(-)-Anomalin ((-)-Praeruptorin B)   中文名称: (-)-白花前胡素 B

Sterol regulatory element-binding proteins (SREBPs)

Praeruptorin B 抑制 SREBP 活性并降低细胞内脂质水平。研究表明 Praeruptorin B 可以显着抑制 SRE 荧光素酶活性，并且这种影响具有剂量依赖性。即使浓度较高，Praeruptorin B 的细胞毒性也很低。 Praeruptorin B 还大幅下调 SREBP-1c 和 SREBP-2 的表达 [1]。 Praeruptorin B 还表现出对 UGT1A9 活性的显着抑制作用 [2]。

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前胡素A（PA）和B（前胡素B/PB）是从白花前胡中分离得到的两个重要化合物，已被报道具有多种生化和药理活性。本研究旨在确定PA和PB对重要II期药物代谢酶尿苷5'-二磷酸葡萄糖醛酸基转移酶（UGTs）亚型活性的抑制作用。体外UGT孵育系统用于测定PA和PB对各种UGT亚型活性的抑制潜力。通过计算机模拟对接来解释PA和PB对UGT1A6活性的抑制差异。测定抑制行为，并使用体外抑制动力学参数（Ki）和PA体内暴露水平的组合进行体外-体内外推。前喷发素A（100μM）对UGT1A6和UGT2B7的活性抑制最强，100μM PA分别抑制了97.8%和90.1%的活性。计算机模拟对接研究表明，氢键相互作用对PA对UGT1A6的抑制作用比PB更强。前喷发素A非竞争性抑制UGT1A6的活性，竞争性抑制UGT2B7的活性。PA对UGT1A6和UGT2B7的抑制动力学参数（Ki）分别为1.2和3.3μM。PA对UGT1A6和UGT2B7的抑制[I]/Ki值分别为15.8和5.8，表明PA在体内对这两种UGT亚型具有很高的抑制潜力。因此，密切监测PA与主要经历UGT1A6或UGT2B7催化代谢的药物之间的相互作用是非常必要的。[2]

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前喷发素A对UGT1A6和UGT2B7的活性抑制作用最强[2]
首先使用100微摩尔的PA和Praeruptorin B/PB来筛选对UGT亚型的抑制潜力（图1和图2）。在测试的UGT亚型中，PA和PB对UGT1A1、UGT1A3、UGT1A8和UGT1A10的活性没有或可以忽略不计的抑制潜力。100微摩尔的PA抑制了97.8%的活性（p < 0.001),但100 μM的PB没有抑制UGT1A6的活性。对于UGT1A7，其活性被抑制了68.3%（p < 0.001). PA和PB均对UGT1A9的活性表现出显著的抑制作用（p < 0.001). 100微摩尔的PA抑制了90.1%的UGT2B7活性（p < 0.001),100 μM的PB对UGT2B7的活性抑制了52.8%（p < 0.05). 在测试的UGT亚型中，PA对UGT1A6和UGT2B7的活性抑制作用最强。

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氢键和疏水相互作用有助于前胡素A对UGT1A6的强烈抑制[2]
通过MODELLER程序构建了UGT1A6的三维结构，并在能量最小化后获得了最精细的模型。此外，配体PA和Praeruptorin B/PB被对接到UGT1A6的空腔中，活性空腔中的氨基酸残基如图3所示。配体PA和PB与UGT1A6的结合口袋由残基Gln34、Asp35、His38、Thr106、Ala107、Thr110、Glu111、Tyr112、Asn114、Asn115、Ala152、Arg172、Ile274、Gly275、Gly276、Met309、Ser311和Phe393组成。在结合袋中，抑制剂PA与Gly276的N原子形成一个氢键（图4）。此外，一些残基与PA形成疏水接触，包括疏水残基Ala152、Met309和Phe393；极性非带电残基Gln34、Tyr112、Asn114和Asn115；以及带极性电荷的残基Asp35、His38和Glu111，如图5A所示。前喷发蛋白B与UGT1A6没有形成氢键相互作用。前喷发蛋白B通过以下氨基酸残基与UGT1A6发生疏水相互作用：疏水残基Ala107、Ile274、Met309和Phe393；极性非带电残基Gln34、Thr106、Tyr112、Asn114、Asn115和Gly275；以及带极性电荷的残基Asp35、His38和Glu111（图5B）。

