Antidiabetic activity of Armillaria mellea polysaccharides: Joint ultrasonic and enzyme assisted extraction

Graphical abstract

Abstract

Armillaria mellea polysaccharides (AMPs) were obtained by ultrasonic assisted extraction (U), enzyme assisted extraction (E) and ultrasonic-enzyme assisted extraction (UE), respectively. The yield of UE-AMPs (6.32 ± 0.14%) was 1.64 times higher than that of U-AMPs (3.86 ± 0.11%) and 1.21 times higher than that of E-AMPs (5.21 ± 0.09%); meanwhile, the highest total sugar content and the lowest protein content were found in UE-AMPs. AMPs obtained from the three extraction methods had the same monosaccharide composition but in different proportions, allowing UE-AMPs to have the most potent antioxidant activity. The antidiabetic activity of UE-AMPs was investigated in streptozotocin (STZ)-induced diabetic mice. UE-AMPs, when given by gavage, greatly prevented weight loss, increased water intake, and considerably decreased blood glucose levels in diabetic mice, which were dose-dependent (P < 0.05). In addition, UE-AMPs also had a positive effect on the reduction of lipid levels in the blood, oxidative damage and liver function impairment. The pathological observation by hematoxylin-eosin staining (HE) revealed that UE-AMPs protected the organs of mice from diabetic complications (liver disease and nephropathy). Hence, our findings demonstrate that UE-AMPs are a suitable choice for improving diabetes and its complications and have great application prospects in the fields of natural medicine and functional food.

1. Introduction

People's health is seriously affected by diabetes mellitus, one of the most common endocrine diseases. It is caused by the pancreas releasing insufficient or inadequate insulin, which results in an increase in blood glucose levels [1]. In the treatment of diabetes mellitus and its complications, a variety of antidiabetic drugs have been used, including glucosidase inhibitors, insulin secretagogues, insulin sensitizing agents, and so on [2], [3]. However, these drugs have negative side effects and toxicity, and their costs are too high for people living in low-income developing nations to afford [4]. For the development of reasonably safe and affordable anti-diabetic drugs, researchers have been focusing more on natural medicinal plant polysaccharides with various biological activities [5]. Studies in the past revealed that polysaccharides extracted from Trichosanthes peel [6], Fructus corni [7], Grifola frondosa [8], and blue honeysuckle [9] showed adequate efficacy in the treatment of diabetes.

The hazel mushroom, Armillaria mellea (Vahl) P. Kumm, is a member of the subphylum Bsaidiomycotina, order Agaricales, Tricholomataceae, and genus Armillariella [10]. It is active from June to the end of September, grows in groups of trees, stumps, or buried wood bases, and has a saprophytic or parasitic growth mode. A lot of Armillaria mellea can be found in the northeast and southwest regions of China, including Heilongjiang, Jilin, and Tibet. Armillaria mellea is the symbiotic fungus of Rhizoma Gastrodiae, which is a high-value crop. The nutritional value of Armillaria mellea has also attracted attention and played an influential role in the development of new natural medicinal products.

The majority of Armillaria mellea's chemical components are polysaccharides, sterols, proteins, fatty acids, sesquiterpenoids [11], non-hallucinogenic indole compounds, triterpenoids [12], adenosine compounds [13], and others [14]. Armillaria mellea polysaccharides (AMPs) have antioxidant, hypolipidemic, anti-dizziness, immune enhancing, and protective properties on macrophages and bone marrow cells [14], [15], [16], [17], [18]. It is a relatively rare study that has demonstrated the therapeutic effects of AMPs on diabetes mellitus as one of the central active components of Armillaria mellea.

It has been reported that AMPs can be obtained by simple methods, such as ultrasonic assisted extraction [19] or enzyme assisted extraction [20]. The ultrasonic assisted extraction method can increase the frequency and speed of material molecular movement through the cavitation effect, increase the solvent penetration, and improve the dissolution rate of natural components [21], which has the advantages of being fast, efficient, and energy-saving compared with traditional extraction processes [22], [23]. Guo et al. [24] prepared Lepidium meyenii Walp polysaccharides by multimodal ultrasound. Their results showed that the energy aggregation alternation of dual-frequency ultrasound under specific conditions increased the yield by 44.90% and enhanced the immunological activity of polysaccharides. Xu et al. [25] found that the structure and antioxidant properties of blackcurrant fruit polysaccharides were changed after different intensities of ultrasonic treatment. The enzyme assisted extraction method uses papain, cellulase, pectinase or their composite enzymes formed in a certain ratio to hydrolyze the fiber components of plant cell walls, thus improving the extraction efficiency, which has the characteristics of low energy consumption, high efficiency and high specificity [26]. Jia et al. [27] found that enzyme assisted extraction can improve both the yield and antioxidant properties of polysaccharides compared to traditional hot water extraction. The result was further boosted by adopting a compound enzyme extraction process.

