Asarone Pharmacokinetics and Bioavailability Test

In recent years, some research publications indicate that the active components of Traditional Chinese Medicine (TCM) are becoming good choices for curing cancers and minimizing side reactions.Pharmacokinetics (PK), which is mainly used for investigating absorption,distribution,metabolism,and excretion of drugs in vivo

The application of Pharmacokinetics on TCM has the three advantages:

(1)Identifying and screening multi-components of TCM could clearly explain its effects;

(2)Clarifying and explaining the combination mechanism of active components in decoction;

(3)Showing and revealing the dynamic process of active components in vivo.

Asarone is based on a-asarone (2,4,5-trimethoxy-1-alkenylbenzene, also known as a-Asarone), the main active ingredient in the traditional Chinese medicine Acorus calamus. It is artificially synthesized to relieve cough and asthma. Anti-epileptic drugs. Since the blood concentration of the drug after entering the human body is very low, it is difficult to carry out pharmacokinetic studies and bioavailability determination. A quantitative detection method with high sensitivity and strong specificity must be found. Bioavailability refers to the extent and speed at which the active ingredients of the drug are released and absorbed from the preparation into the systemic circulation. Medicilon can provide customers with bioavailability research services.

Recent research results indicate that a rapid, sensitive, and effective method, high-performance liquid chromatography (HPLC) combined with mass spectrometry (MS), is useful for screening and identifying the components of a formula.

Established a reversed-phase high performance liquid chromatography method to determine the concentration of Asarone in human serum for the determination of the bioavailability of Asarone. Method: Shimpack CLCODS (150mm×6.0mm, 10μm) was used as the chromatographic column, methanol-water (80:20) was used as the mobile phase, the flow rate was 1ml/min, the fluorescence excitation wavelength was 265nm, the emission wavelength was 365nm, and the column temperature was 40°C. Results: The lowest detectable concentration of Asarone was 0.2ng/ml, and the linear range was 1～200ng/ml; intraday RSD≤6.74%(n=3), daytime RSD≤4.95%(n=5). Conclusion: This method is simple, sensitive, accurate, and suitable for the study of the pharmacokinetics and bioavailability of Asarone.

Experimental method

(1) Reagents and instruments

Asarone reference substance (provided by Guilin Second Pharmaceutical Factory, batch number: 981006); Asarone tablet 1, Asarone tablet 2 (both developed by Hubei Provincial Drug Inspection College, batch number: 20000301, 20000405); Asarone Injection (Guilin Second Pharmaceutical Factory, batch number: 981006); Methanol (Excellent grade, Beijing Chemical Factory); other reagents are of analytical grade. LC-6A type high performance liquid chromatography (HPLC) instrument, RF 535 type fluorescence detector (Shimadzn, Japan).

(2) Chromatographic conditions

Column: Shimpack CLC ODS (150mm×6.0mm. 10μm): Guard column: Shimpack G ODS (10mm×4.0mm, 10μm); mobile phase: methanol-water (80:20); flow rate: 1ml/min; column temperature ：40℃; Fluorescence excitation wavelength: 265nm, emission wavelength: 365nm.

(3) Blood sample processing

Take 0.2ml of serum, add 2.5ml of dichloromethane, vortex for 2min, then centrifuge at 20,000g for 10min, transfer all the lower layer to another test tube with a pointed bottom, place it in a 35℃ water bath, and evaporate dry with nitrogen. After the residue was dissolved in 50 μl methanol, 20 μl was injected.

(4) Preparation of standard curve

Accurately weigh 50 mg of Asarum reference substance, place it in a 50 ml measuring flask, dissolve it with methanol and dilute to the mark, and prepare a standard stock solution with a concentration of 1 mg/ml. Before use, it is diluted with methanol into a series of standard working solutions of a certain concentration, and the appropriate amount is accurately measured and added to 0.2ml of blank human serum to prepare a standard serum with a concentration of 1, 5, 25, 50, 100, 200 ng/ml , Follow the above blood sample processing method for processing.

(5) Calculation

Calculate the content of Asarone by external standard method.

Results

(1) Chromatographic behavior

Under chromatographic conditions, the peak shape of Asarum is symmetrical and steep, without interference from endogenous peaks, and the retention time is 7.68 min.

(2) The standard curve and the linear range

The concentration of Asarone (Y) is in the range of 1～200ng/ml, and it has a good linear relationship with the peak height (X). The regression equation is Y=20.87X-7.98, r=0.9998. The lowest detectable concentration in serum was 0.2ng/ml.

(3) Recovery rate and precision test

Within the concentration range of the standard curve, five concentrations of 2.5, 10, 40, 75, and 150 ng/ml were selected for the method recovery test. Each concentration was tested in parallel for 3 times in the day and 5 times in parallel in the day. It can be seen that the average recovery rate is (101.18±1.77)%, and RSD=1.75%.

Application

Using the determination method established in this article, the bioavailability of Asarone tablets using different production processes was studied. That is, 18 healthy volunteers were given single-dose cross-administration of Asarone Tablets 1 (120mg) and Asarone Tablets. After dose 2 (120mg) and asarone injection (16mg), the concentration of asarone in the subjects’ serum was measured. After calculation, the absolute bioavailability of Asarone Tablet 1 is (5.4±1.6)%, and Asarone Tablet 2 is (9.5±2.5)%. Taking Asarone Tablet 1 as the reference preparation, the relative bioavailability of Asarone Tablet 2 is (181.9±15.2)%. It is proved that the bioavailability of Asarone Tablet 2 (modified by the production process) has increased by 81.9%.

Asarum has a maximum absorption at a wavelength of 258nm, but its concentration in human blood is extremely low, only reaching the ng/ml level, and the use of ultraviolet detectors cannot meet the measurement requirements. According to the characteristics of the chemical structure of Asarone (benzene ring containing 3 methoxy groups and 1 propenyl group), the fluorescence detection method is used, and the sensitivity is greatly improved, which is higher than that of thin layer chromatography and HPLC-UV method. It can quickly enter the red blood cells, and its concentration in whole blood, blood cells and plasma is the highest in blood cells.” Therefore, when separating serum, be careful not to hemolysis.

Asarone has a low melting point, and the temperature should not exceed 35°C when the dichloromethane is heated and evaporated. In addition, the concentration range determined by this method is wide, and the low concentration quantification often has a large error. Therefore, the standard curve can be divided into two concentration ranges during operation to reduce the measurement error.

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