Lingonberries were harvested at the light ripe red stage of ripening from the Greater Higgnan Mountains area of Heilongjiang Province, China. The fresh fruits were frozen and then stored at −40 °C prior until further analysis.

Fresh fruits were homogenised with chilled 80% acetone (1:2; w/v) using a chilled JJ-2 Tissue disintegrator for 5 min. The sample was then further homogenised using a FA25 Superfine Homogeniser for an additional 3 min [14]. The homogenates were filtered and evaporated. The solvents were evaporated to dryness under vacuum and the residue was diluted in distilled water. After separating the precipitate by filtration; the remaining solution was further separated on an adsorptive resin column (X-5; Nankai University, Tianjin, China) [15] and eluted with water, 20%, 30%, 40%, 50%, 60%, 80% and 95% EtOH to produce eight fractions. The 30%-EtOH fraction was dried under vacuum, recrystallized from EtOH, and a red powder was obtained containing the ALB.

Male and female Kunming mice (24 ± 2 g) of clean grade were provided by the Harbin Medical University Animal Center (SCXK20020002, Harbin, China). The animals were conditioned for a week at 23 ± 1 °C with a constant humidity of 55% ± 5% under a cycle of 12 h of dark [16], and had free access to food and tap water according to GLP. All experimental protocols used in this experiment have previously been approved by the School of Medical Science, Harbin Medical University.

A total of 60 mice were used for these experiments, with 10 mice per group. Mice were randomly assigned to one of the six following treatment groups: normal control, model, positive control (Leucogen 1.4 mg/kg body weight/day) and ALB-H(igh), M(edium), L(ow) dose (200, 100, 50 mg/kg body weight/day). Different doses of ALB dissolved in double distilled water were administered intragastrically to the female and male mice for 14 consecutive days. The animals were divided into six groups: Positive group was administered Leucogen tablets (Ji Bei er Medicine Co. Ltd., Jiangsu, China, 10 mg/pellet, batch number: 110304). Model and normal groups were orally administered with distilled water. All mice, except the Normal control group, were exposed to 6.0 Gy ⁶⁰Co γ-ray whole-body radiation on day 15 after administration of DDW or ALB. The animals were monitored daily for the development of symptoms of radiation sickness and mortality.

The radiation centre of the Heilongjiang Academy of Agricultural Sciences was used for the irradiation experiments. Mice were restrained in well-ventilated boxes and exposed to whole-body γ-radiation (6 Gy), at a dose rate of 1.01 Gy/min at a source-to-animal distance (midpoint) of 160 cm.

Blood cell count (leucocytes-WBC, erythrocytes-RBC, hemoglobin-HGB and thrombocytes-PLT) was determined by use of hemocounter (Haematology Analyser 6318K, Nihon-Konden, Tokyo, Japan) on the 7th day after irradiation. For this purpose blood samples were taken from the eyeballs of animals.

Animals were given ALB (50, 100, 200 mg/kg b.wt.) prior to 6.0 Gy whole-body irradiation. On day 7 post-irradiation, animals were sacrificed and the spleen and thymus were removed. The spleen and thymus indices were calculated by dividing organ weight by BW [17]:

At day 7 after radiation the mice were injected intravenously via a lateral tail vein with India ink stain (Beijing Zhongxi Chemical Factory, Beijing, China) at a dose of 0.1 mL/10 g body weight (1:4 dilluted with PBS). Blood samples (20 μL of each) were taken from the retroorbital venous plexus at 2 min (t₁) and 10 min (t₂) intervals respectively after India ink staining injection and mixed with 2 mL 0.1% Na₂CO₃ in tube for measuring the optical density with spectrophotography at 600 nm (OD₁ for 2 min and OD₂ for 10 min) [18]. The PI was calculated according to:

Sterile spleens were obtained at day 21. The spleen was pulverized through a 200-mesh filter and washed with 5 mL phosphate-buffered saline (PBS). The spleen cell suspension was washed three times in PBS in RPMI-1640 culture medium with 10% fetal calf serum and the cell concentration was adjusted to 2 × 10⁶/mL in 96-well plates with 90 μL/well. Experiments were repeated three times. Concanavalin A (Con A, 15 μg/well) was added to the wells. Culture medium was added to negative control wells in place of Con A. The cells were cultivated in 5% CO₂ at 37 °C for 72 h and 5 mg/mL methylthiazolyl tetrazolium (MTT) salt was added after 4 h. The cell suspension was acidified with 10% sodium dodecyl sulfate after 72 h to enable the purple crystals to completely dissolve. The optical density (OD) was determined at 570 nm [5]. The proliferation index = (value for experimental well − value for the control well/value for control well) × 100%.

Femur heads were obtained from mice at day 21. They were washed with 5 mL PBS and centrifuged at 1,000 rpm for 10 min. The supernatant was discarded and the sediment was applied to a slide covered with serum. The smear was fixed in methanol solution for 10 min and then stained with Giemsa for 10–15 min. The presence of micronuclei was observed in five evenly-dispersed visual fields under a high-power microscope. Bone marrow polychromatic erythrocytes appeared blue and mature red blood cells appeared orange. Most micronuclei were round or oval-shaped, single or multiple, and their edges were smooth and regular. The micronuclei rates (‰) were calculated with the number of micronuclei in 1,000 polychromatic erythrocytes [19].