It is worth mentioning that ultrasound-enzyme assisted extraction was applied for efficiency and sustainability [28]. In the experiment by Encalada et al., compared to the use of a single extraction method, ultrasound-hemicellulase led to the highest pectin-enriched fraction yield (27.1%) and extracted almost the whole pectin content of discarded carrots [29]. Similar results have been found by Velyamov et al. [30]. Besides, improvements in anti-proliferative activity [31] and antiglycation activity [32] using the combined ultrasonic assisted extraction and enzyme assisted extraction compared to each method alone have been recently reported. However, the combination of ultrasonic and enzyme for the extraction of AMPs has rarely been reported in the literature, which is of great practical value for the study of AMPs. Hence, the objective of this study was to investigate the positive effect of UE method on AMPs extraction, and the antidiabetic activity of UE-AMPs was demonstrated.

In this study, AMPs were obtained by three different extraction methods: enzyme assisted extraction, ultrasound assisted extraction, and ultrasound-enzyme assisted extraction. The structures and antioxidant properties of the AMPs obtained by the three different extraction methods were characterized. Given the high effectiveness of UE, UE-AMPs were gavaged to diabetic mice induced by streptozotocin (STZ) in order to investigate the antidiabetic activity of AMPs.

2. Materials and methods

2.1. Materials and reagents

Armillaria mellea was purchased from a medicine market in Changbai Mountain, Jilin, China, and identified according to the identification standard of Pharmacopeia of the People's Republic of China. The material was washed in water after removing impurities and dried in an oven at 55 °C until constant weight, then crushed and sieved through a 60mesh sieve. Then pretreated with petroleum ether and 95% ethanol to remove some colored materials and small molecule materials. Cellulase (400U/mg), papain (200U/mg), streptozotocin (STZ), metformin hydrochloride (MH) were purchased from Beijing Biotopped Technology Co., LTD (Beijing, China). Chromatographic grade monosaccharide standards, such as mannose, rhamnose, glucose, galactose, xylose, arabinose, and fucose, were purchased from Sigma-Aldrich (St. Louis, MO, USA). All other reagents were analytically pure from local suppliers.

2.2. Extraction and purification of AMPs

(1) U-AMPs: According to Li et al. 's method with some modifications [33]. The pretreated Armillaria mellea sample of 1.0 g was mixed with 20 mL of distilled water in a beaker. Then the polysaccharides were extracted by an ultrasonic bath (KQ-400KDE, Kunshan Ultrasonic Instrument co., LTD, Kunshan, Jiangsu, China) at 70 °C and the ultrasonic power was 280 W for 40 min. (2) E-AMPs: The extraction of E-AMPs was performed according to the previously reported method with some minor modifications [34]. The 1.0 g of Armillaria mellea sample was weighed, the compound enzyme was cellulase: papain = 1:1, the amount of compound enzyme added was 2.3%, the material-liquid ratio was 1:20 g/mL, and the pH of the aqueous solution was 5. The extraction was carried out at 40 °C for 140 min. (3) UE-AMPs: UE-AMPs were extracted using an ultrasonic bath with a power of 280 W at 70 °C for 40 min, followed by the enzyme complex extraction at 40 °C for 40 min. Other conditions were consistent with those of (2) E-AMPs extraction.

Referring to Chen et al.'s method and simply modifying it [6], after extraction, the supernatant was precipitated with anhydrous ethanol at 4 °C for 12 h to obtain a final concentration of 80% (v/v). Centrifugation at 4000 r/min for 15 min for sediment collection, free protein was removed several times using a Sevage reagent (chloroform: normal butanol = 4:1, v/v) method, and depigmented with AB-8 macroporous resin. The AMPs yield was calculated as follows:

$$\mathrm{Y}\mathrm{i}\mathrm{e}\mathrm{l}\mathrm{d}\left(\%\right)=\frac{m}{M}\times 100$$

where m is the weight of the AMPs, and M is the weight of Armillaria mellea sample.

Finally, the polysaccharides were purified using a DE-52 cellulose (3.5 cm × 20 cm) column, eluted first with distilled water, then with 0.1 mol/L and 0.3 mol/L of sodium chloride solution at a flow rate of 0.8 mL/min. The eluent was collected, and the polysaccharide content of each tube was monitored by the phenol sulfuric acid method. The major fraction (one fraction eluted with distilled water) containing carbohydrates was collected and concentrated. Three polysaccharides were obtained by freeze-drying for the following study.

2.3. Chemical composition and pH determination of AMPs

Total sugar content was determined using the phenol–sulfuric acid method with glucose as the standard [35]. Protein content was determined using the Bradford method, and a standard curve was made using bovine serum albumin (BSA) [36]. A pH meter (FE28-CN, Mettler Toledo, Switzerland, Zurich) was used to determine the pH values of AMPs at a concentration of 0.2 mg/mL.

