3.1. In Vitro StudiesC. asiatica (gotu kola) is a reputed plant species for its traditional use in ayurvedic and Chinese medicines [52], and its positive effects on brain aging have been generally attributed to its two major triterpene saponosides; asiatic and madecassic acids as well as their heterosides; asiaticoside and madecassoside, respectively. For instance, the hydroalcoholic extract of the plant was tested in vitro against acetylcholinesterase (AChE), the key enzyme taking a critical role in the pathogenesis of Alzheimers disease (AD). Since deficit in the level of acetylcholine (ACh), which is hydrolyzed by AChE, has been identified in the brains of AD patients, inhibition of AChE as well as its sister enzyme butyrylcholinesterase (BChE) has become a rational target in drug development against AD [53]. The extract was found to inhibit AChE with 50% of inhibition rate at 150 μg/mL concentration by the spectrophotometric method of Ellman [54]. In our study on the ethanol extracts prepared from the aerial parts of C. asiatica of both Turkish and Indian origins along with the standardized gotu kola extract (containing 10.78% of total asiaticoside and madecassoside) imported from China, we comparatively examined inhibitory potential of these three extracts against AChE, BChE, and tyrosinase (TYRO) at 50, 100, and 200 μg/mL concentrations [48]. As aforementioned that cholinesterases are the important enzymes for AD treatment, TYRO has become an important target for Parkinson’s disease (PD) since this enzyme plays a role in neuromelanin formation in the human brain and could be significant in occurrence of dopamine neurotoxicity associated with neurodegeneration linked to PD [55]. According to our results obtained at 200 μg/mL, only the standardized extract was found to inhibit AChE (48.28±1.64%), whereas the ethanol extracts of the plant samples from Turkey and India exerted 46.95±0.94% and 70.30±3.77% against BChE, respectively, and a notable inhibition against TYRO (42.83±4.21% and 56.20±3.17%, resp.).
Awad et al. investigated inhibitory property of C. asiatica extract towards glutamic acid decarboxylase (GAD) and γ-aminobutyric acid transaminase (GABA-T), which are the enzymes responsible for GABA metabolism and found out that the extract stimulated the activity of GAD over 40% [56]. On the other hand, the leaf extract of C. asiatica growing in China was shown to display neuroprotection through enhancing phosphorylation of cyclic AMP response element binding protein (CREB) in neuroblastoma cells in Aβ(1–42) proteins found within the amyloid plaques occurring in the brains of AD patients [57]. In another study [58], effect of the aqueous leaf extract of the plant on monomers or oligomers that lead to formation of Aβ(1–42) proteins in AD via aggregation was examined using both thioflavin-T test and transmission electron microscope; however, it was observed not to cause any inhibition on aggregation of the monomers and oligomers. Inhibitory activity of the aqueous extract of C. asiatica that contained 84% of asiaticoside was tested by the radioenzymatic assay against phospholipase A2 (PLA2), which play role in neuropsychiatric diseases. The findings pointed out to the fact that the extract could inhibit Ca2+-independent PLA2 and cytosolic PLA2 [59]. The ethanol extract of the plant was observed to cause an increase in neurite development in human SH-SY5Y cell lines at 100 μg/mL concentration, whereas its aqueous extract did not lead any increase in the same cells [60]. Then, the subfractions of the ethanol extract were also tested further in the same assay for neuritic development, and the most effective subfraction was demonstrated to have a nonpolar chemical nature. According to the results of that study, the authors concluded that C. asiatica extract might be beneficial in prevention of neuronal damage.
Lee et al. studied neuroprotective potential of thirty six derivatives of asiatic acid prepared by various structural modifications and tested in primary cell culture consisting of rat cortical neurons exposed to glutamate, which is known as a neurotoxin [61]. Three of the compounds displayed higher protective activity than asiatic acid per se and also significantly reduced production of glutamate-induced nitric oxide (NO) as well as levels of glutathione, glutathione peroxidase, and some other related enzymes.
