Antihyperglycaemic and organic protective effects on pancreas, liver and kidney by polysaccharides from Hericium erinaceus SG-02 in streptozotocin-induced diabetic mice

Abstract

The present work was designed to investigate the antihyperglycaemic and protective effects of two Hericium erinaceus intracellular polysaccharide (HIPS) purified fractions (HIPS1 and HIPS2) from mycelia of H. erinaceus SG-02 on pancreas, liver and kidney in streptozotocin (STZ)-induced diabetic mice. The supplementation of HIPS1 and HIPS2 significantly decreased the blood glucose (GLU) levels; suppressed the abnormal elevations of alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CRE) levels in serum; improved the antioxidant enzymatic (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT)) activities; and attenuated the pathological damage to these organs. The HIPS1 showed superior effects in antihyperglycaemia and organic protection than HIPS2 possible owing to the abundant functional groups (-NH2, -COOH and S=O) in HIPS1, indicating that H. erinaceus SG-02 could be used as a functional food and natural drug for the prevention of diabetes and its complications.

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Antihyperglycaemic and organic
protective eects on pancreas,
liver and kidney by polysaccharides
from Hericium erinaceus SG-02 in
streptozotocin-induced diabetic
mice
Chen Zhang1, Juan Li2, Chunlong Hu3, Jing Wang4, Jianjun Zhang1, Zhenzhen Ren1,
Xinling Song1 & Le Jia1
The present work was designed to investigate the antihyperglycaemic and protective eects of two
Hericium erinaceus intracellular polysaccharide (HIPS) puried fractions (HIPS1 and HIPS2) from
mycelia of H. erinaceus SG-02 on pancreas, liver and kidney in streptozotocin (STZ)-induced diabetic
mice. The supplementation of HIPS1 and HIPS2 signicantly decreased the blood glucose (GLU) levels;
suppressed the abnormal elevations of alkaline phosphatase (ALP), alanine aminotransferase (ALT),
aspartate aminotransferase (AST), urea nitrogen (BUN) and creatinine (CRE) levels in serum; improved
the antioxidant enzymatic (superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase
(CAT)) activities; and attenuated the pathological damage to these organs. The HIPS1 showed superior
eects in antihyperglycaemia and organic protection than HIPS2 possible owing to the abundant
functional groups (-NH2, -COOH and S=O) in HIPS1, indicating that H. erinaceus SG-02 could be used as
a functional food and natural drug for the prevention of diabetes and its complications.
Diabetes mellitus (DM), the most significant chronic disease, which is characterized by hyperglycaemia
and is associated with disturbances in carbohydrate, protein and fat metabolism, has become a global public
health issue due to its prevalence and high morbidity1. Clinically, DM can be classied into two types: type I
(insulin-dependent DM) and type II (non-insulin-dependent DM). e former (type I) is always accompanied
with complications such as vision loss, renal failure and nerve damage, while the latter (type II) is characterized
by peripheral insulin resistance and impaired insulin secretion, leading to cardiovascular system diseases2. Many
factors have potential eects that raise the risk of DM, including viral infections, autoimmune diseases, unnatural
diets, environmental factors, and other variables3–6. Recently, increasing literature reports of both clinical and
experimental studies have demonstrated that oxidative stress plays a vital role in the progression of DM and its
complications7–11. Streptozotocin (STZ), depending on its biotoxicity on pancreatic β-cells, which play important
roles in the homeostasis of blood glucose through insulin synthesis, has been commonly used as an agent for
experimentally inducing DM10–12. e cellular mechanisms of β-cell destruction may be correlated with local
reactive oxygen species (ROS) and nitric oxide (NO), which are induced by cytokine stimulation. In addition, the
liver and kidneys, the detoxifying organs of the body, are aected by STZ-induced oxidative stress, which causes
structural damage. Antioxidant agents can increase the expression of antioxidant enzymes and alleviate oxidative
stress in both serum and organs, which can enhance the resistance to DM13–16. erefore, the antihyperglycaemic
eects of some antioxidants should be investigated.
1College of Life Science, Shandong Agricultural University, Taian, 271018, China. 2Chinese Academy of Agricultural
Sciences, Beijing, 100081, China. 3College of Forestry, Shandong Agricultural University, Taian, 271018, China. 4The
Central Hospital of Taian, Taian, 271000, China. Chen Zhang, Juan Li and Chunlong Hu contributed equally to this
work. Correspondence and requests for materials should be addressed to L.J. (email: jiale0525@163.com)
Received: 13 February 2017
Accepted: 24 August 2017
Published: xx xx xxxx
OPEN

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Clinically, insulin injection and oral administration of hypoglycaemic agents are two traditional therapies
for treating DM17. Among such treatments, the control of postprandial hyperglycaemia is critical in early ther-
apy for DM18. One therapeutic approach to decrease postprandial hyperglycaemia is to retard the absorption
of glucose by inhibiting carbohydrate-hydrolysing enzymes, such as α-amylase and α-glucosidase, in digestive
organs19. e inhibition rate for carbohydrate-hydrolysing enzymes could be regarded as a preliminary indicator
for the eectiveness of therapeutic agents. Recently, common oral hypoglycaemic agents, such as sulfonylureas
and biguanides, have been blamed for their side eects during long-time use and yield little therapeutic ecacy
against diabetic complications17. Hence, alternative medicines and natural therapies with non-toxic properties
have gained further academic attention. As an alternative approach, mushrooms have been widely used in med-
ical applications due to the abundant bioactive compounds they contain, which have potential anticarcinogenic,
anti-inammatory, hypolipidaemic, antidiabetic and hepatinica activities20. Interestingly, it has previously been
demonstrated that polysaccharides from mushrooms, such as Pleurotus djamor7, Phellinus baumii21 and Agrocybe
cylindracea13, have signicant eects in the treatment of DM and its complications.
