Jungle Honey Enhances Immune Function and Antitumor Activity

Abstract

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.

Full text

Hindawi Publishing Corporation
Evidence-Based Complementary and Alternative Medicine
Volume 2011, Article ID 908743, 8pages
doi:10.1093/ecam/nen086
Original Article
Jungle Honey Enhances Immune Function and
Antitumor Activity
Miki Fukuda,1Kengo Kobayashi,1Yuri ko Hi r o n o,1Mayuko Miyagawa,1Takahiro Ishida,1
Emenike C. Ejiogu,2Masaharu Sawai,3Kent E. Pinkerton,4and Minoru Takeuchi1
1Department of Biotechnology, Faculty of Engineering, Kyoto Sangyo University, Kyoto, Japan
2Origins Japan Co. Ltd, Nagano, Japan
3Takara Shuzo Co. Ltd, Kyoto, Motoyama, Kamigamo, Kita-ku, Kyoto 603-8555, Japan
4Center for Health and the Environment, University of California Davis, California, USA
Correspondence should be addressed to Minoru Takeuchi, mtakex@cc.kyoto-su.ac.jp
Received 16 June 2008; Accepted 17 December 2008
Copyright © 2011 Miki Fukuda et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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 (1mg/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.
1. Introduction
Natural products are known to have biological activity,
and we have previously investigated the effect of natural
products on immune function [1,2]. Honey contains various
vitamins, minerals and amino acids as well as glucose and
fructose and is popular as a natural food [3–6]. There is a
wide variety of honey (Manuka honey, Pasture honey, Jelly
bush honey and Jungle honey, etc.), and the varieties are
due to components of the flower sources. Honey is used
not only as natural food but also as traditional medicine for
health care, in beauty products and antiinflammatory skin
care. One variety, Jungle honey, is collected from timber and
blossom by wild honeybees that live in the tropical forest
of Nigeria. Jungle honey is used as traditional medicine or
preventive medicine to treat colds, skin inflammation and
burn wounds as well as for general health care.
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 [7–11]. It has been reported
that flavonoid and phenol acid show antibacterial activity
[12–14]. It was reported in a clinical experiment that when
wound infected with gram positive and gram negative
bacteria were treated with honey, infection was more quickly
eradicated [15–17].
It has been reported that Manuka honey increased IL-
1β,IL-6,andTNF-αproduction from Mono Mac6 cells or
human monocytes [18,19], and the active component was
5.8 kDa, which increased production of these cytokines via
TLR4 [20]. In addition, it was reported that oral intake
of honey augmented antibody productions in primary and
secondary immune responses against thymus-dependent and
thymus-independent antigens [21].

