PHYTOCHEMISTRY AND ANTICANCER BIOACTIVITY OF SECONDARY METABOLITES OF CRYPTOCARYA CRASSINERVIA AND C. APAMIFOLIA (LAURACEAE)

• Main Author: Nugraheni, Dian
• Format: Dissertations
• Language: Indonesia
• Subjects: Kimia
• Online Access: https://digilib.itb.ac.id/gdl/view/70513
• Institution: Institut Teknologi Bandung

Cryptocarya is one of the large genera in the Lauraceae family, which consists of about 350 species. The Cryptocarya plants, locally known in Indonesia as ”huru” or ”medang”, have economic values for building materials and perfumes. They have been used as traditional medicines. Phytochemical studies of Cryptocarya that have been reported generally include stem bark and wood. Meanwhile, research on leaf tissue is still limited. Only 68 species (19%) worldwide have been reported for their phytochemicals, 27 of which are species that exist in Indonesia. The study showed that this plant contains ?-pyrone, flavonoids, alkaloids as major components and lactones, stilbenes, phenylpropanoids, lignans, neolignans, coumarins, terpenoids, steroids, carboxylic acid derivatives, amides derivatives, polyketides as other components. Cryptocarya Indonesia is unique because it can produce compounds of stilbene, phenylpropanoid, neolignan, and amide derivatives that have never been discovered in Cryptocarya outside of Indonesia. Meanwhile, lactones, coumarins, and polyketides have not yet been identified in Cryptocarya Indonesia. Compounds from this genus have various activities, including anticancer, antibacterial, antiviral, antihyperglycemic, antiinflammatory, and antiAlzheimer's. Anticancer studies of those compounds have been examined on several cancer cells, including P-388, KB, NCl-H460, SF-268, MCF-7, SF-268, HT-29, and Ca9-22, which exhibit cytotoxicity that ranges from very active to inactive. Other anticancer studies have also been reported using the inhibitory properties of compounds from Cryptocarya towards tyrosine kinase. Based on this background, this research aimed to study the phytochemical and activity aspects of Indonesian Cryptocarya plants. The first objective was to study the phytochemicals of C. crassinervia’s leaves and C. apamifolia’s stem bark. Furthermore, the second objective was to study the anticancer activity of compounds isolated through cytotoxicity against murine leukemia cells P-388, inhibition towards RTK in vitro, and the interaction of isolated compounds with RTK in silico.Leaf samples of C. crassinervia were collected from Bogor Botanical Gardens, while the stem bark of C. apamifolia was obtained from Werekopa, Fak-Fak, Papua. The isolation and purification of compounds were carried out in several steps, including maceration of dried powder plants, followed by separation and purification using various chromatographic techniques (VLC, MPLC, GCC, RC, and PTLC). Meanwhile, the structure elucidation of the isolated compounds was carried out based on spectroscopic data from NMR, HRMS, CD, UV, and IR. In vitro analysis, the cytotoxic properties of the isolated compounds against P-388 cells were tested by the MTT method, while the inhibitory properties of tyrosine kinase were tested by the Hennek method. Meanwhile, in silico analysis of the interaction of isolated compounds with EGFR was carried out by the molecular docking method using Autodock Vina®. From the phytochemical studies conducted, nine compounds from two Indonesian Cryptocarya species (C. crassinervia and C. apamifolia) were successfully isolated and elucidated. Those compounds included one ?-pyrone, namely cryptocrassinervione (249), three glycosylated flavonols, i.e. kaempferol-3-O-rhamnosyl-2-O-apiofuranoside (250), afzelin (251), quercitrin (78), two flavanones, namely (S)-pinocembrin (23) and (±)-chartaceone A (27), and three stilbenes, i.e. pinosylvin (252), pinosyslvin monomethylether (201), and reflexanbene I (253). Compound 249 is a cinnamoyl hexaketide, which is presumably a development of the previously reported cinnamoyl pentaketide from this genus. The discovery of compound 249 revealed that the structural modification of the compound was still ongoing. Compound 249 also confirms that ?