All of the experimental data at least in triplicate are expressed as the means ± SD. Statistical significance was evaluated using the two-tailed, unpaired Student’s t-test for comparisons between two means. A value of p < 0.05 was considered to indicate statistical significance.

ALB administration was not associated with any mortality throughout the whole observation period. Figure 1 shows the growth curve of body weight in mice. The average weight of mice in each group at the 14th days (before irradiation) compared with the beginning of weight was increased. However, the rate of increased body weight between the groups was no significant difference; this proves that the selected dose of administration group and positive control group in mice has no acute toxicity [20].

Seven days after irradiation, the body weight of the control group (no radiation) continued to increase, the other five groups showed weight decreases compared with the previous body weight. The radiation model group showed varying degrees of hair loss, reduction in activity, food and water intake decreased, the tail roots appeared stained and so on. Compared with the radiation model, ALB-H group and the positive control group showed reduced rates of weight loss. These results indicated that ALB-H and Leucogen can alleviate the weight loss due to the radiation. ALB-treated irradiated groups has not shown any significant dose-dependency.

The variations of the mean values for WBC, RBC, PLT and HGB parameters after exposure are shown in Table 1, which demonstrated a significant (p < 0.01) decrease for the model group exposed to 6 Gy radiation, compared with the unirradiated control group. A significant (p < 0.01) increase in the total WBC, RBC, PLT and HGB count compared to the model group was recorded for the Leucogen and ALB groups; among of them the in crease of RBC was the most highest, and was insignificantly different from the unirradiated control group (p > 0.05). Increase of WBC counts was not high, especially in the ALB-M and ALB-L groups (p > 0.05). The peripheral blood counts in the Leucogen group were similar to those of the ALB-M group (Table 1). These results suggest that ALB could reduce the effects of radiation on hemograms in mice.

Hematopoietic tissues and the immune system are highly sensitive to radiation. Radiation damage to the hematopoietic system affects the hematopoietic stem cells and reduces their cell proliferation capacity [21]. Inhibition of hematopoiesis is one of the clinical symptoms of radiation injury. Radiation injury induced by radiation is usually associated with immune system disease and a decline in white and red blood cells [22]. The results of the current study found that WBC, RBC, PLT, and total HGB in the peripheral blood of irradiated mice were increased by ALB, thus improving hematopoietic function and survival.

Total-body irradiation of mice with 6 Gy 60Co γ-rays significantly reduced thymus and spleen indices (p < 0.01). The spleen indices were significantly higher in all ALB-treated irradiated groups, in a dose-dependent manner, compared with the model group (p < 0.05, p < 0.01). The thymus indexes in the ALB 200 mg/kg and 100 mg/kg groups were significantly higher than that in the irradiated control group (p < 0.01) (Table 2). These results suggest that ALB exerted a protective effect by increasing immune organ indices.

The PI value obtained in mice pretreated differently is shown in Table 2. Significant decrease in the rate of carbon clearance in irradiated mice was observed compared with normal control (p < 0.01). Combined with ALB preteatment, there were no significant differences (p > 0.05) in the decrease in the rate of carbon clearance induced by radiation. ALB 50, 100, 200 mg/kg revealed more strongly an accentuation of the phagotrophy function. The rate of carbon clearance in groups treated with ALB was elevated compared with the model group (p < 0.05, p < 0.01), among them, the group with ALB 50 mg/kg showed the greatest PI value.

To understand the immunomodulatory activity of ALB from lingonberry, we investigated its effects on the proliferation of spleenic cells. The irradiated mice groups were obviously found to display significantly decreased proliferation of splenocytes compared with that of normal control (p < 0.05, p < 0.01). All of the ALB test doses (50–200 mg/kg) significantly increased the proliferation of spleenic cells in a dose dependent manner compared with the model group, but less than positive control group (Figure 2). Con-A (5 μg/mL) stimulated spleenocyte proliferation was significantly enhanced by Leucogen at 1.4 mg/kg and cellular proliferation was increased up to three fold in Con-A treated cells, compared to the model group.

The incidence of micronuclei (Mn) in the bone marrow was significantly increased by irradiation (p < 0.01). The bone marrow micronuclei rates in the 200 mg/kg ALB, 100 mg/kg ALB and were significantly lower than in the model group, (p < 0.05). The bone marrow micronuclei rate in the 50 mg/kg ALB group was similar to that in the positive groups with Leucogen (Figure 3). These results suggest that high doses of ALB inhibited the production of radiation-induced micronuclei.

Micronuclei formation, which assesses the degree of DSBs in DNA, is one of the best assays for the evaluation of genome instability induced by ionizing radiation [23]. This method is performed in vitro (e.g., in human cultured lymphocytes) and in vivo (e.g., in mouse bone marrow cells and peripheral blood leucocytes). Higher genome instability results in a higher frequency of Mn. Pretreatment of human culture lymphocytes with ALB statistically significant reduced the Mn frequencies that were increased by IR. These ALB did not increase the frequency of Mn in non-irradiated human lymphocyte cultures treated at IR protective doses. The bone marrow is damaged at the molecular level by radiation, which causes chromosome aberrations and increased development of micronuclei [24,25]. Micronucleus detection can therefore be used as a diagnostic biological indicator of radiation damage, accurately reflecting the degree of chromosome damage and repair capacity. The bone marrow micronuclei rates detected in the current study showed that ALB significantly reduced DNA damage.