Baicalin   中文名称: 黄芩苷

Natural product; carnitine palmityl transferase 1 (CPT1); NF-κB; Autophagy

通过改变活性氧 (ROS)、Toll 样受体 (TLR) 2 和 TLR4、NF-κB、Bax 和 Bcl-2 等多种介质的合成，黄芩苷可提供针对再灌注损伤 (IRI) 的保护。促炎细胞因子 TLR2/4、MyD88、p-NF-κB 和 p-IκB，以及与 IκB 表达相关的 NF-κB 产生增强，均会受到治疗的抑制 [1]。 MTT测定用于评估细胞活力。与对照细胞相比，用粉末酶处理的SH-SY5Y细胞的活力要低得多。与单独使用粉状酶处理的细胞相比，单独使用黄芩苷（5、10 和 20 μM）可以剂量依赖性方式增强细胞活力 [2]。

在两个季度（10 和 100 毫克/公斤）内，黄芩苷显着降低血尿素氮 (BUN) 和 Scr 浓度，同时还定量预防肾功能丧失。采用0-3分分级系统，评估黄芩苷引起的组织损伤。与假手术组相比，用 10 和 100 mg/kg 黄芩苷治疗仅导致 MDA 含量略有增加，SOD 活性略有降低，这表明当不再注射时黄芩苷会增加氧化作用 [1]。

热位移分析[3]
热位移测定如前所述 进行。对于温度依赖性热位移测定，将50µL脂肪变性Hela细胞或CPT1A过表达大肠杆菌的裂解物（3 mg/mL）与100µMbaicalin/黄芩苷在36至76°C的每个温度点孵育4分钟。样品在4°C下以20000 g离心10分钟，以分离上清液和托盘。将12µL上清液与3µL 5x加载缓冲液混合，然后在10%SDS-PAGE上分离，用于CPT1A的免疫印迹分析。对于剂量依赖性热位移测定，将50µL裂解物（3 mg/mL）与不同浓度的baicalin/黄芩苷（0至1000µM）在52°C下孵育4分钟。通过离心分离上清液，并如上所述对CPT1A进行免疫印迹分析。

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CPT1活性测定[3]
CPT1活性测定是根据之前报告的修改方案进行的，该方案基本上测量了氘标记的肉碱（[D9]-肉碱）向氘标记的棕榈酰肉碱（[D9-棕榈酰肉碱）的转化。反应缓冲液由150 mM KCl、2 mM EDTA、4.5 mM谷胱甘肽、1 mg/mL BSA、0.1 mM棕榈酰辅酶A和0.1 mM[D9]-肉碱在PBS中组成。按指示浓度加入baicalin/黄芩苷。我们使用纯但无活性的重组CPT1A建立蛋白质浓度标准曲线，并在进行CPT1A酶测定时使用蛋白质印迹来估计各种细胞、组织和大肠杆菌裂解物中CPT1A的量。然后使用这些数字将CPT1A活性标准化为绝对值（每m g CPT1A每分钟棕榈酰肉碱的nmol转换）。为了测试细胞CPT1A的活性，将HeLa细胞在冰上用0.1%的Triton X-100溶解在PBS中，并将裂解物调节至2 mg/mL。通过向100µL反应缓冲液中加入20µL裂解物开始酶促反应，并在37°C下孵育10分钟。反应结束时，加入0.3 nmol[D3]-棕榈酰肉碱作为内标，加入600µL预冷甲醇，在冰上提取小分子代谢物。将反应混合物在4°C下以20000 g离心5分钟。然后取出400µL上清液，与600µL蒸馏水混合，制成1mL最终样品体积，用于LCMS测定[D9]-棕榈酰肉碱。LC-MS系统由AB SCIEX 5500三重四极杆质谱仪和岛津DGU-20A液相色谱仪组成，该液相色谱仪配有6个安捷伦柱。缓冲液梯度为100%-0缓冲液A（100%水，0.1%甲酸）和0%-100%缓冲液B（100%乙腈，0.1%甲酸。通过比较[D9]-棕榈酰基肉碱和[D3]-棕榈酰肉碱的峰面积来计算[D9]棕榈酰基肉碱的绝对浓度。在有或没有100µM丙二酰辅酶A处理的情况下平行制备两个反应，CPT1A的活性计算为有和没有丙二酰CoA处理时[D9]-棕榈酰肉碱的差异。为了测试原代肝细胞中的CPT1A活性，将肝细胞在冰上用0.1%的Triton X-10溶解在PBS中，并将裂解物调节至2mg/mL。通过向100µL反应缓冲液中加入10µL裂解物开始酶反应，并在37°C下孵育10分钟。为了检测线粒体中的CPT1A活性，使用培养细胞线粒体分离试剂盒分离线粒体。然后，在冰上用0.1%的Triton X-100在PBS中裂解线粒体，然后将裂解物调节至0.2mg/mL。如上所述，使用10µL线粒体裂解物测定CPT1A活性。为了测试小鼠肝脏的CPT1A活性，用0.1%的Triton X-100均质化组织，以获得2mg/mL的裂解液。如上所述，使用10µL的肝脏裂解液来测定CPT1A的活性。为了测试大肠杆菌的重组CPT1活性，如上所述，使用过表达每个CPT1构建体的大肠杆菌的10µL裂解物（1L大肠杆菌培养物用含有0.1%曲顿-100的50mL PBS裂解）来测定CPT1活性。

