Preventive Effects of AHCC on Carbon Tetrachloride
Induced Liver Injury in Mice
Buxiang SUN, Koji WAKAME, Tomomi MUKODA, Atsushi TOYOSHIMA, Tsutomu
KANAZAWA and Kenichi KOSUNA.
Amino Up Chemical Co., Ltd., High Tech Hill Shin-Ei, 363-32,
Shin-Ei, Toyohira-ku, Sapporo 004, Japan.
Natural Medicine 51 (4), 310-315. 1997 **Translated from Japanese
In the natural world, it is said that approximately 5,900 genera,
6,400 kinds of mycelia inhabit. Lingzhi Ganoderma lucidum, reishi),
zhuling (Polyporus umbellatus, chorei) are popular Chinese traditional
medicine derived from fungi of basidiomycetes family. Those mycelia
are identified to contain various physiologically active substances,
such as polysaccharide with b-1, 3-glucan structure. It has been
reported its activities; anti-tumor activity, accommodation activity
on immune system, hypoglycemic activity, etc. Recent advance of
culturing techniques has enabled artificial culture of basidiomycetes.
Active Hexose Correlated Compounds (AHCC; from Amino Up Chemical
Co., Ltd.) is a mixture containing polysaccharide obtained by
culturing in a liquid culture tank followed by enzyme reactions
and hot water extraction.
AHCC has been observed and reported its bioactivity as Biological
Response Modifiers (BRM) or nonspecific immunoreactive activator. Especially in clinical studies, the effects of AHCC have been
reported; improvement of adult diseases such as diabetes or hepatic
disease, cancer cell atrophy and inhibition of metastases in tumor
patients, the survival time prolongation, reducing side effects
caused by chemotherapy, etc.
Although there are many clinical studies reported, actual pharmacological
mechanism is known only a little. To investigate its pharmacological
mechanism as the first step, we prepared an acute carbon tetrachloride
hepatitis model in mice which symptom is said to be similar to
drug liver injury in human. This is the report of the investigation
of the effect of AHCC on liver function change and drug metabolizing
enzymes in liver.
Materials and Methods
Animals. Male ICR mice of 7 weeks old, body weight 34-38
gm. were purchased from Nippon Clea Co., Ltd. The animals were
kept free access to food (CE-2; Nippon Clear) and water for one
week as pre-breeding period.
Materials. All the material drugs were purchased from
Wakopure Industry Co., Ltd. and Sigma Chemical Co. AHCC freeze-dried
powder was supplied by AMINO UP CHEMICAL CO., LTD.
Preparation of food and its administration- AHCC was dissolved
in water at 50% concentration. Mice were treated with the solution
by oral at the dose of 1g/kg/day for three days. On the forth
day, 20% solution of CCl4 diluted with olive oil (Japan Pharmacopoeia)
was administered i.p. at the dose of 2ml/kg/day for five days.
Those mice were divided into four groups with five mice each:
control group with normal mice, injured group treated with CCl4,
AHCC treated group, AHCC and CC14 co-administrated group (CCl4
was administered after AHCC treatment).
Blood and organ preparation- On the final day of CCl4
administration, mice were given free access to water not giving
any food. 12 hours later, blood sample was collected via carotid.
The samples were centrifuged (3,000 r.p.m,, 5 min) and serum was
obtained. The liver removed was rinsed with in ice-cold saline
solution (0.9%) and weighted. Livers were immediately stored at
-80'C. Serum analysis - Glutamate pyruvate transaminase (GPT)
activity in serum was measured by Rate method (GPT-UV Test Wako),
Albumin was measured by BCG method (Albumin-B Test Wako), Total
protein (TP) was measured by Lowry method, Globulin (G) was calculated
by TP-A, Triglyceride (TG) was measured by Acetylacetone method
(Triglyceride Test Wako)
Hepatic enzyme preparation- Frozen stored livers were
homogenated at ice by adding 4 times of 0.154MKCI-0.05M Tris (pH
7.4)-lmM EDTA. This liver homogenize was first centrifuged l,000r.p.m.,
5°C for 5 min, then lO.OOOr.p.m. for 5°C for l5min., finally
the supernatant fraction was obtained. Then the supernatant fraction
obtained by lO.OOOr.p.m. was centrifuged 38,000r.p.m., 5°C
for 60min and supernatant (cytosol) fraction was obtained for
GST measurement. Microsome fraction was obtained by adding twice
as much as the liver weight of cold 0.154MKCI-0.05M Tris (pH 7.4)-lmM
EDTA-20% glycerol solution in the precipitate gained from 38,000r.p.m.
