Immunostimulating effect of polysaccharides isolated from Ma-Nuo-Xi decoction in cyclophosphamide-immunosuppressed mice

Journal Pre-proof Immunostimulating effect of polysaccharides isolated from MaNuo-Xi decoction in cyclophosphamide-immunosuppressed mice

Duo Jie, Tingting Gao, Zhongshu Shan, Jiayin Song, Ming Zhang, Olga Kurskaya, Kirill Sharshov, Lixin Wei, Hongtao Bi PII:

S0141-8130(19)39219-0

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.042

Reference:

BIOMAC 14069

To appear in:

International Journal of Biological Macromolecules

Received date:

12 November 2019

Revised date:

3 December 2019

Accepted date:

5 December 2019

Please cite this article as: D. Jie, T. Gao, Z. Shan, et al., Immunostimulating effect of polysaccharides isolated from Ma-Nuo-Xi decoction in cyclophosphamideimmunosuppressed mice, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.042

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© 2018 Published by Elsevier.

Journal Pre-proof

Immunostimulating effect of polysaccharides isolated from Ma-Nuo-Xi Decoction in cyclophosphamide-immunosuppressed mice Duo Jie,1,5,# Tingting Gao,2,# Zhongshu Shan,3 Jiayin Song,4 Ming Zhang,1 Olga Kurskaya,6 Kirill Sharshov,6 Lixin Wei,1,7,* Hongtao Bi 1,* 1

Qinghai Provincial Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation,

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Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining 810008, China 2

Department of Psychology, School of Public Health, Southern Medical University, Guangzhou, Department of Orthopaedics, People’s Hospital of Qinghai Province, Xining 810007, China Tianjin Key Laboratory of Architectural Physics and Environmental Technology, Tianjin

-p

4

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510515, China 3

University, 92 Weijin Road, Nankai District, Tianjin 300072, PR China

State Key Laboratory of Tibetan New Drug Development, Institute of Tibetan Medicine of

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5

Qinghai Province, Xining, 810003, China

Department of Experimental Modeling and Pathogenesis of Infectious Diseases, Federal

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6

Research Center of Fundamental and Translational Medicine, Novosibirsk, 630000, Russia, CAS Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology,

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7

Chinese Academy of Sciences, Xining, 810001, China

These authors contributed equally

*

Corresponding Author.

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#

Correspondence: Hongtao Bi and Lixin Wei Lixin Wei

Key Laboratory of Tibetan Medicine Research, Northwest Institute of Plateau Biology, CAS, 23 Xin’ning Road, Xining 810008, China Tel./Fax: +86-971-6143900 E-mail address: [email protected] Hongtao Bi Qinghai Provincial Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation, Northwest Institute of Plateau Biology, CAS, 59 Xiguan Road, Xining 810001, China Tel./Fax: +86-971-6143765 E-mail address: [email protected]

1

Journal Pre-proof Abstract Ma-Nuo-Xi Decoction (MNXD) is well-known in Tibetan medicine to be in line with the theory of treatment determination based on syndrome differentiation. However, the components responsible for its immunomodulating effect are unknown. In this study, three polysaccharide components—MNXD-P, MNXD-BD-P, and MNXD-AD-P—were isolated from MNXD and its basic and auxiliary prescription decoctions, of which MNXD-BD-P is composed

of

β-(1,4)-D-glucan

and

RG-I pectin,

MNXD-AD-P contains

mainly

of

α-(1,4)-D-glucan and some amount of arabinogalactan and/or arabinorhamnogalactan, and MNXD-P contains components of both MNXD-BD-P and MNXD-AD-P. And treatment with

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these polysaccharides could significantly improve the host’s specific and non-specific

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immunity, including cellular and humoral immunities, as well as promote recovery from

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myelosuppression in cyclophosphamide (CTX)-immunosuppressed mice. To our knowledge, this is the first report on chemical and immunoactivity study on polysaccharides from

Keywords:

Ma-Nuo-Xi

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carbohydrate drugs from them.

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traditional Tibetan medicine compounds, which may provide a new idea for development of

Decoction;

Polysaccharide;

Immunostimulating

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Cyclophosphamide-induced immunosuppression; Tibetan medicine

2

effect;

Journal Pre-proof 1. Introduction Ma-Nuo-Xi Decoction (MNXD), originally sourced from the Tibetan medical classic work rGyud-bzhi written by Yuto Yuandan-gombe (748765 A.D.), is the original prescription for hundreds of Tibetan-formulated medicines and as such is known as the “Mother of Decoctions” in Tibetan medicine [1, 2]. The basic prescription of MNXD includes Inula racemosa Hook.f., Tinospora sinensis (Lour.) Merr., and Rubus biflorus Buch., while the auxiliary prescription includes Zingiber officinale Rosc., which can be changed to other single

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or multiple medicinal materials depending on the diseases being treated [3]. According to the

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records in rGyud-bzhi and Somaradza, a classic Tibetan medical work from the mid-13th century A.D., treatment with MNXD relieved exterior syndrome and induced perspiration,

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and it was used to treat plagues, fever, rheumatoid arthritis, and other diseases [3].

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Pharmacological studies testing carbon clearance, delayed type hypersensitivity (DTH), and chicken RBC phagocytosis assays in mice have found that MNXD possesses

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immunomodulating activity[1, 4]. However, the components responsible for this

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immunomodulating effect are unknown.

