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Research Article

Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin

Mohamed M.E. Metwally, Alaa Abdel-Fattah, Hossam S. El-Beltagi and Mostafa A. Ameen
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Background and Objective: Liver injury becomes a health threatening problem which needs the search for safe and effective natural liver protection agents. The purpose of this study was to evaluate the hepatoprotective effect of casein glycomacropeptide (CGMP) which contains branched amino acids against oxidative stress induced in liver damage by CCl4. This was compared with two known polyphenols namely pterostilbene and curcumin. Materials and Methods: Two experiments were carried out. In the first, feeding the additives with different concentrations coincided with CCl4 injection for 5 weeks. In the second, rats were injected with CCl4 for 5 weeks to develop liver fibrosis before starting feeding CGMP and then both feeding CGMP and CCl4 injection were continued for another 5 weeks. Results: Feeding CGMP (100 mg kg1) and pterostilbene (40 mg kg1) significantly improved liver functions. In the second trial, CGMP (150 mg kg1) significantly improved the functions of fibrotic liver and greatly increased the antioxidant enzymes catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GPx) than the polyphenols ingredients. CGMP also restored the kidney normal functions after CCl4 treatment. Conclusion: CGMP which is a fraction of milk casein alleviated the rat's oxidative stress caused by CCl4 injection in both experiments. CGMP greatly increased the antioxidant enzymes compared with the two polyphenols and proved to be a hepatoprotective product at 100-150 mg kg1 that can be used to alleviate liver injury at any stage.

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Mohamed M.E. Metwally, Alaa Abdel-Fattah, Hossam S. El-Beltagi and Mostafa A. Ameen, 2020. Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin. International Journal of Dairy Science, 15: 10-21.

DOI: 10.3923/ijds.2020.10.21



Oxidative stress, which is caused by number of risk factors such as viruses, drugs and alcohol, causes chronic diseases such as atherosclerosis, types of cancers, diabetes 2, cardiovascular diseases and strokes and contributes to initiation and progression of liver injury1. Dietary antioxidants such as polyphenols are used for combating oxidative stress through removal of free radicals, scavenging reactive oxygen species (ROS) and metal ions catalyzing oxidation and regulating CYP 450 enzyme1. They are considered a safe treatment compared to drugs which have problems of efficacy, safety and price. Plant polyphenols are divided into two major classes, namely flavonoids and non-flavonoids2. The non-flavonoids are formed of aromatic ring with one or more of hydroxyl groups (-OH), curcumin and pterostilbene are members of this class. Curcumin (diferuloylmethane (is a hydrophobic polyphenol naturally present in turmeric which is a rhizome from the herb Curcuma longa and is usually used as a spice, giving flavor and natural coloring to food. Pterostilbene (trans-3,5-dimethoxy-4-hydroxystilbene) is a phytoalexin found in grapes, blueberries and peanuts. It present in Vitis vinifera and Pterocarpus santalinm leaves and also in extract of the heartwood of Pterocarpus marsupium. Both compounds were used in number of researches to combat rats liver fibrosis which was developed by number of methods. Curcumin was used to treat liver fibrosis developed by Dioxin, CCl4, dimethylnitrosamine (DMN) or by bile duct ligation3-6. Pterostilbene was also experimented to overcome rats liver fibrosis developed by DMN, acetaminophen and by CCl47-9.

Milk proteins are rich source of bioactive peptides (BAPs). Many BAPs resist degradation by intestinal proteases in the intestinal membrane, survive gastrointestinal and plasmic degradation reaching human blood circulation and to reach their target organ intact. Milk peptides have been identified in gastrointestinal tract (GIT) and human blood as they emerged intact in the human circulation to reach the required organ10. Casein glycomacropeptide (CGMP) has a unique structure since it is rich in branched amino acids and poor in aromatic amino acids which makes it very proper to treat liver aliment.

CGMP constitutes 20-25% of milk whey proteins. CGMP is a heterogenous peptide formed of 64 amino acids, high in branched amino acids valine and isoleucine, low in methionine and free from aromatic acids. The peptide contains phosphorylation and glycosylation sites. About 50-60% of CGMP composition is carbohydrate moieties formed of five saccharides. Most important functional group is sialic acid (N-acetyl neuraminic acid) which constitutes 4.7% of CGMP. This bioactive peptide has a wide range of biofunctional and health promotion aspects as reviewed by Córdova-Dávalos et al.11. It helps in immune system maturation in infants, modulates the defense system in vivo, inhibits virus and bacterial adhesion to cells, inhibits gastrointestinal secretion, promotes the growth of beneficial bacteria (e.g., Bifidobacteria), inhibits cholera and E. coli enterotoxin cell binding12. CGMP prevents colorectal cancer in rats, protects the weaning piglets against E. coli infection causing diarrhea and enhances calcium absorption causing the increase of calcium content of femur in low calcium diet13,14. CGMP has hypoallergenic effect and since it is low in methionine and high in branched valine and isoleucine amino acids make it a candidate to be used for control of liver diseases15. Hydrolyzed CGMP with papain enzyme showed hepatoprotective effects in vitro by protecting human hepatic carcinoma (HepG2) cells from oxidative stress initiated by H2O2. The effect was done by activating Nrf2 and heme oxygenase-1 (HO-1) expression in the cells16.

