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.

(±)-Praeruptorin A (PA) is the major bioactive component in Peucedani Radix (Chinese name: Qian-hu), and exhibits dramatically anti-hypertensive effect typically through acting as a calcium channel blocker. The current study aims on the characterization of the metabolic profiles of PA in vitro and in vivo using high performance liquid chromatography (HPLC) coupled with hybrid triple quadrupole-linear ion trap mass spectrometry (Q-trap-MS) and time-of-flight mass spectrometry (TOF-MS). A total of 12 phase I metabolites (M1-12) in rat liver microsomes (RLMs), 9 phase I metabolites (M1-3, M5-6 and M9-12) in human liver microsomes (HLMs), 2 hydrolyzed products in rat plasma (M11 and M12), none metabolite in human plasma, none metabolite in rat intestinal bacteria, 7 metabolites (M1, M4-7, M13 and M15) in PA-treated rat urine and 6 metabolites (M1, M4-7 and M15) in PA-treated feces were detected and tentatively identified using predictive multiple reaction monitoring-information dependent acquisition-enhanced product ion (predictive MRM-IDA-EPI) mode in combination with enhanced mass spectrum-information dependent acquisition-enhanced product ion (EMS-IDA-EPI) mode in the mass spectrometer domain, respectively, while TOF-MS was adopted to confirm the identification. Further, 2 glucuronidated metabolites (M13-14) in RLMs and none metabolite in HLMs of cis-khellactone (CKL), which was the main actual form of PA in vivo, were generated, while its sulfated product was not observed in either rat liver S9 fractions (RS9) or human liver S9 fractions (HS9). Oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation were demonstrated to be the predominant metabolic types for PA in vitro and in vivo. Judging from the decrement of peak areas, PA was metabolized quickly in both RLMs and HLMs, indicating extensively hepatic first-pass elimination. Taken together, the metabolic fates of (±)-praeruptorin A in vitro and in vivo were elucidated in current study, and Q-trap-MS coupled with LightSight™ software can be adopted as a useful tool for quick detection and identification of metabolites in complex biological matrices. [2]

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This is the first report on the metabolic characterization of Praeruptorin A/PA in vivo and CKL in vitro. The metabolites were detected and tentatively identified using high performance liquid chromatography coupled with triple quadrupole linear ion trap mass spectrometry and LightSight™ software using predictive MRM-IDA-EPI in combination with EMS-IDA-EPI mode. Metabolic pathways including hydrolysis, oxidation, intra-molecular transacylation and glucuronidation were observed in vitro to generated 14 metabolites (M1–14). On the other hand, six (M1, 5–7 and 13) and five (M1 and 5–7) metabolites in M1–13 were detected in PA-treated urine and fecal samples, respectively, along with an additional oxidated product (M15) forming by a step-wise oxidation at C-4′ position. The crucial hydrolysis in plasma and CYP450-catalyzed transformation, rather than gut flora, were revealed to be responsible for the low bioavailability in rats, while the oral bioavailability of PA in human beings is expected to be higher than that in rats. In addition, the present study also suggested that predictive MRM-IDA-EPI coupled with EMS-IDA-EPI mode, which were developed using LightSight™ software, can be utilized as a useful tool for quick detection and identification of metabolite 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.

Praeruptorin A is a member of coumarins.
(+)-Praeruptorin A has been reported in Peucedanum japonicum, Prionosciadium thapsoides, and Ligusticum lucidum with data available.

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(-)-Praeruptorin A has been reported in Peucedanum japonicum and Prionosciadium thapsoides with data available.

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(±)-Praeruptorin A has been previously demonstrated to be able to relax vascular smooth muscles as the main bioactive constituent of P. praeruptorum roots. However, the action characteristics and underlying mechanisms of the enantiomers remain unclear. In the present study, we found that both (+)-praeruptorin A and (−)-praeruptorin A showed a concentration-dependent relaxation of isolated rat aortic rings with functional endothelium contracted by high K+, and (+)-praeruptorin A was more potent than (−)-praeruptorin A. Of note, endothelium removal and pretreatment with l-NAME or MB significantly attenuated the relaxant effect of (+)-praeruptorin A but not (−)-praeruptorin A, and resulted in the relaxant potencies of the two enantiomers tending to be same. These findings strongly suggested that (+)-Praeruptorin A exerted both endothelium-dependent and -independent relaxation of vascular smooth muscles, and (−)-praeruptorin A only exerted endothelium-independent one.