2.4. Structural and compositional characterization

2.4.1. Fourier transform-infrared (FT-IR) spectrometry

The 2 mg sample was subjected to KBr compression and analyzed by FT-IR spectrophotometer (Nicolet iS50, Thermo Fisher Scientific, China) at a wave number of 4000–500 cm⁻¹.

2.4.2. Scanning electron microscope

Scanning electron microscopy (SEM) images of AMPs were obtained by field emission scanning electron microscopy (SU8010, Hitachi LTD., Japan). The gold sprayed samples were observed under high vacuum conditions using SEM at an accelerating voltage of 5 kV. The magnifications of the images were 500x, 1000x and 5000x.

2.4.3. Monosaccharide composition

The monosaccharide composition was determined from previous literature with slight modifications [37]. The samples after PMP derivatization were stored at 4 °C for HPLC analysis. Chromatographic analysis was performed on a 1260 Infinity II Prime HPLC system (Aglient, USA) using a HS PDA detector (Aglient, USA). For the HPLC analysis, the separation was performed on an Agilent ZORBAX Eclipse Plus C18 column (5 μm × 4.6 mm × 100 mm) operating at 30 °C with a flow rate of 1.0 mL/min and a loading volume of 2 μL. The mobile phase consisted of acetonitrile and potassium phosphate buffer (PBS, 0.1 mol/L, pH = 6.7, 17:83, v/v). The mixed standards consisted of mannose, rhamnose, glucose, arabinose, fucose, galactose and xylose. PDA detection was performed at 245 nm.

2.5. In vitro antioxidant activity assays

The antioxidant properties of AMPs were evaluated by DPPH radical scavenging activity and hydroxyl radical scavenging activity. The procedure of the assay was performed according to the previously described method [38]. Ascorbic acid and AMPs samples were dissolved in water for both assays, then diluted into a series of solutions with varying concentration gradients.

2.6. Animals and treatment

Considering the high efficiency of UE method, UE-AMPs were selected as anti-diabetic drugs for STZ-induced diabetic mice experiments.

A total of 60 male C57BL/6 mice (SCXK(京)2021–0006, weighing 20 ± 2 g) were purchased from Charles River (Beijing, China), and raised in a common animal lab. The housing environment was room temperature 22 ± 2℃, humidity: 55 ± 5%, 12 h of alternating light and dark. The experimental procedures were in accordance with the Regulations of the Experimental Animal Administration issued by the State Committee of Science and Technology of China. After 7 d of acclimatization feeding and 12 h of fasting without water, 10 mice were randomly selected as the control group, and the remaining 50 mice were injected intraperitoneally with streptozotocin (STZ) at a dose of 120 mg/kg. Fasting blood glucose (FBG) was determined after fasting without water for 72 h. Mice with FBG ≥ 11.1 were determined to have successful diabetes mimicry. The date of gavage was calculated as week 0 of successful mimicry. 10 mice from each group were administered by gavage every morning at regular intervals. The dosing amounts were: Control group (CON, n = 10): saline 200 μL; Diabetic model group (DM, n = 10): saline 200 μL; Positive control group (PC, n = 10): metformin hydrochloride (MH) 100 mg/kg/d; UE-AMPs low dose group (UE-AMPs-L, n = 10): UE-AMPs 100 mg/kg/d; UE-AMPs medium dose group (UE-AMPs-M, n = 10): UE-AMPs 200 mg/kg/d; and UE-AMPs high dose group (UE-AMPs-H, n = 10): UE-AMPs 300 mg/kg/d. The experiments were conducted for 28 d. The mice were fed and watered freely, and the bedding was changed once a day except for the testing requirement. The water and food intakes of them were monitored daily. In addition, their body weight at week 0 and week 4 were recorded.

2.7. Physiological analysis

2.7.1. Fasting blood glucose values

Fasting blood glucose values of all mice were measured once a week at a fixed time when gavage was started, and blood glucose values was measured using test strips and blood glucose meters (GA-3, Sinocare Inc., Hunan, China) after 8 h of fasting without water. The rate of fasting glucose reduction was calculated for the control and drug gavage groups with the following equation:

$$\mathrm{R}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{o}\mathrm{f}\mathrm{f}\mathrm{a}\mathrm{s}\mathrm{t}\mathrm{i}\mathrm{n}\mathrm{g}\mathrm{g}\mathrm{l}\mathrm{u}\mathrm{c}\mathrm{o}\mathrm{s}\mathrm{e}\mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\left(\%\right)=\frac{A_0-A}{A_0}\times 100$$

where A₀ is the fasting blood glucose values at week 0, and A is the fasting blood glucose values at week 4.

2.7.2. Glucose tolerance

After the last fasting glucose measurement was completed, the gavage was kept for two days. Fasting without water for 12 h before measurement of glucose tolerance. The gavage glucose dose was 2.0 g/kg, and the blood glucose values were measured at 0 min, 30 min, 60 min, 90 min and 120 min after gavage for 30 min, respectively.

