Globin Peptide MG Pharma Incorporation

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Master The Health, From Saito to World

Master The Health, From Saito to World

Globinpeptide

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In 1989, MG Pharma found that Globin Peptide (Globin Digest) had the strongest effect to decrease neutral fat (triglyceride) among several food protein digests. One of the active component of Globin Peptide was proved to be tetra peptide, Val-Val-Tyr-Pro (VVYP).

Globin Peptide

Other physiological functions of Globin Peptide are still being investigated and its anti-diabetic and anti-hypertensive effects have been found. The action mechanisms for anti-diabetes are being determined. (Click Figures to Enlarge)

We already reported that Globin Peptide prevented the intra-abdominal fat (visceral fat) accumulation in dietary obese mice.

Based on the above effects, Globin Peptide has become a health food ingredient utilized for preventing and treating metabolic syndrome.

All That Began with Pig's Weight Loss!

Fat Balance Test The pig had lost his weight after ingesting the protein digests which were under development. Actually, that experiment was failure but it led to the birth of our current product “Globin Peptide” by reverse thinking.

We have got several world patents about decreasing the triglyceride levels by food protein digests including Globin Peptide.

In 1998 we finally had gotten the FOSHU status for our product “Napple Drink” containing Globin Peptide as an active ingredient.

 

■ Action of Globin Peptide

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■ Clinical Effects * Inhibition of Postprandial Hypertriglyceridemia
  * Decrease of Fasting Blood Triglyceride
  * Decrease of Body Fat
  * Decrease of Fasting Blood Triglyceride in Type 2 Diabetes Mellitus
  * Decrease of Fasting Blood Triglyceride in Hyperlipidemic Patients
  * Improvement of Blood Glucose in Type 2 Diabetic Patients
  * Improvement of Blood Pressure in Mild Hypertensive Patients
  * Action Mechanisms
■ Effects * In vitro Pancreatic Lipase
  * Lipoprotein Lipase and Hepatic Triglyceride Lipase
  * Insulin Secretion
  * Mouse Hepatic PPAR-α Activity
  * Fatty Acid β-Oxidation
  * Adipogenic Differentiation of Fibroblast
  * Visceral Fat Accumuration in High Calorie Diet
  * Rebound of Blood Cholesterol, TG or Glucose
  * Blood Glucose Elevation in Mice GTT
  * Blood Glucose Level in Diabetic Mice
  * Blood Pressure in SHR
■ Safety * Clinical
  * Toxicological
■ Product
   
■ References
   
■ Patents
   

 

■ Clinical Effects

Clinical studies of Globin Peptide were carried out not only by us but also by third-party organizations including foreign companies and Japanese goverment.

● Inhibition of Postprandial Hypertriglyceridemia

Inhibition of Postprandial Hyperlipidemia by Globin Peptide

We shows the data abstracted from one of our articles “Hypotriglyceridemic effects of globin digest on subjects with borderline hyperlipidemia” (Kagawa K et al: J Jpn Soc Nutr Food Sci, 52, 71-77 (1999) in Japanese).

[METHODS] This clinical test was carried out using single-blind crossover trial with six of borderline hyperlipidemic Japanese. Washout period was 7 days. These subjects ingested thick soup containing 40g of butter with or without 1g of globin peptide.

[RESULTS] As shown the above figure, Globin Peptide significantly inhibited the increased serum triglyceride levels from 1hr to 6hr after ingestion of fat meal, and also decreased AUC (0-6hr) by 42% of control group (p<0.01) .

 

● Decrease of Postprandial Remnant-like Lipoprotein

Decrease of Postprandial Remnant-like Lipoprotein by Globin Peptide

There is considerable evidence to indicate that elevated levels of plasma triglyceride-rich lipoprotein remnants are associated with increased risk of premature cardiovascular disease, and then with a direct effect of remnant lipoproteins on arterial lipid accumulation and lesion formation. Increased plasma remnant-like lipoprotein-cholesterol (RLP-C) levels are clearly associated with increased risk of disease and can be significantly reduced by lipid-lowering therapy (Twickler TB et al:Circulation,109,1918 (2004) ). Terefore we determined RLP-C levels after fat-load with or without globin peptide(See above Methods).

[RESULTS] Globin Peptide inhibited postprandial increased RLP-C level at early period, and decreased its AUC(0-6hr) to 50% of control.

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● Decrease of Fasting Blood Triglyceride

Decrease of Fasting Blood Triglyceride by Globin Peptide

[PROTOCOL]
Subjects : 89 Chinese volunteers (Control: 44, Experiment: 45)
Design : Randomized placebo controlled study
Dosage : 1500 mg/day (each 500 mg, three times a day) for 70 days
[RESULTS] Significant Reduction in Serum Triglyceride (p<0.05)

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● Decrease of Body Fat

Decrease of Body Fat by Globin Peptide

[PROTOCOL] Same as above
[RESULTS] Body fat after 42 days ingestion of globin peptide significantly decreased 2.0% compared with pre-ingestion (p<0.05), and finally its reduction was 2.4% on 70th day. After 28 days follow-up periods further 0.9% reduction in body fat was observed, but its difference between control and Globin Peptide groups was not statistically significant.