Many herbs contain polysaccharides that are believed to be bioactive ingredients involved

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in immunomodulation [5-9]. These findings have encouraged the isolation of polysaccharides from MNXD, as well as from its basic and auxiliary prescriptions. In this study, we used a cyclophosphamide (CTX)-immunosuppressed mouse model to evaluate the influence of polysaccharides on the immunostimulating ability of MNXD. Macrophages, lymphocytes and natural killer (NK) cells are considered as the important components of the host defense against invading pathogens, especially the macrophages play an important role in the initiation and regulation of the immune response by interacting with lymphocytes and acting as phagocytic, microbiocidal, and tumoricidal effector cells [8]. Therefore, macrophage function, NK cytotoxic activity, lymphocyte proliferation, splenic T-Lymphocyte subpopulations, serum cytokines, and quantitative hemolysis of SRBC (QHS) assays were used to determine polysaccharide influence. 3

Journal Pre-proof 2. Materials and Methods 2.1. Plant materials and chemicals I. racemosa, T. sinensis, R. biflorus, and Z. officinale were purchased from Qinghai Fukang Pharmaceutical Co. Ltd. (Xining, Qinghai, China) on October 15, 2018, and identified by Prof. Yuzhi Du, Northwest Plateau Institute of Biology, CAS in Xining, China. The herbarium samples were numbered as IR20181015 for I. racemosa, TS20181015 for T. sinensis, RB20181015 for R. biflorus and ZO20181015 for Z. officinale, and deposited at

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Qinghai Key Laboratory of Tibetan Medicine Pharmacology and Safety Evaluation. CTX was purchased from Jiangsu Hengrui Medicine Co. (Lianyungang, Jiangsu, China).

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Sheep red blood cells (SRBCs), chicken red blood cells (CRBCs), guinea pig complements,

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RPMI 1640 cell culture medium, and Hank’s balanced salt solution (HBSS) were purchased

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from Gibco BRL (Grand Island, NY, USA). Lipopolysaccharide (LPS), concanavalin A (ConA), 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT), analytical

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standard dextrans (50 kDa, 25 kDa, 12 kDa, 5 kDa, and 1 kDa) for GPC, and the limulus

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amebocyte lysate (LAL) assay kit were purchased from Sigma (St. Louis, MO, USA). Mouse IL-2, IL-4, and INF-γ enzyme-linked immunosorbent assay (ELISA) kits were purchased

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from Invitrogen (Carlsbad, CA, USA). TNF-α and IL-1β ELISA kits and anti-CD4 and anti-CD8 antibodies were purchased from eBioscience (San Diego, CA, USA). The NO kit was purchased from Beyotime (Nantong, Jiangsu, China). DEAE-Cellulose was acquired from Shanghai Hengxin Chemical Reagent Co. Ltd. (Shanghai, China). A TSK-Gel G3000PWXL column (7.8 mm i.d. × 30 cm) with a guard column was obtained from TOSOH Co. (Tokyo, Japan). 2.2. Preparation of MNXD and its polysaccharides The MNXD prescription is composed of I. racemosa, T. sinensis, R. biflorus, and Z. officinale in a weight ratio of 20:20:6:5. The basic MNXD prescription is composed of I. racemosa, T. sinensis, and R. biflorus in a weight ratio of 20:20:6, and the auxiliary MNXD prescription only contains Z. officinale [10, 11]. According to the tradition of 4

Journal Pre-proof “multi-medicinal material decocted together”, herbal materials were mixed as described above, ground into powders, and decocted in boiling water at a ratio of 1:20 (w/v) for 3 h. The decoctions were collected by centrifugation at 10,000 rpm for 30 min to remove insoluble residues and then concentrated to certain volumes to generate the full prescription decoction (MNXD), the basic prescription decoction (MNXD-BD), and the auxiliary prescription decoction (MNXD-AD). Their polysaccharide components were obtained by precipitation with 80% ethanol at 4°C overnight and purification by using a DEAE-Cellulose column with M

NaCl

elution.

The

polysaccharides

from

MNXD,

MNXD-BD,

and

of

0.5

MNXD-AD—named MNXD-P, MNXD-BD-P, and MNXD-AD-P—were stored at -80ºC

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before use.

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2.3. General methods

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Total carbohydrate content was determined by the phenol-H2SO4 method using glucose as a standard [12]. Uronic acid content was determined by the m-hydroxydiphenyl

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colorimetric method using galacturonic acid as a standard [13]. Protein content was

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determined by the Coomassie brilliant blue method using bovine serum albumin as a standard [14]. Visible absorbance was recorded with a UV–Vis spectrophotometer (Model SP-752,

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China).

Fourier-transform infrared (FT-IR) spectra were obtained on a Thermo IS50 FT-IR spectrometer with a DTGS detector in the range of 4004000 cm−1. The sample was measured as a film on a KBr disc. Morphological analyses of the polysaccharide samples were conducted using a field emission scanning electron microscope (FESEM; SU-8010, Hitachi, Japan). The molecular weight (MW) distributions of the polysaccharide samples were determined using high-performance gel permeation chromatography (HPGPC) on a TSK-GEL G3000 PWXL column (7.8 mm × 300 mm, 10 μm), and the monosaccharide compositions of the polysaccharide samples were analyzed by high-performance liquid chromatography (HPLC) using a Bonshell ASB C18 column (100 × 4.6 mm i.d., 2.7μm, 90Å) 5

