The leaves of H. rhamnoides were dried in a well-ventilated shade and pulverized. The sample powder was extracted using 70% aqueous ethanol. The extract was prepared by repeating the manufacturing process thrice under the same conditions.

The 70% aqueous ethanol extract was evaporated (EYELA N-1000, Tokyo Rikakikai Co. Ltd., Tokyo, Japan)under reduced pressure at 40℃ and completely dried using a freeze dryer. The fruits of H. rhamnoides were juiced and freeze dried (Lyoph-Pride 20, IlShinBioBase, Dongducheon, Korea).

The crude extracts were stored at 70℃ in deep freezer. The mixture used in the experiment was prepared by combining leaf and fruit extracts of H. rhamnoides in a ratio of 6 : 4.

Specific pathogen-free male mice (5 weeks old) were purchased from Orient Bio, Inc., Ltd. (Seongnam, Korea). After one week of isolation and acclimation, healthy animals without any significant weight loss were chosen for the experiment.

The mice were allowed free access to pelleted diets (Cargill Inc., Minneapolis, MN, USA), and water and light were maintained on a 12 h cycle. The mice were housed in a controlled condition with a temperature of 23 ± 3℃, relative humidity of 50 ± 10%, ventilation frequency of 10–15 times/h, lighting time of 12 h, and an illumination of 150– 300 lux.

After a week-long adaptation period, healthy animals were selected and divided into seven groups according to the randomization method (n = 10 per group) - G1; normal, G2; normal + 100 ㎎ of HLF/㎏ of body weight, G3; normal + 200 ㎎/㎏ HLF, G4; normal + 200 ㎎/㎏ positive control substance, red ginseng concentrate (6 years old, solid content 64%, ginsenoside Rg1+Rb1+Rg3 5.5 ㎎ /g, Hong Sam Jeong PLUS, Korean Red Ginseng Corp., Inc., Daejeon, Korea), G5; immunosuppressed + negative control, G6; immunosuppressed + 100 ㎎/㎏ HLF, and G7; immunosuppressed + 200 ㎎/㎏ HLF.

Throughout the experimental period, the mice were fed an AIN-93G diet purchased from Research Diets Inc. (New Brunswick, NJ, USA) and allowed free access to food and water.

To induce immunosuppression, 100 ㎎ /㎏ of cyclopho-sphamide (CPA, Sigma-Aldrich, St. Louis, MO, USA) was intraperitoneally injected one day before administration of the test substance in the immunosuppressed test groups (G5, G6, and G7).

One day after CPA administration, the test and positive control substances were orally administered at a specific time daily for four weeks.

To confirm the effect of immunosuppression, the health status of the experimental and dead animals was checked daily. The weight (FOB5K.3NS, Kern, Balingen, Germany) of mice was measured weekly during the experimental period.

The experimental animals were fasted for 16 h before sacrifice, and blood was collected from the orbitals of the mice after anesthesia with tribromoethanol diluted with tert- Amyl alcohol.

Blood was collected in a separate BD Vacutainer serum tube (Becton Dickinson, Holdrege, NE, USA), incubated at room temperature for 30 min, centrifuged at 3,000 rpm for 20 min, and stored in a deep freezer until analysis.

Blood was collected from the retro-orbital venous plexus using microcapillary tubes treated with K2-EDTA (HSU- 2900000, Marienfeld Superior, Lauda-Königshofen, Germany).

Blood collected in capillaries was mixed for 20 min using a Roller Mixer (Seo Kwang Co., Ltd., Seoul, Korea) and then analyzed for hematological parameters, including white blood cells (WBC), lymphocytes, neutrophils, eosinophils, basophils, and monocytes, using an automated hematology analyzer HEMAVET (Drew Scientific, Miami Lakes, FL, USA).

Serum levels of interleukin (IL)-4, IL-6, IL-12, and tumor necrosis factor (TNF)-α were measured using ELISA kits (R&D Systems, Minneapolis, MN, USA) according to the manufacturer’s instructions.

After addition of RPMI 1640 culture medium (Hyclone Laboratories, Logan, UT, USA), the removed spleen was pulverized through a 40 ㎜ stainless steel mesh (BD Falcon, Franklin Lakes, NJ, USA) to prepare a single-cell solution. RPMI 1640 medium was added to the pulverized single-cell solution, centrifuged at 4℃ and 1,200 rpm for 5 min, and red blood cells were removed with a red blood cell lysis buffer (Sigma-Aldrich, St. Louis, MO, USA) to obtain spleen cells.