细胞培养与实验设计[2]
SH-SY5Y细胞在37°C的RPMI-1640培养基 中培养，该培养基补充了15%的胎牛血清，空气中含有95%的空气和5%的二氧化碳，湿度饱和。在60~70%的融合后，SH-SY5Y细胞分为：（i）对照组，在RPMI-1640培养基中孵育；（ii）凝血酶组，根据我们的预实验，凝血酶诱导（40U/L）6小时；（iii）黄芩苷组，在凝血酶诱导前用baicalin/黄芩苷（5μM、10μM或20μM）处理2小时。

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细胞活力测定[2]
如前所述，使用MTT法测量细胞存活率[24]。简而言之，向每个孔中加入15μl MTT溶液（5mg/mL），并在37°C下孵育4小时。去除上清液后，向每个孔中加入80μL DMSO。使用微孔板读数器在492nm处测量吸光度。所有实验均一式三份。

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流式细胞术检测细胞凋亡[2]
不含EDTA的胰蛋白酶消化法收获细胞，在PBS中洗涤两次。用AnnexinV/异硫氰酸荧光素（FITC）和碘化丙啶（PI）染色后，立即用流式细胞仪分析细胞。

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SILAC-ABPP SILAC[3]
ABPP实验是根据先前报告改编的方案进行的。Hela细胞在含有10%SILAC FBS、1%青霉素-链霉素和100µg/mL[13C6.15N4]L-精氨酸-HCl和[13C6.15N2]L-赖氨酸-HHCl或L-精氨酰-HCl和L-赖氨酸酯-HCl的SILAC DMEM中传代10次。在ABPP实验之前，细胞被血清饥饿24小时，并用1 mM游离脂肪酸处理24小时，以诱导脂滴的积累。收集细胞并在-80°C下储存以供进一步实验。将冷冻细胞颗粒重新悬浮在含有0.1%Triton X-100的PBS中，超声处理，并通过100000 g超速离心45分钟将其分离为可溶性和不溶性部分。使用酶标仪上的BCA蛋白测定法（Pierce™BCA蛋白测定试剂盒，赛默飞世尔科技公司）测定可溶性蛋白浓度。将裂解物调节至2 mg/mL，用2 mM黄芩苷（10µL的200 mM DMSO储备）或DMSO处理，用200µM黄芩苷探针（10µL的20 mM DMSO库存）或DMSO标记，然后在365 nm的紫外线辐射下冰上放置1小时。将轻和重探针化的蛋白质组以1:1的比例均匀混合，并立即用氯仿-甲醇沉淀。然后将沉淀物用1.2%的SDS/PBS重新悬浮，并与300µM叠氮化生物素、100µM TBTA、1 mM TCEP和1 mM 7 CuSO4混合1小时。反应后，用氯仿甲醇再次提取蛋白质组以去除多余的试剂。用冷甲醇洗涤蛋白质界面，用1.2%SDS/PBS溶解，用PBS稀释5倍。溶解的蛋白质在室温下与链霉抗生物素蛋白珠（100µL浆液）一起旋转孵育3小时。然后将珠粒用5mL PBS洗涤三次，用5mL水洗涤三次后转移到螺旋顶Eppendorf管中。富集的蛋白质在6 M尿素/PBS中变性，在65°C下用10 mM二硫苏糖醇还原15分钟，在35°C下在黑暗中搅拌20 mM碘乙酰胺（Sigma-Aldrich）阻断30分钟。用950µL PBS稀释反应物，并在1400g下离心3分钟。去除上清液。然后向珠子中加入200µL 2 M尿素/PBS、2µL 100 mM氯化钙水溶液和4µL胰蛋白酶（20µg在40µL胰蛋白缓冲液Promega中复溶）的预混合溶液，并在37°C下搅拌过夜。第二天，将混合物转移到生物旋转过滤器中，通过离心（1000g）将消化后的溶液洗脱到低粘附管中。洗脱液用5%甲酸酸化。