Immunoregulation Effect of Red Ginseng Aqueous Extract on Mice

Fangyi ZHANG, Suicheng GUO

1. Department of Traditional Chinese Medicine, Nanyang Medical College, Nanyang 473061, China; 2. Department of Digestive Medicine, First Affiliated Hospital of Nanyang Medical College, Nanyang 473058, China

Abstract [Objectives] The paper was to observe the effects of red ginseng aqueous extract on immune function of mice. [Methods] The aqueous extract of red ginseng was obtained by water extraction and concentration, and 10 mL of aqueous extract was equivalent to 2.86 g of raw materials. Total 240 mice were randomly divided into blank control group, low dose, medium dose and high dose groups of red ginseng aqueous extract, 60 mice each group. Mice in low dose, medium dose and high dose groups were intragastrically administered with 0.24, 0.48, 1.43 g/kg red ginseng aqueous extract once a day respectively, and those in blank control group were intragastrically administered with equal volume of deionized water at the dose of 0.1 mL/10 g once a day for consecutive 30 d. The immunoregulation effects of red ginseng aqueous extract on mice were explored by organ/body weight ratio measurement, delayed type hypersensitivity (DTH) reaction, Con A-induced spleen T lymphocyte transformation test, antibody-producing cells test, half hemolytic value (HC50) test, carbon particle clearance test, phagocytosis test of chicken red blood cells by macrophages and NK cell activity test. [Results] Compared with the blank control group, the degree of toe swelling in low dose group and medium dose group of red ginseng aqueous extract [(0.62±0.14) mm, (0.53±0.12) mm vs. (0.36±0.10) mm] significantly increased (P<0.05). The ability of T lymphocytes proliferation (0.173±0.054, 0.189±0.063 vs. 0.098±0.012) in low dose group and high dose group significantly increased (P<0.05). The number of haemolytic plaque (137.49×103±24.73×103, 148.43×103±27.53×103vs. 112.96×103±26.28×103) in medium dose group and high dose group significantly increased (P<0.05). The phagocytosis rate (35.67%±3.82%, 49.26%±6.54%, 57.92%±7.36% vs. 24.34%±4.22%) and phagocytosis index (0.72±0.23, 0.82±0.15, 0.91±0.26 vs. 0.35±0.11) of low dose, medium dose, high dose groups significantly increased (P<0.05). However, there was no statistical difference in spleen/body weight ratio, thymus/body weight ratio, half hemolytic value, carbon particle clearance and NK cell activity. [Conclusions] Red ginseng aqueous extract could enhance the immunity of mice.

Key words Ginseng, Red ginseng, Immunoregulation, Mice

1 Introduction

Red ginseng is the dried roots and rhizomes of cultivated products ofPanaxginsengC.A.Mey., with the functions of reinforcing vital energy, returning to blood pressure, and tonifying qi and controlling blood, which is mainly used for bodily weakness, faint pulse and cold extremities, qi dysfunction in blood control, metrostaxis[1-2]. The effects of red ginseng aqueous extract on the immunity of mice were observed from the aspects of cellular immunity, humoral immunity, NK cell activity and mononuclear macrophage function.

2 Materials and Methods

2.1Materials

2.1.1Cell strains. Mouse T lymphocyte cell YAC-1 was purchased from Shanghai Yansheng Industrial Co., Ltd.

2.1.2Experimental animals. A total of 240 4-week-old SPF healthy male ICR mice with the body weight of (20±2) g were purchased from Beijing Charles River Laboratories Co., Ltd., and the production license number was SCXK (Beijing) 2016-0011. The mice were kept in the research center animal room of Nanyang Medical College, and the animal use certificate was SYXK (Wan) 2017-0036. The animal room was a barrier system with artificial light under 12 h light-dark cycle; the temperature was controlled at (20-22) ℃, the relative humidity was 40%-70%, and the ventilation rate was 15 times/h. The mice were raised in plastic cages with free access to food and water.

