**2. Results**

### *2.1. Collagen Is a Basic Component of the GSCM*

### 2.1.1. Amino Acid Analysis

According to the amino acid analysis, the content of hydroxyproline (Hyp) in the GSCM was 86.3 residues, proline (Pro)—91.3 residues per 1000 residues (Table 2). The Hyp percentage in the studied specimen was 10.13 weight %.

The specimen also contained a large amount (330 per 1000 residues) of glycine (Gly). The weight percentage of Gly was 22.18%. This finding is related to the fact that a molecule of collagen consists of a triple helix formed by three polypeptide helical strands, and each helical chain is formed by three-residue-long repeats, with glycine as one of the three residues. Thus, the primary structure of collagen is characterized by a large content of glycine. The high content of glutamic acid (Glu) in the specimen is explained by the presence of proline (Pro) since Pro is synthesized from glutamic acid.

**Table 2.** Amino acid composition of the *D. gigas* CM.

\* Percentage of amino acids to the mass of the test sample.


**Abstract**

Green tea has been used as a medicine in East Asia for thousands of years. Plant-derived compounds called flavanols, which are included in green tea, may have potentials to help maintain healthy brain function. In this chapter, we review the effects of flavanols, e.g. epicatechin (EpiC), on cognitive ability in the pond snail, *Lymnaea stagnalis*. In this decade, the Lukowiak's group has tested the effects of EpiC on cognition ability in *Lymnaea*. In a *Lymnaea* model system, they showed that EpiC and EpiC-containing foods have a rapid and activity-dependent effect enhancing the formation of long-term memory (LTM) following operant conditioning of aerial respiratory behavior. In the last part of this chapter, we also introduce our study for the effects of EpiC on LTM formation in another model system in *Lymnaea*. This study showed that EpiC increases the persistence of LTM formed by classical conditioning of feeding behavior, and suggested that EpiC alters some electrophysiological properties of a neuron in the feeding system.

**Keywords:** Green tea-derived catechins, Epicatechin, Operant conditioning, Classical conditioning, Learning and memory, Long-term memory, *Lymnaea*

#### **1. Introduction**

Green tea is one of the most popular beverages in the world. It is made from *C. sinensis* leaves and include many kinds of phytochemicals. Compounds called flavanols, which are included in green tea, are candidates for the active ingredient that has been used as a medicine in East Asia for thousands of years. An extract, called Sinecatechins, of green tea leaves is also used as botanical drug approved by the FDA in USA [1]. Flavanols (catechins) belonging to the group of polyphenols [2] are contained in green tea (mg/100 g): 26.05 (−)-epigallocatechin-3-gallate (EGCG), 7.57 (−)-epicatechin-3-gallate (ECG), 16.02 (−)-epigallocatechin (EGC) and 6.16 (−)-epicatechin (EpiC) [3]. Recently, it is reported that flavanols may have potentials to help maintain healthy brain function in both vertebrates and invertebrates [4–8].

Gastropod mollusks such as *Aplysia*, *Limax*, *Hermissenda* [9–14] and *Lymnaea* [15–19] are excellent model animals for understanding the causal neuronal mechanisms of learning and memory. In this decade, the Lukowiak's group in University of Calgary has tested the effects of EpiC on cognition in *Lymnaea stagnalis.* In a *Lymnaea* model system, they showed that EpiC and EpiC-containing foods have a

rapid and activity-dependent effect enhancing the formation of long-term memory following operant conditioning of aerial respiratory behavior.

*Lymnaea* is an aquatic pulmonate snail and can breathe either with its lung or skin. It approaches water surface and gets air into the lung through opening its pneumostome (**Figure 1A**). The Lukowiak's group employed a protocol of operant conditioning of aerial respiration to investigate the cognitive function in *Lymnaea*. In a hypoxic environment in which the frequency of aerial respiration in *Lymnaea* increases, applying repeated tactile stimulus to pneumostome as a negative reinforcement (training session; TS) reduces the number of attempted pneumostome openings in *Lymnaea*, and then the behavioral change persists for 3 hours or longer.

A single 30-min training session (0.5 h-TS) results in intermediate-term memory (ITM) that persists for up to 3 hours, whereas two 30-min training sessions with a 1 hour rest interval (2 h-TS) results in long-term memory (LTM) that persists for 24 hours [21]. ITM depends on the translation of existing mRNA transcripts but does not require mRNA transcription. LTM requires both the translation of mRNA and the formation of new mRNA transcripts [22]. Thus, a 2 h-TS could drive the process of mRNA transcription in addition to translation of mRNA.