如图2c所示，进行了一系列使用黄芩苷探针的SILAC-ABPP实验，可以与天然黄芩苷化合物竞争或不竞争。简而言之，轻蛋白质组总是用DMSO处理，并通过紫外线诱导的光交联用baicalin/黄芩苷BP探针标记，然后与如下所述处理的重蛋白质组混合。在“BP对照”实验中，重蛋白质组用DMSO处理，并用紫外线照射的空白DMSO溶液标记。在“紫外线对照”实验中，重蛋白质组用DMSO处理，并用黄芩苷BP探针标记，但没有紫外线辐射。在“CP对照”实验中，重蛋白质组用DMSO处理，并用黄芩苷CP探针（即不含二苯甲酮部分）在紫外线照射下标记。在“竞争”实验中，重蛋白质组与黄芩苷竞争，并用黄芩苷BP探针用紫外辐射标记。三个对照实验旨在消除与探针间接和/或非特异性结合的假阳性靶标。每个对照和竞争实验进行三次重复，所有定量的蛋白质都列在数据集S1中。

Renal ischemia-reperfusion model [1]
Rats were randomly divided into five groups of six rats each: (i) sham group; (ii) IR + saline group; (iii) IR + baicalin (1 mg/kg) group; (iv) IR + baicalin (10 mg/kg) group; and (v) IR + baicalin (100 mg/kg) group. Renal IRI was induced by clamping the left renal artery for 45 min plus a right nephrectomy [18]. Rats were anesthetized through an intraperitoneal injection of pentobarbital sodium (40 mg/kg body weight). After a median abdominal incision, the left renal arteries were clamped for 45 min with serrefine. After clamp removal, adequate restoration of blood flow was checked before abdominal closure. The right kidney was then removed. Sham-operated animals underwent the same surgical procedure without clamping.

Saline-treated animals received intraperitoneal injections of 1 mL 0.9% sterile NaCl 30 min before renal clamping. baicalin-treated rats received intraperitoneal injections of baicalin, diluted in sterile saline to 1, 10, or 100 mg/kg body weight 30 min before renal clamping. After the operation, the rats were kept on a warming blanket for 12 h with food and water available. All animals were sacrificed 24 h after surgery with an overdose of pentobarbital sodium, and their blood and kidneys harvested.

