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Efficacy of black garlic extract on anti-tumor and anti-oxidant activity enhancement in rats

Open AccessPublished:March 30, 2021DOI:https://doi.org/10.1016/j.nutos.2021.03.005

      Summary

      Objective

      The current study aimed to investigate the effect of oral administration of aqueous aged garlic extract (AGE) on the anti-tumor activity of diethylnitrosoamine (DEN)-induced hepatocarcinogenic rats for 7 weeks.

      Methods

      Forty-five male Wistar rats were randomly divided into five groups (n = 9) as follows: Groups A (control); B (DEN-group) received a single intraperitoneal (i.p.) injection of DEN and a dose of and Carbon tetrachloride (CCl4) two weeks later; C, D, and E received the same carcinogenesis protocol as B plus AGE oral doses 150, 300, and 600 mg/Kg/d, respectively, for 7 weeks. Liver function tests (aspartate aminotransferase (AST), alanine aminotransferase (ALT), total bilirubin (TBIL), and serum and liver glutathione reductase (GR) activity, and serum Trolox Equivalent Antioxidant Capacity (TEAC) were measured.

      Results

      The present study revealed that administration of AGE doses 300 mg/Kg/d (group D) and 600 mg/Kg/d group (E), for 7 weeks, significantly (p < 0.01) decreased liver weight, relative to that in group B, with significantly decreased (p < 0.01) serum ALT, AST, and TBIL levels. It also showed that AGE improved liver and serum GR activity levels and serum TEAC levels than in group B.

      Conclusion

      This finding suggests that AGE has remarkable hepatoprotective and antioxidant effects in DEN-induced hepatocarcinogenic rats. Further studies are needed to demonstrate effective various doses of AGE in human. The mechanisms underlying the active principle responsible for the anti-tumor activity and need to be elucidated in future studies.

      Keywords

      1. Introduction

      Cancer is one of the most common causes of mortality. Liver cancer in men/women is the fifth/seventh most frequently diagnosed cancer worldwide, but the second/sixth most frequent cause of cancer deaths. Although there are many cancer treatment strategies, the therapeutic outcome of liver cancer remains very poor. Many compounds have been tested with proven efficacies against experimentally-induced hepatocarcinogenesis [
      • Al-Rejaie S.S.
      • Aleisa A.M.
      • Al-Yahya A.A.
      • Bakheet S.A.
      • Alsheikh A.
      • Fatani A.G.
      • et al.
      Progression of diethylnitrosamine-induced hepatic carcinogenesis in carnitine-depleted rats.
      ]. Some of these compounds include star anise [
      • Yadav A.S.
      • Bhatnagar D.
      Chemo-preventive effect of Star anise in N-nitrosodiethylamine initiated and phenobarbital promoted hepato-carcinogenesis.
      ], silymarin [
      • Ramakrishnan G.
      • Raghavendran H.R.B.
      • Vinodhkumar R.
      • Devaki T.
      Suppression of N-nitrosodiethylamine induced hepatocarcinogenesis by silymarin in rats.
      ], and garlic [
      • Kweon S.
      • Park K.-A.
      • Choi H.
      Chemopreventive effect of garlic powder diet in diethylnitrosamine-induced rat hepatocarcinogenesis.
      ].
      Garlic (Allium sativum L.) is one of the most important medicinal plants intensively used in traditional medicine for over 4000 years to treat several disorders, including arthritis, diabetes, and infectious diseases [
      • Santo S.E.
      • Keiss H.
      • Meyer K.
      • Buytenhek R.
      • Roos T.
      • Dirsch V.
      • et al.
      Garlic and cardiovascular diseases.
      ,
      • Hammami I.
      • Nahdi A.
      • Amara S.
      • Ammar A.B.
      • El May M.V.
      • Mauduit C.
      Open access scientific reports.
      ]. However, some people are reluctant to ingest raw garlic due to its unpleasant odor and taste [
      • Banerjee S.
      • Mukherjee P.K.
      • Maulik S.
      Garlic as an antioxidant: the good, the bad and the ugly.
      ]. Therefore, many types of garlic preparations, including dehydrated garlic powder, garlic oil, garlic oil macerate and AGE have been developed to reduce these attributes without losing biological functions [
      • Ryu J.H.
      • Kang D.
      Physicochemical properties, biological activity, health benefits, and general limitations of aged black garlic: A review.
      ].
      ABGEs have emerged one of the fastest-growing health-oriented food products in the East Asian regional market, with no strong odor or harsh irritating taste, but a fruit-like sweetness created from ordinary fresh garlic processed (aging) in a temperature (65–8 °C)- and humidity (70–80%)-controlled room for a month [
      • Wang D.
      • Feng Y.
      • Liu J.
      • Yan J.
      • Wang M.
      • Sasaki J.-i.
      • et al.
      Black garlic (Allium sativum) extracts enhance the immune system.
      ]. During the aging process, the levels of beneficial compounds such as pyruvate and S-allylcysteine (SAC) increase [
      • Shin J.H.
      • Lee C.W.
      • Oh S.J.
      • Yun J.
      • Kang M.R.
      • Han S.-B.
      • et al.
      Hepatoprotective effect of aged black garlic extract in rodents.
      ]. SAC is a cysteine-derivative with anti-carcinogenic [
      • Liu Z.
      • Li M.
      • Chen K.
      • Yang J.
      • Chen R.
      • Wang T.
      • et al.
      S-allylcysteine induces cell cycle arrest and apoptosis in androgen-independent human prostate cancer cells.
      ] and antioxidant activities [
      • Chung L.Y.
      The antioxidant properties of garlic compounds: allyl cysteine, alliin, allicin, and allyl disulfide.
      ,
      • García E.
      • Santana-Martínez R.
      • Silva-Islas C.A.
      • Colín-González A.L.
      • Galván-Arzate S.
      • Heras Y.
      • et al.
      S-allyl cysteine protects against MPTP-induced striatal and nigral oxidative neurotoxicity in mice: participation of Nrf2.
      ]. Furthermore, another ABGE-derived component, S-allylmercaptocysteine (SAMC), an antioxidant only present in ABGE, inhibits cell growth and promotes apoptosis in several cancer cell lines [
      • Xiao J.
      • Ching Y.P.
      • Liong E.C.
      • Nanji A.A.
      • Fung M.L.
      • Tipoe G.L.
      Garlic-derived S-allylmercaptocysteine is a hepato-protective agent in non-alcoholic fatty liver disease in vivo animal model.
      ]. No report of toxicity have been documented with aged garlic medications [
      • Macan H.
      • Uykimpang R.
      • Alconcel M.
      • Takasu J.
      • Razon R.
      • Amagase H.
      • et al.
      Aged garlic extract may be safe for patients on warfarin therapy.
      ].
      Limited studies on the benefits of ABGE-enhanced anti-tumor activity in liver cancer have been conducted. Therefore, the present study aimed to investigate the effects of orally administered AGE on the anti-tumor activity enhancement in DEN-induced hepatocarcinogenic rats for 7 weeks.