The spleen cells were suspended in complete RPMI 1640 medium (10% FBS, 100 units/㎖ penicillin, and 100 ㎎/ ㎖ streptomycin), and the cell number was determined using a hemocytometer. The proliferation of spleen cells was determined using the CellTiter 96 AQueous ONE Solution Assay Kit (Promega, Madison, WI, USA).

Isolated spleen cells were seeded into a 96 well plate (1 × 10⁵ cells/well) and cultured in a humidified CO₂ incubator (311-TIF, Thermo Fisher Scientific Inc., Waltham, MA, USA) at 37℃ (5% CO₂) for 48 h with complete RPMI 1640 medium, complete RPMI 1640 medium containing 5 ㎍/㎖ concanavalin A (Con A, Sigma-Aldrich, St. Louis, MO, USA), or 100 ㎍ /㎖ lipopolysaccharide (LPS, Sigma-Aldrich, St. Louis, MO, USA).

After 48 h of culture, 20 ㎕ of ONE solution was added to each well and the cells were further cultured for 2 h. The absorbance was then measured at 490 ㎚ using a SpectraMax M2 microplate reader (Molecular Devices, San Jose, CA, USA).

Spleen cells were dispensed into a 48 well plate at 2.5 × 10⁵ cells/well to measure the cytokines produced and secreted by spleen cells. Cells were stimulated with 0.5 ㎍/㎖ Con A or 100 ㎍/㎖ LPS.

After 48 h of culture, the cell culture medium was collected, centrifuged, and the supernatant was taken. IL-2, IL-5, IL-6, and interferon (IFN)-γ levels were measured using the corresponding ELISA assay kit (R&D Systems, Minneapolis, MN, USA) as per the manufacturer’s instructions.

Data are expressed as the means ± SEM. The results were analyzed by Student’s t-test and one-way analysis of variance (ANOVA) using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA). Statistical significance was set at 5% (p < 0.05).

Because it is very difficult to test the exact effects of immunomodulators in healthy humans, a mouse model of immunosuppression has been established to evaluate the effects of immunomodulators in vivo (Huang et al., 2007; Zhang et al., 2021).

CPA is a cytotoxic chemotherapeutic drug that plays an important role in the treatment of tumors, and it has been reported to suppress the immune function of mice when administered at a certain dose for a short period (Wang et al., 2011; Zhu et al., 2018). Here, to investigate the effect of HLF on the survival of immunosuppressed animals, the number of animals was measured daily, and the survival rate is shown in Fig. 1.

Fig. 1.  Survival rate of each group. G1; normal + control group, G2; normal + 100 ㎎/㎏ H. rhamnoides leaf and fruit mixture (HLF), G3; normal + 200 ㎎/㎏ HLF, G4; normal + 200 ㎎/㎏ positive control substance (red ginseng concentrate), G5; immunosuppressed + control group, G6; immunosuppressed + 100 ㎎/㎏ HLF, G7; immunosuppressed + 200 ㎎/㎏ HLF.

The survival rates of the test groups G1, G2, G3, and G4 were 100%, whereas the survival rate of the G5 group, which was immunosuppressed using CPA, was 60%. The survival rates of G6 and G7, which were administered 100 and 200 ㎎ /㎏ HLF, respectively, after inducing immunosuppression were 100% and 80%, respectively.

The results of this experiment in immunosuppressed mice showed that HLF intake had protective effects against immune suppression.

The weight of the experimental animals was measured during the experimental period, and the results are presented in Table 1.

The weight in the normal control group (G1) increased sequentially over time, indicating a normal change in weight. The immunosuppressed control group (G5) showed significant weight loss compared with G1 in the first week.

After the first week, some animals died because of severe weight loss caused by immunosuppression. In contrast, there was no significant difference in weight in the second and third weeks compared to that in G1. However, at week 4, the weight in G5 was significantly lower than that in G1. In the normal state, HLF administration (G2 and G3) resulted in significant weight loss compared to G1 (Table 1). The daily weight gain in G1 was 0.13 ± 0.01 g, while that in G2 and G3 was significantly lower (0.06 ± 0.02 and 0.06 ± 0.01 g, respectively).