2.7.3. Serum collection and measurement of organ indices

Referring to Zen et al. [39], after the last gavage, the blood was extracted from the eyes by fasting without water for 12 h. The liver, kidney and spleen were removed, rinsed repeatedly with saline, and the surface was blotted with filter paper and weighed to calculate the organ index of mice. The organ index is equal to the ratio of organ weight (mg) to body weight (g). The rates of decrease in the liver index and kidney index and the rates of increase in the spleen index were calculated from the organ indices with the following equations:

$$\mathit{Rate}ofliverindexreduction(\%)=\frac{A_{l0}-A_l}{A_{l0}}\times 100$$

$$\mathit{Rate}ofkidneyindexreduction(\%)=\frac{A_{k0}-A_k}{A_{k0}}\times 100$$

$$\mathit{Rate}ofspleenindexelevation(\%)=\frac{A_s-A_{s0}}{A_{s0}}\times 100$$

where A_l0 is liver index of diabetic model group, A_l is liver index of drug gavage group, A_k0 is kidney index of diabetic model group, A_k is kidney index of drug gavage group, A_s0 is spleen index of diabetic model group, A_s is spleen index of drug gavage group.

2.7.4. Blood biochemical analyses

The assay kits for total cholesterol (TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-c) low density lipoprotein cholesterol (HDL-c), superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), alanine transaminase (ALT), and aspartate transaminase (AST) were purchased from the Nanjing Institute of Biological Sciences (Nanjing, China), and assayed according to the kit instructions.

2.7.5. Histopathological observation

Following the previous approach with minor modifications [40], the liver and kidney were dissected from the mice, placed in 10% paraformaldehyde, and stored at 4 °C for 12 h. The organs were removed from the paraformaldehyde, treated with different concentrations of alcohol and dehydrating agents in order of dehydration, and solidified and cut into 4 μm slices for backup; HE staining: the prepared slices were stained in hematoxylin-eosin staining solution for 3 min, followed by another dehydration, and then the slices were sealed for microscopic observation and photo collection after drying.

2.8. Statistical analysis

All experimental data were in triplicate without special instructions. Statistical analysis of the data was done using SPSS (version 26.0) software. The differences were calculated using a one-way analysis of variance (ANOVA) test followed by Duncan's test. The results were expressed as mean ± SD. P < 0.05 was considered statistically significant.

3. Results and discussion

3.1. Yield and chemical composition of AMPs obtained by different extraction methods

Table 1 illustrates that UE-AMPs had the highest extraction yield among the three strategies. During the extraction process, the cavitation effect is responsible for producing more energy, resulting in the remarkable synergistic effect between the enzyme complex and ultrasound [41]. The result is an enhanced ability of AMPs to enter the solvent due to this effect. UE-AMPs had the highest total sugar content (71.76%) among the three methods, which may exhibit better biological activity. Duan et al. [42] extracted comfrey polysaccharides by employing four extraction methods. Among them, the UE method had higher extraction yields compared with other extraction methods. Similarly, Li [34] and Chen et al. [43] also showed that UE method was effective in increasing polysaccharide yields and obtaining better bioactive polysaccharides.

Table 1.

Yield and chemical composition of AMPs obtained by different extraction methods.

Polysaccharides	Yield (%)	Total sugar (%)	Protein (%)	pH
U-AMPs	3.86 ± 0.11c	67.69 ± 1.74ab	2.36 ± 0.31a	5.43 ± 0.07a
E-AMPs	5.21 ± 0.09b	65.32 ± 2.39b	2.48 ± 0.09a	4.96 ± 0.05b
UE-AMPs	6.32 ± 0.14a	71.76 ± 1.48a	1.79 ± 0.17b	5.59 ± 0.09a

U-AMPs, E-AMPs, UE-AMPs is the AMPs obtained by ultrasound assisted extraction, enzyme assisted extraction, ultrasound-enzyme assisted extraction. All data are expressed as means ± SD. Different letters in the same column indicate significant differences (P < 0.05).

3.2. Structural characterization of AMPs obtained by different extraction methods

As shown in Fig. 1, the strong absorption peak near 3354 cm⁻¹, 2923 cm⁻¹, 1597 cm⁻¹ and 1417 cm⁻¹ belonged to the O-H bending vibration, C-H stretching vibration, C = O stretching vibration, and C-H variable angle vibration, which were found in three kinds of AMPs. All three kinds of AMPs had strong absorption peaks near 1077 cm⁻¹, indicating the presence of pyranose. These were the characteristic absorption peaks of polysaccharide. In other words, no significant differences were observed in the plots of the three polysaccharides.

Fig. 1.

FT-IR spectrum of AMPs obtained by different extraction methods.

