Extraction of polysaccharides from Lentinula edodes grown in Astragalus membranaceus supplemented bed and determination of cytotoxicity in colorectal carcinoma cell line HCT-116

Author: Bunu Tamang

Tamang, Bunu, 2021 Extraction of polysaccharides from Lentinula edodes grown in Astragalus membranaceus supplemented bed and determination of cytotoxicity in colorectal carcinoma cell line HCT-116, Flinders University, College of Medicine and Public Health

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The chemical constituents and nutritional composition of mushrooms alter significantly when grown on different substrate conditions. With reference to this fact, Shanxi Yulongxiang Agricultural Development Co. Ltd., China, has grown Shiitake mushrooms in the substrate beds supplemented with an edible medicinal plant Astragalus membranaceus and expected to have some bio-active components transferred from A. membranaceus into the mushroom. The so grown Shiitake mushrooms in the beds supplemented with Astragalus under specific conditions (not mentioned here to protect IP of the company) have been named as HAS-A and HAS-B in this project. Since one of the major bio-active components in both Shiitake and Astragalus are polysaccharides, the major aim of this project was to determine any changes in yield, content and anti-proliferative effects of polysaccharides extracted from HAS-A and HAS-B when compared to that of Shiitake grown under controlled conditions. Interestingly, the yield of polysaccharides in HAS-B and HAS-HAS-A rose significantly higher in every extraction method (~45% w/w and ~31%w/w more than that of controlled shiitake, respectively). However, the anti-proliferative activity was totally dependent on the methods and conditions of extraction. For instance, microwave assisted (MA) and ultrasound-enzyme assisted (UEA) extracts of HAS-B have a comparatively higher anti-proliferative activity of HCT-116 than that of controlled Shiitake when determined by MTT assay and Flow-Cytometry. But the polysaccharide extracts from HAS-B and Shiitake with other extraction methods viz. microwave-ultrasound assisted (MUA), microwave-ultrasound-enzyme assisted (MUEA) and microwave-enzyme assisted (MEA) extraction have no significant difference in inhibiting proliferation of HCT-116. Moreover, the modified UEA extraction method in this study showed significantly higher anti-proliferative activity of HCT 116 compared to those reported in the previous literature. For example, this study showed ~ 89%, ~77% and ~ 94% inhibitory effect of HCT 116 with 1.6 mg/ml of UEA Shiitake, HAS-A and HAS-B polysaccharides, respectively while the literature reported a maximum inhibition of only ~71% with 1.8 mg/ml. Meanwhile, all the enzyme assisted methods of extraction had significantly higher purity when compared to that of non-enzymatic extractions. The monosaccharides composition in HAS-A and HAS-B were mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (GlcAc), galacturonic acid (GalAc), glucose (Gluc), galactose (Gal), xylose (Xyl), arabinose (Ara) and fucose (Fuc). This composition was found similar to that of Shiitake but the amount of glucuronic acid and glucose was significantly higher in HAS-A and HAS-B when compared to Shiitake. In contrast to previous findings (which suggest maximum anti-proliferative activity with higher molecular weight polysaccharides, > 50 k Da), our findings showed the maximum cytotoxic effect of HAS-B and Shiitake against HCT 116 in the molecular weight range of ~2.5 – 832.7 k Da and ~ 2.5 – 10.5 k Da, respectively. In conclusion, this project has demonstrated that Shiitake mushrooms grown in the beds supplemented with A. membranaceus produce a higher amount of polysaccharides and these polysaccharides have higher anti-proliferative activities when compared to those produced under controlled conditions. Innovative green extraction technique such as UEA extraction method can be used for the efficient, effective and economic extraction of bio-active polysaccharides from mushrooms. The optimization of the UEA extraction method and subsequent efficient purification in the future would further help to potentiate the activity of HAS-B polysaccharides, eventually leading to the development of effective complementary treatment to overcome the drawbacks of current chemotherapy.

Keywords: Lentinula edodes, Astragalus membranaceus supplement, HAS-A/HAS-B polysaccharides, extraction, chemical composition, cancer cytotoxicity

Subject: Biotechnology thesis

Thesis type: Masters
Completed: 2021
School: College of Medicine and Public Health
Supervisor: Prof. Wei Zhang