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All mice (C57BL/6j) were maintained in a temperature-controlled barrier facility with a 12-h light/dark cycle and were given free access to food and water. Only male animals were used in this study. The study was stratified randomized block according to the weight. Five mice per group were chosen to reach statistical significance. The research used the random, contrast and single-blinded test. Dietary interventions with a high-fat diet (60 % calories from fat, Research Diets Inc.) or a chow diet (10% calories from fat, Research Diets Inc.) were started at the age of 6 weeks for wild-type littermates and maintained for 24 weeks(11). The daily intragastric administrations of 400 mg/kg baicalin (40 mg/mL saline stock) (12) were started after 12-week dietary intervention and maintained for another 12 weeks before these mice were analyzed for metabolic changes and steatosis-related symptoms. [3]

The dose was chosen based on a previous pharmacokinetic study that measured the final plasma concentration of baicalin as 0.8 μg/mL 6 h after intake [3].
Due to its poor bioavailability, a relatively high (but safe) dose of baicalin was applied in our animal studies, and based on the previous pharmacokinetics analysis, its plasma steady-state concentration is predicted at 0.8 μg/mL after an oral dose of 400 mg/kg (41). It is therefore conceivable that optimization of the structure/activity relationship of baicalin by medicinal chemistry, together with more detailed structural insights of CPT1–baicalin interaction, may yield new synthetic antiobesity drugs with improved pharmacodynamics and pharmacokinetics. In this regard, we have preliminarily explored the activity of an acetylated derivative, Ac-baicalin, which is predicted to have improved bioavailability. The results showed that structural modification of the flavonoid core by acetylation disrupted the binding of CPT1A and that loss of CPT1A activation abolished the lipid-reducing effect of Ac-baicalin (SI Appendix, Fig. S20).[3]

[1]. The protective effect of Baicalin against renal ischemia-reperfusion injury through inhibition of inflammation and apoptosis. BMC Complement Altern Med. 2014 Jan 13;14:19.

[2]. Baicalin protects against thrombin induced cell injury in SH-SY5Y cells. Int J Clin Exp Pathol. 2015 Nov 1;8(11):14021-7.

[3]. Chemoproteomics reveals baicalin activates hepatic CPT1 to ameliorate diet-induced obesity and hepatic steatosis. Proc Natl Acad Sci U S A. 2018;115(26):E5896-E5905.

Baicalin is the glycosyloxyflavone which is the 7-O-glucuronide of baicalein. It is an active ingredient of Chinese herbal medicine Scutellaria baicalensis. It has a role as a non-steroidal anti-inflammatory drug, an EC 3.4.21.26 (prolyl oligopeptidase) inhibitor, a prodrug, a plant metabolite, a ferroptosis inhibitor, a neuroprotective agent, an antineoplastic agent, a cardioprotective agent, an antiatherosclerotic agent, an antioxidant, an EC 2.7.7.48 (RNA-directed RNA polymerase) inhibitor, an anticoronaviral agent and an antibacterial agent. It is a glucosiduronic acid, a glycosyloxyflavone, a dihydroxyflavone and a monosaccharide derivative. It is functionally related to a baicalein. It is a conjugate acid of a baicalin(1-).
Baicalin has been reported in Scutellaria prostrata, Scutellaria scandens, and other organisms with data available.
See also: Scutellaria baicalensis Root (part of).

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Background: Renal ischemia-reperfusion injury (IRI) increases the rates of acute kidney failure, delayed graft function, and early mortality after kidney transplantation. The pathophysiology involved includes oxidative stress, mitochondrial dysfunction, and immune-mediated injury. The anti-oxidation, anti-apoptosis, and anti-inflammation properties of baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, have been verified. This study therefore assessed the effects of baicalin against renal IRI in rats. Methods: Baicalin was intraperitoneally injected 30 min before renal ischemia. Serum and kidneys were harvested 24 h after reperfusion. Renal function and histological changes were assessed. Markers of oxidative stress, the Toll-like receptor (TLR)2 and TLR4 signaling pathway, mitochondrial stress, and cell apoptosis were also evaluated. Results: Baicalin treatment decreased oxidative stress and histological injury, and improved kidney function, as well as inhibiting proinflammatory responses and tubular apoptosis. Baicalin pretreatment also reduced the expression of TLR2, TLR4, MyD88, p-NF-κB, and p-IκB proteins, as well as decreasing caspase-3 activity and increasing the Bcl-2/Bax ratio. Conclusions: Baicalin may attenuate renal ischemia-reperfusion injury by inhibiting proinflammatory responses and mitochondria-mediated apoptosis. These effects are associated with the TLR2/4 signaling pathway and mitochondrial stress. [1]

