Previous work has shown that red wines, grape juices, and other grape products cause endothelium-dependent relaxation (EDR) of blood vessels in vitro by increasing nitric oxide production. In this paper we describe the isolation and characterization of some of the compounds responsible for EDR activity. Concord grape seeds were extracted with methanol and the compounds were separated by Toyopearl TSK HW-40S chromatography. Resulting fractions (primarily phenolic acids, catechins, and proanthocyanidins) were further separated semipreparatively by reversed-phase HPLC, and peaks were collected and bioassayed for EDR activity using the rat aorta preparation. EDR-active compounds were subsequently characterized by HPLC retention times and electrospray-ion-trap mass spectrometry. The compounds exhibiting the most EDR activity were proanthocyanidin trimers, tetramers, pentamers, and polymers and their gallates, as well as a dimer gallate (EC50 values in the range of 0.6-2.5 microg catechin equivalents/mL). These compounds should be useful for in vitro and in vivo studies, particularly as they relate to improvement of cardiovascular function.

We have shown in previous work that extracts of grape seeds (GSE) and skins, grape juice, and many red wines exhibit endothelium-dependent relaxing (EDR) activity in vitro. This EDR activity involves endothelial nitric oxide (NO) release and subsequent increase in cyclic GMP levels in the vascular smooth muscle cells. The NO/cyclic GMP pathway is known to be involved in many cardiovascular-protective roles. The current study focuses on the isolation and identification of EDR-active compounds (procyanidins) from GSE. Crushed Concord grape seeds were extracted with methanol and the extract was separated into seven fractions (A-G) on a Toyopearl TSK-HW-40 column. EDR-active fractions (D-G) were further separated into 25 individual compound peaks by HPLC, 16 of which were EDR active (threshold for relaxation ranged between less than 0.5 microg/mL and greater than 4 microg/mL). Procyanidin identification was accomplished by electrospray-ion trap mass spectrometry (ES-ITMS), MS/MS, and by tannase treatment and acid thiolysis, followed by HPLC and ES-ITMS of the products. Activity of isolated procyanidins tended to increase with degree of polymerization, epicatechin content, and with galloylation. These EDR-active compounds (many of which also possess antioxidant activity), individually or in the form of wines, juices, or nutritional supplements, may be useful in preventing or treating cardiovascular diseases.

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A 63-year-old Caucasian woman was referred to our diabetes clinic for evaluation of an unexplained drop in HbA1c in the past year without an apparent cause. She was diagnosed with type 2 diabetes (T2D) 5 years earlier upon routine laboratory screening. At the time of diagnosis, she was asymptomatic with a BMI of 29.5 kg/m2. She had had two uncomplicated pregnancies. Soon after her T2D diagnosis, she was started on therapy with a fixed combination of glyburide and metformin t.i.d. (2.5 mg + 400 mg, respectively; increased later to 5 mg + 400 mg). She also had hypertension treated with enalapril 20 mg/day. Since then, her HbA1c had been measured every 6 months and remained stable at 59 mmol/mol (7.5%) on the higher dose of glyburide. With no apparent reason, her HbA1c dropped to 42 mmol/mol (6%) during the past year. This low value, within the nondiabetic range, was confirmed 6 months later. She did not report any symptomatic hypoglycemia or a change in BMI. Blood glucose self-monitoring carried out twice weekly indicated poor glycemic control inconsistent with her HbA1c values. Her general practitioner recommended decreasing her antidiabetic therapy by half, but her HbA1c was still 42 mmol/mol (6%) 3 months later. At time of presentation, mild anemia was present. Upon obtaining further, it was discovered that she had started therapy with dapsone for pemphigus 15 months earlier.