Professor Hu Qiuhui, Nanjing University of Finance and Economics: Effects of different extraction processes on the structural characteristics and immune activity of polysaccharides of Pleurotus eryngii

Analysis results of basic physical and chemical indexes of 3 kinds of Pleurotus eryngii polysaccharides

As shown in Table 1, the extraction rates of the three polysaccharides were 3.
13%, 3.
65% and 4.
57%, respectively
.
The results showed that there was no significant difference
in the extraction rate of the three extraction methods.
In the absence of significant difference in extraction rate, ultrasonic extraction time is shorter and the extraction temperature is low compared with hot water extraction method, because ultrasonic waves can destroy fungal cell walls and promote the dissolution
of polysaccharides.
The protein and total sugar mass fractions of U+H were 4.
44% and 46.
79%, respectively, which were higher than those of U and H
.
Ultrasound has a mechanical effect, thereby promoting the dissolution of protein, and then extracted with hot water after ultrasonic treatment, which further promotes the dissolution
of polysaccharides and proteins in Pleurotus eryngii.
The presence of uronic acid in all 3 polysaccharides indicates the presence of acidic polysaccharides in polysaccharides
.
In addition, there was no significant difference
in sulfate content between U, H and U+H.

2.
Ultraviolet spectroscopy results of 3 polysaccharides of Pleurotus eryngii

Nucleic acids and proteins have characteristic absorption peaks
at wavelengths of 260 nm and 280 nm, respectively.
The UV absorption spectra of U, H, and U+H are shown
in Figure 1.
The three polysaccharides had absorption peaks at 280 nm, indicating the presence of protein in the polysaccharides, which corroborated with the results of protein mass fraction analysis
.

Results of Fourier transform infrared spectroscopy of 3.
Polysaccharide Pleurotus eryngii

This is shown
in Figure 2.
The resulting peaks are typical absorption peaks
for polysaccharides.
The infrared spectrum of the polysaccharide has 1 wide and strong peak around 3 400 ^cm-1, indicating the presence of hydroxyl groups in the
polysaccharide.
The wider absorption peak around 3 500 cm-1 is caused by the ^O-H telescopic vibration of the hydroxyl group, so the presence of absorption in this region in the infrared spectrum indicates that U, H and U+H contain hydroxyl groups, indicating that these substances are polysaccharides
.
The characteristic peaks around 2 930 ^cm-1 and 2 360 cm-1 are caused
by ^C-H telescopic vibrations.
Strong absorption at 1 650 ^cm-1 corresponds to the telescopic vibration
of the carbonyl group.
However, the wide and strong absorption peak at 1 405 ^cm-1 can be attributed to symmetrical deformation
of proteins—CH3 and —CH2.
The absorption peak at 1 260 ^cm-1 may be caused
by C-O-C tensile vibration.
Furthermore, absorption peaks between 1 150 cm-1 and 950 ^cm-1 can be attributed to tensile vibrations
of phosphate.
The three polysaccharides had strong absorption peaks at 1 100~1 010 ^cm-1, indicating the presence of pyranosacchase ring structure
.

4.
Analysis results of monosaccharide composition of 3 kinds of oyster mushroom polysaccharides

The composition of monosaccharides from the three Pleurotus eryngii samples is shown
in Figure 3.
When the retention time was 29 min, all three polysaccharides had large absorption peaks, and compared with the standard, it was confirmed that the substance was glucose, indicating that U, H and U+H were mainly composed of
glucose.
The absorption peak was mannose at 13 min, galactose at 34.
18 min, xylose at 37.
80 min, and fucose
at 42.
16 min.
According to the analysis of monosaccharide composition, the three polysaccharides are all composed of mannose, glucose, galactose, xylose and fucose, but the amount ratio of substances is different
.
As shown in Figure 3, the ratio of mannose, glucose, galactose, xylose and fucose in U is 8.
60:80.
90:7.
41:2.
68:0.
57
.
The content ratios of mannose, glucose, galactose, xylose and fucose of the other two polysaccharides H and U+H were 9.
36:79.
72:8.
18:2.
60:0.
42 and 6.
75:82.
66:7.
14:2.
30:1.
12
, respectively.
The difference may be related to
the different extraction methods.

