A Water-Soluble Polysaccharide from the Fibrous Root of Anemarrhena asphodeloides Bge. and Its Immune Enhancement Effect in Vivo and in Vitro

Background The fibrous roots of Anemarrhena asphodeloides Bge. (FRAAB) are byproducts of the rhizome of Anemarrhena asphodeloides. Some studies have revealed secondary metabolic small molecules in FRAAB, but there are few reports on the polysaccharides of FRAAB (PFRAAB). Aim of the Study. The present study aimed to investigate the preliminary characterization and underlying mechanism of immune stimulation of PFRAAB. Materials and Methods The crude polysaccharide of FRAAB was obtained by hot water extraction and alcohol precipitation, and PFRAAB was purified by a diethylaminoethyl-52 (DEAE-52) cellulose chromatographic column and graphene dialysis membrane. The preliminary characterization of PFRAAB was studied by ultraviolet (UV) scanning and Fourier Transform Infrared Reflection (FTIR). The molecular weight and composition of PFRAAB were analysed by high-performance gel permeation chromatography (HPGPC) and high-performance liquid chromatography (HPLC), respectively. The immune stimulation of PFRAAB was investigated by using cyclophosphamide- (CCP-) treated mice and RAW264.7 cells. Results A water-soluble PFRAAB was obtained with a molecular weight of 115 kDa and was mainly composed of arabinose (ara), galactose (gal), glucose (glc), and mannose (man). Compared with CCP-induced mice, PFRAAB significantly (p < 0.05 or p < 0.01) increased the spleen and thymus index, ameliorated injury to the spleen and thymus, and evaluated immunoglobulin levels. In addition, PFRAAB also increased the secretion of nitric oxide (NO), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), and IL-6 in RAW264.7 cells and upregulated the expression of toll-like receptor 4 (TLR4), Myd88, nuclear factor kappa-B (NF-κB) P65, p–NF–κB P65, IKB-α, and p-IKB-α. Conclusion PFRAAB possesses immune stimulation activity and can be used as a potential resource for immune-enhancing drugs. Our present study provides a scientific basis for the comprehensive development of Anemarrhena asphodeloides medicinal plant resources.


Introduction
e immune system plays an important role in human health and is considered a protective barrier against ageing or apoptotic cells in the body, foreign pathogenic microorganisms, and other external factors [1]. Immunosuppression refers to immune dysfunction, which is commonly caused by bacterial or viral infection, chemical drugs, or negative emotions. Diseases such as upper respiratory tract infection, intestinal infection, or even tumour could be caused by immunosuppression [2,3]. In immunosuppressive organisms, the growth of immune organs, including the thymus and spleen, is inhibited, and the levels of natural killer (NK) cells and immunoglobulin in the serum decrease [4]. In addition, the level of CD3/CD4 lymphocytes also decreased due to immunosuppression [5]. RAW264.7 cells, one of the macrophages, play an important role in the innate immune system. RAW264.7 cells can directly engulf pathogens or indirectly secrete inflammatory factors such as TNF-α and interleukin-6 (IL-6) to improve the body's immunity [6]. RAW264.7 cells are the ideal in vitro model and can be used to investigate the mechanism of immune enhancement [7].
Currently, levamisole and glucans have been used as immunomodulators in treating immunosuppression in the clinic, but there are still some side effects, such as neutropenia and fever [8]. Fortunately, polysaccharides, one of the most important plant secondary metabolites, have attracted a great deal of scientific interest for their good merits of immune enhancement and few side effects [9]. e dried rhizome of Anemarrhena asphodeloides Bge. (AAB), which belongs to the Liliaceae family, is a traditional Chinese medicine (TCM) with antipyretic, hypoglycaemic, and immunoregulatory effects [10][11][12]. e dried rhizome of AAB was also approved as food by the National Health Commission of People's Republic of China in 2018. AAB is mainly distributed in Hebei, Anhui, and Shanxi, and the total output of the rhizome of AAB was approximately 50,000 tons per year. In the clinical practice of TCM, only the rhizome of AAB is used. e fibrous roots of dried rhizome of AAB (FRAAB) are the byproducts in the processing of dried rhizome of AAB and account for 1/3 of the total output of dried rhizome of AAB. However, the FRAAB were usually discarded during the primary processing. Studies have shown that there are still some effective active ingredients in FRAAB [13]. If the FRAAB could be used reasonably, it would provide a new drug source and reduce the waste of medicinal plant resources.
In the present study, a water-soluble polysaccharide from FRAAB (PFRAAB) was extracted and purified, and the preliminary characterization and monosaccharide composition of PFRAAB were studied. e immune enhancement effect of PFRAAB was studied on cyclophosphamide-(CCP-) induced immunosuppressive mice. e pathological changes in the spleen and thymus, the level of WBCs, NKcell activity, immunoglobulin, and CD3/CD4 T lymphocytes in different experimental groups were used to estimate the in vivo immune enhancement of PFRAAB. Furthermore, RAW264.7 cells were used to investigate the underlying mechanism of immune enhancement of PFRAAB related to TLR4-NF-κB.
To the best of our knowledge, this is the first study to reveal the properties and immune enhancement effect of PFRAAB.