      2. Materials and methods

      This experimental study was conducted at the King Fahad Medical Research Center (KFMRC) and Food & Nutrition Department, Faculty of Home Economics, King Abdul-Aziz University- Jeddah, Saudi Arabia, from January to August 2017.

      2.1 Diet and chemicals

      Diet was obtained from Grain Silos and Flour Mills Organization (Western Province, Jeddah, Saudi Arabia). SAC and DEN were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). CCl4 was purchased from Fluka® Chemie AG (CH-9470, Buchs, Switzerland). Liver function tests (ALT, AST, and TBIL) kits were purchased from ELITech Group Co. (SEPPIM S.A.S., Zone Industrielle, 61500 SEES, France). GR activity and TEAC assay kits were purchased from BioVision, Inc. (Mountain View, CA, USA).

      2.2 Preparation of black garlic

      Black garlic was obtained from Abazeer Trading Establishment (Al-Mousadiah Plaza, No. 3), Jeddah, Saudi Arabia. It was imported from Ui-Seong Black Garlic Farming Association, Korea. The black garlic was created, according to a previously described method [
      • Wang D.
      • Feng Y.
      • Liu J.
      • Yan J.
      • Wang M.
      • Sasaki J.-i.
      • et al.
      Black garlic (Allium sativum) extracts enhance the immune system.
      ], with modifications [
      • Kim I.
      • Kim J.-y.
      • Hwang Y.-J.
      • Hwang K.-A.
      • Om A.-S.
      • Kim J.-h.
      • et al.
      The beneficial effects of aged black garlic extract on obesity and hyperlipidemia in rats fed a high-fat diet.
      ]. Briefly, the 30 g unpeeled garlic packed in airtight containers was aged at 65–80 °C temperature and 70–80% humidity levels for 30 days in a chamber, without additives or additional treatments. The black garlic was manually peeled, mixed with 10 ml of distilled water. The paste was transferred with 100 ml distilled water and blended. The homogenate was extracted with distilled water for 2 h at 100 °C. After cooling to room temperature, the AGE solution was centrifuged at 3500 rpm for 20 min supernatant collected. The supernatant was filtered, frozen at −80 °C overnight, then lyophilized (freeze-dried) using a lyophilizer, and stored at −20 °C till subsequent use and analysis.

      2.3 Chemical analysis

      SAC amount in the extracts were measured using High-performance liquid chromatography (HPLC) HP 1100 chromatograph (Agilent Technologies, Palo Alto, CA, USA) with ultraviolet (UV) detector analysis and analytical conditions: column size (15 cm x 4.6 mm I.D., 5 μm, USA), column temp (37°C), flow rate (0.4 ml/min), mobile phase [eluent (A), 20 mmol/l sodium dihydrogen phosphate plus 10 mmol/L heptanesulfonic acid with water. Eluents B and A were mixed with acetonitrile in the ratio 1:1(v/v)], and detection was at a wavelength of 208 nm.

      2.4 Experimental animals and treatments

      A total of 45 adult male Wistar Albino rats (initial body weight between 120–145 g) were obtained from the animal house of KFMRC, King Abdul-Aziz University. The rats were housed in plastic cages and environmentally maintained at 25 ± 2 °C and 12-h light/dark cycle. Rats were kept on a commercial standard laboratory diet and tap water during the whole experimental period.
      In this study, hepatocarcinogenesis was induced in rats as reported [
      • Columbano A.
      • Endoh T.
      • Denda A.
      • Noguchi O.
      • Nakae D.
      • Hasegawa K.
      • et al.
      Effects of cell proliferation and cell death (apoptosis and necrosis) on the early stages of rat hepatocarcinogenesis.
      ,
      • el-Demerdash E.
      • el-Denshary E.-D.
      • Al-Gharabli N.
      • Osman A.
      Probucol and liver efficiency during chemically-induced hepatocarcinogenesis.
      ,
      • Mansour M.A.
      • Al-Ismaeel H.
      • Al-Rikabi A.C.
      • Al-Shabanah O.A.
      Comparison of angiotensin converting enzyme inhibitors and angiotensin II type 1 receptor blockade for the prevention of premalignant changes in the liver.
      ]. The rats received a single dose of i.p. DEN (200 mg/kg) and CCl4 (2 ml/kg i.g) two weeks later. The rats were treated with various doses of AGE (150 and 300 mg/kg) according to previous studies used concentrations of AGE (100, 200, or 400 mg/kg) were given five days a week for 4 weeks [
      • Kim M.H.
      • Kim M.J.
      • Lee J.H.
      • Han J.I.
      • Kim J.H.
      • Sok D.-E.
      • et al.
      Hepatoprotective effect of aged black garlic on chronic alcohol-induced liver injury in rats.
      ], and AGE (0, 200, 400 and 800 mg/kg, for 2 weeks [
      • Wang X.
      • Jiao F.
      • Wang Q.-W.
      • Wang J.
      • Yang K.
      • Hu R.-R.
      • et al.
      Aged black garlic extract induces inhibition of gastric cancer cell growth in vitro and in vivo.
      ]. Another previous study also used concentrations of AGE (100–600 mg/kg) daily for 8 weeks [
      • Thomson M.
      • Al-Qattan K.K.
      • Divya J.
      • Ali M.
      Anti-diabetic and anti-oxidant potential of aged garlic extract (AGE) in streptozotocin-induced diabetic rats.
      ].