Hericium erinaceus (H. erinaceus), an edible fungus that inhabits mountainous areas, has been used in tradi-
tional folk medicine and medicinal cuisine in the northeast territories of Asia22. Previous pharmacological studies
have conrmed that H. erinaceus has potential medicinal applications in the treatment and prevention of hyper-
glycaemia, chronic bronchitis, cancer, arteriosclerosis, hypertension, hypercholesterolemia and leucopenia23. In
recent years, basic studies on the chemical identication of the active ingredients of H. erinaceus have progressed,
and polysaccharides have been identied as the main eective components. Submerged cultures have become a
main source of polysaccharides, having the advantage of shorter incubation times and higher production levels
compared with common cultures22, 24. To the best of our knowledge, up to this point, there have been no reports
regarding any protective eects of mycelia polysaccharides from H. erinaceus SG-02 on pancreas, livers and kid-
neys in STZ-induced diabetic mice.
In this article, two new intracellular polysaccharides were successfully isolated from mycelia of H. erinaceus
SG-02. eir antihyperglycaemic and inhibitory eects on carbohydrate-hydrolysing enzymes and their pro-
tective eects on pancreas, livers and kidneys in STZ-induced diabetic mice were investigated. Moreover, their
monosaccharide compositions and infrared spectra were also analysed.
Results
Purication of H. erinaceus intracellular polysaccharide (HIPS). As shown in Fig.1, two elution
peaks, HIPS1 and HIPS2, were puried via DEAE-52 anion-exchange chromatography. HIPS1 was a neutral pol-
ysaccharide, as it was eluted with distilled water, while HIPS2 was an acidic polysaccharide, as it was eluted with
a 0.3 mol/L NaCl solution25. Individual peaks of both HIPS1 and HIPS2 were puried using a Sephadex G-100
cellulose column (Fig.1B and C), demonstrating that the two fractions were both homogeneous and pure. e
yields of HIPS1 and HIPS2 were 21.62 ± 0.74% and 32.75 ± 1.21%, respectively.
Figure 1. Elution proles of HIPS1 and HIPS2. (A) Elution proles of HIPS by DEAE-52 cellulose column
chromatography with gradient of NaCl solution (0, 0.2, 0.3 and 0.5 mol/L), (B) Elution proles of HIPS1, and
(C) HIPS2 on Sephadex G-100 cellulose column.

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Ultraviolet (UV) spectroscopy, Gas chromatography (GC) and Fourier transform infrared (FT-IR)
analysis of polysaccharides. e UV scanning spectrums of HIPS1 and HIPS2 showed no absorption at
280 and 260 nm, indicating the polysaccharides were puried and free of nucleic acids and proteins.
e monosaccharide compositions of HIPS1 and HIPS2 are shown in Fig.2. HIPS1 was composed of xylose
(Xyl) (3.02%), mannose (Man) (4.36%), galactose (Gal) (9.41%) and glucose (Glc) (83.21%), with molar ratios of
1:1.2:2.6:23.1 (Fig.2B), while HIPS2 contained Man, Gal and Glc, with mass percentages of 23.40%, 50.34% and
26.27%, and molar ratios of 1:2.15:1.12 (Fig.2C), respectively.
Figure 2. e gas chromatographs and FT-IR spectrums of polysaccharides. Gas chromatographs of (A)
standard monosaccharides, (B) HIPS1 and (C) HIPS2; FT-IR spectrums of (D) HIPS1 and (E) HIPS2. Peaks:
(1) Rha, (2) Rib, (3) Ara, (4) Xyl, (5) Man, (6) Gal and (7) Glc; UV scanning spectrums of (F) HIPS1 and (G)
HIPS2.

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FT-IR spectra of HIPS1 and HIPS2 are shown in Fig.2. It is obvious that both HIPS1 and HIPS2 exhibited
typical major -OH broad stretching bands for carbohydrates at approximately 3500–3400 cm−1. e absorption
peaks at 1637.85 and 1637.37 cm−1 are attributed to the stretching vibration of C=O. e bands at approximately
1618 cm−1 indicate the presence of -NH2 in the two samples. e absorption peak for HIPS1 at 2030.49 cm−1 is
a characteristic absorption peak for C≡C26, and the bands at approximately 1450 cm−1 indicate the presence of
-COOH groups in HIPS127. e band at approximately 1153 cm−1 suggests valent vibrations of the C-O-C bond
and a glycosidic bridge in HIPS123, while α-glucan28 had an additional band at 1026 cm−1. e characteristic
absorption peak at 866.03 cm−1 indicates the presence of a furan ring in HIPS113, while the additional weak peak
at approximately 620.83 cm−1 describes S=O vibrations in HIPS126, 29.
