P2X3 purinocceptor [1]
P2X7 purinocceptor [1]
NFκB (Phospho-NFkB p65) [2]

In HepG2 cells, treatment with Gardenoside at concentrations of 10 and 20 μM for 24 hours showed no substantial decrease in cell viability, while concentrations of 30, 40 and 50 μM were significantly toxic to HepG2 cells (p<0.05, p<0.01) [2].
In HepG2 cells with FFA-induced steatosis, Gardenoside at 10 μM decreased TG content by 36%, and at 20 μM decreased TG content by 46% compared to the FFA group (p<0.01) [2].
In HepG2 cells, Oil Red O staining visually showed that Gardenoside decreased the lipid droplets in cells cultured with FFAs [2].
In HepG2 cells, FFAs increased TNF-α, IL-6, IL-1β by 4.44, 4.98, 5.78-fold compared to control cells. Gardenoside at 10 μM significantly reverted TNF-α by 45.87%, IL-6 by 24.05%, IL-1β by 45.22% compared to FFA group; at 20 μM, Gardenoside reverted TNF-α by 51.19%, IL-6 by 43.28%, IL-1β by 46.47% compared to FFA group [2].
In HepG2 cells, Western blot analysis showed that FFAs induced the activity of Phospho-NFkB p65, and this was reverted by Gardenoside at 10 and 20 μM for 24 hours [2].
In CCI rats, qRT-PCR showed that the mRNA expression of P2X3 and P2X7 receptors were both significantly increased after sciatic nerve CCI (p<0.01 or p<0.05). Gardenoside administration fully inhibited this sciatic nerve CCI induced mRNA up-regulation of both P2X3 and P2X7 receptors. The mRNA level of P2X3 and P2X7 receptors in the Gardenoside administered CCI group was similar to the controls and was significantly different from the CCI group (p<0.05) [1].
In CCI rats, Western blot showed that the protein expression level of P2X3 and P2X7 receptors were both increased after sciatic nerve CCI. Gardenoside administered CCI group showed a significant decrease of P2X3 and P2X7 receptor expression compared to the CCI group [1].
In CCI rats, Western blot showed that the levels of p-ERK and p-p38 proteins were significantly increased in CCI group than in control or sham (p<0.01). The expression of p-ERK and p-p38 protein was decreased after treatment with Gardenoside and was obviously lower than the model levels (p<0.05) [1].

In CCI rats, Gardenoside administration (5 mL of 300 μM Gardenoside solution with 1% sodium carboxymethyl cellulose, gavage once per day for 14 days) significantly improved the sciatica by partially restoring the decrease of MWT and TWL. MWT in the Gardenoside-administered group showed an increase from the seventh day after the induction of sciatic nerve CCI (p<0.05). TWL showed significant improvement in the CCI groups at day five (16.1±2.5 s vs. 13.7±2.4 s, p<0.05) and more significant improvement at day seven (17.7±1.9 s vs. 11.2±1.9 s, p<0.01) after Gardenoside administration [1].
In CCI rats, ELISA analysis showed that the levels of iNOS, IL-1β, and TNF-α were remarkably increased in CCI group compared to control or sham. After treatment with Gardenoside, the expressions of iNOS, IL-1β, and TNF-α were notably decreased in rats of CCI (p<0.05) [1].

Cell viability was determined by MTT assay. HepG2 cells were plated in 96-multiwell culture plates at 1×10^5 cells per well. After 24 hours of plating, the medium was discarded and fresh medium containing 0, 10, 20, 30, 40 and 50 μM Gardenoside were added. After 24 hours of incubation, cell viability was determined by colorimetry using MTT. Insoluble formazan crystals were dissolved in DMSO and measured at 570 nm with a microplate reader [2].
To determine intracellular TG level, HepG2 cells were plated in 6-well plates at 1×10^6 cells per well. 10 and 20 μM Gardenoside and 500 μM FFAs were added at the same time for 24 hours. The TG level of the cells was measured by using TG assay kit. The cell lysate protein concentrations were measured using the BCA method [2].
Oil Red O staining: HepG2 cells were washed three times with PBS, then fixed with 10% formalin for 1 hour. Cells were washed with 60% isopropanol briefly and incubated with 60% filtered Oil Red O solution for 30 minutes at room temperature after washing three times with distilled water. Then, cells were stained with hematoxylin. Cells with Oil Red O Staining were observed under a microscope [2].
Cytokine assay: Samples of supernatant of HepG2 cells were collected after incubation with Gardenoside and FFAs for 24 hours. The anti-inflammatory cytokines TNF-α, IL-6 and IL-1β were measured by ELISA kits [2].
Western blot analysis: Proteins were extracted from the cells using extraction buffer. Protein extracts were separated on 10% polyacrylamide gels and electrophoretically transferred onto polyvinylidene fluoride membrane. After blocking, the membranes were incubated with primary antibodies (Phospho-NFkB p65, NFkB p65, β-actin), and then with horseradish peroxidase-conjugated IgG. Blots were developed using ECL Detection Kit [2].
qRT-PCR: The L4-L5 dorsal root ganglions on the right side were harvested at day 15 and washed with PBS. mRNA was extracted using trizol. cDNA was synthesized using FastQuant RT Kit (with gDNase) according to the manufacturer's instructions. Expression levels of P2X3 and P2X7 receptor mRNA were measured by real-time PCR system using SYBR Green and normalized to expression level of β-actin [1].
Western blot: Total proteins were separated by SDS-PAGE and transferred to PVDF membrane. The PVDF membrane was immersed in 5% milk powder in TBST buffer, and blocked for 1 hour. Membranes were incubated with corresponding antibodies including rabbit anti-P2X3 antibody (1:1000), rabbit anti-P2X7 antibody (1:1000), rabbit anti-ERK antibody (1:2000), rabbit anti-p-ERK antibody (1:2000), rabbit anti-p38 antibody (1:2000), rabbit anti-p-p38 antibody (1:2000), and anti-β-actin (1:1000) overnight at 4°C. The membranes were washed and incubated with anti-rabbit IgG-HRP secondary antibody (1:1000) at room temperature for 1 hour [1].