      2.5 Experimental design

      One-week post-adaptation to laboratory conditions, 45 adult male Wistar Albino rats (9 rats/cage) were randomly divided into five groups.

      2.5.1 Group A (negative control)

      Nine rats received a single i.p. injection of normal saline (2.5 ml/kg).

      2.5.2 Group B (DEN-control)

      Nine rats were injected with a single i.p DEN injection (200 mg/kg b.wt.). Two weeks later, each rat was given a single CCl4 dose (2 ml/kg b.wt. i.g.).

      2.5.3 Group C

      Nine rats received the same carcinogenesis induction protocol as group B plus daily low-dose AGE oral administration (150 mg/kg) for 7 weeks.

      2.5.4 Group D

      Nine rats received the same carcinogenesis induction protocol as group B plus daily middle-dose AGE oral administration (300 mg/kg) for 7 weeks.

      2.5.5 Group E

      Nine normal rats received the same carcinogenesis induction protocol as group B plus daily oral administration of high-dose AGE (600 mg/kg) for 7 weeks.

      2.6 Measuring body weight gain and relative organ weight

      Bodyweight was measured at the beginning and weekly throughout the study, using an electronic balance, and recorded. The body weight gain (BWG) and relative organ weight (ROW) coefficients were calculated at the end of the experimental period, according to the following equations:
      ROW (%) = Wet weight of organ (g)/Final body weight of rat (g) × 100


      BWG (%) = (Final body weight − Initial body weight)/Initial body weight × 100


      2.7 Blood collection

      At the end of the experimental period (11 weeks), all rats were euthanized using diethyl ether as ane-sthesia, after a 13 h fasting period. Blood samples were collected in test tubes and allowed to coagulate at room temperature. Each sample was placed in a clean dry centrifuge tube. The blood was centrifuged at 3000 rpm for 15 min at 4 °C. The sera were quickly collected, divided into aliquots in properly labeled Eppendorf tubes, and stored at −20 °C until used for biochemical analysis.

      2.8 Biochemical analyses

      2.8.1 Determination of liver function tests

      Liver function tests (ALT, AST, and TBIL) were determined using an automated clinical chemistry analyzer (ELITech Group Co., Flexor EL200, France), according to the manufacturer's instructions.

      2.8.2 Determination of GR activity

      Serum and liver GR activity were assayed using a commercial kit (Mountain View, CA, USA). The analysis was performed, according to the manufacturer's instructions.

      2.8.3 Determination of Trolox equivalent antioxidant activity (TEAC)

      TEAC was assayed using a commercial kit, following the manufacturer's instructions, using a microplate reader (ELx808 Ultra Microplate Bioelisa Reader, Bio-Tek Instruments Inc., USA).

      2.9 Liver and spleen tissue isolation

      Immediately after collecting blood samples, rats were sacrificed by cervical dislocation and their liver and spleen quickly isolated, washed gently with cold saline (0.9% NaCl), blotted dry with filter paper, and weighed. The spleen and part of the liver tissue were fixed in 10 % neutral buffered formalin for histopathology.
      The other liver part (0.1 g) was homogenized in 1.0 ml in ice-cold assay buffer (10% (w/v) using a homogenizer. The samples were centrifuged at 10,000 rpm for 15 min at 4 °C. The supernatants were collected and stored at −80 °C for GR activity measurement.

      2.10 Liver and spleen tissue examination

      A small part of 10 % formalin-fixed liver and spleen specimens from each group were dehydrated in alcohol series, cleared in xylene, and paraffin block-embedded. After routine processing, paraffin sections of each tissue were cut into 3 μm sections, placed on glass slides, and stained with Haematoxylin and Eosin (H&E) stain. H&E staining was examined using a polarizing microscope (BX51TF; Olympus, Tokyo, Japan).
      CD10 and caspase-3 expressions were evaluated using immunohistochemistry (IHC) and the labeled streptavidin-biotin-peroxidase (LSAB) method [
      • Bratthauer G.L.
      Labeled Avidin Binding (LAB) method. Immunocytochemical methods and protocols.
      ]. Briefly, liver sections from the different groups were deparaffinized in xylene, dehydrated in graded alcohol, and washed with distilled water. The slides were blocked with 3% peroxidase for 20 min and rinsed. The slides were divided and incubated with CD10 and caspase-3 antibodies for 30-min. The slides were incubated with primary antibodies (anti-CD10 and anti-caspase-3) for 30 min except for the negative control group (A). The slides were incubated with biotinylated horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min, followed by 3,3-diaminobenzidene (DAB) as a chromogen for 10 min. The slides were counterstained using Mayer's Haematoxylin for 1 min. The slides were examined using a polarizing microscope.