Though the known beneficial effect of polyphenols in treating liver fibrosis, they have number of limitations which limit their therapeutic application. They have poor solubility and bioavailability and they oxidize fast which limits their effect17,18. Their concentration determines their reaction, so they act as antioxidants at low concentration and as pro-oxidants at high concentration because of its fast oxidation19. Therefore, this study was carried out to determine the hepatoprotective effect of CGMP against oxidative stress in rat's liver developed by CCl4 injection and be compared with the effect of the two polyphenols, pterostilbene and curcumin.


Chemicals: Lacprodan® CGMP-20 product containing 95% CGMP with above 78% protein concentration, was a gift from Arla Foods Ingredients Group P/S Company (Sønderhøj, Denmark). Pterostilbene (pTeroPure®) is a product containing 99% of pure pterostilbene and was obtained from Source Naturals®, Inc. (Santa Cruz, California). A commercial curcumin was purchased from local market. Chemical kits were purchased from The Egyptian Company for Biotechnology (SAE), which were imported from MDSS GmbH-Schiffgraben 41 30175 Hannover, Germany. Carbon tetrachloride was obtained from Merck Company (Darmstadt, Germany) with 99.8% purity. All laboratory chemicals used were of analytical grade.

Determination of curcumin purity: Purity of curcumin sample was determined by HPLC according to Wichitnithad et al.20 at Food Safety and Quality Control lab (FSQC), Faculty of Agriculture, Cairo University, Egypt. The eluent was monitored with a fluorescence detector with excitation at 374 nm.

Animal treatments: The study was carried out in January and February during 2019 at Experimental Surgery and Biological Center, Faculty of Medicine, Cairo University, Egypt. Eighty eight male adult Wistar albino rats weighing 200-210 g were used. Animals had free excess to the rodent chow diet which consisted of 23% protein, 3.73% fat and 3.58% fibers and water. The followed experimental protocol was approved by Cairo University Ethics Committee for the Care and Use of Experimental Animals in Education and Scientific Research (Protocol number: CU-II-F-106-18). Animals were maintained at 25±2°C and 50-60% relative humidity under a 12 h light-dark cycle. All rats were adapted for 3 days before the beginning of the experiments.

Experimental design: The study was carried out in two experiments9:

•  The first experiment was carried out by simultaneously injecting rats with CCl4 and feeding the tested product for 5 weeks. First experiment included 56 rats divided into 7 groups (8 rats per group). The tested products were curcumin (100 mg kg1 b.wt.), pterostilbene (15 and 40 mg kg1 b.wt.) and CGMP (50 and 100 mg kg1 of rat's body)
The second experiment was carried out by first developing liver fibrosis in the experimental rats (32 rats divided into 4 groups, 8 rats/group) by their injection with CCl4 for 5 weeks before starting feeding then the rats were fed on150 mg kg1 b.wt., of CGMP with the continuation of CCl4 injection for another 5 weeks. This was compared with feeding rats with 150 mg kg1 of CGMP coincided with CCl4 injection. Each experiment included 8 rats/group

The injected CCl4 was a mixture of CCl4 and olive oil at a ratio of 1:4. The rats were intraperitoneally injected twice a week with CCl4 mix at a dose rate of 1 mL kg1 b.wt. The tested ingredients were given daily to rats by oral gavage with the specific concentration. Results were compared with a negative control and with CCl4 injected rats.

At the end of the experiment, rats were fasted overnight. Blood samples were collected from the retro-orbital venous plexus puncture into eppendorf tubes. Serum was separated from the clotted samples by centrifugation at 3000 rpm for 15 min, then the clear serum was stored at -20°C until analysis. The animals were sacrificed by cervical dislocation after collection of blood. The liver, kidney and spleen were dissected out, rinsed with distilled water, blotted dry on a filter paper, weighed and stored at -20°C for histological examination.

Biochemical analysis in the serum: Concentration of serum enzymes of alanine transaminase (ALT) was determined according to Van der Sluijs Veer and Soons21, aspartate transaminase (AST) was according to Young22 and alkaline phosphatase (ALP) was according to Belfield and Goldberg23. Total, direct and indirect bilirubin were determined according to Walters and Gerarde24. Creatinine, urea and uric acid were colorimetrically determined in the serum25,26. Lactate dehydrogenase (LDH) activity was determined using the kinetic ultraviolet method of Van der Heiden et al.27.