Inhibitory eects on α-amylase and α-glucosidase activities. e inhibitory eects of HIPS1 and
HIPS2 on α-amylase and α-glucosidase activities were investigated in the present work. As shown in Fig.3, the
inhibitory activities of HIPS2 on α-amylase and α-glucosidase were lower than those of HIPS1. e α-amylase
inhibition rate of HIPS1 (53.27 ± 2.16%) was maximal at a concentration of 5 mg/mL, which was 32.09% higher
than that for HIPS2. The 50% inhibitory concentration (IC50) value for HIPS1 on α-amylase activity was
3.42 ± 0.29 mg/mL, and the inhibition rate declined at concentrations greater than 5 mg/mL. At a concentration
of 6 mg/mL, the α-glucosidase inhibition rate of HIPS1 was 36.64 ± 1.83%, which was 14.21% higher than that
for HIPS2.
Eects of HIPS1 and HIPS2 on blood glucose (GLU) levels, body weights, and pancreas, liver
and kidney indices. e results for GLU levels, body weight, and pancreas, liver and kidney indices in
STZ-induced diabetic mice are listed in Table1. Before treatment, the GLU levels of the diabetic mice were
markedly increased than those of normal mice (Table1, P < 0.05). Aer treatment, the GLU levels in the six dose
groups were signicant decreased than that in the MC groups, indicating that the pathological increases could be
suppressed by HIPS1 and HIPS2 administration at dierent doses (P < 0.05). Specically, compared with the MC
group, the nal GLU levels in the H-HIPS1, M-HIPS1, L-HIPS1, H-HIPS2, M-HIPS2 and L-HIPS2 groups were
reduced by 51.65 ± 9.11, 38.77 ± 5.48, 34.21 ± 6.54, 37.05 ± 6.80, 32.56 ± 9.67 and 28.93 ± 5.94%, respectively.
Additionally, both HIPS1 and HIPS2 showed eects in reducing GLU levels but the values remained dierent in
comparison with that in NC groups among the test doses.
e body weights of pre-treatment and post-treatment diabetic mice were also determined. At the onset of
the experiments, there was no signicant dierence (P < 0.05) between the body weights of normal and diabetic
mice, while the diabetic mice without any treatment had emaciated bodies compared with the seven treatment
Figure 3. e inhibition rates of HIPS1 and HIPS2 on α-amylase and α-glucosidase. e values are reported as
the means ± SD (n = 3).

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groups at the end of the study. Specically, the body weights of mice in the NC groups were signicantly (P < 0.05)
increased during the treatment. However, the diabetic mice in the MC group exhibited signicant weight loss
compared with the NC group (Table1, P < 0.05). When compared with that in the MC groups, the mice in
H-HIPS1, M-HIPS1, L-HIPS1, H-HIPS2 and M-HIPS2 groups showed signicant improvements on the body
weights (P < 0.05). And the results also indicated that HIPS1 showed stronger eects in improving the body
weights, which was reected by the paralleled values in comparison with that in the NC groups.
Furthermore, the indices for the pancreas, liver and kidneys in the mice were also investigated, and the results
are listed in Table1. Signicant increases in the pancreas, liver and kidney indices were observed in the diabetic
mice (MC) group compared with those in the NC group (P < 0.05). However, the three indices were decreased
aer treatment with HIPS1 and a high dose of HIPS2. Especially in the H-HIPS1 group, the pancreas, liver and
kidney indices of the mice were reduced by 32.01 ± 2.16, 22.71 ± 2.94 and 39.11 ± 3.32%, respectively, compared
with those in the MC group. However, HIPS2 was much less eective than HIPS1 in restoring the organ indexes,
especially at the low dose.
Eects of HIPS1 and HIPS2 on serum biochemistry. e activities and levels of albumin (ALB), alka-
line phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN)
and creatinine (CRE) in serum are clinically used as biochemical markers for early liver and kidney damage. As
shown in Table2, mice exhibited liver and kidney damage aer injection with STZ, as evidenced by signicant
increases in the serum levels of ALP, ALT, AST, BUN and CRE (P < 0.05) and decreased ALB levels. Dramatically,
when compared with that in the MC groups, the activities of ALT, AST and ALP, as well as the levels of BUN and
CRE were signicantly (P < 0.05) decreased, while the ALB levels were increased by treatment with HIPS1 at the
three tested dosages. However, only the high dose of HIPS2 (600 mg/kg) was eective in maintaining the above
eectiveness. Particularly, the serum biochemical parameters for mice in the H-HIPS1 groups were similar to that
in normal mice, indicating the high dose of HIPS1 was much more eective than the other doses of HIPS1 and
HIPS2. In the present work, HIPS1 could suppress the abnormal elevations of ALP, ALT, AST, BUN and CRE lev-
els and the decline in ALB levels in diabetic mice, indicating that the polysaccharides extracted from H. erinaceus
SG-02 had potential protective eects against STZ-induced liver and kidney damage. Additionally, supplementa-
tion with glibenclamide could achieve eects similar to supplementation with HIPS1 (Table2).
