Preparation of S-Allylcysteine-Enriched Black Garlic Juice and Its

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Preparation of S-allyl cysteine-enriched black garlic juice and its antidiabetic effects in streptozotocin-induced insulin-deficient mice Jun Ho Kim, Su Hyun Yu, Yun Jeong Cho, Jeong Hoon Pan, Hyung Taek Cho, Jeong Ho Kim, Hyejin Bong, Yeojin Lee, Moon Han Chang, Ye Jin Jeong, Garam Choi, and Young Jun Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04948 • Publication Date (Web): 21 Dec 2016 Downloaded from http://pubs.acs.org on December 26, 2016

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Journal of Agricultural and Food Chemistry

Preparation of S-allyl cysteine-enriched black garlic juice and its antidiabetic effects in streptozotocin-induced insulin-deficient mice

Jun Ho Kim,∥ Su Hyun Yu,∥ Yun Jeong Cho, Jeong Hoon Pan, Hyung Taek Cho, Jeong Ho Kim, Hyejin Bong, Yeojin Lee, Moon Han Chang, Ye Jin Jeong, Garam Choi, Young Jun Kim*

Department of Food and Biotechnology, Korea University, Sejong, 30019, Republic of Korea

*

Corresponding author

Tel: +82-44-860-1435

∥

Fax: +82-44-865-0220

E-mail: [email protected]

First two authors equally contributed to this work.

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ACS Paragon Plus Environment


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Abstract

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S-allyl cysteine (SAC), produced in large amounts during the aging process of garlic via

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enzymatic hydrolysis, is known as a key compound responsible for the multiple pharmacological

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activities of aged black garlic. In this study, we investigated the effects of enzyme- and high

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hydrostatic pressure (HHP)-assisted extraction on the content of the bioactive compounds,

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including SAC, in black garlic juice (BGJ) and evaluated the antidiabetic effects of SAC-

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enriched BGJ in streptozotocin (STZ)-treated mice. The aging process increased the contents of

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SAC, total polyphenols, and flavonoids in garlic juice. More importantly, pretreatment of

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pectinase cocktail with HHP resulted in a greater increase in those compounds during aging.

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Enzyme-treated BGJ reduced hyperglycemia and improved islet architecture and β-cell function

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in STZ-treated mice. Moreover, these effects were more potent than those of BGJ prepared by

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the conventional aging process. These findings provide useful information for the production of

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black garlic with improved bioactivities.

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Keywords: S-allyl cysteine, aged black garlic, pectinase, high hydrostatic pressure, antidiabetic

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effect

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Introduction Black garlic, a type of processed garlic prepared by aging the whole bulbs of raw garlic at

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a high temperature under controlled humidity, has been shown to display multiple

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pharmacological activities, including antioxidant, anticancer, antiatherogenic and

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hepatoprotective properties.1-4 It has been reported that black garlic is more pharmacologically

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effective than raw garlic, which may result from the physicochemical changes induced by the

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non-enzymatic browning reactions and enzymatic hydrolysis that occur during the aging

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process.5, 6 Non-enzymatic browning reactions, such as Maillard reaction, caramelization and

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chemical oxidation of phenols, are associated with the formation of heat-induced antioxidants in

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processed foods.7 An increase in the content of S-allyl cysteine (SAC) during the aging of garlic

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is also a critical change responsible for the improved biological activities of garlic. SAC is

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formed owing to the enzymatic hydrolysis of γ-glutamyl-S-allyl cysteine (GSAC) with γ-

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glutamyl transpeptidase. The biological activities of SAC have been extensively studied using in

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vitro and in vivo models.8 Recently, it has been shown that total polyphenol, flavonoid, and SAC

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contents of garlic increase steadily with an increase in aging time, resulting in enhanced

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antioxidant activity of aged garlic.1, 5

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Potent antioxidant ability of SAC has been demonstrated, particularly in streptozotocin

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(STZ)-induced diabetic animal models. Saravanan and Ponmurugan9 reported that the protective

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effects of SAC (150 mg/kg) against STZ-induced oxidative damage in the liver and kidney are

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comparable to those of gliclazide (5 mg/kg), a well-known antioxidant and antihyperglycemic

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drug. Moreover, administration of SAC improved glucose metabolism by modulating hepatic

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glycolysis and gluconeogenesis10 and alleviated diabetes-related erectile dysfunction by reducing

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the generation of reactive oxygen species in STZ-treated rats.11 Because SAC is the most 3



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abundant organosulfur compound in aged black garlic, these findings highlight black garlic as a

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novel promising candidate for diabetes control.