Journal Pre-proof with 1-phenyl-3-methyl-5-pyrazolone (PMP) precolumn derivatization, which were described in our previous study [15]. The HPGPC and HPLC were carried out by using a LC-10ADvp HPLC pump (Shimadzu, Tokyo, Japan) equipped with a refractive index RID-10A detector and an SPD-10Avp UV-VIS detector (Shimadzu, Tokyo, Japan). Fifty milligrams of polysaccharide sample were dissolved in 0.5 mL of deuteroxide (99.8% D) in an NMR tube. The 13C NMR analysis was performed on a Bruker Avance 600 MHz spectrometer (Germany), and the spectra were recorded using a Bruker 5 mm broadband

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observe probe at 20°C and 150 MHz for 13C. 2.4. Immunostimulating capacity assay

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2.4.1. Animals and treatment

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Eight-week-old male BALB/c mice were obtained from Beijing Vital River Laboratory

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Animal Technology Co., Ltd., China (animal production license number: SCXK (Jing) 2012-0001). The mice were housed in standard cages with wood shavings in a room with

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carefully controlled ambient temperature (25°C) and artificial lighting from 8:00 am to 8:00

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pm. The mice were fed standard laboratory chow and distilled water ad libitum. All animal experiments were in compliance with the ARRIVE guidelines, carried out in strict accordance

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with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 8023, revised 1978), and approved by the committee of Northwest Plateau Institute of Biology, CAS for animal experiments. After one-week acclimatization, the mice were randomly divided into seven treatment groups (n = 10): (1) Normal, (2) Model, (3) Levamisole (an immunopotentiating agent used as positive drug), (4) MNXD, (5) MNXD-P, (6) MNXD-BD-P, and (7) MNXD-AD-P. CTX (50 mg/kg per day) was intraperitoneally injected into mice on days 13, and then 0.2 mL of the drugs was orally administered to all mice for 14 days. The dose of levamisole was 10 mg/kg per day, and the dose of MNXD was equivalent to 1.6 g/kg per day of the original medicinal materials (its clinical equivalent dosage) [10, 11]. The doses of the polysaccharides MNXD-P, MNXD-BD-P, and MNXD-AD-P were 0.268 g/kg, 0.178 g/kg, and 0.036 g/kg once a day, 6

Journal Pre-proof respectively, and these doses were equivalent to the polysaccharide yields from 1.6 g/kg, 1.44 g/kg, and 0.16 g/kg per day of the original medicinal materials, respectively. Mice in the normal and model groups were orally administered 0.2 mL sterile water once daily. 2.4.2. Immunostimulating capacity assay Twenty-four hours after the last drug administration, blood samples from the ophthalmic venous plexus were collected. Peripheral white blood cell (WBC), red blood cell (RBC), and platelet numbers were counted using a Coulter LH780 Hematology Analyzer. The

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concentrations of IL-2, IL-4, and INF-γ in the serum were determined using ELISA kits. Finally, the mice were weighed and then sacrificed via decapitation. The spleens and

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thymuses were immediately isolated and weighed, and the organ weight-body weight ratio

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indices were calculated.

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CRBCs were used to assess macrophage phagocytosis, and the procedure was performed as described in our previous study [6]. Briefly, peritoneal cells were immunized by CRBCs,

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stained with 4% Giemsa-PBS solution, and then counted with an inverted microscope

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(Olympus IX-73, Tokyo, Japan) to determine the phagocytic rate and the phagocytic index. The phagocytic index and phagocytic rate were defined as follows: phagocytic rate = number

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of macrophages that engulfed SRBCs out of 100 macrophages, phagocytic index = number of internalized SRBCs in 100 macrophages. Peritoneal macrophage suspensions were prepared and cultured following the procedure described in our previous study [8]. Cells from the peritoneal exudate (PEC) were collected from the immunosuppressed mouse by peritoneal lavage with cold PBS. PECs were washed twice and resuspended in RPMI-1640 medium. Peritoneal macrophages were further isolated by incubating the PECs (1×107/well) in a 6-well plate at 37 °C in a humidified atmosphere with 5% CO2 for 3 h to allow the peritoneal macrophages to adhere. After removing the non-adherent cells with cold PBS, the macrophage monolayer was collected. The adherent cell population was >95% macrophages. The macrophage suspension was added into 96-well

7

Journal Pre-proof microwell plates for a concentration 4×105 cells/well and cultured for 48 h, and then culture supernatants were harvested for an ELISA to detect cytokine secretion. The NO level in the culture supernatant of mouse peritoneal macrophages was determined by the Griess method using a commercial kit according to the instruction of the manufacturer. The NK cytotoxic activity assay was performed as described by Wang et al [8]. Briefly, splenocytes (effector cells, E) at a concentration of 5 × 105 cells/mL and YAC-1 cells (target cells, T) were mixed in the same well of 96-well round-bottom plates, resulting in an E:T ratio

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of 100:1. Following a 20 h incubation at 37 °C in a humidified 5% CO2 incubator, the plates

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were centrifuged (400 × g, 5 min), 50 μL of MTT solution (2 mg/mL) was added to each well,

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and the plate was incubated for another 4 h and subjected to an MTT assay. NK cell activity was calculated using the following equation: NK cytotoxic activity (%) = (AT− (AS− AE))/AT ×

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100%, where AT is the absorbance value of control target cells, AS is the absorbance value of

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test samples, and AE is the absorbance value of control effector cells. The ConA and LPS-induced lymphocyte proliferation assays were carried out as

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described in our previous study [5]. Briefly, a splenocyte suspension was prepared and adjusted to a concentration of 5 × 106 cells/mL, and an aliquot of 100 µL of this suspension of

