Angelicae pubescentis radix (APR) is widely applied in treating rheumatoid arthritis in China. Coumarins are the major active compounds of APR extract including columbianetin, columbianetin acetate, osthole, and columbianadin. The
Traditional Chinese Medicine (TCM) has been crucial to prevent and treat diseases. TCM was applied to treat the illness depending on the theory of Traditional Chinese Medicine, which contains viscera-state theory [
Angelicae pubescentis radix (APR) originates in the root of
Phytochemical study has demonstrated that coumarins are the main constituents in APR [
Although the pharmacokinetics or tissue distributions of osthole, columbianadin, and columbianetin in other plant extracts have been studied in previous literature [
Columbianetin, columbianetin acetate, osthole, and columbianadin were obtained from National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Acetonitrile (HPLC grade) was purchased from Dikma Technologies Inc. (Dikma, USA). Methanol (HPLC grade) and ethyl acetate (analytical grade) were purchased from Tianjin Concord Science Co. Ltd. (Tianjin, China). Formic acid was purchased from Merck (Darmstadt, Germany). Deionized water was purified with a Milli-Q Academic ultra-pure water system (Millipore, Milford, MA, USA).
The liquid chromatography of Waters UPLC (Waters, America) system was used with the photodiode array detection (PDA). Chromatographic separation was performed on an ACQUITY BEH C18 column (4.6 mm × 100 mm, 1.7
For the preparation of the extract, 2 kg APR was cut into pieces and soaked with 75% ethanol (16L) for 2 h. The filtrate was concentrated by using a rotary evaporator under reduced pressure. The dry APR extract (100 mg) was accurately weighed in triplicate. The sample was dissolved with 5 mL of 70% methanol by using an ultrasonic bath for 30min and then cooled at room temperature. The supernatant of 2 mg/mL APR extract was injected into the UPLC-PDA system. The chromatography conditions of UPLC-PDA for separation and determination of four coumarins in APR extract were slightly modified according to previous studies [
The UHPLC-PDA chromatograms with APR extract sample (a) and standard solution of four compounds (b). 1: columbianetin, 2: columbianetin acetate, 3: osthole, 4: columbianadin.
Four stock solutions of columbianetin, columbianetin acetate, osthole, and columbianadin (1 mg/mL) were prepared in methanol and stored in 4°C, individually. The chemical structures of columbianetin, columbianetin acetate, osthole, and columbianadin are shown in Figure
Chemical structures of columbianetin, columbianetin acetate, osthole, and columbianadin.
The tissue samples were homogenized with 2-fold volume of 0.9% normal saline. The tissue homogenate was processed by liquid-liquid extraction. An aliquot of 200
According to the USFDA bioanalytical method validation guidelines [
Female Sprague-Dawley rats (250~280g) were fed under standard housing conditions and were acclimated at breeding room for at least one week beforehand. The rats were fasted for 12h before the experiment. All rats experiments were realized in strict accordance with the Guidelines for the Care and Use of Laboratory Animals and were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine (Tianjin, China). The 18 rats were divided randomly into three groups (n=6 per group). The rats were sacrificed by cervical dislocation at 4, 6, and 8 h after oral administration of 6 g/kg APR extract, respectively. The heart, liver, spleen, lung, kidney, uterus, ovary, and muscle were rapidly excised, rinsed, wiped, and weighed. All tissues were stored at -80°C until analysis.
The representative chromatograms consist of blank tissues homogenate, blank biosample with standard substances, and tissues samples after oral administration of APR extract as shown in Figure
Representative tissue chromatogram of (a) blank heart tissue, (b) blank heart tissue spiked with the standard compounds at LLOQ, and (c) real samples after administration of APR extract. 1: columbianetin, 2: columbianetin acetate, 3: osthole, 4: columbianadin.
The calibration curves were over the range of 1.6-20000 ng/mL, and the correlation coefficients (r) were more than 0.99. These indicated that calibration curves exhibited good linearity to quantify the four coumarins. The lower limit of quantitation (LLOQ) of four analytes was set at 1.6 ng/mL with the S/N value approximating 5.
The results of the precision and accuracy of the four analytes are summarized in Table
The precision and stabilities of columbianetin acetate, columbianetin, osthole, and columbianadin.
