Characterization and Evaluation of the Pro-Coagulant and Immunomodulatory Activities of Polysaccharides from Bletilla striata.

Bletilla striata is widely used for stanching bleeding. In this study, polysaccharides from B. striata (BSP) were extracted by hot water. Four polysaccharides named BSP-1-BSP-4 were fractionated using DEAE-52 cellulose. BSP fractions contained sulfate, and the degrees of substitution of BSP-3 and BSP-4 were 1.59 and 1.70, respectively. Analysis of monosaccharide composition showed that four polysaccharides were mainly composed of mannan and glucose. The in vitro results showed that BSP-1-BSP-4 elicited pro-coagulant capacities by shortening the activating partial thromboplastin time, prothrombin time, and thrombin time and elevating the fibrinogen content. Immunomodulatory activity was evaluated by MTT assay, the pinocytic capacity and NO production. Although BSP fractions did not affect RAW 264.7 cell viability, they, especially BSP-2, enhanced the immunomodulatory activity by increasing the pinocytic capacity and NO production. Overall, BSP may be developed as a potential coagulant with immunomodulatory effects.

Introduction

Bletilla striata (Family Orchidaceae) is a perennial herb that is mainly distributed in China, including Guangxi, Guizhou, Yunnan, and Sichuan provinces. The Chinese Pharmacopoeia (2020 edition) states that B. striata stanches bleeding, reduces inflammation, promotes tissue regeneration, and relieves hematemesis, hemoptysis, and detumescence.[1] Several components, such as triterpenoids, saponins, steroidals, flavonoids, polysaccharides, and polyphenols, have been isolated from B. striata. Polysaccharides from B. striata (BSPs) are one of the major bioactivity-contributing components due to their various beneficial activities, including wound healing,[2] hemostasis,[3,4] anti-angiogenesis,[5] anti-oxidation,[6] anti-inflammation,[7] anti-hepatic,[8] anti-fibrotic,[8] and immunomodulatory properties.[9] Inspired by a research indicating that purified BSP possesses hemostatic effects,[10] we aimed to fractionate promising polysaccharides with the highest pro-coagulant activity.

The BSPs extracted by hot water are composed of mannose and glucose with a molar ratio of 3:1 and can be regarded as glucomannan polymers with a molecular size of 135 kDa.[11,12] Although these polysaccharides have been preliminarily identified by infrared (IR) and nuclear magnetic resonance spectroscopies, with regard to detailed structural information, further characterization is still pending. The X-ray photoelectron spectroscopy (XPS) analysis has received increasing attention for chemical composition evaluation and quantitative analysis of polysaccharides.[13] By using XPS analysis, Wang et al.[14] confirmed that −SO3 groups (S6+, high binding energy of 168.7 eV) are abundant in Artemisia sphaerocephala polysaccharides; a similar observation was reported in another work.[15] Thus, in the present work, XPS analysis was used to obtain the fine structural information of BSP for the first time.

The concept of “immunothrombosis” has been proposed, wherein the immune system uses clotting factors to cause the coagulation of hemolymph and protect them against invading microorganisms.[16,17] Peng et al. found that BSPF2 significantly induced the proliferation of spleen cells from Balb/C mice and exhibited immunological activity.[9] In this regard, the present study was designed to explore the immunomodulatory activity of BSP in vitro by using RAW 264.7 cells.

Herein, BSP was extracted by hot water and isolated using DEAE-52 cellulose chromatography. Physiochemical characteristics were determined through ultraviolet–visible (UV–vis) spectroscopy, IR spectroscopy, XPS analysis, and high-performance liquid chromatography (HPLC). In addition, assays of biological activities including in vitro pro-coagulant and immunomodulatory function were conducted. The obtained results would provide scientific basis for comprehensive utilization of BSP.

Materials and Methods

Plants and Chemicals

B. striata was obtained from Yunnan Province (east longitude: 97°31′–106°11′; north latitude: 21°8′–29°15′, China) in January 2019 and authenticated by Prof. Guangshu Wang, School of Pharmaceutical Sciences, Jilin University, Changchun, China.