-pyrone is a marker compound from the genus Cryptocarya. The identification of compounds 23 and 27 proves that Cryptocarya only produces flavanones with a non-oxygenated B ring. Compound 27, which was discovered outside of Indonesia, explains that Cryptocarya Indonesia is also capable of producing this compound. Compound 27, an arylheptanoid-modified flavanone in ring A, and compound 253, a combination of stilbenes and monoterpenes, both have interesting skeletons that shed light on the capacity of this genus to produce compounds with distinctive skeletons. Meanwhile, the presence of compounds 201, 252, and 253 also affirms the existence of stilbenes, which only exist in Indonesian Cryptocarya. Furthermore, the same compound 253 and similar compound 250, which have also been obtained from other genera in the Lauraceae family (Lindera and Litsea), indicate a significant relationship between Cryptocarya and these two genera. The discovery of compounds 249 and 250 is very prominent because they are new compounds that have never been found before. The results of the isolation significantly contribute to the phytochemical and chemotaxonomic data of compounds in the genus Cryptocarya, specifically Indonesian Cryptocarya. From the activity studies, compound 249 was very active cytotoxic (IC50 4.8 ?M), compound 23 was moderate (IC50 34.6 ?M), and three other compounds (78, 250, and 252) were inactive (IC50 > 50.0 ?M) against P-388 murine leukemia cells. Compound 249, which exhibited the strongest activity, had an ?-pyrone ring suspected significantly contribute to the cytotoxicity towards P-388 cells.The compound framework’s effect also shows that compound 23 from the flavanone derivatives has better activity than the flavonol derivatives (78, 250, 251). The least polar compound is compound 249, which is followed by compound 23, and so on, with compounds 78, 250, 251 having more polarity due to the presence of their hydroxyl and glycoside groups. An increase in the observed polarity coincides with a decrease in cytotoxic activity. These findings imply that molecular polarity affects an isolated compound's cytotoxicity against murine leukemia P-388 cancer cells. The isolated compounds were also tested for inhibition against tyrosine kinase receptors (EGFR, HER2, HER4, IGF1R, InsR, KDR, PDGFR?, and PDGFR?). Compounds ?-pyrone (249), flavonoids (23, 78, 250, 251), and stilbenes (201, 252, 253) at a concentration of 10 ?M present selective inhibition of EGFR with a moderate percentage of EGFR inhibition and weak/no inhibition active against seven other tyrosine kinases. Among these compounds, compound 249 provided the greatest inhibition of EGFR (55%). Flavonoid and stilbene derivatives are only active at inhibiting the same receptor by 35 to 49%. The presence of ?-pyrone units is strongly suspected of having an important role in the inhibition. In addition, the isolated molecules are aromatic compounds that have hydroxy groups, which have the possibility of forming hydrogen bonds with receptors. This is supposed to affect the compound's ability to inhibit EGFR. Through molecular docking with RTK, the isolated compounds (23, 78, 201, 249, 250, 251, 252, and 253) occupy positions on the active site of EGFR. Some compounds (23, 201, 249, 251, and 252) are oriented similarly to erlotinib (commercial inhibitors), and several compounds (23, 78, 201, 249, 250, 251, and 252) have similar interactions with both or one of the key EGFR residues (Met769 and Thr766). Docking scores of the isolated compounds also showed good affinity values between -7.7 and -9.6 kcal/mol.. Meanwhile, the hydrogen bonds of those compounds have a distance of 2.70?3.19 Å, allowing for strong interactions in the EGFR active site. Compound 249, which has the greatest inhibition of EGFR in its in vitro studies, exhibits a similar orientation to erlotinib with the position of the pyrone ring inserted into the EGFR hinge. This compound also has interactions with two key EGFR residues and has good binding scores and interaction similarity, which are -8.3 kcal/mol and 67%, respectively. The outcomes of the binding of the isolated compounds 249 reveal the possibility that has the same orientation and interactions, such as the commercial inhibitor, erlotinib towards EGFR. According to the results of the in vitro and silico activity studies, a new ?-pyrone compound 249 from C. crassinervia leaves potentially as an anticancer agent.