      2.11 Statistical analyses

      Data were analyzed by analysis of variance (ANOVA) and groups compared by Tukey's test, using the statistical package for social sciences (SPSS) software, version 20.0 (SPSS Inc., Chicago, IL, USA). Differences were considered significant at p < 0.05. All the results were expressed as mean ± standard deviation (SD).

      3. Results

      3.1 SAC determination in AGE by HPLC

      AGE was analyzed using HPLC with UV-detector analysis. The chromatogram showed a good resolution for SAC analysis. No significant interferences were observed and the chromatogram baseline was stable. The SAC peak in the chromatogram was sharp and symmetric with a retention time of 8.54 min. By calculating the peak area, we found that the SAC content in AGE powder was 656.5 μg/g (expressed as dry matter base) and 42.7μg/g in fresh aged black garlic, with a satisfactory coefficient of variation (CV), less than 2% (Fig. 1).
      Fig. 1
      Fig. 1HPLC chromatogram of SAC concentration in ABGE.

      3.2 Effects of AGE doses on DEN-injected rats

      The effects of AGE doses on initial and final body weights, liver and spleen weights and biochemical parameters of DEN-injected rats are shown in Table 1. The DEN (200 mg/kg b.wt.)-injection rats showed a significant increase (p < 0.01) in the liver, spleen, and relative liver and spleen weights in the DEN group (B), compared with rats in the negative control group (A). Also, a significant increase (p < 0.01) was observed in serum liver function tests (ALT, AST, and TBIL) accompanied with a significant decrease (p < 0.01) TEAC, and GR activity and liver GR activity in the DEN group (B), compared to the negative control group (A).
      Table 1Effects of AGE doses on initial and final body weights, liver and spleen weights, and biochemical parameters of DEN-injected rats.
      ParametersGroup A (n = 9)Group B (n = 9)Group C (n = 9)Group D (n = 9)Group E (n = 9)
      Initial body weight (g)138.64 ± 6.01137.89 ± 6.8136.61 ± 7.09134.56 ± 7.28134.70 ± 7.48
      Final body weight (g)377.49 ± 32.55358.79 ± 12.7351.28 ± 36.53376.6 ± 25.57370.56 ± 39.02
      Body weight gain (g)238.8 ± 32.01220.9 ± 16.49214.67 ± 40.69242.04 ± 25.71235.86 ± 38.42
      Weight gain (%)172.56 ± 24.07160.97 ± 19.37158.44 ± 36.58180.53 ± 23.27175.58 ± 31.37
      Relative liver weight (%)2.70 ± 0.263.08 ± 0.15∗∗3.1 ± 0.292.76 ± 0.17##2.70 ± 0.23##
      Relative liver weight (%)2.70 ± 0.263.08 ± 0.15∗∗3.1 ± 0.292.76 ± 0.17##2.70 ± 0.23##
      Spleen weight (g)1.08 ± 0.101.40 ± 0.41∗∗1.21 ± 0.391.13 ± 0.12##1.01 ± 0.12##
      Relative spleen weight (%)0.29 ± 0.040.39 ± 0.12∗∗0.35 ± 0.110.3 ± 0.02##0.27 ± 0.02##
      Serum ALT (U/l)78.8 ± 5.26142.4 ± 4.80∗∗123.8 ± 7.30101.7 ± 6.90##82.6 ± 7.03##
      Serum AST (U/l)97.05 ± 4.6192.06 ± 7.57∗∗145.82 ± 10.55112.3 ± 6.48##94.34 ± 5.45##
      Serum TBIL (mg/dl)1.6 ± 0.735.04 ± 0.97∗∗3.86 ± 0.972.42 ± 0.88##1.93 ± 0.37##
      Serum GR (mU/ml)47.6 ± 3.6814.92 ± 1.80∗∗27.3 ± 1.30##38.73 ± 2.88##51.43 ± 3.65##
      Liver GR (mU/g protein)56.02 ± 4.1813.90 ± 3.70∗∗29.9 ± 4.60##45.52 ± 4.30##55.86 ± 4.49##
      Serum TEAC (nmol/ml)49.86 ± 2.8513.47 ± 3.0∗∗24.79 ± 4.18##43.8 ± 5.30##52.07 ± 3.14##
      Data presented are mean ± SD. ∗p<0.05 vs. the negative control group (A). ∗∗p<0.01 vs. the negative control group (A). #p<0.05 vs. group B (DEN-control); ## p<0.01 vs. group B (DEN-control) within each column. Groups: (A) negative control (A), (B) DEN-control (rats with DEN), (C) rats with DEN+AGE (150 mg/kg/d), (D) rats with DEN + AGE (300 mg/kg/d), and (E) rats with DEN + AGE (600 mg/kg/d).
      On the other hand, oral AGE administration in doses, 300 mg/Kg/d (group D) and 600 mg/Kg/d (group E), significantly decreased (p < 0.01) liver and relative liver and spleen weights, compared to those of the DEN group (B). Otherwise, the liver function tests (ALT, AST, and TBIL) levels significantly decreased (p < 0.01) in groups D and E, compared to the DEN group (B). GR activity (liver and serum) and serum TEAC level significantly increased (p < 0.01) post-AGE oral administration in groups C, D, and E, compared to those in the DEN group (B).