3.2. In Vivo Studies Neuroprotective effect of C. asiatica and its major triterpene saponosides has been extensively studied through different experimental models on animals such as passive avoidance and elevated-plus labyrinth tests for memory enhancing effect [62]. A research was carried out in rats to determine effect of the aqueous extract of C. asiatica on intracerebrovascular streptozocin-induced memory associated with sporadic type of AD by applying the extract at doses of 100, 200, and 300 mg/kg (b.w.) and measuring some oxidative stress parameters such as glutathione, superoxide dismutase (SOD), and catalase (CAT) [63]. While a clear dose-dependent improvement was observed in memory-related behaviors in the rat group administered the extract at 200 mg/kg (b.w.) dose, a serious decrease in malondialdehyde (MDA) and an increase in glutathione and CAT levels were recorded, which led to a final suggestion by the authors that C. asiatica extract has a positive effect on memory that is also related to its remarkable antioxidant effect. The same research group subjected this extract to passive avoidance and spontaneous locomotor activity behavioral tests using pentylenetetrazole-(PTZ-) induced memory loss in rats at 100 and 300 mg/kg (b.w.) doses [64]. Following the behavioral tests, MDA and glutathione levels were determined in the rat brains as oxidative stress markers, which significantly contribute to neurodegeneration. Accordingly, the extracts at the tested doses caused a notable improvement in all test parameters.
In another study by Rao et al. [65], enhancing effect of C. asiatica extract on learning and memory was examined during 15 days at 200, 500, 700, and 1000 mg/kg (b.w.) doses by oral administration to mice. Open area, light/dark compartment, and radial-armed labyrinth tests were applied as experimental models, while AChE activity and dendritic arborization development were taken into consideration as biochemical markers. According to the findings, the extract displayed improving effect in radial-armed labyrinth test, whereas it did not cause any change in locomotor activity. On the other hand, extract administration resulted in an increase in AChE activity and dendritic arborization in CA3 neurons located in hippocampus. Thus, the authors concluded that the extracts may positively influence neuronal morphology, especially in young adult mice. In a similar study performed by the same researchers, the fresh leaf extract of C. asiatica was given to adult mice at 2, 4, and 6 mL/kg doses during 2, 4, and 6 weeks, respectively [66]. After these periods, the removed brains of mice were investigated under microscope, which pointed out to the evidence that the extract given at 6 mL/kg dose during 6 weeks caused a significant augment in dendritic arborization in neurons. These authors came to another similar conclusion that the juice obtained by pressing the fresh leaves of C. asiatica tested in the same experimental model in mice also enhanced dendritic arborization [67]. Besides, C. asiatica extract was shown to reduce levels of β-amyloid plaques in hippocampus in mice [68].
Shinomol and Muralidhara investigated effect of C. asiatica extract against oxidative stress and mitochondrial dysfunction induced by 3-nitropropionic acid, a fungal-derived neurotoxin, in the brains of male prepubertal mice, and the extract was found to diminish oxidative stress remarkably through influencing the parameters such as MDA and radical oxygen species [69]. In a related study on rats, C. asiatica extract was reported to have a protective effect against mitochondrial damage occurred in PD by means of improving oxidative stress parameters [70]. Anticonvulsant effect of the crude material and extracts prepared from C. asiatica, also known as “brahmi” in Hindu, was determined in PTZ-induced convulsion model in rats and compared with fenitoin as the reference drug [71]. The data indicated that the crude material of the plant exerted a mild level of anticonvulsant effect at 500 mg/kg dose, while the methanol extract had superior effect to that of the crude material at 3rd and 6th hs. The extract prepared with propylene glycol also produced a dose-dependent anticonvulsant activity at 500 and 1000 mg/kg (b.w.) doses. Similarly, Ganachari et al. demonstrated in vivo anticonvulsant effect of the hydroalcoholic extract of C. asiatica against PTZ- and strychnine-induced opistotonus convulsions at 100 mg/kg (b.w.) [72]. Moreover, the extract was observed to reduce lipid peroxidation and spontaneous locomotor activity, whilst it potentiated pentobarbital-induced sleeping duration and diazepam-induced hyperactivity. In another paper [73], the ethyl acetate fraction of C. asiatica as well as combination of the fraction with some antiepileptic drugs including fenitoin, valproate, and gabapentin individually was administered intraperitoneally to the mice with convulsion induced by PTZ and found that the combinations