Sterol regulatory element-binding proteins (SREBPs)

Praeruptorin B suppresses SREBP activity and decreases intracellular lipid levels. Studies have demonstrated that Praeruptorin B can dramatically suppress SRE luciferase activity, and this impact is dose-dependent. Even at greater concentrations, the cytotoxicity of Praeruptorin B was low. Praeruptorin B also drastically downregulates the expression of SREBP-1c and SREBP-2 [1]. Praeruptorin B also demonstrates considerable inhibitory effects on the activity of UGT1A9 [2].

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Praeruptorin A (PA) and B (Praeruptorin B/PB) are two important compounds isolated from Bai-hua Qian-hu and have been reported to exert multiple biochemical and pharmacological activities. The present study aims to determine the inhibition of PA and PB on the activity of important phase II drug-metabolizing enzymes uridine 5'-diphospho-glucuronosyltransferase (UGTs) isoforms. In vitro UGT incubation system was used to determine the inhibition potential of PA and PB on the activity of various UGT isoforms. In silico docking was performed to explain the inhibition difference between PA and PB towards the activity of UGT1A6. Inhibition behaviour was determined, and in vitro-in vivo extrapolation was performed by using the combination of in vitro inhibition kinetic parameter (Ki ) and in vivo exposure level of PA. Praeruptorin A (100 μM) exhibited the strongest inhibition on the activity of UGT1A6 and UGT2B7, with 97.8% and 90.1% activity inhibited by 100 μM of PA, respectively. In silico docking study indicates the significant contribution of hydrogen bond interaction towards the stronger inhibition of PA than PB towards UGT1A6. Praeruptorin A noncompetitively inhibited the activity of UGT1A6 and competitively inhibited the activity of UGT2B7. The inhibition kinetic parameter (Ki ) of PA towards UGT1A6 and UGT2B7 was calculated to be 1.2 and 3.3 μM, respectively. The [I]/Ki value was calculated to be 15.8 and 5.8 for the inhibition of PA on UGT1A6 and UGT2B7, indicating high inhibition potential of PA towards these two UGT isoforms in vivo. Therefore, closely monitoring the interaction between PA and drugs mainly undergoing UGT1A6 or UGT2B7-catalyzed metabolism is very necessary. [2]

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Praeruptorin A exerted the strongest inhibition on the activity of UGT1A6 and UGT2B7 [2]
One hundred micromolar of PA and Praeruptorin B/PB was firstly used to screen the inhibitory potential on the UGT isoforms (Figs 1 and 2). Among the tested UGT isoforms, PA and PB showed no or negligible inhibition potential on the activity of UGT1A1, UGT1A3, UGT1A8, and UGT1A10. One hundred micromolar of PA inhibited 97.8% activity (p < 0.001), but 100 μM of PB did not inhibit the activity of UGT1A6. For UGT1A7, the activity was inhibited by 68.3% (p < 0.001). Both PA and PB exhibited significant inhibition on the activity of UGT1A9 (p < 0.001). One hundred micromolar of PA inhibited 90.1% activity of UGT2B7 (p < 0.001), and 100 μM of PB exerted inhibition of 52.8% activity of UGT2B7 (p < 0.05). Among the tested UGT isoforms, PA exerted the strongest inhibition on the activity of UGT1A6 and UGT2B7.

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Hydrogen bonds and hydrophobic interactions contribute to the strong inhibition of UGT1A6 by Praeruptorin A [2]
The three-dimensional structure of UGT1A6 was constructed via MODELLER program, and the most refined model was obtained after energy minimization. Furthermore, the ligands PA and Praeruptorin B/PB were docked into the cavity of UGT1A6, and the amino acid residues in the active cavity were shown in Fig. 3. The binding pocket of ligands PA and PB to UGT1A6 is composed of residues Gln34, Asp35, His38, Thr106, Ala107, Thr110, Glu111, Tyr112, Asn114, Asn115, Ala152, Arg172, Ile274, Gly275, Gly276, Met309, Ser311, and Phe393. In the binding pocket, inhibitor PA formed one hydrogen bond to the N atom of Gly276 (Fig. 4). In addition, some residues formed hydrophobic contacts to PA, including hydrophobic residues Ala152, Met309, and Phe393; polar non-charged residues Gln34, Tyr112, Asn114, and Asn115; and polar charged residues Asp35, His38, and Glu111, which were shown in Fig. 5A. Praeruptorin B did not form hydrogen bond interaction with UGT1A6. Praeruptorin B made hydrophobic interaction with UGT1A6 through the following amino acids residues: hydrophobic residues Ala107, Ile274, Met309, and Phe393; polar non-charged residues Gln34, Thr106, Tyr112, Asn114, Asn115, and Gly275; and polar charged residues Asp35, His38, and Glu111 (Fig. 5B).