2 Evidence-Based Complementary and Alternative Medicine
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.
2. Methods
2.1. Preparation of Jungle Honey. Jungle honey (JH) was a gift
from Nihon origins Co. Ltd (Nagano, Japan). JH was har-
vested in the forest areas around the Nsukka area of Enugu
state, Nigeria [22]. Generally, the bees were Apis mellifera
adonsonii. The main plant species that the bees collected
from nectar were Pentaclethra macrophylla,Chrysophyllum
albidum and Milicia excela. Briefly, the components of JH
in 100 g were 900 mg protein, 1400 mg glucon acid, 657 mg
amino acid, 213 mg mineral and 3.46 mg vitamin. JH was
dissolved with distilled water, freeze dried and then adjusted
to 10 mg/mL with PBS(−). JH was steriled by 0.22-μm
filtration (Millipore, MA, USA) and then stored at 4◦Cbefore
use. The endotoxin unit of JH was found to be 2.7EU/mL
using the limulus amebocyte lysate assay kit (Canbrex, MD,
USA). The mice did not immunologically respond to this
unit of endotoxin.
2.2. Mice. 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 ad libi-
tum. This study was approved by the committee for animals
in Kyoto Sangyo University. Mice were intraperitoneally (i.p.)
injected seven times with JH at a dose of 1 mg/mouse/day.
Control mice received PBS(−)[Ca
2+,Mg
2+-free Dulbecco’s
phosphate buffered saline (Nissui Pharmaceutical, Tokyo,
Japan)].
2.3. Analysis of Peritoneal Cells (PC). 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 ×106cells/mL in FACS buffer (PBS
containing 100 μg/mL CaCl2/MgCl2, 0.01% sodium azide
and 1% FCS). The newly appearing cell population found via
Dot Plot analysis after JH exposure was sorted by FACS. The
sorted cell population was suspended at 5 ×105cells/mL in
R(+) (RPMI1640 containing 10% fetal calf serum, 100 U/mL
penicillin, 100 μg/mL streptomycin). The cell suspension
(200 μLof5×105cells/mL) was put on a slide glass using
Cyto Spin and centrifuged at 185 g for 5 min. After the slide
glasses were dried in a dryer, the cells were fixed for 3min by
methanol, followed by Giemsa stain. Giemsa-stained samples
were observed using a light microscope.
2.4. Chemotaxis Assay Using EZ-TAXIScan. 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 ×106cells/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-μmdeep
microchannel. Neutrophils (1 μLof2×106cells/mL) were
put into a hole with which the device is held together
with a stainless holder, and 1μLof10
−6MN-formyl-
methionyl-leucyl-phenylalanine (fMLP) or 1 mg/mL JH was
put into a contra-hole. The holder assembly was filled with
R(+) and incubated for 30 min at 37◦C. A charge-coupled
device (CCD) camera was used to record the migration of
neutrophils toward the high concentration of each sample.
CellsinimageswereanalyzedbyTAXIScanAnalyzer2.
2.5. Antitumor Activity. 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 μg/mL streptomycin]. After 2-3 days, LL/2 were obtained
with trypsin-EDTA (0.25% trypsin : 0.02% EDTA =1:1,
Nacalai tesque) and washed with MEM(−)(D-MEM con-
taining 100 U/mL penicillin and 100 μg/mL streptomycin).
Hemocytometer and trypan blue dye exclusion testing were
used to determine LL/2 total number and viability. Mice
injected with JH or PBS(−) were inoculated intraperitoneally
with LL/2 (4 ×105cells/0.2 mL/mouse). After 4 weeks,
antitumor activity was evaluated by tumor incidence and
weight. Tumor tissues were fixed in 10% neutral buffered
formalin fixative and paraffin embedded. Sections (4 μm)
were stained with H&E.
2.6. Production of Reactive Oxygen Species. PC (1 ×
105cells/100 μL) were incubated with Hydroethidine (HE,
Polysciences, PA, USA, final concentration 10 μM) or 2,7-
Dichlorofluorescin diacetate (DCFH-DA, Sigma, MO, USA,
final concentration 20 mM). After a shaking incubation at
37◦C for 30 min, the cells were washed twice and resus-
pended in 200 μL of PBS(+) (Dulbecco’s phosphate buffered
saline) and then analyzed using FACS.
2.7. Fractionation of Jungle Honey. 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