2.1.3Instruments. The instruments used in the test included carbon dioxide incubator (Sanyo, Japan), clean bench (Ruijie, Tianjin), ME203E electronic analytical balance (Mettler Toledo, USA), microscope (Olympus, Japan), desktop centrifuge (Sanyo, Beijing), constant temperature water box (Yuhua, Gongyi), spectrophotometer (Feile, Nanjing), and microplate reader (Molecular Devices, USA).

2.1.4Drugs and reagents. The 6-year-old red ginseng was purchased from Fusong County, Jilin Province, which was identified asPanaxginsengC.A.Mey. by professor Zhao Tihao at Nanyang Medical College, conforming to the requirements ofChinesePharmacopoeia2015editionfor red ginseng. The reagents used included RPMI 1640 culture solution (Gibco, USA, No.11885084); calf serum (Gibco, USA, No.16010159); penicillin-streptomycin(Gibco, USA, No.15140122); chicken red blood cell (Zeweil, Nanjing, No.01067); SRBC (Bersee, Beijing, No.180605); ConA (Solarbio, Beijing, No.C6218); complement (guinea pig serum, Solarbio, Beijing, No.K091765); Hank’s solution (pH 7.2, Gibco, USA, No.14175095); Venzier’s reagent (Rongbai, Shanghai, No.Z11092); agarose (Solarbio, Beijing, No.Q16752); lithium lactate (Sinopharm, No.20170813); INT (Sigma, USA, No.YK2017070902); NADH (Hualan, Shanghai, No.71085); and PMS (Canspec, Shanghai, No.SZ185746).

2.2Methods

2.2.1Preparation of red ginseng aqueous extract. Approximately 100 g of red ginseng were crushed by a pulverizer, added with 1 000 mL of double distilled water, and heated to boil; after reflux extraction for 2 h, the solution was filtered, and the filtrate was rotarily evaporated to dark brown viscous liquid, namely red ginseng aqueous extract. Each 10 mL of water extract was equivalent to 2.86 g of red ginseng raw materials.

2.2.2Grouping and administration. Total 240 mice were randomly divided into four groups: blank control group, low dose, medium dose and high dose groups of red ginseng aqueous extract, 60 mice each group. Mice in low dose, medium dose and high dose groups were administered intragastrically with red ginseng aqueous extract at the doses of 0.24, 0.48 and 1.43 g/kg, respectively, and those in blank control group were administered intragastrically with equal volume of deionized water at the dose of 0.1 mL/10 g once a day for consecutive 30 d. Each group was randomly divided into four subgroups. Mice in subgroup 1 were tested for organ/body weight ratio, half hemolytic value (HC50), delayed type hypersensitivity and antibody-producing cells; mice in subgroup 2 were carried out carbon particle clearance test; mice in subgroup 3 were conducted phagocytosis test of chicken red blood cells by macrophages; and mice in subgroup 4 were conducted NK cell activity test and ConA-induced T lymphocyte transformation test.

2.2.3Delayed type hypersensitivity test. After the last administration, 15 mice were randomly selected from each group and were intraperitoneally injected with 2% SRBC at the dose of 0.2 mL/mouse. The thickness of left hind toe was measured at 4 d post hypersensitivity, and the same site was measured three times to get the average value. The measuring site was injected subcutaneously with 20% SRBC at the dose of 20 μL/mouse. The thickness of left hind toe was measured at 24 h post injection, and the same site was measured three times to get the average value. The difference between two measurements of toe thickness (degree of toe swelling) represented the degree of delayed type hypersensitivity.

2.2.4Measurement of organ/body weight ratio. At the end of delayed type hypersensitivity test, the same batch of mice in Section2.2.3were weighed and executed. The spleen and thymus were taken, sucked dry by filter paper, and weighed. The spleen/body weight ratio and thymus/body weight ratio were calculated.