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● Decrease of Fasting Blood Triglyceride in Type 2 Diabetes Mellitus

Decrease of Fasting Blood Triglyceride in Type 2 Diabetes Mellitus by Globin Peptide

[PROTOCOL]
Period: each 4 Weeks of guava tea, Globin Peptide, and combination
Subjects: 30 Japanese Diabetic Patients
Design: Well Controlled Study
Dosage of Globin Peptide: 2000 mg/day (1000 mg twice a day)
[RESULTS] Significant Reduction in Fasting Serum Triglyceride (p<0.05) and Increase of HDL-Cholesterol

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● Decrease of Fasting Blood Triglyceride in Hyperlipidemic Subjects

Decrease of Fasting Blood Triglyceride in Hyperlipidemic Patients by Globin Peptide

[PROTOCOL]
Subjects : 4 Japanese volunteers
Design : Well controlled study
Dosage : 2000 mg/day (each 1000 mg, twice a day) for 6 month
[RESULTS] Significant Reduction in Serum Triglyceride

 

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● Improvement of Blood Glucose in Type 2 Diabetic Subjects

Improvement of Fasting Blood Glucose in Type 2 Diabetic Patients by Globin Peptide

[PROTOCOL]
Subjects : 100 Chinese type 2 diabetic patients (Control: 50, Experiment: 50)
Design : Randomized placebo controlled study
Dosage : 2 g/day (each 1 g, two times a day) for 30 days
[RESULTS] Stastically Significant reduction of fasting blood glucose level (p<0.05)

Inhibition of Blood Glucose Elevation 2 hr after meal in Type 2 Diabetic Patients

[RESULTS] Significant Inhibition of postprandial blood glucose elevation (2 hr after meal) (p<0.05)

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● Improvement of Blood Pressure in Mild Hypertensive Patients

Improvement of Blood Pressure in Mild Hypertensive Patients by Globin Peptide

[PROTOCOL]
Subjects : 89 Chinese volunteers (Control: 44, Experiment: 45)
Design : Randomized placebo controlled study
Dosage : 1500 mg/day (each 500 mg, three times a day) for 70 days
[RESULTS] Significant Reduction of Diastolic pressure in Mild Hypertensive Patients (p<0.05)

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● Action Mechanisms

Action Mechanisms of Globin Peptide

Mechanisms of triglyceride (TG) lowering effect of Globin Peptide are as follows; Globin Peptide
1) decrease lipid absorption by inhibition of pancreatic lipase,
2) enhances elimination of TG from blood by activation of lipoprotein lipase and hepatic triglyceride lipase, and enhances degradation of fatty acid by activation of β-oxidation in mitochondria and by proliferation of peroxisome containing catalase, and
3) inhibits hypertrophy of adipocytes and differentiation of fibroblasts to adipocytes.

We are showing various evidence as more fully described below.

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■ Effects

Recently we are studying with a focus on antidiabetic and antihypertensive effects of Globin Peptide.

● In vitro Pancreatic Lipase

Inhibition of in vitro Pancreatic Lipase by Globin Peptide

When it was showed that Globin Peptide has inhibited fecal excretion of fat in pig using single blind test, we studied the effect of Globin Peptide on pancreatic lipase in vitro.
[INCUBATION SYSTEM] 34 u/mL of pancreatic lipase (type-VI-S, pig origin, 10000 µ/mg), 40 µmole/mL of triolein, 0.22 mg/ml of taurocholate sodium, 2 mg/mL of arabic gum, and 4, 20. 40 mg/mL of Globin Peptide in TES-NaCl buffer (pH 7.0).
[RESULTS] 40 mg/mL of Globin Peptide significantly inhibited pig pancreatic lipase activity to 60% of control (no addtion of Globin Peptide).
Globin Peptide Concentration used in this experiment are correspondent to its concentration calculated from 1g dose of Globin Peptide and volume of human gastric jucie.

This result suggest that Globin Peptide reduces fat absorption by inhibition of pancreatic lipase.

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● Lipoprotein Lipase and Hepatic Triglyceride Lipase

Activation of Lipoprotein Lipase and Hepatic Triglyceride Lipase by Globin Peptide

Lipoprotein lipase (LPL) plays a central role in lipoprotein metabolism by catalyzing the hydrolysis of both dietary and endogenous triglycerides in chylomicrons and very low density lipoprotein particles. Hepatic triglyceride lipase (HTGL) localizes on hepatic endotherial cell surface and catbolize mainly chylomicrons from portal vein. Increase of these enzyme activities decrease blood triglyceride levels. We studyed whether Globin Peptide enhances these lipases in post-heparin plasma.
[DESIGN] Ten subjects (normolipidemic 7, borderline lipidemic 2, hyperlipidemic 1), 2-period crossover study including high fat (40 g) diets with of without Globin Peptide and 2-wk washout periods. Blood was collected 170min after meal.

