Antihypertensive Angiotensin I-Converting Enzyme Inhibitory Activity and Antioxidant Activity of Vitis hybrid-Vitis coignetiae Red Wine Made with Saccharomyces cerevisiae

Abstract

A Vitis hybrid-Vitis coignetiae red wine was vinified by fermentation of a mixture of a Vitis hybrid-Vitis coignetiae must with Saccharomyces cerevisiae KCTC 7904 at 25℃ for 10 days. The Vitis hybrid-Vitis coignetiae red wine showed high antihypertensive angiotensin I-converting enzyme (ACE) inhibitory activity (67.8%) and antioxidant activity (76.7%). The antihypertensive ACE inhibitor in the Vitis hybrid-Vitis coignetiae red wine was partially purified by solid phase extraction chromatography, and its ACE inhibitory activity yielded an IC₅₀ of 1.8 mg/mL. Six kinds of oligopeptides, including five new kinds, were contained in the partially purified ACE inhibitor fraction from the red wine after 10 days of fermentation. Antioxidant activity decreased significantly from 76.7% to 40.5% when the post-fermentation period was prolonged to 30 days.

Many studies have reported the health benefits of red wine [1-6]; however, only a few have investigated the cardiovascular and anti-dementia functionalities of red wines [7]. In a previous study [8], the effect of Vitis coignetiae on the quality and anti-hypertensive effects of a new Vitis hybrid (Sheridan) red wine were investigated. In this study, the angiotensin I-converting enzyme (ACE) inhibitory and antioxidant activities of Vitis hybrid-Vitis coignetiae red wine were investigated.

V. coignetiae (Wild grape) and Vitis hybrid (Sheridan) grapes harvested in 2010 were purchased from a commercial market. Saccharomyces cerevisiae KCTC 7904 from the Laboratory of Food Biotechnology at Paichai University (Daejeon, Korea) was used for preparing the red wines. The ACE used in this study was extracted overnight from rabbit lung acetone powder (Sigma Chemical Co., St. Louis, MO, USA) using 100 mM sodium borate buffer (pH 8.3) containing 300 mM NaCl at 4℃. Hippuric acid-histidine-leucine and DPPH were also purchased from Sigma Chemical. Unless otherwise specified, all chemicals were of analytical grade.

The Vitis hybrid grapes were crushed, filtered to prepare juice, and then supplemented with V. coignetiae (10%, w/v). The mixture was again crushed and adjusted to 24° brix by adding sugar. After adding 150 ppm K₂S₂O₅, the mixture was left to settle for 5 hr and then inoculated with 1% S. cerevisiae KCTC 7904, which had been incubated in must for 24 hr. The complex musts were fermented for 10 days at 25℃ and filtered [9].

ACE inhibitory activity was assayed according to a modified method of Cushman and Cheung [10]. A mixture containing 100mM sodium borate buffer (pH 8.3), 300mM NaCl, 3 units ACE, and 50 µL sample (1 mg of the freeze-dried extracts was dissolved in 50 µL of 100 mM sodium borate buffer, pH 8.3) was preincubated for 10 min at 37℃. The reaction was initiated by adding 50 µL hippuric-histidine-leucine at a final concentration of 5 mM and terminated after 30 min of incubation by adding 250 mL 1.0 N HCl. The hippuric acid liberated was extracted with 1 mL of ethyl acetate, and 0.8 mL of the extract was evaporated to dryness using a Speed Vac Concentrator (EYELA, Tokyo, Japan). The residue was then dissolved in 1 mL sodium borate buffer, and the absorbance was measured at 228 nm to estimate ACE inhibitory activity using the following equation:

Inhibition activity (%) = [1 - (A - B)/(C - D)] × 100

where A is the absorbance of the solution containing the ACE, substrate, and sample; B is the absorbance of the solution containing ACE and the sample without the substrate; C is the absorbance of the solution containing ACE and the substrate without the sample; and D is the absorbance of the solution containing only the substrate.

Fibrinolytic activity was assayed as follows [11]. We added 0.5 mL of each sample to 3 mL of the substrate solution (0.6% fibrin in 0.1M McIlvaine's buffer, pH 7.0) and incubated them at 40℃ for 10 min. The reaction was stopped by adding 3 mL 0.4M trichloriacetic acid for 30 min and the mixture was filtered with Whatman No. 2 filter paper (Whatman International Ltd., Clifton, NJ, USA). We then placed a reaction mixture of 1 mL of filtrates, 5mL 0.4M Na₂CO₃, and 1 mL 1 N Folin reagent at room temperature for 30 min. The amount of tyrosine released from the fibrin was determined from a tyrosine standard curve based on absorbance measurements at 660 nm. One unit of activity was defined as the production of 1 µg of tyrosine per minute for a 1 mL sample.

