Author: Bunu Tamang
Tamang, Bunu, 2025 Biorefinery Process and Health-promoting Product Development of Hengshan Astragalus Shiitake (Lentinula edodes) Mushroom, Flinders University, College of Medicine and Public Health
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Hengshan Astragalus Shiitake (HAS) is a new strain of Shiitake mushroom, developed by our industry partner, Shanxi Yulongxiang Agricultural Development Co., Ltd., Datong, China. Our previous work has shown that the new HAS mushroom has a modified polysaccharide and protein composition compared to the control Shiitake mushroom. Extracts of HAS have significantly enhanced antiproliferative activity in vitro against the human colorectal cancer (CRC) cell line, HCT-116. Thus, the current PhD project aimed to develop a natural, effective, and safer treatment for CRC by optimising the bioactive components from the novel HAS mushroom, while also utilising an environmentally friendly green biorefinery process to produce other value-added products, generating minimal or zero waste. There were three primary objectives: 1) Optimize extraction and purification conditions of HAS using a microwave-assisted, green extraction technique, to generate a highly bioactive product; 2) Investigate the anticancer efficacy and mechanisms of action of a semi-purified HAS extract in vitro and in vivo, alone and in combination with a standard of care therapy; 3) Optimise the effective use of HAS green biorefinery extraction by-products with minimal waste, to produce additional health products.
The extraction of HAS was carried out at three different temperatures (40 °C, 60 °C, and 100 °C) and time durations (5, 10, and 20 min) using a green process, microwave-assisted water-based extraction. The extraction temperature and duration significantly changed the antiproliferative activity of the crude extracts of both HAS and Shiitake against human CRC cells HCT-116 in vitro. The optimal extraction condition that provided the highest antiproliferative activity was 60 °C for 5 min, while the higher extractions temperature led to degradation of the proteins and loss of bioactivity. This is the first time that changes in antiproliferative activity from Shiitake mushrooms have been shown to be temperature-sensitive and that degradation occurs at higher extraction temperatures. The crude extracts were enriched for the assumed bioactive molecules (high molecular weight polysaccharides and proteins) using an ethanol precipitation and membrane dialysis method. Further purification using DEAE-Sepharose CL-6B ion exchange column was performed, using the bioactivity-guided assay against HCT-116. Although ethanol precipitation enriched the polysaccharides and proteins, the bioactivity of these enriched extracts was lower compared to its crude form. In contrast, the membrane dialysis process significantly enriched the antiproliferation activity nearly 5-fold compared to the crude extract. With the ion exchange column purification, the highest antiproliferative activity against HCT-116 cells was achieved in the fraction eluted at 0.2M NaCl. This is the first time this level of antiproliferative activity has been observed against any cancer type in response to Shiitake mushroom extracts isolated from the fruiting bodies.
Given the enrichment and purification yield from the membrane-dialyzed HAS extract, this was used for assessment of its safety and anticancer efficacy in vivo. Maximum tolerated dosing (MTD) studies were conducted to identify the safe dose for the HAS extract in mice. The tumour growth inhibition efficiency and pro-immune properties of HAS compared to Shiitake extracts was assessed in an immune competent mouse model of CRC (CT-26 in BALB/c mice). Following this, synergistic effects of a combination treatment of HAS and the standard of care chemotherapy 5-Fluracil (5-FU) for tumour growth inhibition were also assessed. HAS extract demonstrated a significantly higher anticancer effect in vivo compared to the control Shiitake extract, with a ~2-fold reduction in tumour growth. The combination therapy showed approximately a 7-fold reduction in tumour volume compared to the control group, demonstrating a strong synergistic effect of HAS and 5-FU together. The underlying effect of HAS on critical genes involved in apoptotic pathways were also assessed in human CRC cells in vitro and mouse cells in vivo. There was a significant increase in the NOXA/MCL1 ratio in HAS treated CRC cells compared to vehicle control, Shiitake, and 5-FU treated groups, both in vitro and in vivo, suggesting that NOXA is one of the critical mediators for HAS-induced apoptosis. The immunomodulation activity of the HAS and Shiitake extracts was also investigated in mouse plasma samples using the Bio-plex Pro™ mouse cytokine 7-plex system. Interestingly, the plasma from the mice treated with the combination therapy showed elevated levels of pro-inflammatory cytokines, including IL-2, IL-12, and INF-γ, compared to the vehicle control. This elevation was not observed in the groups treated with HAS and 5-FU alone, indicating an activated immune response only with the combination treatment. Treatment with HAS in combination with standard of care therapy poses a possible translation pathway into Phase I clinical trials for better treatment management of CRC in the near future. This could improve patient outcomes for those undergoing therapy for CRC, while maintaining quality of life by reducing the dose of toxic chemotherapies administered.
The clean, uncontaminated biomass after microwave-assisted water extraction of HAS was used for biorefinery processing to generate additional high-value health products, including chitin and chitosan. This study has developed a relatively simple and environmentally friendly process involving microwave-assisted extraction and deacetylation, with significantly reduced chemical use, time, and energy compared to conventional processes. The obtained chitin has a high degree of acetylation, ~80%, and a more potent oil absorption capacity in vitro than commercially available animal-sourced chitins. The potential application of this non-animal-derived chitin and chitosan could be formulated into cholesterol/oil-reducing supplements for weight and metabolism management. The non-animal source also means the vegan and vegetarian population can consume this product.
In Summary, this project developed valuable health-promoting products from a novel Hengshan Astragalus Shiitake mushroom using an effective and efficient green biorefinery process. The antiproliferative, pro-apoptotic and pro-immune bioactivity of HAS polysaccharide and protein-rich extract has potential translational applications in cancer treatment and prevention. Vegan and vegetarian options for managing metabolic disorders could be developed with the novel HAS chitins and chitosan generated in this project. Overall, using environmentally friendly extraction techniques has the potential to generate a paradigm shift in the mass production of a wide range of beneficial health products.
Keywords: Hengshan Astragalus Shiitake, green extraction, polysaccharide-protein enriched fraction, antiproliferative activity, immunomodulation activity, colorectal carcinoma, in vitro, in vivo, fungal chitin/chitosan
Subject: Biotechnology thesis
Thesis type: Doctor of Philosophy
Completed: 2025
School: College of Medicine and Public Health
Supervisor: Prof. Wei Zhang