Natural product from Peucedanum praeruptorum Dunn.; NO/cGMP

(-)-Praeruptorin A has been reported in Peucedanum japonicum and Prionosciadium thapsoides. (±)-Praeruptorin A can effectively relax ileum and tracheal smooth muscles. (+)-Praeruptorin A can improve the vascular hypertrophy by decreasing the area of smooth muscle cells (SMCs), collagen content and [Ca2+]i in SMCs [1].

Praeruptorin A is a coumarin compound naturally occurring in the roots of Peucedanum praeruptorum Dunn., a commonly used traditional Chinese medicine for the treatment of certain respiratory diseases and hypertension. Although previous studies indicated the relaxant effects of (+/-)-praeruptorin A on tracheal and arterial preparations, little is known about the functional characteristics of the enantiomers. In the present study, the two enantiomers were successfully isolated and identified by using a preparative Daicel Chiralpak AD-H column, and their relaxant effects on aorta rings were observed and compared. (+)-Praeruptorin A showed more potent relaxation than (-)-praeruptorin A against KCl- and phenylephrine-induced contraction of rat isolated aortic rings with intact endothelium. Removal of the endothelium remarkably reduced the relaxant effect of (+)-praeruptorin A but not that of (-)-praeruptorin A. Pretreatment of aortic rings with N(omega)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of nitric oxide synthase) or methylene blue (MB, a soluble guanylyl cyclase inhibitor) resulted in similar changes of the relaxant effects of the two enantiomers to endothelium removal. Molecular docking studies also demonstrated that (+)-praeruptorin A was in more agreement to nitric oxide synthase pharmacophores than (-)-praeruptorin A. On the other hand, the two enantiomers of praeruptorin A could slightly attenuate the contraction of rat aortic rings induced by internal Ca(2+) release from sarcoplasmic reticulum (SR). These findings indicated that (+)-praeruptorin A and (-)-praeruptorin A exerted distinct relaxant effects on isolated rat aorta rings, which might be mainly attributed to nitric oxide synthesis catalyzed by endothelial nitric oxide synthase. [1]

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Effects of (±)-Praeruptorin A enantiomers on the contraction of rat aortic rings induced by KCl or PE [1]
The aortic rings were pre-contracted with high concentration of KCl (60 mM) or PE (1 μM), respectively. Once the plateau was attained, (+)-Praeruptorin A and (−)-praeruptorin A (1–30 μM for KCl-induced contraction, 3–100 μM for PE-induced contraction) were accumulatively added, respectively. The concentration–relaxant response curves were obtained. In endothelium-intact aortic rings, the enantiomers of (±)-praeruptorin A had no significant effect on the basal tension at concentrations of 1–100 μM (data not shown), but both of them showed concentration-dependent relaxation against the contraction induced by KCl (60 mM) or PE (1 μM) (Fig. 5A and C). For KCl-evoked contraction, the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 12.1 ± 1.3 μM and 20.9 ± 0.8 μM, and the Emax values were 90.7 ± 1.4% and 68.6 ± 5.2%, respectively. For PE-evoked contraction, the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 35.4 ± 3.6 μM and 45.8 ± 2.5 μM, and the Emax values were 86.3 ± 2.6% and 79.8 ± 2.4%, respectively. The findings indicated that (+)-praeruptorin A was more potent than (−)-praeruptorin A against KCl- or PE-induced contraction of rat aortic rings with intact endothelium.

In endothelium-denuded aortic rings, (+)-Praeruptorin A and (−)-praeruptorin A also showed concentration-dependent relaxation of the contraction induced by KCl or PE (Fig. 5B and D). For KCl-evoked contraction, the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 22.7 ± 1.5 μM and 22.8 ± 2.9 μM, and the Emax values were 65.6 ± 3.5% and 65.3 ± 8.1%, respectively. For PE-evoked contraction, the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 42.9 ± 4.1 μM and 44.0 ± 1.0 μM, and the Emax values were 70.2 ± 3.9% and 69.1 ± 3.0%, respectively. The findings suggested that endothelium removal remarkably attenuated the relaxant effects of (+)-praeruptorin A but not (−)-praeruptorin A, and the action of (+)-praeruptorin A involved endothelium-dependent and -independent relaxation.