      3.3 Histopathological observations

      3.3.1 IHC of and H&E-stained liver sections of the different groups

      Hepatocytes of the control group showed normal histological patterns using &E-stained. It consisted of ill-defined lobules identified by the presence of central veins (CV) and peripherally located portal areas. The cells had light-stained cytoplasm and uniformly sized, rounded central nuclei. Portal areas showed branches of portal vein (PV), hepatic artery (HP), and bile duct (BD), Fig. 2A. Compared to the negative control group (A), liver sections of the DEN group (B) revealed marked presence of (karyomegaly) in the form of enlarged active nuclei and abnormal BD proliferation, some dark small nuclei (pyknotic), known as programmed cell death (apoptosis), were observed among hepatocytes. Also, liver sections from this group showed an increased inflammatory cell infiltrate, especially near the CV, Fig. 2B1 and B2).
      Fig. 2
      Fig. 2Photomicrographs of liver sections stained with H&E of the different groups. Liver sections of group (A) showed normal hepatocyte with equal normal size nuclei (black arrows) also normal BD (black arrows). B1. Liver section of group (B) showed pyknotic and abnormal BD proliferation (white arrows). B2. Liver section of group (B) near the CV, showed nuclear enlargement (karyomegaly). C. Liver section group (C) showed normal hepatocytes and nuclei (black arrows) with less karyomegaly (white arrow). D. Liver section of group (D) showed almost normal structure with regular arrangement of hepatic cell cords (black arrow). E. Liver section of group (E) showed normal BD and nuclei (black arrows). (H&E stain ×1000).
      Liver examination in the DEN-injected ABGE-treated (150 mg/kg b. wt., group C) rats revealed a marked decrease in hepatocytes exhibiting large nuclei (karyomegaly), necrotic regions, and cell infiltrates, compared to the DEN group (B), Fig. 2C. Liver sections from the DEN-injected ABGE-treated (300 mg/kg b. wt., group D) rats showed nearly similar results as group C. Hepatocytes with karyomegaly decreased, Fig. 2D. The DEN-injected rats orally administered AGE (600 mg/kg b. wt., group E), showed a more improved histological structure, normal BD, and nuclei, compared to the DEN group (B), Fig. 2E.
      Hepatocytes in the negative control group (A) showed weak CD10 expression in the cell cytoplasm and walls of blood sinusoids from CD10 IHC staining, Fig. 3A. liver examination in the DEN group (B) revealed high positive CD10 expression in transformed hepatocytes either near the CV or portal areas, Fig. 3B. There was no caspase 3 expression in transformed hepatocytes, near the CV or portal areas, Fig. 4B.
      Fig. 3
      Fig. 3Photomicrographs of CD10-immunostaining for liver sections of the different groups. A. Liver sections of group (A) showed weak expression of CD10 at the borders of hepatocytes along blood sinusoids (thin black arrows). B. Liver sections of DEN group (B) showed high positive expression of CD10 in the cytoplasm of transformed pre-neoplastic cells (white arrows). C. Liver sections of group (C) showed decrease in intensity and cells (white arrows) expressed CD10. D. Liver sections of group (D) showed shrunken and less cells expressing CD10. E. Liver sections of group (E) showed that expression of CD10 in liver cells was similar to those of Group (A). (CD10 immunostaining ×1000).
      Fig. 4
      Fig. 4Photomicrographs of caspase-3 immunostaining for liver sections of the different groups. A. Liver sections of group (A) showed faint (weak) staining in cell cytoplasm and walls of blood sinusoids (black arrows). B. Liver sections of DEN group (B) showed absence of caspase 3 expression in transformed hepatocytes either near central vein or portal areas. C. Liver sections of group (C) stained for caspase 3 notice few cells showed positive staining of hepatocytes which enter into apoptosis indicating cytotoxic similar effect. D. Liver sections of group (D) stained for caspase 3 notice more hepatocytes showed positive stainin. E. Liver sections of group (E) stained for caspase 3 notice increase number of hepatocytes showed positive staining indicating cell death or apoptosis. (Caspase-3 immunostaining ×1000).
      Liver examination in group C showed decreased CD10, Fig. 3C, and increased caspase-3 expressions, Fig. 4C, compared to the DEN group (B). Similarly, a decrease CD10 expression was observed in liver sections from group D, Fig, 3D, while apoptotic marker caspase-3 increased, Fig. 4D, compared to the DEN group (B). Liver sections of group E showed weak CD10 expression in the cells, similar to group A, and increased caspase-3 expression, Fig. 4E.

      3.3.2 H&E-stained spleen sections of the different groups

      Histopathological examination of the spleen tissue of the negative control group (A) showed red (black stars) and white pulps with lymphoid follicles, dark outer regions rich in lymphocyte, and light-stained germinal center indicating the presence of central arterioles, Fig. 5A. Spleen tissue of the DEN group (B) rats showed a marked decrease in white pulp size with lymphocyte depletion and (increased blood content) red pulp congestion, Fig. 5B.
      Fig. 5
      Fig. 5Photomicrographs of spleen sections stained with H&E of the different groups. A. Control splenic sections group (A) showed red pulp (black stars) and white pulp with lymphoid follicle which has outer dark regions rich in lymphocyte (white arrow) and light stained germinal center (white star) notice the presence of central arterioles (black thin arrows). B. Splenic sections of group (B) showed notice the marked decrease in white pulp volume (square) compared to control also red pulp (white stars) showed more red blood corpuscles compared to the dark stained lymphocytes seen in negative control group (A). C. Splenic sections of group (C) showed normal appearance with increase size of white pulp (white arrows). D. Splenic sections of group (D) showed normal size or even proliferating white pulps (black arrows) with active germinal centers (white stars) the red pulp (black stars) also showed normal appearance. E. Splenic sections of Group (E) showed normal size and structure of white pulp with outer cortex rich in lymphocytes (black arrows) and large active germinal center (white star) the red pulp (black star) also showed decreased in red pulp congestion (H&E stain ×200).
      Spleen examination in group C showed well-defined preservation of normal spleen histology with increased white pulp size, Fig. 5C. White pulp with lymphocyte depletion and congestion (increased blood content) in red pulp is shown in Fig. 5C.
      Similarly, group D liver examination showed normal-sized white pulp rich in lymphocytes and decreased red pulp congestion. The spleen in group E rats revealed a marked activation in white pulp histological architecture. The lymphoid masses are rich in active lymphocytes and macrophage, which looked as white cells in the center with decreased red pulp congestion, Fig. 5E.

