Phenolics and triterpenoids from stem bark of Calophyllum lanigerum var. austrocoriaceum (Whitmore) P. F. Stevens and their cytotoxic activities

Abstract Genus Calophyllum is well-known for its phenolic constituents, especially coumarins, which have shown to have a wide range of significant biological activities. In this study, four known phenolic constituents and two triterpenoids have been isolated from the stem bark of Calophyllum lanigerum. The compounds were two pyranochromanone acids are known as caloteysmannic acid (1), isocalolongic acid (2), a simple dihydroxyxanthone, namely euxanthone (3), one coumarin named calanone (4), and two common triterpenoids, friedelin (5), and stigmasterol (6). Chromanone acids were reported for the first time in this Calophyllum species. Cytotoxic evaluations were carried out on n-hexane extract (87.14 ± 2.04 µg/mL; 81.46 ± 2.42 µg/mL) followed by the chromanone acids (1 [79.96 ± 2.39 µM; 83.41 ± 3.39 µM] & 2 [57.88 ± 2.34; 53.04 ± 3.18 µM]) against two cancerous cell lines, MDA-MB-231 and MG-63 cell lines, respectively. The results showed that all tested samples exhibited moderate cytotoxicity. Graphical abstract


Introduction
Calophyllum is one of the genera categorized under the Calophyllaceae family, which consists of approximately 200 species (Tjahjandarie et al. 2021;Zailan et al. 2022).Studies on the genus Calophyllum have been extensively discovered and continue to pique researchers' attention to date.The discovery of new and novel phytochemicals from the group of xanthones and coumarins, especially pharmacologically active compounds in the genus Calophyllum, were extensively reported in recent findings (Daud et al. 2021;Karunakaran et al. 2022;Lizazman et al. 2022;Tjahjandarie et al. 2022).Research on the discovery of new novel compounds will be highly beneficial for the phytochemical database of the genus Calophyllum.
Calophyllum lanigerum is a tropical forest tree that is endemic to Bornean Island especially in the state of Sarawak, Malaysia.Anti-HIV active calanolides were discovered in this species.Calanolides such as calanolide A, calanolide B, calanolide C, and calanolide D were isolated from the fruits and twigs, while calanolide E1 and calanolide E2 were isolated from the stem bark of this species (Kashman et al.1992;McKee et al. 1996;Nahar et al. 2020).Previous pharmacological studies on this species reported that calanolide A has shown promising anti-HIV activity in inhibiting HIV-1 replication (Kashman et al. 1992).
Several phytochemicals were isolated from the stem bark of C. lanigerum and some of them were examined for their cytotoxic effects against MDA-MB-231 and MG-63 cell lines, respectively.In this study, two pyranochromanone acids known as caloteysmannic acid (1), isocalolongic acid (2), and a simple dihydroxyxanthone, namely euxanthone (3) (see Figure 1), were firstly reported from the stem bark of C. lanigerum along with a coumarin called calanone (4), and two common triterpenoids, friedelin (5), and stigmasterol (6).

Result and discussion
The n-hexane stem bark extract of C. lanigerum was repeatedly subjected to gravity column chromatography using silica and Sephadex LH-20 to afford compounds 1-6.Structural elucidation of the compounds 1-6 were achieved with the aid of NMR (1D & 2D), MS and the spectral data of compounds 1-6 were reported and consistent in previous works of literatures (Gustafson et al. 1994;Nasir et al. 2013;Lim et al. 2015;Wong et al. 2018;Lizazman et al. 2022).The 1 H-NMR spectra for compounds 1-6 are provided in the supplementary material as Figures S1-S6.The peak purity UV spectra (HPLC-DAD) for compounds 1 and 2 are shown in supplementary material as Figures S8-S9.

Cytotoxicity assessment
Compound 1 (caloteysmannic acid) and compound 2 (isocalolongic acid) as well as the n-hexane extract of C. lanigerum, were tested for their cytotoxicity against two cancerous cell lines, which are human breast adenocarcinoma (MDA-MB-231) and human osteosarcoma (MG-63) cell lines, respectively, with the concentration ranges of 500 µg/mL to 7.8125 µg/mL after 20 h of treatment.Both compounds 1 and 2 displayed cytotoxicity towards the tested cell lines, MDA-MB-231 (IC 50 : 79.96 ± 2.39 µM and 57.88 ± 2.34 µM) and MG-63 (IC 50 : 83.41 ± 3.39 µM and 53.04 ± 3.18 µM) whereas the n-hexane crude extract displayed cytotoxicity with IC 50 value of 87.14 ± 2.04 µg/mL and 81.46 ± 2.42 µg/mL, respectively.The results of the tested compounds and n-hexane extract were compared with those of positive controls such as 5-Fluorouracil (5-FU) and curcumin.Both of positive controls exhibited moderate cytotoxicity towards tested cell lines.According to the recommendation of the American National Cancer Institute, an IC 50 value of less than 10 µM (compound) and 30 µg/mL (extracts) is considered to be cytotoxic against cancer cells (Daud et al. 2021;Karunakaran et al. 2022).Thus, based on the results obtained, it can be safely deduced that the two tested compounds (1 and 2) and n-hexane extract did portray cytotoxicity effect in comparison with the positive controls.The IC 50 values of the respective samples and positive controls are shown in Table 1.
According to Lim et al. (2015), they reported that caloteysmannic acid (1) and isocalolongic acid (2) exhibited significant cytotoxicity against HeLa cancer line with IC 50 values of 7.3 ± 0.2 µM and 7.8 ± 0.2 µM, respectively.The consistency of cytotoxic activity exhibited by both compounds (1 and 2) can be observed in the tested cytotoxicity in the study conducted.Both compounds showed similar cytotoxic IC 50 values regardless the type of tested cancer cell lines.The observed cytotoxic effect displayed by the pyranochromanone acids may be due to the presence of hydroxyl, 3-phenylpropanoic acid and pyrano moieties at C-5, C-6, C-6a, and C-10a positioned in its nucleus structure (see Figure 1).Furthermore, the difference in the substitution at C-13, which are mono-substituted phenyl (1) and propyl (2) moieties, does not influence much on the inhibitory activities.More preclinical studies need to be carried out on this pyranochromanone acids with various cancerous cell lines to explore its potential as an anticancer candidate based on its reported cytotoxic activities.

Experimental
See Supplementary Data.

Conclusion
This study afforded six phytochemicals, which are two pyranochromanone acids are known as caloteysmannic acid (1), isocalolongic acid (2), a simple dihydroxyxanthone, namely euxanthone (3), a coumarin called calanone (4), and two common triterpenoids, friedelin (5), and stigmasterol (6), from the n-hexane extract Calophyllum lanigerum.The n-hexane extract and both compounds, namely caloteysmannic acid (1) and isocalolongic acid (2), were evaluated for their cytotoxic effect against two cancerous cell lines, MDA-MB-231 and MG-63 cell lines, respectively.The n-hexane extract and the compounds exhibited moderate cytotoxic activity towards the tested cell lines.Both compounds displayed similar cytotoxic activities despite the difference in the pattern of substitution at C-13, which are mono-substituted phenyl (1) and propyl (2) moieties.Overall, it is suggested that the two pyranochromanone acids need to be subjected to more preclinical studies on selected toxicity towards targeted and non-targeted cancerous cell lines to explore its potential as anticancer candidate.

Figure 1 .
Figure 1.compounds isolated from the stem bark of Calophyllum lanigerum.
a Positive control substances.