Crocin effect

As has been pointed out earlier in this chapter, the dietary consumption and historical medicinal use of carotenoids has been well documented. In the modern age, in addition to crocin, 3.7, and norbixin, 3.8, several carotenoids have become extremely important commercially. These include, in particular, astaxanthin, 3.6 (fish, swine, and poultry feed, and recently human nutritional supplements) lutein, 3.4, and zeaxanthin, 3.3 (animal feed and poultry egg production, human nutritional supplements) and lycopene, 3.2 (human nutritional supplements). The inherent lipophilicity of these compounds has limited their potential applications as hydrophilic additives without significant formulation efforts in the diet, the lipid content of the meal increases the absorption of these nutrients, however, parenteral administration to potentially effective therapeutic levels requires separate formulation that is sometimes ineffective or toxic (Lockwood et al. 2003). 

We reported the effects of ethanol extract of C. sativus and its purified components on the central nervous system in terms of learning behaviors in mice and the LTP in the dentate gyrus of hippocampus in anesthetized rats and in the CA1 region of rat hippocampal slices [11-13], This review also discusses the values of folk medicines in modulating apoptotic cell death, together with our recent data of crocin s effect on neuronal cell death. 

We already indicated that intravenous injection of ethanol blocked the LTP induced by tetanic stimulation [18]. However, when saffron crude extracts were injected intracerebroventricularly, the blocking effect of ethanol on the LTP decreased dose-dependently [19]. Moreover, crocin prevents the ethanol-induced impairment of memory acquisition in ST and SD tests [20], From these results it is easily suggested that crocin antagonized the blocking effect of ethanol on the induction of LTP. 

Fig. (3). Effects of crocin and its analogues on the LTP-blocking effect of ethanol.

Effects of crocin on the induction of LTP in the CA1 region of rat hippocampal slices... 

The effect of crocin on the LTP-suppressing effect of ethanol was investigated (Fig. (4)). The potentiation induced by strong tetanic stimulation in the presence of 20 mg/kg crocin and 30% of ethanol (15 ml/kg) was significantly larger than that in the presence of 30% of ethanol (15 ml/kg) alone, indicating that crocin clearly attenuates the action of ethanol [21]. 

B Summary ofthe effects of ethanol and crocin on the induction of LTP. The magnitude ofLTP was evaluated with die population spike amplitude 30 min after tetanic stimulation. The numbers of observations in each group are shown in parentheses. All data are represented as the mean SEM. p<0.01 vs. control, p<0.05 vs. 30% of ethanol (IS ml/kg) alone. Duncan s multiple range test. 

Effects of ethanol and crocin on non-NMDA receptor-mediated synaptic potentials in hippocampal slices... 

The synapic potential mediated by non-NMDA receptors was recorded in normal ACSF. When ethanol (10-50 mM) was added to the perfusing medium, no significant change in non-NMDA receptor-mediated synaptic potential was observed. However, the addition of ethanol at a higher concentration (100 mM) induced a small reduction in non-NMDA receptor-mediated synaptic potential (Fig.(5)A). The reduction in non-NMDA response rapidly occurred after the addition of 100 mM ethanol and reached a steady state within 10 min. After washing out the ethanol, the response gradually returned to the normal level. When 10 pM crocin was added 10 min prior to the ethanol, the non-NMDA response was similarly reduced in the presence of 100 mM ethanol (Fig.(5)B). Crocin (10 pM) did not significantly affect the inhibitory effect of 100 mM ethanol on non-NMDA response (Fig.(5)C) [22],... 

A) Representative experiment showing the effect of ethanol on non-NMDA reseptor-mediated response. (B) Representative experiment showing die influence of crocin on ethanol-induced inhibition of non-NMDA response. Crocin (10 iM) was applied 10 min prior to ethanol (white bar). (C) Concentration-effect curves for ethanol inhibition of non-NMDA response in the absence (O) or presence ( ) of 10 pM crocin. 

In order to confirm the possible interaction of ethanol and crocin on NMDA receptors, we also performed whole-cell patch recording with primary cultured hippocampal neurons and measured membrane currents induced by the application of NMDA in a voltage-clamped condition. Application of 100 pM NMDA induced an inward current of 100.2 9.8 pA (n=10) at a holding potential of -60 mV. The NMDA-induced inward current was not affected by 10 pM CNQX (data not shown), but was completely abolished by 30 pM APV, supporting the fact that the response was mediated by NMDA receptors. Ethanol inhibited NMDA-induced currents in a concentration-dependent manner. Crocin (10 pM) had no effect on NMDA-induced currents by itself (data not shown), but attenuated the inhibitory effect of ethanol on NMDA-induced currents. The concentration-effect curve for ethanol was shifted to the right by the presence of crocin [22]. 

