Scientific Studies on the Effectiveness and Mechanism of Biobran MGN-3

by Hiroaki Maeda (Edited by Chris Gutch PhD) Sept 2003
   

1. 

INTRODUCTION
   
  Fibers and other indigestible components in foods are valued as substances deeply involved in human health which have the effect of maintaining homeostasis and perform a therapeutic role through actions different from essential nutrients. Their main actions are improvement of lipid metabolism, improvement of sugar metabolism, improvement of intestinal environment and inhibition of the toxicity of hazardous substances in the diet.

Dietary fibers derive mainly from plants, algae and microorganisms, and many are sugar polymers, i.e. polysaccharides. There have been a number of reports concerning the immunostimulatory action of polysaccharides in foods and microorganisms for fermentation, leading to their description as Biological Response Modifiers (BRM). Such polysaccharides include zymozan (β-1-3-glucan), a cell wall component of beer yeast, chitin and a cell wall component of baker's yeast (α-1-6-mannan), and cell wall components of mushrooms such as shiitake mushroom, suehirotake mushroom and kawaratake mushroom (β-1-3-glucan), etc. Some of these are used in cancer treatments as immunostimulators, administered mainly intravenously. These components are characterized by the fact that they generally have high molecular weight (0.5-1 million dalton) and their effects are only slight if given orally.

Based on such research background concerning polysaccharides and immunoenhancement, Biobran MGN-3 (Biobran) has been developed as a food with immunostimulatory action designed to work by oral intake.
   

2.

DEVELOPMENT OF RICE BRAN ARABINOXYLAN DERIVATIVE (Biobran MGN-3)
   
  Dietary fiber components of rice bran are cellulose, lignin and hemicellulose. Hemicellulose is further categorized into high molecular weight insoluble hemicellulose A and low molecular weight soluble hemicellulose B. Hemicellulose is a heteroglycan with a highly branched and complicated sugar composition of mainly arabinose and xylose, and, as other components, rhamnose, galactose, mannose, glucose and uronic acid, etc. Hemicellulose B contains a relatively small amount of a molecule with a polymerization degree of approximately 200, which can be absorbed through the intestinal wall after oral intake.

Partial decomposition of hemicellulose B was accomplished by using enzymes able to break down carbohydrates and derivatives were obtained with strong immunostimulatory action, especially on natural killer cells (NK cells), to produce the chemical structure in Figure 1 below.
   
 
  Figure 1
   
  In industrial production, the enzymes used to make the derivatives are a carbohydrase complex that contains α, β-glucosidase, α, β-galactosidase, β-1-4-glucosidase and β-1-3-glucosidase produced outside the fungus body of shitake fungus.
   

3.

PHYSIOLOGICAL ACTIVITY OF RICE BRAN ARABINOXYLAN DERIVATIVE (Biobran)
   
 

(1) 

Immuno Regulation
   
 
(a) Enhancement of human NK cell activity by modified arabinoxylan from Rice Bran (Biobran MGN-3)
   
 
(i) In vitro test
   
  Rat-derived NK cells were incubated with K-562 tumor cells, which were the target cells, in the presence of Biobran MGN-3, and the amount of the K-562 cells dissolved was measured by 51Cr-release assay. The dissolved amount increased concentration-dependently between Biobran MGN-3 concentrations of 25 μg/ml and 100 μg/ml, which confirmed that the increase of NK activity was due to Biobran MGN-3.
   
 
  Figure 2
   
(ii) In vivo test (rat)
   
  Biobran MGN-3 was given orally to Sprague-Dawly rats and after two weeks the change of NK cell activity was measured. Rats were divided into three groups with doses of 0.5 mg, 5 mg and 50 mg/kg/day, with five rats per group. NK cell activities increased dose-dependently with the administration of Biobran MGN-3. Compared with the control group, the activities increased by 119%, 130% and 142% for the 0.5 mg, 5 mg and 50 mg dose groups, respectively. In the 50 mg dose group, the activity increased by 132% compared with the control group within three days of the dose initiation. The increase of NK cell activities by Biobran MGN-3 could be attributed to the increased dissolving power of NK cells but also depended on the number of NK cells. Some difference in effect was observed between male and female rats, and the manifestation of the effect was more remarkable in female rats (Figure 3).
   
 
  Figure 3
   
(iii) Actions on human
   
  A 60-day continuous intake test of Biobran MGN-3 was conducted on 24 healthy subjects (15 women and 9 men, average 34 years old) and the change in NK cell activities was observed. The subjects were divided into three groups with intake amounts of 15 mg/kg/day, 30 mg/kg/day and 45 mg/kg/day, with 8 subjects per group. Twenty cc of blood was collected from each participant: prior to the intake of Biobran MGN-3, after one week, after one month, after two months (at the termination of the test) and one month after the termination and NK activities were measured. In the 30 mg and 45 mg intake groups, NK activities increased approximately two-fold one week after initiation of intake and reached three-fold in two months. In the 15 mg intake group, the activity rapidly increased from one month after initiation and reached almost the same level as in the 30 mg and 45 mg groups in two months. One month after the termination of the intake NK activities returned to the level before the intake (Figure 4). These results suggested that the intake of 15-45 mg/kg/day of Biobran MGN-3 influences NK activities in human.
   
 
  Figure 4: Time and dosage dependence of natural killer (NK) cell activation by Biobran MGN-3 against K562 tumour cells…_.._.._.._ = 45mg/kg/day; …………=30mg/kg/day; _______=15mg/kg/day
   
(iv) Mechanism of Biobran MGN-3 action on NK cells
   
  Both in vitro and in vivo tests confirmed that the number of cytotoxic granules increases in NK cells stimulated by Biobran MGN-3. The increase of binding ability to target cells was also investigated. NK cells of a human who took 45 mg/kg/day of Biobran MGN-3 for 30 days were incubated with K-562, which were the target cells, and the increase of the binding ability was measured. After NK cells and K-562 tumor cells were incubated at 4ºC for one hour, 200 NK cells were measured and the binding rate with K-562 was calculated. The binding rate of NK cells to the target cells (K-562) in a subject who took Biobran MGN-3 significantly increased to 38.5% compared with 9.4% before intake (Figure 5). The typical photograph that indicates binding is shown in Figure 6.
   
