Baicalin is one of the major bioactive constituents of Scutellariae Radix, but the biotransformation of it is poorly understood. In this paper, the metabolism of baicalin in rat was studied. Nine metabolites including one new compound were isolated and identified structurally. The plausible scheme for the biotransformation pathways of baicalin in the rats was deduced. And the main metabolites were evaluated for their antioxidation and anti-inflammation biological activities for the first time.
Scutellariae Radix, the root of
It is well known that the process of drug metabolism affects therapeutic effects of drug. The biotransformation of baicalin is poorly understood, and that is due in part to difficulties that have been encountered in obtaining enough amounts to identify the structure of the metabolites and study the bioactivities of them. Although some works on the metabolism of baicalin have been investigated with the development of chromatography-spectrographic technology, because of the lack of metabolites quantitatively, many questions about the biological activities of the metabolites still remained after administration [
Baicalin was isolated and purified from Scutellariae Radix according to the method reported previously [
Seven-week old Wistar rats (five males and five females), weighing 180 to 210 g, were used for the experiments. For isolation of metabolites, the rats were orally administered baicalin (500 mg/Kg) suspended in 0.5% CMC-Na solution with repeated dosing three times, then urine and feces were obtained by using a metabolic cage for 48 h. All samples were stored below −20°C until use.
Urine samples from rats were combined, filtrated, and adjusted to PH 4 with HCl and performed by chromatographic separation on a resin column eluting with water (part I), C2H5OH/H2O (3 : 7) (part II), and C2H5OH/H2O (9 : 1) (part III). Part III was subjected to ODS column eluting with CH3CN/H2O gradiently (from 95% H2O to 65% H2O) to obtain the compounds
Feces samples from rats were combined, suspended in the water and adjusted to PH 7 with NaHCO3-saturated aqueous solution, and then filtrated. The filtrate was adjusted to PH 4 with HCl and subjected to a resin column eluting with water (part I), C2H5OH-H2O (3 : 7) (part II), and C2H5OH-H2O (9 : 1) (part III). Then part II and part III were combined and subjected to Sephadex LH-20 eluting with C2H5OH-H2O (from 100% H2O to 30% H2O): C2H5OH-H2O (2 : 8) fraction from LH-20 gave
Semipreparative high-performance liquid chromatography was performed on an ODS column (YMC-pack ODS-A, 10 × 250 mm, 5
The NOE correlations on the NOESY spectra of metabolites
The plausible scheme for the biotransformation pathways of baicalin in the rats.
The effects of baicalin (
Livers were obtained from rats and disposed of the blood. Twenty g of the livers was divided into pieces and prepared into homogenate with physiological salt solution by refiner about 8000 r/min and then added physiological salt solution to 400 mL to be 5% tissue homogenate. Saline was used for blank; extract of ginkgo biloba leaves injection 250 mg/mL was used for positive control; baicalin (
All the numbered tubes were added with 1.5 mL 5% tissue homogenate, reference substance flavonoids extracts of ginkgo, and metabolites of baicalin. Negative control was physiological saline. All the tubes were incubated at 37°C for 1 h, and 1.5 mL 20% trichloroacetic acid was added. All the tubes were mixed, and standing for 10 min, centrifuged at 3000 rpm for 10 min. The supernatant was added with 0.67% thiobarbituric acid and heated in boring water for 10 min. The cool solution was tested on spectrophotometer at 532 nm to obtain data. Based on the standard curve of malonaldehyde bis(diethyl acetal), malonaldehyde (nmol/mL) was calculated by multiplying parameter 68.89.
Wistar rats (weight 200 to 250 g) were used for experiments. The rats were decapitated and treated with celiac injection with 15 mL RPMI1640 culture medium. Irrigating solution was centrifuged at 1000 r/min for 10 min. The supernatant was discarded. The deposition was suspended in RPMI1640 culture medium (10% FBS) and incubated for 1 h, and nonadherent cell is discarded. Cells on the wall were washed by RPMI1640 culture medium (10% FBS), dyed by nigrosine, and counted on blood counting chamber. The cell density was adjusted to 60 × 104 cells/cm2. The cells were incubated overnight. The culture medium was changed to serum-free medium. LPS (lipopolysaccharide), different metabolites of baicalin, and L-NAME (NO synthase inhibitor) were added. After 24 h of incubation, the contents of
Baicalin was orally administered to rats. The collected urine and feces samples were extracted and analyzed as described in experimental part. In addition to baicalin, a total of 8 metabolites including baicalin (
Compared with reference substances,
Compared with baicalin, there are two groups of the data of glucuronide on the 1HNMR spectrum of
Compared with the 1HNMR spectrum of baicalin,
Compared with the 1HNMR spectrum of baicalin, there was no glucuronide signal but one methyl proton in these three metabolites. The molecular weight of them (m/z: 307.1 [M+Na]+, 282.9 [M-H]−) also showed m/z 14 more than baicalin. All of them showed A2A’B2 coupled system for ring-C without any substituent that was the same as baicalin. On the 1HNMR spectrum of
Compared with baicalin,
Based on the structures of these metabolites, a plausible scheme for the biotransformation pathways of baicalin in the rats was shown in Figure
The effects of baicalin (
Figure
The inhibition of metabolites on LPS-induced NO yielding.
As described above, baicalin was metabolized to baicalein, glucuronide, and methylated products though metabolism of intestinal bacteria and enterohepatic circulation. Although studies on the metabolism of drugs have been investigated with the development of LC-MS, the bioactivities on metabolites are still lacking of investigation. The results of bioactive experiments of these metabolites in our experiments demonstrated that baicalein and glucuronide showed significant potential on antioxidation and affections on LPS-induced NO yielding compared with reference substance. However, when substituents were replaced by alkyl, bioactivity was depressed. To our knowledge, this is the first time that the metabolites of baicalin were investigated on the antioxidation and anti-inflammation biological activities. These results suggested though baicalein had good biological availability, it could not be used for its instability, while glucuronides of baicalein will be the perspective lead compounds for their good stability and bioactivities.
The authors thank Mr. Wenjun Pan of Shenyang Pharmaceutical University for his experimental assistance. This work is partly supported by Shandong Provincial Natural Science Foundation (no. ZR2009CQ014) and Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry (no.