Abstract
Artocarpus altilis (breadfruit) is an economically important species of the Moraceae family found throughout the tropics. While breadfruit pulp is widely used and processed into various food products, the peel waste is currently underutilized. Our chemical study of breadfruit peel has led to the isolation of the major constituent cyclocommunol (1). Using 1 as the starting material, we synthesized nine cyclocommunol derivatives (2–10) and tested their antibacterial and antitumor activities. Compounds 1, 3, 4, and 9 have weak antibacterial activity against several Gram-(+) and Gram-(–) bacteria. In addition, compounds 7 and 9 showed moderate cytotoxicity against the MCF-7 breast cancer cell line, whereas compound 4 showed comparable cytotoxicity against the NCI-H460 lung cancer cell line. The study showed that the underutilized breadfruit peel waste may be used as a source of compounds with pharmaceutical potential.
Keywords: Artocarpus altilis, cyclocommunol, semi-synthesis, antibacterial, antitumor
Graphical Abstract
1. Introduction
Artocarpus plants are a group of trees and shrubs belonging to the Moraceae family commonly found in Southeast Asia and the Pacific. They produce many phenolic compounds, such as prenylated flavonoids (Chen et al. 1993; Lan et al. 2013), many of which were reportedly to have biological activities, including potent antioxidant, anti-inflammatory, antitumor, antifungal, and/or antibacterial activities (Soifoini et al. 2021; Jalal et al. 2022; Dada et al. 2023). In addition, they also produce lectins, steroids and triterpenes (Jagtap and Bapat 2010; Soifoini et al. 2021). One of the widely distributed Artocarpus plants is breadfruit (A. altilis), which has been used as a food crop for over 3000 years in Oceania, and currently is grown in 90 countries (Turi et al. 2015). The fruit contains high amounts of carbohydrates, phosphorus, and calcium (Mehta et al. 2023); therefore, it is commonly processed into various food products and snacks. The leaf and fruit extracts have been reported to have good antibacterial activities against various bacteria, such as Staphylococcus aureus, Pseudomonas aeruginosa, Streptococcus mutans and Enterococcus faecalis (Pradhan et al. 2013). The methanol (MeOH) extract of the pulp, which contains a high amount of quercetin, has been shown to induce apoptosis in several cancer cell lines including human breast cancer MCF-7 cells (Jalal et al. 2019). While breadfruit pulp is widely used and processed into various food products, the peel waste, which contains about 40% starch, is currently underutilized. It is either discarded or used as animal feed (Ragone 2011). To this end, we explored the potential of breadfruit peel as a source of bioactive compounds. Our preliminary study showed that the ethyl acetate (EtOAc) extract of breadfruit peel has moderate antibacterial activities against several bacteria. While the fruit peel has been reported to contain several different types of polyphenols (Soifoini et al. 2021), the active constituents of the peel with antibacterial activity were unknown. Here we report the isolation and identification of the major antibacterial compound in breadfruit peel, as well as the synthesis of nine derivatives of the natural product and the evaluation of their antibacterial and antitumor activities.
2. Results and discussion
To explore the potential of breadfruit peel to be a source of bioactive compounds, we prepared various extracts of the peel and examined their antibacterial activity. Briefly, the breadfruit peel was dried and pulverized, and the powder was extracted with MeOH. The MeOH extract was then partitioned successively with n-hexane, dichloromethane (CH2Cl2), EtOAc, and water (H2O) to yield n-hexane, CH2Cl2, EtOAc, and H2O extracts. Each extract was evaluated for their antibacterial activity against Propionibacterium acnes, P. aeruginosa, and Staphylococcus epidermidis. The results showed that only the EtOAc extract had moderate antibacterial activity against all three tested bacteria at a concentration of 20 mg/mL with S. epidermidis being the most sensitive and P. acnes being the least sensitive strains (Fig. 1). The EtOAc extract was then fractionated by SiO2 vacuum liquid chromatography (VLC) with a gradient eluent of n-hexane–EtOAc, EtOAc, and MeOH to afford 5 fractions. The major fraction (EA-4) was subsequently purified by SiO2 column chromatography with gradient eluent of n-hexane–EtOAc to give 1 (174 mg, 0.017% from dried peel).
Figure 1.
Agar-based disc diffusion assay of breadfruit peel extracts against several bacteria. (a) Propionibacterium acnes; (b) Pseudomonas aeruginosa; and (c) Staphylococcus epidermidis. (1) MeOH extract; (2) n-hexane extract; (3) EtOAc extract; (4) CH2Cl2 extract; (5) H2O extract; (+) positive control: tetracycline was used for P. acnes and P. aeruginosa, whereas chloramphenicol for S. epidermidis; (–) negative control (MeOH).
