Hypolipidemic Effects of the Edible Mushroom Auricularia polytricha in a Mouse Model

Edible Macrofungi, referred to as mushrooms, are considered as helpful food for reducing the risks of hyperlipidemia (Das et al., 2021; Hadwan et al., 2021). Their sporocarps or fruiting bodies are composed of a high content of fibers, microelements, proteins, and low-fat content (Muszynska et al., 2017). The significant importance of the mushroom as anti-tumor (Wasser, 2002), beneficial in confirming humoral and cellular immunity (Volman, 2009; Liu et al., 2020), regulating fat and cholesterol in the blood (Neyrinck et al.,2009; Huang et al., 2021) were proved. Furthermore, the edible mushroom was healthy when it was used by human being (Valverde et al., 2015).

The genus Auricularia is known as wood ear and belongs to Auriculariales order and Auriculariaceae family. Their species are eatable and therapeutic and have been cultivated in diverse regions of the world. In Iraq, many edible macrofungi have been reported over the last decades. A. polytricha (Al Anbagi, 2014; Al Anbagi and Al-Khesraji, 2022; Al Anbagi et al., 2019; 2022; 2024; Marie et al., 2023; Sulaiman et al., 2024). Different genetic polymorphisms may be expected between the collected macrofungi (Al Anbagi et al., 2024).

Species such as Auricularia polytricha and Auricularia auricular are the fourth most significant cultivated macrofungal species around the world specially in China and many other Asian countries (Zhang et al., 2011; Li et al., 2014; Xiao et al., 2018; Lu et al., 2018). The isolates of A. polytricha have been described by owning unique gelatinous fruiting bodies easily to be seen with naked eyes. This edible fungus is frequently consumed in diverse parts of the world, especially in some parts of Asia, (Xiao et al., 2018). It also has been described as a valuable source of enzymes and organic acids (Zborowski et al., 2003; Abd Razak et al., 2020). Furthermore, A. polytricha has numerous pharmacological properties, positive effects on animal health through reducing the fat accumulation and hyperlipidemia as well as enrich the diversity of gut microbiota. The mechanisms of reducing lipids level by macrofungi could attribute to decrease serum TC, TG and LDL-C levels, enhance hepatic fat accumulation and shift the microbial composition and reduce the relative abundance of gut microbiota, which might be associated with hyperlipidemia (Fang et al., 2021). A variety of biological actions, including antioxidant, anticancer, immunomodulatory, hyperlipidemic, antidiabetic, anticoagulant, and hepatoprotective properties, are attributed to the bioactive substances found in Auricularia mushrooms, primarily polysaccharides (Miao et al., 2020).

In human body, Lipoproteins are important cellular and extracellular molecules with multifaceted structural and functional roles. Their metabolites are key components for cellular communication and metabolism (Amin et al., 2019). However, using lipoproteins as a nutrition supplement to improve human health is a significant goal for many studies.

With the continuous improvement of living standards, the aims of this work are to determine effects of A. polytricha on the total amount of cholesterol, triglyceride and lipoprotein profile [high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very low-density lipoprotein (VLDL)] in mice when A. polytricha was supplemented to the diet as a nutrient source.

The strain of Auricularia polytricha, AP1, was obtained from Juncao Research institute located in Fujian Agricultural and Forestry University, Fujian, China. The A. polytricha mycelial growth was re-cultured under sterile conditions at the laboratories of the Department of Organic Farming, Plant Protection Directorate, Ministry of Agriculture, Baghdad, Iraq. Propagations were made using the prepared mycelia culture. The fungus was cultured and produced the fruiting bodies as described by Zhanxi (2007). Briefly, the culturing substrate was made by grinding corn cobs 39%, wheat straw 39%, wheat bran 20%, calcium carbonate 2% and water 60% The substrate was packed in polyethene bags 20×40 cm and pasteurized at 90ºC for 5 hours using an autoclave. A. polytricha spawn was prepared via sucking wheat seeds in distilled water for 6 hours and autoclaved these seeds twice and inoculated them by discs from the fungal culture. Then the bags with the substrate were inoculated with previously prepared A. polytricha spawn 2% and then incubated at 25-27°C until the bags were completely colonized by A. polytricha mycelia. Inoculated substrates were transferred into incubation cabinet and placed on tablets (200 cm × 30 cm) for 20 days to produce fungal fruit bodies. The conditions of the incubation cabinet were at 18°C and 90–95% relative humidity with a 12-hour interval photoperiod. Adequate ventilation was supplemented to prevent the increase of carbon dioxide, which will prevent the fruit body development. Later, fresh fruiting bodies of the strain were collected and used as a protein source in diet fed mice with protein percentage 9.13-10.22% (Liang et al., 2019).

