Chemical profiling of the cytotoxic triterpenoid-concentrating fraction and characterization of ergostane stereo-isomer ingredients from Antrodia camphorata

https://doi.org/10.1016/j.jpba.2011.09.007 Get rights and content

Abstract

Antrodia camphorata (AC), also known as Antrodia cinnamomea, an endemic species in Taiwan, is one of the treasured medicinal mushrooms. AC is traditionally used for its chemopreventive biofunctions. In this investigation, we report a convenient method for concentrating the antiproliferative active triterpenoid-rich fraction (FEA), from ethanolic extract of AC (EEAC). A series of stereo-isomers of zhankuic acids (18) from the FEA was purified by HPLC using an efficient acidic solvent system. The structures of compounds 18 were elucidated based on spectroscopic data analysis, and the absolute configuration of α-chiral carboxylic acid at C-25 in the structures was assigned based on reaction with (R)- and (S)-1-(9-anthryl)-2,2,2-trifluoroethanol. Major ingredients of FEA (eight ergostanes 18 and two lanostanes 910) were further characterised by high-performance liquid chromatography-photodiode array detection/mass spectrometry (HPLC-PDA/MS). Compounds 18 and their pair mixture forms (antcin K, antcin C, zhankuic acid C, and zhankuic acid A) were subjected to anti-proliferative assay against three human leukemia cell lines. Among them, the derivatives with carbonyl group at C-3 showed cytotoxicity with IC50 values ranging from 16.44 to 77.04 μg/ml.

Highlights

► Method concentrating antiproliferative triterpenoid fraction (FEA) was developed. ► A series of stereo-isomers of zhankuic acids from the FEA was purified by HPLC. ► The structures of compounds were elucidated based on spectroscopic data analysis. ► Eight ergostanes and two lanostanes from FEA were characterised by HPLC-PDA/MS. ► Pure compounds and their pair mixtures were subjected to antiproliferative assay.

Introduction

Antrodia camphorata (AC, also known as Antrodia cinnamomea), by name Chang-Chih, is an endemic fungus in Taiwan. Hundreds years before, the Taiwanese aborigines found that the wild AC had a special effect to relief hangover syndrome. It was also used in folk medicine for the treatment of food and drug detoxication, diarrhea, abdominal pain, hypertension, skin itching, and cancer [1], [2]. Because of the potential pharmaceutical value of its biologically active ingredients, the fruiting bodies of AC are regarded as one of the health treasure troves of Taiwan [3]. Due to scarcity in nature and the difficulty in its artificial cultivation, the average price of AC is higher than the Truffle (Tuber magnatum), which is considered as one of the most expensive mushrooms worldwide. Several research groups have studied the phytochemical constituents of AC fruiting bodies and their pharmacological activities [4]. However, few studies have focused on analyzing the active components of AC fruiting bodies and their pharmacological mechanism of action [5], [6], [7], [8].

In our previous study, we reported that the ethanolic extract from wild fruiting bodies of A. camphorata (EEAC) could induce HL 60 cell apoptosis via histone hypoacetylation, up-regulation of histone deacetyltransferase 1, and down-regulation of histone acetyltransferase activities [9]. Moreover, after fractionation of EEAC and cytotoxicity evaluation of different fractions, we found that the ethyl acetate fraction (FEA), which showed characteristic 1H NMR signals of triterpenoids, was the cytotoxic active fraction of EEAC. In this bio-guided fractionation procedure, the cytotoxic components of EEAC can be attributed to FEA. The aforementioned results encouraged us to carry out further chemical and cytotoxic analyses targeting the active fraction, FEA. In the current study, the chemical profile of FEA active components (the triterpenoid-rich fraction from A. camphorata) was illustrated by isolation, purification, and structural elucidation of the major ergostane and lanostane derivatives by NMR and HPLC-PDA/MS.

Chiral centers present in the skeleton of bioactive botanical secondary metabolites, which are generated by specific enzyme systems in biosynthesis, are always an important issues in drug discovery. The dramatic effect of chirality on the activity and/or toxicity of any therapeutic entity was always explored and monitored. Zhankuic acids, the most abundant triterpenoid of AC, were reported to be present in a mixture of a stereo-isomeric pairs with a chiral center at C-25 [10]. So far, only zhankuic acid A was successfully separated into two isolated peaks using capillary electrophoresis [11], however other zhankuic acids, such as antcin K, antcin C, zhankuic acid C, zhankuic acid A, etc., have never been obtained in a pure form utilizing the isolation procedures embedded by previous studies [4]. Separating chiral components of isomeric mixture is crucial for understanding the mechanism of action of each isomer and the adverse effect of the undesired isomer on human body [12], [13]. The lack of studies on separating pure isomers of ergostane triterpenes from AC was the driving force to investigate the possibility of developing an efficient method for their separation. In this current study, purification and structural elucidation for a series of pure stereo-isomers of ergostane triterpenes (18) are described herein, and their chemical profiling and cytotoxic activities were also investigated.

Section snippets

General experimental procedures

Melting points were determined using a Fisher-Johns melting point apparatus (Thermo Fisher Scientific Inc., Rockford, USA), and the values presented are uncorrected. Optical rotations were measured with a JASCO DIP-370 digital polarimeter (JASCO Inc., Tokyo, Japan). UV spectra were obtained on a JASCO V-530 UV-Vis spectrophotometer (JASCO Inc., Tokyo, Japan). The IR spectra were measured on a Mattson Genesis II spectrometer (Thermo Fisher Scientific Inc., Rockford, USA). 1H and 13C NMR spectra

Anti-proliferative effect of AC fractions obtained using a variety of sample preparation methods

Several preparation methods have been developed over the past few years by different research groups for offering AC extracts. We summarised the extraction and partition methods published in the previous papers [1], [2], [10], [14], [15], [16], [17]. It was stated in publications that these extraction methods were designed to yield the maximum possible amount of extracts for use in future applications. Developing a high yield process is especially desirable because of the high price of the raw

Conclusions

We developed a convenient fractionation procedure for concentrating the anti-proliferative triterpenoids from AC fruiting bodies. The chemical profile of this triterpenoids-rich fraction was illustrated by HPLC, NMR, and HPLC-PDA/MS. Triterpenes (FEA) were the major constituents of AC fruiting bodies, accounting for about 22% of the original crude material total weight and nearly 20% of the total FEA weight was zhankuic acid A. Using a combination of extraction, fractionation, and HPLC, an

Acknowledgements

The authors would like to thank Dr. Hung-Liang Lay, the National Pingtung University of Science and Technology, Taiwan, for the identification of wild fruiting bodies of Antrodia camphorata. This work was supported by the National Science Council (NSC 98-2324-B-037-001 and 98-2321-B-037-060) and the Department of Health (DOH100-TD-C-111-002), Executive Yuan, Taiwan.

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These authors contributed equally to this study.

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