caused an additive effect producing a higher anticonvulsant activity than each of the drugs. Additionally, neurotoxicity of the fraction and each combination was established by rotarod test, and combination of the extract with gabapentin was less neurotoxic. In the light of this evidence, the authors stated that conjoint use of the ethyl acetate fraction of C. asiatica with epileptic drugs might be beneficial for epileptic patients. In another study [74], De Lucia et al. reported anticonvulsant and sedative activities of the hydroalcoholic extract of C. asiatica in rats using elevated-plus labyrinth and PTZ-induced convulsion models, and the extract was also shown to exert low toxicity by chronic application with the LD50 value of 675 mg/kg (b.w.). Anticonvulsant activity of the hexane, chloroform, ethyl acetate, water, and n-butanol extracts prepared from C. asiatica was determined using PTZ-induced convulsion model in male Wistar rats, and effect of the extracts was also searched on Na+/K+, Mg2+, and Ca2+-ATPase activity [75]. The results pointed out to an increase in activity of three types of ATPases in the extract-administered groups accompanied by anticonvulsant activity. Anxiolytic activity of the hexane, ethyl acetate, and methanol extracts of C. asiatica and asiaticoside was tested using elevated-plus labyrinth, open area, social interaction, locomotor activity, and new cage models in rats [76]. The results indicated that only the methanol and ethyl acetate extracts of the plant along with asiaticoside displayed anxiolytic activity in elevated-plus labyrinth test. In another paper [77], sedative effect of C. asiatica was mainly attributed to brahmoside and brahminoside, the triterpene derivatives, whereas anxiolytic activity was suggested to be partly resulted from the interaction with cholecystokinin receptors (CCKB), a group of G protein-coupled receptors which are considered to take a potential place in modulation of anxiety, nociception, and memory.
C. asiatica extract was administered orally to old rats during 60 days at 300 mg/kg (b.w.) dose per day, and the cortex, hypothalamus, striatum, cerebellum, and hippocampus regions of the rat brains were investigated in terms of lipid peroxidation and protein carbonyl (PCO) contents [78]. The researchers made a statement that the extract may be showing a neuroprotective effect in old rats by way of bringing about a significant decrease in PCO contents and lipid peroxidation. Radical scavenging effect of the chloroform-methanol (4 : 1) extract of the plant was examined in monosodium glutamate-treated Sprague-Dawley female rats at 100 and 200 mg/kg doses [79]. Following the extract administration, a significant increase was observed in SOD and CAT levels, whereas glutathione level was not influenced. Flora and Gupta reported that the flavonoid fraction of C. asiatica demonstrated a protecting effect against lead acetate-induced neurotoxicity in mice through antioxidant mechanisms [80]. In another paper, asiatic acid, one of the major triterpene derivatives in C. asiatica, administered orally at 30, 75, and 165 mg/kg (b.w.) doses, was shown to have neuroprotective property in mice with permanent cerebral ischemia by evaluating infarct volume and behavioral changes between 1st and 7th days [81]. In the same study, the compound was additionally investigated in HT-22 cells exposed to oxygen glucose in terms of cell viability and mitochondrial membrane potential. Asiatic acid considerably diminished the infarct volume by 60% and 26% at the 1st and 7th days, respectively, which improved neurological status at 24 h after ischemia. The authors concluded that asiatic acid, which might be mediated to some extent by decreasing blood-brain barrier permeability as well as reduction in mitochondrial damage, could be useful for cerebral ischemia treatment.
Probable improving effect of C. asiatica extract at 150 and 300 mg/kg (p.o.) doses was assessed against colchicine-induced memory using Morris water maze and plus-maze performance tests in male Wistar rats as well as oxidative damage parameters such as lipid peroxidation, nitrite, reduced glutathione, glutathione-S-transferase, SOD, and as a biochemical parameter, AChE activity [82]. The 25-day chronic administration of the extract caused a significant improvement in memory and oxidative damage parameters along with AChE activity. On the other hand, asiaticoside from C. asiatica exerted a neuroprotective effect against PD by reversing neurotoxicity induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) in rats via balancing dopamine and antioxidant mechanism [83].
Antidepressant activity of C. asiatica was evaluated using its triterpene fraction in cortex, hippocampus, and thalamus regions of rat brains by determining the corticosterone levels [84]. The triterpene fraction created a momentous diminution in corticosterone level and a notable increase in amount of monoamine-related neurotransmitters.