 
_
Figure 5: Percentage of conjugate formation between natural killer (NK) cells and K562 target cells.   Figure 6: Natural killer (NK) effector-tumour targets conjugate formation.
   
  Ghoneum M. (Drew Univ., USA):INT. IMMUNO THERAPY XIV (2) PP.89-99, 1998
   
(b) In vitro effect of Biobran MGN-3 on macrophage group cell activity
   
  The effect of Biobran MGN-3 on macrophage group cells in inducing the production of pharmacological mediators has been investigated. TNF-a, IL-6 and NO were assayed as mediators. Macrophage cells were incubated with various concentrations of Biobran MGN-3 (1-100µg/ml) and supernatants were collected for assay of mediators. TNF-a was assayed by cytotoxicity on L929, IL-6 by cytosis on B13.19 and NO colorimetrically by reaction with Griess reagant. LPS was used as a positive control.
   
 
(i) Using Murine macrophage cell line RAW264.7, Biobran MGN-3 showed strong activity on the three mediators at concentrations greater than 10µg/ml, as did LPS.
   
(ii) Murine peritoneal macrophages(C3H/He). The effect of Biobran MGN-3 against the macrophage originating from peritoneal cavity of normal mouse is showed in Figure 7. Again Biobran MGN-3 showed strong activity at concentrations above 10µg/ml.
   
(iii) Human macrophage cell line U937. Biobran MGN-3 induced strong activity as measured by production of cytokines TNF-a and IL-6, equivalent to LPS at 100µg/ml.
   
  The results show that Biobran MGN-3 is a potent substance for activating either normal mouse or human macrophage. The studies suggested active concentrations of over 10µg/ml.
   
 
  Figure 7: Murine peritoneal macrophages (C3H/He mice)
   
  Matsuura M. (Jichi Medical School, JAPAN) : Report of Jichi Medical school
   
(c) Immunostimulation and cancer prevention
   
  Several studies have established the excessive cancer risk for workers exposed to various chemicals common in the workplace. A study was designed to examine the immune alteration associated with exposure to toxic chemicals and the possibility of counteracting chemical toxicity using Biobran MGN-3.
   
  Eleven individuals who had been exposed to chemicals in the workplace participated in the study. The participants demonstrated immune dysfunction as indicated by: low levels of natural killer (NK) cell activity (10.2±4.2 LUs), lymphocyte blastogenic responses to T-cell mitogens (PHA, 39060±12517cpm and COMA, 36224±11922cpm) and B-cell mitogen (PWM, 16550±6330cpm), compared to control responses. Subjects received Biobran MGN-3 at a dose of 15mg/kg/day for four months. Treatment with Biobran MGN-3 increased NK cell activity 4 and 7 fold at two and four months respectively, while T and B-cell functions were 130-150% higher than base line values.
   
  Ghoneum M. (Drew Univ., USA) : The abstract of the 7th International Congress on Anti-Aging & Biomedical Technolgies Conference Proceedings Manual, 1999
   
(d) Production of TNF-α and IF-γ from Human PBL by Biobran MGN-3 Modified Arabinoxylan from Rice Bran.
   
  The mechanism by which Biobran MGN-3 elevates NK cytotoxic activity has been investigated. This was done by testing the action of Biobran MGN-3 on the levels of both tumor necrosis factor-α, (TNF-α) and interferon-γ (IFN-γ) secretions and on the expression of key cell surface receptors.
   
  Peripheral blood lymphocytes were treated with Biobran MGN-3 at concentrations of 0.1 mg/ml and 1 mg/ml, and supernatants were subjected to enzyme-linked immunosorbent assay. Results showed that Biobran MGN-3 is a potent TNF-α inducer and the effect was dose-dependent. Biobran MGN-3 concentration at 0.1mg/ml and 1 mg/ml increased TNF-α production by 22.8- and 47.1-fold, respectively. Biobran MGN-3 also increased production of IFN-γ but at lower levels as compared to TNF-α. With respect to key cell surface receptors, Biobran MGN-3 increased the expression of CD69, an early activation antigen at 16 hours after treatment. Furthermore, the interleukin-2 receptor CD25 and the adhesion molecule ICAM-1 (CD54) were up-regulated after treatment with Biobran MGN-3. Treating highly purified NK cells with Biobran MGN-3 also resulted in increased levels of TNF-α and IFN-γ secretion in conjunction with augmentation of NK cell cytotoxic function. Furthermore, addition of Biobran MGN-3 to interleukin-2-activated NK cells resulted in a synergistic induction of TNF-α and IFN-γ secretion.
   
  Ghoneum M. (Drew Univ., USA), Jewett A. (UCLA, USA) : Cancer Detection and Prevention Vol.24/Issue 4, 2000
   
(e) The effect of modified rice-bran arabinoxylan on NK activity of human peripheral blood lymphocytes.
   
  The effects of Biobran MGN-3 and its molecular fractionates on NK activity have been investigated High molecular weight fraction (M.W. 10-50 kDa) obtained by gel filtration technique using Sephadex G-25 and G-75 was added to human peripheral blood lymphocytes. After 3-days incubation, NK activity was determined. Fluorescence-labeled K-562 cell line was used as the target cells and the activity was determined gy fluorescence method using Tere Scan. The same experiment was conducted in the presence of IL-2.
   
  In these experiments no significant differences were found in the activation of NK cells either by BioBan Biobran MGN-3 or by the high molecular weight fraction. However, when IL-2 was added at the same time and incubated, increased NK activity was observed compared to the addition of IL-2 by itself. This indicates that Biobran MGN-3 activates NK cells in the presence of IL-2 and that such activity is also present in the high molecular weight fraction.
   
  Ueda Y., Shimomura C. (Chiba Univ., JAPAN) : The abstract of the 2002 Annual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry
   

(2)

Anti-Viral Effect
   
  Anti-HIV activity in Vitro of Biobran MGN-3
   
  Anti-HIV activity of Biobran MGN-3 has been evaluated in vitro. First, the inhibitory action against the production of the HIV-1 p24 antigen was evaluated. Mononuclear cells collected from three healthy subjects were incubated with the HIV-1 SF strain at 37ºC for one hour in the presence of Biobran MGN-3. The Biobran MGN-3 concentrations were 0-100 μg/ml. Biobran MGN-3 concentration-dependently inhibited the production of the HIV-1 p24 antigen (Figure 8).
   
