**6. Conclusions**

Psychotherapy appears efficacious in enabling sufferers of psychological trauma to better cope with the memories of their traumatic experience, with the reconstruction of the traumatic memories (Peres et al., 2007). Emotionally-charged memories are subjective representations of an event, often distorted and distant from the original episode, but salient in their significance to the individual (Creamer et al., 2005). Although there is a marked degree of inter individual variability in the processing of memory of life-events and basic emotions, we postulate that the re-interpretation and reconstruction of traumatic memories will be efficacious in relieving PTSD symptomatology. This process will influence the neural networks sub serving these experiences, leading to the formation of new memories that are less fragmented and available for narrative expression, an idea that is consistent with neuroimaging and clinical observations. Understanding the neural processes associated with successful response to psychotherapy may point to specific mechanisms that can be

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**5. Discussion**

All humans have variation in genetic material, even identical twins by the time of born, and this variation give us the uniqueness. We inherit our genes from our parents, so the members of the same family share majority of their genetic material involving its variations. The variation in DNA that cause wrong in required genetic code translates into a specific protein called pathogenic variant or mutation when linked to phenotypic particularly if they occur within the protein coding sequence of the gene. Variations are caused by the environment and genetic factors. Several disorders arise from multiple effectors like environmental, lifestyle, and

genetic factors. From these diseases is behavioral genetics neuropsychiatric, such as schizophrenia, depression in which a genetic fault transferred from parents to sons through familial genes causing elevated the risk of a particular disease. SNPs can affect more than 90% of genetic variation and are responsible for the occurrence of differences between the humans. Despite the presence of SNPs, there is no relation to modify or change cellular function that is to say have no effects, and at the same time, many SNPs were found to participate in the initiation of disease like cancer or act as prophylaxis against a certain disease or even impact the responses to medications. Depending on the SNPs' position in the genome, we can classify them into that happen with exome (coding region), intron (non-coding region), and between adjacent genes (intergenic region). Because there is much of non-coding DNA (99%) that lead to harboring majority of SNPs occurring in these segments. However, only small part (1%) of genome is considered important to represent exome that can contribute existing mutations, and SNPs that have a big outcome on several diseases. Non-coding genome includes many categories of regulatory factors such as promoters, enhancers, silencers, and insulators. Each one of them provides binding sites for proteins, carries out transcription, activates transcription, suppresses transcription, and controls transcription process, respectively.

SNPs are very important and used in various studies such as estimating the tendency to disease and predicting genetic lesion; and are also used as biomarkers since they can occur near disease genes for complicated diseases, but not always. In complex disease, which means a pathological circumstance of the body due to a defect in a number of genetic and environmental aspect, SNP can affect that person has toward a specific disease. Nowadays, various methods have been established and assembled to identify known or unknown SNPs through two categories which are genotyping and scanning sequence, respectively.


*2.5. HPLC-PDA and UPLC-QTOF-MS Analysis*

The anthocyanins extract powders were dissolved in water with 10% acetic acid and 1% phosphoric acid. Then, all samples were centrifuged at 9339× *g* (12,000 rpm) at 4 ◦C for 10 min, and the supernatants were used for HPLC-PDA and UPLC-QTOF-MS analysis.