[RESULT] The triglyceride level after fat meal was 174±31 mg/dL in control (without Globin Peptide) group and 127±22 mg/dL (73% of control) in Globin Peptide group. It was confirmed that Globin Peptide inhibits postprandial hyperlipidemia. In control group LPL activities (control) was 241±22 µg/ml, and HTGL was 16.4±1.7 µmol/ml/hr. As shown in figure, Globin Peptide increases LPL (155% of control, p<0.01) and HTGL (122%, p<0.05). In addition significant positive coreration between blood triglyceride levels and LPL activities was found, and in Globin Peptide group the correlation line was sifted to left. Globin Peptide appear to lower the increased triglyceride level by increae of LPL and HTGL.

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● Insulin Secretion

Increase of Insulin Secretion by Globin Peptide

Globin Peptide enhanced lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities catabolizing triglyceride as menthioned above. It is not well unknown the mechanism that cause expression of these enzymes or/and its exocytotic secretion to plasma memmbranes, but insulin may be one of its candidates. So we determined blood insulin level in six normal subjects after oral ingestion of Globin Peptide using cross-over trial.

[RESULTS] Insulin AUC of control was 8.7±1.14 µuniti/mL/3hrs. Globin Peptide of 3g significantly increased AUC to 183% of control (p<0.05). Increased insulin level by Globin Peptide may cause inhibition of postprandial triglyceride elevation.

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● Mouse Hepatic PPAR-α Activity

Enhancement of Mouse Hepatic PPAR-alfa Activity by Globin Peptide

We examined the effect of globin peptide on fatty acid metabolism, particularly mitochondorial and peroxisomal fatty acid oxidations in liver.

After 10-20 days feeding of high fat diet in which protein was replaced by globin peptide in 16-50%, hepatic catalase activity as a marker of peroxisome, increased nearly to the same extent as the case of addition of 0.125% hypolipidemic drug clofibrate in mice.

Very long chain fatty acids (C20 or more) are oxidized in peroxisome. The enzymes in peroxisomes do not attack shorter chain fatty acids. Octanoyl and acetyl groups are subsequently removed from the peroxisomes in the forms of octanoyl and acetyl carnitine, and both are further oxidized in mitochondria.(from Harper's Biochemistry)

Our result shows that globin peptide enhances very long fatty acid catabolism probably via increased PPAR-α activities. Recently it was confirmed that Globin Peptide increased PPAR-α expression itself in hepatocyte in vitro.

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● Fatty Acid β-Oxidation

Enhancement of Fatty Acid beta-Oxidation by Globin Peptide

Fatty acids are converted to acetyl CoA by β-oxidation in mitochondria and/or in peroxisomes, and acetyl CoA are used in Krebs cycle and finally some of carbon atoms separated from fatty acids are excreted into exhalation. Therefore, radioacitivities in expired gas after administration of C14-labeled triolein exhibits extent of β-oxidation of fatty acids.

Globin Peptide significantly increased the radioactivities in exhalation compared to that of control especially at early time of oil ingestion.

These results suggest that Globin Peptide enhances β-oxidation in mitochondria and peroxisomes.

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● Adipogenic Differentiation of Fibroblast

Inhibition of Hypertrophy of Adipocytes by Globin Digest

Adipose tissue is no longer considered just an energy storage organ, but a real endocrine organ, which releases bioactive mediators called adipocytokines. Adipocytes, as well as other cells present within fat tissues, are capable of releasing numerous adipocytokines leading to the pathogenesis of metabolic syndrome in obese individuals.

Adipose tissue growth occurs by both hyperplasia and hypertrophy. The mature adipocytes have little capacity for cell division, and the hyperplastic capacity of adipose tissue resides in a population of fibroblast-like adipocyte precursor cells.

Globin Peptide significantly suppressed the differentiation of 3T3-LI mouse fibroblasts into mature adipocytes. This finding was a result of the cooperative study with Central Research Institute, Itoham Foods Inc. The value of the vertical axis in the graph is proportional to the number of mature adipocytes. The low values in the Globin Peptide groups mean that fibroblasts are not easy to differentiate into the mature adipocytes.

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● Visceral Fat Accumuration in High Calorie Diets

Inhibition of Increased Visceral Fat in High Fat Diet by Globin Digest

The fundamental cause of obesity is a long-term imbalance in energy intake and expenditure leading to the increase in body mass including the accumulation of subcutaneous and visceral fat. Although general obesity is an important risk factor for many diseases, several clinical studies have demonstrated that the increase in visceral fat, which is the fat located in the viscera, is most strongly related to many health conditions, including an insulin resistance, type 2 diabetes, cardiovascular disease, stroke, and metabolic syndrome.