2.2.5Antibody-producing cell test. At the end of delayed type hypersensitivity test, the same batch of mice in Section2.2.3were executed to get spleen. The spleen was added with Hank’s solution, and ground into 8 mL cell suspension. The medium was dissolved by heating, and mixed with equal volume of Hank’s solution, then packaged into small test tubes, 0.5 mL each tube. Afterwards, the tubes were added with 50 μL of 10% SRBC solution, and mixed evenly. Approximately 20 μL of spleen cell suspension were spread on the glass slide with agarose thin layer. After agarose was solidified, the slides were placed horizontally in a slide frame and incubated for 1.5 h in an incubator. The complement diluted with SA buffer solution (1∶8) was added into grooves of slide frame, and continuously incubated for 2.0 h, to count the number of hemolytic plaque. The number of antibody-producing cells was represented by the number of hemolytic plaque.

2.2.6Half hemolytic value test. At the end of delayed type hypersensitivity test, the same batch of mice in Section2.2.3were executed to remove eyeballs. Approximately 1.5 mL of blood were placed in a centrifuged tube for 1 h, then centrifuged at 2 000 r/min for 10 min to collect the supernatant. The serum was diluted 300 times with SA buffer; subsequently, 100 μL of diluted serum was loaded into 96-well plate, and successively added with 50 μL of 10% SRBC and 100 μL of complement (diluted with SA buffer at the ratio of 1∶8); after incubated in water bath at 37 ℃ for 30 min, the reaction was terminated by placing in ice bath. Afterwards, the solution was centrifuged at 1 500 r/min for 10 min, and 50 μL of supernatant was added with 150 μL of Venzier’s reagent; half hemolytic pores were set; 12.5 μL of 10% SRBC was added with Venzier’s reagent to 0.2 mL then loaded into another 96-well plate, which was thoroughly mixed and placed at room temperature for 10 min. The blank control group was used as blank, and the optical density (OD) of samples was measured by microplate reader at 540 nm. Hemolysin content was expressed asHC50, andHC50=ODvalue of samples/ODvalue of half hemolysis of SRBC×dilution ratio.

2.2.7Mouse spleen T lymphocyte transformation test induced by ConA. After the last administration, 15 mice were randomly selected from each group. After execution, the spleen was taken and placed in a sterile plate, and ground into single-cell suspension by adding appropriate amount of Hank’s liquid; after rinsed with Hank’s liquid for three times, the solution was centrifuged at 1 000 r/min for 10 min. The number of live cells (more than 95%) was counted by Trypan blue staining, and the concentration was adjusted to 2×107cells/mL. The cell suspension was inoculated into 24-well plate, 1 mL suspension each well; one well was added with 75 μL of ConA solution (equivalent to 7.5 μg/mL), and another well was used as control; the plate was then incubated in an incubator for 72 h. Approximately 0.7 mL of supernatant was moved from each well, and 0.7 mL of serum-free RPMI1640 medium was added; subsequently, 50 μL of MTT (5 mg/mL) solution was added into each well, and the plate was incubated for 4 h. Accurately 1 mL of acid isopropanol was added to completely dissolve purple crystals, and blew for blending. TheODvalue was measured by microplate reader at 570 nm. T lymphocyte proliferation ability=ODwith ConA-ODwithout ConA