Preparation of liver homogenate: Before dissection, the liver tissue was rinsed with a phosphate buffered saline (PBS) solution at pH 7.4 containing 0.16 mg mL1 heparin. One gram of the tissue was homogenized in 10 mL cold buffer (50 mM potassium phosphate, pH 7.5) using ART-MICCRA D-8 homogenizer, Germany. The homogenate was then centrifuged at 8210 rpm for 4 min at 5°C by Sigma laboratory centrifuges, Germany. The content of malondialdehyde (MDA) in the liver homogenate was measured at 534 nm using UV2300 spectrophotometer (Techcomp, Shanghai, China) according to the method of Ohkawa et al.28. The activity of glutathione peroxidase (GPx) was measured according to the method of Paglia and Valentine29, superoxide dismutase (SOD) was according to the method of Nishikimi et al.30 and catalase was according to the method of Aebi31. Other rat's organs, after rinsing were weighed.

Histopathological examination: Liver samples were fixed in 10% buffered neutral formaldehyde solution and embedded in paraffin wax for histological examination. Sections of 5-6 μm in thickness were stained with hematoxylin and eosin stain8 and examined with a light electric microscope with a magnification power of 400 X.

Hepatosomatic index was calculated according to the following equation32:

Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin

Statistical analysis: A randomized complete block design with one factor was used for analysis all data with 3 replications for each parameter. The results were expressed as mean±standard deviation and the treatments means were compared by least significant difference (LSD) test using Assist at program33,34.


Purity of curcumin sample: The chromatogram of curcumin contained 3 peaks (Fig. 1) which agrees with literature which points out that turmeric sample constitutes of 3 components.

Effect of curcumin, pterostilbene and CGMP on liver and kidney of CCl4-treated rats: Table 1 presents the effect of CCl4 on liver parameters. CCl4 increased serum enzymes ALT, AST and ALP by 4.45, 5.13 and 2.57 times the control values and elevated indirect and total bilirubin by 1.65 and 1.94 factors, respectively. On the other hand, CCl4 reduced the antioxidant enzymes GPx, CAT and SOD by 0.38, 0.45 and 0.21 factors. CCl4 increased LDH and MDA by a ratio of 1.9 and 9.24, respectively. Feeding additives overcame the adverse effects of CCl4, particularly pterostilbene (40 mg) and CGMP (100 mg) which resulted in values better than the other additives which showed moderate improvements.

Table 2 shows the effect of ingredients feeding of rats with fibrosed liver. Feeding CGMP (150 mg) by both methods of feeding greatly overcame the adverse effect of CCl4 injection. Both reduced liver enzymes, LDH and MDA and increased the antioxidant enzymes GPx, CAT and SOD. Table 3 presents the effect of CCl4 on kidney activity and the improvement obtained by additives feeding. CCl4 caused the elevation of uric acid and urea.

Table 1:
Effect of feeding curcumin, pterostilbene and CGMP on liver functions of rats injected with CCl4
Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Means with different superscripts in the same row are significantly different at p<0.05

Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Fig. 1:
Chromatogram of curcumin sample

Table 2:
Effect of feeding CGMP on liver functions of rats injected with CCl4 either for 5 weeks before feeding the glycopeptide or both coincided together
Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Means with different superscripts in the same row are significantly different at p<0.05, *Rats were injected with CCl4 for 5 weeks before feeding the glycopeptide then both injection and feeding continued for another 5 weeks

Table 3:
Effect of feeding curcumin, pterostilbene and CGMP on kidney functions of rats injected with CCl4
Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Means with different superscripts in the same column are significantly different at p<0.05, *Rats were injected with CCl4 for 5 weeks before feeding the glycopeptide then both injection and feeding continued for another 5 weeks

Table 4:
Effect of feeding curcumin, pterostilbene and CGMP on the weights of body organs and hepatosomatic index (HSI) of rats injected with CCl4
Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
PS: Pterostilbene, *Rats were injected with CCl4 for 5 weeks before feeding the glycopeptide then both injection and feeding continued for another 5 weeks

Feeding the various additives restored kidney normal function with both methods of CCl4 treatment. All additives exerted a recovery effect by reducing the concentration of uric acid and urea to be close to the normal concentrations. Once again, pterostilbene (40 mg) and CGMP (100 mg) were the most effective. CGMP (150 mg) showed the improved effect in both methods of feeding. Table 4 shows the body organs weight of treated rats. Injected CCl4 increased body organs weight. Pterostilbene (40 mg) and CGMP (100 and 150 mg) once again recovered almost the normal weight of body organs and the HSI (%).

Histopathological examination of the liver and kidney tissues: Light microscopic study of control rats showed normal histology of hepatic cells with preserved nucleus, cytoplasm and hepatocytes which were arrayed in well-formed nucleus cords around the central vein (Fig. 2). CCl4 injection induced severe morphological and histological deformations in the liver which revealed congestion of central vein and hepatic sinusoids as well as focal hepatic necrosis associated with inflammatory cells infiltration, collagen fibers deposition, haemorrhage, inflammatory cells infiltration in the portal triad, hyperplasia of epithelial lining bile duct, fibroplasia in the portal triad and fatty change of hepatocytes.