Compound 1 was isolated as a yellow powder. Its HRESIMS showed a characteristic molecular ion peak [M+H]+ at m/z 353.1019 (calc. m/z 353.1018) representing a molecular formula of C20H17O6+. The 1H NMR spectrum of 1 (Tables S1 and S2) showed resonances of two methyl groups at δH 1.70 (3H, s) and 1.96 (3H, s) and two doublets at δH 5.49 (1H, d, J = 9 Hz) and 6.21 (1H, d, J = 9 Hz), suggesting the presence of a prenyl group attached to a tertiary carbon. Three aromatic proton resonances were observed at δH 6.45 (d, J = 2 Hz), 6.65 (dd, J = 2 and 8 Hz) and 7.73 (d, J = 8 Hz) as well as two singlets at δH 6.27 (s) and 6.54 (s), indicating the presence of a 1,2,4-trisubstituted and a 1,2,3,5-tetrasubstituted benzene moieties. The 13C NMR spectrum of 1 showed resonances of 20 carbons, which based on the chemical shifts and HSQC correlations were predicted to belong to a prenylated flavonoid. Further 2D NMR analysis, including HMBC and NOESY, suggested that 1 is a 4´,5,7-trihydroxy prenylated flavonoid, cyclocommunol (Fig. 2). The chemical structure of 1 was subsequently confirmed by comparing the data with those reported in the literature (Tables S1 and S2) (Lin and Sheh 1992; Sengul et al. 2009). Cyclocommunol (1) was first isolated from Artocarpus communis (Lin and Sheh 1992). It has been reported to have antibacterial and antitumor activities against several cancer cell lines. Cyclocommunol reportedly kills cells via a caspase-dependent apoptotic manner, down-regulation of the phosphorylation/expression of Akt/mTOR and Mcl-1, generation of reactive oxygen species, and/or induction of autophagy (Soifoini et al. 2021). To explore the therapeutic potential of its derivatives, we synthesized nine cyclocommunol derivatives (2-10) (Fig. 2) and tested their antibacterial and antitumor activities.
Figure 2.
Chemical structure of cyclocommunol and synthetic schemes to derivatives 2–10.
Compound 2 was synthesized from 1 by reacting it with methyl iodide in the presence of K2CO3 (Noviany et al. 2021). The trimethyl product (55% yield) was purified chromatographically and the chemical structure was confirmed by NMR and HRESIMS. The HRESIMS of 2 showed a molecular ion peak [M+H]+ at m/z 395.1493, which is 42 atomic mass units higher than that of 1. The 1H NMR spectrum of 2 (in CDCl3) showed three methoxy proton resonances at δH 3.94 (3H, s), 3.92 (3H, s), and 3.84 (3H, s) (Table S3), whereas the 13C NMR spectrum also showed three methoxy carbon resonances at δC 56.4, 55.8, and 55.6 (Table S4), consistent with the methylation of all three free hydroxy groups in 1.
Compounds 3 and 4 were synthesized from 1 by esterification with acetic anhydride in the presence of 4-dimethylaminopyridine (DMAP). The reaction gave 3 (7,4´-diacetyl cyclocommunol) (m/z 437.1229) and 4 (5,7,4´-triacetyl cyclocommunol) (m/z 479.1331) in 35% and 65%, respectively. The products were separated by SiO2 column chromatography using hexane-EtOAc (4:1) as a mobile phase. The structures of compounds 3 and 4 were assigned based on comparisons of their 1H and 13C NMR data with those of 1. The less sterically hindered C-7 and C-4´ hydroxy groups seem to be more susceptible to modifications than the C-5 hydroxy group. In addition, the hydrogen bonding between the C-5 hydroxy group (δH ~12.7) and the neighboring ketone is expected to reduce the reactivity of the hydroxy group.
Compound 5 was prepared by treating 1 with prenyl bromide and K2CO3 to give 7,4´-diprenyl cyclocommunol (5) (m/z 489.2270) in 68% yield. The positions of the substituents at C-7 and C-4´ are consistent with those observed in compound 3. Compound 6 was synthesized by treating 1 with benzyl bromide in the presence of K2CO3 in 98% yield. The reaction went smoothly, and a fully substituted product (m/z 623.2439) was obtained. Our first attempt to derivatize 1 with 3-trifluoromethyl benzoic acid, in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI•HCl) and DMAP, only provided a mono-substituted product 7 (m/z 525.1153). While the result was somewhat disappointing, we learned that the C-4´ hydroxy group is more reactive than the C-7 hydroxy group. Nevertheless, upon repeating the reaction under the same condition, we did obtain the fully substituted compound 8 (m/z 869.1438) with a reasonable yield of 46%. The reaction of 1 with benzyloxyacetic acid in the presence of EDCI•HCl and DMAP only gave the disubstituted product 9 (m/z 649.2077) in 30% yield. Similar to compounds 3 and 5, the positions of the substituents were determined to be at C-7 and C-4´. Finally, the reaction of 1 with benzoyl chloride in pyridine gave compound 10 (m/z 561.1535). All products were purified by SiO2 column chromatography and/or HPLC and the chemical structures were characterized by 1H and 13C NMR (Tables S1 to S4) as well as HRESIMS.