The scanning electron microscope images of three kinds of AMPs taken under various multiples are displayed in Fig. 2, which demonstrated the stark differences in the apparent structure among the three kinds of AMPs. U-AMPs had an uneven appearance, a compact and irregular arrangement, and a filamentous microstructure. Chen et al. [19] obtained AMPs with a similar lengthy shape using ultrasound assisted extraction. This might be due to changes in plant tissues caused by ultrasonic cavitation effects. E-AMPs were messy particle structures with small holes, which might have been left by enzyme-catalyzed hydrolysis of the cell wall. A closely spread lamellar surface with an uneven surface and holes made up the microstructure of UE-AMPs. The Armillaria mellea tissue was changed further and became homogenous and regular as a result of the synergistic interaction between ultrasonic cavitation and enzymatic hydrolysis. From this, we could infer that the microscopic morphology of polysaccharides is closely related to the extraction method.

Fig. 2.

SEM image of AMPs obtained by different extraction methods. a-c, d-f, g-i are U-AMPs, E-AMPs, UE-AMPs at 500×, 1000 × and 5000×, respectively.

3.3. Monosaccharide composition of AMPs obtained by different extraction methods

As shown in Fig. 3A, the HPLC spectra of the monosaccharide mixed standard showed that the peak order is mannose, rhamnose, glucose, galactose, xylose, arabinose, and fucose, respectively. As seen in Fig. 3B-D, the AMPs obtained by the three extraction methods all contain mannose, rhamnose, glucose, galactose, and fucose, which is consistent with the findings of Duan et al. [42]. However, the ratio of monosaccharide composition of AMPs was different in each extraction method (Fig. 3 and Table. 2). UE-AMPs obtained by the combined action of ultrasound and complex enzymes showed better biological activity, as demonstrated in the next experiments. Interestingly, this is similar to the previous experimental findings that the UE method helped obtain fractions with superior biological activity [31], [32].

Fig. 3.

HPLC spectra of AMPs obtained by different extraction methods. A, B, C, D represent monosaccharide reference, U-AMPs, E-AMPs and UE-AMPs, respectively. (1, mannose; 2, rhamnose; 3, glucose; 4, galactose; 5, xylose; 6, arabinose; 7, fucose).

Table 2.

Monosaccharide composition of AMPs obtained by different extraction methods.

Polysaccharides	Monosaccharide composition (molar ratio)
	Man	Rha	Glc	Gal	Fuc
U-AMPs	1.44	1	6.61	3.84	9.57
E-AMPs	1.83	1	10.21	2.29	15.51
UE-AMPs	1.43	1	11.54	2.51	5.97

U-AMPs, E-AMPs, UE-AMPs is the AMPs obtained by ultrasound assisted extraction, enzyme assisted extraction, ultrasound-enzyme assisted extraction.

3.4. In vitro antioxidant activity of AMPs obtained by different extraction methods

In Fig. 4, the antioxidant activity of AMPs was evaluated by (A) DPPH radical scavenging activity and (B) hydroxyl radical scavenging activity, which were dose dependent in both assays. UE-AMPs exhibited greater DPPH radical scavenging activity in the assay range of 0.00625–4 mg/mL, as shown in Fig. 4A, with a scavenging rate of up to 76.72 ± 0.59%. Fig. 4B illustrated that the hydroxyl radical scavenging rate of AMPs was much lower than that of ascorbic acid, as reflected by a maximum hydroxyl radical scavenging rate of 97.94 ± 0.60% for ascorbic acid, while AMPs were much smaller than this value. Notably, UE-AMPs exhibited stronger hydroxyl radical scavenging activity compared to U-AMPs and E-AMPs. The radical scavenging activity of polysaccharides was positively correlated with the hydroxyl radical donating capacity of their hydroxyl groups [44], [45]. Besides, it is widely acknowledged that the antioxidant activity of polysaccharides is related to various factors, such as chemical composition, monosaccharide composition and molecular weight [46]. The AMPs obtained by the three extraction methods had different monosaccharide composition ratios, which might lead to variations in monosaccharide sequences, glycosidic bonds, and polysaccharide configurations, thus exhibit different antioxidant properties. An interesting finding was that the combination of ultrasonic and enzyme assisted extraction resulted in AMPs with stronger antioxidant activity compared to the use of a single extraction method.

Fig. 4.

In vitro antioxidant activity of AMPs obtained by different extraction methods. (A) DPPH radical scavenging activity and (B) hydroxyl radical scavenging activity.

3.5. Antidiabetic activities in diabetic rats

3.5.1. Effects of UE-AMPs on body weight, food and water intake in diabetic mice

The mice in the CON group had black and bright hair, dry padding, a good mental state, and liveliness, while the DM group showed the typical “three more, one less” symptoms of diabetes, such as increased intake of water and food, increased urination, and weight loss. The mice treated with metformin hydrochloride (MH) and UE-AMPs improved to varying degrees, and the coat color and mental state also improved.

According to Table. 3, compared with the CON group, all the mice injected with STZ showed significant weight loss and increased water intake (P < 0.05), which was consistent with the symptoms of “three more and one less.” During the experiment, the weight of mice in the control group increased by 6.02 g, while that in the DM group decreased by 2.54 g. Weight loss in all groups improved after 4 weeks of intragastric administration and was significantly different from the DM group (P < 0.05).