5.
Molecular weight analysis results of 3 kinds of polysaccharides of Pleurotus eryngii

The results are shown in
Figure 4.
The elution peaks of U, H and U+H are all multiple peaks, but none of them are single symmetry peaks, indicating that none of the three polysaccharides are uniform polysaccharides
.
According to the standard molecular mass, the linear regression equation for fitting dextran is lg mw=-3.
392 5 t+29.
413 (^R2=0.
993 3).

The chromatographic peaks of U appeared at 5.
26, 10.
90 min and 13.
21 min, with molecular weights of 13 152, 281 and 53 kDa, and molecular mass distribution ratios of 83.
61%, 3.
02% and 13.
37%,
respectively.
H peaks appeared at 5.
62, 10.
89 min and 13.
16 min, with molecular masses of 10 232, 281 and 61 kDa, and molecular mass distribution ratios of 93.
15%, 1.
94% and 4.
90%,
respectively.
U+H peaks appeared at 5.
59 min and 13.
17 min, with molecular weights of 10 471 kDa and 60 kDa, respectively, and molecular mass distribution ratios of 98.
59% and 1.
40%,
respectively.

6.
Proliferative effect of 3 kinds of polysaccharides on RAW264.
7 cells

It can be seen from Figure 5 that U has a toxic effect on macrophages at low mass concentrations (5, 10 μg/mL), possibly because under ultrasound, large molecular weight polysaccharides are continuously extracted, and with the extension of ultrasound time, some macromolecular weight polysaccharides will be degraded into small molecular weight polysaccharides
.
Different molecular weight polysaccharides exhibit different receptor affinities for macrophages, while macromolecular weight polysaccharides usually show strong stimulating activity
.
U has a promoting effect on macrophage proliferation in the range of 50~200 μg/mL
.
H had no toxic effect on macrophages in the experimental mass concentration range, and H had a significant promoting effect on macrophage proliferation in the mass concentration range (50~200 μg/mL) (P<0.
05).
<b13> U+H can significantly promote macrophage proliferation in the test mass concentration range, and has a dose-effect relationship in the range of 0~25 μg/mL, reaches a maximum at 25 μg/mL, and has a proliferation effect in the range of 50~200 μg/mL but is not concentration-dependent
.
In this study, the effects of three polysaccharides on RAW264.
7 cells showed that U, H and U+H can activate immune cells
.

7.
Effects of 3 polysaccharides on the phagocytic capacity of RAW264.
7 cells

It can be seen from Figure 6 that compared with macrophages without polysaccharides added to the medium, the three polysaccharides have a significant effect on the phagocytic ability of macrophages (P<0.
05).
<b10> The phagocytosis rates of U were 173.
87% and 193.
45% at 25 and 200 μg/mL, respectively, and the phagocytosis effect was the best
.
The phagocytosis rate of H was 127.
27% at low mass concentration (5~50 μg/mL), and 151.
34% and 163.
64%
at high mass concentration (100 and 200 μg/mL), respectively.
That is, the effect of H on phagocytosis at low concentrations was significantly lower than that at high concentrations (P<0.
05).
<b13> U+H has the best phagocytosis effect at low mass concentration (5~25 μg/mL), up to 187.
62%, and the phagocytosis effect decreases significantly (P<0.
05)<b14> under the conditions of 50, 100 and 200 μg/mL.
There were obvious differences in the effect of three polysaccharides of the same mass concentration on the phagocytosis rate of macrophages, and it was speculated that the difference in phagocytic rate was related
to the structural difference of polysaccharides.

Conclusion