This result can be explained by the study of Jeong et al. (2015), which suggests that H. rhamnoides leaf extract inhibits the accumulation of fat in the differentiation process of 3T3-L1 cells and can prevent adipogenesis through the activation of AMP-activated protein kinase alpha (AMPKα) and inhibition of adipogenesis transcription factors.

AMPK plays an important role in promoting the oxidation of fatty acids and reducing the accumulation of adipose tissue. Adipocytokines produced and secreted by adipocytes are involved in various signaling processes. Their levels increase in the presence of obesity, suggesting a characteristic condition of chronic inflammation. Notable adipocytokines include adiponectin, leptin, TNF-a, IL-6 and IL-12. Adiponectin and leptin stimulate the fatty acid oxidation of adipose tissue by activating AMPK.

They also serve as precursors to the inflammatory process, along with other cytokines such as IL-6, IL-12, and TNF-a, and play an important role in various immune-related diseases (Gil et al., 2008; Kubota et al., 2007; Ouchi et al., 2012)

Blood samples were collected from the experimental animals and the distribution of leukocytes, which play a crucial role in the immune system, was analyzed (Table 2).

The total leukocyte count in the blood decreased following HLF treatment and immunosuppression induction, but there were no statistically significant differences. The leukocyte composition in the blood of the normal control group (G1) was as follows: 70.06 ± 1.89% lymphocytes and 24.93 ± 1.35% neutrophils, with eosinophils, basophils, and monocytes present in small percentages in a normal distribution. In the immunosuppressed group (G5), the proportion of lymphocytes significantly decreased and the proportion of neutrophils significantly increased. HLF administration decreased the proportion of lymphocytes and increased the proportion of neutrophils in both the normal (G2, and G3) and immunosuppressed (G6, and G7) groups. Neutrophils are rapidly activated phagocytes that are responsible for immediate inflammatory responses and effective bacterial clearance.

Jeong et al. (2020) showed that blueberry yeast-fermented powder increases the levels of neutrophils, which are immune cells in the blood, compared with a control group treated with CPA alone.

This result suggests that blueberry yeast-fermented powder may positively strengthen the immune system by increasing the levels of immune cells in the blood. Similarly, HLF was expected to increase the proportion of neutrophils and enhance their function, which is supported by our findings.

Cytokines are produced by various immune cells and play key roles in immune regulation by regulating cell activation, growth, and differentiation. They can be classified according to various criteria, and those produced and secreted by helper T (Th) cells, which are the major immune cells, can be classified into Th1 and Th2 cytokines.

Representative Th1 cytokines include IL-2, IL-12, IFN-γ, and TNF-α, which mainly act on cell-mediated immune responses. IL-4, IL-6, Il-8, and transforming growth factor (TGF)-β are representative Th2 cytokines that mainly act on humoral immune responses mediated by B cells. In this study, the serum levels of the Th1 cytokines IL-12 and TNF-α and the Th2 cytokines IL-4 and IL-6 were measured to investigate the effects of HLF on the production and secretion of cytokines. Serum IL-12, IL-4, and IL-6 levels were not significantly altered by immunosuppression or HLF administration.

On the other hand, serum TNF-α levels significantly decreased following HLF administration under normal conditions. In the case of immunosuppression, TNF-α levels were significantly increased by the administration of 200 mg/kg HLF (Table 3).

Kim et al. (2022) investigated the immunomodulatory effects of an enzymatic hydrolysate from porcine placenta, and showed that TNF-α levels significantly decrease in response to CPA treatment when compared to the levels in the normal group. In contrast, in the experimental groups treated with porcine placental enzymatic hydrolysate, all experimental groups showed significantly higher levels than the control group, showing similar results to those of HLF in this study.

TNF-α is a representative proinflammatory cytokine that acts on macrophages to promote inflammation and as a co-stimulator for activating T cells and B cells. Based on these results, it can be concluded that TNF-α levels increase when HLF is administered in a state of immunosuppression, thereby regulating the immune system.

Splenocytes were isolated from the spleens of experimental animals in each test group, and their proliferation rate was investigated. Normal proliferation rates were observed, with no significant differences between the test groups (data not shown).

