Gas chromatography (GC) fingerprinting and immunomodulatory activity of polysaccharide from the rhizome of Menispermum dauricum DC

Preparation of polysaccharide extracts

Distilled water was used in the process of extracting crude polysaccharides from the rhizome of M. dauricum under the optimal extraction parameters, which were determined through response surface optimization. Following the completion of three rounds of extraction, the aqueous extract was subjected to a vacuum and concentrated. After that, four times the volume of ethanol was introduced into the solution to precipitate the polysaccharide, and afterward, the mixture was left to stand at 4 °C for an entire night. The precipitate was collected and deproteinized by means of the Trichloroacetic acid (TCA)-n-butanol method and then subjected to freeze-drying to produce a total polysaccharide fraction (tMDP) (Yang et al., 2022).

Complete acid hydrolysis

Hydrolysis of 10 mg of tMDP was performed using 3 mL of 2 mol/L trifluoroacetic acid (TFA) in a vial that was sealed at 110 °C for 6 h. To get rid of the TFA residue, the mixture was combined with 1 mL of methanol, and then it was rotary-evaporated thrice under a vacuum until it was completely dry. The hydrolyzed polysaccharide samples were prepared for derivatization (Liu et al., 2015b).

Preparation of derivatization and GC analysis

The GC method was employed to measure the composition of the monosaccharides. The hydrolyzed tMDP and monosaccharide standards were added with hydroxylamine hydrochloride, pyridine, and inositol hexaacetate, and stirred at 90 °C for 30 min. Then, acetic anhydride was introduced and the reaction continued for 30 min at 90 °C for acetylation. A GC examination was performed on the products of the reaction. The temperature program was set to 170 °C for 3 min, 170 °C to 178 °C at a rate of 0.5 °C/mins for 3 min, and elevated to 210 °C for 5 min at a rate of 2 °C/mins (Yang et al., 2022).

Extraction and purification of polysaccharides

tMDP was extracted from the S10 batch (highest PCA score) of the rhizome of M. dauricum by the method above, and the TCA-n-butanol method was used to remove protein. Subsequently, the purification of tMDP was done utilizing the Sephadex G-50 column (1.75 cm × 66 cm), Sephacryl S-100 column (1.75 cm × 66 cm), and DEAE-52 cellulose column (5.5 cm × 30 cm), and A white purified polysaccharide (MDP) was obtained by collecting the major fraction and lyophilizing it (Yang et al., 2022).

Cell culture

Beina Chuanglian Biotechnology Co., Ltd. (Beijing, China) provided the RAW264.7 cells utilized in this study. At a temperature of 37 °C, the cells were incubated in DMEM that had been supplemented with streptomycin sulfate (100 g/ml), penicillin (100 units/mL), and 10% fetal bovine serum. The environment of the incubator was humidified and contained 5% carbon dioxide.

Cell viability test

By conducting the Methyl thiazolyl tetrazolium (MTT) test in vitro, we examined the effect that varying concentrations of MDP had on the RAW264.7 cells’ viability. In general, 96-well microplates were seeded with RAW264.7 cells at a density of 1 × 10⁴ cells per well. After the cells had been cultured at 37 °C for 24 h with 5% carbon dioxide, they underwent a second round of incubation at 37 °C for another 24 h with MDP samples at increasing dosages (0, 10, 50, 100, 200, and 400 µg/mL) or LPS (1 µg/mL, positive control). Following the initial incubation, 20 μL of the MTT solution with a concentration of 5 mg/mL was introduced into each well, and the plates were subsequently re-incubated at 37 °C for a further 4 h in the medium. After that, the medium was carefully aspirated, and each well was then treated using 150 μL of DMSO so that the dissolving crystals could be dissolved. At last, to ascertain the absorbance at 570 nm, we made use of a microplate reader (BioTek Instruments Inc., Winooski, VT, USA) (Wang et al., 2017a).

Cell viability % = (OD treatment group)/(OD control group) × 100%.