To drive aerial respiration in *Lymnaea*, a 3-neuron central pattern generator (CPG) was shown to be both necessary and sufficient (**Figure 1B**) [23, 24]. Subsequently, it was shown that one of the three CPG neurons, Right Pedal Dorsal 1 (RPeD1), is a necessary site for LTM formation, extinction and reconsolidation of the memory [18, 22, 25]. It is also possible to utilize a semi-intact preparation where aerial respiratory behavior and neuronal activity can be simultaneously studied [26, 27].

*Lymnaea* can be classically, as well as operantly, conditioned and LTM can be formed by the following learning procedures [28, 29]. Conditioned taste aversion (CTA), which is a classical conditioning, is based on pairing sucrose as a conditioned stimulus (CS) with an aversive chemical unconditioned stimulus (UCS) such as KCl, which inhibits feeding and evokes a withdrawal response. After ten trials, the feeding response of trained snails to sucrose became significantly weaker than that of control snails, and this associative memory lasted for more than 2 weeks [30].

#### **Figure 1.**

*Pneumostome in* Lymnaea *and the neural circuit including in aerial respiratory behavior. (A)* Lymnaea *with the opened pneumostome. (B) Schematic drawing of the central pattern generator (CPG) to drive aerial respiration. Depolarization of Right Pedal Dorsal 1 (RPeD1) activates input3-interneuron (IP3I) via a biphasic effect (inhibition followed by excitation). Subsequently, IP3I excites both RPeD1 and a group of motor neurons (VI/J cells) involved in pneumostome opening. IP3I also inhibits visceral dorsal 4 interneuron (VD4), which is involved in pneumostome closing. The combined inhibitory input from both RPeD1 and IP3I causes burst firing of VD4. These figures are reproduced from [20] with permission.*

*Green Tea-Derived Catechins Have Beneficial Effects on Cognition in the Pond Snail DOI: http://dx.doi.org/10.5772/intechopen.99789*

#### **Figure 2.**

*Neuronal circuit corresponding to the feeding behavior. Each neuron is indicated by an abbreviation (see [31]). Modulatory function is indicated by yellow and initiating function by orange. Central pattern generator (CPG) interneurons and motoneurons active during the three phases of the feeding rhythm are indicated by green (P = protraction), blue (R = rasp) and red (S = swallow). Neurons labeled with two colors have two functions. Dots indicate inhibitory chemical synapses, bars excitatory chemical synapses and resistor symbols electrotonic (electrical) synapses. This figure is reproduced from [31] with permission.*

Individual neurons in *Lymnaea* can be identified as the neuronal circuit corresponding to the feeding behavior (**Figure 2**). An identified spontaneously active pair of neurons, the cerebral giant cells (CGCs), has been shown to both modulate the neuronal network underling the feeding behavior and be necessary for LTM and its retrieval following CTA training [31, 32]. The most significant CGC synaptic connections are with the neuron 1 medial (N1M) cell, an interneuron in the CPG that co-ordinates rhythmic feeding movements [33–36].

In the last part of this chapter, we introduce our study for the effects of EpiC on LTM formation in the feeding system in *Lymnaea*.

#### **2. Enhancing effects of epicatechin on memory formation by operant conditioning of aerial respiratory behavior**

To date, the most detailed information on effects of EpiC in *Lymnaea* has been obtained from experiments using operant conditioning of aerial respiratory behavior. In this paradigm, snails are subjected to a protocol that the pneumostome receives a weak tactile stimulus whenever the snail attempts to open the pneumostome in a hypoxic environment. The number of attempted pneumostome openings is recorded for each snail for 30 minutes. To determine whether memory is formed following the training session (TS), an identical procedure is performed 24 hours later, which is called a memory test (MT). The number of attempted pneumostome openings in the MT is compared with that in the TS, and long-term memory (LTM) is evaluated if the number of attempted openings in the MT is significantly lower than that in the TS [25, 37].