Materials and Chemicals.
e FRAAB were collected from Bozhou City (Anhui Province, China) in October 2021 and were identified by Professor Chuanshan Jin. e voucher specimen is kept in the Laboratory of Natural Pharmaceutical Chemistry (Anhui University of Chinese Medicine, Hefei, Anhui, China). e FRAAB samples were dried at 50°C until the water content was less than 10%, and then the samples were smashed into powder (∼80 mesh). Antibodies against TLR4, Myd88, NF-κB p65, p-NF-κB p65, IKB-α, and p-IKB-α were purchased from Abcam (Beijing, China); TAK-242 (TLR4 inhibitor) was purchased from Apexbio (Houston, USA). e distilled water was filtered with a Milli-Q water purification system (Milford, MA, USA). e Sevage reagent was composed of chloroform and n-butanol with a volume ratio of 1 : 4, and chloroform and n-butanol were purchased from Tianjin Siyou Company (Tianjin, China).

Extraction and Purification of PFRAAB.
e powder of FRAAB was accurately weighed, and distilled water was added to the FRAAB powder at a ratio of 20 : 1 (liquid-solid). e mixture was refluxed in a heat water bath 3 times for 1 h each time. After refluxing, the extraction was collected and concentrated to 1/10 of the original volume, and the concentrated extraction was precipitated with ethanol at a final concentration of 80% (v/v) and then stored at 2°C for 12 h to obtain the biomacromolecules. Subsequently, the precipitates were treated with sevage reagent to remove the protein, and crude polysaccharides (CP) were obtained. e CP solution was dialyzed by a graphene dialysis membrane with a retained molecular weight of 3.5 kDa. Finally, the refined PFRAAB was prepared by freeze drying for further experiments. e ratio of the yield from the FRAAB powder to the refined PFRAAB was 2.31%.

UV Scanning of PFRAAB.
For UV spectrum analysis, a total of 2.12 mg PFRAAB was weighed precisely and dissolved in 25 mL distilled water, and the PFRAAB solution (0.085 mg mL −1 ) was scanned by a UV-8000 ultraviolet spectrophotometer (Yu'an Analysis Instrument Co., Ltd., Shanghai, China) in the wavelength range of 200∼400 nm.
e IR spectrogram of PFRAAB was obtained by the potassium bromide (KBr) method according to Alliouche's methods with modification [14]. Briefly, a total of 100 mg dry KBr was added to the PFRAAB powder (2.0 mg), and then the mixture was pressed and screened by A total of 5.05 mg PFRAAB was weighed precisely and dissolved in 1 mL distilled water to prepare the PFRAAB solution. e PFRAAB solution was filtered with a 0.45 µm membrane prior to HPGPC analysis, and the injection volume was 20 µL. e molecular weight of PFRAAB was calculated according to the standard curve.

Monosaccharide Composition Analysis.
A total of 2 M trifluoroacetic acid solution (TFA) was added to the PFRAAB sample (5 ± 0.05 mg) and hydrolysed at 120°C for 2 hours. en, methanol was added to the mixture, and the acid was removed by evaporation 3 times. e hydrolysed sample was dissolved in sterile water, and the solution was filtered with a 0.22 µm membrane for HPLC analysis. e monosaccharide composition analysis was conducted on high-performance liquid chromatography (HPLC) combined with refractive index (RI) detection and a DionexTM CarboPacTM PA20