Eects of HIPS1 and HIPS2 on GSH peroxide (GSH-Px), superoxide dismutase (SOD), cata-
lase (CAT), and malondialdehyde (MDA). To study the eects of HIPS1 and HIPS2 administration on
enzyme activities and free radical production, GSH-Px, SOD and CAT activities and MDA levels were measured
Group
GLU levels (mmol/L) Body weight (g) Pancreas index
(g/100 g) Liver index
(g/100 g) Kidney index
(g/100 g)Pre-treatment Post-treatment Pre-treatment Post-treatment
NC 4.13 ± 0.21a 4.18 ± 0.12a 25.23 ± 1.21a 34.29 ± 1.12a#1.76 ± 0.13a 15.41 ± 0.89a 4.27 ± 0.19a
MC 13.82 ± 0.15c 15.14 ± 0.56b#24.16 ± 0.95a 25.24 ± 0.97d 2.78 ± 0.27d 23.78 ± 1.76d 9.64 ± 0.71d
PC 14.76 ± 0.17b 5.35 ± 0.41f#23.93 ± 0.32a 32.77 ± 1.13ab#2.31 ± 0.25c 18.16 ± 0.79a 5.82 ± 0.48ab
H-HIPS1 14.05 ± 0.58c 7.32 ± 0.82e#24.89 ± 0.68a 31.38 ± 1.42ab#1.89 ± 0.21ab 18.38 ± 1.06a 5.87 ± 1.03ab
M-HIPS1 13.67 ± 0.32c 9.27 ± 0.17d#25.45 ± 0.86a 30.19 ± 1.17abc#1.92 ± 0.16ab 19.43 ± 1.73ab 6.81 ± 0.92b
L-HIPS1 13.87 ± 0.13c 9.96 ± 0.45cd#24.97 ± 0.79a 28.96 ± 0.59bc#2.23 ± 0.11bc 21.51 ± 0.53bc 7.21 ± 1.27bc
H-HIPS2 13.98 ± 0.49c 9.53 ± 0.47d#25.41 ± 0.47a 29.05 ± 1.51bc#2.21 ± 0.23bc 20.96 ± 0.67ab 8.21 ± 1.52c
M-HIPS2 14.87 ± 0.25b 10.21 ± 0.91cd#25.47 ± 1.18a 29.21 ± 0.92c#2.26 ± 0.22bc 22.15 ± 0.82c 8.54 ± 0.96c
L-HIPS2 13.99 ± 0.31c 10.76 ± 0.34c#25.03 ± 1.31a 27.56 ± 1.21b#2.58 ± 0.15cd 23.21 ± 2.23cd 9.21 ± 1.19cd
Table 1. Eects of HIPS1 and HIPS2 on the GLU levels, body weights, as well as index of pancreas, liver and
kidney. e values are reported as the means ± SD (n = 10 for each group). Means with the same letter are not
signicantly dierent (P < 0.05). #Signicant dierence compared to Pre-treatment (P < 0.05).
Group ALB (g/L) ALP (U/L) ALT (U/L) AST (U/L) BUN (mmol/L) CRE (μmol/L)
NC 35.65 ± 2.47a 121.67 ± 13.66a 37.41 ± 3.56a 116.42 ± 13.36a 6.23 ± 0.19a 60.27 ± 2.19a
MC 12.74 ± 3.78f 183.13 ± 20.14d 126.38 ± 15.37e 217.83 ± 30.55f 8.17 ± 0.21d 93.53 ± 10.38d
PC 30.79 ± 3.80bc 138.21 ± 9.76ab 47.93 ± 7.31ab 142.29 ± 17.45bc 6.57 ± 0.44ab 66.22 ± 2.95ab
H-HIPS1 31.21 ± 3.52b 139.52 ± 10.59b 44.98 ± 5.99a 135.27 ± 16.11ab 6.81 ± 0.33ab 66.32 ± 4.31ab
M-HIPS1 29.28 ± 4.3bc 150.67 ± 8.38bc 62.13 ± 10.96b 151.12 ± 11.65c 7.21 ± 0.11bc 75.22 ± 3.34b
L-HIPS1 23.78 ± 5.62de 159.53 ± 13.55bc 88.74 ± 16.51c 188.67 ± 9.64e 7.86 ± 0.31cd 78.66 ± 4.92c
H-HIPS2 25.95 ± 1.49cd 161.07 ± 15.33bcd 83.77 ± 16.59c 180.29 ± 15.49d 7.95 ± 0.12cd 75.49 ± 7.47c
M-HIPS2 20.87 ± 3.38e 168.42 ± 11.57cd 109.21 ± 18.65d 186.44 ± 13.87de 8.09 ± 0. 28cd 86.08 ± 6.85cd
L-HIPS2 17.99 ± 3.05e 172.69 ± 15.94cd 115.39 ± 20.19de 194.41 ± 22.51e 8.26 ± 0.39cd 87.24 ± 3.77d
Table 2. Eects of HIPS1 and HIPS2 on serum biochemistry in dierent groups. e values are reported as the
means ± SD (n = 10 for each group). Means with the same letter are not signicantly dierent (P < 0.05).

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in the three organs (Fig.4). At the end of the experiment, the GSH-Px, SOD and CAT activities in STZ-induced
diabetic mice (MC groups) were signicantly reduced (P < 0.05), while MDA levels were markedly increased
(P < 0.05) compared with those in the NC groups, indicating that the pancreas, liver and kidney all suered
oxidative stress. As shown in Fig.4, the abnormal physiological and biochemical changes in the three organs of
diabetic mice were restored by HIPS1 and HIPS2 administration. e GSH-Px, SOD and CAT activities in the
liver reached maximum values of 72.75 ± 4.38, 158.68 ± 16.42 and 223.14 ± 26.12 U/mg protein, respectively, in
the H-HIPS1 group, values that were signicantly higher than those in the MC group (20.98 ± 3.26, 65.85 ± 5.31
and 74.96 ± 9.41 U/mg protein), respectively, (P < 0.05 for all), and with no signicant dierence compared to the
NC group (Fig.4A–C). e hepatic MDA levels reached 5.98 ± 0.23 μmol/L in the H-HIPS1 group, which was
reduced by 49.14 ± 6.72% compared with those in the MC groups (Fig.4D).