The binding free energy of inhibitors PA and PB towards UGT1A6 was calculated. Praeruptorin A bound into the binding pocket of UGT1A6 with the binding free energy of −5.52 kcal/mol, and PB got the binding free energy of −5.27 kcal/mol. Hence, the rank of the binding free energy was PA < PB, suggesting that PA exerted stronger inhibition activity towards UGT1A6 comparing with PB, which is equivalent to the experimental result.
UGT1A8 was selected as the representative UGT isoform to understand why PA did not specifically inhibited some UGT isoforms. Praeruptorin A and B were docked the same binding pocket of UGT1A8, and the residues in the binding pocket were shown in Fig. S1 in the Supporting Information. The binding pocket of UGT1A8 was composed of residues Ser36, His37, Phe39, Trp98, Phe109, Phe150, Arg170, Leu304, Gly305, Ser306, Met307, Arg333, Gln354, His369, Gly371, Ser372, His373, Gly374, Phe391, Asp393, and Gln394. In the binding pocket of PA and PB towards UGT1A8, the two inhibitors formed the same hydrogen bond to residue Gly374 (Fig. S2). In addition, the two inhibitors formed hydrophobic contacts to UGT1A8. Praeruptorin A formed hydrophobic contacts to residues Phe39, Phe109, Arg170, Ser306, Gln354, His369, Gly371, His373, Phe391, and Asp393. Praeruptorin B formed hydrophobic contacts to residues Ser36, His37, Phe39, Phe109, Phe150, Arg170, Leu304, Gly305, Ser306, Met307, Gly371, His369, His373, Phe391, and Asp393 (Fig. S3).

Praeruptorin A made interactions to UGT1A8 with binding free energy to be −7.64 kcal/mol. Praeruptorin B exerted similar binding free energy with PA to UGT1A8, with the binding free energy to be −7.63 kcal/mol. Furthermore, PA and PB made similarity interactions towards UGT1A8 in the binding pocket. Praeruptorin A and B were bound into the same binding pocket of UGT1A8, and both formed one hydrogen bond to N atom of Gly374 in the binding pocket. Both PA and PB formed hydrophobic contacts to non-polar residues Phe39 and Phe109. All these results explained why PA did not show specific inhibition towards UGT1A8 in comparison with PB.

Although they were remained heavier than diet-fed mice, mice treated with Praeruptorin B (50 mg/kg) were noticeably lighter than mice treated with a vehicle, indicating that Praeruptorin B may be able to lessen diet-induced obesity (DIO). More notably, mice treated with the same amount of Praeruptorin B showed a significant reduction in both the fat/lean and fat/body weight ratios. Additionally, it was demonstrated that animals treated with prateruptorin B had much lower serum levels of TC and TG than mice fed a high-fat diet. Similar to lovastatin, preruptorin B raises HDL-c and decreases LDL-c. Additionally, praeruptorin B was equivalent to lovastatin in that it dramatically decreased the levels of TC and TG in the liver when compared to mice administered with a vehicle. According to staining results, mice treated with prateruptorin B showed reduced buildup of lipids than mice treated with vehicle. In rats fed a high-fat diet, praeruptorin B dramatically lowers increased fasting blood glucose and insulin levels [1].

Determination of inhibition potential of Praeruptorin A and B on the activity of UGTs [2]
The evaluation of inhibition of PA and Praeruptorin B/PB on UGTs' activity was performed according to the previous literatures (Tang et al., 2016; Liu et al., 2016; Zhang et al., 2016). In vitro incubation mixture was consisted of the following ingredients in the 50 mM of Tris-HCl buffer (pH = 7.4): MgCl2 (5 mM), phase II co-factor UDPGA (5 mM), the probe substrate 4-MU, and recombinant UGTs. The stock solution (20 mM) of PA and PB was prepared by using dimethyl sulfoxide, and various concentration of working solution was prepared through the dilution by using dimethyl sulfoxide. Pre-incubated for 5 min later, UDPGA was added in the incubation mixture to initiate the glucuronidation reaction of 4-MU. The reaction temperature, reaction time, and analytical conditions have been previously described (Tang et al., 2016; Liu et al., 2016; Zhang et al., 2016).

