NF-κB

Barlerin (8-O-Acetyl shanzhiside methyl ester) treatment inhibits TNF-induced nuclear transcription factor B (NF-κB) activation and reduces high-mobility group box-1 (HMGB-1) expression in SH-SY5Y cells. [1]. By preventing High-mobility group box 1 (HMGB1) expression, treatment of H9c2 cells with barlerin (8-O-Acetyl shanzhiside methyl ester) 9 μM prevents TNF-α-induced NF-κB phosphorylation[2].

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Effects of 8-O-acetyl shanzhiside methylester (ND01) on NF-κB activation and HMGB-1 expression [1]
The NF-κB pathway plays a critical role in the secretion of cytokines. The quantity of p50 and p65 was measured in the nucleus. Stimulation with TNF-α led to a robust activation of the NF-κB transcription factor p50/p65. However, this activation was partially blocked by ND01, as shown in fig. 1. [1]
The IκB kinase system as another activation agent of NF-κB was also examined. Stimulation with TNF-α resulted in a marked degradation of IκB. This degradation was inhibited by ND01 (fig. 2). In addition, the phosphorylation of p-IκB was increased by TNF-α and also inhibited by ND01. [1]
Phosphor-NF-κB expression was low in non-TNF-α-stimulated SH-SY5Y cells. However, phosphor-NF-κB expression was significantly increased after TNF-α 20 ng/ml-stimulated SH-SY5Y cells for 120 min. We compared the effect of ND01 on the TNF-α-induced activation of phosphor-NF-κB and HMGB-1 expression level in the SH-SY5Y cells to that of a selective HMGB-1 inhibitor, glycyrrhizin. The results of fig. 2 showed that pre-treatment of SH-SY5Y cells with glycyrrhizin (100 μM) for 120 min., blocked the TNF-α-induced HMGB-1 expression and reduced phosphorylation of NF-κB. Pre-treatment of SH-SY5Y cells with ND01 10 μM blocked TNF-α-induced NF-κB phosphorylation and reduced HMGB-1 expression, as shown in fig. 2. [1]

Barlerin (8-O-Acetyl shanzhiside methyl ester) 40 mg/kg exhibits a notable neuroprotective effect even when administered 4 hours after I/R. In ischaemic brain tissue, barlerin 40 mg/kg inhibits NF-κB activation, reduces HMGB-1 expression, attenuates histopathological damage, and reduces brain swelling[1]. The angiogenesis in the ischaemic brain is markedly accelerated by barlerin (8-O-Acetyl shanzhiside methyl ester), which also enhances functional recovery after stroke. Additionally, compared to vehicle treatment, barlerin significantly boosts vascularization. It raises the levels of Ang1, Tie2, and Akt VEGF as well as their expression[3]. Activated partial thromboplastin, prothrombin, and thrombin times in mice are unaffected by barlerin (8-O-Acetyl shanzhiside methyl ester), but capillary blood clotting time and blood loss volume are significantly shortened. In hyperfibrinolysis mice, it significantly extends the time it takes for euglobulin clots to dissolve[4].

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Inflammatory activation plays a vital role in the pathophysiological mechanisms of stroke and diabetes mellitus (DM), exerts the deleterious effects on the progression of the brain and leads to vascular damage in diabetic stroke. The objectives of this study were to investigate the effects of 8-O-acetyl shanzhiside methylester (ND01) on tumour necrosis factor-α (TNF-α)-stimulated SH-SY5Y cell line in vitro and the experimental ischaemic diabetic stroke model in vivo. TNF-α-stimulated SH-SY5Y cells were pre-incubated with ND01, then analysed protein expression. For in vivo experiment, the diabetic rats were subjected to middle cerebral artery occlusion (MCAO) for 30 min. followed by reperfusion for 23 hr. Treatment of SH-SY5Y cells with ND01 blocked TNF-α-induced nuclear transcription factor κB (NF-κB) activation and decreased high-mobility group box-1 (HMGB-1) expression. ND01 40 mg/kg demonstrated significant neuroprotective effect even after delayed administration at 4 hr after I/R. ND01 40 mg/kg attenuated the histopathological damage, decreased brain swelling, inhibited NF-κB activation and reduced HMGB-1 expression in ischaemic brain tissue. These data show that ND01 protects diabetic brain against I/R injury with a favourable therapeutic time-window by alleviating diabetic cerebral I/R injury and attenuating blood-brain barrier (BBB) breakdown, and its protective effects may involve HMGB-1 and NF-κB signalling pathway.[1]