Similar results were also observed for GSH-Px, SOD and CAT activities in the kidney (Fig.4E–G) and pan-
creas (Fig.4I–K). When compared with the MC group, signicant (P < 0.05) increases were observed aer the
administration of HIPS1 (including the three doses) and HIPS2 (including the high dose and middle dose). e
MDA levels for the kidney (Fig.4H) and pancreas (Fig.4L) also declined aer treatment with the polysaccharides.
Figure 4. Eects of HIPS1 and HIPS2 on GSH-Px, SOD, CAT, and MDA in liver (A–D), kidney (E–H) and
pancreas (I–L). e values are reported as the means ± SD (n = 10 for each group). Means with the same letter
are not signicantly dierent (P < 0.05).

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Glibenclamide-treated mice also manifested signicant increases in SOD, GSH-Px, and CAT activities, and
decreases in the MDA levels in the three organs compared with diabetic mice (P < 0.05).
e relevant indices in the H-HIPS1 group were approximately equal to those in the NC group. is conclu-
sion demonstrated that the high dose of HIPS1 exhibited strong potential antioxidant eects against oxidative
stress in the liver, kidney and pancreas.
Histopathological observations of the pancreas, liver and kidney. Histopathological observations
of the pancreas, liver and kidney were performed using haematoxylin and eosin (HE) staining (Figs5–7).
e eects of HIPS1 and HIPS2 on pancreatic tissues are shown in Fig.5. Severe degeneration in the pancreas
appeared in the STZ-induced diabetic mice, including lymphocyte inltration, hypochromatosis and the disap-
pearance of cell borders in the pancreatic islets (Fig.5B). Aer treatment with HIPS1 at all doses and HIPS2 at
the high and middle doses, improvement in the pancreatic tissues could be observed, which could be evidenced
by decreased inltration and more integrated cell structure in the pancreatic islets compared with the mice in
the MC group (Fig.5D–I). ese results indicated that treatment with polysaccharides from H. erinaceus SG-02
could repair islet damage and improve the structural integrity of pancreatic islet beta-cells and tissues.
As shown in Fig.6A, the hepatocyte morphology was normal, and the hepatic cells were arranged in a reg-
ular manner with abundant cytoplasm, distinct cell borders and normal central nuclei. In the MC group, liver
damage was observed in the mice treated with STZ. e changes in hepatocyte morphology were characterized
by perivascular inammatory cell inltration, fatty degeneration, hypochromatosis and the disappearance of cell
borders (Fig.6B). In the dosage groups, treatment with HIPS1 (600 and 400 mg/kg) and HIPS2 (600 mg/kg)
Figure 5. Eects of HIPS1 and HIPS2 on pancreatic histopathology in STZ-induced diabetic mice
(hematoxylin-eosin staining, 400×). (A) NC, (B) MC, (C) PC, (D) H-HIPS1, (E) M-HIPS1, (F) L-HIPS1,
(G) H-HIPS2, (H) M-HIPS2 and (I) L-HIPS2 & showed the inltration of lymphocytes, #showed the
hypochromatosis and the disappearance of cell borders.

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showed eects in ameliorating the liver damage (Fig.6D,E and G). Especially for mice in the H-HIPS1 groups,
the liver structures were similar to normal liver architectures (Fig.6D).
e changes in kidney cortex histology are shown in Fig. 67. Compared with the mice in the NC groups with
normal architecture (Fig.7A), glomerular augmentation, glomerular swelling, mesangial matrix proliferation and
loss of interstitial space were observed in mice in the MC group aer STZ injection (Fig.7B), indicating that kidney
damage occurred in the diabetic mice. Aer the treatments with HIPS1 at high and middle doses, these pathological
changes were attenuated, which were evidenced by the normal morphology of glomerulus in Fig.7D and C.
Interestingly, the histopathological analysis of the pancreas and liver was consistent with the results of enzyme
activities and MDA levels as biochemical indicators of their function (Table2 and Fig.4). Histological studies of
the kidney cortices of mice in the H-HIPS1 and PC groups demonstrated similarities to those in the NC group,
which echoed the results of the biochemical assays (Table2 and Fig.4).
Acute toxicity. In the acute toxicity assays, the mice treated with HIPS1 and HIPS2 did not exhibit any
gross behavioural changes (including irritation, restlessness, respiratory distress, abnormal locomotion and
catalepsy) or toxic symptoms (such as decreased food intake or shaggy hair) either immediately or during the
post-treatment period compared with the control group, indicating that these polysaccharides were essentially
non-toxic substances.
Discussion
Increasing data indicate enhanced oxidative stress and changes in antioxidant capacity in both clinical and exper-
imental forms of DM that are key mechanisms in the pathogenesis of diabetic complications30. STZ injection
has been used to establish an experimental hyperglycaemia model, owing to its high toxicity in stimulating the
Figure 6. Eects of HIPS1 and HIPS2 on hepatic histopathology in STZ-induced diabetic mice (hematoxylin-
eosin staining, 400×). (A) NC, (B) MC, (C) PC, (D) H-HIPS1, (E) M-HIPS1, (F) L-HIPS1, (G) H-HIPS2,
(H) M-HIPS2 and (I) L-HIPS2. Arrows showed necrotic zones, triangles indicated hypochromatosis, hollow
triangle showed fatty degeneration and *showed the perivascular inammatory cell inltration.