      4. Discussion

      Black garlic is essentially a processed food, obtained through the transformation of fresh garlic, that are intensively used in traditional medicine. SAC is known to be scarce in fresh raw garlic, Kodera et al. reported that fresh raw garlic contains 20 to 30 μg/g [
      • Kodera Y.
      • Suzuki A.
      • Imada O.
      • Kasuga S.
      • Sumioka I.
      • Kanezawa A.
      • et al.
      Physical, chemical, and biological properties of S-allylcysteine, an amino acid derived from garlic.
      ]. However, SAC amount in black garlic is generally five to six times higher than that in raw garlic [
      • Wang D.
      • Feng Y.
      • Liu J.
      • Yan J.
      • Wang M.
      • Sasaki J.-i.
      • et al.
      Black garlic (Allium sativum) extracts enhance the immune system.
      ,
      • Sato E.
      • Kohno M.
      • Hamano H.
      • Niwano Y.
      Increased anti-oxidative potency of garlic by spontaneous short-term fermentation.
      ]. Several analytical methods have been reported for SAC determination like HPLC [
      • Yamazaki Y.
      • Tokunaga T.
      • Okuno T.
      Quantitative determination of eleven flavor precursors (S-alk (en) yl cysteine derivatives) in garlic with an HPLC method.
      ] and thin-layer chromatography (TLC) [
      • Asdaq S.
      • Inamdar M.
      Potential of garlic and its active constituent, S-allyl cysteine, as antihypertensive and cardioprotective in presence of captopril.
      ]. Based on the results of this study, SAC content in AGE powder was 656.5 μg/g (expressed as dry matter base) and 42.7μg/g in fresh aged black garlic, which is much lower than previously mentioned Sasaki et al. [
      • Sasaki J.
      • Lu C.
      • Machiya E.
      • Tanahashi M.
      • Hamada K.
      Processed black garlic (Allium sativum) extracts enhance anti-tumor potency against mouse tumors.
      ], but higher than the level found by Kodera et al. [
      • Kodera Y.
      • Suzuki A.
      • Imada O.
      • Kasuga S.
      • Sumioka I.
      • Kanezawa A.
      • et al.
      Physical, chemical, and biological properties of S-allylcysteine, an amino acid derived from garlic.
      ]. We suggest that the partial disagreement with previous studies could be due to variations in black garlic processing time or analytical methods.
      DEN is a potent hepatocarcinogenic nitrosamine present in tobacco smoke, water, cheddar cheese, cured, and pharmaceutical agents [
      • El-Shahat M.
      • El-Abd S.
      • Alkafafy M.
      • El-Khatib G.
      Potential chemoprevention of diethylnitrosamine-induced hepatocarcinogenesis in rats: Myrrh (Commiphora molmol) vs. turmeric (Curcuma longa).
      ]. DEN is widely accepted experimentally for pre-neoplastic lesions and hepatic tumor induction in rats. Our results showed a significant increase (p<0.01) in liver, serum, and relative liver and spleen weights post-DEN (200 mg/kg b.wt.) injection in the DEN group (B) rats, compared to the negative control group (A) rats. These findings are in line with results from previous studies [
      • Abe R.
      • Okano J.-I.
      • Imamoto R.
      • Fujise Y.
      • Murawaki Y.
      Sequential analysis of diethylnitrosamine-induced hepatocarcinogenesis in rats.
      ,
      • Abdel-Hamid N.M.
      • Nazmy M.H.
      • Abdel-Ghany M.I.
      • Nazmy W.H.
      Cytokines as important playmakers of experimental hepatocarcinogenesis confounded by diabetes.
      ,
      • Jayakumar S.
      • Madankumar A.
      • Asokkumar S.
      • Raghunandhakumar S.
      • Kamaraj S.
      • Divya M.G.J.
      • et al.
      Potential preventive effect of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in rats.
      ]. Our results indicate that daily oral administration of AGE doses, 300 (group D) and 600 mg/Kg/d (group E), showed a significant decrease (p<0.01) in the liver, spleen, and relative liver and spleen weights, compared to the DEN group (B). These observations are consistent partially with the results of previous studies that used garlic oil [
      • Zhang C.-L.
      • Zeng T.
      • Zhao X.-L.
      • Yu L.-H.
      • Zhu Z.-P.
      • Xie K.-Q.
      Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats.
      ] and AGE [
      • Krishnappa S.
      • Satyanarayana M.
      • Rao S.
      • Yathiraj S.
      • Shridhar N.
      • Mukartal S.
      • et al.
      Pathomor-phological studies on the effect of aged garlic extract in aflatoxin b 1 induced liver damage in wistar albino rats.
      ].
      The results of our study showed DEN-induced hepatocellular damage in Wistar rats, clearly evidenced by the significant elevation in the serum levels of liver biochemical parameters (AST, AST, and TBIL) in the DEN group (B). These results were in agreement with other researches [
      • Shahjahan M.
      • Vani G.
      • Shyamaladevi C.
      Effect of Solanum trilobatum on the antioxidant status during diethyl nitrosamine induced and phenobarbital promoted hepatocarcinogenesis in rat.
      ,
      • Pradeep K.
      • Mohan R.
      • Victor C.
      • Gobianand K.
      • Karthikeyan S.
      Protective effect of Cassia fistula Linn. on diethylnitrosamine induced hepatocellular damage and oxidative stress in ethanol pretreated rats.
      ,
      • Rezaie A.
      • Fazlara A.
      • Karamolah M.H.
      • Shahriari A.
      • Zadeh H.N.
      • Pashmforosh M.
      Effects of Echinacea purpurea on hepatic and renal toxicity induced by diethylnitrosamine in rats.
      ]. The activities of serum AST and ALT enzymes are generally accepted as indices of liver injury and this is distinct in rodents [