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8-O-acetyl shanzhiside methylester (ND01) was isolated from the leaves of Lamiophlomis rotata (Benth.) Kudo. In this study, we investigated the anti-myocardial ischemia and reperfusion (I/R) injury effects of ND01 in vivo and elucidated the potential mechanism in vitro. The results indicated that ND01 significantly attenuated hypoxia-induced cytotoxicity in a concentration-dependent manner in H9c2 cells. Treatment of H9c2 cells with ND01 9 μM blocked TNF-α-induced nuclear factor kappaB (NF-κB) phosphorylation by blocking High-mobility group box1 (HMGB1) expression. Treatment of rats with ND01 10mg/kg, (i.v.) protected the animals from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics and reduction of myocardial damage severity. Treatment with ND01 also lowered serum levels of pro-inflammatory factors and reduced High mobility group box-1 protein (HMGB1) and phosphorylated NF-κB expression in ischemic myocardial tissue. Additionally, continuous i.v. of ND01 14 days attenuated cardiac remodeling. These protective effects suggested that ND01 might be due to block of myocardial inflammatory cascades through an HMGB1-dependent NF-κB signaling pathway.[2]

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Reseasrchers investigated whether 8-O-acetyl shanzhiside methylester (ND01) regulates angiogenesis and thereby improves functional outcome after stroke. Adult male rats were subjected to 1 hr of middle cerebral artery occlusion (MCAO) and reperfusion, and treated with or without different doses (5 and 10 mg/kg) of ND01, starting 24 hr after ischaemia and reperfusion (I/R) and by intravenous injection daily for 14 days. Neurological functional tests were performed and cerebral Evans blue extravasation was measured. Angiogenesis and angiogenic factor expression were measured by immunohistochemistry and Western blot, respectively. The results indicated that ND01 significantly promoted angiogenesis in the ischaemic brain and improved functional outcome after stroke. ND01 also significantly increased vascularization compared with vehicle treatment. ND01 increased the expression of VEGF, Ang1, phosphorylation of Tie2 and Akt VEGF. The Ang1/Tie2 axis and Akt pathways appear to mediate ND01-induced angiogenesis. [3]

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The anti-fibrinolytic activity come from 8-O-Acetyl shanzhiside methylester (ASM) one of the highest iridoid glycosides contents in TIG extracted from L. rotata. ASM significantly (P<0.05) shorten CBCT and reduced blood loss volume in vivo, but did not influence mice APTT, PT or TT. In particular, it significantly prolonged ECLT in hyperfibrinolysis mice. It indicated that ASM could inhibit fibrinolysis. ASM was also effective in CBCT, traumatic bleeding volume and ECLT in hyperfibrinolysis mice model. Conclusions: ASM was the major hemostatic compound in L. rotata. The haemostasis mechanism of ASM was achieved by anti-fibrinolytic activity. ASM was a new fibrinolysis inhibitor as iridoid glycoside compound.[4]

NF-κB-binding assay [1]
SH-SY5Y cells (5 × 106) were pre-incubated with 8-O-acetyl shanzhiside methylester (ND01) 10 μM for 22 hr, then incubated with TNF-α (20 ng/ml) for 1 or 2 hr, then washed once with PBS, scraped cells into 1 ml cold PBS and pelleted by centrifugation. Nuclear extracts were prepared as described previously 12. The DNA-binding activity of NF-κB (p50/p65) was determined using an ELISA kit

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8-O-acetyl shanzhiside methylester (ND01) inhibited NF-κB activation and HMGB1 expression [2]
We compared the effect of ND01 on the TNF-α-induced (20 ng/ml for 30 min) activation of NF-κB and HMGB1 in H9c2 cells to a selective HMGB1 inhibitor glycyrrhizin (100 μM) and a NF-κB

Cells are pretreated with Barlerin (8-O-Acetyl shanzhiside methyl ester) in a range of concentrations (1, 3, 9 and 27 27μM) for 24 hours prior to hypoxia. MTT assays are used to assess cell viability[2].