  Next, the inhibitory action against syncytia formation was evaluated. Mononuclear cells from five AIDS patients were incubated with PHA in the presence of Biobran MGN-3 at 37ºC for seven days. Biobran MGN-3 concentrations were 0-100 μg/ml. Biobran MGN-3 concentration-dependently inhibited syncytia formation, and the maximum inhibitory rate was 75% with 100 μg/ml 7) (Table 1).
   
 
 
Table 1

Inhibition of Syncytia Formation by Biobran
Biobran dosage (μg/ml) Syncytia formation (SF)
No. of SF % Inhibition
0
12.5
25
50
100		 42.0±8
25.8±7
21.5±5
15.8±4
10.5±3		 00.0
38.5
50.0
62.5
75.0
Figure 8 __  
   
  Ghoneum M. (Drew Univ., USA):Biochemical and Biophysical Research Communications 243, (1998)
   

(3)

Anti-Tumor Effect
   
 
(a) Study on the growth inhibiting component of cancerous cells in culture cell lines derived from modified rice-bran arabinoxylan
   
  In this study, the growth inhibiting effect of Biobran MGN-3 modified rice-bran arabinoxylan on various cancerous cell lines such as HL60, K562, HLE was investigated, and the potential differentiation-induction of HL60 and K562. Biobran MGN-3 was added to the culture cell lines. After 3-days incubation, the rate of live cells decreased in all cells lines as the amount of Biobran MGN-3 added increased.
   
  After precipitation with ethanol, the precipitate was mixed with distilled water and the supernatant was fractionated using Sephadex G-25 column. It was fractionated into 3 fractions (A,B,C) which were added to the culture cell lines. Growth inhibiting effects were observed with C fraction for HL60 and K562 and with B and C fractions for HLE. In addition, from Giemsa stain and nonspecific esterase stain, potential differentiation-induction was indicated for HL60 and K562. These results indicate that Biobran MGN-3 has components which show growth inhibition of cancerous cells and the potential differentiation-induction for HL60 and K562.
   
  Masada M. (Chiba Univ., JAPAN) : The abstract of the 2002 Annual Meeting of the Japan Society for Bioscience, Biotechnology and Agrochemistry
   
(b) Effectiveness of Biobran MGN-3 against Tumor cell Growth
   
  The direct effect of Biobran MGN-3 on skin cancer cell growth and cytokine production has been evaluated. Incubation of a squamous cell carcinoma [SCC13] cell line with Biobran MGN-3 arrested tumor cell growth (30% decrease in cell number after 48 hours and 50% at 72 hours of culture) as compared to control SCC13 cells grown in a MEM media alone, which continued to increase in cell number.
   
  Employing flow cytometry procedures, analyses showed that after 16 hours of treatment of SCC13 cells with Biobran MGN-3, there was a five-fold increase in intracellular levels of interleukin 10 [ IL-10], but no apparent change in content of interferon-γ [INF-γ]. ELISA analyses showed 8-fold higher levels of IL-10 and a 3-fold increase in IL-12 in the culture media of SCC13 cells. However, little change in INF-γ concentration was detected. The effect of Biobran MGN-3 on other cell lines, such as normal and tumor breast cells and prostate cancer cells, was also evaluated.
   
  These findings indicate that Biobran MGN-3 acts by not only enhancing the host immune function but also through a direct alteration of tumor cell growth and production of cytokines. These findings may offer a mechanism of action which could explain the high clinical success and impressive benefits of Biobran MGN-3 treatment observed by the author over a period of 4 years.
   
  Ghoneum M. (Drew Univ., USA) : The abstract of the 8th International Congress on Anti-Aging & Biomedical Technolgies Conference Proceedings Manual, 2000
   

(4)

Complementary Effect in Cancer Therapy
   
 
(a) Evaluation of NK cell Activity and survival rates in multi-immuno therapies for various cancer patients
   
  A study has been reported which was designed to determine whether or not the administration of Biobran MGN-3 could have an apothanasia effect and improve the Quality of Life (QOL) for 205 progressive and partially metastasized cancer patients in stages late III-IV, after surgery. Participants in the clinical study were hospitalized patients in the Sano Surgical Clinic, Japan. They were treated with complementary alternative medicines and conventional anticancer medicines with low side effects.
   
  The 205 patients hospitalized for 6 months were grouped into two groups, viz. 109 patients (control group) treated with the clinic's standard complementary alternative medicines, and 96 patients who were in addition given Biobran MGN-3 (Biobran MGN-3 group) for one year and a half.
   
  All the patients were measured for natural killer activity as an indication for the variation of immunoparameter. Simultaneously, the QOL of the patients were also checked. The NK cell activities of the patients after surgery were low on average, however by the administration of Biobran MGN-3, NK activity was observed to increase and the apothanasia ratio also increased; the higher the NK activity of patient, the higher the apothanasia ratio that was observed. (Table 2) The findings indicated that NK activity can be used as a pathological index of progressive cancers. QOL improvement was also observed on administration of Biobran MGN-3.
   
  Table 2: Relation between total survival rate, NK activity and survival rates in 2 groups
 
Group Biobran Group Control Group
Total survival rate 52/96 (54.2%) 19/63 (33.9%
NK activity category
Less than 19.9%
20%-40%
More than 40%
17/40 (42.5%)**
18/35 (51.4%)*
17/21 (81.0%)
2/16 (12.5%)
7/25 (28.0%)
10/15 (66.7%)
  % significant to the control group **p<0.01 *p<0.05
   
  Takahara K. (Sano Surgical Clinic, JAPAN) : The Abstract of the 3rd Annual Meeting of the Japanese Society for Complementary & Alternative Medicine & treatment, 2000
   
(b) Immunomodulatory and Anti-Cancer Properties of Biobran MGN-3 in 5 Patients with Breast Cancer
   
  Five patients with breast cancer were given Biobran MGN-3 at 3g/day, and their NK cell activities were measured by 4-hr 51Cr-release assay using K562 tumor cells as targets. The Results showed:
   
 
(i) Patients having a low level of basal NK activity (12.7-58.3%) at effector : to target (E:T) ratios of 12 and 100:1, had their NK activity significantly enhanced by Biobran MGN-3 treatment (41.8-89.5%) at the same E:T ratios.
   