CGMP (100 mg) showed no congestion of central vein, mild Kupffer cells activation, no focal hepatic necrosis, mild collagen deposition, mild fibroplasia in the portal triad, mild hyperplasia of epithelial lining bile duct and mild fatty changes of hepatocytes. Feeding CGMP (150 mg) whether coincided with CCl4 injection or after liver had developed fibrosis resulted in improvements ranged from mild into moderate without much difference between both methods of feeding. All additives mostly alleviated the adverse effects occurred in kidney by CCl4 injection (Fig. 3). Table 5 presents summary of rats liver and kidney histopathology changes occurred by CCl4 injection and the effect of feeding additives. CGMP (100 mg) and pterostilbene (40 mg) showed the most improvements in liver cells while all additives significantly improved the histological structure of kidney tissues.

Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Fig. 2(a-e):
(a) Liver of rats from control group showing the normal histological structure of hepatic lobule, (b) Liver of rats treated with CCl4 showing collagen fibers deposition, haemorrhage and inflammatory cells infiltration in the portal triad, (c) Liver of rats treated with CGMP (100 mg) showing apoptosis of hepatocytes, (d) Liver of rats treated with pterostilbene (40 mg) showing sinusoidal leucocytosis and (e) Liver of rats treated with CGMP (150 mg) after induction of hepatic fibrosis showing appearance of newly formed bile ductuoles and fibroplasia in the portal triad (H and E X 400)

Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
Fig. 3(a-d):
(a) Kidney of rats from control group showing the normal histological structure of renal parenchyma, (b) Kidney of rats treated with CCl4 showing hypertrophy and congestion of glomerular tuft as well as focal renal haemorrhage, (c, d) Kidney of rats treated with pterostilbene (40 mg) and CGMP (100 mg) showing no histopathological changes and (e) Kidney of rats treated with CGMP (150 mg) after induction of hepatic fibrosis showing focal regenerating renal tubules (H and E X 400)

Table 5:
Effect of feeding curcumin, pterostilbene and CGMP on histopathological lesions of liver and kidney of rats injected with CCl4 for 5 weeks before or coincided with the feeding
Image for - Hepatoprotective Effect of Casein Glycomacropeptide as Compared to Pterostilbene and Curcumin
-: No change, +: Mild change, ++: Moderate change, +++: Severe change, *Rats were injected with CCl4 for 5 weeks before feeding the glycopeptide then both injection and feeding continued for another 5 weeks, PS: Pterostilbene


Curcumin sample proved to be pure and composed of three components which agreed with the number listed by literature35. In literature, the commercial sample composed of curcumin I (71-77%), mono-dimethoxycurcumin (curcumin II) (17-19.4%) and bis-demethoxycurcumin (curcumin III) representing 3-9.1% of the sample. Therefore, the chromatogram of the used sample which contained only three peaks proved that the sample was pure enough for running the experiment. The sample composed of 66.9% curcumin I, 19.83% curcumin II and 13.25% curcumin III.

CCl4 injection caused liver bridging fibrosis and led to renal damage. Injured liver released its enzymes such as ALT, AST and ALP which are responsible for protein and amino acids metabolism and gamma-glutamyltransferase (GGT) which is responsible for transferring amino acids across membrane into blood and their concentrations indicate the degree of damage. On liver injury, usually ALT enzyme which mostly presents in liver cells, is released in higher concentration than AST. Moreover, antioxidant enzymes SOD, CAT and GSH-Px which are the main line of defense against oxidation stress caused by ROS components (e.g., superoxide, OH-radicals, H2O2) and reactive nitrogen species (RNS) (e.g., nitrogen dioxide, nitric oxide, superoxide), decreased due to liver injury1.

In the first experiment, CCl4 injection as compared to the control caused significant increase of the concentration (U L1) of the enzymes ALT, AST, ALP and total and indirect bilirubin in blood. This means that liver is not functioning and could not clear bilirubin properly. Feeding the tested ingredients improved the above parameters and the improvement varied according to type of additive and its concentration. CGMP (100 mg) reduced ALT, AST and ALP enzymes by 38.8, 29.6 and 45.1% of the CCl4 values and were reduced by pterostilbene (40 mg) to 39.2, 23.9 and 48.2%, respectively. Both ingredients also reduced indirect and total bilirubin almost matching the control values. The improvement of both pterostilbene and CGMP was concentration dependent.

CCl4 injection significantly increased MDA concentration in blood by ten folds of the control which resulted from lipid peroxidation. Moreover, CCl4 increased the oxidative stress by reducing the concentration of the antioxidant enzymes CAT, GPx and SOD to be 45.1, 38 and 21% of the control, respectively. Also, LDH enzyme concentration increased indicating severe liver cells damage e.g., hemolysis. Feeding the experimental ingredients effectively helped in restoring the concentration of the above enzymes, particularly pterostilbene (40 mg) and CGMP (100 mg) which raised CAT, GPx and SOD enzymes concentrations to reach almost more than 80% of the control and LDH decreased to its normal concentration.