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In this study, we investigated the antidiabetic effects of aged black garlic, prepared by

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pectinase treatment with high hydrostatic pressure (HHP) processing, in STZ-treated mice.

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Pectinase treatment has been shown to increase the recovery of antioxidant compounds from

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natural matrices by catalyzing the hydrolysis of the cell wall polysaccharides.12, 13 HHP has been

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used as a non-thermal alternative to juice pasteurization with improved nutritional and

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antioxidant values.14 Interestingly, Tomlin et al.15 reported that HHP stabilized the pectinase

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cocktail, which maintained activity for longer period than did the pectinase cocktail at

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atmospheric pressure. In addition, HHP has been shown to control the enzymatic and non-

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enzymatic browning reactions that occur during food processing and storage.16, 17 Thus, we

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hypothesized that, during aging of garlic, pectinase pretreatment under HHP condition could

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enhance the formation and/or recovery of the antioxidant compounds, including polyphenols,

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flavonoids, and SAC, which would result in improved antioxidant and antidiabetic activities of

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black garlic. This study was carried out to investigate these effects in STZ-induced insulin-

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deficient mice.

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Materials and methods

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Sample preparation. Fresh garlic was ground using a laboratory blender (Waring Commercial,

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New Hartford, CT, USA), and the commercial pectinase cocktail (Multifect Pectinase FE,

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Genencor International, NY, USA), containing pectinase, cellulase, and hemicellulose, was

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added at 3% (v/v) into the mashed garlic juice. Pressure treatment was performed at 100 MPa 4


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and 55°C for 24 h using a high pressure liquefying extractor (DFS-2L, Toyo Koatsu Co. Ltd.,

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Hiroshima, Japan). The pressure value was selected based on the ABTS radical scavenging

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activity of garlic juice treated at different pressure conditions. After HHP treatment, the garlic

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juice was further incubated in a thermo-hydrostat chamber (TH-G-800, Jeio-Tech, Seoul, Korea)

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at 55°C with 70% relative humidity for 15 days. The aged garlic juice was filtered, freeze-dried,

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and then stored at -20°C until future use. To determine the effect of pectinase treatment with

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HHP, the aged garlic sample was prepared with the same procedure described above, except for

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the enzyme and HHP treatments. The mashed raw garlic juice was also filtered, freeze-dried, and

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compared in this study.

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Determination of SAC, total polyphenols, and total flavonoids. SAC content of garlic juice

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was determined according to the method described previously5 using an HPLC system equipped

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with diode-array detector (Nanospace SI-2, Shiseido, Tokyo, Japan). The analyte was seperated

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using a Hypersil GOLD column (250 mm × 4.6 mm ID, 5 µm; Thermo Scientific, Waltham, MA,

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USA).

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The total polyphenol content was determined using Folin–Ciocalteu’s colorimetric method with

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some modifications as described in our previous report18 and expressed as mg of gallic acid

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equivalents (GAE). Total flavonoid content was measured using aluminum chloride colorimetric

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assay as previously reported19, and expressed as mg of rutin equivalents (RE).

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ABTS radical scavenging assay. The antioxidant activity of garlic juice was measured using the

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ABTS assay as previously described20 with slight modifications. Briefly, 2,2'-azobis (2-

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amidinopropane) dihydrochloride (1 mM) and ABTS (2.5 mM) were dissolved in phosphate5



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buffered saline (PBS, 100 mM). The ABTS solution was heated at 70°C for 30 min and then

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cooled down to room temperature. The diluted ABTS solution (0.98 mL) was mixed with the

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sample solution (0.02 mL), and the control solution was mixed with an equal amount of water.

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The absorbance was measured at 734 nm after 20 min. The activity was expressed as vitamin C

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equivalents antioxidant capacity.

