Antiasthmatic Effect, Quantitative Analysis And Stability Of Labdane Diterpenoids From Coleus Forskohlii

Qiaoruisu is the dried herb of Coleus forskohlii (Willd) Briq, which belongs to the Labiatae family. The plant is indigenous to India. Only few amounts of the wild species were found in China, so the plant has been cultivated in Yunnan and Hunan Province. The main active constituents in C. forskohlii are labdane diterpenoids, including forskolin and isoforskolin. Forskolin, the main component in Indian species, has attracted much attention for its multifaceted pharmacological effects. Isoforskolin, the principal active component from China-origin C. forskohlii, possesses similar activity to forskolin of activating adenylyl cyclase, and exerts antispasmodic and ocular hypotensive effects. By far, Qiaoruisu drug preparations available in our country are used for the treatment of cough and asthma. Although there have been several studies on the antiasthmatic components in C. forskohlii, the activities of most of the labdane diterpenoids have not been reported. Existing methods for the quantitative analysis of C. forskohlii often use a single chemical marker or select several markers but not based on the related pharmacological investigation. Besides, the adopted sample preparation methods are generally labour-intensive, time-consuming and require large volumes of solvents. There have been a few studies on the in vitro stability and metabolism on forskolin, but none on isoforskolin has been reported by now. Therefore, the present study is to investigate the in vitro antiasthmatic effects of nine labdane diterpenoids isolated in our laboratory, to improve the quality evaluation method for the crude drug, and to study the in vitro stability and metabolism of forskolin and isoforskolin. The results provide useful information for the further development and application of domestic C. forskohlii.1. In vitro antiasthmatic effect of labdane diterpenoids in C. forskohliiThe antiasthmatic effects of nine labdane diterpenoids were investigated on histamine-induced spasm of isolated guinea pig tracheas. Forskolin, isoforskolin, forskolin G, forskolin J, forskolin A and forskolin I showed significant relaxation effects on spastic tracheas, of which EC50 values are 1.94±0.82,3.21±1.08,8.15± 1.96,3.16±2.76,12.04±5.95 and 29.24±4.32 μg/mL, respectively. The first five diterpenoids had maximum relaxation rates of over 60%, showing potent spasmolytic effects. Forskolin H,6-acetyl-l-deoxyforskolin and 1,6-diacetyl-9-deoxyforskolin had no significant spasmolytic effect.2. Determination of labdane diterpenoids in C. forskohliiFive antiasthmatic labdane diterpenoids (forskolin, isoforskolin, forskolin G, forskolin J and forskolin A) were set as chemical markers for the quality assessment of C. forskohlii. A matrix solid-phase dispersion (MSPD) method was established and optimized as the sample preparation method. C18 was chosen as the sorbent, and ethyl acetate:methanol (1:4, v/v) as the elution solvent. The quantification of the diterpenoids was achieved by HPLC-ELSD, and the identification of the five compounds was performed by HPLC-MS/MS. The HPLC-ELSD method provided good specificity, linearity, sensitivity, precision, accuracy, repeatability and stability. The proposed MSPD-HPLC-ELSD method is simple, efficient, less solvent-consuming, and applicable to the quality assessment of C. forskohlii.3. In vitro stability and metabolism of main active components in C. forskohliiA systemic study was performed on in vitro stability of forskolin and isoforskolin. (1) The hydrolysis products of forskolin and isoforskolin in basic solution were isolated and identified. The two compounds shared a common hydrolysis product of deacetylforskolin (forskolin D). (2) An HPLC-DAD method was established to determine the original drugs and main hydrolysis products of forskolin and isoforskolin in pH 1.5-8.5 buffer solutions. The degradation processes of both forskolin and isoforskolin fitted first-order kinetic equations. Isoforskolin had better stability than forskolin. (3) The stability of forskolin and isoforskolin in simulated gastric solution (SGF) and simulated intestinal solution (SIF) was investigated. In SGF, isoforskolin was stable but forskolin was not. In SIF, both compounds were stable. (4) An HPLC-MS/MS method was established to determine the metabolic stability of forskolin and isoforskolin in human liver microsome. The half-lives of forskolin and isoforskolin in human liver microsome were 14.7 min and 26.4 min, respectively.