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Involvement of NO/cyclic guanosine monophosphate (cGMP) pathway in the relaxation of (±)-Praeruptorin A enantiomers against the contraction of rat aortic rings induced by KCl. [1]
To ascertain whether NO/cGMP pathway was involved in the relaxant effects of (±)-praeruptorin A enantiomers, l-NAME (100 μM, an inhibitor of NO synthase) and MB (10 μM, an inhibitor of soluble guanylyl cyclase (sGC) for catalyzing the formation of cGMP) were added 30 min prior to the application of KCl in endothelium-intact rat aortic rings, respectively. l-NAME or MB itself had no effect on the basal tension of rat aortic rings at the test concentration (data not shown). In l-NAME-pretreated aortic rings, the IC50 values of (+)-Praeruptorin A and (−)-praeruptorin A were 21.0 ± 4.1 μM and 22.4 ± 1.6 μM, and the Emax values were 70.3 ± 8.5% and 64.2 ± 7.6%, respectively (Fig. 6A). In MB-pretreated aortic rings, the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 20.2 ± 0.5 μM and 22.1 ± 1.1 μM, and the Emax values were 80.0 ± 1.3% and 70.2 ± 4.2%, respectively (Fig. 6B). Similar to endothelium removal, either l-NAME or MB pretreatment remarkably attenuated the relaxation of (+)-praeruptorin A but not (−)-praeruptorin A against KCl-induced contraction, and the relaxant effects of the two enantiomers tended to be same. The findings suggested that NO/cGMP signaling pathway substantially participated in the relaxation of (+)-praeruptorin A rather than (−)-praeruptorin A.

To clarify whether the sensitivity of the relaxant potency of (+)-Praeruptorin A to NO blockades be due to a synergy between NO (or cGMP) and (+)-praeruptorin A, we observed the effects of (±)-praeruptorin A on the acetylcholine-evoked relaxation (a NO-dependent relaxation). The results showed that neither (+)-praeruptorin A nor (−)-praeruptorin A can influence the relaxant effect of acetylcholine on the isolated rat aorta rings with intact endothelium. The IC50 values for acetylcholine alone, acetylcholine plus (+)-praeruptorin A (30 μM) and acetylcholine plus (−)-praeruptorin A (30 μM) were 4.0 ± 0.7 μM, 4.3 ± 0.6 μM, and 4.2 ± 0.4 μM, respectively. The findings demonstrated that (+)-praeruptorin A exerted the relaxant effects not by a synergy with NO.

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Effects of (±)-Praeruptorin A enantiomers on the contraction of rat aortic rings induced by intracellular Ca2+ release [1]
In the Ca2+-free K–H solution, PE (1 μM) could induce a transient contraction of endothelium-denuded rat aortic rings due to the release of intracellular Ca2+. As shown in Fig. 7, (±)-Praeruptorin A enantiomers only slightly attenuated PE-induced contraction with near potencies. The contraction ratio (T2/T1) of aortic rings in control and (+)-praeruptorin A (3 μM, 10 μM, 30 μM)-treated groups were 93.3 ± 2.1%, 92.2 ± 2.5%, 88.7 ± 3.2%, and 81.9 ± 4.3%, respectively. T2/T1 values in control and (−)-praeruptorin A (3 μM, 10 μM, 30 μM)-treated groups were 94.6 ± 3.7%, 91.8 ± 1.9%, 85.8 ± 3.5%, and 80.1 ± 5.1%, respectively. Heparin (100 μg/ml), a selective IP3R inhibitor, significantly inhibited the contraction induced by PE. The T2/T1 values for control and heparin-treated group were 98.1 ± 7.5% and 6.5 ± 1.3%, respectively. The findings indicated that the (±)-praeruptorin A enantiomers could slightly attenuate the contraction of rat aortic rings induced by IP3R (inositol-1,4,5-trisphosphate receptor)-mediated internal Ca2+ release.

