Neuroprotective activity in vivo and HPLC determination of 6-hydroxykynurenic acid (6-HKA), an active constituent of Ginkgo biloba L. leaves extract

The commercial refined extract of Ginkgo biloba L. leaves can protect brain tissue against cerebral ischemia/reperfusion (I/R) injury [1], an activity commonly attributed to the antioxidant activity of its flavonoids and proanthocyanidins. However, preliminary experiments identified 6-hydroxykynurenic acid (6-HKA) as a major contributor to this activity. For 6-HKA quantification by HPLC (Agilent XDB-C8 column (5 um, 4.6X250 mm); acetonitrile, water, methanol gradient; 1 ml/min; 25 °C; UV 350nm), pulverised drug material was extracted with 70% MeOH at 60 °C on the ultrasonic bath for 45 min, filtered, concentrated to dryness under reduced pressure, and re-dissolved in exactly 10 ml of H₂0/CH₃CN/MeOH (94:5:1). Pure 6-HKA was measured under identical conditions to generate a calibration curve by peak area in order to calculate the content in the leave samples. For the in vivo assay, male Sprague-Dawley rats were divided into 6 groups (sham: 8, placebo: 16, 6-HKA 2.5 mg/kg: 16, 6-HKA 5 mg/kg: 14, 6-HKA 10 mg/kg: 10, nimodipine 10 mg/kg: 8). All non-sham animals were subjected to cerebral I/R injury by occluding the middle cerebral artery with a nylon suture that was removed after 2h of ischemia to establish reperfusion. After recovery from anaesthesia, the rats were returned to their cages with free access to water and food. Death rates: placebo 50%, 6-HKA 2.5 mg/kg 50%, 6-HKA 5 mg/kg 43%, 6-HKA 10 mg/kg 20%, nimodipine 0%. All animals were sacrificed 24h after reperfusion. Coronal brain sections were stained and the infarct ratio calculated. Brain slices were stored in liquid N₂ and powdered in frozen state. The level of Ca²⁺ in the brain tissue was measured using a commercial assay kit. Under treatment of cerebral I/R injury with 6-HKA, the death rate of the test animals decreased, their neurological dysfunctions were ameliorated, and both average infarct size and Ca²⁺ concentration in the brain tissue were significantly reduced as compared to the placebo group.

Keywords: Ginkgo biloba L., 6-HKA, neuroprotective activity, cerebral ischemia, reperfusion injury.

References:

[1] Hu B, Sun S, Mei G, Chen L, Tong E. Protective effects of Ginkgo biloba extract on rats during cerebral ischemia/reperfusion. Chin Med J (Engl) 2002; 115: 1316 – 1320