The microsome fraction was applied for the measurements of Protein,
P450, LPO, UDP-GT, ERDM and AHH.
Lipid peroxidation (LPO) measurement- The measurement
was based on Yagi method. 0.05ml of liver microsome fraction was
centrifuged 3,000r.p.m., lOmin with 0.5ml of 1/12N H2S04 4.0ml,
10% phosphotungstic acid aqueous solution, and TBA reagent was
added in the precipitate for 60min heating in boiling water. Obtained
fluosubstance was extracted by n-butanol and measured its fluorescence
intensity with spectrophotofluorometer.
The measurement of P450 content- The measurement was based on
Omura and Sato method. Two cells of enzyme solution 3ml which
is equivalent to liver weight (250mg) were prepared with hydrosulfite
sodium OlM in each cell. One of them were treated with CO for
30 seconds, and another was used as the control. The content of
P450 was calculated from the highest peak of 450nm.
The measurement of each enzyme activity- Glutathione S-transferase
(GST) measurement was measured by Habig method. Uridine phosphate
glucuronyl transferase (UDP-GT) by Mulder method, Arylhydrocarbon
Hydroxylase (AHH) by Dehnen method, Benzyloxyresorufin 0-dealkylation
(BROD) by Lubet method, Eryhromycin N-demethylase (ERMD) by Wrighton.
Histopatbological examination- The liver was fixed in formalin
and the prepared slide was stained with haematoxylin-eosin (HE).
Statistical analysis- All the data were expressed as means
ąSD. Statistical analysis was performed by ANOVA method and significant
difference was judged by Kruskall-wallis test. A P value less
than 0.05 was considered as significant difference.
1. General condition in mice
In CCI4 administration group, some toxicity was observed: the
decrease of active movement, hair xanthosis, and inhibition of
body weight gain. On the other hand, in the AHCC and CCI4 combined
treatment group, those symptoms were reduced. Comparing to the
CCI4 group, which showed liver weight gain by more than 70%, a
significant inhibition in liver weight gain was observed in AHCC
2. Biochemical parameter change in serum and microsome
Comparing to the serum GPT activity increased to 369.5 (IU) in
CCI4 group, it was 164.7 (IU) in AHCC co-administration group. Not any significant change in triglyceride, albumin or globulin
LPO in liver microsome fraction in CCI4 group was 287 nmol/ml
which was more than twice as much as in control group. On the
other hand, LPO in AHCC co-administration group was 152 nmol/ml,
the lipid hyperoxidation was significantly inhibited. AHCC administration
group also showed lower lipid hyperoxidation comparing to control
3. Phase I drug metabolizing enzyme activity
Cytochrome P450 content ia liver microsome franction was decreased
in CCI4 group, however, AHCC co-administration inhibit P450 reduction. P450 content in AHCC group was increased compared with control
group. By the measurement of drug metabolizing enzyme activities,
BROD activity was not observed. Also, ERDM activity was remarkably
inhibited in CCI4 group and induced in AHCC group, however, there
was no significant difference between AHCC co-administration group
and control group. AHH activity was tending to be induced in CCI4 group and AHCC
group, the significance is not clear as in co-administration group
4. Phase u drug metabolizing enzyme activity
The measurement of drug metabolizing enzymes showed that CCI4
suppressed GST activity and UDP-GT activity. This effect was not
observed when AHCC was administrated together. Additionally, GST
activity was induced in AHCC group .
5. Histopathological changes
In CCI4 group, widespread centrilobular necrosis and inflammatory
cell infiltration were observed. On the other hand, in CCI4 and
AHCC co-administration group, centrilobular necrosis was rarely
observed and small part of inflammatory cell infiltration was
As a liver poisonous substance, which induces experimental liver
injury, CCI4 is generally used and known to induce acute liver
injury by short-term administration.
Acute liver injury model was used in this experiment to find
a protecting effect of AHCC on liver. AHCC is known to have immune
stimulate effect or anti-tumor effect as bioactivity, and it is
expected to have another various activities in plant polysaccharide
like AHCC. It is considered that this experiment made one of its
It was observed the degradation of appetite, weight loss, auxesis
of liver, sGPT level increase, etc. in CCI4 induced liver injury
mice prepared for this experiment.