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generation of H2O2 in β-cells, which may induce lipid peroxidation and the depletion of antioxidant enzymes, and
even worse, organic damage31–33. Previous publications have reported decreases in antioxidant enzyme activities,
including GSH-Px, SOD and CAT, in the pancreas, liver and kidney of diabetic mice, in agreement with our pres-
ent conclusions34. Interestingly, the reductions in enzyme activities and the increase of MDA levels were markedly
suppressed aer treatment with HIPS1 or HIPS2 (Fig.4), demonstrating that the polysaccharides may inhibit oxi-
dative damage to pancreatic, hepatic and nephritic tissue. Pancreas, one of the most vital organs of polysaccharide
metabolism, maintains homeostasis of blood glucose through insulin synthesis. STZ exhibits toxicity on pancre-
atic β-cells, leading to pancreatic damage, and results in disordered insulin secretion32, 33. As sensitive organs, the
liver and kidneys have a great capacity to detoxify toxic substances, and they play pivotal roles in glucose metab-
olism in vivo31. erefore, the pathological damages that are inicted on the liver and kidneys by hepatotoxic and
nephrotoxic agents appear to be fatal in diabetes35, 36. Furthermore, in diabetic animals, the oxidative stress that is
induced by free radicals can aect the functions of the pancreas, liver and kidneys37. In the present work, as shown
in Figs5–7, the STZ-induced diabetic mice exhibited serious damage to the pancreas, liver and kidneys, including
cell necrosis, hypochromatosis, fatty degeneration and glomerular proliferation. Interestingly, this damage was
considerably recovered by HIPS1 and HIPS2 administration, indicating that the damage to the pancreas, liver
and kidneys in STZ-induced diabetic mice can be protected and repaired by intervention with HIPS1 and HIPS2.
High blood glucose levels are the main characteristic of DM1. In STZ-induced diabetic mice, high GLU levels
and increased uptake of food and water indicate the successful establishment of the model38. One therapeutic
approach for decreasing hyperglycaemia is to retard the absorption of glucose by inhibition of carbohydrate
hydrolysis enzymes, such as α-amylase and α-glucosidase. Hence, it is important to investigate the inhibition
of α-amylase and α-glucosidase to analyse the antihyperglycaemic eects19, 39. In the present study, HIPS1 and
Figure 7. Eects of HIPS1 and HIPS2 on kidney cortex histopathology in STZ-induced diabetic mice
(hematoxylin-eosin staining, 400×). (A) NC, (B) MC, (C) PC, (D) H-HIPS1, (E) M-HIPS1, (F) L-HIPS1, (G)
H-HIPS2, (H) M-HIPS2 and (I) L-HIPS2. Arrows showed glomerular augmentation and triangles showed
mesangial matrix proliferation, while *showed the loss of interstitial space.

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HIPS2 produced inhibitory eects against the two enzymes at dierent concentrations, and HIPS1 was more
eective than HIPS2, apparently due to the dierent structures of the polysaccharides. Nevertheless, Chiba noted
that the inhibitory eects may be associated with the individual structures of the enzymes40.
Blood has direct contact with most of the tissues in the animal body, and pathological changes are likely to be
reected in levels of some proteins and enzymes in blood samples21. Enhanced ALB levels and decreased activi-
ties of ALP, ALT and AST in serum have been used clinically as biochemical markers for liver damage41, 42. BUN
and CRE, endogenous by-products that are released into body uids and excreted by glomerular ltration, have
been widely used in the clinic to reect the physical status of the kidneys43. In the present study, changes in the
activities of ALB, ALP, ALT, and AST and in serum levels of BUN and CRE were suppressed by HIPS1 and HIPS2
administration, indicating the stabilization of plasma membranes and repair of STZ-induced pancreatic, hepatic
and nephritic damages. ese results are also consistent with our previous studies8.
e present ndings suggest that HIPS1 and HIPS2 from H. erinaceus SG-02 are novel bioactive compounds
with potential anti-diabetic and organic protection eects. Moreover, HIPS1 was much more ecient than HIPS2
in preventing the diabetes-induced organs damages, with the possible mechanism might be due to the mono-
saccharide composition and bond types. Figure2 shows that HIPS1 contains Xly and a high percentage of Glc
in the monosaccharide composition compared with HIPS2. Furthermore, HIPS1 contains more active radicals
and functional groups, such as -NH2, -COOH and S=O, than HIPS2. ese structural characteristics might
contribute to the scavenging capacities of ROS. Moreover, HIPS1 and HIPS2, especially HIPS1, could indirectly
relieve oxidative damage to the pancreas, liver and kidneys by improving the antioxidant enzyme activities and
reducing MDA levels (Fig.4), possibly resulting in the amelioration of tissue necrosis and inammatory damage
in these organs. Otherwise, the repair capacities of HIPS1 and HIPS2 on the organs may involve a reduction of
insulin secretion or stimulation of insulin release44 and may indirectly participate in inammatory pathways45,
possibilities that require further study.