Animals and Experimental Groups.
A total of 60 healthy Kunming mice (18 ± 2 g, Laboratory Animal Certificate no. SCXK2021-128) were purchased from the Experimental Animal Center of Anhui Medical University (Anhui, China). All of the mice were kept under a temperature of 23-25°C and humidity of 50-60%. e animal house was in a 12/12 h dark and light cycle (lights on 6:30 am). e mice had free access to standard laboratory food and water. All of the mice were reared adaptively for 4 days before the establishment of the immunosuppressive model. e immunosuppressive animal model was established by intraperitoneal injection with CCP (100 mg kg −1 ) once a day for 3 consecutive days. ymosin (TM) was selected as the positive control drug at a dose of 10 mg kg −1 . e mice injected with CCP were randomly divided into 5 groups: the model group (CCP group), positive drug (TM, 10 mg kg −1 ) group, PFRAAB low-dose (PFRBBA-L, 125 mg kg −1 ) group, PFRAAB medium-dose (PFRBBA-M, 250 mg kg −1 ) group, and PFRAAB high-dose (PFRBBA-H, 500 mg kg −1 ) group.
ere were 10 mice in each group. Another 10 healthy mice were selected as the normal control (NC) group. All mice in the PFRAAB groups and positive drug group were given corresponding doses of PFRAAB or TM at 9:00 am and 17:00, and each mouse in the normal control group was administered the same volume of physiological saline. e administration lasted for 14 days.
All animal experiments were approved by the animal ethics committee of Anhui University of Traditional Medicine (Animal Ethics no. AHTCM-2021125).

Determination of Peripheral White Blood Cell (WBC) Count, NK-Cell Activity, and Immunoglobulins in Serum.
On the 14th day of PFRAAB administration, peripheral blood was collected from the eyes of each mouse, and the white blood cells were counted by an automatic blood cell analysis instrument (Mindry, BC-5180 CRP, Wuhan, Hubei, China). e determination of natural killer cell activity was conducted by Han's method with some modification [15]. YAC-1 cells and splenic lymphocytes were selected as the target cells and effector cells, respectively. YAC-1 cells were seeded in a 96-well plate at a density of 2 × 10 5 cells per well, and then splenic lymphocyte cells were added to the corresponding well at a density of 1 × 10 7 cells per well. e 96well plates were incubated at 37°C with 5% CO 2 for 20 h. e orbital blood of each mouse was collected and kept at 25°C for 2 h. en, the blood was centrifuged at 3500 r•min −1 for 10 min, and the serum was obtained. e levels of IgA, IgM, and IgG were measured under the guidance of the manufacturer's instructions.

2.4.3.
e Spleen and ymus Index and HE Staining. e mice were sacrificed by cervical dislocation, and then the weights of the spleen and thymus of each mouse were measured. e spleen or thymus index was the ratio of spleen or thymus weight (mg) to body weight (g). Pathological research was carried out by haematoxylin and eosin (HE) staining. In short, the spleen and thymus tissues were fixed in 10% (v/v) neutral buffered formaldehyde for 1 week. en, the spleen and thymus samples were washed with PBS (1%) and cut into 5 μm slices with a freezing microtome. e slices were stained with HE and scanned by fluorescence microscopy (Nikon, Shanghai, China).

Measurement of T Lymphocyte Subsets.
e splenic lymphocytes of the mice were isolated from the spleen of each mouse. Briefly, the spleen was gently ground Evidence-Based Complementary and Alternative Medicine with lymphocyte separation medium, and then the mixture was filtered with a nylon net (∼200 mesh). e filtrate was transferred to a microcentrifuge tube with 500 μL RPMI-1640 medium and centrifuged at 4°C and 2000 r min −1 for 30 min. e upper layer of the suspension was washed with 10 mL RPMI-1640 medium and centrifuged at 4°C and 1000 r·min −1 for 10 min. e supernatant was discarded, and the sublayer was resuspended in RPMI-1640 medium. e concentration of splenic lymphocytes was adjusted to 1 × 10 5 per millilitre, a total of 1 mL of cell suspension was added to a microcentrifuge tube, and 5 μL of anti-CD3/CD4 was added to the tube. Each tube was shaken and incubated at 37°C with 95% O 2 and CO 2 for 10 min. Finally, the cells were washed twice with 500 μL PBS and analysed by flow cytometry (Navios 3 L 10C, Beckman, USA), and the data were analysed with FlowJo v. 09.