Many metabolic diseases are caused by disruption of lipid homeostasis. Sterol regulatory element-binding proteins (SREBPs) are a family of nuclear transcription factors that are associated with lipid de novo synthesis, thereby, SREBPs have been considered as targets for the treatment of metabolic diseases. In this study, we identified Praeruptorin B as a novel inhibitor of SREBPs. HepG2 cells were used to verify lipid-lowering effects of praeruptorin B. The expression of SREBPs, as well as their target genes was markedly suppressed. Furthermore, we found that Praeruptorin B inhibits the proteins expression of SREBP by regulating PI3K/Akt/mTOR pathway. In praeruptorin B-treated high fat diet (HFD)-fed obese mice, HFD induced lipid deposition, hyperlipidemia and insulin resistance were significantly ameliorated, and SREBPs and related genes in liver were down-regulated. These findings suggest that praeruptorin B exerts lipid-lowering effects through SREBPs regulation and could serve as a possible therapeutic option to improve hyperlipidemia and hyperlipidemia-induced comorbidities. [1]

The metabolic elimination pathway of PA and Praeruptorin B/PB has been initially investigated in the previous literatures. The experiment carried out by Song et al. showed that PA can be quickly metabolized in human, and oxidation, hydrolysis, intra-molecular acyl migration and glucuronidation are main metabolic elimination pathway of PA (Song et al., 2014). After the incubation of PB with human liver microsome phase I incubation mixture, many phase I metabolites were formed (Song et al., 2011). All these results indicate the potential interaction between PA and PB with DMEs. This study firstly used in vitro determination system to investigate the inhibition behaviour of PA and PB on the activity of various isoforms of UGTs. The results showed the strongest inhibition potential of PA on the activity of UGT1A6 and UGT2B7. In silico docking method was used to explain why the inhibition potential of PA was stronger than PB on the activity of representative UGT isoform UGT1A6. Both PA and PB can be docked into the activity cavity of UGT1A6 through hydrogen bonds and hydrophobic interactions. Praeruptorin A formed one hydrogen bond to UGT1A6, and PB formed no hydrogen bond to UGT1A6. Praeruptorin B made more hydrophobic contacts towards UGT1A6 in comparison with PA. Therefore, the hydrogen bonds mainly contribute the stronger inhibition of PA on UGT1A6.

The in vivo drug–drug interaction magnitude can be predicted by using the combination of in vitro inhibition kinetic parameter (Ki) and in vivo exposure concentration. The plasma concentration can reach approximately 7500 ng/mL (19 μM) after i.v. administration of 5 mg/kg of PA in rats with liver cirrhosis. Using this value, the [I]/Ki value was calculated to be 15.8 and 5.8 for the inhibition of PA on UGT1A6 and UGT2B7. Based on the above standard for [I]/Ki, the in vivo inhibition magnitude of PA towards UGT1A6 and UGT2B7 will be very strong.

UGT1A6 plays a core function in the metabolism of both xenobiotics and important endogenous substances. For example, UGT1A6 is the key enzyme catalyzing the glucuronidation metabolism of neurotransmitter serotonin (Sakakibara et al., 2015). Additionally, UGT1A6 can detoxify the carcinogenic arylamines and aryl hydrocarbons (Bock and Kohle, 2005). UGT2B7 plays an important role in catalyzing the glucuronidation metabolic reaction of many clinical drugs, including mycophenolic acid and 3′-azido-3′-deoxythymidine (zidovudine, AZT) (Frymoyer et al., 2013; Uchaipichat et al., 2008). UGT2B7 can also conjugate some endogenous substances, including bile acids, androgens, and estrogens (Gall et al., 1999). Therefore, the strong inhibition of PA towards these two UGT isoforms will significantly disrupt the metabolism of these substances.

In conclusion, the present study investigated the inhibition of UGT isoforms by PA and Praeruptorin B/PB, and the strong inhibition of PA on the activity of UGT1A6 and UGT2B7 was demonstrated. Highly possible in vivo inhibition magnitude of PA on UGT1A6 and UGT2B7 was also predicted, indicating the necessary monitoring of the interaction between PA and drugs mainly undergoing UGT1A6 or UGT2B7-catalyzed metabolism. [1]

[1]. Praeruptorin B improves diet-induced hyperlipidemia and alleviates insulin resistance via regulating SREBP signaling pathway. RSC Adv., 2018, 8, 354–366.

[2]. The Inhibition of UDP-Glucuronosyltransferase (UGT) Isoforms by Praeruptorin A and B. Phytother Res. 2016 Nov;30(11):1872-1878.

[3]. Determination of Anomalin and Deltoin in Seseli resinosum by LC Combined with Chemometric Methods. Volume 66, pages 677–683, (2007)

Anomalin has been reported in Angelica cincta, Seseli grandivittatum, and other organisms with data available.