3-Hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitory activity was assayed spectrophotometrically by measuring the rate of decrease in absorbance at 340 nm due to the oxidation of NADPH [11]. A 0.5 mL volume of the reaction mixture contained the following: 50 µM potassium phosphate buffer, pH 7.0, 2 µM dithiothreitol, 0.3 µM NADPH, 0.15 µM HMG-CoA, and 100 µg of protein (enzyme). Two reaction mixture aliquots were preincubated in 2 mm light-path glass cuvettes for 5 min at 37℃. For the assay, we added HMG-CoA to one reaction mixture aliquots and added HMG-CoA with each red wine extract to the other reaction mixture aliquot. The aliquots were assayed at 37℃ in a recording spectrophotometer. The initial velocity of the reaction was measured, and the net rate of NADPH oxidation was determined by subtracting the rate of oxidation in the absence of HMG-CoA from the rate observed when both substrates were present. Thus, we calculated the HMG-CoA reductase inhibitory activity as follows:

HMG-CoA reductase inhibitory activity (%) = [1 - (A₃₄₀ of sample - A₃₄₀ sample of blank)/(A₃₄₀ of control - A₃₄₀ control of blank)] × 100

Antioxidant activity was assayed as follows using DPPH [11]. A 0.8 mL DPPH solution (12.5 mg DPPH dissolved in 100 mL ethanol) was added to 0.2 mL of sample, shaken for 10 sec, and left for 10 min. We then determined the absorbance at 525 nm. The antioxidant activity was calculated as: [1 - (absorbance of reaction mixture - absorbance of sample alone)/absorbance of blank] × 100.

The ACE inhibitor contained in the Vitis hybrid-Vitis coignetiae red wine was partially purified by solid phase extraction chromatography as follows. Fifty mL of concentrated Vitis hybrid-Vitis coignetiae red wine was applied to a C₁₈ solid phase extraction (Sep-Pak C₁₈ Cartridge; Waters Co., Milford, MA, USA), and the peptides in the active fraction were analyzed by liquid chromatography-mass spectrometry (MS). The active fractions were separated by 12% SDS-PAGE and in-gel digested, as described previously [12]. The digests were resolved in 15 mL 0.02% formic acid in 0.5% acetic acid. The peptide samples (10 mL) were concentrated on an MGU30-C₁₈ trapping column (LC Packings, San Francisco, CA, USA) and subjected to a nanocolumn (10 cm, 75 µm i.d., C₁₈ reverse-phase column, Proxeon Biosystems, Staermosegaardsvej, Denmark) at a flow rate of 120 nL/min. Subsequently, the peptides were eluted from the column by applying a gradient of 0~65% acetonitrile for 80 min at the same flow rate. All MS and MS/MS spectra were acquired in data-dependent mode using a LCQ-Deca ESI ion trap mass spectrometer (Thermo Finnigan Co., San Jose, CA, USA). The MS/MS spectra were identified using SEQUEST ver. 3.3 software (Thermo Finnigan Co.).

Functional properties of Vitis hybrid (Sheridan)-V. coignetiae red wine

After 10 days of fermentation, the Vitis hybrid-V. coignetiae red wine yielded an ethanol content of 13.5% and 67.8% antihypertensive ACE inhibitory activity (Table 1). Furthermore, the antioxidant activity was 76.7%, but no or very low levels of fibrinolytic and HMG-CoA reductase inhibitory activity were detected.

Table 1.

Physiological functionality of Vitis hybrid (Sheridan)-Vitis coignetiae red wine

HMG-CoA, 3-hydroxy-3-methyl-glutaryl-CoA; n.d., not detectable.

ACE inhibitory peptides and antioxidant activity

After the partial purification of the ACE inhibitor from the Vitis hybrid-V. coignetiae red wine by systematic solvent extraction and solid phase extraction chromatography, an active fraction with an IC₅₀ of 1.8 mg/mL was obtained. Six kinds of oligopeptides, including five new kinds, were contained in the red wine after a 10 day fermentation (Table 2). Therefore, we chemically synthesized these oligopeptides, and we plan to identify the antihypertensive ACE inhibitory peptides after determining their ACE inhibitory activity. Recently, various ACE inhibitors with antihypertensive effects [13] have been isolated from Korean traditional alcoholic beverages [12] and sake and its by-products [9]. Almost all ACE inhibitors are peptides or a complex of glycoprotein and polyphenol [14], except for triterpene [11]. Many different sequences of ACE inhibitors have been reported, ranging from tripeptides to oligopeptides [9].

Table 2.

Peptide analysis of the Vitis hybrid-Vitis coignetiae red wine after 0 and 10 days of fermentation

Although the peptide sequences of the ACE inhibitor from this red wine were longer and thus were degraded more easily in the stomach by some proteases and more difficult to absorb in the intestine, this is the first report of unique antihypertensive ACE inhibitory oligopeptides in red wine. In a previous study [8], we demonstrated the antihypertensive action of a partially purified ACE inhibitor using the spontaneous hypertensive rat.

The antioxidant activity of the Vitis hybrid-V. coignetiae red wine decreased significantly from 76.7% to 40.5% during a 30 day post-fermentation period and remained unchanged thereafter (data not shown), although its ACE inhibitory activity increased about 12% during a 60 day post-fermentation period in a previous study [8].