The anti-obesity effect of Globin Peptide on the high-fat diet-induced obesity model (upper figure) and high-carbohydrate diet-induced obesity model (lower figure) was examined. The mice were fed a high-fat diet (50% calories as fat) or a normal diet (5% calories as fat) for 2 weeks. The level of the visceral fat in the high-fat diet group increased about twice of that in the normal diet group. The levels of the visceral fat and body weight in the high-fat diet with 4% or 8% Globin Peptide group significantly decreased to the same levels as those in the normal diet group.

Inhibition of Increased Visceral Fat in High Carbohydrate Diet by Globin Digest

By contrast, the levels of the visceral fat and body weight in the high-fat diet with 12.5% Globin Protein (undigested protein) group did not differ compared with those in the high-fat diet group (data not shown). These results indicate that the anti-obesity effect of Globin Peptide is the action by the peptide form, and is not the action by the protein form or amino-acid composition.

As is the case with the high-fat diet, the high-carbohydrate diet causes the obesity. The mice were given 30% sucrose in the drinking water as a high-carbohydrate diet for 5 weeks. The level of the visceral fat in the high-carbohydrate diet with 8% Globin Peptide group decreased as compared with that in the high-carbohydrate diet group.

These results indicate that Globin Peptide suppressed the visceral fat accumulation and weight gain in the high-fat diet-induced obesity model and high-carbohydrate diet-induced obesity model.

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● Rebound of Blood TG, Glucose, or Cholesterol

Inhibition of Rebound Elevation of TG and Glucose by Globin Digest

It has been confirmed that Globin Peptide had the preventive effect on the rebound of the blood TG, glucose, or cholesterol in the clinical trials.

In the upper figure, the preventive effect of Globin Peptide on the rebound of the blood TG and glucose in the clinical trial in the United States is shown. All of 30 subjects in the trial were obesity patients. Before the trial, subjects stayed in the hospital, and the levels of the body weight, body fat, blood TG, glucose, and cholesterol in the subjects were reduced to the desirable levels by the diet therapy and exercise therapy for 2 months. After that, subjects returned home, and performed the home therapy of the low-calorie diets with or without Globin Peptide for 12 weeks. The changes in the blood parameters before and after the home therapy were examined. The levels of the blood TG and glucose in the placebo group rebounded. By contrast, the levels of the blood TG and glucose in the Globin Peptide group did not rebound, and significantly decreased more than them before the administration of Globin Peptide.

The patients of the hypercholesterolemia are treated with the cholesterol-lowering statin drugs, such as mevalotin. The blood cholesterol level in the patients is reduced to the desirable level by the statin drugs, but rebounds after stopping the administration of statin drugs. For this reason, the patients of the hypercholesterolemia need to take the statin drugs over a long period of time, and statin drugs are called "the drugs taken throughout a lifetime". However, it has been reported globally that the administration of statin drugs for a long period of time increases the incidence of the side effects, such as a muscle disorder, liver function disorder, and neuropathy.

Inhibition of Rebound Elevation of Cholesterol by Globin Digest

On the other hand, the diet therapy of the low-cholesterol diets is recommended to the patients of the hypercholesterolemia by the doctors. However, it is difficult for patients to continue the diet therapy, because the quality of life (QOL) of the patients worsens, for example, the low-cholesterol diets are not delicious or only a patient takes a different diet in a family.

In the lower figure, the effect of Globin Peptide on the rebound of the blood cholesterol in the patients of the hypercholesterolemia after stopping the administration of statin drugs was examined. Before the trial, the patients of the hypercholesterolemia took the statin drug (mevalotin) for 4 weeks, and the blood cholesterol level in the patients decreased to near the normal level of 220 mg/dL.

After that, the patients took Globin Peptide every day for 6 weeks after stopping the administration of the statin drugs. The blood cholesterol level in the patients without the administration of Globin Peptide returned to near the prior level of 270mg/dL at 2 weeks after stopping the administration of the statin drugs. However, the blood cholesterol level in the patients with the administration of Globin Peptide was maintained in the normal level at 6 weeks after stopping the administration of the statin drugs.

These results indicate that the administration of Globin Peptide maintained the blood cholesterol level in the normal level after stopping the administration of the statin drugs without the diet therapy, and the side effects of the statin drugs may be prevented by stopping the administration of the statin drugs.

As stated above, Globin Peptide suppressed the rebound of the blood TG, glucose, or cholesterol after stopping the therapy or drug administration. If Globin Peptide is used for the treatment of the lifestyle-related disease, the side effects by the drug chronic administration will be reduced by stopping the drug administration, and the necessity for dietary restriction will be reduced. The administration of Globin Peptide may become the treatment method which takes the patient's QOL into consideration to the first.

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● Blood Glucose Elevation in Mice Glucose Tolerance Test (GTT)

Inhibition of Elevated Blood Glucose in Mice GTT by Globin Peptide

The effect of Globin Peptide on the carbohydrate metabolism was examined. The mechanism of the effect of Globin Peptide on the carbohydrate metabolism is still under examination. The oral administration of 0.5 g/kg or 2 g/kg Globin Peptide decreased the elevation of the blood glucose at 30 min and 60 min after glucose administration in the normal mice, ICR mice.