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Effects of K+ channel blocker on the relaxation of (±)-Praeruptorin A enantiomers against the contraction of rat aortic rings induced by PE [1]
To recognize whether K+ channel opening was involved in the relaxant effects of (±)-praeruptorin A enantiomers, TEA (5 mM, a putative K+ channel blocker) was added 30 min prior to the application of PE (1 μM) in endothelium-denuded rat aortic rings in the standard K–H solution.
Fig. 8A showed that the two enantiomers could attenuate PE-induced contraction with near potencies, and the IC50 values of (+)-Praeruptorin A and (−)-praeruptorin A were 42.9 ± 4.1 μM and 44.0 ± 1.0 μM, respectively. TEA pretreatment did not alter the relaxant effects of the enantiomers, and the IC50 values of (+)-praeruptorin A and (−)-praeruptorin A were 43.1± 3.8 μM and 44.1 ± 6.4 μM, respectively (Fig. 8B). The findings suggested that K+ channel opening was not involved in the relaxant effects of (±)-praeruptorin A enantiomers.

Incubation of Praeruptorin A/PA and CKL in intestinal bacteria [2]
The Brain Heart Infusion (BHI) medium and intestinal microflora solution were prepared following the protocols proposed in reference. The biotransformation of PA or CKL by rat intestinal bacteria was performed in a 0.5 mL incubation system, containing 50 μL the intestinal microflora solution, 5 μL PA or CKL stock solution in DMSO (final concentration of 25 μmol/L for either compound), and 445 μL BHI medium. The incubation was anaerobically carried out at 37 °C in a GasPak EZ Anaerobe Pouch system for 4 h and quenched by the addition of 500 μL of ice-cold methanol. 0 min incubation, reaction without microflora solution and incubation without PA/CKL were performed as the controls. Each reaction was conducted in triplicate. The samples were then successively processed by centrifugation and filtration. Subsequently, the filtrate was subjected for the LC-UV–MS/MS analysis.

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Phase I metabolism of Praeruptorin A/PA in RLMs and HLMs [2]
The metabolism of PA in hepatic microsomal proteins was implemented in a total of 200 μL incubation solution containing PA (final concentration: 25 μmol/L), RLMs or HLMs (1 mg/mL), a NADPH-regenerating system (4 mmol/L MgCl2, 1 mmol/L β-NADP+, 1 mmol/L G-6-P and 1 U/mL G-6-PD) and 100 mmol/L potassium phosphate buffer (pH 7.4) at 37 °C. The reactions were terminated at 0, 5, 10, 15, 20, 30, 60, 90, 120 min, respectively, by pouring 200 μL ice-cold methanol and vortexing to mix thoroughly. Subsequently, each incubate was centrifuged at 15,000 × g for 10 min at 4 °C and the resultant supernatant was then filtered through 0.45 μm Nylon membrane filter before being subjected for HPLC-UV analysis. Samples that were incubated for 0 min, 60 min without β-NADP+, 60 min without PA, and 60 min with denatured liver microsomal proteins, served as controls. Each reaction was performed in triplicate. For the sake of PA metabolites identification, incubates obtained from 20 min reactions were chosen and loaded onto the LC-UV–MS/MS system. To identify the enzyme(s) that involved in PA hydrolysis in the absence of the NADPH-regenerating system, HLMs or RLMs were preincubated with carboxylesterase inhibitor, BNPP (500 μmol/L) or PMSF (500 μmol/L), for 5 min prior to the addition of PA to initiate incubation for another 20 min. 0 min incubation and 20 min reaction without chemical inhibitors were used as controls. Each experiment was performed in triplicate.

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Hydrolysis of Praeruptorin A/PA using pooled rat plasma, human plasma, hCES1 and hCES2 [2]
The activities of hCES1 and hCES2 were verified using their respective probe substrates, imidapril and irinotecan (CPT-11), following the procedures proposed in literature. Pooled rat plasma was freshly prepared from five drug-free male SD rats and the pooled human plasma was obtained as a present from Macau Red Cross. PA (25 μmol/L) was incubated with rat plasma (final content: 1 mg/mL), human plasma (final content: 1 mg/mL), hCES1 (final content: 1 unit) or hCES2 (final content: 1 unit) in potassium phosphate buffer solution (pH 7.4, 100 mmol/L) for 60 min at 37 °C with the final incubation volume of 200 μL. There were two types of control samples, including incubation with denatured plasma protein and incubation in the absence of PA. Each reaction was processed in triplicate. Incubations were terminated by the addition of equal volume of ice-cold methanol. Precipitated proteins were removed by centrifugation. The supernatant fluid was filtered and an aliquot (10 μL) of filtrate was injected into the LC-UV–MS/MS system.