Concentration |
Intra-day precision | Inter-day precision | Autosampler for 24 hours stability | ||||
---|---|---|---|---|---|---|---|
Accuracy(%) | RSD(%) | Accuracy(%) | RSD(%) | Accuracy(%) | RSD(%) | ||
Columbianetin | 80 | 95.9 | 5.20 | 94.2 | 8.71 | 99.8 | 7.83 |
500 | 93.9 | 0.35 | 96.2 | 2.11 | 97.9 | 2.89 | |
2000 | 93.8 | 0.74 | 97.3 | 1.91 | 96.0 | 9.48 | |
|
|||||||
Columbianetin acetate | 80 | 93.6 | 3.05 | 93.5 | 7.32 | 97.4 | 2.55 |
400 | 92.6 | 3.04 | 95.6 | 0.85 | 96.7 | 7.63 | |
2000 | 91.8 | 5.71 | 90.4 | 1.89 | 85.4 | 3.14 | |
|
|||||||
Osthole | 160 | 97.3 | 5.29 | 98.3 | 4.32 | 99.2 | 5.28 |
500 | 89.2 | 0.91 | 91.6 | 1.67 | 97.1 | 12.8 | |
2000 | 91.9 | 2.68 | 93.6 | 2.64 | 88.4 | 5.39 | |
|
|||||||
Columbianadin | 160 | 92.6 | 5.77 | 93.8 | 6.54 | 97.1 | 5.73 |
500 | 93.4 | 0.51 | 95.8 | 2.32 | 99.1 | 3.30 | |
2000 | 93.7 | 0.74 | 96.9 | 1.47 | 95.6 | 8.74 |
The results of recovery are listed in Table
The recoveries of columbianetin, columbianetin acetate, osthole, and columbianadin.
Tissues | columbianetin | columbianetin acetate | ||||||||||
Low | Middle | High | Low | Middle | High | |||||||
Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | |
(%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | |
|
||||||||||||
Heart | 81.8 | 3.96 | 94.6 | 1.72 | 93.8 | 0.69 | 92.8 | 3.80 | 82.8 | 4.74 | 83.8 | 2.55 |
Liver | 87.8 | 2.67 | 88.7 | 0.72 | 94.2 | 1.57 | 85.7 | 7.03 | 82.1 | 10.5 | 87.1 | 2.97 |
Spleen | 89.5 | 4.09 | 99.1 | 1.55 | 97.4 | 1.93 | 80.8 | 9.63 | 80.7 | 3.92 | 93.2 | 1.19 |
Lung | 97.7 | 2.58 | 99.4 | 8.71 | 97.8 | 1.46 | 84.3 | 10.3 | 85.8 | 9.46 | 86.1 | 5.15 |
Kidney | 97.4 | 4.64 | 95.8 | 5.80 | 95.5 | 0.61 | 84.6 | 4.71 | 81.1 | 4.75 | 91.7 | 3.02 |
Uterus | 93.0 | 8.54 | 95.7 | 8.83 | 97.3 | 3.65 | 97.6 | 1.02 | 86.2 | 4.13 | 88.4 | 7.43 |
Ovary | 99.3 | 5.82 | 92.6 | 3.28 | 97.9 | 7.40 | 83.1 | 10.4 | 85.7 | 5.75 | 89.5 | 5.21 |
Muscle | 92.1 | 6.42 | 93.9 | 0.35 | 93.8 | 0.74 | 91.3 | 6.04 | 98.3 | 3.22 | 90.1 | 5.71 |
|
||||||||||||
Tissues | osthole | columbianadin | ||||||||||
Low | Middle | High | Low | Middle | High | |||||||
Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | Recovery | RSD | |
(%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | (%) | |
|
||||||||||||
Heart | 91.2 | 7.70 | 98.6 | 2.75 | 94.3 | 3.55 | 82.1 | 1.83 | 80.2 | 4.73 | 88.4 | 1.13 |
Liver | 93.1 | 4.42 | 94.6 | 5.87 | 93.1 | 5.93 | 81.3 | 4.84 | 81.9 | 1.92 | 84.3 | 5.68 |
Spleen | 95.1 | 6.40 | 95.7 | 7.61 | 90.9 | 3.33 | 81.0 | 3.11 | 83.1 | 6.19 | 91.9 | 2.00 |
Lung | 94.3 | 9.37 | 99.8 | 1.38 | 92.3 | 5.74 | 96.5 | 7.32 | 89.0 | 1.32 | 90.0 | 3.05 |
Kidney | 94.9 | 10.88 | 97.1 | 4.08 | 95.5 | 7.42 | 92.2 | 1.73 | 95.3 | 1.26 | 91.4 | 0.81 |
Uterus | 88.2 | 10.51 | 96.5 | 2.33 | 99.9 | 5.11 | 94.3 | 9.57 | 99.2 | 2.72 | 96.5 | 7.75 |
Ovary | 93.8 | 3.76 | 94.7 | 6.11 | 93.9 | 5.63 | 96.8 | 3.41 | 98.4 | 4.09 | 98.4 | 7.47 |
Muscle | 97.3 | 5.29 | 89.2 | 0.90 | 91.9 | 2.68 | 92.6 | 5.77 | 98.2 | 4.76 | 97.6 | 3.20 |
The stability of processed samples which were kept in an autosampler for 24 h is shown in Table
The tissue distributions of the four coumarins were investigated at 4, 6, and 8 h after given orally 6.0 g/kg APR extract to female rats. The examined tissues included the heart, liver, spleen, lung, kidney, uterus, ovary, and muscle. The concentrations of four coumarins in tissues are presented in Table
The concentrations of columbianetin, columbianetin acetate, osthole, and columbianadin in tissues (ng/mL)(n=6).