When snails were given a 0.5 h-TS, which do not usually form LTM lasting 24 hours or more, in the presence of 15 mg/L EpiC, the memory persisted until 24 hours after that training. Thus, EpiC can promote LTM formation by driving the process of mRNA transcription in addition to mRNA translation. EpiC also enhances LTM formation. When snails were operantly conditioned in EpiCcontaining pond water (15 mg/L) by a 2 h-TS (TS1, TS2; **Figure 3A**), which typically results in memory lasting only 24 hours in pond water without EpiC (control group, TS1 versus MT, n = 12, no significance; **Figure 3B**), they formed LTM lasting at least 72 hours (EpiC group, TS1 versus MT, n = 12, *P* < 0.01; **Figure 3C**) [4, 38]. Moreover, following a 2 h-TS in EpiC-containing pond water, snails were received the MT in standard pond water (i.e. no EpiC) at 96 hours, 1 week and 2 weeks

#### **Figure 3.**

*Epicatechin (EpiC) enhances LTM formation and diminish the rate of extinction following operant conditioning. (A) A timeline of the experiment is shown. (B–E) White and gray bars show snails trained without EpiC or with EpiC, respectively. Snails were either operantly conditioned in pond water (B) or EpiCcontaining pond water (C). (D, E) In an observation (Obs) and extinction sessions (Ext1–3), the number of pneumostome openings was calculated for each snail without tactile stimulation in hypoxic water for 30 minutes. (B, C) n = 12, \*\*P < 0.01 compared with TS1. (D, E) n = 12, \*\*P < 0.01 compared with Obs. These figures are produced from [4] with permission.*

after the 2 h-TS [38]. The snails maintained in a very long-term memory lasting for 2 weeks or longer. Memory formed in the presence of EpiC is resistant to forgetting, which is not dependent on EpiC being present in the MT. In addition, snails received extinction sessions (Ext1–3). In extinction sessions, snails are allowed to freely perform aerial respiration in the same hypoxic environment as TS, and the learned association between the pneumostome opening and weak tactile stimulus can be extinguished. Snails trained in the presence of EpiC were also more resistant to extinction of memory (EpiC group, Obs (pre-training) versus Ext2, n = 12, *P* < 0.01; **Figure 3E**), including blocking "the original memory" by overwriting a newer memory [39, 40], than control snails (control group, Obs (pre-training) versus Ext2, n = 12, no significance; **Figure 3D**) [4].

Exposure to EpiC does not alter locomotor activity and spontaneous aerial respiratory behavior themselves in *Lymnaea* compared to naïve snails [4]. Thus, studying the effects of EpiC could exemplify specificities for drugs that directly interact with neuronal signaling pathways for LTM formation and persistent.

In mice, EpiC improves retention of spatial memory by enhancing angiogenesis [8]. Oral intake of EpiC via gavage increases the level of EpiC and its metabolites in rat plasma and brain [41], and then EpiC could influence brain functions. In *Lymnaea*, EpiC can easily absorb via skin into the body cavity, and subsequently may contact to the CNS by an open circulatory system.

Additionally, it is reported that EpiC also has a rapid and activity-depend effect on promoting LTM formation in *Lymnaea* [4, 5, 38]. Fernell et al. demonstrated that

#### *Green Tea-Derived Catechins Have Beneficial Effects on Cognition in the Pond Snail DOI: http://dx.doi.org/10.5772/intechopen.99789*

EpiC must be present during operant conditioning or applied with 1 hour immediately after training in order for EpiC to cause LTM enhancement [42]. However, EpiC exposure at 1 hour after training did not result in enhanced memory formation. Thus, One hour after training is important for LTM formation. It is widely known that there is an important period for encoding and consolidation of the memory following learning [43]. In *Lymnaea*, the consolidation period persists for about 1 hour. LTM requires both regulation of gene activity and new protein synthesis, whereas ITM requires only new protein synthesis. Previously, it was shown in *Lymnaea* that applying a cold block (10 minutes in 4°C pond water) immediately after training blocks only LTM formation, but not block ITM formation [44, 45]. Thus, these results suggest that EpiC could alter the gene activity for LTM.

A specific mechanism by which EpiC enhances memory formation has not been clarified. However, there have been many studies in mammals suggesting various ways in which dietary flavonoids may exert such beneficial effects on the CNS [8]. EpiC is known as antioxidant, protecting neurons from injury caused by oxidative stress [46]. EpiC can be photo-inactivated by exposure to ultraviolet light (UV). Exposed to the sun for 6 hours, EpiC changes the molecular conformation by breaking the cyclic ether through a radical mechanism [47]. But no significant change was observed in the antioxidant activities of EpiC upon 6 h beta-UV radiation. Following photo-inactivation of EpiC, memory enhancement did not occur. Photoinactivation of foods containing EpiC also blocked their ability to enhance LTM [7]. Thus, enhancing effect of EpiC on memory formation in *Lymnaea* is less likely to be caused by antioxidant properties of EpiC and may be directly due to affecting the signaling pathway required for memory formation in neurons. This does not mean that antioxidant properties do not act against oxidative damage and therefore protective effect may contribute to retain memory over long-term.

