| Vascular dementia (VD) is the second most common cause of dementia in the elderly, after Alzheimer's disease (AD). VD is defined as a loss of cognitive function resulting from ischemic or hemorrhagic brain lesions due to cerebrovascular disease. Vascular dementia is not a single disease, but a series of conditions with different pathological correlates and physiopathological mechanisms. It is incontrovertible that VD is the second largest causeof dementia in the elderly, representing 15-20% of all cases of dementia worldwide. As the populations are increasingly aging in most parts of the world—including China and India where more than two billion people live—it is likely that stroke, vascular cognitive disorder, and VD will become the silent epidemic of the 21st century. Currently, no standard treatment and agents exists for vascular dementia, and little is known about primary and secondary prevention, apart from direct extrapolation from work in stroke.Previous studies from our laboratory showed that metabolin of ICA seem to possess estrogen-like activity, ICA plus PNS exerted a significant prophylactic and therapeutic effect in AD model rats and Herba Epimedii (HE) plus Radix Notoginseng (RN) crude extract combination possess HUO XUE HUA YU effects.Based on the pathological mechanisms of Vascular dementia in both traditional Chinese medicine and modern medical sciences, as well as the recent studies on the therapeutic effects of Herba Epimedii and Radix Notoginseng on either the VD patients or the VD animal models, we adopted the modern isolation methods to study the active components of these two herbs. Icariin (ICA) and Panax notoginseng saponins (PNS) were extracted from HE and RN respectively, and by combinedadministration of the two extracts, we further observed their therapeutic effects on improving the learning and memory capabilities in a rat VD model, in an attempt to provide some experimental evidence for the preventive and therapeutic effects, as well as the relevant mechanisms of the two herbs in VD treatment.1. Studies on extraction and purification of the PNS 1.1 Studies on extraction of the PNSThe affecting factors such as content of solvent, the amount of solvent, time and the frequency of the extraction were studied by orthogonal experiment of extraction. The optimum extraction result was obtained in 70% ethanol of 10-fold as much as the medicine with 3 times of extraction for 90 min. 12 Studies on purification of the PNSSelect the optimal technology of separation and purification of the PNS by macroporous resins. The optimal technological conditions were as follows: crude liquid(0.3 g/ml) flew through the adsorption column by 50% adsorption capacity of macroreticular resins, resin was eluted successively with 5 times volume water, 10 times volume 70% ethanol, adsorption flow rate is 2.5% loading amount and eluting flow rate is 5% loading amount.2. Effects of ICA combined with the PNS on spacial learning and memory capabilities induced by cerebral ischemia reperfusion in rats.After successfully trained by 8-arm radial electric maze test, the rats were repeatedly induced cerebral ischemia and reperfused for twice, combined with intraperitoneal injection of sodium nitroprusside to establish the VD model followed by one week of drug treatment. Spacial learning and memory ability were evaluated by 8-arm radial electric maze and Step-down passive avoidance test after a period of drug therapy. The result showed the learning and memory abilities of rats by cerebral ischemia reperfusion significantly decreased compared with that in the sham-operated group, as judged by the 8-arm radial electric maze test (P<0.05) and step-down passive avoidance test (P<0.05^ P<0.01). The combination (ICA+PNS 2.5+20 x 5+40 ^ 10+80 mg/kg, ig ) significantly protected and improved all theevaluated indexes of the model groups after three weeks(P<0.05> P<0.01).). ICA orPNS treatment only shown a slight but not significant reduction compared with thosein vehicle group.3. Mechanism studies of ICA combined with the PNS on improving speciallearning and memory capabilities in VD model rats.3.1 Effects of ICA combined with the PNS on SOD activity and MDA level inbrain of VD model rats.The brain of above rats were sampled after the final behavioral test. The activity of SOD and the level of MDA in the brain was determined after Homogenate. The results showed the combination (ICA+PNS 2.5+20> 5+40. 10+80 mg/kg, ig) could decreased the content of MDA in the brain(P<0.05 -. P<0.01) and the combination(ICA+PNS 5+40 mg/kg, ig) could elevate the activity of SOD in the brahXPO.OS). Administration of PNS alone also significantly decreased the content ofMDA(/><0.05).32, Anti-apoptotic studies of ICA combined with the PNS on hippocampal CA1 pyramidal cell in VD model ratsA set of rats underwent the same experimental protocols as behavioral experiment were administrated 10 days. Thereafter, the brains were embedded in paraffin and representative serially coronal sections were obtained. Tissue sections were routinely stained with hematoxylin and eosin (HE) and Adjacent sections were used to detect fragmented DNA in apoptotic cells(TUNEL). The results showed the combination(ICA+PNS 2.5+20^ 5+40> 10+80 mg/kg, ig) clearly decreased the number ofTUNEL positive cells, indicated that ICA+PNS posses anti-apoptotic and neuroprotective effects on CA1 hippocampal neurons of the ischemic brain.CONCLUSION1. ICA combined with PNS could significantly protect and improve the spatial learning and memory abilities of VD model rats induced by cerebral ischemia reperfusion, which suggests that the combination may have preventive and therapeutic effects on VD.2. ICA combined with PNS can elevate SOD activity and decrease MDA content of the brain induced by cerebral ischemia reperfusion in rats, which suggests that preventing oxidative stress function may is one of the mechanisms by which the combination improve spatial learning and memory abilities.3. ICA combined with PNS could clearly decreased the number of TUNEL positive cells on CA1 hippocampal neurons induced by cerebral ischemia reperfusion. The anti-apoptotic and neuroprotective effects might be one of the mechanisms by which the combination exerts the spatial learning and memory abilities -improving effect...
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