Standard sugars including mannose (Man), ribose (Rib), rhamnose (Rha), glucuronic acid (Glu A), galacturonic acid (Gal A), glucose (Glu), galactose (Gal), and arabinose (Ara) were purchased from Sino-pharm Chemical Reagent Co., Ltd. (Shanghai, China). T-series dextran standards including T-10, T-40, T-70, T-100, and T-500 kDa were obtained from the National Institutes of Food and Drug Control (Beijing, China). 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and lipopolysaccharide (LPS) were acquired from Sigma-Aldrich (St Louis, USA). The nitric oxide (NO) assay kit was supplied by Beyotime Biotechnology (Jiangsu, China). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were provided by Sciencell (Shanghai, China). Trypsin–EDTA was purchased from Gibco, Life Technologies (Grand Island, USA). Reagents and instruments for assay of activating partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), and fibrinogen (FIB) were obtained from Innova Medical Technology Co. (Jiangsu, China). Yunnan Baiyao and normal saline were acquired from a local hospital (Jilin, China). Standard human plasma [Fresh armed State Drug Administration (prospective) 2006, 3401635] was provided by Dade Behring Marburg Gmbh (Marburg, Hesse, Germany). All other chemicals and reagents used were of analytical grade.

Extraction of Polysaccharides

Fresh tubers of B. striata were washed and cut into slices, which were extracted based on a previous study[12] with the following conditions: an extraction temperature of 70 °C, extraction time of 1 h, and liquid-to-solid ratio of 30: 1 (mL/g). Two extraction solutions were merged, filtered with gauze, and concentrated by rotary evaporation under reduced pressure. The solutions were precipitated twice with four volumes of 95% ethanol at 4 °C overnight and lyophilized to obtain crude BSP.

Purification and Isolation of Polysaccharides

Crude BSP powder was dissolved in deionized water and deproteinized with Sevag solution (chloroform and n-butanol at a ratio of 4:1).[18] Protein content was calculated using Bradford assay.[19] After removal of the Sevag reagent, the purified BSP solution was dialyzed (MD10, Viskase, Darien, IL, USA) in distilled water for 72 h to remove small molecular impurities. The solution in the dialysis bag was concentrated, dried, and subjected to a DEAE-52 cellulose column (2.6 cm × 30 cm). The polysaccharides were eluted stepwise with a gradient concentration of NaCl solution (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, and 0.7 M) at a flow rate of 1.0 mL/min (10 mL/tube) and sequentially named as BSP-1, BSP-2, BSP-3, BSP-4, and BSP-5 (0.4–0.7 M NaCl). BSP-5 was shelved owing to its low content. The eluting fractions were dialyzed, concentrated, and lyophilized. The separated BSPs were stored. Total polysaccharide content was assayed by the phenol sulfuric acid method at 490 nm.[20] Uronic acid content was determined by meta-hydroxydiphenyl assay at 520 nm.[21]

Physicochemical Properties of Polysaccharides

Sulfate Group Identification

The sulfate group (−SO3H) content was detected by the BaCl2–gelatin turbidity method[22] with slight modifications. In brief, 2 g of BaCl2 was dissolved in a 0.3% gelatin solution prepared in hot water (60–70 °C). About 5 mg of each BSP-1–BSP-4 was sealed with 4 mL of HCl (1 M) and hydrolyzed at 105 °C for 12 h. The hydrolysate was dried under a nitrogen atmosphere and dissolved in 1 mL of water, then 1 mL of HCl (1 M) and 0.5 mL of barium chloride–gelatin (5 mg/mL) were added. The mixture was fully shocked and then incubated for 20 min at 25 °C. In standard curve determination, 0.2 mL of the polysaccharide solution was added to measure sulfur content (S%). Degree of substitution (DS) was calculated using the following equation:

UV–Vis and IR Spectrum Analysis

The UV–vis spectra of BSP-1–BSP-4 solutions (1 mg/mL) were recorded by a UV–vis spectrophotometer (L5S, INESA Analytical Instrument Co., Ltd., Shanghai, China) from 200 to 800 nm.[23]

The IR spectra of BSP-1–BSP-4 fractions were recorded using KBr compressed into tablets (1:100) through an FTIR-650 Fourier transform infrared spectrophotometer (Gangdong Sci. & Tech. Development Co., Ltd., Tianjin, China) within the range of 4000–400 cm–1.[24] Second-derivative IR spectra obtained by the OMNIC 8.0 Savitzky–Golay derivative were used to distinguish overlapping peaks and obtain high-resolution peaks.[25]

X-ray Photoelectron Spectroscopy Analysis

XPS analysis was carried out on a bioemulsifier film deposited on a glass slide by using a PHI5600 photoelectron spectrometer (Physical Electronics, Eden Prairie, MN, USA) to reveal the chemical states of the elements in BSP-1–BSP-4 fractions. The emitted photoelectrons were detected by a hemispherical analyzer set at an angle of 45°. The total acquisition time was 2 min and 0.3 s. Core-level spectroscopy with a constant pass energy mode of 20 eV was equipped with an energy step size of 0.05 eV.[26] Data analysis was performed by XPS PEAK 4.1 software.