Sciatica model (Bennett and Xie's unilateral sciatic nerve CCI model): Rats were anesthetized by intraperitoneal injection of chloral hydrate (200 mg/kg). The sciatic nerve on the right side was exposed by creating a skin incision and cutting through the connective tissue between the biceps femoris and gluteus superficial muscles. Four 4-0 chronic gut ligatures were lightly tied around the sciatic nerve with 1 mm intervals, to just occlude without influence to the epineural blood flow. After suture, penicillin was administrated to prevent infections. In the Sham group, the sciatic nerve was just exposed without ligation [1].
Gardenoside administration: In the gardenoside-administrated groups, 5 mL Gardenoside solution (300 μM Gardenoside with 1% sodium carboxymethyl cellulose) was administrated through gavage once per day for 14 days. In the other groups, same amount of 1% sodium carboxymethyl cellulose solution was administrated with the same frequency and duration [1].
MWT evaluation: Mechanical withdrawal threshold was measured using classical von Frey filaments. Rats were placed in transparent chamber with a von Frey filament grid on the bottom. Von Frey filaments with a series of pressures (0.13, 0.20, 0.33, 0.60, 1.30, 3.60, 5.00, 7.30, 9.90, and 20.1 g) were used. Ten observations were made for each rat under each pressure. The stimulus intensity required to produce a response 50% of the measure times were recorded [1].
TWL evaluation: Thermal withdrawal latency was measured by using automatic heat pain stimulation. Rats were placed in transparent chamber and thermal radiation was applied onto the footpads. The withdraw time from the beginning of thermal radiation was considered as TWL. Maximum 30 seconds thermal radiation was applied to avoid thermal injury [1].

[1]. Gardenoside suppresses the pain in rats model of chronic constriction injury by regulating the P2X3 and P2X7 receptors. J Recept Signal Transduct Res. 2018 Jun;38(3):198-203.

[2]. Inhibitory Effect of Gardenoside on Free Fatty Acid-Induced Steatosis in HepG2 Hepatocytes. Int J Mol Sci. 2015 Nov 20;16(11):27749-56.

Gardenoside has been reported to be found in Oldenlandia herbacea var. herbacea, Gardenia jasminoides, and other organisms with available data.

---

Gardenoside may be able to relieve CCI-induced neuropathic pain by regulating the P2X3 and the P2X7 expression on the ischiadic nerve [1].
The analgesic effect of Gardenoside may be depended on activating the P2X3/P2X7 receptors, and Gardenoside may be used as a drug for treating neuropathic pain [1].
Gardenoside has a protective effect on FFA-induced cellular steatosis in HepG2 cells, indicating that Gardenoside might be a potential therapeutic herb against NASH by suppressed supernatant inflammatory cytokine production and intracellular NFκB activity [2].
The "two-hit hypothesis" is the most important pathogenesis of NAFLD. The first hit is the development of hepatic steatosis via accumulation of triglycerides in hepatocytes, and the "second hit" involves hepatic injury, inflammation, which are closely associated with oxidative stress in the liver [2].

C17H24O11

404.3659

404.131

C, 50.50; H, 5.98; O, 43.52

24512-62-7

442423

Off-white to light yellow solid powder

1.6±0.1 g/cm3

672.8±55.0 °C at 760 mmHg

118-120ºC

242.1±25.0 °C

0.0±4.7 mmHg at 25°C

1.643

-4.66

6

11

6

28

649

9

O([C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H])[C@]1([H])[C@]2([H])[C@]([H])(C(C(=O)OC([H])([H])[H])=C([H])O1)C([H])=C([H])[C@@]2(C([H])([H])O[H])O[H]

XJMPAUZQVRGFRE-AYDWLWLASA-N

InChI=1S/C17H24O11/c1-25-14(23)8-5-26-15(10-7(8)2-3-17(10,24)6-19)28-16-13(22)12(21)11(20)9(4-18)27-16/h2-3,5,7,9-13,15-16,18-22,24H,4,6H2,1H3/t7-,9-,10-,11-,12+,13-,15+,16+,17-/m1/s1

methyl (1S,4aS,7S,7aS)-7-hydroxy-7-(hydroxymethyl)-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4a,7a-dihydro-1H-cyclopenta[c]pyran-4-carboxylate

NSC325664; NSC 325664; NSC-325664; Gardenoside

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 (~247.30 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.18 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.

---

Solubility in Formulation 2: ≥ 2.5 mg/mL (6.18 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.

---

View More

Solubility in Formulation 3: ≥ 2.5 mg/mL (6.18 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.

---

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