A New Triterpene From Uncaria macrophylla and Its Antitumor Activity

On our ongoing investigation, a new oleanolic triterpene, 3β,6β,19α-trihydroxy-12-oleanen-28-oic acid (1) was obtained from the chloroform-soluble portion of the 90% alcohol-water extract of the stem bark of Uncaria macrophylla. Its structure was elucidated by extensive spectroscopic methods, including 1D and 2D (1H-1H COSY, HSQC and HMBC) NMR and HR-ESI-MS. The cytotoxicities of the compound was evaluated against two cancer cell lines of MCF-7 and HepG2 by the MTT method, and the compound exhibited weak activities with the IC50 values of 78.2 µg/mL and 73.9 µg/mL.


Introduction
The genus Uncaria is widely distributed in tropical regions, including Southern Asia, Africa and South America [1], and most plants of the genus Uncaria have been used as important sources of OPEN ACCESS medicinal natural products in the family of Rubiaceae. Many species of the genus Uncaria have been used for curing fevers, nervous disorders, apasmolytic, analgesic, hypertension [2], cancer, arthritis, diabetes, inflammation [3][4][5]. Previous studies show that alkaloids, triterpenes and flavones [6][7][8] are the most widespread of the secondary metabolites isolated from the genus Uncaria. As a part of our systematic isolation of phytochemical constituents, a new triterpene (1, Figure 1) was isolated from the chloroform-soluble fraction of the alcohol-water extract of U. macrophylla. In this paper, the isolation, structure elucidation by extensive spectral methods including 1D, 2D NMR and MS and the antitumor activities are presented for the first time.

Structural Identification
Compound 1 (Figure 1) was isolated as a white solid from the chloroform extracts of Uncaria macrophylla. The HR-ESI-MS spectrum revealed a molecular ion peak at 511. 3383  C-NMR spectrum suggested compound 1 was an oleanane pentacyclic triterpene posessing a Δ 12, l3 moiety [9]. Furthermore, signals at 182.65 in the 13 C-NMR spectrum together with the IR absorption at 1699.17 cm −1 indicated the presence of an carboxyl group in 1. The 1 H-NMR signals at δ 4.92 and δ 3.26 together with the 13 C-NMR spectral data at δ 80.39, 69.22 and 82.75 suggested compound 1 to be a trihydroxy substituted pentacyclic triterpene [10].
Consequently, the structure 1 was established and determined to be 3β,6β,19α-trihydroxy-12-oleanen-28-oic acid (Figure 1), and it was named uncarioleanic acid. Compound 1 was evaluated for its inhibitory ability against two cancer cell lines (MCF-7 and HepG2) in vitro. The results showed that compound 1 was weakly effective in inhibiting the growth of MCF-7 and HepG2, with IC 50 values against the two cell line of 78.2 µg/mL and 73.9 µg/mL while those of cisplatin were 7.5 µg/mL and 8.2 µg/mL, respectively.

Extraction and Isolation
The air-dried and powdered sample (5 kg) was extracted successively with 90% EtOH-H 2 O (50 kg, 70 °C, 2 hours). The extracts were dried under reduced pressure using a rotary evaporator to yield petroleum ether (32.3 g), chloroform (31.8 g), ethyl acetate (15.6 g) and water (8.5 g) extracts. The chloroform extract was chromatographed over a silica gel column using a stepwise gradient system petroleum ether-acetone. The eluted fraction A (petroleum ether/acetone 70:30, 3.2 g) was then subjected to a Sephadex LH 20 column chromatography using chloroform-methanol (40:60), the obtained fraction D (160 mg) was subjected to reverse chromatography (ODS) with a gradient of MeOH-H 2 O (35:65) to get compound 1 (8 mg).  Table 1.

Bioassays
Antitumor activity was assayed on MCF-7 and HepG2 cells using cisplatin as positive control. Cells were plated in the appropriate media on 96-well plates in a 100 μL total volume at a density of 6 × 104 cells/mL. The final concentrations of each compound were 0.625, 1.25, 2.5, 5.0, 10 μg/mL. The plates were incubated at 37 °C in 5% CO 2 for 72 h. Cell viability was determined based on the mitochondrial conversion of MTT to formazan.

Conclusions
A new triterpene, 3β,6β,19α-trihydroxy-12-oleanen-28-oic acid (1) was isolated from the stem bark of U. macrophylla. The isolation of the new compound was a new addition to the molecular diversity of U. macrophylla. Compound 1 exhibited weak antitumor activity.