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.

(+)-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].

[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.

(-)-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]

C21H22O7

386.39518

386.136

C, 65.28; H, 5.74; O, 28.98

14017-71-1

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

9821539

Typically exists as solid at room temperature

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-YRCPKEQFSA-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-/m1/s1

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

(-)-Praeruptorin A; 14017-71-1; Isopteryxin; [(9R,10R)-10-acetyloxy-8,8-dimethyl-2-oxo-9,10-dihydropyrano[2,3-f]chromen-9-yl] (Z)-2-methylbut-2-enoate; (+/-)-Praeruptorin A; 73069-25-7; (9R,10R)-10-(acetyloxy)-8,8-dimethyl-2-oxo-2H,8H,9H,10H-pyrano[2,3-h]chromen-9-yl (2Z)-2-methylbut-2-enoate; 10-(Acetyloxy)-9,10-dihydro-8,8-dimethyl-2-oxo-2H,8H-benzo(1,2-b:3,4-b')dipyran-9-yl 2-methyl-2-butenoate (9R-(9alpha(Z),10alpha))-;

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.)