CCl4 injection of fibrotic rats raised the concentration of ALT, AST and ALP enzymes and direct and indirect bilirubin. Feeding CGMP (150 mg) improved the above results by reducing the ALT, AST and ALP. Indirect and total bilirubin turned into almost the normal values. Feeding CGMP (150 mg) coincided with CCl4 resulted in improvement almost similar to the effect on fibrotic rats. CCl4 injection of fibrotic rats reduced the concentration of CAT, GPx and SOD to reach 38.8, 22.6 and 9.8% but increased MDA and LDH in blood by 1127 and 264% of the control values. Feeding fibrotic rats with CGMP (150 mg) attenuated the adverse effect of CCl4 injection by recovering CAT, GPx and SOD to reach 90.4, 72.5 and 68.8% and decreased MDA and LDH to reach only 386.2 and 121.1% of the control values. The improvement of CAT, GPx and SOD in non-fibrotic rats fed with CGMP (150 mg) was very close to the above improvements with values of 96, 75 and 80% of the control. Therefore the differences were not significant between both methods of CCl4 injection.

In another words, the percent increase of CAT, SOD and GPx from CCl4 values were 232, 700 and 320% by CGMP (150 mg) of fibrotic rats compared to 193, 329 and 230% of pterostilbene (40 mg). Moreover, CGMP (150 mg) in non-fibrotic rats resulted 247, 819 and 334% increase of CAT, SOD and GPx, respectively. This means that CGMP restored the concentration of the antioxidant enzymes by many folds of pterostilbene values. Moreover, the increase of MDA and LDH was reduced to be 34.25 and 45.8% in fibrotic rats and to reach 20 and 44.9% of the CCl4 values with the early feeding. Therefore, the hydrolysis step is not required for the use of CGMP as suggested by Li et al.16.

The improvement of kidney functions varied between the additives. Once again, CGMP (100 mg) and pterostilbene (40 mg) significantly decreased creatinine, urea and uric acid as both ingredients were the most effective in improvements. In the second experiment which dealt with fibrotic rats, the feeding on CGMP (150 mg) improved kidney's functions of fibrotic rats by reducing their increase by 87.3, 67.8 and 84.8% of the CCl4 values for uric acid, creatinine and urea, respectively. Similarly, CGMP (150 mg) improved kidney's functions of rats fed the additive coincided with CCl4. These results proved that CGMP (150 mg) improvement of liver and kidney injury with CCl4 was almost similar whether the liver was already fibrosed or at its initiation.

Literature pointed out that workers experimented different concentrations of pterostilbene and curcumin, Lee et al.7 recommended the use of 20 mg kg1 of pterostilbene while El Sayed et al.8 tried 50 mg kg1 to overcome the effect of one injection of acetaminophen. Curcumin was tried4-6 by 10 and 100 mg kg1 and up to 200 mg kg1. The behaviour of curcumin was not as expected comparing with the reported in literature as workers pointed out that there are factors limiting curcumin effect, namely low solubility and the fast oxidation. Although that, the findings of curcumin in this study are significantly agreed with El-Desoky, et al.36 as curcumin mitigated the effects on functions of liver and kidney and the profile of concentrations of lipids. It almost completely reversed the enzymatic and non-enzymatic indicators of oxidative stress in tartrazine-fed rats.

Pterostilbene has strong antioxidant and anti-inflammatory properties contribute to its diverse pharmacological effects, including inhibition of hepatic stellate cells activation and secretion of profibrogenic cytokines during the development of liver fibrogenesis7. The antioxidant activity of pterostilbene was proved by Satheesh and Pari37 as the lower activities of SOD, CAT, GPx, glutathione-S-transferase and reduced glutathione as well as the elevated levels of lipid peroxidation were normalized in diabetic rats after treatment with pterostilbene at a dose of 40 mg kg1 for 6 weeks, which is compatible with our findings in this study.

CGMP increased the antioxidant defense systems by restoring CAT, GPx and SOD concentration with higher ratio than polyphenols. This means that CGMP proved to be a hepatoprotective agent at 100-150 mg kg1 that can be used to alleviate liver injury at any stage and be more effective and persistent as antioxidant agent than polyphenols. Actually, CGMP is better hepatoprotective agent than polyphenols because it contains the branched amino acids valine (9.1%), leucine (2-4%) and isoleucine (11.3%) and low in aromatic amino acids. Branched amino acid (BA) was found to reduce hepatic viral load of patients by enhancing interferon signaling. BA decreases in serum of cirrhotic patients while aromatic acids increase. BA in rats injected with CCl4 suppresses hepatocyte apoptosis, enhances hepatocyte regeneration and increases hepatocyte growth factors suppressing the oxidative stress38.

CGMP revealed improvements compatible with many of studies as Cheng et al.39 found that CGMP and its hydrolysate with papain have antioxidant activity against H2O2-induced oxidative stress in macrophages by alleviating ROS generation and restoring the activities of endogenous antioxidants. Song et al.40 also found a GMP-derived peptide possess potent antioxidant and anti-inflammatory activities and ameliorates hepatic insulin resistance and type 2 diabetes. CGMP is one of the bioactive components of whey protein isolate which previously showed protective effects against liver toxicity induced by DMN in rats41. Similarly, Kume et al.42 proved a potential whey protein as a potent nutraceutical against hepatitis and liver fibrosis. Therefore, based on current findings, CGMP probably has a vital role in these effects.

