Two new methoxylated flavones isolated from Casimiroa edulis La Llave and their MMP-9 inhibitory activity

Abstract Casimiroa edulis La Llave is known to contain unusual 5,6-dimethoxyflavones bearing a variously oxygenated B-ring. Phytochemical investigation of the leaves and the roots of C. edulis achieved the isolation of two new methoxylated flavones, named casedulones A (1) and B (2), together with 12 known analogues. Their unique structures were established with the aid of spectral analyses and total syntheses. Pre-treatment with 20 µM of 1 and 2 suppressed MMP-9 expression in LPS-mediated THP-1 cells, indicating that the characteristic flavonoids in C. edulis could be potential anti-angiogenics for cancer prevention. Graphical Abstract


Introduction
Casimiroa La Llave, classified into Zanthoxyloideae of Rutaceae family, consists of 10 species and distributes from Mexico to Central America (Esposito et al. 2011;WFO: World Flora Online 2022). C. edulis La Llave, an evergreen tree ranging from 3 to 12 m tall, is well-known to bear edible fruits called 'white sapote' that is now harvested in Wakayama and Okinawa prefectures, Japan. Its seeds have been handed down as a Mexican folk medicine for the treatment of insomnia, arthritic pain, and hypertension (Kincl et al. 1956); thus, early phytochemical studies were focused on the seeds to yield a variety of chemical constituents such as alkaloids (Aebi 1956;Meisels and Sondheimer 1957), limonoids (Sondheimer et al. 1959), and flavonoids (Dreyer and Bertelli 1967). The subsequent studies revealed the occurrence of furocoumarins in the leaves (Rizvi et al. 1985;Nagai et al. 2014); whereas there have been few reports of the phytochemicals in the roots (Maiti et al. 2007;Tun et al. 2020).
In the current study, we dealt with the isolation of 14 flavonoids including two new methoxylated flavones, named casedulones A (1) and B (2), from the leaves and/or the roots of C. edulis. Herein described are the structure elucidation and the total syntheses of 1 and 2, as well as the evaluation with the lipopolysaccharide (LPS)-mediated MMP-9 expression in human acute monocytic leukaemia (THP-1) cells for finding out anticancer products.

MMP-9 inhibitory activity
Anticancer screenings with cultured human promyelocytic leukaemia (HL-60) cells have led to the discovery of zapotin and its analogues identified in C. edulis as cytotoxic principles (Mata-Greenwood et al. 2001;Maiti et al. 2007). According to the successive research, propounded is the idea that the apoptotic effect is associated with protein kinase C (PKC) signalling pathway (Toton et al. 2016). Meanwhile, a recent pharmacological study has demonstrated that nobiletin (5,6,7,8,3 0 ,4 0 -hexamethoxyflavone), abundantly present in the citrus peels, suppressed MMP-2 and MMP-9 expressions via extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) pathways in human osteosarcoma cells (Cheng et al. 2016). It is noteworthy that no clinical signs of toxicity were observed after administration of zapotin (40 mg/kg body weight/day) in rats for three days (Strawa et al. 2021). Likewise, the oral intake of nobiletin also did not lead to adverse side effects at the concentration of 40 mM for a period of 3 to 20 weeks (Goh et al. 2019). As far as we surveyed, polymethoxyflavones did not exhibit serious toxicity at the effective concentration.
The efficacy of zapotin-type polymethoxyflavones on the MMPs expression has never been investigated; therefore, we examined the effectiveness of the newly isolated 1 and 2 against mRNA and protein expression, and also enzymatic activity of MMP-9 in THP-1 cells elicited by LPS. As shown in Figure S5, 1 suppressed LPSinduced MMP-9 mRNA (A) and protein (B), and activity (C) by 79%, 77% and 84%, respectively. In addition, 2 suppressed them rather strongly by 97%, 95% and 95%, respectively.

Experimental
See supplementary material.

Conclusion
Twelve 5,6-dimethoxylated flavones (1 and 3-13) were isolated from the leaves of Casimiroa edulis, and seven related compounds (2-5, 8, 9 and 14) were isolated from the roots. The structures of two new zapotin-type flavonoids, named casedulones A (1) and B (2), were elucidated by comparison with the literature values and by means of 2D-NMR experiments. The new flavones were finally synthesized to confirm the structures. Both 1 and 2 inhibited MMP-9 mRNA and protein expression, and enzymatic activity in LPS-mediated THP-1 cells according to their pre-treatment. MMP-9 is involved in metastasis and infiltration of cancer cells; therefore, 1 and 2 could be seed compounds for developing cancer preventive agents.

Funding
The author(s) reported there is no funding associated with the work featured in this article.