2.2.8NK cell activity test. Yac-1 cells were digested with trypsin, and the concentration was adjusted to 4×105cells/mL with RPMI 1640 complete medium. The spleen was taken aseptically from the same batch of mice in Section2.2.7, to prepare spleen cell suspension. The cell suspension was rinsed with Hank’s solution for three times, and then centrifuged at 1 500 r/min for 10 min. The solution was resuspended with RPMI 1640 complete culture medium. The number of live cells (95% or more) was counted by trypan blue staining, and the concentration was adjusted to 2×107cells /mL, ensuring that the ratio of effector cells to target cells was 50∶1. The target cells and effector cells were added into U-shaped 96-well plate, 100 μL each cell. The target cell natural release wells were added with 100 μL of target cells and culture medium, respectively; the maximum target cell release well was added with 100 μL of target cells and 1% NP40, and three parallel wells were set. After incubated at 37 ℃ for 4 h, the 96-well plate was centrifuged at 1 500 r/min for 5 min; 100 μL of supernatant was absorbed from each well, and placed in ELISA plate; the ELISA plate was added with 100 μL of LDH matrix liquid (nitrotetrazolium chloride 6.6×10-4mol/L, 5-methylphenazinium methosulfate 2.8×10-4mol/L, sodium lactate 5×10-2mol/L and NAD I 1.3×10-3mol/L dissolved in 0.2 mol/L Tris-HCl buffer, pH 8.2), and reacted for 10 min; afterwards, each well was added with 30 μL of 1 mol/L HCl to terminate the reaction. TheODvalue was measured by microplate reader at 492 nm. NK cell activity (%)=(ODof reaction well-ODof natural release well)/(ODof maximum release well-ODof natural release well)×100%.

2.2.10Phagocytosis test of chicken red blood cells by macrophages. After the last administration, 15 mice were randomly selected from each group and intraperitoneally injected with 1 mL of 5% hematocrit chicken red blood cell suspension; after 1.5 h, the mice were executed by dislocation and intraperitoneally injected with 2 mL of normal saline. The mice were rotated, and skin of middle abdominal wall was cut open; 1 mL of peritoneal lotion was absorbed and dropped onto two slides; after incubated at 37 ℃ for 20 min, the slides were rinsed with normal saline to remove uncoated cells, then dried. The slides were fixed with methanol-acetone (1∶1), stained with 4% giemsa-phosphate buffer for 3 min, then rinsed with distilled water, and dried. Total 100 phagocytes were counted by oil immersion lens, and phagocytosis rate and phagocytosis index were calculated. Phagocytosis rate (%)=number of macrophages swallowing chicken red blood cells/number of counted macrophages×100%; phagocytosis index=total number of swallowed chicken red blood cells /number of counted macrophages.

3 Results and Analysis

3.1Comparisonoforgan/bodyweightratioofmiceDuring the administration, mice in each group were good in behavioral signs, with bright hair, normal food and water intake. Compared with the blank control group, there was no significant difference in thymus/body weight ratio and spleen/body weight ratio among red ginseng aqueous extract groups (P>0.05), indicating that red ginseng aqueous extract had no significant effect on organ/body weight ratio of mice (Table 1).

Table 1 Effects of red ginseng aqueous extract on organ/body weight ratio of mice (mg/g, n=15)

3.2Effectsofredginsengaqueousextractoncellularimmunityofmice

3.2.1Delayed type hypersensitivity. Compared with the blank control group, the degree of toe swelling in low dose and medium dose groups of red ginseng aqueous extract significantly increased (P<0.05), indicating that red ginseng aqueous extract enhanced delayed type hypersensitivity reaction of mice (Table 2).

3.2.2ConA-induced T lymphocyte transformation. Compared with the blank control group, the T lymphocyte proliferation in low dose and high dose groups of red ginseng aqueous extract significantly increased (P<0.05), indicating that red ginseng aqueous extract promoted the ability of ConA-induced T lymphocyte transformation (Table 2).

Table 2 Effects of red ginseng aqueous extract on toe swelling and T lymphocyte proliferation of mice n=15)

3.3Effectsofredginsengaqueousextractonhumoralimmunityofmice

3.3.1Antibody-producing cells. The number of hemolytic plaque in medium dose and high dose groups of red ginseng aqueous extract was higher than that in blank control group (P<0.05 orP<0.01), indicating red ginseng aqueous extract could increase the number of antibody-producing cells in mice (Table 3).

3.3.2Half hemolytic value. There was no significant difference inHC50between various red ginseng aqueous extract groups and blank control group (P>0.05), indicating that red ginseng aqueous extract had no significant impact onHC50of mice (Table 3).

