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

By modifying the synthesis of several mediators, including as reactive oxygen species (ROS), Toll-like receptors (TLR) 2 and TLR4, NF-κB, Bax, and Bcl-2, baicalin provides protection against reperfusion injury (IRI). The pro-inflammatory cytokines TLR2/4, MyD88, p-NF-κB, and p-IκB, as well as the enhanced production of NF-κB in correlation with IκB expression, are all inhibited by treatment [1]. The MTT assay was used to assess cell viability. When compared to control cells, the vitality of SH-SY5Y cells treated with powdered enzyme was much lower. When compared to cells treated with powdered enzyme alone, baicalin (5, 10, and 20 μM) alone enhanced cell viability in a dose-dependent way [2].

For two quarters (10 and 100 mg/kg), baicalin dramatically decreased blood urea nitrogen (BUN) and Scr concentrations while also quantitatively preventing renal function loss. Using a 0–3 point grading system, the tissue damage caused by baicalin was evaluated. Treatment with 10 and 100 mg/kg baicalin resulted in only a minor increase in MDA content and a slight lowering of SOD activity when compared to the sham group, suggesting that baicalin increases oxidation when reinjection is removed [1].

Thermal shift assay [3]
The thermal shift assay was performed as previously described. For the temperature-dependent thermal shift assay, 50 µL of lysates (3 mg/mL) from steatosis Hela cells or CPT1A-overexpressing E.coli were incubated with 100 µM of baicalin at each temperature point from 36 to 76 °C for 4 min. The samples were centrifuged at 20,000 g for 10 mins at 4 °C to separate the supernatant and pallet. 12 µL of the supernatant was mixed with 3 µL of 5x loading buffer and then separated on a 10 % SDS-PAGE for immunoblotting analysis of CPT1A. For the dose-dependent thermal shift assay, 50 µL of lysates (3 mg/mL) were incubated with various concentrations of baicalin (between 0 to 1000 µM) at 52 °C for 4 min. The supernatant was isolated by centrifugation and subjected to immunoblotting analysis of CPT1A as described above.

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CPT1 activity assay [3]
The CPT1 activity assay was performed according to a modified protocol reported previously, which basically measures the conversion of deuterium-labeled carnitine ([D9]-carnitine) to deuterium-labeled palmitoylcarnitine ([D9]-palmitoyl-carnitine). The reaction buffer is composed of 150 mM KCl, 2 mM EDTA, 4.5 mM glutathione, 1 mg/mL BSA, 0.1 mM palmitoyl-CoA and 0.1 mM [D9]-carnitine in PBS. Baicalin was added as indicated concentrations. We used the pure but inactive recombinant CPT1A to establish a protein concentration standard curve and use western blot to estimate the amount of CPT1A in various cell, tissue and E.coli lysates when we performed the CPT1A enzymatic assay. These numbers were then used to normalize CPT1A activity to an absolute scale (conversion of nmol of palmitoyl-carnitine per min per m g of CPT1A) To test cellular CPT1A activity, HeLa cells were lysed in PBS with 0.1% of Triton X-100 on ice, and the lysates were adjusted to 2 mg/mL. The enzymatic reaction was started by adding 20 µL of lysates to 100 µL of the reaction buffer and incubated at 37 °C for 10 mins. At the end of reaction, 0.3 nmol of [D3]- palmitoyl-carnitine was added as internal standard and 600 µL of precooled methanol was added to extract the small-molecule metabolites on ice. The reaction mixture was centrifuged at 20,000 g for 5 mins at 4 °C. Then 400 µL of the supernatant were taken out and mixed with 600 µL of distilled water to make 1 mL of the final sample volume for measurement of [D9]-palmitoyl-carnitine by LCMS. The LC-MS system is composed of an AB SCIEX 5500 triple-quadrupole mass spectrometer and a SHIMADZU DGU-20A liquid chromatography instrument with an 6 Agilent column. The buffer gradient is 100%-0 Buffer A (100% water, 0.1% formic acid) and 0%-100% Buffer B (100% acetonitrile, 0.1% formic acid) for 10 mins. The absolute concentration of [D9]-palmitoyl-carnitine is calculated by comparing the peak areas of [D9]-palmitoyl-carnitine and [D3]-palmitoyl-carnitine. Two reactions were prepared in parallel with or without 100 µM of malonyl-CoA treatment and the activity of CPT1A was calculated as the difference in [D9]-palmitoyl-carnitine between with and without malonyl-CoA treatment. To test CPT1A activity in primary hepatocytes, hepatocytes were lysed in PBS with 0.1% of Triton X-10 on ice, and the lysates were adjusted to 2 mg/mL. The enzymatic reaction was started by adding 10 µL of lysates to 100 µL of the reaction buffer and incubated at 37 °C for 10 mins. To test CPT1A activity from mitochondria, the mitochondria were isolated by Mitochondria Isolation Kit for Cultured Cells. Then the mitochondria were lysed in PBS with 0.1% of Triton X-100 on ice, and then the lysates were adjusted to 0.2 mg/mL. 10 µL of mitochondrial lysates were used to assay CPT1A activity as described above. To test CPT1A activity from mouse livers, the tissues were homogenized with 0.1 % of Triton X-100 to obtain the lysates at 2 mg/mL. 10 µL of liver lysates were used to assay CPT1A activity as described above. To test recombinant CPT1 activity from E. coli, 10 µL of lysates (1L E. coli cultures lysed by 50mL PBS with 0.1% triton-100) of E. coli overexpressing each CPT1 construct were used to assay CPT1 activity as described above except that the measurement of [D9]-palmitoyl-carnitine was performed without malonyl-CoA treatment as the control in calculating the CPT1 activity.