(+)-Praeruptorin A and (−)-Praeruptorin A were dissolved in PEG400. [1]
To investigate the effects of compounds on the contraction induced by intracellular Ca2+ release, the aortic rings were exposed to Ca2+-free solution, and PE (1 μM) were used to induce the first transient contraction (T1). Thereafter, the rings were washed twice with standard Krebs–Henseleit solution (at least 40 min of incubation period for refilling the intracellular Ca2+ stores) and then twice with Ca2+-free K–H solution (15 min of incubation period). Then PE was used to induce the second transient contraction (T2) in the absence or presence of (+)-Praeruptorin A and (−)-Praeruptorin A, added 30 min before PE application. Heparin (100 μg/ml) was used as a positive control. The ratio of the second contraction to the first contraction (T2/T1) was calculated [1].

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Preparation of urine and fecal samples [2]
Male SD rats (220 ± 15 g) were acclimated in laboratory for one week prior to the experiments, housed in separate cages at a temperature of 23 ± 1 °C with a 12 h light/dark cycle and 50% relative humidity, free access to standard diet and water. All the rats were fasted over night before the experiments yet free access to water.
Three rats as PA-group were orally administered of Praeruptorin A/PA in a 50% aqueous 1,2-propylene glycol solution (v/v) while the control group (3 rats) was treated with the solvent (50% aqueous 1,2-propylene glycol solution, v/v). The urine and fecal samples were collected over 0–48 h after treatment and pooled within group. Pooled urine from PA-group and control group were diluted with 3 folds of methanol, vortexed and centrifuged to remove precipitate and then introduced to the LC-UV–MS/MS system, respectively. At the meanwhile, pooled fecal samples from PA-group or control group were extracted with acetonitrile at 10 mL per gram fecal sample for 30 min using ultrasonic water bath and then filtered through 0.45 μm membrane before analysis.

(±)-Pemphigustin A (PA) is the main bioactive component of Peucedanum praeruptorum, exhibiting a significant hypotensive effect, typically acting as a calcium channel blocker. This study aimed to characterize the in vitro and in vivo metabolomics profile of Peucedanum praeruptorum A using high-performance liquid chromatography-triple quadrupole-linear ion trap mass spectrometry (HPLC-Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). Twelve phase I metabolites (M1-12) were detected in rat liver microsomes (RLMs), nine phase I metabolites (M1-3, M5-6, and M9-12) were detected in human liver microsomes (HLMs), two hydrolysis products (M11 and M12) were detected in rat plasma, no metabolites were detected in human plasma, no metabolites were detected in rat intestinal bacteria, seven metabolites (M1, M4-7, M13, and M15) were detected in PA-treated rat urine, and six metabolites (M1, M4-7, and M15) were detected in PA-treated feces. These metabolites were preliminarily identified in the mass spectrometry domain using Predictive Multiple Reaction Monitoring-Information-Dependent Acquisition-Enhanced Product Ions (Predictive MRM-IDA-EPI) mode and Enhanced Mass Spectrometry-Information-Dependent Acquisition-Enhanced Product Ions (EMS-IDA-EPI) mode, and confirmed by Time-of-Flight Mass Spectrometry (TOF-MS). In addition, two glucuronidated metabolites (M13-14) of cis-khellactone (CKL, the main form of PA in vivo) were generated in rat liver microsomes (RLM), while no metabolites were detected in human liver microsomes (HLM), and no sulfation products were observed in rat liver S9 fraction (RS9) and human liver S9 fraction (HS9). The study showed that oxidation, hydrolysis, intramolecular acyl migration and glucuronidation are the main metabolic pathways of PA in vitro and in vivo. Based on the reduction of peak area, PA is rapidly metabolized in both RLM and HLM, indicating that there is a wide first-pass effect in the liver. In summary, this study elucidates the metabolic pathways of (±)-praeruptorin A in vitro and in vivo, and confirms that Q-trap-MS combined with LightSight™ software can be used as an effective tool for rapid detection and identification of metabolites in complex biological matrices. [2] This is the first report on the metabolic characteristics of Praeruptorin A/PA in vivo and CKL in vitro. High-performance liquid chromatography-triple quadrupole linear ion trap mass spectrometry (HPLC-MIL-IDA-EPI) combined with LightSight™ software was used to detect and preliminarily identify metabolites using predictive MRM-IDA-EPI and EMS-IDA-EPI modes. In vitro experiments observed metabolic pathways including hydrolysis, oxidation, intramolecular transacylation, and glucuronidation, generating 14 metabolites (M1-14). On the other hand, six metabolites (M1, M5-7, and M13) and five metabolites (M1 and M5-7) from M1-13 were detected in PA-treated urine and fecal samples, respectively, along with an additional oxidation product (M15) formed by stepwise oxidation at the C-4′ position. The study indicates that key hydrolysis and CYP450-catalyzed transformation in plasma, rather than gut microbiota, are the main reasons for the low bioavailability of PA in rats; however, the oral bioavailability of PA in humans is expected to be higher than in rats. Furthermore, this study also demonstrates that the method combining predictive MRM-IDA-EPI and EMS-IDA-EPI modes developed using LightSight™ software can serve as an effective tool for the rapid detection and identification of metabolites in complex biological matrices. [2]

