Research of triterpenoids and carotenoids in Cosmos bipinnatus Cav. raw materials

Abstract

Introduction. Triterpenoids and carotenoids are natural biologically active substances produced by plant cells. Triterpenoids have inherent antitumor, anti-inflammatory, antimicrobial, hepato- and cardioprotective activity. Carotenoids are important antioxidants that prevent the development of many diseases, maintain health and improve the quality of human life. Valuable types of pharmacological action of substances of terpene nature explain the attention to them of scientists all over the world. The sources of these substances are representatives of the aster family (Asteraceae), in particular Cosmos bipinnatus Cav. Scientists from Thailand, Nigeria, India and other countries of the world paid attention to the study of terpene derivatives of this plant. Cosmos bipinnatus Cav. cultivated in Ukraine as an ornamental plant. Compounds of terpene nature in the raw materials of Cosmos bipinnatus Cav., which was grown in our country, had not been studied before, so it was appropriate to study these substances for further standardization of raw materials and the development of herbal medicinal products based on them. Materials and methods. For the study, we used grass and flowers of Cosmos bipinnatus Cav., which were collected during the flowering phase of the plant in July - August 2021/2022 in the Kharkiv region (Ukraine). For the extraction of triterpene compounds, 0.05 g of raw material was placed in a vial with a capacity of 2 ml, an internal standard and 0.6 ml of solvent (methylene chloride) were added. As an internal standard, tridecane was used at the rate of 50 μg per sample, followed by the calculation of its concentration. The sample was kept for 3 hours at a temperature of 50°C in an ultrasonic extractor or at room temperature for a day. The extract was poured into a vial with a capacity of 2 ml and concentrated by purging (100 ml/min) with pure nitrogen to a residual extract volume of 10 μl. The introduction of the sample (3 μl) into the chromatographic column was performed in splitless mode for 0.5 min. The following chromatographic conditions were observed during the analysis: Agilent Technologies 6890 chromatograph with 5973 mass spectrometric detector, chromatographic column – capillary DB-5, internal diameter 0.25 mm, length 30 m; velocity of carrier gas (helium) 1.2 ml/min; evaporator temperature 350ºС, thermostat temperature was programmed from 50º to 320ºС at a rate of 4 deg/min. The NIST05 and WILEY 2007 mass spectra library with a total of more than 470,000 spectra were used for component identification in conjunction with AMDIS and NIST identification programs. The internal standard method was used to calculate the quantitative content. The content of components (C, mg/kg) was calculated according to the formula: C=К1*К2, where К1=П1/П2 (П1 – the area of the peak of the substance under investigation, П2 – the area of the peak of the standard); K2=50/M (50 is the mass of the internal standard (μg), which was introduced into the sample, M is the weight of the sample (g)). Identification of components was carried out by retention time and compliance of UV spectra with the substance-standard. For the extraction of carotenoids, 1.00 g of raw material was placed in a 100 ml flask and 30 ml of a mixture of hexane-acetone-ethanol-toluene (10:7:6:7), 2 ml of a 40% methanol solution of potassium hydroxide was added and left in a dark place at 16 hours at room temperature. After that, 30 ml of hexane and 10% sodium sulfate solution were added to the flask, the solution was vigorously shaken and left in a dark place for 1 h to separate the phases. The hexane phase was used for research. Analysis of carotenoids was performed by HPLC according to a known method. Research results. 5 triterpene compounds and 5 carotenoids, of which 4 pentacyclic and 1 tetracyclic saponin, were identified in the grass of Cosmos bipinnatus Cav. 7 triterpenoids, including 5 pentacyclic and 2 tetracyclic saponins, and 6 carotenoids were found in the flowers of this plant. Among the pentacyclic saponins, β-amyrin (45.20 mg/kg) and α-amyrin (23.55 mg/kg) prevailed in the grass of the studied plant, β-amyrin (19.37 mg/kg), lupeol dominated in the flowers (14.72 mg/kg) and α-amyrin (13.21 mg/kg). Among the carotenoids, β-carotene (70.15 mg/kg) prevailed in the grass of Cosmos bipinnatus Cav., in the flowers of the plant, β-carotene (78.50 mg/kg), lutein (52.15 mg/kg) and violaxanthin (40, 72 mg/kg). The triterpenoids 24-methylenecycloartanol, taraxasterol and the carotenoid neoxanthin were identified in the flowers of Cosmos bipinnatus Cav. The obtained results are important for substantiating the complex use of raw materials of Cosmos bipinnatus Cav. Conclusions. The conducted studies prove the presence of important triterpenoids and carotenoids in the grass and flowers of Cosmos bipinnatus Cav., their content allows predicting the pharmacological effect of the studied raw material and the prospect of developing medicines based on it.

Keywords: Cosmos bipinnatus Cav., triterpenoids, carotenoids, GC/MC, HPLC.