Molecular Weight Analysis

HPLC (Elite P230IIHPLC, Elite Analytical Instruments Co., Ltd., Dalian, China) with a gel chromatographic column was used to determine the molecular weights (Mw) of BSP-1–BSP-4 fractions as previously reported.[27] The chromatographic conditions were as follows: a column temperature of 50 °C, flow rate of 1.0 mL/min, RID temperature of 35 °C, and total run time of 30 min. The dextran standards were applied to prepare a calibration curve for determining the molecular weight of the samples.

Monosaccharide Composition Analysis

Based on a previous report,[28] 20 mg of the samples were hydrolyzed to monosaccharides by adding 2 M TFA (2 mL) into a reactor and kept at 110 °C for 5 h. The hydrolytic liquid was evaporated thoroughly, and the obtained powder sample was re-dissolved in methanol. This process was repeated three times to remove any residual TFA. The hydrolyzed samples (0.2 mL) and monosaccharide standards dissolved in water were mixed with 0.3 mol/L NaOH solution (0.2 mL). The mixture was added with 0.5 M PMP (0.2 mL) dissolved in methanol solution and heated in a water bath for 1 h at 70 °C. The solution was cooled and added with 0.3 M hydrochloric acid solution (0.2 mL) to neutralize. The solution was extracted using 1 mL of chloroform and swirled for 30 s. The solution was centrifuged, and the chloroform layer was discarded. The process was repeated three times. The final supernatant was filtered using a 0.22 μm membrane. In brief, 10 μL of the samples after derivatization were injected into Ultimate 3000 HPLC (Thermo, USA) coupled with a Supersil ODS2 column (5 m, 4.6 mm × 250 mm) and an Ultimate 3000 diode array detector (DAD, Thermo) to determine the monosaccharide composition of BSP-1–BSP-4 fractions. The chromatographic conditions were as follows: mobile phase consisting of PBS (pH = 6.8) and acetonitrile (82: 18, v/v), a flow rate of 0.8 mL/min, column temperature of 30 °C, detector wavelength of 245 nm, and run time of 85 min. The monosaccharide composition was confirmed by comparing the retention time of the standard sugars. The area normalization method was used to calculate the molar ratio.

In Vitro Coagulant Activity Analysis

All samples were prepared at concentrations of 50, 100, 200, 400, and 800 μg/mL and dissolved in normal saline (0.9%) for subsequent analysis. APTT, PT, TT, and FIB contents were determined using a previously described method with some modifications.[29] For determination of APTT, 100 μL of the APTT reagent, 100 μL of plasma, and 100 μL of the sample solution were mixed and incubated for 3 min at 37 °C. The solution was added with 100 μL of calcium chloride (37 °C) and analyzed by a coagulometer (Infinite M200, Shanghai, China). For determination of PT or TT, the samples and plasma (100 μL) were mixed and incubated for 1 min at 37 °C. Then, the solution was added with a PT reagent (200 μL) or pre-warmed TT reagent (100 μL), respectively. Clotting time was recorded.

Fibrinogen was detected in accordance with the kit’s instruction. In brief, 100 μL of plasma and 100 μL of the sample were added with a dilute solution (0.9 mL), and then the mixture was incubated for 30 s at 37 °C. Clotting time was recorded after mixing the FIB regent (50 μL) and the above solution (0.1 mL).[30] FIB content was calculated using a standard curve.

Normal saline served as the blank control. Yunnan Baiyao, a well-known Chinese medicine that has been used for hemostasis in China for approximately 100 years, was used as the positive control.[31,32]

In Vitro Immunomodulatory Activity Assay

Cell Culture

RAW 264.7 cells were purchased from Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). After recovery, cells in the humidified 5% CO2 incubator (at 37 °C) were cultured in DMEM containing 10% fetal bovine serum and 1% each of penicillin and streptomycin. Cell passages were performed with 0.25% trypsin–EDTA when cells reached a density of 80–90%.