Polyphenols not only oxidize fast but also their therapeutic effect depends on number of factors. Generally, bioavailability and activity of polyphenols depend on foods structure and their interactions with other food constituents particularly the proteins, lipids and carbohydrates. They can even slow down digestion of carbohydrates through inhibition of digestive enzymes43. High doses of polyphenols may also pose a certain risk to subjects already suffering from oxidative stress and inflammation as polyphenols could also act as prooxidant44. Currently, many phenolics do not have a clear health statement. Neither, FDA nor European Food Safety authority has identified the ideal dietary intake of phenolic diets nor their safety limits45.

Further studies are required to explore the mechanism which through CGMP can prevent liver fibrosis and to promote the pivotal role of CGMP as antifibrotic and antioxidant agent. Based on the findings of this study, CGMP is suggested asa promising protective agent for alleviating the oxidative stress and liver damage effectively with better antioxidant activity than polyphenols.


Results proved that curcumin, pterostilbene and casein glycomacropeptide (CGMP) significantly attenuated the hepatotoxic effect of CCl4 injection on liver and kidney functions. Pterostilbene (40 mg) and CGMP (100 mg) produced the highest improvement. The additives helped liver cells to be recovered and be intact, therefore reduced the release of ALT, AST and ALP enzymes and opposed peroxidation by reducing MDA release and recovering the antioxidants enzymes. CGMP (150 mg) alleviated liver injury whether at its initiation or after development of fibrosis and effectively opposed the oxidative stress that human exposed to far more than polyphenols.


This study revealed the protective effect of CGMP in alleviating the oxidative stress and liver damage that can be beneficial for treating the chronic liver diseases. This study will help the researcher to discover the novel role of CGMP in improving and recovering fibrotic liver and to consider CGMP as a promising agent for treating liver diseases with distinctive effect than the known plant polyphenols.


We thank Arla Foods Ingredients Group P/S Company for providing the gift sample of Lacprodan® CGMP-20 and its cooperation for achieving this study.


1:  Li, S., H.Y. Tan, N. Wang, Z.J. Zhang, L. Lao, C.W. Wong and Y. Feng, 2015. The role of oxidative stress and antioxidants in liver diseases. Int. J. Mol. Sci., 16: 26087-26124.
CrossRef  |  Direct Link  |  

2:  Stagos, D., G.D. Amoutzias, A. Matakos, A. Spyrou, A.M. Tsatsakis and D. Kouretas, 2012. Chemoprevention of liver cancer by plant polyphenols. Food Chem. Toxicol., 50: 2155-2170.
CrossRef  |  Direct Link  |  

3:  Salim, A.B., A. Zohair and A.A.K. Abou-Arab, 2013. Protective effect of turmeric on 2,3,7,8-tetra chlorodibenzo-P-dioxin (TCDD) induced oxidative stress and hepatotoxicity in rats. J. Applied Sci. Res., 9: 1790-1797.
Direct Link  |  

4:  Shu, J.C., Y.J. He, X. Lv, G.R. Ye and L.X. Wang, 2009. Curcumin prevents liver fibrosis by inducing apoptosis and suppressing activation of hepatic stellate cells. J. Nat. Med., 63: 415-420.
CrossRef  |  Direct Link  |  

5:  Weerawatanakorn, M., S.C. Hsieh, M.L. Tsai, C.S. Lai and L.M. Wu et al., 2014. Inhibitory effect of tetrahydrocurcumin on dimethylnitrosamine-induced liver fibrosis in rats. J. Funct. Foods, 7: 305-313.
CrossRef  |  Direct Link  |  

6:  Kabirifar, R., Z.A. Ghoreshi, A. Rezaifar, F. Binesh, K. Bamdad and A. Moradi, 2018. Curcumin, quercetin and atorvastatin protected against the hepatic fibrosis by activating AMP-activated protein kinase. J. Funct. Foods, 40: 341-348.
CrossRef  |  Direct Link  |  

7:  Lee, M.F., M.L. Liu, A.C. Cheng, M.L. Tsai, C.T. Ho, W.S. Liou and M.H. Pan, 2013. Pterostilbene inhibits dimethylnitrosamine-induced liver fibrosis in rats. Food Chem., 138: 802-807.
CrossRef  |  Direct Link  |  

8:  El-Sayed, E.M., A.M. Mansour and M.E. Nady, 2015. Protective effects of pterostilbene against acetaminophen‐induced hepatotoxicity in rats. J. Biochem. Mol. Toxicol., 29: 35-42.
CrossRef  |  Direct Link  |  