Tissues | columbianetin | columbianetin acetate | osthole | columbianadin | ||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|
4h | 6h | 8h | 4h | 6h | 8h | 4h | 6h | 8h | 4h | 6h | 8h | |
Heart | 735.0±316.5 | 814.6±192.3 | 86.9±80.2 | 77.4±41.2 | 175.0±50.0 | 45.3±40.7 | 183.5±56.0 | 227.4±39.3 | 135.5±28.6 | 0.00±0.00 | 54.3±36.8 | 0.0±0.0 |
Liver | 1467.0±913.4 | 2032±514 | 325.4±262.5 | 183.3±22.2 | 1677.0±42.4 | 94.0±86.4 | 669.3±286.5 | 1352±529.0 | 257.8±24.3 | 273.2±185.4 | 908.4±738.6 | 46.8±51.3 |
Spleen | 533.1±266.9 | 529.1±243.6 | 171.0±9.9 | 47.4±30.6 | 180.9±0.0 | 42.8±53.4 | 191.2±48.5 | 206.6±67.6 | 153.5±42.6 | 0.00±0.00 | 0.00±0.00 | 12.2±24.4 |
Lung | 653.0±306.1 | 647.4±227.5 | 95.3±72.0 | 76.6±46.9 | 94.5±70.9 | 16.9±17.7 | 275.0±101.6 | 188.3±49.0 | 125.8±24.7 | 0.00±0.00 | 0.00±0.00 | 0.0±0.0 |
Kidney | 810.3±460.5 | 933.7±295.8 | 222.1±41.5 | 104.7±45.7 | 180.9±0.0 | 50.5±40.9 | 221.2±86.1 | 264.1±19.6 | 162.5±38.2 | 136.3±60.9 | 0.00±0.00 | 142.2±47.4 |
Uterus | 565.1±129.8 | 582.6±130.5 | 155.1±34.8 | 140.5±49.2 | 428.3±47.0 | 77.7±76.9 | 261.0±57.0 | 382.6±83.5 | 167.0±42.3 | 133.7±44.6 | 0.00±0.00 | 0.00±0.00 |
Ovary | 614.6±341.2 | 604.9±132.2 | 91.4±16.3 | 204.8±42.6 | 292.9±145.0 | 98.6±38.8 | 358.5±78.3 | 403.5±97.6 | 224.0±20.2 | 118.7±68.5 | 126.0±9.7 | 0.00±0.00 |
Muscle | 712.2±176.4 | 663.3±239.3 | 117.4 ±92.8 | 55.0±10.7 | 79.7±49.4 | 32.9±31.7 | 154.7±9.7 | 118.1±82.9 | 126.4±7.0 | 0.00±0.00 | 0.0±0.0 | 0.00±0.00 |
The tendency graph of columbianetin, columbianetin acetate, osthole, and columbianadin in eight tissues at 4, 6, and 8 h after oral administration of APR extract (n=6).
A sensitive, reliable, and fast method was established and validated for the simultaneous tissue distribution of osthole, columbianetin, columbianetin acetate, and columbianadin after oral administration of APR extract in rat. The tissue distribution provides a process to understand the pharmacological activity of APR
The data used to support the findings of this study are included within the article.
The authors declare that they have no conflicts of interest.
Shujing Chen and the first author contributed equally to this study.
This research was supported by the National Natural Science Foundation of China (81503213), Special Program of Talents Development for Excellent Youth Scholars in Tianjin, Tianjin Research Program of Application Foundation and Advanced Technology (15JCYBJC29300), and PCSIRT (IRT-14R41).