To investigate the effect of HLF on B cell proliferation in the spleen, B cell activity was induced by LPS treatment and the degree of proliferation in each test group was investigated. The number of live cells in the control group without LPS treatment was 0.978 ± 0.008.

In the case of LPS treatment, the number of live cells (1.198 ± 0.007) significantly increased compared to that in the control group, indicating that B cells were strongly activated by LPS. Under normal conditions, B cell proliferation was decreased by 100 ㎎/㎏ HLF, but B cell proliferation was effectively increased by 200 ㎎/㎏ HLF. Cell proliferation in the G5 group, which had suppressed immunity, was significantly inhibited compared to that in the normal control group (G1), with a value of 0.987 ± 0.012. When HLF was administered (G6 and G7), however, cell proliferation was significantly increased to 1.147 ± 0.029 and 1.247 ± 0.032, respectively (Table 4). These results suggest that HLF enhances the humoral immune function of B cells.

T cells are involved in cell-mediated immunity and are activated by Con A. To investigate the effect of HLF on the proliferation of T cells in the spleen, cells were activated by Con A and then cell proliferation was investigated. The number of live cells in the control group without Con A treatment was 0.799 ± 0.005, and the number of live cells in the Con A-treated group (G1) was significantly increased to 1.041 ± 0.004, indicating that T cells were activated by Con A. Cell proliferation in G5, which had suppressed immunity, was significantly decreased (0.0828 ± 0.004) compared to that in the normal control group (G1). The HLF-treated groups showed significantly increased T cell proliferation in both the normal and immunosuppressed states (Table 5).

This indicates that HLF may enhance the cell-mediated immune function of T cells. Lee et al. (2019) reported that Platycodon grandiflorum extract enhances immunity by improving the proliferative ability of T cells. Our results suggest that HLF enhances immune function through the proliferation of splenocytes and may prevent infection, and that it is a potential candidate for use in immune enhancement.

Furthermore, HLF may be a promising immunogenic substance that enhances humoral immune responses to various diseases.

To investigate the effect of HLF administration on cytokine production and secretion by LPS-treated and Con A-treated splenocytes, the culture supernatant of splenocytes was collected and the contents of IL-2, IFN-γ, IL-5, and IL-6 were measured. When splenocytes were stimulated with LPS, there was a significant increase in IL-2 production and secretion, while IFN-γ production and secretion significantly decreased. IL-5 and IL-6 production and secretion were not induced by LPS treatment (Table 6).

IL-2 production and secretion were also significantly increased when splenocytes were stimulated with Con A, while IFN-γ production and secretion were significantly decreased, and IL-5 and IL-6 production and secretion were unchanged (Table 7).

Under normal conditions, IL-6 production and secretion by LPS-induced splenocytes were significantly increased by the administration of HLF (200 ㎎/㎏). IL-2 production and secretion by Con A-treated splenocytes were significantly decreased by administration of 100 or 200 ㎎ /㎏ HLF (Tables 6 and Table 7).

In the immunosuppressed state, IL-2 production and secretion by LPS-induced splenocytes were significantly decreased in the group treated with HLF (100 ㎎/㎏). These results indicate that HLF decreases the production and secretion of the proinflammatory cytokine IL-2 and increases the production and secretion of the anti-inflammatory cytokine IL-6, thereby regulating the production and secretion of cytokines to exert immunomodulatory effects.

In immune responses, Th1 cells primarily promote inflammatory responses by producing cytokines like IL-2 and IFN-γ. In contrast, Th2 cells produce cytokines such as IL-5 and IL-6 to promote anti-inflammatory responses (Lee et al., 2018; Moghbeli et al., 2021).

In the immuno- suppression condition, a decrease in IL-2 levels was observed, suggesting a potential reduction in Th1 cell activity. This could indicate that the inflammatory response might have been suppressed.

Conversely, an increase in IL-6 levels under normal conditions could indicate an increase in Th2 cell activity, suggesting a possible enhancement of the anti-inflammatory response.

These results may suggest a shift in the Th1/Th2 balance towards Th2, or anti-inflammatory responses. This could indicate that HLF may regulate the immune response by suppressing Th1 cell activity and promoting Th2 cell activity, thereby adjusting the Th1/Th2 balance. However, this interpretation needs to be further validated through additional experiments and data analysis.