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NF-κB activation and HMGB-1 expression in TNF-α-stimulated SH-SY5Y cells [1]
Human neuroblastoma (SH-SY5Y) cells were were cultured and maintained in F12+ DMEM (1:1, v/v) media, supplemented with 10% phosphate buffered saline (PBS) and 1% penicillin/streptomycin. Cells were kept at 37°C in a humidified 5% CO2/95% O2 incubator. The dissociated cells were seeded in poly-l-lysine-coated plates at a density of 5 × 105/cm2 and cultured in DMEM, supplemented with 10% (v/v) horse serum, 5% (v/v) foetal bovine serum, 100 U/ml penicillin and 0.1 mg/ml streptomycin. The fresh medium was changed twice weekly. [1]
For the experiment of TNF-α-stimulated SH-SY5Y cell lines in vitro, SH-SY5Y cells (5 × 106) were pre-incubated with 8-O-acetyl shanzhiside methylester (ND01) (9 μM) or HMGB-1 inhibitor, glycyrrhizin (100 μM) for 120 min., and then incubated with TNF-α (20 ng/ml) for 30 min. and cultured in a CO2 incubator for 12 hr. Cells were washed twice with ice-cold PBS on ice and lysed in NP40 lysis buffer 50 mM Tris, pH 7.4, 250 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM Na3VO4, 1% NP-40 and 0.02% NaN3) supplemented with 1 mM PMSF and 1 × protease inhibitor cocktail. Equal amounts of cell protein (50 μg) were separated by SDS–PAGE and analysed by Western blot using specific antibodies to HMGB-1, IκB, phosphor-IκB-α, phosphor-NF-κB and proliferating cell nuclear antigen (PCNA, loading control). Optical densities of the bands were scanned and quantified with a Gel Doc 2000. Data were normalized against those of the corresponding PCNA bands. Results were expressed as fold increase over control.

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8-O-acetyl shanzhiside methylester (ND01) attenuated hypoxia-induced cytotoxicity [2]
The results of cell viability were shown in Fig.1. Exposed to hypoxia for 6 h, there were only 52.0 ± 8.9% viable cells as compared to the control cells. 8-O-acetyl shanzhiside methylester (ND01) (3, 9 and 27 μM) prevented cells from hypoxia-induced damage in a concentration-dependent manner, restored cell survival to 63.7 ± 6.0%, 69.9 ± 8.6% and 74.4 ± 9.6%.

Rats: In saline, Barlerin (8-O-Acetyl shanzhiside methyl ester) is made. Adult male rats are given an hour of middle cerebral artery occlusion (MCAO) and reperfusion. They are then given different doses of 8-O-acetyl shanzhiside methylester (ND01) (5 and 10 mg/kg) intravenously every day for 14 days, starting 24 hours after the I/R (ischaemia and reperfusion). In addition to measuring cerebral Evans blue extravasation, neurological functional tests are carried out[3].
\nMouse: In saline, Barlerin (8-O-Acetyl shanzhiside methyl ester) is made. The five groups (saline group, Hemocoagulase, 0.34 KU/kg, intravenous, ASM-L, 100 mg/kg, ASM-M, 250 mg/kg, and ASM-H, 500 mg/kg) of male Balb/C mice (20 to 25g) are assigned at random. Five minutes before sodium pentobarbital (200 mg/kg, i.p.) is used to anesthetize the patient, the drugs and the vehicle are injected through the vena cauda. After the injection, blood is drawn from the heart 20 minutes later. Prothrombin time, thrombin time, fibrinogen, and activated partial thromboplastin time are measured[4].\n