(ii) The augmentation in NK activity was detected as early as 1-2 weeks post treatment and was further increased with continuation of administering Biobran MGN-3.
   
(iii) Two patients who participated early in the study (6-8 months) went into complete remission.
   
  Ghoneum M. (Drew Univ., USA) : The abstract of An American Association for Cancer Research Special Conference, 1995
   
(c) NK immunomodulatory function in 27 cancer patients by Biobran MGN-3, a modified arabinoxylan from rice bran
   
  Biobran MGN-3 immunomodulatory function was examined in 27 cancer patients. The patients had different types of advanced malignancies: 7 patients had breast CA, 7 prostate, 8 multiple myeloma (MM), 3 leukemia and 2 cervical CA. All patients were under treatment with conventional therapy and were also given 3g of Biobran MGN-3 daily, then NK activity was examined at 2 weeks, 3 months and 6 months. Activity of NK cells was examined by 51Cr-release assay using K562 tumor cells as targets, at effector : taget ratios from 12:1-100:1. The results showed that:
   
 
(i) Patients had a low level of basal NK activity.
   
(ii) Treatment with Biobran MGN-3 caused a remarkable increase in NK activity after 2 weeks. The percentage of induction were as follows: breast CA 154-332%, prostatic 174-385%, leukemia 100-240%, MM 100-537%, and cervical CA 100-275%.
   
(iii) Enhancement of NK activity continue to rise at 3 months and 6 months after treatment.
   
  Ghoneum M. (Drew Univ., USA): The abstract of 87th Annual Meeting of the American Association for Cancer Research, 1996
   
(d) A case in which Biobran MGN-3 was used as a supplemental treatment when curing metastasis from lung cancer
   
  A case in which a favourable outcome was obtained as a result of using Biobran MGN-3 as a supplement in treatment of a lung cancer which had spread from the lungs to a wide area of bones is presented in this report.
   
  The patient was a 67 year old male. In August, 1996, he had consulted a doctor because of a drastic decrease in body weight and complained of severe coughing and expectoration. The diagnosis was a complication of lung cancer (squamous cell carcinoma) and tuberculosis (M. tuberculosis). After preferentially treating the tuberculosis with antibiotics, treatment of the lung cancer by irradiation was initiated in October in parallel with the tuberculosis treatment. In December of the same year, the lower half of his right lung was excised, removing the tumor. After irradiation treatment, he left hospital in January, 1997.
   
  In June of the same year, he consulted the doctor again, complaining of pain in the right breast. After diagnosis by bone scintigram, multiple osteo-metastasis was confirmed. The tumor had spread mainly to the ribs of the right chest, but there was also a wide dispersion to the bones of the entire body. From July, sustained-release morphine morphine was administered as analgesic. Meanwhile, administration of Biobran MGN-3 was started at 3g per day from the end of May. From January, 1998, his pain decreased. While Biobran MGN-3 was continually administered, the slow-release morphine was gradually reduced and in June, the morphine was stopped altogether. The tumor marker ICPP was 16.8ng/ml when the recurrence was confirmed and gradually reduced to 7.6ng/ml in December 1997 and 6.7ng/ml in June 1998. A remarkable improvement was shown in the bone scintigram and an obvious retraction of the tumor spread to the bones was confirmed. NK cell activity was as low as 9.0% when the disease recurred but it gradually increased and is now maintained at a high level.
   
  Sobajima T. (Hoshigaoka Kosei Nenkin Hospital, JAPAN) : The Abstract of the 2nd Annual Meeting of the Japanese Society for Complementary & Alternative Medicine & Treatment, 1999
   
(e) Applications of Biobran MGN-3 in Post Conventional Therapy
   
  The concept of Tumor Dormancy therapy is becoming a major concept of cancer therapy in Japan. The basic therapeutic policy is to prolong the patient's life while maintaining a high quality of life. Dr Tunekawa performs the therapy when this is the patient's wish and he regards improvement of quality of life an important therapeutic objective. He reported that he has thirty-four cancer patients receiving a combination of dormant chemotherapy and complementary and alternative medicine (CAM), and described the therapeutic course for three of these:
   
 
(i) Patients (primary disease): Gastric cancer in 3, pulmonary cancer in 3, malignant lymphoma in 2, colon (rectal) cancer in 6, breast cancer in 3 and others in 17
   
(ii) Treatment period: 6-18 months
   
(iii) Case studies
 
1. T.S. (60), F, gastric cancer (stage IV), cancerous peritonitis: The patient was operated on for scirrhous gastric cancer in January 2000. In February 2000, she developed cancerous peritonitis and underwent gastrectomy at stage IV. CA19-9 was 108. In August 2000, she visited our clinic with complaints of abdominal pain, constipation, anemia and anorexia. CA19-9 was 390 and NK activity was 25.6. She received a combination of TS1 and holistic therapy. Biobran MGN-3 3 g/day was given for immune enhancement. She responded 1 month later with CA19-9 being 63. She showed a steady decrease in tumor markers and increase in NK activity. In August 2001, 11 months later, CA19-9 became 25 and NK activity was 51.5. Almost no subjective symptoms persist and she is now well nourished.
2. F.A. (46), F, breast cancer, metastases to the lumbar vertebra and uterine body: The patient was operated on for breast cancer in July 1998 and was treated with hormones and anticancer agents. She was found to have metastases of the lumbar vertebra in March 2001 and uterine body in April 2001, and underwent hysterectomy in May 2001. She was discharged home on Taxol and Paraplatin. In July 2001, she visited Dr. Tunekawa with a complaint of bone pain. At that time, CA was 153, NCC-ST was 439 and NK activity was 9.3. Paraplatin was continued and holistic therapy was started. Biobran MGN-3 3 g/day was given for immune enhancement. Two months later, CA became 18, NCC-ST was 28.9, NK activity was 22.0 and pain was resolved. Subsequently, she showed a steady decrease in tumor markers and increase in NK activity. In July 2002, CA was 14, NCC-ST was 3.2, NK activity was 59, there was no pain and bone scintigraphic findings were less remarkable. Now she is well nourished and is enjoying playing the drums.
   