Compounds 1–10 were evaluated for their antibacterial activity against S. aureus, Escherichia coli, P. aeruginosa, Salmonella enterica using an agar diffusion assay (Fig. 3). P. acnes was not tested as our preliminary study showed that the EtOAc extract of breadfruit peel did not show significant activity against this strain. The results indicated that 1, 4, and 9 were found to have low antibacterial activity against the four tested bacterial strains, whereas 3 was active against E. coli, S. aureus, and P. aeruginosa. On the other hand, 7 was only active against E. coli and S. aureus. However, the synthetic derivatives appear to have lower antibacterial activity than 1, as judged from their inhibition zones (Fig. 3). Conversely, 2, 5, 6, 8, and 10 did not show any antibacterial activity against the tested bacterial strains. Most synthetic derivatives that contain ester side chains at C-7 and/or C-4´ showed antibacterial activity, whereas compounds with ether side chains have no antibacterial activity.
Figure 3.
Antibacterial activity test of compounds 1–10 on agar-based disc diffusion assay. Each disc was impregnated with 2 μL of compound solution (6 mg/mL). Compounds 1–10 are numbered as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, respectively. Apramycin (+) (0.5 mg/mL, 2 μL) and DMSO (−) (2 μL) were used as positive and negative controls, respectively.
Cytotoxicity evaluation of compounds 1-10 against the MCF-7 breast cancer cell line and the NCI-H460 lung cancer cell line showed that only 7 and 9 have moderate cytotoxicity against the MCF-7 cell line, with IC50 values of 77 and 33 μM, respectively, whereas 4 showed detectable cytotoxicity against the NCI-H460 cell line with an IC50 value of 76 μM (Table 1). Other compounds were considered inactive with IC50 value of more than 100 μM. Unfortunately, based on these results alone, it is not immediately clear why only 7 and 9 were active against MCF-7 cell, whereas 4 is more active against NCI-H460 cell. Therefore, further investigations are warranted to obtain more understanding of the structure-activity relationship of cyclocommunol and its derivatives.
Table 1.
In vitro cytotoxicity test of compounds 1-10 against cancer cell lines.
| Comp. | IC50 (μM) | |
|---|---|---|
| MCF-7 | NCI-H460 | |
| 1 | >100 | >100 |
| 2 | >100 | >100 |
| 3 | >100 | >100 |
| 4 | >100 | 75.6 |
| 5 | >100 | >100 |
| 6 | >100 | >100 |
| 7 | 76.9 | >100 |
| 8 | >100 | >100 |
| 9 | 32.7 | >100 |
| 10 | >100 | >100 |
| 5-fluorouracil | 29.1 | 63.4 |
3. Experimental Section
4. Conclusions
The present study showed that cyclocommunol (1) is one of the major bioactive components of breadfruit peel. Using 1 as a precursor, we synthesized nine cyclocommunol derivatives (2–10) and tested their antibacterial and antitumor activities. Compounds 1, 4, and 9 demonstrated weak antibacterial activity against S. enterica, E. coli, S. aureus, and P. aeruginosa, whereas compound 3 was active against S. aureus, E. coli, and P. aeruginosa. In addition, compounds 7 and 9 showed moderate cytotoxicity against the MCF-7 cell line, with IC50 values of 77 and 33 μM, respectively, whereas compound 4 was active against the NCI-H460 lung cancer cell line with an IC50 value of 76 μM.
Supplementary Material
Acknowledgments
The authors thank Tasya Aurellia, Nurunnisa F. Amalia, and Cindy Z. Ulayya for collecting samples and providing technical assistance.
Funding
This work was supported by Institute for Research and Community Service, University of Jambi, Indonesia under grant 249/UN21.11/PT.01.05/SPK/2022 and the Oregon State University College of Pharmacy Faculty Development Funds. Arash Samadi and Leigh E. Skala were supported by Grant T32 AT010131 from National Center for Complementary and Integrative Health (NCCIH). The content is solely the responsibility of the authors and does not represent the official views of NCCIH or the National Institutes of Health (NIH).
Footnotes
Declaration of interest statement
The authors declare that they have no conflict of interest.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/xxxxxxxx
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