Jungle honey (JH) is collected from timber and blossom by wild honey bees that live in the tropical forest of Nigeria. JH is used as a traditional medicine for colds, skin inflammation and burn wounds as well as general health care. However, the effects of JH on immune functions are not clearly known. Therefore, we investigated the effects of JH on immune functions and antitumor activity in mice. Female C57BL/6 mice were injected with JH (1 mg/mouse/day, seven times intra-peritoneal). After seven injections, peritoneal cells (PC) were obtained. Antitumor activity was assessed by growth of Lewis Lung Carcinoma/2 (LL/2) cells. PC numbers were increased in JH-injected mice compared to control mice. In Dot Plot analysis by FACS, a new cell population appeared in JH-injected mice. The percent of Gr-1 surface antigen and the intensity of Gr-1 antigen expression of PC were increased in JH-injected mice. The new cell population was neutrophils. JH possessed chemotactic activity for neutrophils. Tumor incidence and weight were decreased in JH-injected mice. The ratio of reactive oxygen species (ROS) producing cells was increased in JH-injected mice. The effective component in JH was fractionized by gel filtration using HPLC and had an approximate molecular weight (MW) of 261. These results suggest that neutrophils induced by JH possess potent antitumor activity mediated by ROS and the effective immune component of JH is substrate of MW 261.
Natural products are known to have biological activity, and we have previously investigated the effect of natural products on immune function [
It is generally known that honey has antibacterial activity that has been reported to be due to its high osmolarity, acidity and presence of hydrogen peroxide and unidentified substances from floral sources [
It has been reported that Manuka honey increased IL-1
Honey may provide the basis for the development of novel therapeutics for patients with wounds. Therefore, the purpose of this study was to investigate the effects of Jungle honey on immune function and antitumor activity in mice.
Jungle honey (JH) was a gift from Nihon origins Co. Ltd (Nagano, Japan). JH was harvested in the forest areas around the Nsukka area of Enugu state, Nigeria [
Female C57BL/6 mice were used at 8–10 weeks. Ten mice were used in each group. Mice were obtained from Japan SLC (Shizuoka, Japan). They were housed in transparent plastic cages with stainless wire lids in the animal facility of Kyoto Sangyo University (Kyoto, Japan). They were maintained under standard conditions, with a dark period from 8 pm to 8 am, and water and food were provided
Peritoneal cells were analyzed using Fluorescence Activated cell Sorter (FACS) Calibur (Becton-Dickinson, CA, USA). After seven injections of JH, PC were collected by peritoneal lavage from the mice with cold PBS. PC were pooled into plastic tubes and centrifuged at 185 g for 10 min. The pelletted cells were resuspended at 1 × 106 cells/mL in FACS buffer (PBS containing 100
Neutrophils were obtained from guinea pig peripheral blood. Blood was diluted twice with PBS. To precipitate red blood cells, peripheral blood was added in equal parts to 3.5% Dextran in saline and incubated at room temperature for 30 min. The leucocyte-rich supernatant was centrifuged at 400 g for 30 min on a Ficoll-Paque Plus (GE Healthcare, Tokyo, Japan) density gradient. The pellet was hemolyzed by hypotonic lysis. Fractionated neutrophils were centrifuged at 185 g for 10 min and resuspended at 2 × 106 cells/mL in R’(+)(RPMI1640 containing 0.1% bovine serum albumin, HEPES). A chemotaxis assay for neutrophils was evaluated with EZ-TAXIScan. Time-lapse images of neutrophils during chemotaxis were obtained using EZ-TAXIScan equipped with a six channel chamber (GE Healthcare). This chamber consists of an etched silicon substrate and a flat glass plate, both of which form two compartments with a 4-
Lewis Lung Carcinoma/2 (LL/2) cells were used as tumor cells. LL/2 cells were maintained in a 10-cm dish (BD Falcon, CA, USA) at 2- to 3-day intervals using MEM(+) [D-MEM (Nacalai tesque, Kyoto, Japan) containing 10% fetal calf serum, 100 U/mL penicillin and 100
PC (1 × 105 cells/100
JH (100 mg/mL) was fractionized from Fr. 1 to Fr. 5 by gel filtration using a Shodex OHpak SB-802 HQ column and HPLC (LC-20AD, RID-10A, SPD-20A, CB-20A, Simazu, Japan). Elution was carried out with PBS(−) at a flow rate of 1 mL/min for 30 min. Standards curves were traced using polyethylene glycols, which had MW of 3930, 1020, and 106 (Polymer Laboratories, Germany), and LCsolution GPC (Shimau), under the same conditions in HPLC. The MW of JH was estimated using the polyethylene glycol standard curves. Each fraction of JH was freeze dried and then adjusted to a concentration of 10 mg/mL with PBS(−).