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Animal study. Male C57BL/6J mice (Central Lab. Animal Inc., Seoul, Korea), 8 weeks of age,

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were housed with a 12-h light/dark cycle and fed standard pellet diet during the experimental

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period. All mice were acclimatized under laboratory conditions for 1 week and randomly divided

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into six treatment groups: 1) non-STZ + vehicle, 2) STZ + vehicle, 3) STZ + raw garlic juice (GJ)

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at 200 mg/kg (GJ 200), 4) STZ + aged black garlic juice (BGJ) at 200 mg/kg (BGJ 200), 5) STZ

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+ pectinase- and HHP-treated black garlic juice (PBGJ) at 100 mg/kg (PBGJ 100) and 6) STZ +

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PBGJ at 200 mg/kg (PBGJ 200). Mice were orally administered all compounds with vehicle

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(normal saline) once daily for 31 days. After 2 weeks of oral administration, diabetes was

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induced by an intraperitoneal injection with either 50 mg/kg STZ (dissolved in citrate buffer, 50

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mM, pH 4.5) or citrate buffer alone for four consecutive days. After the experimental periods

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animals were euthanized using an overdose of avertin (2,2,2-tribromoethanol). Blood was

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collected from the heart, and pancreas was collected for further analysis. Figure 1A shows a

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timeline of the experiment. The care and treatment of animals were done in compliance with the

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Institutional Animal Care and Use Committee at Korea University.

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Measurement of glucose and insulin levels. Random-fed blood glucose level was determined

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before and after STZ injection with a glucometer (OneTouch Ultra 2, LifeScan, Inc., Milpitas, 6


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CA, USA) using tail tip blood. Cumulative incidence of diabetes was calculated by dividing the

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number of hyperglycemic mice (non-fasting glucose level ≥ 250 mg/dL) by total number of

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mice at each time point. For oral glucose tolerance test (OGTT), mice were administered orally 2

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g/kg of glucose solution after a 16-h fast. Blood samples were drawn at 0, 15, 30, 60, and 90 min

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following the administration for the measurements of glucose and insulin. Plasma insulin was

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measured using an ELISA kit (Millipore Co., Billerica, MA, USA). The area-under the glucose

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curve and insulin curve were calculated to evaluate the response to an OGTT.

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The pancreas was homogenized in acidified ethanol and incubated for 16 h at 4°C. The extracts

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of pancreas were centrifuged at 3,000 g for 10 min at 4°C, and then the insulin content of the

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supernatant was determined using an ELISA kit (Millipore) following the manufacturer's

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instructions.

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Histological and immunohistochemical staining. Pancreata were fixed overnight in 10%

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neutral formalin and embedded in paraffin. Tissue sections were deparaffinized and dehydrated

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in a series of xylene and ethanol washes, and stained with Hematoxylin and Eosin (H&E). The

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sections were permeabilized with 0.1% Triton X-100 for 10 min and blocked in 5% goat serum.

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The sections were incubated overnight at 4°C with either 1:100 rabbit anti-insulin (Cell

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Signaling Technology, Danvers, MA, USA ) and mouse anti-glucagon (Abcam, Cambridge, UK).

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The slides were then washed with PBS and incubated with the appropriate secondary antibodies:

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Alexa Fluor 595 goat anti-rabbit and 488 anti-mouse IgG (1:200; Invitrogen, CA, USA). The

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slides were washed again in PBS and mounted using an Antifade Mounting Medium (Vector

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Laboratories, Burlingame, CA, USA). Images of islets were acquired using a fluorescence

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microscope (Carl Zeiss AG; Oberkochen, Germany). The area of islet was determined by using 7



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Image J software (ImageJ v1.32j, National Institutes of Health).

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Apoptosis measurement. β-Cell apoptosis was measured by TUNEL and active caspase-3

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staining of the pancreas sections. TUNEL assay was performed by using In situ cell death

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detection kit (Roche Diagnostics, Basel, Switzerland), as specified by the manufacturer. Cells

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with blue (nuclei) and green (TUNEL) nuclear granulation were visualized using a fluorescence

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microscope. Anti-cleaved caspase-3 (Cell Signaling Technology) antibody was used at a

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concentration of 1/800 for the immunofluorescence staining of active caspase-3. The percentage

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of apoptosis was calculated by dividing the number of TUNEL- or active caspase-3-positive cells

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by the total number of cells stained nuclei. Four mice per condition and 5-7 islets per tissue

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section were counted.

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Statistical analysis. All statistical analyses were performed by one-way ANOVA using the SAS

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software (SAS Institute Inc., Cary, NC). The repeated measure was used for the statistical

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analysis of the values of body weight and blood glucose over time. Multiple comparisons were

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performed with a Tukey-Kramer adjustment. Results are presented as mean ± SE. A value of P

Preparation of S-Allylcysteine-Enriched Black Garlic Juice and Its

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