Cell Viability Analysis

MTT assay was used to assess the effect of BSP-1–BSP-4 fractions on the RAW 264.7 cells viability.[33] The sample powder was dissolved in DMEM and diluted to 25, 50, 100, 200, 400, and 800 μg/mL. After ultrasonic dissolution, the sample solution was stored at −20 °C. The cells (100 μL) were plated into 96-well plates (1 × 105 cells/well) for 12 h and treated with 25, 50, 100, 200, 400, and 800 μg/mL BSP-1–BSP-4 for 24 h. The blank control group was added with DMEM without BSP, and the positive group was added with lipopolysaccharide (LPS, 25 μg/mL). The cells were added with 15 μL of MTT (5 mg/mL) in a 5% CO2 incubator at 37 °C for another 4 h. The supernatant was discarded, and formazan was dissolved in 150 μL of DMSO. The mixture was shaken for 10 min to ensure complete dissolution of the purple crystals. Optical density was recorded at 490 nm by using a spectrophotometric plate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The absorbance of the blank control (0 μg/mL) was considered as 100%. Five independent wells for each concentration were analyzed.

Nitric Oxide and Phagocytosis Analysis

RAW 264.7 cells were plated into 96-well plates as described in Section . The supernatant was collected to determine NO production by using a NO assay kit. The supernatant was mixed with an equal volume of Griess Reagent I (1% sulfanilamide) and Griess Reagent II (0.1% N-1-naphthylethylenediamine dihydrochloride in 5% phosphoric acid) at room temperature for 10 min. Absorbance at 540 nm was recorded by a spectrophotometric plate reader, and NO production was calculated by comparison with NaNO2 standards.

Neutral red phagocytosis assay was conducted to evaluate the phagocytic capacity of RAW 264.7 cells.[34] After removal of the supernatant, the non-adherent cells were removed by washing twice with PBS. Each well was then added with 100 μL of neutral red solution and incubated for 1 h. The solution was discarded, and the cells were washed twice with PBS to remove residual neutral red solution. The cells were broken down by lysate solution (ethanol and 1.0 mol/L acetic acid in a 1:1 ratio). The plates were kept overnight at room temperature. Absorbance at 490 nm was recorded using a spectrophotometric plate reader.

Statistical Analysis

GraphPad Prism 6.01 software (LaJolla, CA, USA) was used for analysis. One-way analysis of variance (ANOVA) and Bonferroni multiple comparisons tests were used for intergroup comparisons. A value of p < 0.05 was considered statistically significant.

Results and Discussion

Extraction, Purification, and Isolation of BSP

The yield of BSP using hot water reached 45.55%. After purification procedures including ethanol sedimentation, deproteination, and dialysis, the purified BSP was separated on anion exchange DEAE-52 cellulose and eluted with distilled water (BSP-1), 0.1 M NaCl (BSP-2), 0.2 M NaCl (BSP-3), and 0.3 M NaCl (BSP-4) to obtain four fractions with the ratio of 3:10:3:2 (Figure ). The contents of polysaccharides in the abovementioned fractions reached 93.3, 93.5, 96.23, and 90.21%, respectively (Table ). Meanwhile, a small amount of protein was detected, and BSP-2 had the highest content of uronic acids among the fractions (Table ).

Figure 1

Elution profile of polysaccharides on the DEAE-52 cellulose column.

Table 1

General Components in BSP-1–BSP-4 Fractions

polysaccharide	uronic acids (%)	proteins (%)	total carbohydrates (%)
BSP-1	2.47 ± 0.15	1.05 ± 0.19	93.3 ± 3.75
BSP-2	3.54 ± 0.19	0.46 ± 0.13	93.5 ± 5.15
BSP-3	1.10 ± 0.7	0.67 ± 0.12	96.23 ± 4.18
BSP-4	2.37 ± 0.31	1.32 ± 0.46	90.21 ± 6.16

Sulfate Group Identification

The results of the BaCl2–gelatin turbidity method showed that BSP-1 and BSP-2 became turbid under a black background compared with the blank control group. The turbidity degrees of BSP-3 and BSP-4 were higher than those of BSP-1 and BSP-2. These observations suggested that BSP-1–BSP-4 fractions contained sulfate and that BSP-3 and BSP-4 had higher sulfate content than BSP-1 and BSP-2. The DS of BSP-1–BSP-4 decreased in the following order: BSP-4 (1.70) > BSP-3 (1.59) > BSP-1 (0.29) > BSP-2 (0.03). Overall, these results demonstrated that BSP belonged to natural polysaccharides with a sulfate radical.

