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Title: Biotechnology-driven in vitro cultivation of fortified mycelium of Fomitopsis officinalis : in search for adjuvants for prophylactic of noncommunicable diseases
Abstract:
Introduction The same progress that enabled the development of modern medicine, has also brought new challenges for the human body. According to World Health Organization the challenges in question are primarily: particulate matter air pollution, metabolic disturbances, and affective disorders that lead to unhealthy behavioral patterns. Diseases that result from abovementioned risk factors are chronic, noncommunicable and lack a single pathogenic agent that could become a target for therapeutic interventions. Therefore, the classic medical model of restoring health to the body (treatment) does not cope well with civilization diseases, and their contribution to the loss of health, as well as mortality, has been steadily growing for the last 30 years. An alternative, more promising approach is prevention. Since prevention requires individuals to develop health–promoting habits, this approach may prove particularly difficult to implement in people most at risk of civilization diseases, because of the comorbidity of somatic and affective disorders. Hence, a form of support that would at the same time address both classes of problems would be of great use. The classic "one compound – one biological target" pharmacological model may not be able to meet this task. Therefore, in search of new solutions, some researchers, as well as the public, turn towards nature and – in ; recent years more frequently – towards medicinal mushrooms. Aims of study The aim of this dissertation was to scientifically approach the question of whether Fomitopsis officinalis – an arboreal medicinal mushroom, known for thousands of years for its health–promoting properties – is a promising target for such pursuits. Due to its status of an endangered species, any attempt to use Fomitopsis officinalis in the prevention of civilization diseases must be based on material obtained in vitro. This approach opens a possibility of enrichment (fortification) of the material during cultivation. Therefore, the second goal of this study was to investigate the potential impact of fortification with zinc or magnesium salts on the composition and properties of Fomitopsis officinalis. Methods In order to answer the above questions, the following methods were used: 1) mycochemistry analyses: HPLC – to measure the levels organic compounds, FAAS – to measure the levels of bioelements in the obtained material; 2) in vitro tests: DPPH – to investigate antioxidant activity, MTT – to study anti–cancer activity in the lung (A549), prostate (DU 145) and skin (A375) cancer cell lines, and a mouse macrophage cell line RAW 264.7 – to examine the effect of the material on inflammation; 3) molecular biology methods: PCR and sequencing – to confirm the identity of Fomitopsis officinalis sp ; ecies obtained from in vitro cultures, qRT–PCR – to measure the level of mRNA expression in mouse hippocampus after long–term dietary supplementation with lyophilized material, Western Blot – to measure the level of proteins in RAW 264.7 murine macrophage cells and rat hippocampus after chronic administration of 3 different doses (100 mg/kg, 250 mg/kg, 500 mg/kg) of the material; 4) an artificial digestive system Gastroel 2014 – to study the bioavailability of organic compounds and bioelements; 5) in vivo studies: elevated plus maze (EPM) and open field tests – to assess an anxiolytic–like effect in rodents, forced swimming test (FST) – to profile stress coping strategies in rats, novel object recognition test (NOR) – to test long–term memory of mice, and the attentional set– shifting test (ASST) – to test executive functions of rats. In vivo studies were performed on male CD1 mice and Wistar rats, i.e. on outbred strains. Animals' diet was supplemented with 2 mg (mice) or 5 mg (rats) of lyophilisate per 1 g of chow, in order to allometrically adjust the daily dose in rodents to a commercially available preparation of another medicinal mushroom – the lion's mane. For this reason, in studies on dietary supplementation, lyophilisate of this species was used as positive control. In addition, both species were compared to the pink oyster mushroom – a popular arboreal e ; dible mushroom, as it showed a profile of indole and phenolic compounds composition similar to that of Fomitopsis officinalis. Results During the first stage of the project, in vitro cultivation of Fomitopsis officinalis was optimized in the conditions of the Department of Pharmaceutical Botany, Faculty of Pharmacy, Jagiellonian University Medical College mushroom laboratory; and, the species identity of the obtained material was confirmed. Subsequently, it was shown that: 1) a precursor for serotonin (5–hydroxy–L–tryptophan), a precursor for dopamine (phenylalanine), and a flavonoid with antioxidant properties (catechin) are present in Fomitopsis officinalis grown in vitro, but not in the fruiting bodies of Fomitopsis officinalis of natural origin; 2) Fomitopsis officinalis from in vitro cultures contains: 2 times more provitamin D2 (ergosterol) and zinc, 3 times more iron and slightly more magnesium than the fruiting bodies of Fomitopsis officinalis of natural origin; 3) fortification of Fomitopsis officinalis with zinc or magnesium sulphates has a positive effect on the bioavailability of 5–hydroxy–L–tryptophan, while fortification with magnesium – on the bioavailability of phenylalanine; 4) digestion of the fruiting bodies leads to a ~2.5–fold decrease in their antioxidant activity, while the activity of mycelium from in vitro cultures decreases ~10 times; 5) on ; ly Fomitopsis officinalis from in vitro cultures showed anti–cancer activity in lung cancer cell lines, while fruiting bodies were more active in skin cancer. In the next step, it was observed that at lower concentrations (50 µL): 1) compared to lion's mane and Fomitopsis officinalis fortified with zinc or magnesium, ono–fortified Fomitopsis officinalis increased the level of induced cyclooxygenase (COX-2) in mouse macrophages; and, 2) fortification with magnesium, caused an increase in TLR4 receptor levels. At higher concentrations (100 µL): 1) zinc–fortified Fomitopsis officinalis increased the level of non– induced cyclooxygenase (COX-1) and TLR4 receptor; and, 2) the effect of zinc–fortified Fomitopsis officinalis on TLR4 was enhanced in the absence of inflammation. The above profile clearly showed that the mechanism of Fomitopsis officinalis immunostimulating action is modulated by fortification; and, that the effect of Fomitopsis officinalis on the immune response is stronger than that of lion's mane. In vivo studies on the impact of dietary supplementation with Fomitopsis officinalis on affective functions showed a positive effect on anxiety–like behavior of both mice and rats in the elevated plus maze test; and, an increase in exploratory/orientation behavior (rearing) in the open field test. In addition, in the forced swimming test, Fomitopsis officinalis se ; lectively increased the occurrence of one of the forms of active stress coping (swimming) at the expense of another form (climbing), while a passive form of stress–coping (immobility) remained unaffected. This effect was present in the absence of any supplementation–induced locomotor activity changes (measured in the open–field test), which suggests that the forced swimming test results can be interpreted in terms of modulation of distress response. In addition, both oyster mushroom and lion's mane reduced passive coping to an extent analogous to a second generation antidepressant (fluoxetine). With regard to cognitive effects, in vivo studies have shown that lion's mane slightly improves long–term memory in mice, and that weight gain: 1) may have a negative effect on this cognitive function in control chow–fed animals; 2) causes a decrease in the expression of genes coding mTOR and GSK3B kinases in mouse hippocampus, regardless of supplementation. In the attentional set–shifting test, no effect of experimental manipulations was observed, although half of the rats supplemented with Fomitopsis officinalis failed to complete the task, which requires reinforcement learning. Ex vivo studies of protein levels in rat hippocampus showed: 1) a decrease in the phosphorylated form of a receptor for neurotrophic factors (p-TrkB), as a result of Fomitopsis officinalis suppleme ; ntation; 2) lower protein levels of a transcription factor responsible for oxidative stress response and neurotoxicity (NRF2), as a result of lion’s mane supplementation. Conclusions The present study suggests that in vitro cultivated Fomitopsis officinalis has a favorable biological activity profile in terms of anti–cancer, immunostimulating and anxiolytic–like effects, with no effect on cognitive functions; and, a potentially adverse effect on motivation to learn from reinforcement that is dependent on individual differences. Due to a very complex biochemical composition, attributing the above effects to specific mechanisms would require a separate set of extensive studies.
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Rada Dyscypliny Nauki farmaceutyczne
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Muszyńska, Bożena ; Krakowska, Agata
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Last modified:
Nov 14, 2024
In our library since:
Apr 8, 2024
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http://dl.cm-uj.krakow.pl:8080/publication/5065
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| Edition name | Date |
|---|---|
| ZB-139421 | Nov 14, 2024 |
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