Isolation of anticancer and anti-trypanosome secondary metabolites from the endophytic fungus Aspergillus flocculus via bioactivity guided isolation and MS based metabolomics
Introduction
Endophytes are microbes that are harboured inside plant internal tissues without triggering any immediate, apparent negative effects [1]. Piling evidence points to their possible involvement in the biosynthesis of plant natural products, or even that they might be the sole producers of several other groups of novel pharmacologically active and structurally diverse secondary metabolites [2]. Advanced methods in natural products drug discovery have provided an access to a rich source of novel drug leads having the advantage of optimizing yield production levels via the large-scale cultivation of the microorganisms. Aspergillus is a fungal genus belonging to Ascomycetes fungi that are found in several terrestrial and marine organisms. The endophytic Aspergillus flocculus was first isolated from the stem of the Egyptian medicinal plant Markhamia platycalyx. Roots, stems, barks, and leaves of Markhamia species are used in traditional medicine to treat various ailments. In Africa, Markhamia plant parts were traditionally used for treating microbial and parasitic diseases, anaemia, diarrhoea, backache, sore eyes, pulmonary troubles, gout, scrotal elephantiasis, rheumatoid arthritis, and superficial skin condition [3]. In terms of chemistry, Markhamia species produce a myriad of metabolites i.e. antitumor naphthofurandione and naphthoquinones from M. tomentosa and M. hildebrandtii respectively [4], cytotoxic sterols from M. zanzibarica [5], anti-parasitic triterpenoids musambins A–C and their glycosides in M. lutea [6] in addition to polyphenols [7,8]. For thorough review on bioactive metabolites from the terrestrial Aspergillus endophyte, our previous publication ought to be consulted [9]. Continuing our interest in the metabolites profiling of plant fungal endophytes, we report herein on the isolation of bioactive anticancer and antitrypanosomal metabolites from A. flocculus guided by metabolomics modern metabolomics tools.
In a preliminary screening for anticancer and antimicrobial active agents from endophytic fungi, A. flocculus an endophyte associated with the stem of M. platycalyx (Bignoniaceae) exhibited toxicity against prostate cancer cell line (PC3) and chronic myelogenous leukemia (K562) (Figs. 1–3S) [9]. Cancer Research UK recorded 356,860 cancer cases in 2014 with estimated deaths of 163,444 which is more than 45% mortalities. Prostate cancer was the fourth most common cause of cancer death in the UK with more than 11,000 deaths recorded in 2016 [10]. According to the International Agency of Research on Cancer, an estimated 1.1 million men worldwide were diagnosed with prostate cancer in 2012, considered as 15% of the cancers diagnosed in men. Between 2009 and 2013, leukemia was the fifth most common cause of cancer death in men and the sixth in women. In 2017, 24,500 people in US are estimated to die from leukemia (14,300 males and 10,200 females). In addition, there is approximately more than 380,000 people living with, or in remission from, leukemia [11]. Human African Trypanosomiasis (HAT), or sleeping sickness, is a vector-borne parasitic disease, caused by the parasitic protozoan T. brucei. Trypanosomiasis is one of the most neglected tropical diseases that occurs in sub-Saharan Africa and is regarded as being life threatening if left untreated. HAT affects remote rural communities in isolated regions with inadequate access to suitable health services, with many cases that could not be diagnosed or reported and ultimately affecting the true statistics of disease prevalence in Africa [12]. All available medications used for HAT treatment must be taken by injection over a long-time thus requiring medical facilities and specialized staff that unfortunately often do not exist in rural infected areas. Adverse effects are severe and sometimes fatal [13]. Eradication of HAT is considered though possible by World Health Organization (WHO) [14]. Consequently, reliable methods for diagnosis, novel, safe, effective, and easy-to-apply drugs are still warranted [15]. Historically, natural products have been recognized as a rich source of compounds that have played a potential role in ailments treatment and for maintaining a better health status. The large structural diversity of natural products along with their myriad of biological effects has served as “lead” compounds for drug design programs. In fact, Food and Drug Administration (FDA) reported that 34% of discovered drugs between 1981 and 2010 were derived from natural products [16]. Moreover, the recent technological advances in high throughput screening along with metabolomics and dereplication studies has led to a paradigm shift in natural products drug discovery. Metabolomics is the technology designed to provide general qualitative and quantitative profile of metabolites in biological systems at different status conditions, with many applications in aspects related to drug discoveries, particularly when coupled to bioactivity assay to expedite the identification of bioactive agents. Particularly of value among the different metabolomics platforms, liquid chromatography coupled to mass spectrometry LCMS can generate an informative rich dataset that can assist in the tentative identification of natural products classes present in crude extracts prior to an intensive isolation attempts. For pinpointing active components, multivariate data analysis is normally employed to correlate bioactivity and LCMS profiles [17]. In this study, we used a molecular interaction correlation network that uses the Pearson correlation coefficient to link the chemical and biological profile of either active extracts or fractions. Thus, the metabolites contributing to the bioactivity can be putatively identified before extensive isolation attempts.
Section snippets
Fungal material
The endophytic fungus was isolated from the fresh stems of Markhamia platycalyx family (Bignoniaceae) collected in October 2010 from Al-Zohriya gardens (Al-Zamalek, Giza, Egypt). Plant material was authenticated by Dr. Therese L. Yousef, senior expert at Orman Garden and with the voucher specimen (No. 633) deposited. Plant material was cut into small pieces, washed with sterilized demineralized water, then thoroughly surface sterilized with 70% isopropanol for 1–2 s and ultimately air dried
Metabolite profiling, dereplication study and multivariate data analysis of the crude extracts
We have previously developed a high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) method for profiling of fungal endophytes [17] which we apply herein for the profiling of A. flocculus culture. To provide a comprehensive coverage of A. flocculus metabolome, fungal extract was analysed in both positive and negative ion electrospray ionization (ESI) MS modes as changes in ESI polarity can often circumvent or significantly alter competitive ionization and suppression
Conclusion
In conclusion, results provided a realistic, compound-based rational for the anticancer and anti-trypanosomal principles present in Aspergillus flocculus. Additionally, they pointed to an additional evidence for the efficacy and complementarity of LC-MS metabolites profiling when coupled to bioassays in the field of natural product based drug discovery, to speed up the traditional lengthy processes of identifying an active principle by consecutive isolation from crude extracts. The emerging
Acknowledgements
Financial support for Dr. Ahmed Tawfike by sector of missions, Ministry of Higher Education, Egypt, is greatly appreciated. Thanks are due to Dr. Rothwelle Tate, Strathclyde Institute of Pharmacy and Biomedical Science for his guidance through the molecular biological procedures used for the phylogenetic identification of endophytic fungi.
Conflict of interest
The authors declare no conflict of interest.
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Current address: Molecular Discovery Group, Department of Computational and Analytical Science, Rothamsted Research, Harpenden, AL5 2JQ England, UK.