Analysis of UV–Vis and IR

Figure shows weak absorption peaks at 260 and 280 nm, indicating that the BSP-1–BSP-4 fractions contained a small amount of nucleic acids and proteins.[35] These results are consistent with the quantitative analysis in Table .

Figure 2

UV–vis spectra of BSP-1, BSP-2, BSP-3, and BSP-4.

The IR analysis indicated that all polysaccharides exhibited similar absorption peaks in Figure a. In particular, the characteristic absorption peaks were as follows: the absorption peak near 3414 cm–1 corresponding to the O–H stretching vibration, the peak at 2925 cm–1 corresponding to the C–H stretching vibration in −CH2, the peak at 1643 cm–1 corresponding to the asymmetric stretching vibration of COO–,[36] the peak at 1421 cm–1 that was considered as the −OH bending vibration, and the peak at approximately 1066 cm–1 that was due to the C–O–C symmetric stretching vibration of carbohydrates.[37] These results demonstrated that all the BSP fractions possessed the typical structural characteristics of polysaccharides.

Figure 3

(a) IR spectra and (b) second-derivative IR spectra of BSP-1, BSP-2, BSP-3, and BSP-4.

Second-derivative IR spectroscopy is a method to distinguish overlapping peaks and obtain high-resolution peaks;[25] this method was used to compare differences in peaks especially within 2000–500 cm–1 to explore the fine structures of BSP-1–BSP-4.[38] In Figure b, the peak at 1730 cm–1 was assigned to the C=O of the carbonyl group in ester, and BSP-2 showed the highest absorption;[39] the peak at 1150 cm–1 was assigned to the bending vibrations of C–O;[40] and the peak at 879 cm–1 was related to β-glucosidic bonds.[41] The most important peak of BSP fractions at 1252 cm–1 could be attributed to the asymmetric stretching vibration of S=O, and the peak at 816 cm–1 represented the symmetrical vibration of C–O–S that was associated to a C–O–SO3 group; BSP-4 exhibited the strongest absorption.[42,43]

Analysis of XPS

XPS analysis was conducted to determine the presence of the valence state of different elements of BSP-1–BSP-4. Figure shows the survey spectra of four elemental components assigned to 168.5 (S), 283.4 (C), 400.1 (N), and 532.1 eV (O);[44] in particular, C 1s and O 1s had strong binding energy and were the main components of the polysaccharides. Furthermore, small amounts of nitrogen and sulfur were detected.

Figure 4

Wide-survey XPS spectra of BSP-1, BSP-2, BSP-3, and BSP-4.

In addition to content analysis, the curve fitting of C 1s, O 1s, N 1s, and S 2p is shown in Figure a. The C 1s peak could be divided into three positions: the peaks at 284.5 eV were assigned to non-functionalized carbon (C–C and C–H);[45] the peaks at 286.4 eV were due to C–O, C–N, or C–S bonds;[46] and the peaks centered at the binding energy of 287.7 were attributed to O–C=O or HN–C=O.[15] Changes in the intensity ratio of carbon contributions are found in Figure a. BSP-1, BSP-3, and BSP-4 had a strong intensity at 284.5 eV, and BSP-2 and BSP-4 had a strong intensity at 286.4 eV compared with the other peaks.

Figure 5

XPS analysis of (a) C 1s, (b) O 1s, (c) N 1s, and (d) S 2p spectra of BSP-1–BSP-4.

The O 1s spectra are shown in Figure b and could be decomposed into two positions at 531.5 and 532.6 eV. The peak at 531.4 eV was associated with O double bonded to C in carboxylic acid, ester, or amide, while the O 1s peak at 532.6 eV arose from the alcohol, hemiacetal, or acetal group.[14,47,48] These findings indicated that the contents of C–O and C=O bonds were relatively high in BSP-2, consistent with the results of glucuronic acid in Table and ester carbonyl group at 1730 cm–1 in the IR analysis. BSP-2 had a strong intensity at 286.4 and 532.6 eV, which might be related to the acetyl oxygen group.[10,48]

Nitrogen and sulfur were also detected in the samples (Figure c,d). The N 1s peak was divided into two parts, at 400.2 and 399.1 eV. The peak at 399.1 eV was related to the NH2 or NH group,[49] and the peak at 400.1 eV was attributed to NH–C=O bonds.[50] The weak N 1s peak might be due to the protein residuals or glycoprotein complex. In addition to C, O, and N, S was the first element found in the BSP fractions. With regard to the S 2p peak, the peak at the binding energy 168.5 eV was associated with SO3– bonds that possessed negatively charged groups.[49] BSP-3 and BSP-4 had stronger intensity at 168.5 eV than BSP-1 and BSP-2, suggesting that they possessed higher sulfate content. These results were consistent with the turbidity experiment in Section .