Evidence-Based Complementary and Alternative Medicine 3
carried out with PBS(−)ataflowrateof1mL/minfor
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(−).
2.8. Expression of IL-1βmRNA. Aliquots of obtained PC
(1 ×105cells/100 μL/well) were cultured with or without
JH fractions (final concentration 500 μg/mL) in 96-well flat
bottom culture plates (Becton-Dickinson, MA, USA) at
37◦Cfor24hin5%CO
2. After 24 h, total RNA was iso-
lated by acid guanidinium thiocyanate-phenol-chloroform
assay. Total RNA was transcribed to cDNA with MLV
reverse transcriptase (Invitrogen, CA, USA). Oligonucleotide
primers were used from published cDNA sequences of IL-
1β(250 bp) and β-actin (268 bp) (house-keeping gene). PCR
was performed for 30 cycles using the following primer
pairs: β-actin sense (5-GCATTGTTACCAACTGGGAC-3)
and β-actin antisense (5-TCTCCGGAGTCCATCACAAT-
3); IL-1βsense (5-AGCTACCTGTGTCTTTCCCG-3)and
IL-1βantisense (5-GTCGTTGCTTGGTTCTCCTT-3). The
amplification profile consisted of denaturation at 94◦Cfor
30 s, primer annealing at 56◦C for 30 s and extension at
72◦C for 30 s. PCR products were visualized using ethidium
bromide after 8% polyacrylamide gel electrophoresis. Data
on the expression in IL-1βmRNA were quantified by Scion
image.
2.9. Statistical Analysis. Allvaluesareexpressedasmean±
SE. Comparisons between control and JH-injected mice were
made with the Student’s t-test. Any P-values <.05 were
considered statistically significant.
3. Results
3.1. Increases of the Number of PC by Jungle Honey. The
number of PC was significantly (P<.001) increased in JH-
injected mice (5.13 ±0.28 ×106cells/mouse) compared to
control mice (1.17 ±0.11 ×106cells/mouse).
3.2. Induction of New Cell Populations of PC by Jungle
Honey. New cell populations in JH-injected mice were found
at FSC 120–400, SSC 200–800 by Dot Plot analysis of
FACS (Figure 1(b)) compared to control mice (Figure 1(a)).
An isolated, new cell population was found, as is shown
in Figure 1(c) and the isolated cells were found to be
neutrophils (Figure 1(d)) by Giemsa stain. The new cell
population in JH-injected mice was observed by a light
microscope and was identified as neutrophils by their
morphology (Figure 1(d)).
3.3. Enhancement of Chemotaxis for Neutrophil by Jungle
Honey. Forty neutrophils migrated in 30 min in the JH-
treated group compared to 13 neutrophils in the non-
treated group (Figure 2(a)-i, ii). The velocity of the migrating
neutrophils was 0.17 ±0.01 μm/s in the JH-treated group
and 0.04 ±0.01 μm/s in the non-treated group (Figure 2(b)).
The radian of neutrophils was 0.39 ±0.04 rad in JH-
treated group and 0.09 ±0.03 rad in the non-treated
group (Figure 2(b)). JH resulted in significantly (P<.001)
increased numbers, velocity, and radiation of migrated cells.
Therefore, JH showed chemotactic activity for neutrophils.
3.4. Inhibition of LL/2 Tumor Growth by Jungle Honey. The
incidence of LL/2 tumors was 20% in JH-injected mice
and 100% in control mice (Figure 3(a)). The mean tumor
weight was 0.02 ±0.02 g in JH-injected mice and 2.57 ±
1.05 g in control mice (Figure 3(b)). These results reveal
that JH inhibited tumor incidence and growth (Figure 4). In
histological findings of control tumor tissue, necrotic areas
were recognized (Figure 4(a)-i, iii), but there were few infil-
trations of neutrophils (Figure 4(a)-ii, iv). In JH injected-
tumor tissue, massive necrotic areas (Figure 4(b)-v) and
infiltration by many neutrophils were observed (Figure 4(b)-
vi). Hemorrhagic necrotic areas and a disassociation between
tumor cells were found (Figure 4(b)-vii, viii).
3.5. Increases of ROS Production in PC by Jungle Honey. The
ratio of control was 1.0. The ratio of O−
2was 1.16 and the
ratio of H2O2was 1.13 in the JH treated group. The ratio
of O−
2or H2O2producing cells were significantly (P<.001)
increased in JH-injected mice compared with control mice
(Figure 5). Therefore, Reactive Oxygen Species (ROS) may
be associated with antitumor activity.
3.6. Enhancement of IL-1βmRNA Expression in PC by Jungle
Honey Fractions. IL-1βmRNA expressions by JH and each
fraction from Fr. 1 to Fr. 5 were 0.94 ±0.06, 0.67 ±0.12, 1.11
±0.13, 0.42 ±0.05, 0.58 ±0.04 and 0.64 ±0.05, respectively
(Figure 6(a)). IL-1βmRNA expressions were significantly (P
<.001) increased by JH or Fr. 2 (Figure 6(b)).
4. Discussion
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 ∼4-fold in JH-injected mice compared with con-
trol mice. This result suggests that JH induces cell migration.
Although the effects of other types of honey on PC numbers
are not yet reported, mice treated with other natural products
(i.e., hydroalcoholic extract from Chenopodium ambrosioides
or aqueous extract from Orbignya phalerata Mart)havebeen
shown to have increased PC as well [23,24].
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

4 Evidence-Based Complementary and Alternative Medicine
0
200
400
600
800
1000
0 200 400
FSC-H
600 800 1000
SSC-H
(a) Control
0
200
400
600
800
1000
0 200 400
FSC-H
600 800 1000
SSC-H
(b) Jungle honey
0
200
400
600
800
1000
0 200 400
FSC-H
600 800 1000
SSC-H
(c) Isolated cell population
(×400)
(d) Isolated cell population
Figure 1: Induction of new cell populations of PC by Jungle honey. (a) Control. (b) Jungle honey. (c) Isolated cell population. (d) Isolated
cell population.
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 [25].
This result agrees with our present report.
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, polysac-
charide from Ganoderma lucidum have chemotactic activity
for neutrophils in the Boyden chamber assay [26].
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
cellswereusedassyngeneictumorcells,whichhavelow-
tumor antigen and inhibit the immune system as well as
human cancer [27]. The incidence and the mean weight of
LL/2 tumors were decreased in JH-injected mice compared
to control mice. These results suggest that JH has a preventive
effect on tumor growth. Tumor weight was found decreased
by royal jelly, propolis or polyphenol in propolis [28–30].
The tumor tissue was infiltrated by many neutrophils at
massive necrotic areas in JH-injected mice. Therefore, it was
suggested that the neutrophils were involved with inhibition
of tumor growth.
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