FRAAB Exerted Immune Enhancement Activity in Vitro
2.5.1. Cell Viability. RAW264.7 cells were cultured in DMEM containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin and incubated at 37°C with 5% CO 2 for 24 h. Cell viability was assayed by CCK-8 assay. Briefly, the cells were cultured in 96-well plates (5 × 10 5 cells per well) for 24 h and then treated with a series concentration of PFRAAB (12.5 to 400 μg•mL −1 ) and incubated for 24 h. Finally, CCK-8 solution (10 μL) was added to each well, and the optical density (OD) was measured at 540 nm. e cells without PFRAAB were selected as the blank control group. e ratio of the OD value of the sample group to the OD value of the blank group was calculated as the cell viability.

Phagocytosis Assay.
e phagocytosis assay was carried out according to Pan's methods with some modifications [16]. e cell density was adjusted to 5 × 10 5 cells per well and treated with 50, 100, and 200 μg•mL −1 PFRAAB or LPS (1 μg•mL −1 ) for 24 h. After 24 h, the supernatant was discarded, and neutral red stain (100 μL) was added to each well. After coincubation for 1 h, the cells were washed with PBS 3 times, and 100 μL of cell lysate was added. e absorbance was measured at 540 nm with a microplate reader. e phagocytosis rate was calculated according to the following formula.
Phagocytosis index (PI) � A1/A0 (A0: the absorbance of the blank control, A1: the absorbance of the sample). Polymyxin B (PMB), an LPS activity inhibitor, was used to determine whether there was LPS in PFRAAB [17]. RAW264.7 cells were pretreated with or without PMB (100 μg•mL −1 ) for 1 h. en, PFRAAB or LPS was used to stimulate RAW264.7 cells to secrete NO. e level of NO was determined by ELISA kits as above.

RNA Extraction and RT-PCR Analysis.
e mRNA expression of iNOS, TNF-α, IL-1β, and IL-6 was evaluated by real-time reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from RAW264.7 cells by TRIzol ® reagent (Sparkjade Biotechnology Co., Ltd., Shandong, China), and the RNA was reverse transcribed into cDNA by a SPARKscript II RT Plus kit (with cDNA Eraser). en, the levels of IL-1β, IL-6, iNOS, and TNF-α expression were amplified by 2 × SYBR Green qPCR Mix (with ROX) according to the manufacturer's instructions. GAPDH was used as the internal reference, and the relative expression was calculated by the 2 −△△Ct method. e primers and amplification sequences are listed in Table 1.

Western
Blotting. Protein expression was detected by western blotting. After being treated with different concentrations of PFRAAB (50, 100, and 200 μg•mL −1 ) and LPS (1.0 μg•mL −1 ) for 24 h, the RAW264.7 cells were lysed in RIPA to extract proteins. e same amount of protein was subjected to SDS-PAGE to transfer to a PVDF membrane. en, the membranes were incubated with specific antibodies against TLR4, Myo88, NF-κB P65, p-NF-κB P65, IKB-α, and p-IKB-α at 4°C overnight, and the secondary antibody was applied for 1 h at 37°C.

Statistics.
e data of the experiments are presented as the mean ± SD. One-way ANOVA was used to compare multiple groups, and statistical analysis was performed with SPSS 21.0 statistical software (IBM, New York, NY). Differences between the two groups were compared using Dunnett's tests and were considered significant when p < 0.05.

UV and FTIR Scanning Results.
e UV spectrum showed a smooth curve in the range of 200-400 nm except for a weak absorption at approximately 250 nm (Figure 1(a).
According to the FTIR results, the peaks at 3407.65 cm −1 and 2929.39 cm −1 were characteristic of the stretching vibrations of -OH and C-H, respectively. e wave number at 1735 cm −1 was the -C�O stretching vibration of -COOH.

e Molecular Weight of PFRAAB.
e linear equation was Y � −0.1917x + 12.108 (R 2 � 0.9934), which was generated according to dextran standards with series molecular weights, as mentioned in Section 2.3.4. e molecular weight of PFRAAB was 115 kDa.