Furthermore, K+ channels also participate in the regulation of muscle contractility and vascular tone. Direct activation of K+ channels on arterial smooth muscle cells should hyperpolarize the cell membrane, and inhibit Ca2+ influx and smooth muscle contraction. In the present study, TEA (a nonselective K+ channel blocker) pretreatment did not alter the relaxant effects of (±)-Praeruptorin A enantiomers, suggesting that K+ channel opening might be not involved in the relaxation of the enantiomers.

In conclusion, both (+)-Praeruptorin A and (−)-praeruptorin A can produce a concentration-dependent relaxation of isolated rat aortic rings contracted by KCl. The action of (+)-praeruptorin A is more potent than (−)-praeruptorin A. The most important reason for the difference is probably that (+)-praeruptorin A but not (−)-praeruptorin A can well agree to the pharmacophores of eNOS, and activates NO/cGMP signaling pathway. [1]

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Due to the powerful capability of CYP450s and plasma for the metabolism of Praeruptorin A/PA, the observation of CKL as the major product and the slight glucuronidation of CKL in RLMs (the decrement of CKL was less than 10% calculating by the peak areas), the mainly actual form of PA in rats were tentatively characterized as CKL, which is consistent with our previous report. On the other hand, the potent actual form of PA in human would include the proto type and CKL. In sight of the lower activity observed for CKL, the therapeutic effect of PA could last for a longer time in human beings than rats. However, M15, a step-wisely oxidated product of PA, was observed as one of the main metabolites in PA-treated urine, indicating that this metabolites might offer a significant contribution for the PA in vivo process and should be taken in account in the further studies. The active component is likely to become traceful in the body owing to absorption/distribution barrier and/or biotransformation hurdle, and the interference from endogenous substances always makes it hard to detect and identify the drug-related components in biological samples. Hence, high sensitive and selective techniques are imperiously demanded to meet the needs of metabolic characterization. In current study, the limit of detections of PA and its metabolites were less than 2 nmol/L using the developed predictive MRM-IDA-EPI method. Moreover, due to the employment of precursor-product ion transitions, this method is expected to possess high selectivity. However, the key point for the characterization of metabolic profile using the proposed method lies on the prediction prior to the LC–MS/MS measurement. In current study, the presumption was performed on the basis of knowledge obtained for angular-type pyranocoumarin metabolism and the common metabolic pathways that might occur for the xenobiotics in vivo. In fact, some softwares, PALLAS MetabolExpert (CompuDrug) and METEOR MetabolicExpert (Lhasa) for instance, are available for the prediction of metabolites in silico, which could promote the speculation for in vivo metabolism. On the other side, EMS-IDA-EPI mode, the LODs of which were less than 100 nmol/L for either PA or its metabolites, was also introduced to avoid the miss of potential metabolites during prediction, in spite that none additional product was afforded. Therefore, the proposed method provides a preferable analytical choice for the characterization of metabolites in biological matrices, while EMS-IDA-EPI could adopted as the complementary tool at the meanwhile. [2]

C₂₁H₂₂O₇

386.40

386.136

C, 65.28; H, 5.74; O, 28.98

73069-25-7

(-)-Praeruptorin A;14017-71-1; 21499-23-0; 73069-25-7; 73069-27-9

38347607

White to off-white solid powder

1.3±0.1 g/cm3

486.8±45.0 °C at 760 mmHg

211.5±28.8 °C

0.0±1.2 mmHg at 25°C

1.574

4.18

0

7

5

28

720

2

C/C=C(/C)\C(=O)O[C@H]1[C@H](C2=C(C=CC3=C2OC(=O)C=C3)OC1(C)C)OC(=O)C

XGPBRZDOJDLKOT-NXIDYTHLSA-N

InChI=1S/C21H22O7/c1-6-11(2)20(24)27-19-18(25-12(3)22)16-14(28-21(19,4)5)9-7-13-8-10-15(23)26-17(13)16/h6-10,18-19H,1-5H3/b11-6-/t18-,19-/m0/s1

(Z)-(9R,10S)-10-acetoxy-8,8-dimethyl-2-oxo-2,8,9,10-tetrahydropyrano[2,3-f]chromen-9-yl 2-methylbut-2-enoate

(-)-Praeruptorin A (+/-)-Praeruptorin A DL-Praeruptorin A

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 (~258.80 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.47 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 (6.47 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 (6.47 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.)