计算了抑制剂PA和PB对UGT1A6的结合自由能。前喷发素A以-5.52的结合自由能结合到UGT1A6的结合口袋中 kcal/mol，PB的结合自由能为-5.27 kcal/mol。因此，结合自由能的等级为PA < PB，表明PA对UGT1A6的抑制活性比PB更强，这与实验结果相当。
选择UGT1A8作为代表性的UGT亚型，以了解为什么PA没有特异性抑制某些UGT亚基。前爆发素A和B与UGT1A8的同一结合袋对接，结合袋中的残留物如支持信息中的图S1所示。UGT1A8的结合口袋由残基Ser36、His37、Phe39、Trp98、Phe109、Phe150、Arg170、Leu304、Gly305、Ser306、Met307、Arg333、Gln354、His369、Gly371、Ser372、His373、Gly374、Phe391、Asp393和Gln394组成。在PA和PB对UGT1A8的结合口袋中，这两种抑制剂与残基Gly374形成了相同的氢键（图S2）。此外，这两种抑制剂与UGT1A8形成疏水接触。前喷发蛋白A与残基Phe39、Phe109、Arg170、Ser306、Gln354、His369、Gly371、His373、Phe391和Asp393形成疏水接触。前喷发蛋白B与残基Ser36、His37、Phe39、Phe109、Phe150、Arg170、Leu304、Gly305、Ser306、Met307、Gly371、His369、His373、Phe391和Asp393形成疏水接触（图S3）。

前喷发蛋白A与UGT1A8相互作用，结合自由能为-7.64 kcal/mol。前喷发素B与PA的结合自由能与UGT1A8相似，结合自由能为-7.63 此外，PA和PB在结合袋中对UGT1A8产生了相似的相互作用。前喷发素A和B结合到UGT1A8的同一结合口袋中，并在结合口袋中与Gly374的N原子形成一个氢键。PA和PB都与非极性残基Phe39和Phe109形成疏水接触。所有这些结果都解释了为什么与PB相比，PA对UGT1A8没有表现出特异性抑制作用。

尽管它们仍然比饮食喂养的小鼠重，但用 Praeruptorin B (50 mg/kg) 治疗的小鼠明显比用载体治疗的小鼠轻，表明 Praeruptorin B 可能能够减轻饮食诱导的肥胖 (DIO)。更值得注意的是，用相同量的 Praeruptorin B 治疗的小鼠的脂肪/瘦肉和脂肪/体重比均显着降低。此外，研究表明，与高脂肪饮食的小鼠相比，用 prateruptorin B 治疗的动物血清 TC 和 TG 水平要低得多。与洛伐他汀类似，preruptorin B 会升高 HDL-c 并降低 LDL-c。此外，与给予赋形剂的小鼠相比，praeruptorin B 与洛伐他汀相当，它显着降低了肝脏中 TC 和 TG 的水平。根据染色结果，与用媒介物治疗的小鼠相比，用普拉普托林 B 治疗的小鼠显示出脂质积聚减少。在喂食高脂肪饮食的大鼠中，葡萄球菌前蛋白 B 显着降低升高的空腹血糖和胰岛素水平 [1]。

前胡素A和B对UGTs活性抑制潜力的测定[2]
根据之前的文献（Tang等人，2016；Liu等人，2016）对PA和Praeruptorin B/PB对UGTs活性的抑制作用进行了评估。体外培养混合物由以下成分组成 mM Tris-HCl缓冲液（pH = 7.4):MgCl2（5 mM）、II相辅因子UDPGA（5 mM”）、探针底物4-MU和重组UGT。PA和PB的储备溶液（20mM）通过使用二甲亚砜制备，并通过使用二甲基亚砜稀释制备各种浓度的工作溶液。预孵化5 min后，将UDPGA加入孵育混合物中以启动4-MU的葡糖醛酸化反应。反应温度、反应时间和分析条件已在之前描述过（Tang等人，2016；Liu等人，2016，Zhang等人，2017）。

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

92.04

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

注意： 本产品在运输和储存过程中需避光。

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.86 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (5.86 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (5.86 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
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10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
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