Methods
Ethics statement. All experiments were performed in accordance with the guidelines and regulations of the
ethics committee of the Shandong Agricultural University and the Animals (Scientic Procedures) Act of 1986
(amended 2013).
All experimental protocols were submitted to and approved by the ethics committee of the Shandong
Agricultural University in accordance with the Animals (Scientic Procedures) Act of 1986 (amended 2013).
Materials. e H. erinaceus SG-02 strain used in this experiment was preserved in the Fungi and Application
Laboratory of Shandong Agriculture University (Taian, China) and was maintained on a potato dextrose agar
(PDA) slant. e diagnostic kits for analysing SOD, GSH-Px, and CAT activities and MDA levels were purchased
from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). e standard monosaccharide samples,
including rhamnose (Rha), ribose (Rib), arabinose (Ara), Xyl, Glc, Man and Gal, were provided by the Merck
Company (Darmstadt, Germany) and the Sigma Chemical Company (St. Louis, MO, USA). All other reagents and
chemicals used in the present work were analytical reagent grade and were supplied by local chemical suppliers.
Isolation and purication of polysaccharides. H. erinaceus SG-02 was initially maintained on PDA
slants and then inoculated into 500 mL of liquid medium containing glucose (10 g), peptone (1.5 g), yeast extract,
(2 g), KH2PO4 (1 g) and MgSO4 (0.5 g). Aer 25 days of fermentation, the mycelia of H. erinaceus SG-02 were
collected and cleaned three times with distilled water. e dry mycelia were shattered and extracted with distilled
water at 80 °C for 3 h and centrifuged at 3000 rpm for 10 min, aer which the precipitate was discarded. e
supernatant was mixed with anhydrous ethanol (4 °C overnight) to obtain the crude polysaccharide, which was
deproteinized using Sevag reagent (chloroform/n-butanol, 5:1, v/v) and labelled as HIPS.
HIPS (1 g) was dissolved in 15 mL of distilled water and puried on a DEAE-cellulose column (26 × 400
mm) at a ow rate of 2.0 mL/min and eluted with sodium chloride solutions at concentrations of 0, 0.2, 0.3
and 0.5 mol/L. e major eluate was then collected separately and puried by gel permeation chromatography
(Sephadex G-100 column AQ3, 1.3 × 50 cm). e above steps were repeated twenty times, and the main fractions
were lyophilized by vacuum freeze-drying (Labconco, USA) for further study.
UV spectroscopy, GC and FT-IR analysis of polysaccharides. e UV scanning spectrum was deter-
mined by microplate spectrophotometer (Dynex Spectra MR, Dynex Technologies, USA)
e monosaccharide composition was determined by GC (GC-2010, Shimadzu, Japan) according to the
method of Sheng, et al.46. e monosaccharide compositions were identied by comparing the retention times
with the standards, and the monosaccharide contents were calculated using area normalization methods.
Infrared spectra of the samples were recorded using an infrared spectrometer (Nicolet 6700, ermo Fisher
Scientic, USA) with a range of 4000–400 cm−1 47.
Inhibition assay on α-amylase and α-glucosidase activities. e HIPS fractions were stored at
−80 °C prior to the assay of α-amylase inhibition. e reaction mixture, including 0.2 mL of α-amylase (6 U/
mL) and 0.2 mL of polysaccharide solution (1.0 to 6.0 mg/mL, dissolved in 0.2 mol/L phosphate buer, pH 6.6),
was activated by adding the starch substrate solution (0.4 mL, 1%, w/w) and was processed at 37 °C for 10 min,
followed by termination with 2 mL of DNS reagent (1% 3,5-dinitrosalicylic acid and 12% sodium potassium tar-
trate in 0.4 mol/L sodium hydroxide)48. e sample was maintained in boiling water for 10 min and then diluted
with 15 mL of distilled water in an ice bath. e α-amylase activity was determined by measuring the absorbance
at 540 nm. e IC50 value was dened as the concentration of polysaccharide that produced an inhibition rate of
50% under the assay conditions19.

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AInhibition rate (%)(1(AA)/ )100 (1)
cij
=− −×
where Ac was the absorbance of the mixture that includes 0.2 mL of phosphate buer, 0.4 mL of starch solution
and 0.2 mL of α-amylase solution; Ai was the absorbance of the mixture that includes 0.2 mL of sample, 0.4 mL of
starch solution and 0.2 mL of α-amylase solution; and Aj was the absorbance of the mixture that includes 0.4 mL
of phosphate buer, 0.2 mL of α-amylase solution and 0.2 mL of sample.
Inhibition of α-glucosidase activity was measured according to reported methods with some modications19.
e α-glucosidase (0.2 mL, 3.75 U/mL) was premixed with the samples (dissolved in 0.2 mol/L phosphate buer,
pH 6.8) at various concentrations (0.5 to 6.0 mg/mL), and 6 mmol/L p-nitrophenyl-α-D-glucopyranoside (in
phosphate buer) as a substrate was added to the mixture to initiate the reaction. e reaction was incubated
at 37 °C for 30 min and stopped by adding 6 mL of 0.1 mol/L sodium carbonate. e α-glucosidase activity was
determined by measuring the absorbance at 400 nm.