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● Blood Glucose Level in Diabetic Mice

Decrease in Blood Glucose Level of Diabetic Mice by Globin Peptide

The effect of Globin Peptide on the high blood glucose in the model of type 2 diabetes, KKAy mice, was examined. The blood glucose level in KKAy mice was decreased at 2 h after the oral administration of 0.5 g/kg or 2 g/kg Globin Peptide. The blood glucose level in KKAy mice returned to the level before administration at 24 h after the administration of Globin Peptide (data not shown). These results indicate that Globin Peptide decreased the blood glucose level in the insulin resistant diabetes.

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● Blood Pressure in SHR

Decrease in Blood Pressure of SHR by Globin Peptide

The blood pressure level in the normal rats, SD rats, was not changed after the oral administration of 1 g/kg Globin Peptide. However, the blood pressure level in the spontaneously hypertensive rats, SHR rats, was decreased at 2 h or 4 h after the oral administration of 1 g/kg Globin Peptide. The reduction level of the blood pressure in SHR rats was about 15 mgHg from 163 mgHg of the prior level at 4 h after the administration of Globin Peptide. The antihypertensive effect of Globin Peptide on the blood pressure in SHR rats weakened at 8 h after the administration of Globin Peptide (data not shown). These results indicate that Globin Peptide decreased the blood pressure level in the hypertensive state without affecting the normal blood pressure level.

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■ Safety

● Clinical
    1. No hepatic and renal toxicities were observed in high dose ingestion (3g Globin Peptide/day) for 2 months.
    2. Antigenicity: No case report was found.
    3. Addiction: No addiction of Globin Peptide effects were observed.

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● Toxicological
    1. Acute Toxicity: male mice, Lethal Dose (p.o.)
        Results: over 10g/kg body weight
    2. Subacute Toxicity: male rats, Globin Peptide 4g/kg/day (p.o.) for 3 months
        Results: No toxic reactions were observed.
    3. Mutagenic Studies:
  • Chromosomal Aberration Test on Mammalian Cells
  • Reverse Mutation Test on Bacteria
  • Micronucleous Test on Mice
      Results: No clastogenic activity in vivo and in vitro was found.

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■ Product

    1. Trade Name: BOREP GD
    2. Appearance: cream to light brown powder
    3. Protein: > 85%
    4. Average peptide length: 3 - 5
    5. Ash: < 6%
    6. Moisture: < 5%
    7. Active Peptide (Var-Var-Tyr-Pro): > 0.6%
    8. Processing: heat- and acid-stable
    9. Solubility: highly soluble and clear in water

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■ References

    1. Matsutaka H, Fukuhama C, Isono M, Fujii K, Kagawa K
      1. Jpn J Pharmacol, 58 (Supple I), 80P (1992).
        "Effect of oligopeptides on lipid metabolism. 3) Effect of oligopeptides from various protein digestants on lipid abnormalities in serum and liver."
        [Abstract] We found out the serum triglyceride-lowering effect of oligopeptides from various protein digestants, in which globin digest showed the maximum activity. Their mode of action is dose-dependent and does not show species specificity. Additional effects of oligopeptides on lipid metabolism such as hepatic triglyceride lipase activity, fatty acid synthesis and fat deposition in adipose tissue have been studied. In this report, we examined the effect of oligopeptides on fatty acid metabolism, particularly mitochondorial and peroxisomal fatty acid oxidation in liver. After 10-20 days feeding of high fat diet in which protein was replaced by globin digest in 16-50%, hepatic catalase activity was increased nearly to the same extent as the case of addition of 0.125% clofibrate in mice.

    2. Watanabe S, Matsutaka H, Numata M, Nakamura T, Fukuhama C, Noma S, Kagawa K
      1. Jpn J Pharmacol, 61(Suppl Ⅰ), 79P (1993).
        "Effect of oligopeptides on lipid metabolism. 4) in vivo and in vitro effects on adipocyte differentiation."
        [Abstract] We already showed that oligopeptides from various protein digestants affect triglyceride level in blood, triglyceride lipase activity, fatty acid synthesis and fatty acid oxidation in liver, and fat deposition in adipose tissues. 0ne of protein digestants, globin digest (GD), has the maximum activity. DNA content in adipose tissue of obese mice increased 1.5 times the level of control diet group after 3 weeks feeding of high fat diet, whereas the increase of DNA was depressed by subsitution of 25% protein for GD in the diet. Its inhibitory effect on adipocyte conversion was also observed in vitro examination with Swiss mouse 3T3‐L1 cells. When cells were treated with 10, 20, 40, and 80μg/ml of GD for 48hr before and after induction by insulin, 1-methyl-3-isobutylxanthine and dexamethasone for 48hr, GDPH activities were dramatically decreased depending on its concentration in culture medium. We also examined some of oligopeptide fractions obtained by reversed-phase chromatography. In addition, in vivo treatment of GD increased in vitro lipolytic activity of adipose tissue. These results suggest that some oligopeptide fraction of globin digest inhibits adipocyte differentiation and some oligopeptide enhances lipolytic activity in adipocyte.