  Tunekawa H. (Tokai Society for Promotion of Holistic Medicine, JAPAN): Abstract from Biobran Workshop in Berlin, 2002
   
(f) Assessment of Biobran MGN-3 in Treatment of Progressive Cancer
   
  Dr Mizukami has experience of giving Biobran MGN-3 to 97 advanced cancer patients. The kinds of cancer were stomach cancer, large intestine cancer, breast cancer, lung cancer, pancreatic cancer, liver cancer, bile duct cancer, pharyngeal cancer, ovarian cancer, glandulae cervicales uterine cancer, corpus uterine cancer, renal cancer, thyroid cancer, prostatic cancer, cancer of the oral cavity, multiple myeloma, etc. Although the patients had already received operations, chemotherapy, radiotherapy, etc. in large hospitals, the progress of most patients was poor. They had suffered metastasis and recurrences, and requested immunotherapy and visited Dr Mizukami's hospital. In almost all examples of Biobran MGN-3 use, neither chemotherapy nor radiation were used at the same time. Clinical observation and inquiry were carried out in detail regarding the Quality of Life (QOL) of patients taking Biobran MGN-3. Note was taken of phenomena common to patients taking Biobran MGN-3.
   
  There were some cases in which the QOL clearly improved after taking Biobran MGN-3. Generally, although the QOL of advanced cancer patients tends to fall in a straight line with progress of time, patients taking Biobran MGN-3 showed this tendency to a reduced extent, and had a tendency to live longer with good QOL. Examples of long-term survival were also seen.
   
  The following concrete observations were made concerning QOL:
 
(i) Although control of sharp pain is not easy and morphine is used in many cases for advanced cancer patients, some of those who took Biobran MGN-3 did not need to use morphine. Even when morphine was used, a tendency to use smaller quantities of morphine was observed.
(ii) Overall a decreased tendency to feel languid was observed.
(iii) A decreased tendency for loss of appetite was observed.
(iv) A tendency to be able to stay at home and to feel good just before death was observed.
(v) Retention of clear consciousness even just before death, and a tendency to be able to talk to their family was observed.
   
  Dr Mizukami concluded that for advanced cancer patients, the fact that QOL does not decrease so readily when Biobran MGN-3 is taken, is important for future cancer medical treatment.
   
  Mizukami O. (Health Promotion Research Institute New Life Layman Foundation, JAPAN) : Abstract from Biobran Workshop in Berlin, 2002
   

(5)

Effect of Apoptosis
   
  Biobran MGN-3 sensitizes Human T cell leukemia cells to Death Receptor (C95)-induced Apoptosis
   
  In this study, the effect of Biobran MGN-3 on death receptor-induced apoptosis in human leukemic HUT 78 cell line was investigated. HUT 78 cells were pretreated with Biobran MGN-3 and then were incubated with agonistic antibody against death receptor (Fas, CD95). Apoptosis was determined by propidium iodide technique using FACScan. Activation of caspase 3, caspase 8 and caspase 9 was determined by flow cytometry. Mitochonodrial membrane potential was measured with DIOC6 dye using FACScan. Expression of CD95 and BCl-2 were measured by flow cytometry.
   
  Biobran MGN-3 was found to enhance anti-CD95 –induced apoptosis in a dose-dependent manner. Increased cell death was correlated with increased depolarization of mitochondrial membrane potential and increased activation of caspase 3, caspase 8 and caspase 9. Biobran MGN-3 treatment had no effect on the level of expression CD95 but it caused down regulation of BCl-2 expression. These results suggest that Biobran MGN-3 increases the susceptibility of cancer to undergo apoptosis mediated by death ligands, which may be relevant to anti-cancer activity.
   
  Ghoneum M. (Drew Univ., USA) : Cancer Letter, 2003
   

(6)

Activation of Vital Defence
   
 
(a) Active oxygen radical scavenging by Biobran MGN-3
   
  Investigations on scavenging of oxygen radicals by Biobran MGN-3 and its fractionates have been reported. Biobran MGN-3 was fractionated into components using a Sephadex G-25 column. Each component was named in descending order as L (L > 10,000 molecular weight), M (10,000 > M > 3,000 molecular weight), and S (3,000 molecular weight> S).
   
  Active enzyme scavenging activity was evaluated by measuring the superoxide anion radical (•O2) scavenging activity, hydroxyl radical scavenging activity of the Fenton reaction (•OH), and the scavenging activity of the hydroxyl radical generated by ultraviolet irradiation.
   
  The results of the measurements are shown in Table. The S (low molecular weight) fraction excelled all others in the inhibition of •OH generation caused by •O2 and ultraviolet irradiation. High scavenging activity was observed in all fractions for the scavenging activity of •OH generated in the Fenton reaction . (Table 3 )
   
  Table 3
  Scavenging Activity of Biobran MGN-3 on Active Oxygen radicals
(•O2 and •OH and UV induced •OH)
   
 
Kind of Active Oxygen and SOD activity Scavenging ratio of Superoxide anion radical (%) SOD activity (U/ml) Scavenging ratio of UV induced Hydroxyl radical (%)
20 2.0
(mg/ml)		 0.2 20 2.0
(mg/ml)		 0.2 20 2.0
(mg/ml)		 0.2
BioB

BioB-L

BioB-M

BioB-S		 64.6

39.9

49.5

90.4		 23.0

10.4

15.6

68.1		 4.4

0

0

26.4		 7.6

5.0

7.2

70.5		 0.9

0.8

1.4

15.7		 0

0

0

2.6		 94.9
(72.6)
97.2
(41.8)
97.0
(45.4)
96.5
(71.0		 78.9
(35.9)
34.4
(16.5)
68.4
(9.9)
55.1
(54.9)		 3.3
(11.5)
3.3
(1.0)
8.7
(3.9)
4.2
(19.6)
  ·O2:HPX-XOD reaction,·OH:Fenton reaction
  ·OH by UV light reaction:365nm,4×103J/m2/min×5
   
  Tazawa K. (Toyama Medical and Pharmaceutical Univ., JAPAN) : Biotherapy Vol.14, 2000
   
(b) A basic study of arabinoxlan compound (Biobran MGN-3) on the activation of vital defenses.
   
