Three New Sesquiterpene Aryl Esters from the Mycelium of Armillaria mellea

Three new sesquiterpene aryl esters and eight known compounds were isolated from the EtOH extract of the mycelium of Armillaria mellea. The structures of new compounds were established by analysis of their spectroscopic data. Some of the isolates showed cytotoxicity to a variety of cancer cell lines, including MCF-7, H460, HT-29, and CEM.


Introduction
Armillaria mellea (Tricholomataceae) is a fungus symbiotic with the Chinese medicinal herb "Tianma" (Gastrodia elata Blume). The fruiting bodies of A. mellea have been used in Traditional Chinese Medicine for the treatment of hypertension, headache, insomnia, dizziness, and vertigo. Recently, the cultured mycelium of A. mellea became a health food in Taiwan and China and its tablets are used to treat geriatric patients with palsy, headache, insomnia, dizziness, and neurasthenia [1][2][3][4][5]. Previous chemical studies of A. mellea reported the isolation of a number of sesquiterpene aromatic esters, in which the tricyclic 5-6-4 protoilludane or protoilludene alcohols were esterified with orsellinic acid or its derivatives [1][2][3][6][7][8][9][10][11][12]. Some of the sesquiterpene aryl esters exhibited cytotoxic activities against human cancer cells. Among these sesquiterpene aryl esters, arnamial showed cytotoxicity against MCF-7, CCRF-CEM, HCT-116, and Jurkat T cells, but melledonal C only showed cytotoxic activity against CCRF-CEM cells [11]. Armillaridin was reported to exhibit cytotoxicity against MCF-7, HeLa, K562, and Jurkat T cells [12]. In addition to inhibiting the growth of the cancer cells, armillaridin also enhanced radiosensitivity of human esophageal cancer cells and there might be potential to integrate armillaridin with radiotherapy for esophageal cancer treatment [13]. Moreover, 4-O-methylarmillaridin showed cytotoxicity against MCF-7 and Jurkat T cells and dehydroarmillyl orsellinate showed cytotoxicity against MCF-7 and K562 cells [12]. Recently, armillarikin was reported to inhibit growth and induce apoptosis in human leukaemic K562, U937, and HL-60 cells [14]. In this paper, we describe the isolation and structural elucidation of three new sesquiterpene aryl esters from the mycelium of A. mellea, as well as the cytotoxic activities of the isolated components [15].
Compound 3 gave a molecular formula of C24H33ClO7 deduced from FABHRMS and NMR (Table 1) spectra. Its 13 C-NMR spectrum displayed 24 signals, including a methoxyl signal at δ 55.6 and a carbonyl signal at δ 169.0, and the signals for C-3, C-4, and C-8-C-15 were similar to those in 2.
In the aromatic region, the signals resembled those in 2 except that the signal for C-4′ shifted from δ 102.6 to 99.6. In the HMBC spectrum (Figure 2), the methoxyl protons showed correlation to C-5′, which indicated that the methoxyl group was attached to C-5′ carbon of the aromatic ring. The 1 H-NMR spectrum of 3 was also similar to that of 2 except for the signals of H2-1, H-2, H-5, and H2-6 as well as one additional signal at δ 3.89 (s) attributed to a methoxyl group. Signals for H2-1 shifted downfield to  ). Therefore, the benzoyloxy group in 3 was suggested to be linked to C-1, which was confirmed by the HMBC correlation of H2-1 to C-1′. The stereochemistry of 3 was established by the NOESY spectrum and the coupling constant of H-2 and H-3. The NOESY cross-peaks of H3-14/H-9,H-12b (δ 1.52), H-9/H-6b (δ 1.54), H-3/H-12a (δ 1.97), and H-5/H3-8,H-6a (δ 1.71) and the large coupling constant of 10.8 Hz for H-3 revealed that its relative configuration was the same as that in 2. Accordingly, the relative structure of 3 was determined and it was named melleolide R.
The isolated compounds from A. mellea were tested in vitro for cytotoxicity to a variety of human cancer cell lines including MCF-7, H460, HT-29, and CEM and the results are summarized in Table 2. Compounds 2-4 and 6-9 showed cytotoxicity to MCF-7 cells, in which compound 2 was most cytotoxic. Compounds 1, 4, and 6-9 exhibited comparable cytotoxicity against H460 cells. Compounds 1 and 6 showed stronger cytotoxicity to HT-29 cells than other tested compounds. Compounds 1, 3, and 5-7 showed comparable cytotoxicity to human leukemia cells. Among all tested compounds, 6 exhibited cytotoxicity to all of these cancer cells. Compounds 1-5 and 7-9 showed selective cytotoxicity and compounds 10 and 11 were inactive to these cancer cell lines.

Source of Organism
The strain of the fungus A. mellea (# BCRC 36361) was purchased from the Food Industry Research and Development Institute, Hsinchu, Taiwan.

Fermentation of Organism
The strain BCRC 36361 was inoculated into 1 L of the medium (1.0% glucose, 1.0% oat powder, 0.1% peptone, 0.1% yeast extract, pH 4.5) in a 2-L Hinton flask at 25 °C on a rotary shaker (120 rpm) for six days. The mycelium was aseptically transferred to a 500-L fermenter containing 400 L of the above medium and incubated at 25 °C for ten days.

Cell Culture
Four cancer cell lines, MCF-7, H460, HT-29, and CEM, were derived from the American Type Culture Collection (Manassas, VA, USA) and were maintained in DMEM or RPMI medium supplemented with 2 mM L-glutamine and 10% heat-inactivated fetal bovine serum (FBS) under standard culture conditions. The cell viability and cell number were determined by the Trypan Blue dye-exclusion method.

Cancer Cell Cytotoxicity Assay
To assess cell viability, the alamar blue (AB) assay (dye purchased from Biosource International, Nivelles, Belgium) was used as previously described [20]. This involved aspirating medium at the end of each treatment period and adding 100 µL of fresh medium containing 10% v/v AB to control and treated wells. Plates were incubated at 37 °C for 6 h prior to measuring the absorbance at 540 nm and at 595 nm wavelengths using a spectrophotometric plate reader. Experimental data were normalized to control values.

Conclusions
Three new sesquiterpene aryl esters, melleolides N (1), Q (2), and R (3), together with eight known compounds 4-11 were isolated from the EtOH extract of the mycelium of Armillaria mellea. Nine isolates showed cytotoxicity to a variety of cancer cell lines, including MCF-7, H460, HT-29, and CEM. Among the isolates, compound 6 exhibited cytotoxicity to all of these cancer cells and compounds 1-5 and 7-9 showed selective cytotoxicity.