=− −×Inhibition rate (%)(1(AA)/A) 100 (2)
ijc
where Ac was the absorbance of the mixture that includes 0.2 mL of phosphate buffer, 0.2 mL of
p-nitrophenyl-α-D-glucopyranoside solution and 0.2 mL of α-glucosidase solution; Ai was the absorbance of
the mixture that includes 0.2 mL of sample, 0.2 mL of p-nitrophenyl-α-D-glucopyranoside solution and 0.2 mL
of α-glucosidase solution; and Aj was the absorbance of the mixture that includes 0.2 mL of phosphate buer,
α-glucosidase solution and 0.2 mL of sample.
Animal experiments. Ninety Kunming strain mice (4 weeks old, weight: 20 ± 2 g, purchased from Taibang
Biological Products Ltd. Co. (Taian, China)) were acclimated for one week under the following conditions: tem-
perature (23 ± 1 °C), humidity (55 ± 5%) and a 12-h light-dark cycle, during which time they had free access to
food and water ad libitum. Aer the acclimation, the mice were induced to become diabetic by an intraperito-
neal injection with 120 mg/kg STZ (freshly dissolved in sodium citrate buer, pH 4.5), while the NC group was
injected intraperitoneally with citrate buer49. e GLU levels in the blood samples obtained from the tail of
each mouse were measured 48 h aer the last STZ injection using an ACCU-CHEK blood glucose metre (Roche,
Basel, Switzerland). Mice with GLU levels greater than 13.3 mmol/L were considered successful models of dia-
betes50 and were randomly allocated to the MC group, the PC group and six dosage groups (including L-HIPS1,
L-HIPS2, M-HIPS1, M-HIPS2, H-HIPS1 and H-HIPS2), which contained ten mice per group. Dierent groups
were administered dierent solutions intragastrically as follows:
NC group (n = 10): normal control. MC group (n = 10): diabetic control + distilled water. PC group (n = 10):
diabetic control + glibenclamide (10 mg/kg). H-HIPS1 group (n = 10): diabetic control + HIPS1 (600 mg/
kg). M-HIPS1 group (n = 10): diabetic control + HIPS1 (400 mg/kg). L-HIPS1 group (n = 10): diabetic con-
trol + HIPS1 (200 mg/kg). H-HIPS2 group (n = 10): diabetic control + HIPS2 (600 mg/kg). M-HIPS2 group
(n = 10): diabetic control + HIPS2 (400 mg/kg). L-HIPS2 group (n = 10): diabetic control + HIPS2 (200 mg/kg).
e treatments lasted for two weeks. At the end of the experiment, all mice were weighed and sacriced using
a diethyl ether anaesthetic treatment aer fasting overnight51.
e organic index for the pancreas, liver and kidneys was calculated as follows: (organ weight)/(body weight)
(g/100 g)29.
ALT, AST and ALP activities, along with the levels of ALB, BUN and CRE, were measured using an automatic
biochemical analyser (ACE, USA).
The pancreas, liver and kidneys were removed rapidly, weighed, and homogenized (1:9, w/v) in
phosphate-buered solution (PBS, 0.2 mol/L, pH 7.4). Aer centrifugation at 4 °C for 20 min (5000 rpm), the
supernatants were collected and assayed for the hepatic/nephritic activities of GSH-Px, SOD, and CAT and MDA
levels according to the commercial kit instructions7.
Fresh pancreas, liver and kidney tissue masses were xed in 4% formaldehyde solution (pH 7.4) for more
than 24 h, embedded in paran and then cut into slices using a microtome. e slices were stained with HE and
photographed using an optical microscope (400 × magnication)7.
Acute toxicity. Acute toxicity was determined by the method of Chao et al.52. Briey, een Kunming strain
mice (4 weeks old, weight: 20 ± 2 g) were randomly divided into one control group, which received the normal
saline solution (0.9%), and two dosage groups, which received isometric HIPS1 or HIPS2 solutions at the nal
concentration of 2000 mg/kg by gavage. e mice were observed continuously for gross behavioural changes,
toxic symptoms and mortality during the 14-day feeding period.
Statistical analysis. Data were statistically analysed using two-way ANOVA and the T-test (SAS, USA).
Data were expressed as the means ± SD (standard deviation). Dierences were considered statistically signicant
if P < 0.05.
Conclusions
e present study demonstrated that HIPS1 and HIPS2 from H. erinaceus SG-02 exerted protective eects on the
pancreas, liver and kidney in STZ-induced diabetic mice based on the results of biochemical and histopatholog-
ical analyses. Especially, the HIPS1 was much more ecient in preventing the diabetes-induced organ damages
owing to the functional groups (-NH2, -COOH and S=O). ese results may provide a mechanistic basis for the
use of H. erinaceus SG-02 as potentially natural and functional foods and drugs for the prevention and alleviation
of diabetes and its complications.

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Acknowledgements
is work was supported by grants from Mushroom Technology System of Shandong Province (SDAIT-07-05)
and Fundamental Research Funds for Central Non-prot Scientic Institution (1610132016041).
Author Contributions
Chen Zhang and Le Jia designed the research. Chen Zhang, Chunlong Hu and Jianjun Zhang analyzed data.
Chen Zhang and Jianjun Zhang performed research. Chen Zhang, Chunlong Hu, Zhenzhen Ren and Xinling
Song prepared gures and table. Chen Zhang wrote the manuscript. Juan Li and Jing Wang contributed to the
improvements of the English language. All authors were involved in checked the paper and contributed to the
preparation of the nal manuscript. All authors read and approved the nal manuscript.
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