Table 3 Effects of red ginseng aqueous extract on number of hemolytic plaque and HC50 of mice n=15)

3.4Effectsofredginsengaqueousextractonphagocytosisofmononuclearmacrophages

3.4.1Carbon particle clearance. There was no significant difference in carbon particle clearance between various red ginseng aqueous extract groups and blank control group (P>0.05), indicating that red ginseng aqueous extract had no significant impact on phagocytosis of mononuclear macrophages (Table 4).

Table 4 Comparison of carbon particle clearance of mice n=15)

3.4.2Ability of macrophages swallowing chicken red blood cells. The phagocytosis rate and phagocytosis index of different red ginseng aqueous extract groups were significantly higher than those in blank control group (P<0.01), indicating that red ginseng aqueous extract enhanced the ability of macrophages swallowing chicken red blood cells (Table 5).

Table 5 Comparison of macrophages swallowing chicken red blood cells n=15)

3.5EffectsofredginsengaqueousextractonNKcellactivityThere was no significant difference in NK cell activity between various red ginseng aqueous extract groups and blank control group (P>0.05), indicating that red ginseng aqueous extract had no significant impact on NK cell activity of mice (Table 6).

Table 6 Comparison of NK cell activity (%, n=15)

4 Discussion

The effects of red ginseng aqueous extract on immune function of mice were observed in the test. The results showed that after continuous intragastric administration of red ginseng aqueous extract for 30 d, low dose and high dose of red ginseng aqueous extract promoted ConA-induced T lymphocyte proliferation, while low dose and medium dose of red ginseng aqueous extract enhanced the degree of toe swelling, indicating that they enhanced the cellular immunity. The antibody production was increased in both medium and high dose groups, indicating that they enhanced the humoral immune function. Different dose groups increased the number of chicken red blood cells swallowed by peritoneal macrophages, increased phagocytosis rate and phagocytosis index, indicating that red ginseng aqueous extract enhanced the function of mononuclear macrophages. However, red ginseng aqueous extract had no significant effect on spleen/body weight, thymus/body weight,HC50, carbon particle clearance and NK cell activity of mice. According to the criteria for testing immunity enhancement inTechnicalSpecificationforHealthFoodInspectionandEvaluation(2003 edition)[3], it could be determined that red ginseng aqueous extract enhanced the immunity of mice.

Ginseng has the pharmacological effects of regulating nervous system, improving hematopoietic function of bone marrow, promoting metabolism, delaying aging, expanding blood vessels, improving the body’s resistance,etc., and has good effects on cardiovascular diseases, diabetes mellitus and neurasthenia[4-8]. It has clinical therapeutic effects on hypertension, angina pectoris, coronary atherosclerosis, chronic hepatitis, tumor and many other diseases[9]. Studies showed that red ginseng aqueous extract inhibited ConA-mediated T lymphocyte proliferation and phagocytosis of neutral red by macrophages, indicating that it had strong immunosuppressive effect on normal mice[10]. Red ginseng aqueous extract regulated Th2 response by inhibiting nuclear transcription factors such as NF-κB p65, thereby inhibiting the airway inflammation in asthmatic mice[11]. Red ginseng aqueous extract inhibited airway inflammation by reducing the levels of alveolar lavage fluid in asthmatic mice IL-4, IL-5, IL-14, TGF-1, VEGF, immunoglobulin E and hydroxyproline in lung tissue[12]. Red ginseng aqueous extract induced neovascularization by activating PI3K/Akt, MEK/ERK and eNOS/NO pathways[13]. Red ginseng aqueous extract had significant protective effect on apoptosis of Aβ25-35induced human neuroblastoma SH-SY5Y by improving cell survival and lowering mitochondrial membrane potential[14]. Our research results demonstrated that red ginseng aqueous extract enhanced the cellular immunity, humoral immune function and mononuclear macrophage function of mice.

Overall, the effects of red ginseng aqueous extract on immune function mainly embodies in enhancement of cellular immunity, humoral immunity and mononuclear macrophage function. However, the specific mechanism of its immunoregulation remains to be further studied.