Table 3.

Effects of UE-AMPs on body weight, food and water intake in diabetic mice.

Group	Body weight (g)		Food intake (g/mice/day)	Liquid intake (mL/mice/day)
	Initial	Final
CON	22.23 ± 1.67a	28.25 ± 1.08a	3.28 ± 0.85b	2.73 ± 0.24d
DM	16.87 ± 1.38b	14.33 ± 1.04c	5.54 ± 1.11a	6.82 ± 0.88a
PC	16.07 ± 1.13b	17.36 ± 1.24b	4.22 ± 0.67a	3.97 ± 0.68c
UE-AMPs-L	15.46 ± 1.29b	16.23 ± 1.08b	4.30 ± 1.09ab	5.56 ± 0.94b
UE-AMPs-M	16.93 ± 1.29b	17.68 ± 0.86b	4.78 ± 0.76 ab	5.42 ± 1.14b
UE-AMPs-H	16.77 ± 1.63b	17.42 ± 1.46b	4.02 ± 1.15 ab	5.12 ± 0.48bc

CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. All data are expressed as means ± SD (n = 10). Different letters in the same column indicate significant differences (P < 0.05).

3.5.2. Effects of UE-AMPs on the levels of blood-glucose in diabetic mice

Diabetes causes the pancreatic islets to lose their normal function of regulating blood glucose, resulting in abnormal blood glucose concentrations in organisms [47]. Table 4 showed that, with the exception of the CON group, all five groups were hyperglycemic, indicating that STZ modeling was successful. Fasting blood glucose levels in the CON group were affected by mental status and fluctuated but did not change much. Blood glucose levels in the UE-AMPs and PC groups were significantly lower after two weeks of gavage (P < 0.05). Until all gavage phases were completed, the PC group had the lowest fasting glucose values because MH increased the activity of enzymes related to the glycolytic pathway by increasing the rate of sugar metabolism and the efficiency of sugar utilization by the organism. As the dose of UE-AMPs increased, the fasting glucose lowering effect on diabetic mice increased, and there was no significant difference between the UE-AMPs-H and PC groups (P > 0.05). This demonstrated that there was a dose-dependent effect of UE-AMPs on improving fasting blood glucose levels in mice, and it could at most exert the same effect as the positive control MH used as a hypoglycemic agent in clinical practice. Therefore, it could be inferred that UE-AMPs had an active effect on improving diabetes.

Table 4.

Effects of UE-AMPs on fasting blood glucose in diabetic mice.

Group	Fasting blood glucose (mmol/L)					Reduction rate (%)
	0	1	2	3	4
CON	5.17 ± 1.18b	4.96 ± 0.72b	5.75 ± 0.62c	5.52 ± 1.13d	5.84 ± 0.45d	-/-
DM	14.11 ± 1.86a	13.68 ± 2.57a	14.69 ± 0.82a	14.18 ± 1.90a	14.89 ± 1.09a	-/-
PC	13.60 ± 2.04a	12.48 ± 1.61a	10.30 ± 0.63b	8.28 ± 1.89c	7.74 ± 1.29c	43.09
UE-AMPs-L	13.51 ± 1.05a	12.29 ± 1.43a	11.08 ± 1.35b	10.18 ± 0.78bc	9.75 ± 1.21b	27.83
UE-AMPs-M	14.47 ± 0.59a	12.44 ± 1.82a	11.23 ± 1.22b	10.98 ± 1.30b	9.25 ± 0.74bc	36.07
UE-AMPs-H	13.91 ± 1.19a	12.28 ± 1.36a	10.76 ± 2.31b	9.17 ± 1.84bc	8.60 ± 1.02bc	38.17

CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. All data are expressed as means ± SD (n = 10). Different letters in the same column indicate significant differences (P < 0.05).

Glucose tolerance is an important indicator of whether the organism can control blood glucose level well, which is an effective means to verify the ability of UE-AMPs to improve the regulation of blood glucose level in diabetic mice [48]. In all groups, glucose values reached a maximum after 30 min of gavage, decreased after 60 min, and basically returned to the pre-gavage values after 120 min (Fig. 5A). However, blood glucose values in diabetic mice in the DM group remained high after 120 min, indicating that diabetic mice in the DM group had impaired glucose tolerance. This was further confirmed by calculating the area under the curve (AUC, Fig. 5B), which is the total amount of elevated blood glucose in mice throughout the glucose tolerance experiment, and the lower the AUC value, the greater the effect of the drug. A significant decrease in AUC value occurred in both the PC and UE-AMPs gavage groups compared with that in the DM group (P < 0.05), demonstrating the significant effect of UE-AMPs on the treatment of glucose tolerance in diabetic mice. Dendrobium officinale polysaccharides (DOP) also had the ability to improve glucose tolerance in diabetic mice, and the AUC of diabetic mice in the DOP gavage groups was significantly reduced [49].