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splenocytes was seeded into each well of a 96-well plate in the presence of either ConA (5 µg/mL) or LPS (10 µg/mL). After preincubation for 48 h at 37 °C in a humidified 5% CO2 incubator, 10 µL of 0.4% MTT was added to each well. The plates were incubated for another 4 h, and then the solution was dissolved in dimethyl sulfoxide (100 µL/well) and measured A570 nm. Splenic T-lymphocyte subpopulations assay was performed as described by Qi et al. [16]. In brief, the splenocyte suspension was adjusted to 1 × 106 cells/mL and incubated with 10 μL of either anti-CD4 or anti-CD8 antibody for 60 min at 4 °C. Cells were then washed twice with PBS and resuspended in 1% paraformaldehyde (PFA). The counts of CD4+ and CD8+ T lymphocytes were determined by using a FACSCalibur (BD Medical Technology, Franklin Lakes, NJ, USA) and CellQuest software, which were expressed as percentages of the total 8

Journal Pre-proof number of T-lymphocytes. Quantitative hemolysis of SRBC (QHS) assay was performed as described by Bin-Hafeez et al. [17]. Mice in all groups were initially sensitized by 0.2 mL of 10% SRBCs suspension on day 12. Five days later, the splenocyte suspension of 1 × 106 cells/mL prepared in PBS was incubated at 37 °C for 1 h after mixing with 1 mL of 0.2% SRBCs and 1 mL of 10% guinea pig serum complement. After centrifugation (3,000 rpm for 3 min), the extent of hemolysis of SRBCs in the supernatant was determined at 413 nm.

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2.5. Statistical analysis All data were expressed as the mean ± standard deviation (SD) of six replicates and

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subjected to analysis of variance for a completely random experimental design. Multiple t

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tests were conducted to identify differences among means (without assuming consistent SD)

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by using GraphPad Prism software (Version 7.00). P < 0.05 was considered statistically

3. Result

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significant.

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3.1. Analysis of the MNXD polysaccharides

The MNXD-P, MNXD-BD-P, and MNXD-AD-P samples were obtained from the full

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prescription decoction, basic prescription decoction, and auxiliary prescription decoction, respectively. MNXD-P and MNXD-BD-P are light brown powders, and accounted for 16.72% and 16.11% of their original medicinal materials weight, respectively. While, MNXD-AD-P is a white powder, accounted for 22.43% of the original medicinal materials (Z. officinale) weight. Due to the process of removing protein and other impurities with strong-bonding ability by DEAE-Cellulose ion exchange chromatography, total sugar contents of the three polysaccharide fractions were determined to be above 95% by the phenol-H2SO4 method, and their protein contents were all below 1% (Table 1). Their UV spectra contained no peaks at 260 nm, indicating no nucleic acid in them. The m-hydroxydiphenyl colorimetric assay showed that the uronic acid contents of MNXD-P and MNXD-BD-P were 6.52% and 6.89%, indicating pectic polysaccharide 9

Journal Pre-proof components in them. The result was further verified by monosaccharide composition assay. The monosaccharide composition analysis indicated both of MNXD-P and MNXD-BD-P were mainly composed of glucose, arabinose, galactose and galactouronic acid, and a small amount of rhamnose. While, MNXD-AD-P, polysaccharide from Z. officinale, contains plenty of glucose (91.83%), small amounts of rhamnose, galactose and arabinose, and almost none of the uronic acid.

HPGPC analysis showed that MNXD-P and MNXD-BD-P both exhibited two main

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elution peaks: one peak was at the position of void volume (≥100 kDa), and the molecular

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weight of another peak was calculated to be Mw 2.45 kDa; MNXD-AD-P exhibited three

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main elution peaks: one peak was at the position of void volume (≥100 kDa), and the elution

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volumes of another two peaks were at the positions of Mw 61.50 kDa and 10.79 kDa. FT-IR spectra (Figure 1A) showed the absorptions at 3290.2 cm-1 and 3292.5 cm-1 caused

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by the O–H stretching vibration, 2925.9 cm-1 caused by the C–H stretching vibration, 1019.5 cm-1 and 1023.3 cm-1 attributed to pyranose ring. The FT-IR spectra for MNXD-P and

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MNXD-BD-P revealed absorptions at 874.1 cm-1, 871.4 cm-1, 817.8 cm-1, and 817.9 cm-1,

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which indicated the presence of both β-glycosidic and α-glycosidic linkages. The spectrum for MNXD-AD-P revealed an absorption at 844.6 cm-1, which indicated the presence of α-glycosidic linkages alone [5, 15, 18]. The scanning electron micrographs (SEM) of MNXD-P, MNXD-BD-P and MNXD-AD-P were shown in Figure 1B at magnifications of 250 and 2000. The surface of MNXD-P is composed of loose scaly and fibrous structures. The scaly structures have some microspores (2-10 μm in diameter), and the diameter of fibrous structures is 0.2-1 μm. The surface of MNXD-BD-P is composed of branching lamellar structures with the 4-15 μm-width. The surface of MNXD-AD-P appears some fragments and short-staples. The fragment is flat and smoot without any microspore, and the diameter of short-staple is 0.2-1 μm. So, the apparent morphology of MNXD-P was probably due to the strength of the 10

Journal Pre-proof intramolecular hydrogen bonds of the components from MNXD-BD-P and MNXD-AD-P, which caused loose scaly and fibrous structures. The

13

C NMR spectra of the MNXD polysaccharides are shown in Figure 2, and the

chemical shifts of the main glycosyl residues are listed in Table 2. MNXD-AD-P mainly contained

an

α-(1,4)-D-glucan

and

some

amount

of

arabinogalactan

and/or

arabinorhamnogalactan, which is mostly consistent with the structural features of polysaccharides isolated from Z. officinale reported by Wang et al. [19], besides the absence