The HPLC system consisted of a pump (LC-20 AT) and a photodiode array (PDA) detector (SPD-10A) (Shimadzu Corp., Tokyo, Japan). The analytical column was the Intertsil ODS-SP C18 column (4.6 × 250 mm, 5 μm, GL Sciences Inc., Tokyo, Japan). Mobile phase A was 10% acetic acid and 1% phosphoric acid in water, and mobile phase B was 100% acetonitrile. Elution was performed at a flow rate of 1.0 mL/min, and the solvent gradient was as follows: 0–8 min, 10% B; 8–12 min, B from 10% to 40%; 12–15 min, 40% B; 15–25 min, from 40% to 10% B. The injection volume was 20 μL and the column was thermostated at 25 ◦C. The analysis wavelengths were 278 nm for monitoring the SMB process and 520 nm for quantifying the anthocyanins [21]. The UV-VIS spectra were scanned from 220 to 800 nm. Cyanidin 3-*O*-galactoside were quantified using cyanidin 3-*O*-galactoside (*y* = 18,674*x* + 11,660, R2 = 0.9994) and cyanidin 3-*O*-glucoside, cyanidin 3-*O*-arabinoside and cyanidin 3-*O*-xyloside were quantified using cyanidin 3-*O*-glucoside (*y* = 25,228*x* + 10,850, R2 = 0.9990). The purity was determined by the area normalization method of these four anthocyanins on HPLC-PDA at 278 nm. The anthocyanin contents were determined by comparing retention times and absorption spectra, and further confirmed by UPLC-QTOF-MS.

A Waters ACQUITY UPLC system coupled with a Xevo G2QTOF mass spectrometer (Waters Corporation, Milford, MA, USA) was used for anthocyanins confirmation as previously described [22]. The separation of anthocyanins was conducted on a HSS T3 column (2.1 mm × 100 mm, 1.8 μm). The mobile phase was (A) water + 0.2% formic acid and (B) acetonitrile + 0.2% formic acid: 0 to 1 min, 5% B; 1 to 2 min, 5% to 40% B; 4 to 8 min, 95% B; 8.1 to 10 min, 5% B. The injection volume was 2 μL and the flow rate was 0.3 mL/min. The ESI parameters were set as follows: negative mode, capillary voltage 2.2 kV, sampling cone voltage 30 V, extraction cone voltage 4 V, source temperature 100 ◦C, desolvation gas (nitrogen) temperature 400 ◦C, cone gas flow rate 20 L/h, desolvation gas (nitrogen) flow rate 800 L/h, collision energies 6 eV and mass range from 50 to 1200 Da. The anthocyanin structure was determined according to its fragmentation pattern of deprotonated and product ions.

#### *2.6. DPPH Free Radical Scavenging Assay*

The ROS scavenging capacity was determined by the DPPH free radical scavenging assay as previously reported [23]. The assay was performed in a 96-well plate using serial dilutions of 5 μL aliquots of each Cyn-3-gal (14 to 449 mg/L), anthocyanins crude extract (2.5 to 80.0 mg/L) and SMB ACN (0.03 to 1.0 g/L). DPPH solution (200 μL, 0.06 mM) was added to each well, and the plate was incubated at room temperature in the dark for 30 min. The absorbance was determined at 520 nm using a Thermo Multiskan MK3 Automated Microplate Reader (Thermo Fisher Scientific, Waltham, MA, USA). The EC50 value was calculated using a calibration curve of Trolox (25 to 250 mg/L) by SPSS.

#### *2.7. ABTS Free Radical Scavenging Assay*

The ABTS•+ scavenging capacity assay was determined as previously described with some modifications [24]. The ABTS•+ solution was produced by reacting aqueous ABTS solution (7 mM) with potassium persulfate (2.45 mM). Diluted ABTS•+ solution with an absorbance of 0.70 ± 0.02 at 734 nm was employed in the analysis. The reactions were performed by adding 4 mL of ABTS•+ solution and 0.4 mL of each Cyn-3-gal (14 to 449 mg/L), anthocyanins crude extract (2.5 to 40.0 g/L) and SMB ACN (0.03 to 1.0 g/L). After 6 min of incubation at room temperature, absorbance values were measured on a spectrophotometer at 734 nm. The EC50 value of ABTS•+ scavenging capacity was calculated using a Trolox calibration curve (25 to 250 mg/L).