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\n\nStreptozotocin (STZ)-induced diabetes model and rat cerebral ischaemia study protocol [1]
\nTwo hundred rats (fasting for 20 hr) were induced by single i.p. STZ at a dose of 50 mg/kg. STZ was diluted in citrate buffer 0.1 M (pH 4.0). After STZ injection for 3 weeks, rats with glycaemia value between 12.0 and 20.0 mM were used. The diabetic rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.). Rectal temperature was recorded and maintained at 37°C throughout the surgical procedure. The operation of MCAO was carried out according to previous procedures with minor modifications 13. The left common carotid artery was occluded, and the branches of the external carotid artery were dissected and divided. The internal carotid artery was followed rostrally, and a 4-0 filament (the diameter of the filament was 0.25, but the diameter of the tip was 0.34 mm to create a globular stopper) was introduced into the internal carotid artery and advanced until resistance was felt. The filament was removed after 30 min. The rats were kept under conditions of controlled temperature (24–25°C) for the first 23 hr after surgery. [1]
\n\nA pilot study was conducted with four different doses of 8-O-acetyl shanzhiside methylester (ND01) (10, 20, 40 or 80 mg/kg) to determine the dose-dependent effect in the acute I/R-treated diabetic rats. It was observed that 8-O-acetyl shanzhiside methylester (ND01) at doses of 20, 40 and 80 mg/kg significantly (p < 0.05) lowered infarct volume and neurological deficit scores of the acute I/R-induced diabetic rats after 23 hr of the experiment. Hence, 8-O-acetyl shanzhiside methylester (ND01) 40 mg/kg was chosen for this study. [1]
\n\nFor therapeutic time-window studies, 40 MCAO diabetic rats were randomly divided into four groups of 10 rats each plus 10 diabetic rats as control. Rats received dose of 40 mg/kg by intravenous bolus injection into the tail vein 2, 4 and 6 hr after reperfusion. Vehicle-treated rats were administered with saline. Neurological deficits were determined 23 hr after ischaemia followed by brain infarct examination. [1]
\n\nFor anti-inflammatory mechanism studies, 54 rats were randomly divided into three subgroups of 18 rats each plus 18 rats as control (non-diabetic). Rats received a dose of 40 mg/kg intravenous bolus injection into the tail vein 30 min. after reperfusion. Diabetic or vehicle-treated rats were administered with saline. All the brain were evaluated by Evans blue extravasation, then analysed HMGB1, IκB, phosphor-IκB-α, phosphor-NF-κB by Western blot and the histopathological damage were evaluated by NeuN staining, especially. [1]
\n\nFor long-term studies, 20 rats were randomly divided into two groups of 10 rats each. Rats received a dose of 40 mg/kg by intravenous bolus injection into the tail vein 30 min. after reperfusion. The vehicle-treated rats were administered with saline. Neurological deficits were determined on days 3, 7 and 14 after I/R. Fourteen days after I/R, eight rats were stained in the 8-O-acetyl shanzhiside methylester (ND01) 40 mg/kg group, and six rats were stained in the vehicle-treated group. The brain infarct was examined according to a previous method. [1]

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\n\nI/R procedure to induce cerebral ischaemia. [3]
\n The body weight of rats was 280–320 g. After 1 week of acclimatization, rats were anaesthetized with chloral hydrate (350 mg/kg, i.p.). The middle cerebral artery occlusion (MCAO) operation was performed according to procedures described previously. The left common carotid artery was occluded, and the branches of the external carotid artery were dissected and divided. The internal carotid artery was followed rostrally and a 4–0 filament (the diameter of the filament is 0.25, but the diameter of the tip is 0.34 mm to create a globular stopper) was introduced into the internal carotid artery and advanced until resistance was felt. The filament was removed after 1 hr. Core body temperature was maintained at 37 ± 0.5°C on the heating pad.
\n\nA total of 160 rats were divided into two groups; each group included four subgroups (each subgroup consisted of 20 rats): non-I/R (sham) group, vehicle-treated group, 8-O-acetyl shanzhiside methylester (ND01) 5 mg/kg group and 8-O-acetyl shanzhiside methylester (ND01) 10 mg/kg group. After 23 hr of reperfusion, all animals were administered an intravenous bolus injection (i.v., via the tail) of the corresponding drug daily for 14 days. The sham and vehicle-treated rats were both given saline. At 7 and 14 days, the first group of animals was used to evaluate nerve behaviour, then to measure permeability of the blood-brain barrier; each subgroup consisted of eight rats. At 7 and 14 days, the second group of animals was used to analyse Western blots, microvasculature density and immunohistochemical staining. Four rats of each subgroup were used to analyse Western blot in ischaemic brain core; the next four rats of each subgroup were used to detect microvasculature density and immunohistochemical staining.[3]

[1]. 8-O-acetyl shanzhiside methylester attenuates cerebral ischaemia/reperfusion injury through an anti-inflammatory mechanism in diabetic rats. Basic Clin Pharmacol Toxicol. 2014 Dec;115(6):481-7.

