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 dosage was chosen based on a previous pharmacokinetic study that determined the final plasma concentration of baicalin to be 0.8 μg/mL 6 hours after ingestion [3]. Due to the low bioavailability of baicalin, we used a relatively high (but safe) dose in our animal studies. Based on previous pharmacokinetic analyses, the steady-state plasma concentration of baicalin after oral administration of 400 mg/kg was expected to be 0.8 μg/mL (41). Therefore, it is conceivable that by optimizing the structure-activity relationship of baicalin through medicinal chemistry and combining it with a more in-depth structural study of the CPT1-baicalin interaction, it is possible to obtain novel synthetic anti-obesity drugs with better pharmacodynamics and pharmacokinetics. To this end, we preliminarily explored the activity of an acetylated derivative, Ac-baicalin, which is expected to have higher bioavailability. The results showed that structural modification of the flavonoid core by acetylation disrupted the binding of CPT1A, and the loss of CPT1A activation eliminated the lipid-lowering effect of Ac-baicalin (SI Appendix, Figure 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 7-O-glucuronide of baicalein, belonging to the glycosidic flavonoid family. It is the active ingredient of the traditional Chinese medicine Scutellaria baicalensis. Baicalin possesses various pharmacological activities, including nonsteroidal anti-inflammatory drugs, EC 3.4.21.26 (prolyl oligopeptidase) inhibitors, prodrugs, plant metabolites, ferroptosis inhibitors, neuroprotective agents, antitumor agents, cardioprotective agents, anti-atherosclerotic agents, antioxidants, EC 2.7.7.48 (RNA-directed RNA polymerase) inhibitors, anticoronavirus agents, and antibacterial agents. It is a glucuronic acid, glycosidic flavonoid, dihydroxyflavonoid, and monosaccharide derivative functionally related to baicalein. It is the conjugate acid of baicalin (1-). Baicalin has been reported to be found in creeping Scutellaria baicalensis, climbing Scutellaria baicalensis, and other organisms with relevant data. See also: Scutellaria baicalensis root (part). Background: Renal ischemia-reperfusion injury (IRI) increases the risk of acute renal failure, delayed recovery of transplanted kidney function, and early mortality after kidney transplantation. Its pathophysiological mechanisms include oxidative stress, mitochondrial dysfunction, and immune-mediated damage. Baicalin, a flavonoid glycoside isolated from Scutellaria baicalensis, has been shown to possess antioxidant, anti-apoptotic, and anti-inflammatory properties. Therefore, this study evaluated the effects of baicalin on renal IRI in rats. Methods: Baicalin was administered intraperitoneally 30 minutes before renal ischemia. Serum and kidney tissue were collected 24 hours after reperfusion. Renal function and histological changes were assessed. Oxidative stress markers, Toll-like receptor (TLR) 2 and TLR4 signaling pathways, mitochondrial stress, and apoptosis were also detected. Results: Baicalin treatment reduced oxidative stress and histological damage, improved renal function, and inhibited pro-inflammatory responses and renal tubular cell apoptosis. Pretreatment with baicalin also reduced the expression of TLR2, TLR4, MyD88, p-NF-κB and p-IκB proteins, reduced caspase-3 activity, and increased the Bcl-2/Bax ratio. Conclusion: Baicalin may alleviate renal ischemia-reperfusion injury by inhibiting pro-inflammatory response and mitochondrial-mediated apoptosis. These effects are related to the TLR2/4 signaling pathway and mitochondrial stress. [1]

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Baicalin is a substance extracted from the dried root of Scutellaria baicalensis Georgi and has been shown to have neuroprotective effects. However, its exact mechanism is not fully understood. This study aimed to investigate the effect of baicalin on thrombin-induced SH-SY5Y cell damage and explore its possible mechanism. SH-SY5Y cells were treated with thrombin alone or pretreated with different concentrations of baicalin (5, 10, 20 μM) for 2 hours and then treated with thrombin. Cells untreated with thrombin and baicalin served as controls. Cell viability was detected by MTT assay. Apoptosis was analyzed by flow cytometry. The mRNA expression level of protease-activated receptor 1 (PAR-1) was detected by real-time quantitative PCR. The protein expression levels of PAR-1, Caspase-3 and NF-κB were detected by Western blotting. Baicalin reduced cell death after thrombin treatment in a dose-dependent manner, while inhibiting NF-κB activation and PAR-1 expression. In addition, baicalin also reduced Caspase-3 expression. The above results suggest that baicalin may prevent cell damage after thrombin stimulation by inhibiting PAR-1 expression and NF-κB activation. [2] Obesity and related metabolic diseases are becoming a global epidemic, leading to rising mortality and high healthcare costs. No effective treatment has yet been found. Based on the observation that baicalin (a flavonoid compound derived from the Chinese herb Scutellaria baicalensis) has unique anti-steatodendronal activity, we performed quantitative chemical proteomics analysis and identified carnitine palmitoyltransferase 1 (CPT1) (a regulatory enzyme of fatty acid oxidation) as the key target of baicalin. Flavonoids directly and selectively activate hepatic CPT1 subtypes, thereby accelerating lipid influx into mitochondria for oxidation. Long-term use of baicalin can improve diet-induced obesity (DIO) and hepatic steatosis, and systematically improve other metabolic disorders. Disruption of the predicted binding site of baicalin on CPT1 can completely eliminate the beneficial effects of this flavonoid compound. We found that baicalin, as an allosteric CPT1 activator, opens up new avenues for the drug treatment of DIO and its related 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.)