The experiment was carried out in a laboratory of the college of Veterinary Medicine, University of Baghdad, Iraq. One hundred and twenty BALB/c female mice, which are more stable to gain and lose the weight and less variability than the male that stated by De Souza et al. (2022). These mice are specific-pathogen-free grade, body weight 20-25 g., and 12-week age, were divided randomly into three groups (40 mice /treatment) and four replicates/treatment, 10 mice each. The three treatments were as being introduced (Table 1). The first group represents the control treatment (0% AP). Mice were housed and adapted to their new living conditions of (25 ± 2) °C, a relative humidity of 60±5 (%) and a 12-hour cycle of light and dark. The mice were fed on a commercial feed for 2 weeks for adaptation. After that, collected fruiting bodies from their cultivation substrates were dried in the room conditions in dark and ground into small particles by grinder to produce fruiting body powder (Figure 1A and B). The prepared diets and their compositions were shown in Table 1. The diet powder (40 mesh) was mixed with water, converted into 2-mm diameter pellets using meat grinder, and oven dried at 60ºC (Figure 1C and D). The treatments were distributed in completely randomized design (CRD).

Table 1: Composition of experimental diets for 100 gm in the three treatments.

AP: Auricularia polytricha

Body weights (g) and food intake (g/d) were recorded daily for eight weeks using a balance scale. Blood samples were collected by cardiac puncture and used for plasma lipid profile. Ten mice from each replicate (four replicates for each treatment) were fasted sacrificed. The mouse liver was immediately separated, washed and weighed to determine the liver index (%) based on the liver mass (g) to the body weight (g) (Liu et al., 2022).

Analysis of samples were collected in heparinized tubes and performed in the same Animal Laboratory. Plasma was separated by centrifugation at 3000 rpm for 10 min at room temperature to obtain mice serum. Liver extraction was extracted following the method of Garvey et al. (1977). Liver tissues were perfused with cold saline, excised, and kept frozen at -70ºC. Plasma total cholesterol, triglycerides (TG), and high-density-lipoprotein (HDL) levels were estimated using enzymatic assay kits according to the manufacturer’s instructions (Sigma, St. Louis, MO). Low-density lipoprotein (LDL) levels were also assessed according to the Friedewald equation (Vieira et al., 2016).

LDL = total cholesterol - (HDL + VLDL) where the concentration of very low-density lipoproteins (VLDL) is calculated by the serum concentration of TG. VLDL= TG/5.

Data was subjected to ANOVA analysis using the SPSS software. (Pallant, 2010) and Duncan's multiple range test, statistical comparisons of treatment means were achieved (Duncan, 1955).

Successfully, fruiting bodies were grown and produced under the previously described conditions and applied to animal diets (Figure 1). During the 8-week feeding trial, the dietary supplementation with A. polytricha to oral fed female mice did not affect the body weights, food intake, and the liver weights. There were no significant differences in these parameters between the treatments of 50% and 100% A. polytricha powder and the control treatment, 0% A. polytricha (Figure 2). The results of this study showed supplementation with AP did not significantly affect animal growth and feed efficiency compared to the control treatment. These findings confirmed previous studies that the dietary supplementation with macofungi have not increased the body weight of animals (Liu et al., 2022). This proved the unique nutritional composition of the edible Auricularia species which have higher amounts of proteins, very low percentage of soluble sugars (Mau et al., 1998), a low level of fat content (Cheung 2013), and a lower percentage of carbohydrates (Cheung 2013; Mau et al., 1998). Consequently, for this reason, the diet with macrofungi could not increase mice’s weights and body fat. The study also showed that total cholesterol, LDL, and VLDL in plasma of mice were decreased with increasing macrofungal concentrations in the regime (Figure 3).

