Cytotoxic and NF-κB and mitochondrial transmembrane potential inhibitory pentacyclic triterpenoids from Syzygium corticosum and their semi-synthetic derivatives

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Abstract

Syzygium is a large genus of flowering plants, with several species, including the clove tree, used as important resources in the food and pharmaceutical industries. In our continuing search for anticancer agents from higher plants, a chloroform extract of the leaves and twigs of Syzygium corticosum collected in Vietnam was found to be active toward the HT-29 human colon cancer cell line. Separation of this extract guided by HT-29 cells and nuclear factor-kappa B (NF-κB) inhibition yielded 19 known natural products, including seven triterpenoids, three ellagic acid derivatives, two methylated flavonoids, a cyclohexanone, four megastigmanes, a small lactone, and an aromatic aldehyde. The full stereochemistry of (+)-fouquierol (2) was defined for the first time. Biological investigations showed that (+)-ursolic acid (1) is the major cytotoxic component of S. corticosum, which exhibited also potent activities in the NF-κB and mitochondrial transmembrane potential (MTP) inhibition assays conducted, with IC50 values of 31 nM and 3.5 µM, respectively. Several analogues of (+)-ursolic acid (1) were synthesized, and a preliminary structure-activity relationship (SAR) study indicated that the C-3 hydroxy and C-28 carboxylic acid groups and 19,20-dimethyl substitution are all essential in the mediation of the bioactivities observed for this triterpenoid.

Introduction

Syzygium (Myrtaceae) is a large genus of flowering plants, containing about 1200–1800 species that are distributed mainly in tropical and subtropical regions.1 Of these, the clove tree (Syzygium aromaticum Merr. & Perry) is used as an important economic plant in the food and pharmaceutical industries with particular use as a spice and a flavoring agent that has antioxidant, antimicrobial, antinociceptive, antiviral, and cytotoxic activities.2 Syzygium cumini (L.) Skeels, known as “jamun” or “jambolan” (black plum) in Hindi, is another important species used widely in several traditional medicine systems for the treatment of diabetes, allergy, arthritis, and other diseases.3 Chemical purification has shown that benzoic acid analogues and flavonoids are the main bioactive components of clove,2 while chalcones and phloroglucinol derivatives have been identified as the major cytotoxic components of S. samarangense Merr. & Perry and S. jambos (L.) Alston, respectively.4, 5, 6 Interestingly, the flowers of S. aromaticum were found to contain large quantities of (+)-oleanolic acid [1.6% (w/w)] and eugenol [89% (w/w) of the clove essential oil], and thus this species is regarded as an important natural source of these both compounds.2, 7

Pentacyclic triterpenes are common secondary plant metabolites, as represented by ursane-, oleanane-, and lupane-type triterpenes, with (+)-ursolic acid, (+)-oleanolic acid, and (+)-betulinic acid being the representative members. These three natural products are attracting increasing interest for their promising bioactivities, including their anticancer potential as mediated by induction of cancer cell apoptosis and suppression of angiogenesis.7, 8, 9 Among these, (+)-ursolic acid was found to induce doxorubicin-resistant HepG2 cell apoptosis through both caspase-dependent and independent pathways.10 Tumor growth was suppressed significantly when 4-week-old athymic BALB/c nude male mice inoculated with DU145 human prostate cancer cells were treated with (+)-ursolic acid (i.p., 200 mg/kg, twice a week) for six weeks,11 and such an antitumor efficacy was found to be mediated through inhibition of NF-κB activation.11, 12 Several clinical studies suggested that ursolic acid has potential for the development of an anticancer drug.13 Two other well-known plant triterpenoids, (+)-betulinic acid and (+)-oleanolic acid, have been reported to inhibit proliferation of melanoma and human gallbladder cancer cells, respectively, and in turn, they suppressed tumor growth in representative tumor xenograft models.14, 15 Mechanistically, both compounds mediated their anticancer potential by induction of apoptosis through activation of the mitochondrial pathway.15, 16

In our continuing search for anticancer agents from higher plants,17 a chloroform partition of the leaves and twigs of Syzygium corticosum (Lour.) Merr. & Perry collected in Vietnam was found to be cytotoxic toward the HT-29 human colon cancer cell line, but the chemical constituents of this plant have not been reported previously. Separation of this extract guided by cytotoxicity for HT-29 cells and NF-κB inhibition yielded 19 known natural products. All compounds isolated were tested for their cytotoxicity against HT-29 cells, and those isolated in sufficient quantities were evaluated further for their cytotoxicity toward other human cancer cell lines and for their NF-κB and mitochondrial transmembrane potential (MTP) inhibitory effects. The effective functional groups of the major active compound, (+)-ursolic acid (1), have been determined, and a preliminary structure-activity relationship (SAR) study for pentacyclic triterpenes and their cancer cell line cytotoxicity and NF-κB and MTP inhibitory activities are reported.

Section snippets

Purification and identification of the bioactive compounds from Syzygium corticosum

The MeOH extract of the leaves and twigs of S. corticosum was partitioned with n-hexane and CHCl3, and all the dried partitions were evaluated for their cytotoxicity toward HT-29 cells. A major known compound, (+)-ursolic acid (1),18 was purified from this extraction process, and it was found to exhibit cytotoxic and NF-κB inhibitory activities. Following this result, the CHCl3 extract was subjected to chromatographic separation guided by HT-29 cells and NF-κB inhibition, and 18 other known

Conclusion

Several triterpenoids, ellagic acid analogues, flavonoids, and megastigmanes have been isolated and identified for the first time from the leaves and twigs of Syzygium corticosum collected in Vietnam, of which (+)-ursolic acid (1) was isolated in a large quantity, with a yield of up to 0.2% (w/w). Thus, S. corticosum can be regarded as a new natural source of (+)-ursolic acid (1), which exhibited cytotoxicity against the MDA-MB-231 human breast cancer cells and potent NF-κB and MTP inhibitory

General experimental procedures

Optical rotations were measured at room temperature on an Anton Paar MCP 150 polarimeter. UV spectra were recorded on a Hitachi U2910 ultraviolet spectrophotometer. ECD measurements were performed using a JASCO J-810 spectropolarimeter. IR spectra were recorded on a Nicolet 6700 FT-IR spectrometer. 1H and 13C, DEPT 90, DEPT 135, HSQC, HMBC, NOESY, and COSY NMR spectra were recorded at room temperature on a Bruker Avance DRX 400, a Bruker Avance II 400, a Bruker Avance III HD 700, or a Bruker

Disclosure statement

The authors declare no competing financial interest.

Acknowledgments

This investigation was supported by grant P01 CA125066 funded by the National Cancer Institute, NIH, Bethesda, MD. The plant sample of Syzygium corticosum was collected under a collaborative agreement between the University of Illinois at Chicago (USA) and the Institute of Ecology and Biological Resources of the Vietnam Academy of Science and Technology, Hanoi (Vietnam). We thank Drs. Appaso Jadhav and Nelson Freitas Fernandes, Division of Medicinal Chemistry and Pharmacognosy, College of

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