TLR4 pathway blocking experiment

RAW264.7 cells were seeded at a density of 1 × 10⁴ cells/well into 96-well microplates for 24 h. Following the adhesion of the cells, various doses of MDP (50, 100, and 200 μg/mL), as well as LPS, were introduced, and TAK-242 was added at the same time to make the final concentration of 20 μg/mL. Next, the concentrations of IL-6, TNF-α, and NO in the supernatant were detected with the aid of ELISA kits (ML BIO Biotechnology, Shanghai, China) that are commercially available. The assays were carried out in compliance with the guidelines that were supplied by the manufacturer, after which the cytokine levels were calculated using the standard curves.

Western blotting analysis

After cultivating the RAW 264.7 cells for 24 h on a tissue culture plate with six wells having a flat bottom at 5 × 10⁶ cells per well, the MDP was added at a final concentration of 50 μg/mL in order to activate these cells for 24 h. The positive control consisted of the cells that had been treated using LPS at a concentration of 1 μg/mL. At the end of the incubation period, all of the conditioned cells were harvested and processed to produce the total proteins. Briefly, samples were prepared by RIPA (radioimmune precipitation assay) with protease and phosphatase inhibitors. The BCA technique was used to determine the protein. After denaturation, 30 μg of protein per well was deposited via electrophoresis onto SDS-PAGE and then transferred onto PVDF membranes. 10% nonfat milk (prepared in TBS supplemented with 0.1% Tween 20) was employed in blocking the membranes at ambient temperature for 4 h. Afterward, the membranes were probed with primary antibodies against P38 (1:750), p-P38 (1:1,000), ERK (1:1,500), p-ERK (1:1,500), JNK (1:1,500), p-JNK (1:2,000), NFκB (1:1,500), p-NFκB (1:1,000), MyD88 (1:1,500), TLR4 (1:1,000), as per the guidelines stipulated by the manufacturer. After being rinsed in TBST, the membranes were subjected to incubation with the secondary antibody (1:5,000) for 1 h at ambient temperature. ECL chemiluminescence detection kit (Millipore, MA, USA) was utilized to analyze the signals, which were subsequently quantified with an Amersham Imager 600 Chemiluminescence imaging system (GE, Boston, MA, USA).

GC fingerprints of tMDP

tMDP was prepared by the optimized extraction conditions of RSM and deproteinization using the TCA-n-butanol method, which was used to establish GC fingerprinting, thus laying the foundation for controlling the quality of the rhizome of M. dauricum. To ascertain the structural characteristics of polysaccharides, it is required and essential to take into account the monosaccharide composition. Compositions of monosaccharides and polysaccharide ratios varied remarkably by the source, which was an essential aspect of the polysaccharide’s biological activity. In the quality control of active polysaccharides throughout the last several years, the monosaccharide composition-associated fingerprints have seen a widespread application. To determine the standard GC fingerprint, GC was utilized to conduct monosaccharide composition analysis on 10 different batches of tMDP. As shown in Fig. 2A, GC fingerprinting of full acid hydrolysates of 10 different batches of tMDP exhibited a high degree of similarity having 11 common peaks. Figure. 2B shows the fingerprint that was used as a reference. The chromatogram of mixed standard monosaccharides depicted in Fig. 2C revealed the presence of seven different components, with peaks 1, 2, 3, 8, 9, 10, and 11 representing rhamnose, arabinose, fucose, mannose, glucose, galactose, and inositol (internal standard), respectively. To sum up the above, tMDP contained six monosaccharides, among which rhamnose, glucose, and galactose were the majority, whereas arabinose, fucose and mannose, were of low content.

The similarity analysis of the GC fingerprints

Based on the professional program “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (TCM)” (Version 2012A), similarity values were computed premised on the GC chromatograms of polysaccharide samples as well as the reference fingerprint. Relative to the reference fingerprint, the similarity across 10 batches of tMDP ranged from 0.943% to 0.998 (Table 3). The great similarity revealed that the previously developed GC fingerprint was particularly suited as one of the multi-dimensional fingerprints for quality control of the rhizomes of M. dauricum.