      • Li Y.
      • Tang Z.-Y.
      • Hou J.-X.
      Hepatocellular carcinoma: insight from animal models.
      ]. Furthermore, our study demonstrated a significant (p < 0.01) reduction in the activity of liver enzymes and TBIL in groups D and E, compared to the negative control group (A). These findings indicated that oral AGE administration could suppress DEN-induced liver damage, which was in agreement with previous studies [
      • Kim M.H.
      • Kim M.J.
      • Lee J.H.
      • Han J.I.
      • Kim J.H.
      • Sok D.-E.
      • et al.
      Hepatoprotective effect of aged black garlic on chronic alcohol-induced liver injury in rats.
      ,
      • Chu Q.
      • Lee D.T.
      • Tsao S.W.
      • Wang X.
      • Wong Y.C.
      S-allylcysteine, a water-soluble garlic derivative, suppresses the growth of a human androgen-independent prostate cancer xenograft, CWR22R, under in vivo conditions.
      ]. AGE reportedly helps in parenchymal cell regeneration in the liver; thus, protecting membrane integrity and minimizing enzyme leakage [
      • Jayakumar S.
      • Madankumar A.
      • Asokkumar S.
      • Raghunandhakumar S.
      • Kamaraj S.
      • Divya M.G.J.
      • et al.
      Potential preventive effect of carvacrol against diethylnitrosamine-induced hepatocellular carcinoma in rats.
      ].
      Our results reported a significant (p < 0.01) decrease in serum and liver GR activities in the DEN group (B), compared to the negative control group (A). These results were in agreement with previous studies [
      • Dewa Y.
      • Nishimura J.
      • Muguruma M.
      • Jin M.
      • Kawai M.
      • Saegusa Y.
      • et al.
      Involvement of oxidative stress in hepatocellular tumor-promoting activity of oxfendazole in rats.
      ,
      • Farombi E.O.
      • Shrotriya S.
      • Surh Y.-J.
      Kolaviron inhibits dimethyl nitrosamine-induced liver injury by suppressing COX-2 and iNOS expression via NF-κB and AP-1.
      ,
      • Chen B.
      • Ning M.
      • Yang G.
      Effect of paeonol on antioxidant and immune regulatory activity in hepatocellular carcinoma rats.
      ]. Our results also demonstrated that serum TEAC value in the DEN group (B) rats significantly (p < 0.01) decreased, compared to the negative control group (A). The lower TEAC levels may be due to decreased antioxidant synthesis or increased antioxidant consumption by oxidants or by modifying the antioxidant property modification by oxidative stress [
      • Olivieri S.
      • Conti A.
      • Iannaccone S.
      • Cannistraci C.V.
      • Campanella A.
      • Barbariga M.
      • et al.
      Ceruloplasmin oxidation, a feature of Parkinson's disease CSF, inhibits ferroxidase activity and promotes cellular iron retention.
      ]. The present results demonstrated that ABGE-treated rat groups (C, D, and E) showed a significant (p < 0.01) increase in serum and liver GR activities, compared to the DEN group (B). These findings indicate that the oral AGE administration may alleviate DEN-mediated oxidative injury, dose-dependently, which was in agreement with previous studies that used SAC (200 mg/Kg b.wt.) [
      • Sundaresan S.
      • Subramanian P.
      Prevention of N-nitrosodiethylamine-induced hepatocarcinogenesis by S-allylcysteine.
      ], garlic [
      • Shaarawy S.M.
      • Tohamy A.A.
      • Elgendy S.M.
      • Elmageed Z.Y.A.
      • Bahnasy A.
      • Mohamed M.S.
      • et al.
      Protective effects of garlic and silymarin on NDEA-induced rats hepatotoxicity.
      ], and garlic oil [
      • Zhang C.-L.
      • Zeng T.
      • Zhao X.-L.
      • Yu L.-H.
      • Zhu Z.-P.
      • Xie K.-Q.
      Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats.
      ]. More so, the present study also showed that serum TEAC levels significantly increased (p < 0.01) post-AGE oral administration in groups C, D, and E, compared to those of the DEN group (B). The improved percentage reduction in GR activity and TEAC values indicate that AGE oral administrated doses can enhance antioxidant activity, therefore, has a protective effect against DEN-induced oxidative stress and may provide the liver with an effective detoxification mechanism.
      In this study, spleen sections from the different groups were examined. The results showed that treating rats with DEN (200 mg/kg i.p) and CCl4 (2 ml/kg i.g) produced marked depletion of splenic lymphocyte contents, white, and red pulps. It is generally accepted that spleen plays a complex role in tumor immunity, which changes at different cancer stages [
      • Zhang S.
      • Li Z.-f.
      • Dun P.
      • Huang C.
      • Rui Z.
      • Liu Z.-w.
      Changes of splenic macrophage during the process of liver cancer induced by diethylnitrosamine in rats.
      ]. AGE administration to DEN-treated animals showed enlargement of splenic white pulps, with the appearance of new cell populations with active light stained nuclei at the marginal zone. Also, in the high-dose group, the germinal center looked larger and contained cells with lighter nuclei, indicating activation.
      In this study, karyomegaly (enlarged nuclei) and some nuclei revealing clear signs of dark small nuclei (pyknotic) were observed in hepatocytes in the DEN group (B). These findings are comparable with an early study, [
      • Poojari R.
      • Gupta S.
      • Maru G.
      • Khade B.
      • Bhagwat S.
      Chemopreventive and hepatoprotective effects of embelin on N-nitrosodiethylamine and carbon tetrachloride induced preneoplasia and toxicity in rat liver.