Regarding organ weight, AHCC significantly inhibit liver weight
gain. Liver of mouse administered CCI4 is known to become fatty
liver because of disorder of lipid metabolism, and olive oil used
as a solubilizer is also a cause of adiposity in liver. As a result
of histopathological examination, adipose degeneration in liver
was significantly inhibited in AHCC co-administrated group more
than in CCI4 group. This result made us think AHCC had some effect
on fatty liver.
For serum parameter, sGPT level, a general index of hepatitis,
which elevate when liver cell is injured, was suppressed. This
result suggests that radical derived from CCI4 has a preventive
effect for attacking liver cell. Liver cytochrome P450 measured
in this research was decreased in CCI4 group. For the cause of
decreasing P450, since P460 content usually depend on the content
of protein, the possibilities are whether heme protein change
of composition or metabolism damaged or the change of P450 active
part, e.g. CO bind inhibition, has happened. On the other hand,
P450 tend to be induced in AHCC group. In AHCC and in CCI4 group,
it was considered that P450 content went to the normal level by
the counteraction of these two substances.
Furthermore, AHH as P450 metabolic enzyme belongs to CYPlA isozyme.
It is a general compound, metabolic enzyme especially for aromatic
compound, and has strong relation to metabolic activation of cancergenesis.
ERDM belongs to CYP3A molecule and has intimate relation to metabolism
of endocrine substances such as antibiotic or hormone. Liver drug
metabolizing enzyme is known as the most important enzymes when
endogenic substances like hormone or xenobiotics are absorbed
and go thorough liver, and also they are known as the enzyme easy
to change its amount or activity when liver was injured. It is
also known that these enzymes are suppressed and cause endocrine
disorder when hepatic failure (liver abortive) is happened, various
general symptoms, feebleness or febricula, arise by adrenal hormone
disorder or sex hormone metabolism disorder. On the other hand,
it is considered that AHCC induce these enzymes and maintain the
balance of biofunctions; detoxication of external substances,
metabolism, metabolism of endogenic substances (hormone). Also,
it was suggested that the dose of AHCC need to have an adjusted
when it is administered with P460 drug metabolize enzyme.
Another type of drug metabolizing enzyme is phase II drug metabolizing
enzyme: GST and UDP-GT. GST strongly relates to detoxification
as glutathione conjugation reaction. That is GST detoxification
hydrophobic compound by protein-bounding and operate bioprotective
reaction against detriments absorbed or taken in the body or formed
by metabolism under normal conditions. UDP-GT is also known to
detoxification various kinds of external exogenous materials or
endogenic substances such as 0-, N-, S- and C-glucuronic acid
conjugate as a reaction of glucuronic acid conjugation. AHCC showed
inhibition effect on declining phase II drug metabolizing enzyme
activity caused by CCI4, also GST activity induced effect was
observed in AHCC administered group. That is one of the protective
mechanism for liver injury caused by CCI4 is to accelerate detoxification
of CCI4. CCI4 becomes trichloromethylradical. This radical is
supposed to form lipid peroxidation followed by hyperoxidation
reaction to damage cytoplasmic or cell membrane which cause liver
injury. LPO level in AHC and CCI4 co-administrated group suppressed
compared more than in CCI4 group, and it was reduced more in AHCC
group than in control group. According to these results, it is
considered that AHCC had another effect to erase radical and prevent
liver injury caused by the radical. In conclusion, AHCC showed protective effect for acute liver injury
in mice regarding general condition, serum parameter, liver drug
metabolize enzymes. The most significant effects of AHCC are the
inhibition of liver auxesis, reducing general poisoning symptom,
and inducement of detoxic enzymes. It is necessary to explicate
the pharmacological functions of these protective effects.
We thank Professor Masuo Hosokawa (Hokkaido University), Dr. Genichiro Nonaka (Advisor, Amino Up Chemical Co., Ltd.), Dr. Morio
Fukuhara (National Institute of Public Health) and all the researchers
of Amino Up Chemical Co., Ltd.
A: albumin, AHCC: active hexose correlatedd compounds, AHH: arylhydrocarbon
hydroxylase, BRM: biological response modifiers, BROD: benzyl
oxyresorufm 0-dealkylation, CCI4: carbon tetrachloride, ERMD:
erythromycin N -demethylase, G: Globulin, GPT: glutamate pyruvate
transaminase, GST: glutathione S-transferase, TG; tryglyceride,
TP: total protein, UDP-GT: uridine phosphate glucuronyl transferase.