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of →2,3,4)-α-D-Manp-(1→ residues and the presence of α-L-Rhap residues (C-1: δ 98.297.9 ppm; C-6: δ 16.7 ppm). The 13C NMR spectrum of MNXD-BD-P exhibited six anomeric

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signals at δ 109.2, 107.4, 103.2, 103.0, 99.4, and 92.4 that were assigned to α-L-Araf,

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β-D-Galp, α-D-GalpA, β-D-Glcp, α-L-Rhap, and unsubstituted β-D-Glcp, respectively, and

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signals at 181.4 and 57.3 suggested that the GalpA residue might exist as a methyl ester [18, 20, 21]. These results indicated that MNXD-BD-P was composed of a short-chain

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β-(1,4)-D-glucan and an RG-I pectin. MNXD-P contained all of the signal characteristics of

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the glycosyl residues found in both MNXD-BD-P and MNXD-AD-P. 3.2. Immunostimulating effect of MNXD polysaccharides

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According to the “Tibetan medicine standards of Qinghai province” and “Drug standards · Tibetan medicine by Ministry of Health PRC” [10, 11], the dose of MNXD was equivalent to 1.6 g/kg per day of the original medicinal materials (its clinical equivalent dosage). The dose of the polysaccharide was calculated using the following equation: polysaccharide dose (g/kg) = the original medicinal materials per day (g/kg) × yield of polysaccharide (%). So, the doses of the polysaccharides MNXD-P, MNXD-BD-P, and MNXD-AD-P calculated based on their yields were 0.268 g/kg, 0.178 g/kg, and 0.036 g/kg once a day, respectively. The quantity of endotoxin in MNXD and its polysaccharides was determined to be less than 0.015 EU/mg (negative) by LAL assay, indicating that the sample was free of endotoxin contamination. As shown in Table 3, after intraperitoneal injection of CTX for 3 days, the body weight, spleen and thymus indices, and peripheral WBC count of 11

Journal Pre-proof mice in the model group were significantly decreased compared to those in the normal group. Following oral administration of MNXD polysaccharides for 14 days, these CTX-induced decreases were ameliorated (P < 0.05), indicating a positive immunostimulatory effect on the atrophy observed in CTX-treated mice. The RBC and platelet count of mice treated with the MNXD polysaccharides was markedly elevated in the treatment groups compared to the model group, suggesting that treatment with the MNXD polysaccharides recovered the CTX-induced myelosuppression observed in the model mice.

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Macrophages are an integral part of the immune system, and they play an important role in the initiation and regulation of the immune response by interacting with lymphocytes and

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acting as phagocytic, microbiocidal, and tumoricidal effector cells [22]. As shown in Figure

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3A, treatment with the MNXD polysaccharides increased the phagocytic rate and the index of

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peritoneal macrophages compared to the model group (P < 0.05), and the phagocytic rates in the groups treated with MNXD-BD-P and MNXD-AD-P were more significantly increased (P

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< 0.05). As shown in Figure 3B, the decrease in macrophage secretion levels of IL-1β, TNF-α,

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and NO induced by CTX were all significantly recovered following treatment with the MNXD polysaccharides (P < 0.05). However, the NO level of the MNXD-P group was

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significantly lower than that of the MNXD group (P < 0.05). In addition to inducing macrophage phagocytosis, the nonspecific immune system also plays an important role in regulating NK cytotoxic activity [23]. As shown in Table 4, although MNXD failed to significantly increase the NK cytotoxic activity in CTX-treated mice, treatment with the MNXD polysaccharides resulted in significantly increased NK cytotoxic

activity compared to the model group (P < 0.05). Lymphocytes are considered one of the important components of the host defense against invading pathogens. Lymphocyte proliferation is a crucial event in the activation cascade of both cellular and humoral immune responses [24]. As shown in Figure 3C treatment with MNXD and its polysaccharide components significantly promoted ConA-induced T-cell and LPS-induced B-cell proliferation in CTX-treated mice (P < 0.05). Moreover, the level of 12

Journal Pre-proof ConA-induced T lymphocyte proliferation in the MNXD-AD-P group and the levels of LPS-induced B lymphocyte proliferation in the MNXD-BD-P and MNXD-AD-P groups were significantly higher than those in the MNXD-P group (P < 0.05). Cytokines act through receptors and are especially important in the immune system. They modulate the balance between humoral and cell-based immune responses and regulate the maturation, growth, and responsiveness of particular cell populations [25]. As shown in Figure 3D, the administration of MNXD and its polysaccharides significantly increased the

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serum concentrations of IL-2, IL-4, and INF-γ in CTX-treated mice (P < 0.05). T-lymphocyte subpopulations are of great importance in T-cell homeostasis and immune

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regulation. CD4+ T-cells (helper T-lymphocytes) and CD8+ T-cells (killer T-lymphocytes) are

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two important subpopulations of T-lymphocytes, and the CD4+/CD8+ ratio is considered a

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marker for both immune senescence and immune activation [26]. As shown in Table 4, treatment with MNXD and its polysaccharide components significantly increased the ratio of

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CD4+/CD8+ in CTX-treated mice (P < 0.05), and the CD4+/CD8+ ratio of the MNXD-BD-P

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group was significantly higher than that of MNXD and MNXD-P (P < 0.05). To further investigate the effect of MNXD polysaccharides on the humoral immune

