Anacardium occidentale L. is used throughout the world to treat type 2 diabetes. In Portugal, a traditional herbal preparation made with stem bark of this species (AoBTHP) has been used for more than 30 years to treat this pathology. The AoBTHP was standardized on total phenolic content, and its hypoglycemic activity was assessed using db/db mice (n = 26) for 92 days. Three doses (40.2, 71.5, and 127.0 mg/kg/day, per os) were tested, and glibenclamide (5 mg/kg/day) was used as positive control. During the study, glycemia was measured under non-fasting or fasting states. In sequence, thin-layer chromatography bioautographic assays were used for the detection of possible alpha- and beta-glucosidase inhibitors. A significant hypoglycemic effect in fasting glycemia in days 31 and 57 was observed with the three tested doses. The 71.5 mg/kg and 127.0 mg/kg AoBTHPs significantly reduced non-fasting glycemia on day 24. The highest dose showed the most significant hypoglycemic effect. Gallic acid was identified as the major alpha- and beta-glucosidase inhibitor. The 127 mg/kg/day AoBTHP dose showed a greater glucose-lowering effect than glibenclamide. For the first time, a standardized AoBTHP was tested using an in vivo diabetes model, and its usage was preclinically validated for type 2 diabetes treatment. The hypoglycemic activity of an AoBTHP can be related to the presence of alpha- and beta-glucosidase inhibitors, such as gallic acid, but other mechanisms can also be involved.
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