  In this study, through an animal experiment, the influence of Biobran MGN-3 as regards its biophylactic activation on survival rate in the lipopolysaccharide (LPS)-induced lethal sepsis model was observed.
   
  In the experiment, BALB/c mice (male, 5-7 weeks old) were used. 20mg/kg and 200 mg/kg of Biobran MGN-3 were dissolved in 0.5ml of PBS, and via an oral zonde, administered every other day for two weeks, in total seven times. 0.5 ml of PBS was administered orally in the same interval for the control group. 200μg/mouse of LPS was administered intraperitoneally 12 hours after the final oral administration, and the conditions of the mice were observed over time. In another experiment, 100 μg/mouse of LPS was administered intraperitoneally in the Biobran MGN-3 group and control group, the mice were euthanized 0, 2, 4, 8 hr after the LPS administrations, and peripheral blood was collected from the heart. Serum was separated, and IL-6 and TNF were measured. IL-6 activity was measured using the B9 cell line and TNF activity was measured by bioassay in the WEHI164-13 cell line.
   
  As shown in Figure 9, when 200 μg/mouse of LPS was administered, the survival rate significantly improved in groups where 20 mg/kg or 200 mg/kg of Biobran MGN-3 was administered everyday, compared with that in the control group (20 mg/kg Biobran MGN-3 group vs. control group, p = 0.0456; 200 mg/kg Biobran MGN-3 group vs. control group, p = 0.0232, by Mantel-Cox test). When 100 μg/mouse of LPS was administered, all the mice survived in groups where 20 mg/kg or 200 mg/kg of Biobran MGN-3 was administered everyday, while 3 out of 10 mice died in the control group.
   
  To establish the mechanism for improvement of the survival rate by Biobran MGN-3, blood concentrations of IL-6 and TNF were measured. In the experimental group where Biobran MGN-3 was administered, compared with the control group, the blood IL-6 level was significantly lower two hours after the administration of LPS (control group 702.9 ± 24.7 ng/ml, Biobran MGN-3 group 403.1 ± 59.6 ng/ml; p < 0.01); however, 8 hr after the administration, it significantly increased (control group 88.5 ± 50.0 ng/ml, Biobran MGN-3 group 441.0 ± 115.0 ng/ml; p < 0.05). Meanwhile, 4 hr after LPS administration, the blood TNF level significantly increased in the Biobran MGN-3 group compared with those in the control group (control group 492 ± 187, Biobran MGN-3 group 1816 ± 307 pg/ml; p < 0.01).
   
  In the LPS-induced lethal sepsis model, multiple organ failure was assumed to be induced by a large amount of inflammatory cytokines (IL-1, 6, TNF-α) released from the reticuloendothelial system cells of the whole body, which leads to death. In this study, significant improvement of the survival rate by the administration of Biobran MGN-3 was observed. Possible causes were that Biobran MGN-3 intake inhibits the production of histotoxic cytokines generated from macrophages or Biobran MGN-3 blocks the route to histotoxicity at the target cell level.
   
  Sudo N., Kubo C. (Kyushu Univ., JAPAN) : The Japanese Journal of Clinical and Experimental Medicine, Vol.78, 1, 2001
   
(c) Reduction in weight loss of mice treated with Cisplatin due to Biobran MGN-3.
   
  In treatment of cancers platinum based drugs frequently cause substantial side effects, such as nausea, vomiting, nephropathy and hypomagesemia due to damage of renal tubules (Lajer & Dangaard 1999). Futhermore, in addition to hearing loss and peripheral neuropathy, myelosuppression is one of the most devastating suppressive side effects (Prestayko et al. 1979) leading to immunocomopromised states. Therefore, any reducution of the side effects of cisplatin would be worthy of achieving. To do this, the effect of Biobran MGN-3 was studied on amelioration of weight loss of mice under tolerable maximal dose of cisplatin.
   
  One week before cisplatin administration, Biobran MGN-3 was started to be administered to two groups of mice at a concentration of 10mg/ml of Biobran MGN-3 (dry weight) in water or by intraperitoneal injection in a volume of 0.1 ml at the same concentration of Biobran MGN-3 in PBS. The dose of 1 mg of Biobran MGN-3 per mouse was calculated from that recommended for human usage (50mg/kg). One shot of cisplatin was administered intraperitoneally in a volume of 0.1 ml at the concentration of 15mg/kg of cisplatin in PBS containing 0.5% DMSO as a vehicle. Two groups of mice received a gavage of water or intraperitoneal administration of PBS and one week later cisplatin was administered to the both groups.
   
  Weight loss after intraperitoneal injection of cisplatin occurred the next day in both groups with and without administration of Biobran MGN-3. The greatest weight loss was observed in both groups at the 5th day after cisplatin treatment, with or without Biobran MGN-3 orally as well as intraperitoneally. The greatest weight loss occurred in mice given cisplatin without Biobran MGN-3 administration. Although loss of body weight in mice given Biobran MGN-3 appeared to be close to the 20% shown for in the groups of cisplatin treatment without Biobran MGN-3, no mice died, nor did any show diarrhea or rectal bleeding, frequent side effects of cisplatin. In the recovery phase, earlier weight gain took place in the groups of mice given Biobran MGN-3 than those without it.
   
 
  Figure 10          
   
 
  Figure 11          
   
  Endo Y., Kanbayashi H. (Mac Master Univ., CANADA) : Pharmacology and Toxicology, 2003
   
(d) The Effect of Biobran MGN-3 on Cisplatin and Adriamycin Induced Toxicity in the Rat
   
  Biobran MGN-3 is derived from rice bran and is produced by the partial hydrolysis of the water soluble hemicellulose fraction of rice bran by carbohydrases derived from Lentius edodes mycelia [US Pat. 5560914]. Biobran MGN-3 has been shown to be a biological response modifier producing an increase in natural killer cell activity in immunocompromised patients [Int. J. Immunother. 14 (1) 1998].
   