PFRAAB Alleviated the Damage to the Spleen and
ymus in CCP-Induced Immunosuppressed Mice. As shown in Figures 2(a) and 2(b), compared with the normal control (NC) group, the spleen and thymus index decreased significantly in the CCP group (p < 0.05). After treatment with PRFAAB, the organ index increased significantly compared with that in the CCP group, especially in the PFRAAB-M and PFRAAB-H groups.
HE staining is shown in Figures 2(c) and 2(d). HE staining of the spleen showed that the boundary of the white and red pulp lymphoid was clear, and the spleen cells were arranged tightly and regularly with clear nuclei in the normal group. In the CCP group, the boundary of the white and red pulp lymphoid was destroyed, and part of the splenic corpuscle was sparse, while this phenomenon was reversed in the PFRAAB-M and PFRAAB-H groups (Figure 2(c). In the histopathological images of thymus HE staining (Figure 2(d), normal cortical morphology and flails can be seen in the normal control group. In the CCP group, the boundary between the medulla and cortex was not clear, and the number of medullae significantly decreased. PFRAAB remarkably alleviated thymus damage.

PFRAAB Increased the Number of Leukocytes in Pe-
ripheral Blood, NK-Cell Activity, and Immunoglobulin in CCP-Induced Immunosuppressed Mice. According to Figure 3(a), the number of WBCs in the peripheral blood of the CCP group mice was 2.37 ± 0.72 (×10 9 /L) and showed a significant decrease compared with the normal control (NC) group. After treatment with PFRAAB, the number of WBCs increased significantly in the PFRAAB-M (p < 0.01) and PFRAAB-H (p < 0.01) groups compared with the CCP group. In addition, NK-cell activity also increased  Evidence-Based Complementary and Alternative Medicine 5 significantly in the PFRAAB-M (p < 0.01) and PFRAAB-H (p < 0.01) groups compared with that in the CCP group (Figure 3(b)). Moreover, compared with the normal control (NC) group, the levels of IgA, IgM, and IgG in the serum of the CCP group significantly decreased (p < 0.01). After treatment with PFRAAB, the level of immunoglobulin showed a significant increase in the PFRAAB group, especially at doses of 250 and 500 μg•mL −1 (p < 0.01 or p < 0.01). Figure 4, compared with the CCP group, the levels of CD3 + and CD4 + significantly (p < 0.05) decreased. After intervention with the positive control drug and PFRAAB (especially in the medium-and high-dose groups), the levels of CD3 + and CD4 + cells increased significantly (p < 0.01).

Effects of PFRAAB on Cell Viability and Phagocytosis in RAW264.7 Cells.
e CCK-8 assay was used to estimate whether PFRAAB showed cytotoxic effects on RAW264.7 cells. According to Figure 5(a), all concentrations of PFRAAB in the experiment improved the cell viability when compared with the blank control (BC) group. In addition, the cell viability increased significantly (p < 0.05 or p < 0.01) when the concentration of PFRAAB was 12.5 to 100 μg•mL −1, and the cell viability was the highest when the concentration of PFRAAB was 100 μg•mL −1 . Unfortunately, the cell viability began to decrease when the concentration of PFRAAB reached 200 μg•mL −1 . erefore, three concentrations (50, 100, 200 μg•mL −1 ) of PFRAAB were chosen for further study.
As shown in Figure 5(b), compared with the blank control (BC) group, phagocytosis was significantly (p < 0.05 or p < 0.01) increased by the three concentrations (50, 100, 200 μg•mL −1 ) of PFRAAB, and the concentration of 100 μg•mL −1 showed the best effect. RAW264.7 Cells. According to Figures 6(a)-6(d), the levels of NO, TNF-α, IL-1β, and IL-6 increased significantly (p < 0.05 or p < 0.01) in the PFRAAB (50, 100, 200 μg•mL −1 ) and LPS groups compared with the blank control group and showed a dose-effect relationship within a certain range. As shown in Figure 6(e), the level of NO decreased significantly in the LPS + PMB group compared with the LPS group, and there was no significant difference in the level of NO whether PMB was added to PFRAAB.   Figure 8, the expression of TLR4, Myd88, p-IkB-α/IkB-α, and p-p65/p65 was significantly ( * p < 0.05 or * * p < 0.01) upregulated by three concentrations (50, 100, 200 μg•mL −1 ) of PFRAAB in a dose-dependent manner. e expression of proteins related to the TLR4 signalling pathway reached the highest when the concentration of PFRAAB was 200 μg•mL −1 . erefore, 200 μg•mL −1 PFRAAB was selected for further experiments.