    3. Kagawa K, Matsutaka H, Fukuhama C, Watanabe Y, Fujino H
      1. Life Sci, 58(20), 1745-1755 (1996).
        "Globin digest, acidic protease hydrolysate, inhibits dietary hypertriglyceridemia and Val-Val-Tyr-Pro, one of its constituents, possesses most superior effect"
        [Summary] Globindigest(GD), prepared from globin by acidic protease treatment,suppressed the elevation of serum triglyceride level in not only total but also chylomicron fraction affter oral administration of olive oil. By screening with this lowerlng activity, we concluded that Val-Val-Tyr-Pro (VVYP) would be most effective constituent having hypotriglyceridemic action in GD. The mode of their action was dose dependent and did not show species specincity. Neither the repression of peristaltic movement of intestine nor the delaying of gastric emptylng wascaused by intake of GD or VVYP, however, the excretion of administered lipid was much more than that of control. Furthermore, administration of GD caused more prominent activation of hepatic triglyceridelipase (HTGL) and the increase of hepatic free fatty acid (FFA) concentration in early phase after administration of fat. From these results, it could be elucidated that GD, and also VVYP, inhibited fat absorption from digestive tract and enhanced activity of HTGL,so that more rapid clearance of dietary hypertriglyceridemia was caused.

    4. Kagawa K, Matsutaka H, Fukuhama C, Fujino H, Okuda H
      1. J Nutr, 128(1), 56-60 (1998).
        "Suppressive Effect of Globin Digest on Postprandial Hyperlipidemia in Male Volunteers"
        [ABSTRACT] We have reported previously that various edible protein digests inhibit dietary hyperlipidemia in mice, rats, pigs and dogs. Of the various digests tested, globin digest had the most potent inhibitory activity, and a tetrapeptide extracted from globin digest, Val-Val-Tyr-Pro, had activity 7000-fold greater than that of the parent digest. In this clinical study, we investigated the influence of globin digest on serum chylomicron triglyceride concentrations as an indicator of the effect of globin digest on fat absorption and catabolism in humans. Parallel and crossover trials were conducted in which men consumed a control high fat diet (25g fat, 7.6g carbohydrate, 1.9g protein and 0.7g sodium chloride) or the same diet supplemented with globin digest. The supplemented dosages were 1 and 4g globin digest. In the parallel trial, 22 men were divided into three groups: control, globin digest 1g and globin digest 4g. The increases in chylomicron triglyceride concentrations at 1 h after ingestion of 1 or 4 g globin digest were significantly lower (P<0.05) compared with the control group. The crossover trial involved six subjects who consumed the control high fat diet and the same diet supplemented with 4g globin digest. Serum chylomicron triglyceride levels increased in both groups at 1 and 2 h after ingestion, but when subjects consumed 4g globin digest the increases were suppressed to 75 (P<0.05) and 42% (P<0.05) of the increases in controls at the corresponding times, respectively. The areas under the curves of chylomicron and serum total triglyceride concentrations during the 4h after ingestion of 4g globin digest were 46 (P<0.05) and 34% (P<0.05) lower, respectively, than when the men consumed the high fat control diet. In these trials, globin digest reduced the increase in serum chylomicron triglyceride concentrations as a result of the ingestion of a high fat diet. This hypotriglyceridemic effect of globin digest may be valuable for preventing obesity and in lowering the incidence of cardiovascular diseases.

       

    5. Kagawa K, Fukuhama C, Fujino H, Okuda H
      1. J Jpn Soc Nutr Food Sci, 52, 71-77 (1999)(in Japanese).
        "Hypotriglyceridemic Effect of Globin Digest on Subjects with Borderline Hyperlipidemia"
        [Summary] It has already been reported that globin digest(GD)exerts a hypotriglyceridemic action in laboratory animals and humans. A tetrapeptide, Val-Val-Tyr-Pro, extracted from GD, has also been shown to be 7000-times more active than the parent GD. We studied the hypotriglyceridemic effect of GD in subjects with borderline hyperlipidemia. In normal subjects, 1g of GD showed sufficient hypotriglyceridemic action. When borderline hypertriglyceridemic subjects were administered 1g of GD as a beverage with 40g of fat, the increases in serum and chylomicron(CM) TGs at 1-5h after the administration were suppressed to 25-50% of the control values(without GD). The areas under the serum TG and CM-TG concentration curves(AUCs;0-6h) were significantly decreased to about 40% of the control, respectively. GD enhanced the elimination rate constants of TG and CM-TG in borderline hypertriglyceridemic subjects, and showed a more effective hypotriglyceridemic action than in normal subjects. GD thus appears to be useful for prevention of cardiovascular disease and atherosclerosis in subjects with borderline hypertriglyceridemia.