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response, changes in the serum hemolysin content in response to exposure to cellular antigen were measured [6]. The results (Table 4) showed that the serum hemolysin level was significantly increased in mice treated with MNXD and its polysaccharides compared to the model group (P < 0.05). 4. Discussion MNXD is well-known in Tibetan medicine to be in line with the theory of treatment determination. It is usually used to improve the patients’ immunity in treating plague, fever, rheumatoid arthritis, and other diseases [27]. Alamgir and Uddin (2010) have reported that a branched arabinogalactan isolated from T. sinensis, one of the herbal components of MNXD, is immunoactive [28]. However, there has yet to be a report on the MNXD polysaccharides and their immunoactivity. 13

Journal Pre-proof In this study, we investigated the composition and structural characteristics of MNXD-P, MNXD-BD-P, and MNXD-AD-P isolated from MNXD and its basic and auxiliary prescription decoctions. The results indicated that MNXD-BD-P might be composed of a β-(1,4)-D-glucan with MW of 2.45 kDa and an RG-I pectin with MW over 100 kDa. MNXD-AD-P mainly contained α-(1,4)-D-glucan and some amount of arabinogalactan and/or arabinorhamnogalactan. MNXD-P contained components of both MNXD-BD-P and MNXD-AD-P. Scanning electron microscopy showed that MNXD-P is composed of loose,

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scaly, and fibrous structures, while MNXD-BD-P is composed of branching lamellar structures and MNXD-AD-P has some fragments and short-staples.

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We characterized the immunomodulatory effects of treatment with MNXD and its

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polysaccharides in CTX-immunosuppressed mice and found that these effects occurred

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through the regulation of immune organs, cells, and cytokines. This suggests that MNXD and its polysaccharides can enhance the host’s specific and non-specific immunity, including

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cellular and humoral immunities. Moreover, peripheral RBC and platelet counts showed that

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treatment with MNXD and its polysaccharides inhibited CTX-induced myelosuppression, which is an important limiting factor in the outcome and recovery of tumor patients receiving

significantly

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chemotherapy. Compared to treatment with MNXD, treatment with MNXD-P was better

in

increasing

body

weight

and

NK

cytotoxic

activity

in

CTX-immunosuppressed mice. Furthermore, compared to treatment with MNXD-BD-P, MNXD-P (MNXD-BD-P with the addition of MNXD-AD-P) was significantly better in increasing body weight and hemolysin formation. For decades, glucans have been studied for their biological and immunological activities. The scientific community has mainly focused on immune reactions, and it has been concluded that glucans represent unique immunostimulants that are active in every species [29]. Previous studies of polysaccharide structure–activity relationships revealed that the β-glucans (including β-1,3-glucan, β-1,6-glucan, β-(1,3)(1,6)-glucan and β-(1,3)(1,4)-glucan) can regulate immune responses when administered alone and can connect innate and adaptive 14

Journal Pre-proof immunity to improve immunogenicity of vaccines, because a set of immunological cell surface receptors have been revealed to recognize β-glucans, including dectin-1, complement receptor 3 (CR3), CD5, lactosylceramide, and so on [30, 31]. Recently, some branched or linear a-1,4-/1,6-glucans isolated from medicinal plants ginseng, Rubus crataegifolius Bge., Hedysarum polybotrys and Aconitum kusnezoffii Reichb. have also been found to exhibit strong immunological activities [32-36]. Besides, pectic polysaccharides, the primary components of plant cell walls with intriguing structural diversity, were reported to possess

of

antitumor and a diverse immunomodulating activity that can mediate both phagocytosis and antibody production [8, 20, 37, 38]. The wide structural diversity of plant cell wall

ro

polysaccharides reflects the different mechanisms exerted on the immune systems. In the

-p

present study, MNXD-P mainly contains α-(1,4)-D-glucan, β-(1,4)-D-glucan and RG-I pectin,

re

in which β-(1,4)-D-glucan and RG-I pectin are from MNXD-BD-P and α-(1,4)-D-glucan is from MNXD-AD-P. So, β-(1,4)-D-glucan and RG-I pectin may take on a major responsibility

lP

for the immunostimulating activity of MNXD-P, and α-(1,4)-D-glucan is a positive

na

contributor to immunostimulating effect of MNXD-P on humoral immunity. These results may provide guidance on design and development of carbohydrate drugs from MNXD, and a

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new idea for modernization of traditional Tibetan medicines. 5. Conclusions

In this study, three polysaccharides (MNXD-P, MNXD-BD-P, and MNXD-AD-P), isolated from MNXD and its basic and auxiliary prescription decoctions, were evaluated to enhance the host’s specific and non-specific immunity, including cellular and humoral immunities through the regulation of immune organs, cells, and cytokines in CTX-immunosuppressed mice. Moreover, the treatment with these polysaccharides inhibited CTX-induced myelosuppression. Composition and structural analysis revealed that MNXD-BD-P was composed of β-(1,4)-D-glucan and RG-I pectin, MNXD-AD-P mainly contained

α-(1,4)-D-glucan

and

some

amount

of

arabinogalactan

and/or

arabinorhamnogalactan, and MNXD-P contained components of both MNXD-BD-P and 15

Journal Pre-proof MNXD-AD-P. Among them, MNXD-P may be a positive contributor to immunostimulating effect of MNXD on cellular immunity, and MNXD-AD-P plays positive role for immunostimulating effect of MNXD-P on humoral immunity. Acknowledgments This work was supported by the National Key R&D Program of China (2018YFC1708006), China Postdoctoral Science Foundation funded project (2019M652982), the Innovation Platform Program (2017-ZJ-Y08), the Natural Science Foundation of Qinghai

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Province (2018-ZJ-902), the International Partnership Program (153631KYSB20160004), and the Central Asian Drug Discovery and Development Center of CAS (CAM201806), Qinghai

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Provincial Health and Family Planning Medical and Health Science and Technology Project

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(2017-wjzdx-07) and Youth Innovation Promotion Association CAS.