PFRAAB Exerted Immune Enhancement Related to the TLR4-NF-Κb Signalling Pathway. As shown in
TAK-242 was used as an inhibitor of TLR4 to suppress the TLR4-NF-κB pathway. e results showed that the secretion of TNF-α, IL-6, and IL-1β in RAW264.7 cells was markedly decreased after treatment with TAK-242 ( Figure 9).

Discussion
e dried rhizome of Anemarrhena asphodeloides is a commonly used medicine in China with antipyretic, antitumour, and immune enhancement effects [18,19]. FRAAB are the byproducts of the dried rhizome of Anemarrhena asphodeloides when processed during primary processing. Currently, a large number of FRAAB have been abandoned with the development of the TCM industry and have not attracted enough attention, which would cause a certain degree of environmental pollution and waste of resources [20]. erefore, studying the chemical substances and bioactivities of FRAAB is of great significance to improve the utilization of medicinal resources of Anemarrhena asphodeloides.
Previous studies showed that the polysaccharides obtained from the dried rhizome of Anemarrhena asphodeloides possessed antioxidant activities, in vitro hypoglycaemic activity, and laxative effects [12,21,22]. In addition, the polysaccharides of AAB exert neuroprotective and immunoregulatory effects, but the molecular mechanism is unclear [10]. Moreover, literature studies have shown that some secondary metabolic small molecules, such as neomangiferin, mangiferin, isomangiferin, timosaponin BII, and timosaponin AIII, all exist in FRAAB and AAB [23], which indicates that macromolecular substances, such as polysaccharides, also exist in FRAAB. However, PFRAAB and their activity have not been reported. In this research, the preliminary chemical properties and immune enhancement of PFRAAB were investigated, which provided a scientific basis for further study of the fibrous roots of Anemarrhena asphodeloides. Our results suggest that PFRAAB can be used as a natural source of immune stimulation agents and provide a scientific basis for the comprehensive utilization of AAB resources.
Polysaccharides, mainly derived from natural products or TCMs, are polymers composed of several monosaccharides and linked by glycosidic bonds [24]. Polysaccharides   possess high polarity and can be extracted with hot water. Meanwhile, the proteins could also be extracted by hot water, which can be removed by alcohol precipitation and membrane separation techniques [25]. In this paper, PFRAAB was extracted and purified from FRAAB. e UV scanning results showed that there was no absorption in the range of 200-400 nm except for the weak absorption at approximately 250 nm, which suggested that protein or nucleic acids were reduced to the lowest and that there were only some uronic acids in PFRAAB. Moreover, HPGPC analysis showed that there was a single and symmetric peak, which indicated that PFRAAB was a homogeneous polysaccharide. e results of HPLC analysis showed that there were uronic acids in PFRAAB, which was in accordance with the results of UV scanning. Immune organs (spleen and thymus), immune cells, and immune proteins constitute the immune system, which is an important barrier to protect the body from the interference of external pathogenic organisms [26]. CCP, a cytotoxic immune suppressive agent, is commonly used as an antitumour agent in the clinic [27]. However, excessive CCP use could lead to the suppression or injury of immune organs, the inhibition of NK-cell activity, and a decrease in the levels of T lymphocytes and immunoglobulin [28]. In this study, the immune organ index and the levels of immune cells and immunoglobulins in CCP-induced immunosuppressed mice were all markedly decreased after treatment with CCP, but this phenomenon could be reversed by PFRAAB at doses of 250 or 500 μg•mL −1 . Our study successfully confirmed that PFRAAB exerted immune enhancement by elevating immune organs, immune cells, and immunoglobulins.
TM, a commonly used immune enhancer, was selected as the positive control. Our results revealed that there was no significant difference between the PFRAAB-H group and the TM group in elevating the number of peripheral blood WBCs, NK-cell activity, and immunoglobulin (IgA, IgG, IgM) levels induced by CCP, although the effect in the PFRAAB-H group was slightly lower than that in the TM group. In the clinic, most immune enhancers, such as TM,  Figure 9: Effects of PFRAAB on the secretion of TNF-α (a), IL-6 (b), and IL-1β (c) in RAW264.7 cells via TLR4. * p < 0.05 and * * p < 0.01 when comparing with the BC group. # p < 0.05, ## p < 0.01 when PFRAAB group compared with PFRAAB + TKA242 group. interferon, and nucleic acids, need to be administered by intramuscular injection to exert their efficacy [29]. e administration mode of PFRAAB was oral administration, and TM and PFRAAB were administered in different ways, which may have different requirements for the drug dose. Moreover, intramuscular injection may bring more pain to patients than oral administration. us, our study proved that PFRAAB was a potential oral immune enhancer. Furthermore, the literature reported that FRAAB contains small molecules with antitumour and neuroprotective effects [30] but the effects of macromolecular substances such as polysaccharides in PFRAAB have rarely been reported. Our research provides a further understanding of FRAAB.
