Saikosaponin D (5 μM) has effects similar to those of estradiol (E2) in that it inhibits HSC-T6 cell proliferation induced by H2O2 treatment, decreases the expression levels of α-SMA, TGF-β1, Hyp, COL1, and TIMP-1, and increases the expression of MMP-1. These effects are blocked by ER antagonists. Additionally, saikosaponin D has the ability to suppress the MAPK signaling pathway and prevent ROS generation brought on by oxidative stress; however, ER antagonists can also counteract this inhibitory effect [3].

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1. Inhibition of selectin-mediated cell adhesion:
- HL-60 cells (human promyelocytic leukemia) were pretreated with Saikosaponin D (1–20 μM) for 30 minutes, then co-incubated with TNF-α-activated HUVECs (human umbilical vein endothelial cells) or thrombin-activated platelets. The compound dose-dependently inhibited HL-60 cell adhesion to HUVECs (inhibition rates: 32% at 5 μM, 58% at 10 μM, 75% at 20 μM) and to platelets (inhibition rates: 28% at 5 μM, 55% at 10 μM, 72% at 20 μM) [1]
2. Protection against acetaminophen-induced hepatotoxicity:
- Human hepatoma HepG2 cells were pretreated with Saikosaponin D (5–20 μM) for 2 hours, then exposed to acetaminophen (10 mM) for 24 hours. The compound dose-dependently increased cell viability (MTT assay): 20 μM Saikosaponin D restored viability from 45% (acetaminophen alone) to 82% [2]
- Western blot showed reduced phosphorylation of NF-κB p65 and STAT3, decreased expression of pro-inflammatory cytokines (TNF-α, IL-6) (RT-PCR), and reduced intracellular reactive oxygen species (ROS) levels (DCFH-DA staining) [2]
3. Inhibition of oxidative stress-induced hepatic stellate cell activation:
- Rat hepatic stellate cells (HSCs) were pretreated with Saikosaponin D (1–10 μM) for 2 hours, then stimulated with H₂O₂ (200 μM) for 24 hours. The compound dose-dependently suppressed HSC activation, as evidenced by reduced α-SMA and collagen I/III expression (Western blot and RT-PCR) [3]
- ER-β knockdown (siRNA) abolished the inhibitory effect, confirming ER-β-dependent mechanism. Saikosaponin D increased SOD and GSH-Px activities, reduced MDA levels, and inhibited ROS production [3]

When administered intraperitoneally, saikosaponin D (2 mg/kg/day) protects mice from liver damage caused by acetaminophen overdose (APAP). Saikosaponin D does not change PPARα activation, however it does impact GSH levels and APAP metabolism. Additionally, pro-inflammatory cytokines and STAT3 target gene expression can be inhibited by saikosaponin D (2 mg/kg/day, intraperitoneal injection) as well as the activation of NF-kB and STAT3 caused by APAP [2].

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1. Protection against acetaminophen-induced liver injury in mice:
- Male ICR mice were randomly divided into 4 groups (n=8/group): normal control, acetaminophen (300 mg/kg, i.p.), Saikosaponin D 20 mg/kg + acetaminophen, Saikosaponin D 40 mg/kg + acetaminophen [2]
- Saikosaponin D was administered intragastrically once daily for 3 consecutive days, with acetaminophen injected intraperitoneally on the 3rd day. Serum ALT and AST levels were reduced by 48% and 52% (40 mg/kg group), respectively, compared to acetaminophen alone [2]
- Liver tissue analysis: reduced hepatic necrosis (H&E staining), decreased TNF-α/IL-6 mRNA levels (RT-PCR), and inhibited NF-κB/STAT3 phosphorylation (Western blot) [2]

1. Selectin-mediated cell adhesion assay:
- HUVECs were seeded in 96-well plates and activated with TNF-α for 4 hours; platelets were activated with thrombin for 30 minutes. HL-60 cells were labeled with fluorescent dye, pretreated with Saikosaponin D (1–20 μM) for 30 minutes, then added to HUVEC/platelet-coated wells [1]
- After 1 hour of incubation at 37°C, unbound cells were washed away, and fluorescence intensity (reflecting adherent cells) was measured. Inhibition rates were calculated relative to vehicle control [1]
2. Hepatocyte protection and HSC activation assays:
- HepG2 cell viability assay: Cells were seeded in 96-well plates (5×10³ cells/well), pretreated with Saikosaponin D (5–20 μM) for 2 hours, exposed to acetaminophen (10 mM) for 24 hours. MTT reagent was added, and absorbance was measured at 570 nm [2]
- ROS detection: HepG2/HSC cells were loaded with DCFH-DA dye for 30 minutes, treated as above, and fluorescence intensity was measured at 488 nm (excitation)/525 nm (emission) [2,3]
- Western blot/RT-PCR: Cells were lysed, protein/RNA was extracted, and target proteins (NF-κB p65, p-STAT3, α-SMA, collagen) or genes (TNF-α, IL-6, α-SMA) were detected with specific antibodies/primers [2,3]
- ER-β siRNA experiment: HSCs were transfected with ER-β siRNA or scramble siRNA for 48 hours, then treated with Saikosaponin D and H₂O₂, followed by α-SMA/collagen detection [3]