Aliquots of obtained PC (1 × 105 cells/100
All values are expressed as mean ± SE. Comparisons between control and JH-injected mice were made with the Student's
The number of PC was significantly (
New cell populations in JH-injected mice were found at FSC 120–400, SSC 200–800 by Dot Plot analysis of FACS (Figure
Induction of new cell populations of PC by Jungle honey. (a) Control. (b) Jungle honey. (c) Isolated cell population. (d) Isolated cell population.
Control
Jungle honey
Isolated cell population
Isolated cell population
Forty neutrophils migrated in 30 min in the JH-treated group compared to 13 neutrophils in the non-treated group (Figure
Enhancement of chemotactic activity for neutrophils by Jungle honey. (a) Image of neutrophil chemotaxis. (b) Dot plots of velocity and direction of neutrophils. Up arrow: Migrated cells, filled circle: control, filled diamond: fMLP (10−6 M), filled triangle: JH (1 mg/mL).
Image of neutrophil chemotaxis
Dot plots of velocity and direction of neutrophils
The incidence of LL/2 tumors was 20% in JH-injected mice and 100% in control mice (Figure
Inhibition of the incidence of LL/2 tumor (a) Tumor weight (a) by Jungle honey.
Incidence of LL/2 tumor
Tumor weight
Histological findings of LL/2 tumor by Jungle honey. (a) Control. (b) Jungle honey.
Control
Jungle honey
The ratio of control was 1.0. The ratio of O2− was 1.16 and the ratio of H2O2 was 1.13 in the JH treated group. The ratio of O2− or H2O2 producing cells were significantly (
Increases of ROS production in PC by Jungle honey. Open square: O2−, filled square: H2O2.
IL-1
Enhancement of IL-1
Fraction by gel filtration
IL-1
Jungle honey (JH) is collected from timber and blossom by wild honeybees that live in the tropical forest of Nigeria, where JH is used as traditional or preventive medicine for colds, skin inflammation and burn wounds as well as general health care. Therefore, we expected that JH would have potential biological, especially immune, activity. Until now, the effect of JH on immunomodulatory activity has been relatively unknown. Therefore, we investigated the effects of JH on immune function and antitumor activity in mice.
We found that the number of peritoneal cells (PC) was increased
To characterize the new cell population found after JH treatment, we investigated surface antigens by FACS. In Dot Plot analysis, a new cell population appeared in the region of FSC 120–400 and SSC 200–800 in JH-injected mice. The percent of Gr-1 surface antigen and the intensity of Gr-1 antigen expression of PC were increased in JH-injected mice (data not shown). Moreover, the new cell population was found to be neutrophils based on morphology. Although the effects of honey on Dot Plot and cell surface antigen of PC were not reported, it was shown that the number of neutrophils was increased in treated mice with propolis [
Our results showed that JH may have chemotactic activity for neutrophils. Therefore, we investigated the chemotactic activity of JH for neutrophils. The velocity and direction of migration were increased by JH compared with the control treatment. These results suggest that JH possesses chemotactic activity for neutrophils. Although there are no reports concerning chemotactic activity of honey, polysaccharide from
Because it was demonstrated that the number of PC and migration of neutrophils were increased by JH, we investigated antitumor activity by immune cells. LL/2 tumor cells were used as syngeneic tumor cells, which have low-tumor antigen and inhibit the immune system as well as human cancer [
To investigate the mechanism of antitumor activity by JH, we examined ROS, a well-known antitumor factor. The ratio of ROS produced by cells was increased in JH-injected mice. Although the effect of honey on cellular ROS production has hereto been unreported, one study reported that H2O2 production was increased in PC treated with extracts of
JH was fractionized from Fr. 1 to Fr. 5 by gel filtration using HPLC to identify the effective component in JH, and IL-1
Our results suggested that JH-induced neutrophils to the peritoneal cavity, and the neutrophils were activated by IL-1
Grant-in-Aid for Scientific Research (C) in Japan Society for the Promotion of Science (Grant no. 20500606).
The authors appreciate Dr Suzette Smiley-Jewell for her efforts to provide technical assistance and suggestions in the preparation of this manuscript.