T lymphocytes play an important role in immune response and immune recognition. CD3 + and CD4 + are helper T cells and the two main T lymphocyte subsets according to their surface markers [31]. Besides, flow cytometry is an established technology used for the determination of different types of cells [32]. e higher the level of CD3 + and CD4 + , the stronger the immune function. e present study showed PFRAAB-M and PFRAAB-H group significantly increased the level of CD3 + and CD4 + , which suggested that PFRAAB exerted good immune enhancement effective.
LPS, a glycolipid present on the surface of Gram-negative bacteria, is recognized as an inflammatory agent [33]. e proinflammatory factors secreted by RAW264.7 cells, such as IL-1β, IL-6, and TNF-α, increased significantly when stimulated by LPS [34]. However, cytokines, including IL-1β, IL-6, and TNF-α, play an important role in mediating and regulating the immune response, and host immunity requires these cytokines when encountering foreign harmful microorganism invasion [35]. e increasing level of IL-1β contributes to the promotion of the proliferation of T lymphocytes and further participates in cellular immunity [36]. IL-6 and TNF-α are mainly produced by macrophages and have immune activation effects to further remove infectious agents [37,38]. In addition, the secretion of cytokines increased when given a low concentration of NO [39]. erefore, moderately increasing the secretion of cytokines is helpful to improve immune function. In the present study, our results showed that the levels of IL-1β, IL-6, and TNF-α secreted by RAW264.7 cells increased significantly after stimulation with PFRAAB but were lower than those of the LPS group, which suggested that PFRAAB has the effect of immune stimulation and improves immune function to a certain extent. Furthermore, the mRNA expression levels of iNOS, IL-1β, TNF-α, and IL-6 in RAW264.7 cells were also elevated by PFRAAB. Moreover, PMB addition did not affect the secretion of NO by RAW264.7 cells, which indicated that there was no LPS in PFRAAB. Our results validated that PFRAAB could exert enhanced stimulation by increasing the levels of NO and cytokines. e toll-like receptor 4 (TLR4) is one of the most important proteins in the immune system and functions in identifying pathogens [40]. Once TLR4 is activated, its downstream proteins, such as myeloid differentiation factor 88 (Myd88), are recruited, and IκB-α is phosphorylated and hydrolysed. Finally, the NF-κB pathway is activated [41], and the production of cytokines such as TNF-α, IL-6, and IL-1β is promoted by NF-κB-p65 [42]. In this study, the expression of TLR4, Myd88, IkB-α, p-IkB-α, p65, and p-p65 was obviously upregulated by PFRAAB. Furthermore, TAK-242 was selected as the inhibitor of TLR4 to investigate the signalling pathway of PFRAAB. e results showed that the levels of TNF-α, IL-6, and IL-1β in RAW264.7 cells were reduced when they were treated with TAK-242, which suggested that the immune enhancement of PFRAAB was related to the TLR4-Myd88-NF-κB signalling pathway.

Conclusion
In summary, a water-soluble polysaccharide was obtained from FRAAB. e preliminary chemical properties and monosaccharide composition were analysed. Subsequently, the immune enhancement of PFRAAB in vivo and in vitro was investigated. e in vivo results showed that PFRAAB could ameliorate the injury to the spleen and thymus of immunosuppressive mice induced by CCP and improve the activity of NK cells and the level of immunoglobulin. Moreover, the in vitro results showed that PFRAAB could improve the phagocytosis of RAW264.7 cells, and with no cytotoxicity, PFRAAB could also stimulate RAW264.7 cells to secrete NO and cytokines. e underlying mechanism of PFRAAB immune-enhancing activity may be related to the TLR4-NF-κB signalling pathway. In summary, the results suggested that PFRAAB showed good immune enhancement and that FRAAB could be used as a source of immune enhancers. is study also supplies a scientific reference for the comprehensive utilization of Anemarrhena asphodeloides Bge.

Data Availability
Data are available upon request to the authors.