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Bai-hua Qian-hu, the dried roots of Peucedanum praeruptorum Dunn (Umbelliferae), is a well-known traditional Chinese medicine, has been officially listed in the Chinese Pharmacopeia. It has been reported to exert multiple biochemical and pharmacological activities, including antipyretic and antitussive activity (Song et al., 2015; Xiong et al., 2012). Praeruptorin A (PA) and B (PB) are two important compounds isolated from Bai-hua Qian-hu and have been reported to exert therapeutic functions, including cardioprotective (Wang et al., 2004; Song et al., 2015) and antiinflammation effects (Yu et al., 2011; Xiong et al., 2012). More and more studies are finding the anti-tumour utilization of PA and PB/Praeruptorin B. For example, PA and its derivatives have been demonstrated to reverse P-glycoprotein-mediated multidrug resistance in cancer cells (Shen et al., 2006; Shen et al., 2012; Fong et al., 2008). Praeruptorin A and B have been reported to exhibit therapeutic function towards gastric cancer (Liang et al., 2010). Therefore, PA and PB are potential drug candidates having high potential for R&D.

Many factors should be considered for the R&D of new drug candidates into the market, and pharmacokinetic factor is one of the most important factors. Metabolism behaviour remains to be the most important factor among the pharmacokinetic properties, and metabolic evaluation contains metabolic pathway identification and metabolic enzyme inhibition potential determination. Drug metabolic enzymes (DMEs) are divided into phase I and phase II DMEs. Uridine 5'-diphospho (UDP)-glucuronosyltransferases (UGTs) are the most important phase II DMEs and have been reported to participate in the metabolic elimination of many xenobiotics (e.g., drugs, herbs, and pollutants) (Atasilp et al., 2016) and endogenous substances (e.g., oestrogen, bilirubin, and bile acids) (Mu et al., 2016; Kallionpaa et al., 2015; Bock, 2015). The inhibition of UGTs' activity will significantly affect the plasma exposure of xenobiotics and endogenous substances. For example, indinavir and sorafenib inhibit UGT1A1-catalyzed glucuronidation of bilirubin, resulting in the elevation of plasma concentration of bilirubin (Zucker et al., 2001; Peer et al., 2012). The inhibition of bisphenol A and phthalates on the activity of UGT isoforms has been utilized to explain the toxicity mechanism of bisphenol A and phthalates (Jiang et al., 2013; Liu et al., 2016).

The present study aims to determine the inhibition of PA and Praeruptorin B/PB on the activity of UGT isoforms. [2]

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Liquid chromatography–chemometric methods [LC-Partial least squares (LC-PLS), LC-principle component regression (LC-PCR) and LC-artificial neural network (LC-ANN)] were developed for the determination of (−)-anomalin (ANO) and deltoin (DEL) in the root and aerial part of Seseli resinosum Freyn et Sint. (Umbelliferae). Firstly, chemometric conditions were optimized by testing different mobile phases at various proportions of solvents with various flow rates in different wavelengths by using a normal phase column to obtain the best separation and recovery results. As a result, a mobile phase consisting of n-hexane and ethyl acetate (75:25 v/v) at a constant flow rate of 0.8 mL min−1 on the above column system at ambient temperature were found to be the optimal chromatographic condition for good separation and determination of ANO and DEL in samples. Multichromatograms for the concentration set containing ANO and DEL compounds in the concentration range of 50–400 ng mL−1 were obtained by using a diode array detector (DAD) system at selected wavelength sets, 300 (A), 310 (B), 320 (C), 330 (D) and 340 (E). Three LC-chemometric approaches were applied to the multichromatographic data to construct chemometric calibrations. As an alternative method, traditional LC at single wavelength was used for the analysis of the related compounds in the plant extracts. All of the methods were validated by analyzing various synthetic ANO–DEL mixtures. After the above step, traditional and chemometric LC methods were applied to the real samples consisting of extracts from roots and aerial parts of S. resinosum. The results obtained by LC-chemometric approaches were compared to each other and with those obtained by traditional LC.[3]

C24H26O7

426.46

426.167

C, 67.59; H, 6.15; O, 26.26

4970-26-7

10251869

White to yellow solid powder

1.2±0.1 g/cm3

524.8±50.0 °C at 760 mmHg

177.5-178.5℃

225.5±30.2 °C

0.0±1.4 mmHg at 25°C

1.573

5.99

0

7

6

31

835

2

C/C=C(/C)\C(=O)O[C@H]1[C@H](C(OC2=C1C3=C(C=C2)C=CC(=O)O3)(C)C)OC(=O)/C(=C\C)/C

PNTWXEIQXBRCPS-IOWUNYDSSA-N

InChI=1S/C24H26O7/c1-7-13(3)22(26)29-20-18-16(11-9-15-10-12-17(25)28-19(15)18)31-24(5,6)21(20)30-23(27)14(4)8-2/h7-12,20-21H,1-6H3/b13-7-,14-8-/t20-,21-/m1/s1

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

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 (234.49 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.86 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 (5.86 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 (5.86 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.)