Phytochemicals from Calophyllum canum Hook f. ex T. Anderson and their neuroprotective effects

Abstract Previous phytochemical investigations reported that Calophyllum spp have biosynthesized a wide range of bioactive phenolics such as xanthones and coumarins. The phytochemical study conducted on the stem bark of C. canum has led to the isolation of eight trioxygenated xanthones namely: 5-methoxytrapezifolixanthone (1), 5-methoxyananixanthone (2), caloxanthone C (3), 1,5-dihydroxy-3-methoxy-4-isoprenylxanthone (4), 6-deoxyisojacareubin (5), euxanthone (6), trapezifolixanthone (7), ananixanthone (8), together with three common triterpenoids, β-sitosterol (9), friedelin (10), and stigmasterol (11). Furthermore, xanthones 1 and 2 were isolated for the first time as naturally occurring xanthones from the plant extract. The structures of these compounds were identified and elucidated using advanced spectroscopic techniques such as 1 D & 2 D NMR, MS, and FTIR. The neuroprotective property of selected compounds was tested through in vitro stroke model. Among all tested compounds, 1 µm of compounds 8, 9, and 10 showed significant neuroprotective activity via reduction of apoptosis by ∼ 50%. Graphical Abstract


Introduction
The genus Calophyllum is a large group of trees that belongs to the Calophyllaceae family which is native to the pantropical region such as Southeast Asia (Zailan et al. 2021). Over the past few years, numerous phytochemical studies conducted on selected Calophyllum species unveiled that this genus is well known for biosynthesizing active compounds from phenolic groups, especially xanthones and coumarins (Taher et al. 2021). Moreover, this genus exhibited extensive biological activities like antimalarial, antiviral, antisecretory, antibacterial, chemopreventive, cytoprotective, analgesic, antitumour-promoting, and cytotoxic activity (Gupta and Gupta 2020). In ethnobotany, various parts of the Calophyllum plant were utilized as traditional remedies in treating malaria, bronchitis, gastric and hepatic disturbances, pain, wound infections, inflammation, rheumatism, varicose, haemorrhoids, chronic ulcer and acts as a diuretic (Zailan et al. 2021).
'Bintangor merah', scientifically known as Calophyllum canum is a 35 m tall tree ranging from medium-sized to high shrubs, inhabiting a well-drained ground and peat swamps up to 1200 m altitude. The latex was utilized for tuba fishing whilst the wood was an essential source of timber. The most recent phytochemical investigation on the C. canum stem bark has led to the isolation of a new xanthone dimer, namely, biscalaxanthone, along with the known trapezifolixanthone, trapezifolixanthone A, taraxerone, and taraxerol (Taher et al. 2021). In addition, few works of literature have reported its biological activities such as cytotoxicity, anti-bacterial, and anti-oxidant (Susanti et al. 2011;Alkhamaiseh et al. 2012;Lim et al. 2016;Ramli et al. 2019;Taher et al. 2021, Zailan et al. 2021. Stroke is a neurological disorder caused by an abrupt interruption of blood flow to the brain resulting in loss of neurological function. There is still no neuroprotective drug licensed for use in treatment of ischaemic stroke (Bharate et al. 2018). The neuroprotective properties of euxanthone (6) have been reported, showing its therapeutic potential in Alzheimer's Disease (Yuan et al. 2018). b-sitosterol (11) was reported to be effective in an animal model of Alzheimer's Disease (Ye et al. 2020).
To date, the scientific report on the phytochemical studies and biological activities of C. canum is limited. Therefore, in the present investigation conducted, we report the isolation and characterization of 11 compounds (1-11) from the stem bark of C. canum. The neuroprotective activity of the selected compounds were reported by using in vitro stroke model.

Neuroprotective activity
The present study utilised a PC12 cell-based stroke model for identifying potential leads from compounds isolated from the stem bark of C. canum. The selected compounds 6-10 were screened for their neuroprotective properties by using an in vitro stroke model (Chua and Lim 2021). All the compounds tested reduced apoptosis compared to untreated control, with ananixanthone (8), b-sitosterol (9), and friedelin (10) showing significant reduction of the same order as the known neuroprotectant DPAT (Supplementary material, Figure S37). This is the first report on the neuroprotective activity of C. canum and its constituents (6-10) by using an in vitro stroke model approach.
Based on the comparison of the structures between the isolated xanthones (6-8) (Supplementary material, Figure S38), the positioning of the free hydroxyl group at C-7 in compound 6 might contribute to apoptosis as the compound did not show any significant activity. This is because the placement of the hydroxyl moiety in the nucleus of xanthones plays a vital role in determining whether a biological activity is enhanced or suppressed (Salman et al. 2019). Nevertheless, the presence of the pyrano ring and the prenyl group within the positional isomers of the xanthones (7 and 8) could attribute towards protective effect in minimizing cell death (Pinto et al. 2021). However, the attachment of prenyl group at C-2 and the fusion of pyrano ring at C-3 and C-4 in compound 8 gave better neuroprotective activity in reducing apoptosis by 41%. Apart from that, the closed ring structure of pentacyclic terpenoid, compound 10 enhanced the neuroprotective effect (reduction in apoptosis by 54%) in the in vitro stroke model compared to the tetracyclic terpenoid structure of compound 9 (48% attenuation of apoptosis) which consists of a side-chain on carbon 17. The neuroprotective effects could be observed due to the structural pattern of the reported terpenoids present in compounds. Therefore, further study of the neuroprotective potential of other phytochemicals from C. canum is necessary to elucidate the structure-activity relationship of the xanthones and terpenoids in order to determine the functional groups that are responsible for the neuroprotective activity.

Experimental
See Supplementary Data.