Fig. 5.

Effects of UE-AMPs on glucose tolerance and AUC in diabetic mice. A: effects on glucose tolerance; B: effects on AUC; CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. Different letters represent significant differences (P < 0.05).

3.5.3. Effects of UE-AMPs on organ index of diabetic mice

The organ index (organ weight/body weight) evaluates how well mice grow and develop. Diabetes elevated the liver and kidney indices while decreasing the spleen indices in mice, as shown in Table 5, and this condition reversed after UE-AMPs were gavaged. There was no significant difference among the PC, UE-AMPs-M, and UE-AMPs-H groups in liver and spleen indices (P > 0.05), which proved that UE-AMPs had a certain alleviating effect on liver and spleen injury in diabetic mice. Moreover, the UE-AMPs also reduced kidney injury in diabetic mice, as evidenced by a significant reduction in kidney index in the UE-AMPs gavage group. The above results proved that UE-AMPs effectively protected the organs of diabetic mice and the effect was positively correlated with the dose.

Table 5.

Effects of UE-AMPs on organ index of diabetic mice.

Group	Liver (mg/g)	Reduction rate (%)	Kidney (mg/g)	Reduction rate (%)	Spleen (mg/g)	Elevation rate (%)
CON	46.58 ± 1.62d	-/-	13.55 ± 0.24e	-/-	3.57 ± 0.39a	-/-
DM	68.34 ± 1.40a	-/-	18.36 ± 0.17a	-/-	2.60 ± 0.52c	-/-
PC	55.06 ± 2.52c	19.43	15.01 ± 0.23d	18.23	3.11 ± 0.26ab	19.61
UE-AMPs-L	59.62 ± 1.44b	12.76	17.97 ± 0.31ab	2.11	2.75 ± 0.36c	5.77
UE-AMPs-M	57.95 ± 3.91bc	15.21	17.44 ± 0.20b	5.02	2.95 ± 0.03ab	13.46
UE-AMPs-H	56.25 ± 1.23c	17.69	15.94 ± 0.15c	13.20	3.04 ± 0.43ab	16.92

CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. All data are expressed as means ± SD (n = 10). Different letters in the same column indicate significant differences (P < 0.05).

3.5.4. Effects of UE-AMPs on blood lipid in diabetic mice

Diabetes often causes hyperlipidemic complications because of the relationship between its pathogenesis and disorders of lipid metabolism [50]. Fig. 6 showed the effect of UE-AMPs on lipids in diabetic mice, and TG and TC contents were significantly lower in both PC and UE-AMPs gavage groups compared to the DM group (P < 0.05), which indicates that UE-AMPs can effectively reduce TG and TC contents in diabetic mice (Fig. 6A). From Fig. 6B, there was no large difference among the UE-AMPs-L, the UE-AMPs-M, and the DM groups (P > 0.05), but HDL-c content in the UE-AMPs-H group (1.17 ± 0.15 mmol/L) was significantly higher than that in the DM group (0.72 ± 0.16 mmol/L). Similarly, LDL-c levels were lower in both the PC and UE-AMPs gavage groups when compared to the DM group (P < 0.05). Both HDL-c and LDL-c contents are important factors in anti-atherosclerosis, and it can be inferred that UE-AMPs might be natural products with hypolipidemic effects, which is the same as the previous findings [17], [51].

Fig. 6.

Effects of UE-AMPs on blood lipids in diabetic mice. A: effects on TG/TC content; B: effects on HDL-c/LDL-c content; CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. Different letters represent significant differences (P < 0.05).

3.5.5. Effects of UE-AMPs on oxidative stress in diabetic mice

The association between the development of diabetes and oxidative reactions has been reported to be significant. The antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD), as well as the antioxidant molecule reduced glutathione (GSH), can inhibit free radical production, and MDA produced by lipid peroxidation can reduce the activity of these enzymes [50]. Diabetic mice in the DM group demonstrated a substantial decrease in CAT and SOD activity, a drop in GSH content, and an increase in MDA content in comparison to the CON group (P < 0.05), suggesting that diabetes induced severe oxidative damage in mice (Fig. 7). After high doses of UE-AMPs gavage (300 mg/kg/d), the activity of CAT was significantly improved and the damage of hydroxyl radicals to the mice's organism was reduced (Fig. 7A). With the increase of the UE-AMPs gavage dose, the GSH content of diabetic mice increased continuously and reached the highest level close to normal, which proved that UE-AMPs could reduce the damage of superoxide anion radicals to the mice's organism and that the effect on the GSH content was dose-dependent (Fig. 7B). The effect of UE-AMPs on SOD activity was not significant because of a slight increase in SOD activity in diabetic mice after gavage with UE-AMPs (Fig. 7C). The MDA content in the PC and UE-AMPs gavage groups was considerably lower than that in the DM group (P < 0.05), whereas the MDA content in the UE-AMPs-H group did not differ significantly from that in the PC group, both of which were close to normal levels (Fig. 7D). The results indicated that UE-AMPs had a significant effect in reducing lipid oxidation and free radical damage to the diabetic mice's organism, which verified the conclusion that AMPs have strong antioxidant properties [52], [53], [54].