Analysis of Monosaccharide Composition

Retention time was compared between the samples and the standard mixture to determine the monosaccharide composition (Table ). BSP-1–BSP-4 had higher proportions of mannose and glucose. In particular, BSP-1 and BSP-4 contained more types of monosaccharides than BSP-2 and BSP-3. BSP-2 had the highest proportion of GalA among the fractions.

Table 2

Monosaccharide Composition (Molar Ratio %) of BSP-1–BSP-4a

	sample
monosaccharide composition	BSP-1	BSP-2	BSP-3	BSP-4
Man	22.34	52.32	3.20	10.35
Rib	0.24	—	—	1.00
Rha	0.23	—	1.20	1.18
GluA	0.89	—	—	—
GalA	—	2.44	0.87	1.21
Glu	27.84	31.26	8.77	6.12
Gal	1.00	1.00	1.00	1.00
Xyl	1.28	—	—	1.06
Ara	—	—	—	0.62
Fuc	0.56	—	—	—

“–” means not present.

Analysis of Molecular Weight

Table shows the molecular weight of three fractions determined based on the calibration curve. The average Mw of BSP-4 was 7.15 × 105 Da, which was the lowest among the fractions.[51]

Table 3

Molecular Weights of BSP-1–BSP-4a

name	BSP-1	BSP-2	BSP-3	BSP-4
Mw (Da)	761,123	843,940	950,302	715,462

Mw means average molecular weight

Analysis of In Vitro Coagulant Activity Assay

The coagulant activities of BSP-1–BSP-4 were assessed by measuring APTT, PT, TT, and FIB. The effect of polysaccharides on APTT was first examined. As shown in Figure a, BSP-1 (0.2–0.8 mg/mL), BSP-2 (0.4–0.8 mg/mL), BSP-3 (0.2–0.8 mg/mL), and BSP-4 (0.2–0.8 mg/mL) exhibited significant pro-coagulant activity (p < 0.05); in particular, 0.8 mg/mL BSP-4 had better pro-coagulant activity that had been found to exceed Yunnan Baiyao. Hence, BSP fractions could activate the intrinsic pathway of coagulation.[52]

Figure 6

Effects of BSP-1–BSP-4 (0.05–0.8 mg/mL) on plasma coagulation time determined by measuring (a) APTT, (b) PT, (c) TT, and (d) FIB in vitro. *p < 0.05 versus normal saline; ^p < 0.05 versus Yunnan Baiyao.

The effect of BSP fractions on PT was then determined. Figure b shows that BSP-1 (0.2–0.8 mg/mL), BSP-2 (0.4–0.8 mg/mL), BSP-3 (0.4–0.8 mg/mL), and BSP-4 (0.4–0.8 mg/mL) significantly shortened the PT than normal saline (p < 0.05). Hence, BSP fractions possessed high pro-coagulant activity by converting prothrombin to thrombin in a short time via the extrinsic coagulation pathway.[53]

Active thrombin can convert soluble fibrinogen into insoluble fibrin, leading to clotting.[54] The acceleration of this process can shorten the clotting time. TT is considered an indicator for common coagulant pathways. In this regard, BSP-1 (0.2–0.8 mg/mL), BSP-2 (0.4–0.8 mg/mL), BSP-3 (0.4–0.8 mg/mL), and BSP-4 (0.2–0.8 mg/mL) reduced the TT compared with normal saline (p < 0.05). The pro-coagulant effect of BSP-1–BSP-4 (0.8 mg/mL) in TT assay was better than that of Yunnan Baiyao (p < 0.05). Hence, BSP fractions might play an important role in TT regulation and were more beneficial to thrombin-mediated fibrin formation.[55]

Given that FIB mainly reflects the content of fibrinogen that produces fibrin, fibrinogen content was determined (Figure d). No significant difference in FIB was observed between BSP-1 and normal saline within the concentrations of 0.05–0.8 mg/mL (p > 0.05). Meanwhile, significant pro-coagulant effects were found in BSP-2 (0.2–0.8 mg/mL), BSP-3 (0.4–0.8 mg/mL), and BSP-4 (0.4–0.8 mg/mL). Hence, BSP fractions affected fibrinolytic systems.