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Baicalin, an extract from the dried root of Scutellaria baicalensis Georgi, was shown to be neuroprotective. However, the precise mechanisms are incompletely known. In this study, we determined the effect of baicalin on thrombin induced cell injury in SH-SY5Y cells, and explored the possible mechanisms. SH-SY5Y cells was treated with thrombin alone or pre-treated with baicalin (5, 10, 20 μM) for 2 h followed by thrombin treatment. Cells without thrombin and baicalin treatment were used as controls. Cell viability was detected by MTT assay. Cell apoptosis was analyzed by flow cytometry. Real-time PCR was performed to determine the mRNA expression of protease-activated receptor-1 (PAR-1). Western blotting was conducted to determine the protein expression of PAR-1, Caspase-3 and NF-κB. Baicalin reduced cell death following thrombin treatment in a dose-dependent manner, with concomitant inhibition of NF-κB activation and suppression of PAR-1 expression. In addition, baicalin reduced Caspase-3 expression. The above findings indicated that baicalin prevents against cell injury after thrombin stimulation possibly through inhibition of PAR-1 expression and NF-κB activation. [2]

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Obesity and related metabolic diseases are becoming worldwide epidemics that lead to increased death rates and heavy health care costs. Effective treatment options have not been found yet. Here, based on the observation that baicalin, a flavonoid from the herbal medicine Scutellaria baicalensis, has unique antisteatosis activity, we performed quantitative chemoproteomic profiling and identified carnitine palmitoyltransferase 1 (CPT1), the controlling enzyme for fatty acid oxidation, as the key target of baicalin. The flavonoid directly activated hepatic CPT1 with isoform selectivity to accelerate the lipid influx into mitochondria for oxidation. Chronic treatment of baicalin ameliorated diet-induced obesity (DIO) and hepatic steatosis and led to systemic improvement of other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 completely abolished the beneficial effect of the flavonoid. Our discovery of baicalin as an allosteric CPT1 activator opens new opportunities for pharmacological treatment of DIO and associated sequelae. [3]

C21H18O11

446.36

446.084

C, 56.51; H, 4.06; O, 39.43p

21967-41-9

21967-41-9;

64982

Light yellow to yellow solid powder

1.7±0.1 g/cm3

836.6±65.0 °C at 760 mmHg

202-205 ºC

297.2±27.8 °C

0.0±3.2 mmHg at 25°C

1.740

0.31

187.12

6

11

4

32

748

5

C1=CC=C(C=C1)C2=CC(=O)C3=C(C(=C(C=C3O2)O[C@H]4[C@@H]([C@H]([C@@H]([C@H](O4)C(=O)O)O)O)O)O)O

IKIIZLYTISPENI-ZFORQUDYSA-N

InChI=1S/C21H18O11/c22-9-6-10(8-4-2-1-3-5-8)30-11-7-12(14(23)15(24)13(9)11)31-21-18(27)16(25)17(26)19(32-21)20(28)29/h1-7,16-19,21,23-27H,(H,28,29)/t16-,17-,18+,19-,21+/m0/s1

(2S,3S,4S,5R,6S)-6-(5,6-dihydroxy-4-oxo-2-phenyl-chromen-7-yl)oxy-3,4,5-trihydroxy-tetrahydropyran-2-carboxylic acid

Baicalin； 21967-41-9; Baicalein 7-O-glucuronide; 7-D-Glucuronic acid-5,6-dihydroxyflavone; Baicalein 7-glucuronide; CHEBI:2981; MFCD00134418; 347Q89U4M5;

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)

DMSO : ≥ 100 mg/mL (~224.03 mM)
H2O : < 0.1 mg/mL

配方 1 中的溶解度: ≥ 2.5 mg/mL (5.60 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 (5.60 mM) (饱和度未知) 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), 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: 20 mg/mL (44.81 mM) in 0.5% CMC-Na/saline water (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
*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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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：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
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