[1]. (+/-)-Praeruptorin A enantiomers exert distinct relaxant effects on isolated rat aorta rings dependent on endothelium and nitric oxide synthesis. Chem Biol Interact. 2010 Jul 30;186(2):239-46.

[2]. Metabolic characterization of (±)-praeruptorin A in vitro and in vivo by high performance liquidchromatography coupled with hybrid triple quadrupole-linear ion trap mass spectrometry and time-of-flight mass spectrometry. J Pharm Biomed Ana. 2014 Mar:90:98-110.

[3]. Effects of praeruptorin A and praeruptorin C, a racemate isolated from Peucedanum praeruptorum, on MRP2 through the CAR pathway. Planta Med. 2013 Nov;79(17):1641-7.

Phytosine is a hydroxybenzoic acid. Phytosine has been reported in hydrangeas (Hydrangea serrata) and large-leaved hydrangeas (Hydrangea macrophylla), and related data have been published. Preptorin A is a coumarin compound. (+)-Preptorin A has been reported in Peucedanum japonicum, Prionosciadium thapsoides, and Ligusticum lucidum, and related data have been published. (-)-Preptorin A has been reported in Peucedanum japonicum and Prionosciadium thapsoides, and related data have been published. Previous studies have shown that (±)-Preptorin A is the main bioactive component of preptorin roots and can relax vascular smooth muscle. However, the characteristics and potential mechanisms of action of its enantiomers remain unclear. In this study, we found that both (+)-praeruptorin A and (−)-praeruptorin A could relax isolated rat aortic rings with functional endothelium in a concentration-dependent manner, with (+)-praeruptorin A exhibiting a stronger relaxing effect than (−)-praeruptorin A. Notably, endothelial removal and pretreatment with L-NAME or MB significantly attenuated the relaxing effect of (+)-praeruptorin A, but had no significant effect on the relaxing effect of (−)-praeruptorin A, ultimately leading to a convergence of relaxing efficacy between the two enantiomers. These results strongly suggest that (+)-praeruptorin A can exert both endothelium-dependent and endothelium-independent relaxing effects, while (−)-praeruptorin A only exerts an endothelium-independent relaxing effect. Furthermore, potassium channels are also involved in regulating muscle contractility and vascular tone. Direct activation of potassium channels on arterial smooth muscle cells leads to cell membrane hyperpolarization and inhibits calcium ion influx and smooth muscle contraction. In this study, TEA (a non-selective K+ channel blocker) pretreatment did not alter the relaxing effect of (±)-praptoline A enantiomers, suggesting that K+ channel opening may not be related to the relaxing effect of these enantiomers. In summary, both (+)-praptoline A and (−)-praptoline A can relax isolated rat aortic rings constricted by KCl in a concentration-dependent manner. The effect of (+)-praptoline A is stronger than that of (−)-praptoline A. The most important reason for this difference is likely that (+)-praptoline A, rather than (−)-praptoline A, binds better to the pharmacophore of eNOS, thereby activating the NO/cGMP signaling pathway. [1] Due to the potent metabolic capacity of CYP450 enzymes and plasma for preptorlin A/PA, and the observation that CKL is the major metabolite in rat liver