Cell culture and experimental design [2]
SH-SY5Y cells were cultured in RPMI-1640 medium supplemented with 15% fetal bovine serum at 37°C in an air atmosphere containing 95% air and 5% CO2 with a saturated humidity. Upon a confluence of 60~70%, the SH-SY5Y cells were divided into: (i) control group, incubated in RPMI-1640 medium; (ii) thrombin group, which was subject to thrombin induction (40 U/L) for 6 h based on our pre-experiment; and (iii) baicalin groups, which were treated by baicalin (5 μM, 10 μM, or 20 μM) for 2 h before induction of thrombin.

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Cell viability assay [2]
Cell viability was measured using MTT assay as previous described [24]. Briefly, 15 μl of the MTT solution (5 mg/mL) was added to each well and incubated for 4 h at 37°C. After removing the supernatant, 80 μL DMSO were added into each well. The absorbance was measured at 492 nm using a microplate reader. All experiments were performed in triplicate.

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Apoptosis detection by flow cytometry [2]
Cells were harvested by trypsinization without EDTA, and washed twice in PBS. After staining with AnnexinV/fluorescein isothiocyanate (FITC) and propidium iodide (PI), the cells were immediately analyzed by flow cytometer.

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SILAC-ABPP The SILAC [3]
ABPP experiments were performed based on protocols adapted from previous reports. Hela cells were passaged 10 times in SILAC DMEM with 10 % SILAC FBS, 1 % penicillinstreptomycin, and 100 µg/mL [13C6,15N4]L-arginine-HCl and [13C6,15N2]L-lysine-HCl or L-arginine-HCl and L-lysine-HCl. Before ABPP experiment, cells were serum-starved for 24 h and treated with 1 mM free fatty acids for 24 h to induce accumulation of lipid droplets. The cells were harvested and stored at -80 °C for further experiments. Frozen cell pellets were re-suspended in PBS with 0.1% Triton X-100, sonicated and separated into soluble and insoluble fractions by ultracentrifugation at 100,000 g for 45 min. The soluble protein concentration was determined using the BCA protein assay (Pierce™ BCA Protein Assay Kit, Thermo Fisher Scientific) on a microplate reader. The lysates were adjusted to 2 mg/mL and treated with 2 mM baicalin (10 µL of 200 mM stock in DMSO) or DMSO, labeled with 200 µM of the baicalin probe (10 µL of 20 mM stock in DMSO) or DMSO, and then put on ice under UV radiation at 365 nm for 1 h. The light and heavy probelabeled proteome were mixed equally in a 1:1 ratio and immediately precipitated with chloroform-methanol. Then the precipitations were re-suspended with 1.2 % SDS/ PBS and combined with 300 µM biotin-azide, 100 µM TBTA, 1 mM TCEP, and 1 mM 7 CuSO4 for 1 h. After this reaction, the proteomes were extracted again with chloroformmethanol to remove redundant reagents. The protein interphase was washed with cold methanol, solubilized with 1.2 % SDS/ PBS and diluted 5x with PBS. The solubilized proteins were incubated with streptavidin beads (100 µL of slurry) at room temperature for 3 h with rotation. The beads were then washed with 5 mL of PBS three times and 5 mL of water three times before being transferred to a screw-top Eppendorf tube. The enriched proteins were denatured in 6 M urea/PBS, reduced with 10 mM dithiothreitol at 65 °C for 15 mins and blocked with 20 mM iodoacetamide (Sigma-Aldrich) at 35 °C for 30mins in dark with agitation. The reaction was diluted with 950 µL of PBS and centrifuged at 1400 g for 3 mins. The supernatant was removed. Then the beads were added with a premixed solution of 200 µL of 2 M urea/PBS, 2 µL of 100 mM calcium chloride in water and 4 µL of trypsin (20 µg reconstituted in 40 µL of the trypsin buffer, Promega) and incubated at 37 °C with agitation overnight. Next day the mixture was transferred to a Bio-spin filter and the digested solution was eluted into a low-adhesion tube by centrifugation (1000 g). The eluents were acidified with 5% formic acid. As illustrated in Fig. 2c, a series of SILAC-ABPP experiments using the baicalin probe with or without competition of the native baicalin compound were performed. Briefly, light proteomes were always treated with DMSO and labeled by the baicalin BP probe via UV-induced photo-crosslinking and then mixed with the heavy proteomes processed as described below. In the "BP control" experiment, heavy proteomes were treated with DMSO and labeled with a blank DMSO solution with UV radiation. In the "UV control" experiment, heavy proteomes were treated with DMSO and labeled with the baicalin BP probe but without UV radiation. In the "CP control" experiment, heavy proteomes were treated with DMSO and labeled with the baicalin CP probe (i.e., without the benzophenone moiety) with UV radiation. In the "competition" experiment, heavy proteomes were competed with baicalin and labeled by the baicalin BP probe with UV radiation. The three control experiments were designed to eliminate false positive targets from indirect and/or non-specific binding to the probe. Three replicates were performed for each control and competition experiment and all the proteins quantified were listed in Dataset S1.

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

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

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.60 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 (5.60 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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: 20 mg/mL (44.81 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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 (Please use freshly prepared in vivo formulations for optimal results.)