[2]. Cardioprotection with 8-O-acetyl shanzhiside methylester on experimental myocardial ischemia injury. Eur J Pharm Sci. 2012 Aug 30;47(1):124-30.

[3]. Effect of 8-O-acetyl shanzhiside methylester increases angiogenesis and improves functional recovery after stroke. Basic Clin Pharmacol Toxicol. 2011 Jan;108(1):21-7.

[4]. A new anti-fibrinolytic hemostatic compound 8-O-acetyl shanzhiside methylester extracted from Lamiophlomis rotata. J Ethnopharmacol. 2016 Jul 1;187:232-8.

8-O-acetylganoside methyl ester has been reported to exist in Barleria lupulina, Lamium garganicum, and other organisms with relevant data. In this study, we observed that 8-O-acetylganoside methyl ester (ND01) significantly improved cerebral ischemia/reperfusion injury in diabetic rats without lowering blood glucose, indicating that ND01 has an immediate neuroprotective effect, but not through lowering blood glucose. Infarct size is an important pathophysiological indicator for assessing the efficacy of cerebral ischemia treatment.¹⁹ Our results (Figure 3) show that even with delayed administration at 2 and 4 hours after ischemia/reperfusion, 8-O-acetylganoside methyl ester (ND01) treatment still significantly reduced the infarct volume following cerebral ischemia/reperfusion injury in diabetic rats. Neuronal degeneration and necrosis have been found to be associated with behavioral deficits; behavioral assessment may reveal the efficacy of treatment.²⁰ This study showed that ND01 treatment reduced neurological function scores (Figure 3). NeuN is a sensitive marker of early damaged neurons in ischemic injury²¹, and our data (Figure 4) showed that a dose of 40 mg/kg of ND01 reduced the reduction of NeuN immunopositive neurons in the ischemic cerebral cortex at 23 hours and 14 days after ischemia/reperfusion. This suggests that ND01 has potential benefits in the treatment of cerebral ischemia^[1]. The updated Preclinical Recommendations²² of the Academic Industry Roundtable on Stroke Treatment outlines potential reasons for translating effective preclinical research results into successful clinical trial results. Therefore, we investigated the therapeutic time window and long-term efficacy of 8-O-acetylganoside methyl ester (ND01) in a diabetic rat model of cerebral ischemia/reperfusion, and our data showed that 8-O-acetylganoside methyl ester (ND01) has significant and long-term neuroprotective effects with a good therapeutic time window. [1] Stroke triggers an inflammatory response that lasts for hours after the stroke and plays a central role in the pathogenesis of neuronal damage in ischemic stroke, especially diabetic stroke. The inflammatory response leads to the late stages of ischemic injury and neurological deterioration through a variety of mechanisms. Diabetes is also an inflammatory disease. This study shows that 8-O-acetylganoside methyl ester (ND01) has significant anti-inflammatory effects (Table 2 and Figure 5), especially ND01 treatment has a long-term benefit for the recovery of neuronal function after cerebral ischemia/reperfusion injury (Table 1). This suggests that the neuroprotective effect of ND01 may be related to its inhibition of the inflammatory response. [1] Myeloperoxidase (MPO) is considered an indicator of neutrophil infiltration and is highly expressed 24 hours after cerebral ischemia. Studies have shown that there is a significant correlation between neutrophil infiltration and infarction formation in cerebral ischemia models. Our results show that ND01 reduces MPO activity in diabetic ischemic brain tissue. This suggests that the neuroprotective effect of ND01 may stem from its inhibition of neutrophil infiltration (Table 2) [1]. Blood-brain barrier (BBB) permeability is significantly increased in diabetic patients, which may be due to BBB dysfunction and/or immature angiogenesis caused by diabetes. BBB disruption also occurs in the early stages of cerebral ischemia (within 24 hours). Our data suggest that ND01 improves diabetic cerebral ischemia/reperfusion injury by reducing BBB disruption [1]. NF-κB activation is associated with phosphorylation of IκB-α and NF-κB in ischemic brain tissue. Decreased NF-κB activation can protect the brain from the effects of NF-κB-dependent gene activation. HMGB-1 is a novel player in ischemic brain tissue. Meanwhile, diabetes significantly increased HMGB levels and led to worse functional outcomes after stroke compared to non-diabetic MCAO rats. All these data suggest that HMGB-1 plays a key role in diabetic stroke. The HMGB-1 signaling pathway is involved in the activation of NF-κB, and NF-κB kinase inhibitors are crucial for HMGB-1. Inhibition of HMGB-1 release in astrocytes can alleviate neuroinflammation and prevent ischemic astrocyte necrosis and NF-κB expression.³⁴ Early inhibition of HMGB-1 and NF-κB upregulation has significant benefits in diabetic cerebral ischemia. Based on these findings, we investigated the anti-inflammatory properties of 8-O-acetylganoside methyl ester (ND01) in diabetic cerebral ischemia and further explored its potential mechanism. 8-O-acetylganoside methyl ester (ND01) significantly inhibited the upregulation of HMGB-1 and NF-κB. These results indicate that inhibition of HMGB-1 and NF-κB expression is involved in the neuroprotective effect of ND01 against diabetic cerebral ischemia injury. Therefore, we believe that the protective effect of ND01 may stem from its inhibition of the inflammatory cascade response through the HMGB-1-dependent NF-κB signaling pathway. [1] In summary, the results of this study indicate that 8-O-acetylganoside methyl ester (ND01) exhibits significant neuroprotective effects in diabetic cerebral ischemia/reperfusion injury, including reducing blood-brain barrier disruption, reducing infarct volume, alleviating brain injury, and decreasing the expression of HMGB-1, phosphorylated IκB-α, and NF-κB in ischemic brain tissue. These effects of ND01 are associated with the inhibition of inflammatory responses. These findings suggest that ND01, as an effective anti-inflammatory lead compound, has potential application value in the treatment of early diabetic cerebral ischemia/reperfusion injury.