       

    6. Kyoichi Kagawa, Hisako Matsutaka, Yasuchika Ban, Chizuko Fukuhama and Hiroaki Fujino
      1. Jpn Pharmacol Ther, 36, 531-540 (2008).
        "Globin digest suppresses postprandial elevation of remnant-like particles by activation of lipoprotein lipase and hepatic triglyceride lipase; the possible involvement of insulin secretion in activation of the lipases"
        [ABSTRACT] Globin digest (GD) that produced from enzymatic hydrolysis of globin protein in hemoglobin suppressed postprandial elevations of chylomicron triglycerides in man. High lipoprotein remnant level after fat ingestion is a risk factor for atherosclerosis. Effect of GD was studied on postprandial remnant-like particles (RLP) as an index of lipoprotein remnant and on lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) activities. Twelve healthy subjects attended to a placebo-controlled crossover study. The subjects ingested a control high fat diet with or without GD. The increases of RLP concentration after ingestion of GD 1 g were significantly lower than the control. The areas under the curves of RLP during 6 h of GD group were 30 - 50% lower than the corresponding control group. Another placebo-controlled crossover trial was performed using the same diet to examine the influence of GD on LPL and HTGL activities in the healthy participants. The LPL and HTGL in the subjects were activated by GD supplementation. Activation of the lipases by GD was indirect action through inner activators. When the subjects ingested GD 3 g, serum insulin levels temporary increased than the control. The activation of lipases seemed to occur through insulin's action. Pancreatic lipase activities were reduced dose-dependently by GD in vitro. Suppression of pancreatic lipase and activation of LPL and HTGL by GD might be contributed to the reduction of postprandial RLP levels. GD seems to be useful for lowering incidence of cardiovascular diseases and atherosclerosis.

       

    7. Chunning Tong, Yuka Sasakawa, Zhou YuHong, Xu HaiBin, Ma Ning, Feng YongQuan, Kaori Yamamoto, Fumiko Nakaoka, Mayumi Nakao, Miki Nakamura, Chizuko Fukuhama and Kyoichi Kagawa
      1. Jpn Pharmacol Ther, 36, 1039-1044 (2008)(in Japanese).
        "I. Effect of Globin Digest on bood glucose and serum insulin - Type 1 diabetes -"
        [ABSTRACT] Globin digest (GD) is a commercially available oligopeptide mixture derived from edible globin proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. In this study, the effect of GD on blood glucose and serum insulin in mice was examined. GD suppressed the elevation of blood glucose in oral glucose tolerance test. Furthermore, in alloxan-induced diabetic mice for 30 days oral administration of GD, it decreased blood glucose levels in oral glucose tolerance test. In streptozotocin-induced diabetic mice GD did not exhibit a significant effect.

       

    8. Yuka Sasakawa, Chunning Tong, Kaori Yamamoto, Fumiko Nakaoka, Mayumi Nakao, Miki Nakamura, Chizuko Fukuhama and Kyoichi Kagawa
      1. Jpn Pharmacol Ther, 36, 1045-1050 (2008)(in Japanese).
        "II. Effect of Globin Digest on bood glucose and serum insulin - Type 2 diabetes -"
        [ABSTRACT] Globin digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. In this study, the effect of GD on blood glucose and serum insulin in the model of type 2 diabetes, KK-Ay mice, was examined. GD decreased the level of blood glucose in KK-Ay mice and suppressed the elevation of blood glucose in oral glucose tolerance test and insulin tolerance test of KK-Ay mice. Furthermore, GD elevated the level of serum insulin in KK-Ay mice and in oral glucose tolerance test of KK-Ay mice, and promoted the use of insulin in insulin tolerance test of KK-Ay mice. The promotion of insulin secretion and its use by GD may be critical for the suppression of blood glucose.

       

    9. Fumiko Nakaoka, Yuka Sasakawa, Kaori Yamamoto, Mayumi Nakao, Miki Nakamura, Chunning Tong, Chizuko Fukuhama, Kyoichi Kagawa
      1. Life Sci, 86, 424-434 (2010).
        "Anti-diabetic effects of globin digest and its active ingredient Leu-Ser-Glu-Leu in ICR mice, streptozotocin-induced diabetic mice and KK-Ay mice"
        [ABSTRACT] Aims: Leu-Ser-Glu-Leu (LSEL) is the main active ingredient of globin digest (GD) that has an anti-diabetic effect. Here, we investigated the anti-diabetic effect of LSEL for the first time. Main methods: The anti-diabetic effects of GD and LSEL in ICR mice, streptozotocin (STZ)-induced diabetic mice and KK-Ay mice were examined. Key findings: GD and LSEL suppressed the elevation of blood glucose in an oral glucose tolerance test (OGTT) in ICR mice, STZ-induced diabetic mice and KK-Ay mice as well as in an oral sucrose tolerance test in ICR mice and in an insulin tolerance test (ITT) in KK-Ay mice. GD and LSEL decreased the blood glucose levels in the basal state in STZ-induced diabetic mice and KK-Ay mice. Furthermore, GD and LSEL elevated the serum insulin levels in an OGTT in ICR mice and KK-Ay mice and promoted the use of insulin in an ITT in KK-Ay mice. GD and LSEL increased the translocation or expression of the glucose transporter 4 in the muscle of ICR mice, STZ-induced diabetic mice and KK-Ay mice and increased the expression of the uncoupling protein 2 (UCP2) in the muscle of ICR mice. Significance: These results indicate that GD and LSEL control blood glucose through the promotion of glucose uptake in the muscle of the mice. The acceleration of glucose uptake by GD and LSEL may be controlled by the promotion of insulin secretion and the up-regulation of UCP2 expression. GD and LSEL seem to be useful for lowering the incidence of hyperglycemia.