The total cholesterol levels were 72.6 and 71.0 mg.100ml^-1 in the treatments, 50 %AP, and 100%AP respectively compared with 95.0 mg.100ml^-1 in the control. The decrease of cholesterol in the mice blood was associated with decreasing in triglycerides, LDL and VLDL. Triglycerides were decreased from 5 and 10 mg.100ml^-1 in the control treatment to 4.20, and 4.10 mg.100 ml^-1 for 50% AP, and 100%AP treatments respectively. LDL, which were 18.77 mg.100 ml^-1 in the control treatment decreased to 17.70, and 17.13 mg.100ml^-1 in 50%AP and 100%AP treatments respectively. VLDL also decreased from 27.90 mg.100ml^-1 in the control treatment to 24.73, and 24.37 mg.100 ml^-1 in AP50%, and AP100% treatments respectively. However, the diet with A. polytricha mushroom increased high-density-lipoproteins (HDL) from 23.13 mg.100 ml^-1 in the control treatment to 24.27 and 24.73 mg.100ml^-1 in AP50%, and AP100% treatment respectively. The mushroom diet in both treatments also decreased the Atherogenic index and Liver total cholesterol of mice after 8 weeks compared with that in the control treatment (Figure 3). In contrast, the mushroom diet increased the ratio of HDL-cholesterol to total cholesterol by 0.334 in the AP50% treatment and by 0.348 in the AP100% treatment. However, no significant difference was found between the effects of these two treatments (Figure 4).

The reduction in total cholesterol, LDL, and VLDL levels in current results was consistent with the previous findings. That is because of promoted fatty acid oxidation and inhibited gluconeogenesis via adiponectin pathway in the liver (Fontes et al., 2022). Decrease the total cholesterol and LDL cholesterol in present findings also similar to results of Budinastiti and his colleagues (2018) who reported the negative effects of water from boiled A. polytricha on the total cholesterol and LDL cholesterol of Wistar rats even after being reused cooking oil. These results may be attributed to the high viscosity of intestinal mucosa of mice (Liu et al., 2021). The activity of lactobacillus enzymes that precipitate and separate cholesterol from bile salt leading to decrease concentrations is not excluded (Lin et al., 1988; Gomez et al., 2021). In this study, increase the HDL cholesterol content supported the previous results as the water extracts of presented species showed anti-hypercholesterolemic effects in rats (Fontes et al., 2022). The increase HDL may be due to the inhibition of VLDL as well as may be resulting from increasing of bile acids contents (Yin et al., 2021). There are several fungus’s nutrient complex constituents which influenced the changing of blood lipid profiles. These included polysaccharides, arkobat acid, niasin, dan flavonoid and fiber (Rowan, and Sullivan, 2002; Zhao et al., 2015). For example, the β-glucan polysaccharide, which is one of the fungus fibers, can impede gastrointestinal lipase enzyme reaction such as pancreatic lipase enzyme which led to reduced absorbance and re-absorbance of fats including cholesterol and fat acid through fat metabolism (Rowan, and Sullivan, 2002). Based on antioxidant assays, the improvement in mice health by diet with A. polytricha can attributed to capacity of radical scavenging of this fungus. The ethyl acetate portion was the highest effective radical scavenger based on antioxidant investigations (Teoh et al., 2018).

According to a previous study, noodles created from A. polytricha can also prevent hyperlipidemia and liver fat buildup that resulted from the HFD and regulate the gut microbiota. A. polytricha noodles may promote the diversity of gut microbiota through the decreasing the relative abundance of Deferribacteres as well as the ratio of Firmicutes to Bacteroidetes and increasing the relative richness of Verrucomicrobia, potentially causative to the prevention of hyperlipidemia (Fang et al., 2021). Furthermore, a previous work suggested that A. polytricha supplement could provide an exceptional protecting substance against NAFLD under regular nutritive recovery. It is treated by reducing the inflammatory response, oxidative stress, and lipid deposition (Chiu et al., 2014). Additionally, polysaccharides from Auricularia auricular can modify the composition of fecal microbes and promote microbial community diversity. Auricularia auricular polysaccharides can boost immunoglobulin levels in mouse serum (Zhao et al., 2015).

In conclusion, the results of this work indicated that the edible mushroom may be promising as complementary dietary strategy for decreasing cholesterol levels in plasma. Otherwise, the results revealed no significant effect of mushrooms on body weights and food intake of mice. The mushroom in the diet caused a significant decrease in triglycerides, cholesterol, very low-density-lipoprotein, low density lipoproteins while the high-density lipoprotein was increased compared to the control treatment.

This work was financed by the Iraqi Ministry of Agriculture and Veterinary Medicine College, Baghdad University, Iraq.

The roles of first, fourth, and fifth authors were designing and achieving the experimentation while the roles of third and fourth authors were data analysis, writing, reviewing, and editing.

Cultivation and using this edible mushroom in Iraq as a food supplement and good nature nutrient food are a unique step for future use of this fungus for improving health.

The authors declare that there is no conflict of interest.