  Aim: To prevent gross pathological changes and weight loss produced by a single dose of cisplatin (CIS) or adriamycin (ADR) by daily oral dosing of 5 or 50mg/kg Biobran MGN-3. Following an acclimation period of 13 days, male Spraque-Dawley rats were selected for test based on body weights and assigned (10 rats/group) to each of the following (dose stated as mg/ml):
 
(1) Biobran 5 gm PO+Veh IP
(2) Biobran 50 gm PO+Veh IP
(3) Biobran control PO+CIS 8mg IP
(4) Biobran 5 gm PO+ CIS 8mg IP
(5) Biobran 50 gm PO+ CIS 8mg IP
(6) Biobran Control PO+ADR 10mg IP
(7) Biobran 5 gm PO+ ADR 10mg IP
(8) Biobran 50 gm PO+ ADR 10mg IP
   
  Rats received oral (PO) Biobran MGN-3 (suspended in distilled water) or vehicle (veh) daily for 11days. The chemotherapeutic agents or veh were administreted to each rat by a single IP injection on Day 3. Rats were observed for clinical sings daily for 11 days. Body weights were recorded every other day. On Day 11, all animals were euthanized by CO2 inhalation and necropsied. Gross appearance of major organs was evaluated and the presence of gastrointestinal damage noted.
   
  Results: Five rats from Group 3, 3 from Group 5, and 1 from Group 4 died between D7 and 11. Rats receiving Biobran MGN-3 at 5 or 50 mg/kg PO showed a statistically significant increase in body weight (+72%). Rats receiving CIS or ADR alone showed a smaller increase in body weight (-1.5%, CIS; +30%, ADR). Rats receiving Biobran MGN-3 at 5 or 50 mg plus CIS or ADR had a significantly greater weight gain than that observed with the chemotherapeutic agent alone (Biobran MGN-3 5 mg in CIS treated rats, +11% and +46% in ADR treated rats). Biobran MGN-3 50 mg in CIS treated rats, +44% and +43% in ADR treated rats.Surviving rats receiving Biobran MGN-3 appeared healthier, gained weight and had a lower incidence of gross intestinal pathology than those taking CIS or ADR without Biobran MGN-3.
   
  Table 4
  Effect of Biobran on Body Weight Loss Induced by Cisplatin and Doxorubicin
 
Treatment (all intraperitoneal) Day 0 Day 3 Day 5 Day 7 Day 9 Day 11
5mg/kg PO+Vehicle
50mg/kg PO+Vehicle
Control PO+Cp8mg/kg
5mg/kg PO+Cp8mg/kg
50mg/kg PO+Cp8mg/kg
Control PO+Dx10mg/kg
5mgn/kg PO+Dx10mg/kg
50mg/kg PO+Dx10mg/kg		 100
100
103
99
101
103
101
101		 100
98
101
99
99
100
100
101		 100
100
82
85
92
88
92
92		 100
97
69
76
90
82
89
87		 100
97
55
68
85
77
86
85		 100
97
57
65
84
76
85
83
  PO-oral, Dx-Doxorubicin, Cp-Cisplatin
   
  Jacoby H. I. (USA) : Journal of Nuturaceuticals, Function & Medical Foods, Vol.3 (4) 2001
   
(e) The effect of Biobran MGN-3 on radiation therapy induced toxicity in the mouse
   
  This study, investigates the modifying effect of Biobran MGN-3 on radiosensitivity as expressed in bone-marrow death caused by total body irradiation. First, with possible clinical application in mind, the authors studied each effect quantitatively across a wide range of radiation dosage between 4.5 Gy and 8.5 Gy.
   
  Four or 5-week-old SPF male BALB/c mice (F2) were the subject of these experiments. In the Biobran MGN-3 groups Biobran MGN-3 was added to the food of F2 mice at 50 mg/kg body weight. There were 10-50 mice per group. Starting at two days after caging, the food was changed in the F2 group to Biobran MGN-3 added food. Four and 5-week-old mice were irradiated after feeding with Biobran MGN-3 for 15 days and 8 days.. Body weight was measured 3 times a week and the number of deaths was checked every day. In some cases mice used in the experiment had been given Biobran MGN-3 for two weeks before irradiation in other cases administration of Biobran MGN-3 was only started after irradiation.
   
  Although in the F2 group, mortality of mice due to bone-marrow death was observed from the seventh day after irradiation, mice tended to die a little later in the Biobran MGN-3 groups. In the F2 group, LD50 was approximately 5.15 Gy and the dose reduction factor (DRF) was approximately 1.14. As for body weights, a tendency towards a heavier body weight was maintained in the Biobran MGN-3 groups. The effect of starting Biobran MGN-3 administration earlier, suggested that it was preferable for Biobran MGN-3 to be given prior to irradiation.
   
  The irradiation dose of mice was calculated as being 1.21-fold of the above dosages at their body center, meaning that LD50 in the control group was equivalent to 6.23 Gy. Though the radioprotective effect of Biobran was DRF = 1.14, which was not a large effect, no side effects were observed in the present study.
   
  Nakatugawa S. (Nagoya Univ., JAPAN) : The Report of Nagoya Univ., 2003
   
(f) Effect of Biobran MGN-3 on Experimental Liver Dysfunction in Rats
   
  This study investigated the effect of Biobran MGN-3 on liver dysfuncution. The effect of Biobran MGN-3 on the development of experimental liver dysfunction in rats induced by galactosamine (GalN) and acetaminophen (AAP) was investigated. To develop experimental liver dysfunction, GalN was administered in Experiments 1-3 and AAP in Experiments 4 and 5.
   