PCA of the GC fingerprints

The GC fingerprints of the rhizome of M. dauricum samples were then presented quantitatively by utilizing PCA. The relative peak area of seven distinctive chromatographic peaks (peaks 1, 2, 3, 4, 8, 9, and 10) in Fig. 2 was chosen in PCA. As depicted by the scree plot (Fig. 3A), the first three PCs had eigenvalues of 4.302, 1.088, and 1.015, explaining 61.45%, 15.55%, and 14.50% of variance correspondingly, representing 91.50% of the total variance. The following equations can be used to explain the principal components:

PC1 = 0.436 * X1 + 0.437 * X2 + 0.451 * X3 + 0.255 * X4 + 0.325 * X8 + 0.387 * X9 + 0.308 * X10

PC2 = −0.318 * X1 − 0.156 * X2 − 0.181 * X3 − 0.735 * X4 + 0.481 * X8 + 0.061 * X9 − 0.256 * X10

PC3 = 0.114 * X1 − 0.237 * X2 − 0.180 * X3 − 0.006 * X4 + 0.362 * X8 − 0.520 * X9 + 0.704 * X10

The PCA results were further analyzed by OriginPro 2021. As per the 3D scores plot of PCA depicted in Fig. 3B, all of the rhizomes of M. dauricum samples were integrated into a group within the 95% confidence ellipse, which indicated that the quality of the rhizome of M. dauricum from Northeast and Shandong of China was similar, which was consistent with the determination of polysaccharide content (Table 1). The high similarity suggested that the GC fingerprint was very suitable as a quality control indicator for the rhizome of M. dauricum. However, the rhizome of M. dauricum polysaccharides from different origins also differed slightly and could be clustered into three categories broadly, among which S1, S2, S4, and S8 were all produced in Shandong, S3, S5, S7, and S10 were from Heilongjiang and Jilin, while S6 and S9 were from Liaoning.

Effect of MDP on the morphology of RAW264.7 cells

tMDP was further purified by cellulose and gel columns to obtain a homogeneous polysaccharide fraction MDP. To examine the impact of MDP on RAW264.7 cells’ morphology, the morphologic changes of RAW264.7 cells were observed under a microscope. As could be observed in Fig. 4A, the cells that belonged to the control group had a round shape and normal structure. Nonetheless, following MDP treatment, there was a remarkable alteration in the structure of the cells. It is quite clear to observe that many of the antennas generated surrounding the cells may enhance the area of contact with exterior substances and were beneficial to the phagocytic uptake. This suggests that concentrations of MDP ranging from 10 to 400 μg/mL are capable of activating macrophages without causing cytotoxic effects.

Effect of MDP on RAW264.7 cell viability

Macrophages are the primary cells implicated in the innate defense mechanism of the immune system. In addition, they are the major cells responsible for inflammation and other immunological processes that occur inside the host. It has been shown that many different polysaccharides stimulate both the activity of cells as well as their proliferation (Wang et al., 2018). As a result, we studied how MDP affected the viability of RAW264.7 cells, and the findings are depicted in Fig. 4B. At concentrations of 10, 50, 100, 200, and 400 µg/mL, the levels of cell viability for the various groups were 120.66%, 123.64%, 118.76%, 130.02%, and 114.73%, correspondingly, which illustrate that MDP has the potential to significantly improve RAW264.7 proliferation.