      ], in which karyomegaly observed in most samples was considered as evidence of pre-neoplastic change.
      Our study noted that CD10 was highly expressed in hepatocytes of the DEN group (B) rats, compared to the negative control group (A), indicating their tendency to proliferate and transform to neoplastic cells. On the other hand, dose-dependent decrease in CD10 expression was observed in the ABGE-treated groups. IHC has been applied extensively to hepatic neoplasms for differential diagnosis of HCC, cholangiocarcinoma, and metastatic carcinoma [
      • Lee H.S.
      • Kim W.H.
      • Kang G.H.
      Hepatocyte expressions in hepatocellular carcinomas, gastrointestinal neoplasms, and non-neoplastic gastrointestinal mucosa: its role as a diagnostic marker.
      ]. CD10 expression has been detected in tumor cells such as breast and liver cancers and thought to be involved in tumor-stromal interactions [
      • Sasaki T.
      • Kuniyasu H.
      • Luo Y.
      • Fujiwara R.
      • Kitayoshi M.
      • Tanabe E.
      • et al.
      Serum CD10 is associated with liver metastasis in colorectal cancer.
      ,
      • Louhichi T.
      • Saad H.
      • Dhiab M.B.
      • Ziadi S.
      • Trimeche M.
      Stromal CD10 expression in breast cancer correlates with tumor invasion and cancer stem cell phenotype.
      ].
      In the present study, the presence of caspase-3 in histology labs was used to detect early apoptotic cells. It was observed that it was low in the control group and present only in few cells and along the blood sinusoidal walls. Also, in the DEN group (B) some cells stained positive as apoptosis is a normal process accompanying tumor formation. Caspase-3 expression was activated in the BGE-treated groups, dose-dependently. A similar effect was observed by [
      • Zhang C.-L.
      • Zeng T.
      • Zhao X.-L.
      • Yu L.-H.
      • Zhu Z.-P.
      • Xie K.-Q.
      Protective effects of garlic oil on hepatocarcinoma induced by N-nitrosodiethylamine in rats.
      ], who demonstrated that DEN administration inhibited caspase-3 expression, but garlic oil activated its expression. The effects displayed by the garlic derivatives include apoptosis induction, regulation of cell cycle progression and, signal transduction pathway modifications, all of which are cancer-reducing events [
      • Pinto J.T.
      • Rivlin R.S.
      Antiproliferative effects of allium derivatives from garlic.
      ,
      • Knowles L.M.
      • Milner J.A.
      Possible mechanism by which allyl sulfides suppress neoplastic cell proliferation.
      ].
      Moreover, the safety of AGE was also determined in several studies. Hoshino et al. investigated that e effects of garlic preparations, including dehydrated raw garlic powder (RGP), dehydrated boiled garlic powder (BGP), and AGE on the gastric mucosa. The result of the study reported that AGE did not cause any undesirable effects. Whereas raw garlic powder caused severe damage, including erosion. BGP also caused reddening of the mucosa [
      • Hoshino T.
      • Kashimoto N.
      • Kasuga S.
      Effects of garlic preparations on the gastrointestinal mucosa.
      ]‏. Badr and Al-Mulhim also showed the significant gastric mucosal healing of the high AGE dose (200 mg/kg) compared to that at dose 100 mg/kg in the gastric mucosal injury induced by indomethacin. The safety of AGE was also determined in this study [
      • Badr G.M.
      • Al-Mulhim J.A.
      The protective effect of aged garlic extract on nonsteroidal anti-inflammatory drug-induced gastric inflammations in male albino rats.
      ].
      However, the present study has some limitations. Therefore, further studies are needed with a positive control group treated with a standard drug to elucidate the mechanisms of action of garlic as well as its efficacy and safety in the treatment of various diseases. Future studies should focus on identifying the major components, aside SAC, responsible for the anti-tumor activity and also aim to elucidate the underlying mechanisms. Further studies could also quantify SAC in different garlic products, with the identification of the effective AGE dose to reduce the supplementation to feasible doses. Further studies are needed to demonstrate effective various doses of AGE in human.
      In conclusion, the results of this study revealed that different AGE doses are effective in enhancing the anti-tumor activity in DEN-induced hepatocarcinogenic male Wistar rats. AGE administration in doses 300 mg/Kg/d (group D) and 600 mg/Kg/d (group E) for 7 weeks, significantly (p < 0.01) decreased liver, spleen, and relative liver weights, compared to that of the DEN group (B), with improved liver function tests by significantly decreased (p<0.01) serum ALT, AST, and TBIL levels. It also showed that AGE improved GR activity (liver and serum) and serum TEAC levels, indicating that AGE oral administration can enhance antioxidant activity. This finding suggests that AGE has remarkable hepatoprotective and antioxidant effects in DEN-induced hepatocarcinogenic rats. Further studies are needed to demonstrate effective various doses of AGE in human. The mechanisms underlying the active principle responsible for the anti-tumor activity and need to be elucidated in future studies.

      Declaration of competing interest

      The authors declare no conflicts of interest.

      Acknowledgments

      The authors would like to express their gratitude to King Khalid University, Saudi Arabia for providing administrative and technical support.

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