  In Experiment 1, Biobran MGN-3 of different concentrations was administered intraperitoneally to the rats and after 1 hour GalN was administered at the rate of 800 mg/kg.
  In Experiment 2, Biobran MGN-3 was administered orally, and fractionated Biobran MGN-3 of high molecular weight and low molecular weight was administered intraperitoneally. After 1 hour, GalN was administered at the rate of 800 mg/kg.
  In Experiment 3, after being heated, hydrolysed and processed with ion exchange resin, Biobran MGN-3 was administered intraperitoneally. After 1hour, GalN was administered at the rate of 800 mg/kg.
  In Experiment 4, Biobran MGN-3 was administered intraperitoneally or orally, and after 1 hour, AAP was administered at the rate of 700 mg/kg.
  In Experiment 5, after being heated, hydrolysed and processed with ion exchange resin, Biobran MGN-3 was administered intraperitoneally, and after 1 hour, AAP was administered at the rate of 500 mg/kg.
   
  In all experiments, rats were dissected 24 hours after administration of GalN or AAP and their serum transaminase (GOT, GPT) levels were determined.
   
  Results
  Experiment 1: In all groups where Biobran MGN-3 was administered, the increases of serum GOT and GPT activities due to GalN-induced liver dysfunction were significantly suppressed, compared with those in the control group. The suppressing effect of Biobran MGN-3 on GalN-induced liver dysfunction peaked at 20 mg/kg and no further change was observed with higher concentration of Biobran MGN-3 in the suppressing effect on GalN-induced liver dysfunction.
  Experiment 2: In all groups where Biobran MGN-3 or high/low molecular weight fractions of Biobran MGN-3 was administered intraperitoneally, the increases of serum GPT activities due to GalN-induced liver dysfunction were significantly suppressed, compared with those in the control group. The suppressing effects was similar to those observed with Biobran MGN-3 itself.
  Experiment 3: In groups where hydrolyzed Biobran MGN-3 was administered, the increases of serum GOT activities due to GalN-induced liver dysfunction were significantly suppressed, compared with those in the control group.
  Experiment 4: In groups of where Biobran MGN-3 was administered intraperitoneally or orally, the increases of serum GOT activities due to AAP-induced liver dysfunction were significantly suppressed, compared with those in the control group.
  Experiment 5: Corresponding to the results of Experiment 3, the effect of hydrolyzed Biobran MGN-3 on AAP was assessed. In groups where hydrolyzed Biobran MGN-3 was administered, the increase of serum GOT activities were significantly suppressed, compared with those in the control group.
   
  Thus, Biobran MGN-3 was confirmed to have a suppressive effect on GalN-induced or AAP-induced liver dysfunction. The active constituent appears not to be hydrolyzed by HCl.
   
  Yamada T. (Chiba Univ., JAPAN): The abstract of the 6th Annual Meeting of Japanese Association for Dietary Fiber Research, 2002
   
(g) Oral administration of Biobran MGN-3 alleviates common cold syndrome in elderly people
   
  For high-risk groups such as the elderly or children, preventive measures against infections, such as influenza vaccination, and drastic precautions against secondary bacterial infection are important. When community-acquired pneumonias that may have developed through exacerbation of the common cold were investigated by age, the risk of secondary infection by bacteria became higher in elderly people over 75 years old. The risk of acquiring concomitant pneumonia is also high in elderly patients with neurological disorders who are at high risk of aspiration. In the elderly whose immunocompetence declines due to various factors, the clinical usefulness of Biobran MGN-3 against the development of the common cold was therefore evaluated.
   
  Elderly subjects were selected from those under the care of "Atreyu Uozaki" a health care facility for the elderly in Kobe, Hyogo, between January and March in 2002, who were not seriously ill, and who consented to the present study. A Biobran MGN-3 fraction (HRB) was used as the test food and rice bran containing mainly the water soluble component, was used as the control (RB).
   
  For the symptoms of common cold (fever, headache, fatigue, chill, cough, sputum, nasal discharge, nasal obstruction, sore throat, chest pain), the number of days when even a single symptom of common cold was manifest was counted. Each symptom was converted to scores depending on its level (no symptom = "0", mild = "1", moderate = "2", severe = "3") and the "common cold symptom score" was calculated by dividing the accumulated score of each subject by the number of intake days.
   
  Results: Among individual symptoms, manifestation frequencies of "cough," "fatigue," "fever" and "sore throat" were high on starting intake of both foods. The number of days when symptoms were manifest was fewer in the HRB group than in the RB group. Looking at the common cold symptom score, the RB group showed a high score in total. Although the score for "nasal symptoms" was lower in RB group, the scores for common symptoms such as "cough," "fatigue" and "fever" were higher. Hence it was concluded that common cold symptoms were less prevalent in the HRB group.
   
  This study demonstrated that when HRB was taken orally by elderly patients with the common cold, the period of symptom manifestation was shortened, aggravation of symptoms was halted, and the necessity for symptomatic treatment was reduced through the extract's immunostimulatory action.
   
  Tazawa K. (Toyama Medical and Pharmaceutical Univ., JAPAN) : Joural of Traditional Medicines, 20 (3), 2003
   

(7)

Anti-Allergology Effect
   
 
(a) Evaluation of the effects of asthma prevention and symptom reduction Biobran MGN-3 in model asthmatic mice
   
  The effect of asthma prevention and symptom reduction by Biobran MGN-3 has been evaluated using TDI-induced asthma model mice.
   
  First, 2 g/litre of Biobran MGN-3 was diluted with drinking water and given daily to the above model mice (BALB/c, female), which were divided into 4 groups (A-D) as follows:
   
  Group A: One month pre-administration of Biobran MGN-3 and administration during TDI sensitization period and challenging period.
  Group B: One month pre-administration of Biobran MGN-3 and administration until the end of TDI sensitization period.
  Group C: Administration of Biobran MGN-3 only during TDI challenging period.
  Group D: control group.
   
  Group B was for the assessment of preventive effect and Group C was for the assessment of symptom reduction effect. The effect of Biobran MGN-3 was evaluated by blood histamine concentrations, the number of eosinophils in BALF, TDI earlobe application test, and blood IgG1, IgG2a, IgE-type specific antibody values at sensitization.
   
  The peak blood histamine concentrations at 7 minutes after TDI challenge were, Group A: 2.5±0.53, Group B: 4.2±0.75, Group C: 4.3±7.8, Group D: 6.4±0.87 (ng/ml), and Biobran MGN-3 administration groups showed significantly lower values compared with the control group. In the sensitization test with various concentrations (0.01-10%) of TD