MDP enhanced immunomodulatory effects via TLR4 in vitro

Macrophages are by far the most important body cells that are associated with the immunological defense system (Cheng et al., 2019). After being activated, they are capable of producing a wide variety of chemokines and cytokines. One of the most important mechanisms that immunoregulators use is to cause the release of several biological components (IL-6, TNF-α, NO, etc.) (Tian et al., 2019). NO is a type of vital molecule that is produced by macrophages, and it played an important function in the modulation of apoptosis as well as the host’s defense against cancer cells and other pathogens (Ren et al., 2019). In addition, NO may also stimulate phagocytosis and lysis in macrophages, two processes that are essential to the immune system. Because of this, the ability of macrophages to release NO is an indication of the effects that polysaccharides have on the functioning of the immune system (Wang et al., 2017a). Inflammation, cancer, and immunological illnesses may all be traced back to the main active molecules inside organisms, such as TNF-α and IL-6, which perform integral roles in the pathogenesis of these conditions (Wang et al., 2017a). If the host is attacked by external pathogens, activated macrophages produce IL-6 and TNF-α for the purpose of mediating the immune system’s response (Sorimachi et al., 1999). Our previous study showed that MDP could promote the production of NO, IL-6 and TNF-α in RAW264.7 cells (Yang et al., 2022). These results suggested that MDP has significant immunomodulatory activities via the mechanism of increasing the release of NO, IL-6, and TNF-α in RAW264.7 cells. However, the mechanism of the in vitro immunomodulatory activity of MDP is unclear.

TLRs have been discovered as key membrane receptors that perform an instrumental function in activating macrophages (Fan et al., 2021). Among TLRs, several investigations have demonstrated that TLR4 is implicated in the generation of cytokines in response to polysaccharide stimulation (Liu et al., 2022b). To further investigate whether MDP exerts immune activity through activation of the TLR4 pathway, TAK-242, an inhibitor of TLR4, was utilized so that additional confirmation of the processes that result in MDP-mediated macrophage activation could be achieved. As depicted in Fig. 5, TAK-242 considerably decreased the levels of TNF-α, NO, and IL-6 (P < 0.05) as opposed to the MDP treatment group, illustrating that the TLR4 pathway was implicated in the MDP-mediated activation of macrophages.

MDP increased the protein expression of the TLR4-MAPK/NFκB signaling pathway

By performing a Western blot analysis, we evaluated how MDP affected the protein expression levels of TLR4 to get a deeper insight into its action mechanism. According to the results shown in Fig. 6A, MDP significantly promoted the expression of TLR4 protein, which is consistent with the above experimental results. Once TLR4 is activated, TLR4 is capable of binding with the MyD88 protein to create the TLR4-MyD88 complex, which then stimulates the phosphorylation of the downstream proteins, such as MAPKs (ERK, JNK, and p38) and NF-κB (Xin et al., 2019). The findings of Western blotting (Fig. 6B) illustrated that the protein expression of MyD88 was remarkably enhanced in RAW264.7 cells following MDP treatment, which indicated that TLR4 protein, after being activated by MDP, combined with a large amount of MyD88 and then activated the downstream pathway.

MAPKs perform crucial functions in immunological and inflammatory responses, including the stimulation of cell proliferation and the regulation of cytokine production via the modulation of transcriptional factors (Yang et al., 2019). Phosphorylation is a common mechanism that underlies the functions that are controlled by MAPKs (Yang et al., 2019). To determine if the MAPK signaling pathway was implicated in MDP-related macrophage activation, the phosphorylation levels of the p38, ERK, and JNK MAPK pathways were examined through western blot analysis. As depicted in Figs. 6D–6F, the phosphorylation levels of ERK, JNK, and p38 were substantially elevated in RAW264.7 cells following stimulation with MDP in contrast with the normal control cells. It has been shown that NFκB is a ubiquitous transcriptional factor that is essential for the activation of macrophages via the stimulation of diverse genes implicated in the modulation of immunological as well as inflammatory responses (Yang et al., 2019). As per the findings of Western blot (Fig. 6C), MDP caused an enhancement in the protein expression of p-NFκB in RAW264.7 cells. In conclusion, following MDP treatment, the key protein of TLR4-MAPK/NFκB signaling pathways including TLR4, MyD88, p- NFκB, p-ERK, p-P38, and p-JNK were up-modulated substantially in RAW264.7 cells. As per these findings, it is evident that MDP may stimulate the TLR4-MAPK/NFκB signaling pathways, which in turn exerts immunomodulatory impacts.