As mentioned above, LTM formation is dependent on altered gene activity and new protein synthesis [48]. It is well-known that the consolidation period following learning plays an important role for LTM formation [20, 49], in which the learning is encoded into memory. EpiC can cause an enhancement of memory formation if snails experience EpiC during training or immediately after training. However, EpiC exposure at 1 hour before training or 1 hour after training was not sufficient to cause memory enhancement [42]. In *Lymnaea*, it is thought that the consolidation period persists for about 1 hour. If the operant conditioning is performed in the presence of environment stressors (exposure to predator kairomones or KCl), it results in strengthening of memory formation. The effects via sensory input from the osphradium (a sensory organ) are dependent on a serotonergic signaling pathway [50]. However, the enhancing effects of EpiC do not require either the input from the osphradium or serotonergic signaling pathway.


**3.3.1 Stable phase equilibria**

For the stable equilibrium study, the isothermal dissolution method was used in this study. The series of complexes of the quaternary system were loaded into clean polyethylene bottles and capped tightly. The bottles were placed in the thermostatic rotary shaker, whose temperature was controlled to (298.15 ± 0.1) K, and rotated at 120 rpm to accelerate the equilibrium of those complexes. A 5.0 cm3 sample of the clarified solution was taken from the liquid phase of each polyethylene bottle with a pipet at regular intervals and diluted to 50.0 cm3 final volumes in a volumetric flask filled with DDW. If the compositions of the liquid phase in the bottle became constant, then equilibrium was achieved. Generally, it takes about 50 days to come to equilibrium.

#### **3.3.2 Metastable phase equilibria**

The isothermal evaporation method was used in metastable phase equilibria study. According to phase equilibrium composition, the appropriate quantity of salts and DDW calculated were mixed together as a series of artificial synthesized brines and loaded into clean polyethylene containers (15 cm in diameter, 6 cm high), then the containers were put into the box for the isothermal evaporation at (308.15 ± 0.2) K. The experimental conditions with air flowing velocity of 3.5-4.0 m/s, relative humidity of 20-30%, and evaporation rate of 4-6 mm/d are presented, just like the climate of the Qaidam Basin. For

Stable and Metastable Phase Equilibria in the Salt-Water Systems 407

K2B4O7·4H2O

Borax

single polarized light (10×10) orthogonal polarized light (10×10) (b) Invariant point (KCl + Borax + K2B4O7·4H2O)

The metastable equilibria solid phases in the two invariant points are further confirmed with X-ray diffraction analysis, and listed in Figure 4, except in the invariant points (NaCl + KCl + Borax) in Figure 4a which shows that the minerals KCl, NaCl, Na2B4O7·10H2O and a minor Na2B4O7·5H2O are existed. The minor of Na2B4O7·5H2O maybe is formed due to the dehydration of Na2B4O7·10H2O in the processes of transfer operation and/or grinding.

Fig. 3. Identification of the invariant points for the solid phase in the reciprocal system (NaCl - KCl – Na2B4O7 - K2B4O7 - H2O) with a polarized microscopy using an oil-immersion method. (a), the invariant point (NaCl + KCl + Na2B4O7·10H2O); (b), the invariant point

No. Visible Ref. Code Chemical Formula Score Scale

KCl NaCl

B4O5(OH)4(Na2(H2O)8 Na2B4O7(H2O)5

(a), the X-ray diffraction photograph and the analytical data for the invariant point

Factor

0.369 0.732 0.030 0.007

Semi-Quant/%

(KCl + Na2B4O7·10H2O + K2B4O7·4H2O).

K2B4O7·4H2O KCl

Borax

True True True True

(NaCl + KCl + Borax)

01-075-0296 01-075-0296 01-075-0296 01-075-0296

metastable evaporation, the solutions were not stirred, and the crystal behavior of solid phase was observed periodically. When enough new solid phase appeared, the wet residue mixtures were taken from the solution. The solids were then approximately evaluated by the combined chemical analysis, of XP-300D Digital Polarizing Microscopy (Shanghai Caikon Optical Instrument Co,. Ltd., China) using an oil immersion, and further identification with X-ray diffraction (X'pert PRO, Spectris. Pte. Ltd., The Netherlands). Meanwhile, a 5.0 cm3 sample of the clarified solution was taken from the liquid phase of each polyethylene container through a filter pipette, and then diluted to a 250.0 cm3 final volume in a volumetric flask filled with DDW for the quantitative analysis of the compositions of the liquid phase. Some other filtrates were used to measure the relative physicochemical properties individually according to the analytical method. The remainder of the solution continued to be evaporated and reached a new metastable equilibrium.