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Competing Interests The authors declare no conflicts of interest.

lP

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[23] D.S.R. Barreira, and C. Munz, Natural killer cell activation by dendritic cells: balancing inhibitory and activating signals. Cell Mol. Life Sci. 68 (2011) 3505-3518. [24] G. Zeng, Y. Ju, H. Shen, N. Zhou, and L. Huang, Immunopontentiating activities of the purified polysaccharide from evening primrose in H22 tumor-bearing mice. Int. J Biol. Macromol. 52 (2013) 280-285. [25] G.A. Cabral, G.A. Ferreira, and M.J. Jamerson, Endocannabinoids and the immune system in health and disease. Handb. Exp. Pharmacol. 231 (2015) 185-211. [26] T. Sainz, S. Serrano-Villar, L. Diaz, T.M. Gonzalez, M.D. Gurbindo, M.I. de Jose, M.J. Mellado, J.T. Ramos, J. Zamora, S. Moreno, and M.A. Munoz-Fernandez, The CD4/CD8 18

Journal Pre-proof ratio as a marker T-cell activation, senescence and activation/exhaustion in treated HIV-infected children and young adults. AIDS 27 (2013) 1513-1516. [27] Suka Ningni-duojie, Millions and Millions of Sarira of Tibetan Medicine, Gansu Nationality Publishing Company, Lanzhou, 1990. [28] M. Alamgir, and S. Uddin, Recent advances on the ethnomedicinal plants as immunomodulatory agents. Ethnomedicine: A Source of Complementary Therapeutics (2010) 227-244.

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[29] P. Sima, J. Richter, and V. Vetvicka, Glucans as New Anticancer Agents. Anticancer Res. 39 (2019) 3373-3378.

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[30] Y. Jin, P. Li, and F. Wang, beta-glucans as potential immunoadjuvants: A review on the

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[31] V. Vetvicka, Glucan-immunostimulant, adjuvant, potential drug. World J Clin. Oncol. 2

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immune-enhancing activity. Int. J Biol. Macromol. 123 (2019) 713-722. [33] W. Ni, X. Zhang, H. Bi, J. Iteku, L. Ji, C. Sun, J. Fang, G. Tai, Y. Zhou, and J. Zhao, Preparation of a glucan from the roots of Rubus crataegifolius Bge. and its immunological activity. Carbohydr. Res. 344 (2009) 2512-2518. [34] W. Cao, X.Q. Li, L. Liu, M. Wang, H.T. Fan, C. Li, Z. Lv, X. Wang, and Q. Mei, Structural analysis of water-soluble glucans from the root of Angelica sinensis (Oliv.) Diels. Carbohydr. Res. 341 (2006) 1870-1877. [35] S. Li, D. Wang, W. Tian, X. Wang, J. Zhao, Z. Liu, and R. Chen, Characterization and anti-tumor activity of a polysaccharide from Hedysarum polybotrys Hand.-Mazz. Carbohydr. Polym. 73 (2008) 344-350. [36] T. Gao, S. Ma, J. Song, H. Bi, and Y. Tao, Antioxidant and immunological activities of 19

Journal Pre-proof water-soluble polysaccharides from Aconitum kusnezoffii Reichb. Int. J Biol. Macromol. 49 (2011) 580-586. [37] M.B. Ye, and B.O. Lim, Dietary pectin regulates the levels of inflammatory cytokines and immunoglobulins in interleukin-10 knockout mice. J Agric. Food Chem. 58 (2010) 11281-11286. [38] S. Li, G. Yang, J. Yan, D. Wu, Y. Hou, Q. Diao, and Y. Zhou, Polysaccharide structure and immunological relationships of RG-I pectin from the bee pollen of Nelumbo nucifera.

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Journal Pre-proof

Captions to illustrations Figure 1. FT-IR spectra and scanning electron micrographs of the MNXD polysaccharides. Figure 2. 13C NMR spectra of the MNXD polysaccharides. Figure 3. Effects of the MNXD polysaccharides on (A) phagocytosis, (B) secretion of IL-1β, TNF-α, and NO from macrophages, (C) lymphocyte proliferation, (D) and serum cytokine concentrations in CTX-treated mice. a P < 0.05 vs. normal group. b P <

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na

lP

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-p

ro

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0.05 vs. model group. c P < 0.05 vs. MNXD group. d P < 0.05 vs. MNXD-P group.