9:  Zhan, W., X. Liao, T. Tian, L. Yu and X. Liu et al., 2017. Study on the effects of blueberry treatment on histone acetylation modification of CCl4-induced liver disease in rats. Genet. Mol. Res., 16: 1-10.
CrossRef  |  PubMed  |  Direct Link  |  

10:  Xu, Q., H. Hong, J. Wu and X. Yan, 2019. Bioavailability of bioactive peptides derived from food proteins across the intestinal epithelial membrane: A review. Trends Food Sci. Technol., 86: 399-411.
CrossRef  |  Direct Link  |  

11:  Córdova-Dávalos, L.E., M. Jiménez and E. Salinas, 2019. Glycomacropeptide bioactivity and health: A review highlighting action mechanisms and signaling pathways. Nutrients, Vol. 11, No. 3.
CrossRef  |  Direct Link  |  

12:  Rong, Y., Z. Lu, H. Zhang, L. Zhang, D. Song and Y. Wang, 2015. Effects of casein glycomacropeptide supplementation on growth performance, intestinal morphology, intestinal barrier permeability and inflammatory responses in Escherichia coli K88 challenged piglets. Anim. Nutr., 1: 54-59.
CrossRef  |  Direct Link  |  

13:  Chen, Q., Y. Liang, C. Zhu, Y. Yan and G. Pang, 2013. Effects of casein glycomacropeptide on the early development of primary colorectal cancer in rats. Food Sci. Hum. Wellness, 2: 113-118.
CrossRef  |  Direct Link  |  

14:  Burns, P., A. Binetti, P. Torti, U. Kulozik and L. Forzani et al., 2015. Administration of caseinomacropeptide-enriched extract to mice enhances the calcium content of femur in a low-calcium diet. Int. Dairy J., 44: 15-20.
CrossRef  |  Direct Link  |  

15:  Jauregui-Rincon, J., E. Salinas-Miralles, N. Chávez-Vela and M. Jiménez-Vargas, 2018. Glycomacropeptide: Biological Activities and Uses. In: Whey-Biological Properties and Alternative Uses, Gigli, I. (Ed.)., IntechOpen Limited, London, ISBN: 978-1-83880-926-3

16:  Li, T., B. Chen, M. Du, J. Song, X. Cheng, X. Wang and X. Mao, 2017. Casein glycomacropeptide hydrolysates exert cytoprotective effect against cellular oxidative stress by up-regulating HO-1 expression in HepG2 cells. Nutrients, Vol. 9, No. 1.
CrossRef  |  Direct Link  |  

17:  Wan, L. and J.G. Jiang, 2018. Protective effects of plant-derived flavonoids on hepatic injury. J. Funct. Foods, 44: 283-291.
CrossRef  |  Direct Link  |  

18:  Halliwell, B., 2012. Free radicals and antioxidants: updating a personal view. Nutr. Rev., 70: 257-265.
CrossRef  |  PubMed  |  Direct Link  |  

19:  Cömert, E.D. and V. Gökmen, 2018. Evolution of food antioxidants as a core topic of food science for a century. Food Res. Int., 105: 76-93.
CrossRef  |  Direct Link  |  

20:  Wichitnithad, W., N. Jongaroonngamsang, S. Pummangura and P. Rojsitthisak, 2009. A simple isocratic HPLC method for the simultaneous determination of curcuminoids in commercial turmeric extracts. Phytochem. Anal., 20: 314-319.
CrossRef  |  Direct Link  |  

21:  Van der Sluijs Veer, G. and J.W.P.H. Soons, 1992. A time-resolved fluoroimmuno assay of the IgM-rheumatoid factor. Eur. J. Clin. Chem. Clin. Biochem., 30: 301-305.
CrossRef  |  Direct Link  |  

22:  Young, D.S., 1990. Effects of Drugs on Clinical Laboratory Tests. 3rd Edn., AACC Press, Washington, DC., ISBN-13: 9780915274536, pp: 6-12
Direct Link  |  

23:  Belfield, A. and D.M. Goldberg, 1971. Revised assay for serum phenyl phosphatase activity using 4-amino-antipyrine. Enzyme, 12: 561-573.
PubMed  |  Direct Link  |  

24:  Walters, M.I. and H.W. Gerarde, 1970. An ultramicromethod for the determination of conjugated and total bilirubin in serum or plasma. Microchem. J., 15: 231-243.
CrossRef  |  Direct Link  |  

25:  Tietz, N.W., 1986. Textbook of Clinical Chemistry. W.B. Saunders, Philadelphia, USA., ISBN-13: 9780721688862, pp: 1271
CrossRef  |  Direct Link  |  

26:  Tietz, N.W., 1990. Clinical Guide to Laboratory Tests. 2nd Edn., WB Saunders, Philadelphia, pp: 566

27:  Van der Heiden, C., B. Ais, W.G. Ardt and C. Rosallsis, 1994. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes, Part 8, IFCC method for LDH. Eur. J. Clin. Chem. Clin. Biochem., 32: 639-655.