Effect of crocin on TNF-a-induced morphological changes and DNA fragmentation in the nucleus of PC-12 cells... 

The effect of crocin on the TNF-a-induced cell death of PC-12 cells is shown in Fig. (6). In serum-free GIT medium conditions, PC-12 control cell morphology remained intact at 24 h (panel A). However, the cells treated for 24 h with TNF-a (500 units/ml) appeared rounded and showed the characteristics of necrotic and/or apoptotic cells (panel B). In the combination with 10 pM crocin, PC-12 cell morphology retained intact neuronal cell morphology at 24 h (panel C). Crocin alone had no effect on the morphology of PC-12 cells (data not shown). 

Fig. (7), TNF-a treatment resulted in an elevation of caspase -3 activity in the cells at 6 h (a 3.9-fold increase), and the elevation lasted for 24 h. Crocin (0.1-10 pM) suppressed the TNF-a-induced activation of caspase-3 in a concentration-dependent manner. Caspase activity in the copresence of 10 pM crocin was near the control level, while the crocin (10 pM) alone had no effect on caspase activity in cells untreated with TNF-a. These results suggest that crocin can suppress the TNF-a-induced cell death of PC-12 cells by blocking the activation of caspase-3. It is possible that the crocin treatment may also suppress upstream signals for caspase-3 activation.

Fig. (8) shows the effects of TNF-a, crocin or their combination on Bcl-2 and Bc1-Xl mRNA levels in PC-12 cells. TNF-a treatment had no effect on the Bc1-Xl mRNA levels at 3 h but significantly decreased the mRNA expression at 18 h, compared to the control (panel A). Crocin (10 pM) alone or its combination with TNF-a appeared to rather enhance the expression of Bc1-Xl mRNA at 3 h. At 18 h, the TNF-a-induced decrease... 

Measurement of cytochrome c revealed that TNF-a significantly increased the cytosolic cytochrome c levels in PC-12 cells at 6 h (Fig. (10)). The increase in cytochrome c was suppressed to near the control level by the presence of 1 or 10 pM crocin. Therefore, our present results show the possibility that crocin has a pharmacological effect that prevents apoptosis of neuronal cells [17]. 

An oral administration of crocin had no effect on memory acquisition in normal mice but improved the ethanol-induced impairment of learning behaviors of mice in passive avoidance performance tasks. This phenomenon resembled that of crude extract of saffron as reported previously. The tendencies of the effect between CSE and crocin are similar to each other. From these results it can be easily speculated that crocin is the most important principle in crude extract of saffron. Other crocetin glucoside esters weakly antagonized the blocking effect of ethanol on the LTP compared to crocin. 

We demonstrated for the first time that crocin selectively antagonizes the inhibitory effect of ethanol on NMDA-receptor-mediated responses in hippocampal neurons. This action of crocin may underlie the antagonism against ethanol-induced memory impairment. Crocin should be useful as a new pharmacological tool for studying the mechanism of ethanol inhibition of NMDA receptor functions. 

Concerning the pharmacological activities of saffron components, the inhibitory effect for the increase of bilirubin in blood [2], and the deterioration activities or cholesterol and triglyceride levels in serum by crocin and crocetin have been reported [3]. Anti-tumor activity of saffron on mice transplanted with sarcoma-180,... 

A single oral administration of crocin had no effect on memory acquisition in normal mice in both ST and SD test (data not shown). In ST test, when crocin was given 10 min before the ethanol treatment the latency increased Fig. (9-A) and the number of successful mice increased at a dose of 200 mg/kg compared to ethanol-treated group in the testing trial Fig. (9-B). In SD test, crocin decreased the number of errors and increased the number of success dose-dependently Fig. (9-C and D). 

Fig. (9). Effect of crocin on 30% ethanol-induced impairment of memory acquisition in ST and SD tests.

Crocin of 50 mg/kg ameliorated the blocking effect of ethanol on the LTP at approximately 84% compared to the control as indicated in Fig. (10). Crocetin gentiobiose glucose ester also antagonized the blocking effect of ethanol on the LTP dose-dependently. But die intensity is not so strong, about a half of crocin when compared the intensity of 50 mg/kg. On the other hand, crocetin di-glucose ester did not remove the inhibitory effect of ethanol on the LTP. 

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