C₁₉H₂₈O₁₂

448.42

448.158

57420-46-9

57420-46-9

162823

White to off-white solid

1.52 g/cm3

634.2±55.0 °C at 760 mmHg

220.0±25.0 °C

0.0±4.2 mmHg at 25°C

1.594

-2.76

5

12

7

31

725

10

O(C(C([H])([H])[H])=O)[C@@]1(C([H])([H])[H])C([H])([H])[C@]([H])([C@]2([H])C(C(=O)OC([H])([H])[H])=C([H])O[C@]([H])([C@]12[H])O[C@@]1([H])[C@@]([H])([C@]([H])([C@@]([H])([C@@]([H])(C([H])([H])O[H])O1)O[H])O[H])O[H])O[H]

ARFRZOLTIRQFCI-NGQYDJQZSA-N

InChI=1S/C19H28O12/c1-7(21)31-19(2)4-9(22)11-8(16(26)27-3)6-28-17(12(11)19)30-18-15(25)14(24)13(23)10(5-20)29-18/h6,9-15,17-18,20,22-25H,4-5H2,1-3H3/t9-,10-,11+,12-,13-,14+,15-,17+,18+,19+/m1/s1

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

8-O-Acetyl shanzhiside methyl ester; 8-O-Acetyl shanzhiside methyl ester; 8-O-Acetylshanzhiside methyl ester; methyl (1S,4aS,5R,7S,7aS)-7-acetyloxy-5-hydroxy-7-methyl-1-[(2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4a,5,6,7a-tetrahydro-1H-cyclopenta[c]pyran-4-carboxylate; 5beta-Dihydro Finasteride; Cyclopenta[c]pyran-4-carboxylic acid,7-(acetyloxy)-1-(b-D-glucopyranosyloxy)-1,4a,5,6,7,7a-hexahydro-5-hydroxy-7-methyl-, methyl ester, (1S,4aS,5R,7S,7aS)-; Umbroside; Barlerin

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 (~223.0 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.58 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 (5.58 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 (5.58 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.)