       

    10. Kyoichi Kagawa and Shin-ichi Hasegawa
      1. Jpn Pharmacol Ther, 34, 1163-1164 (2006)(in Japanese).
        "Single dose toxicity studies of Globin Digest in mice"
        [ABSTRACT] Globin Digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. Single oral toxicity of GD was examined in ICR mice. The LD50 values of GD were more than 10g/kg for both male and female mice. Pathologically, no remarkable change associated with GD administration was observed in any sexes, and is expected to be nontoxic via oral ingestion in humans.

       

    11. Kyoichi Kagawa and Shin-ichi Hasegawa
      1. Jpn Pharmacol Ther, 34, 1165-1172 (2006)(in Japanese).
        "Repeated dose toxicity studies of Globin Digest in rats"
        [ABSTRACT] Globin Digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. 28-day repeated oral toxicity of GD was examined in Wistar rats. No toxic signs associated with of GD were observed up to the highest dose (4g/kg/day).

       

    12. Masahiko Higashikuni, Shizuyo Sudo, Shigeaki Watanabe, Kazuhisa Honda, Masahiro Numata and Kyoichi Kagawa
      1. Jpn Pharmacol Ther, 34, 1173-1178 (2006)(in Japanese).
        "Bacterial reverse mutation assay for Globin Digest"
        [ABSTRACT] Globin Digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. GD was tested in the bacterial reverse mutation assay using S. typhimurium TA98, TA100, TA1535, TA1537, and E. coli WP2uvrA with and without metabolic activation (S9 fraction) by plate incorporation. Five doses between 312.5 and 500 g/plate were used. Reverting colonies of GD supplemented groups increased in dose-dependent manner at the preliminary test. However, the results of amino acid analysis for GD and macroscopic observation of lawns of bacterial colonies indicated that histidine and tryptophan included in GD would have caused an increment of the reverting colonies. To eliminate the influence of these amino acids, the same quantities of histidine and tryptophan in GD were introduced into the reference controls. Both of reference controls and GD group increased in the reverting colonies at almost the same rate. These results suggest that the increment of the reverting colonies in GD group was due to the histidine and tryptophan included in GD. Based on the results mentioned above, it is concluded that GD was not mutagenic to bacteria.

       

    13. Masahiko Higashikuni, Shizuyo Sudo, Shigeaki Watanabe, Kazuhisa Honda, Masahiro Numata and Kyoichi Kagawa
      1. Jpn Pharmacol Ther, 34, 1179-1183 (2006)(in Japanese).
        "In vitro chromosomal aberration assay for Globin Digest"
        [ABSTRACT] Globin Digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. Chinese Hamster Lung cells were exposed to 1.25, 2.5 and 5.0 mg/ml of GD for either 6 hr in the presence of metabolic activation or 6, 24 or 48 hr in the absence of metabolic activation. There was no significant increase in the frequency of chromosomal aberrations at any dose level tested, at any sampling time in the presence or absence of metabolic activation. Under the conditions of this test, GD was not clastogenic at any dose level.

       

    14. Masahiko Higashikuni, Shizuyo Sudo, Shigeaki Watanabe, Kazuhisa Honda, Masahiro Numata and Kyoichi Kagawa
      1. Jpn Pharmacol Ther, 34, 1185-1187 (2006)(in Japanese).
        "In vivo mouse micronucleus assay for Globin Digest"
        [ABSTRACT] Globin Digest (GD) is a commercially available oligopeptide mixture derived from edible proteins developed to effectively lower serum triglyceride levels and to regulate dietary fat metabolism. GD was evaluated in an in vivo mouse micronucleus assay. Male mice were dosed with 0.5, 1.0, 2.0g/kg body weight by oral administration. Mitomycin C was used as a positive control. Results of the assay showed that under the conditions of this test evaluated at 24h after dosing, GD did not induce a significant increase in micronucleated polychromatic erythrocytes at any dose. GD was considered to be negative in the mouse in vivo micronucleus test.

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■ Patents

    1. EU 0420979B1 : Lipid metabolism improving agent and method of use
    2. US 5,723,443 : Lipid metabolism promoting agent and its use
    3. US 5,958,885 : Peptide and formulations thereof inhibiting elevations of triglyceride levels in blood
    4. US 6,046,168 : Peptide inhibits blood triglyceride level
    5. AU-B-30370/89: Lipid metabolism improving agent and its method of use

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