microsomes (RLM), with low glucuronidation of CKL (the reduction in CKL was less than 10% based on peak area), it is preliminarily believed that the main actual form of PA in rats is CKL, which is consistent with our previous reports. On the other hand, the effective actual form of PA in humans may include the original form and CKL. Given the low activity of CKL, the therapeutic effect of PA in humans may last longer than in rats. However, the stepwise oxidation product of PA, M15, was observed to be one of the major metabolites in PA-treated urine, indicating that this metabolite may significantly contribute to the in vivo process of PA and should be considered in future studies. Due to absorption/distribution barriers and/or biotransformation obstacles, active ingredients may become trace in vivo, and interference from endogenous substances always makes it difficult to detect and identify drug-related components in biological samples. Therefore, there is an urgent need for highly sensitive and selective techniques to meet the needs of metabolic characterization. In this study, the detection limits of PA and its metabolites were less than 2 nmol/L using the developed predictive MRM-IDA-EPI method. In addition, the method is expected to have high selectivity due to the use of precursor-product ion transition. However, the key to metabolospectral characterization using this method is the prediction before LC-MS/MS measurement. In this study, the prediction was based on existing knowledge of diagonal pyranocoumarin metabolism and common metabolic pathways of xenobiotics that may occur in vivo. In fact, some software, such as PALLAS MetabolExpert (CompuDrug) and METEOR MetabolicExpert (Lhasa), can be used for computer simulation to predict metabolites, which helps to predict metabolism in vivo. On the other hand, in order to avoid missing potential metabolites during the prediction process, this study also introduced the EMS-IDA-EPI mode, which has a detection limit of less than 100 nmol/L for pyranocoumarin and its metabolites, although this mode does not produce additional products. Therefore, the proposed method provides a better analytical option for the characterization of metabolites in biological matrices, and EMS-IDA-EPI can also be used as a supplementary tool. [2]

C21H22O7

286.4

286.084

C, 65.28; H, 5.74; O, 28.98

21499-23-0

21499-23-0; 14017-71-1; 73069-25-7; 73069-27-9

146694

White to off-white solid powder

1.37g/cm3

540.6ºC at 760 mmHg

206.2ºC

2.66E-12mmHg at 25°C

1.637

2.56

2

5

2

21

385

1

COC1=C(C=C(C=C1)[C@H]2CC3=C(C(=CC=C3)O)C(=O)O2)O

PBILBHLAPJTJOT-CQSZACIVSA-N

InChI=1S/C16H14O5/c1-20-13-6-5-9(7-12(13)18)14-8-10-3-2-4-11(17)15(10)16(19)21-14/h2-7,14,17-18H,8H2,1H3/t14-/m1/s1

(3R)-8-hydroxy-3-(3-hydroxy-4-methoxyphenyl)-3,4-dihydroisochromen-1-one

Phyllodulcin; 21499-23-0; d-Phyllodulcin; 9DDW04R41V; (3R)-8-hydroxy-3-(3-hydroxy-4-methoxyphenyl)-3,4-dihydroisochromen-1-one; UNII-9DDW04R41V; (R)-3,4-Dihydro-8-hydroxy-3-(3-hydroxy-4-methoxyphenyl)-1H-2-benzopyran-1-one; 1H-2-Benzopyran-1-one, 3,4-dihydro-8-hydroxy-3-(3-hydroxy-4-methoxyphenyl)-, (R)-;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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