Overall, BSP is more likely to be a promising coagulant by regulating intrinsic, extrinsic, and common coagulant pathways.

In general, the body’s own coagulation process can transform blood into insoluble fibrin, which is mainly considered to be involved in primary hemostasis. When no hemostatic agent is available to implement hemostasis timely, especially in the battlefield, operating rooms, and emergency rooms, many deaths will occur due to uncontrollable bleeding.[56] Thus, an ideal hemostatic material or agent should be developed. Thus far, a wide variety of polysaccharides have been investigated and applied as hemostatic agents because of their advantages including low price, minimal side effects, and biodegradable properties.[57−59] A previous study reported that BSP hydrogels exhibited pro-coagulant activity.[11] Moreover, most sulfated polysaccharides play an important role in the coagulation pathway via the mechanism that the negatively charged groups of polymeric sulfates could bind to the positively charged groups in proteins.[60,61] Among the BSP fractions, BSP-4 significantly reduced the APTT and TT, which might be ascribed to its higher sulfate content and lower molecular weight.[54,62]

Uronic acids play an important role in coagulation.[63] Charge density increases upon the incorporation of carboxyl groups,[64] thereby enhancing the combination between the base protein and carboxyl groups. In this regard, BSP-2 with the highest uronic acid content exhibited the highest enhancing effect on FIB.

Analysis of Immunomodulatory Activity

The effect of BSP-1–BSP-4 on the RAW 264.7 cells’ viability was measured by MTT assay. As shown in Figure A, all BSP fractions (0.05–0.8 mg/mL) showed no significant inhibition effect (p > 0.05). Pinocytic activity, a prominent feature of macrophage activation,[65,66] was also determined using neutral red assay. Figure B shows that BSP-1 (100 and 200 μg/mL), BSP-2 (50, 100, and 200 μg/mL), and BSP-3 (100 μg/mL) increased the uptake rate compared with the blank control (p < 0.05). BSP-2 possessed a higher uptake rate than the other BSP fractions and was similar to the positive control (100 μg/mL, p > 0.05). A previous study proposed that polysaccharides that are composed of fucose or mannose residues could attach to mannose receptors and trigger pinocytosis.[67] This fact explains why BSP-2 exhibits the best pinocytic capacity among the BSP fractions tested. NO production was also evaluated because NO is an important signal transduction medium in the immune system.[68] As shown in Figure C, BSP-1 (25–800 μg/mL), BSP-2 (25–800 μg/mL), and BSP-4 (50–800 μg/mL) significantly increased the amount of NO production (p < 0.05) compared with the blank control. The BSP-3 group had no enhancing effect on NO production (p > 0.05). Similar to the results of pinocytic activity, 100 μg/mL BSP-2 showed the best effect on increasing NO production among the fractions.

Figure 7

Effect of BSP-1–BSP-4 on (A) cell viability, (B) pinocytic capability, and (C) secretion levels of NO of RAW 264.7 cells. The group without BSP was used as the blank control group, and the group treated with LPS (25 μg/mL) was used as the positive control. *p < 0.05 versus the blank control group.

In addition to mannose residues, uronic acids are associated with immunomodulatory activities of polysaccharides. BSP-2 with an appropriate molecular weight and high uronic acid content might exhibit higher immunomodulatory activity than other BSP fractions, similar to previous reports.[69,70] BSP-3 showed inapparent immunomodulatory activity possibly due to its lowest uronic acid content and highest molecular weight. We will focus on the structure and mechanism of the extracted BSP in a future study.

Conclusions

BSP was extracted by hot water, and four polysaccharides were isolated and obtained using DEAE-52 cellulose. BSP fractions belonged to natural polysaccharides with a sulfate radical, as confirmed by the results of second-derivative IR spectroscopy and XPS analysis. The results of the in vitro coagulant assay revealed that BSP fractions had stronger pro-coagulant activity compared with normal saline. Finally, BSP fractions were observed to possess significant immunomodulatory activity by enhancing pinocytic capacity and NO production. Overall, BSP can be used as natural pro-coagulant and immunomodulatory agents in pharmaceutical and nutraceutical industries.