Fig. 7.

Effects of UE-AMPs on oxidative stress in diabetic mice. A: effects on CAT activity; B: effects on GSH content; C: effects on SOD activity; D: effects on MDA content; CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. Different letters represent significant differences (P < 0.05).

3.5.6. Effects of UE-AMPs on liver function in diabetic mice

One of the complications of diabetes mellitus that has attracted a lot of attention is diabetic liver disease, where the liver plays an important role in the body in metabolism, detoxification, storage and excretion of exogenous and own metabolites [55]. The activities of alanine transaminase (ALT) and aspartate transaminase (AST) can reflect the liver function of the body, which are very effective indicators. As shown in Fig. 8, the activity of ALT was significantly lower in the UE-AMPs-M and UE-AMPs-H groups compared to the DM group (P < 0.05), while the AST activity of diabetic mice in the UE-AMPs gavage group was reduced with no significant difference between the UE-AMPs-H, PC, and CON groups (P > 0.05), indicating that UE-AMPs had the ability to repair damaged hepatocytes and the high dose of UE-AMPs.

Fig. 8.

Effects of UE-AMPs on liver function in diabetic mice. CON, DM, PC, UE-AMPs-L, UE-AMPs-M, UE-AMPs-H represent the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively. Different letters represent significant differences (P < 0.05).

3.5.7. Effect of UE-AMPs on the morphology of liver and kidney tissue in diabetic mice

The liver and kidney are relatively sensitive organs with high detoxifying capacities that play an important role in glucose metabolism. Analyzing the morphological changes in liver and kidney tissues aids in understanding the mechanism of diabetes's effects on the organs [56]. As demonstrated in Fig. 9, the CON group had a healthy liver with regular hepatocyte morphology. Diabetic mice (DM group) showed irregular shapes of liver lobules, deepened colors, and blurred cell boundaries, which proved that diabetes mellitus had seriously damaged hepatocytes in mice. In the UE-AMPs gavage groups, the inflammatory cell infiltration was reduced, the intercellular vacuoles were reduced, and the arrangement tended to be neat.

Fig. 9.

Effects of UE-AMPs on the morphology of liver tissue in diabetic mice. a, b, c, d, e, f represents the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively.

Fig. 10 illustrated that mice in the CON group had a complete overall structure with well-arranged glomerular and tubular structures. In contrast, mice in the DM group had inflammatory cell infiltration and obvious adhesion of intra-glomerular epithelial cells to the glomerular capsule. When compared to the DM group, the renal histomorphology in the UE-AMPs gavage groups improved, with less inflammatory cell infiltration and more regular glomerular and tubular organization. The kidney structure was basically restored in the UE-AMPs-H group.

Fig. 10.

Effects of UE-AMPs on the morphology of kidney tissue in diabetic mice. a, b, c, d, e, f represents the control group, STZ model group, low dose UE-AMPs group, medium dose UE-AMPs group, and high dose UE-AMPs group, respectively.

These results are consistent with previous findings (3.5.3 and 3.5.6). The degree of damage was gradually reduced, and it could be found that the degree of liver and kidney damage improved with the increase in the UE-AMPs gavage dose. A similar phenomenon can be found in Auricularia cornea var. Li. polysaccharide (ACP) [57]. High doses of ACPs had an important role in protecting the liver and kidneys from damage in diabetic mice.

4. Conclusions

In summary, UE effectively increased the yield of AMPs while also improving their chemical composition and monosaccharide composition, resulting in the greatest antioxidant activity of UE-AMPs. The experimental results of STZ-induced diabetic mice showed that UE-AMPs had a significant effect on improving the symptoms of “three more and one less” in mice. In diabetic mice, UE-AMPs had dose-dependent effects on blood glucose control, blood lipid reduction, and oxidative stress resistance. According to the organ index study, liver function study and pathological observation of HE staining, it could be concluded that UE-AMPs protect the organs of mice and avoid the occurrence of liver disease and kidney disease complicated by diabetes. Therefore, UE was an advanced polysaccharide extraction technology, and UE-AMPs could be used as a novel natural active substance for the treatment of diabetic mice in the fields of medicine and food.

CRediT authorship contribution statement

Xiaoyi Li: Methodology, Formal analysis, Investigation, Writing – original draft, Visualization. Jingshu Zhu: Investigation, Formal analysis, Writing – review & editing. Tengyu Wang: Software, Formal analysis. Jiapeng Sun: Investigation. Tianhao Guo: Investigation. Lijuan Zhang: Validation. Guoping Yu: Conceptualization, Supervision, Project administration, Writing – review & editing, Funding acquisition. Xiufang Xia: Resources, Methodology, Project administration.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.