28:  Ohkawa, H., N. Ohishi and K. Yagi, 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem., 95: 351-358.
CrossRef  |  PubMed  |  Direct Link  |  

29:  Paglia, D.E. and W.N. Valentine, 1967. Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med., 70: 158-169.
CrossRef  |  PubMed  |  Direct Link  |  

30:  Nishikimi, M., N.A. Rao and K. Yagi, 1972. The occurrence of superoxide anion in the reaction of reduced phenazine methosulfate and molecular oxygen. Biochem. Biophys. Res. Commun., 46: 849-854.
CrossRef  |  PubMed  |  Direct Link  |  

31:  Aebi, H., 1984. Catalase in vitro. In: Methods in Enzymology, Academic Press, Cambridge, Massachusetts, ISBN: 9780121820053, pp: 121-126
CrossRef  |  Direct Link  |  

32:  Sadekarpawar, S. and P. Parikh, 2013. Gonadosomatic and hepatosomatic indices of freshwater fish Oreochromis mossambicus in response to a plant nutrient. World J. Zool., 8: 110-118.
Direct Link  |  

33:  Snedecor, G.W. and W.G. Cochran, 1994. Statistical Methods. 9th Edn., Iowa State University Press, Ames, Iowa, USA

34:  Silva, F.A.S. and C.A.V. de Azevedo, 2009. Principal components analysis in the software Assistat-statistical attendance. Proceedings of the 7th World Congress on Computers in Agriculture Conference, June 22-24, 2009, American Society of Agricultural and Biological Engineers, Reno, NV., USA -
Direct Link  |  

35:  Li, R., F. Liu, X. Yang, L.Q. Chen and F. Wang et al., 2019. Analysis of bisabolocurcumin ether (a terpene-conjugated curcuminoid) and three curcuminoids in Curcuma species from different regions by UPLC-ESI MS/MS and their in vitro anti-inflammatory activities. J. Funct. Foods, 52: 186-195.
CrossRef  |  Direct Link  |  

36:  El-Desoky, G.E., A. Abdel-Ghaffar, Z.A. Al-Othman, M.A. Habila and Y.A. Al-Sheikh et al., 2017. Curcumin protects against tartrazine-mediated oxidative stress and hepatotoxicity in male rats. Eur. Rev. Med. Pharmacol. Sci., 21: 635-645.
Direct Link  |  

37:  Satheesh, M.A. and L. Pari, 2006. The antioxidant role of pterostilbene in streptozotocin-nicotinamide-induced type 2 diabetes mellitus in Wistar rats. J. Pharm. Pharmacol., 58: 1483-1490.
CrossRef  |  Direct Link  |  

38:  Tajiri, K. and Y. Shimizu, 2018. Branched-chain amino acids in liver diseases. Transl. Gastroenterol. Hepatol., Vol. 3.
CrossRef  |  Direct Link  |  

39:  Cheng, X., D.X. Gao, J.J. Song, F.Z. Ren and X.Y. Mao, 2015. Casein glycomacropeptide hydrolysate exerts cytoprotection against H2O2-induced oxidative stress in RAW 264.7 macrophages via ROS-dependent heme oxygenase-1 expression. RSC Adv., 5: 4511-4523.
CrossRef  |  Direct Link  |  

40:  Song, J.J., Q. Wang, M. Du, T.G. Li, B. Chen and X.Y. Mao, 2017. Casein glycomacropeptide-derived peptide IPPKKNQDKTE ameliorates high glucose-induced insulin resistance in HepG2 cells via activation of AMPK signaling. Mol. Nutr. Food Res., Vol. 61, No. 2.
CrossRef  |  Direct Link  |  

41:  Oryan, A., M.H. Eftekhari, M. Ershad, M.R. Panjehshahin and H.R. Tabatabaei, 2011. Hepatoprotective effects of whey protein isolate against acute liver toxicity induced by dimethylnitrosamine in rat. Comparat. Clin. Pathol., 20: 251-257.
CrossRef  |  Direct Link  |  

42:  Kume, H., K. Okazaki and H. Sasaki, 2006. Hepatoprotective effects of whey protein on D-galactosamine-induced hepatitis and liver fibrosis in rats. Biosci. Biotechnol. Biochem., 70: 1281-1285.
CrossRef  |  Direct Link  |  

43:  Kardum, N. and M. Glibetic, 2018. Polyphenols and their interactions with other dietary compounds: Implications for human health. Adv. Food Nutr. Res., 84: 103-144.
CrossRef  |  Direct Link  |  

44:  Kaulmann, A. and T. Bohn, 2016. Bioactivity of polyphenols: Preventive and adjuvant strategies toward reducing inflammatory bowel diseases-promises, perspectives and pitfalls. Oxid. Med. Cell. Longev., Vol. 2016.
CrossRef  |  Direct Link  |  

45:  Mark, R., X. Lyu, J.J. Lee, R. Parra-Saldívar and W.N. Chen, 2019. Sustainable production of natural phenolics for functional food applications. J. Funct. Foods, 57: 233-254.
CrossRef  |  Direct Link  |  

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