Evidence-Based Complementary and Alternative Medicine 5
(i) Control
0min
(ii) Jungle honey
0min
After 30 m in After 30 m in
(a) Image of neutrophil chemotaxis
0.04
0
Direction (Radian)
0.20.40.6
0.06
0.08
0.1
0.12
0.14
Veloc i t y ( μm/sec)
0.16
0.18
0.2
0.22
0.24
0.26
0.28
(b) Dot plots of velocity and direction of neutrophils
Figure 2: 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 (10−6M), filled triangle: JH (1 mg/mL).
Incidence of LL/2 tumor (%)
40
60
80
100
0Control Jungle honey
20
(a) Incidence of LL/2 tumor
0
1
2
3
Tumo r w eig h t ( g)
Control
∗∗∗
Jungle honey
(b) Tumor weight
Figure 3: Inhibition of the incidence of LL/2 tumor (a) Tumor weight (a) by Jungle honey.
(i) (x200)
(iii) (x200)
(ii) (x400)
(iv) (x400)
(a) Control
(v) (x100)
(vii) (x200)
(vi) (x400)
(viii) (x400) H.E. Stain
(b) Jungle honey
Figure 4: Histological findings of LL/2 tumor by Jungle honey. (a) Control. (b) Jungle honey.

6 Evidence-Based Complementary and Alternative Medicine
0.9Control Jungle honey
1
1.1
1.2∗∗∗ ∗∗∗
Index of ROS production
Figure 5: Increases of ROS production in PC by Jungle honey. Open
square: O−
2,filledsquare:H
2O2.
Refractive index (RI)
0
0 5 10 15
Retention time
20 25 30
min
25000
50000
75000 Fr1 Fr2
M.W.1020
M.W.3910 M.W.106
Fr3 Fr4 Fr5
uv
(a) Fraction by gel filtration
0
0.2
0.4
Control Fr. 1 Fr. 2 Fr. 3 Fr. 4 Fr. 5
Jungle
honey
0.6
0.8
1
1.2
1.4
∗∗∗
∗∗∗
IL-1βmRNA expression/β-actin
(b) IL-1βmRNA expression
Figure 6: Enhancement of IL-1βmRNA expression in PC by Jungle
honey fractions. (a) Fraction by gel filtration. (b) IL-1βmRNA
expression.
ROS production has hereto been unreported, one study
reported that H2O2production was increased in PC treated
with extracts of G. lucidum or P. c o r nuc o p i a e [31]. This
result agrees with our present study. It was reported that
ROS produced by activated neutrophils has tumor cytotoxic
properties as well as preventive action against infection [32–
35]. Since infiltration of many neutrophils was observed
at necrotic areas in JH injected-tumor tissue, there is a
possibility that antitumor activity by JH is due to production
of ROS by infiltrated neutrophils into tumor tissue.
JH was fractionized from Fr. 1 to Fr. 5 by gel filtration
using HPLC to identify the effective component in JH, and
IL-1βmRNA expressions in PC were assayed using each
fraction. IL-1βaugments immune response, functions and
migration capability of neutrophils. JH and Fr. 2 augmented
the expression of IL-1βmRNA in PC. Therefore, the effective
componentwasFr.2inJH,andtheeffective component in
Fr. 2 was estimated to have a MW of 261. In contrast, it was
reported that the effective component of honey or royal jelly
was 55 kDa (Apalbumin-1), and the effective component of
Manuka honey was 5.8 kDa; these two components also were
found to increase TNF-αproduction [19,36–38]. Therefore,
the active component of honey or royal jelly and Manuka
honey differs from the active component of JH.
Our results suggested that JH-induced neutrophils to the
peritoneal cavity, and the neutrophils were activated by IL-
1β, which was produced by JH stimulation. Then ROS from
activated neutrophils was associated with antitumor activity.
In addition, the effectivecomponentinJHwasfoundtohave
a MW of 261.
Funding
Grant-in-Aid for Scientific Research (C) in Japan Society for
the Promotion of Science (Grant no. 20500606).
Acknowledgment
The authors appreciate Dr Suzette Smiley-Jewell for her
efforts to provide technical assistance and suggestions in the
preparation of this manuscript.
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