21

Journal Pre-proof

Table 1. The yields; total sugar, uronic acid, and protein contents; molecular weight distributions; and monosaccharide compositions of the MNXD polysaccharides. Fraction

Yield

Total sugar

Uronic acid

Protein content

MW distribution

Monosaccharide composition (%)

(%)

content (%)

content (%)

(%)

(kDa)

Rha

MNXD-P

16.72

95.16

6.52

0.35

≥100a, 2.45

MNXD-BD-P

16.11

95.12

6.89

0.56

≥100, 2.45

MNXD-AD-P

22.43

96.92

0.98

0.12

≥100, 61.50, 10.79

a

l a

e

r P

Elution peak at the position of void volume of the TSK-GEL G3000 PWXL column.

n r u

o J

22

Glc

Gal

Ara

3.42

f o

11.48

54.99

14.43

15.68

3.80

12.56

52.95

14.68

16.01

1.02

0.29

91.83

3.97

2.89

o r p

GalUA

Journal Pre-proof

Table 2. 13C NMR chemical shifts of the MNXD polysaccharides in D2O. Chemical shift (δ, ppm) Glycosidic linkage C-2

99.7

71.5 73.3 74.5 71.7

60.3

→4,6)-α-D-Galp-(1→

91.8

72.6 72.8 76.4 73.9

71.0

→3)-β-L-Arap-(1→

95.7

69.2 76.1 69.8 60.5

/

→4)- β-D-Glcp-(1→

103.0 74.3 76.9 80.8 77.1

62.0

→4)-α-D-GalpA-(1→

103.2 70.3 71.1 82.7 76.6

181.4

→4)- β-D-Glcp-(1→

103.1 74.4 76.8 80.9 77.2

62.1

→4)-α-D-GalpA-(1→

103.3 70.3 71.2 82.8 76.5

181.4

→4)-α-D-Glcp-(1→

99.9

→4,6)-α-D-Galp-(1→

91.9

C-4

C-5

ro

-p

95.8

lP

→3)-β-L-Arap-(1→

na

MNXD-P

Jo ur

MNXD-BD-P

23

C-6/-COOR

71.4 73.4 74.6 71.8

60.3

72.7 72.7 76.5 73.9

71.1

69.3 76.2 69.8 60.6

/

re

MNXD-AD-P →4)-α-D-Glcp-(1→

C-3

of

C-1

Journal Pre-proof Table 3. Effects of MNXD polysaccharides on body weight; immune organ indexes; and WBC, RBC, and platelet counts in CTX-treated mice. Spleen

Thymus

index

index

Body weight Group (g) (mg/g)

(mg/g)

WBC

RBC

Platelet

(1012/L)

(109/L)

(1011/L)

25.09±1.27

3.29±0.26

1.73±0.26

7.72±0.85

4.53±0.27

5.89±0.67

Model

17.36±1.48a

2.42±0.41a

0.85±0.29a

4.87±0.71a

1.55±0.23a

3.62±0.47a

Levamisole

22.67±1.58b

3.53±0.35b

1.49±0.27b

7.35±0.83b

3.91±0.45b

5.72±0.59b

MNXD

23.55±1.39b

3.73±0.64b

1.48±0.35b

7.61±0.94b

4.19±0.41b

5.68±0.73b

MNXD-P

24.96±1.21bc

3.21±0.51b

1.56±0.34b

7.92±0.91b

4.25±0.34b

5.77±0.82b

MNXD-BD-P

23.22±1.56bd

3.69±0.42b

1.81±0.41b

4.05±0.31b

5.48±0.71b

MNXD-AD-P

22.43±1.71bd

3.64±0.41b

1.65±0.19b

4.01±0.44b

5.59±0.68b

ro

of

Normal

-p

7.89±0.82b 7.76±0.87b

re

The values are presented as mean ± SD, n = 10. a P < 0.05 vs. normal group. b P < 0.05 vs. model

Jo ur

na

lP

group. c P < 0.05 vs. MNXD group. d P < 0.05 vs. MNXD-P group.

24

Journal Pre-proof Table 4. Effects of MNXD polysaccharides on NK cytotoxic activity, CD4+/CD8+ T-cell ratio, and serum hemolysin levels in CTX-treated mice NK cytotoxic activity

CD4+/CD8+ ratio

Group (%)

Serum hemolysin (A413nm)

37.69±4.56

3.52±0.35

0.81±0.02

Model

21.92±2.73a

2.11±0.27a

0.32±0.02a

Levamisole

31.79±3.25b

3.19±0.33b

0.68±0.02b

MNXD

23.72±3.67

2.73±0.31b

0.69±0.01b

MNXD-P

29.29±4.0bc

2.74±0.31b

0.67±0.03b

MNXD-BD-P

26.96±3.32b

3.06±0.29bcd

0.62±0.02bcd

MNXD-AD-P

31.20±3.4bc

2.89±0.34b

-p

ro

of

Normal

0.69±0.02b

Values are presented as mean ± SD, n=10. a P < 0.05 vs. normal group. b P < 0.05 vs. model group.

na

lP

re

P < 0.05 vs. MNXD group. d P < 0.05 vs. MNXD-P group.

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c

25

Journal Pre-proof Highlights 1. This is the first report on chemical and immunoactivity study on polysaccharides from traditional Tibetan medicine compounds. 2. Three polysaccharide components—MNXD-P, MNXD-BD-P, and MNXD-AD-P—were isolated from Ma-Nuo-Xi Decoction (MNXD) and its basic and auxiliary prescription decoctions. 3. Treatment with these polysaccharides could significantly improve the host’s specific and non-specific immunity, including cellular and humoral immunities, as well as promote

of

recovery from myelosuppression in cyclophosphamide (CTX)-immunosuppressed mice.

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4. MNXD polysaccharides were composed of α-(1,4)-D-glucan, β-(1,4)-D-glucan, RG-I pectin

-p

and some amount of arabinogalactan and/or arabinorhamnogalactan. 5. MNXD-P may be a positive contributor to immunostimulating effect of MNXD on cellular

re

immunity, and MNXD-AD-P plays positive role for immunostimulating effect of MNXD-P on

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na

lP

humoral immunity.

26

Figure 1

Figure 2

Figure 3