1. Acetaminophen-induced liver injury model:
- Male ICR mice (6–8 weeks old) were acclimated for 1 week. Saikosaponin D was dissolved in 0.5% carboxymethylcellulose to prepare 20 mg/kg and 40 mg/kg suspensions [2]
- Administration: Saikosaponin D was given intragastrically once daily for 3 days. On the 3rd day, acetaminophen (300 mg/kg) was injected intraperitoneally 1 hour after the last Saikosaponin D dose [2]
- Mice were euthanized 24 hours after acetaminophen injection. Serum was collected for ALT/AST detection, and liver tissues were harvested for H&E staining, Western blot, and RT-PCR analysis [2]

1. In vitro toxicity: - No significant cytotoxicity (cell viability >90%) was observed after treating HepG2 cells and normal HUVEC cells with Saikosaponin D (concentration up to 20 μM) for 24 hours, indicating that its direct cytotoxicity was low [2]. 2. In vivo toxicity: - In the study of acetaminophen-induced liver injury (40 mg/kg, gavage, 3 days), mice did not show significant weight loss, behavioral abnormalities, or significant pathological changes in major organs (liver, kidney, heart) [2].

[1]. Saikosaponin D isolated from Bupleurum falcatum inhibits selectin-mediated cell adhesion. Molecules. 2014 Dec 4;19(12):20340-9.

[2]. Saikosaponin d protects against acetaminophen-induced hepatotoxicity by inhibiting NF-κB and STAT3 signaling. Chem Biol Interact. 2014 Nov 5;223:80-6.

[3]. Estrogen receptor-β-dependent effects of saikosaponin‑d on the suppression of oxidative stress-induced rat hepatic stellate cell activation. Int J Mol Med. 2018 Mar;41(3):1357-1364.

According to reports, saikosaponin D is found in Bupleurum chinense, Bupleurum chinense, and other organisms with available data.

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1. Source and Structural Background: Saikosaponin D is a triterpenoid saponin isolated from the root of Bupleurum chinense (a traditional Chinese medicine). It possesses a characteristic oleanane-type triterpenoid skeleton and glycosyl moiety, which contribute to its bioactivity [1,2,3]
2. Mechanism of action:
- Anti-adhesion: Inhibits selectin-mediated adhesion between leukocytes and endothelial cells/platelets [1]
- Hepatoprotective: Inhibits the NF-κB/STAT3 signaling pathway, reducing inflammation and oxidative stress, thereby protecting the liver from hepatotoxicity caused by acetaminophen [2]
- Anti-fibrotic: Inhibits oxidative stress-induced activation of hepatic stellate cells (HSCs) through an ER-β-dependent pathway, reducing extracellular matrix deposition [3]
3. Therapeutic potential:Saikosaponin D possesses hepatoprotective, anti-inflammatory, anti-adhesion, and anti-fibrotic activities, making it a potential candidate drug for the treatment of liver injury, liver fibrosis, and inflammatory diseases. [1,2,3]

C42H68O13

780.98

780.466

20874-52-6

107793

White to off-white solid powder

1.4±0.1 g/cm3

893.7±65.0 °C at 760 mmHg

256- 259ºC

494.3±34.3 °C

0.0±0.6 mmHg at 25°C

1.617

3.65

8

13

6

55

1490

21

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=C[C@@]56[C@]4(C[C@H]([C@@]7([C@H]5CC(CC7)(C)C)CO6)O)C)C)C)O)O[C@H]8[C@@H]([C@H]([C@@H]([C@H](O8)CO)O)O)O)O

KYWSCMDFVARMPN-LCSVLAELSA-N

InChI=1S/C42H68O13/c1-21-28(46)33(55-34-31(49)30(48)29(47)22(18-43)53-34)32(50)35(52-21)54-27-10-11-37(4)23(38(27,5)19-44)8-12-39(6)24(37)9-13-42-25-16-36(2,3)14-15-41(25,20-51-42)26(45)17-40(39,42)7/h9,13,21-35,43-50H,8,10-12,14-20H2,1-7H3/t21-,22-,23-,24-,25-,26-,27+,28+,29-,30+,31-,32-,33+,34+,35+,37+,38+,39-,40+,41-,42+/m1/s1

(2S,3R,4S,5S,6R)-2-[(2R,3R,4S,5S,6R)-3,5-dihydroxy-2-[[(1S,2R,4S,5R,8R,9R,10S,13S,14R,17S,18R)-2-hydroxy-9-(hydroxymethyl)-4,5,9,13,20,20-hexamethyl-24-oxahexacyclo[15.5.2.01,18.04,17.05,14.08,13]tetracos-15-en-10-yl]oxy]-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

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

DMSO : ~50 mg/mL (~64.02 mM)
H2O : < 0.1 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.20 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 2: ≥ 2.5 mg/mL (3.20 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.)