#### *2.8. Experimental Animals*

Thirty-six Sprague-Dawley (SD) rats (male, weighing 250 ± 20 g, specific pathogenfree) were purchased by Beijing HFK Bioscience Co. Ltd. (Beijing, China). The rats were kept at the animal facility with free access to standard chow and water at 22 ± 2 ◦C, the relative humidity of 45 ± 15% and 12 h light/dark cycle. All animals had adapted for one week before experiments. SD rats were randomly divided into 3 groups (*n* = 12) of sham, Aβ, Aβ + SMB ACN. The group of Aβ and Aβ + SMB ACN were treated by intracerebral ventricle injection of Aβ1–40, and then Aβ + SMB ACN group were administered by intragastric administration with anthocyanins (50 mg/kg anthocyanins 85%) for one month. The experiment was approved by the Animal Ethical and Welfare Committee (No. IRM-DWLL-2017095).

#### *2.9. Preparation of Aβ-Induced Damage Rat Model*

The Aβ1–40 oligomers were prepared according to a previously reported method [25]. Briefly, Aβ1–40 peptide was prepared as a stock solution at a concentration of 1 mg/mL in sterile sodium chorionic solution, followed by aggregation via incubation at 37 ◦C for 4 days.

Rats were anesthetized with 10% chloral hydrate (0.3 mL/100 g i.p.) and the head was symmetrically held in the stereotaxic apparatus (ZS-FD, ZS Dichuang, Beijing, China). The scalp skin was clean shaved and the skull was exposed. One hole was drilled on the skull at coordinates of −0.8 mm posterior to the bregma and 1.4 mm lateral according to the atlas of Paxinos and Watson [26,27]. The needle was injected 4 mm deep into the ventricle with a speed of 1 μL/min for 5 μL and left in place for 5 min after the injection of Aβ1–40. The sham rats were injected with 0.9% saline. The surgical wound was sutured and the animals were returned to their cages with free access to food and water, and allowed to recover for 1 day [28].

#### *2.10. Morris Water Maze*

After one month of intragastric administration with anthocyanins, the Morris water maze (MWM) test was performed as previously described [29]. The circular pool was 150 cm in diameter and 60 cm in depth with an invisible platform (12 cm in diameter and 25 cm in depth). The water in the pool was mixed with black ink. During the experiment, the temperature of the water remained within 22 to 24 ◦C, and all landmarks around the maze remained the same. The MWM included spatial learning and acquisition trials (hidden platform trials) and a spatial probe trial. Before the spatial acquisition trials, each rat was put into the water to adapt for 2 min and the platform was visible, located in the middle of the first quadrant for one day. Then, water was added to the pool with the platform 1 cm below the water surface. During the hidden platform trials, each rat was placed into the water from one of the start positions, facing the wall. The spatial acquisition trials were conducted over four days, with four trials per day. If the animal reached the platform, the timer was stopped. If the animal failed to find the platform within 90 s, the animal was placed on the platform for 15 s to help it learn the platform's location. The spatial probe test was on day 5, and then the platform was removed. The rats were released from the third quadrant (180◦ from the original platform position) and the time spent crossing the target quadrant and the number of times the region in which the platform was previously located was crossed were recorded over a 90 s period. After testing, the rats were dried with a towel to keep them warm. The animals were recorded with a video camera, and the data were analyzed using ANY-maze behavioral tracking software (Stoelting Co., Wood Dale, IL, USA)

#### *2.11. Nissl Staining*

For Nissl staining of the brain, the rats were euthanized by intraperitoneal injection of 0.3% chloral hydrate, and were transcardially perfused with 100 mL of saline (0.9% *w*/*v* NaCl). Then, the brains were removed and put in the 15 mL of 4% paraformaldehyde. Serial coronal hippocampal sections with a thickness of 25 μm were cut using a cryostat (Leica Microsystems, Wetzlar, Germany). The sections were washed twice for 5 min in 0.01 M PBS and incubated in 1% toluidine blue staining solution for 5–10 min at room temperature. Then, the sections were rinsed in distilled water, soaked in 95% ethanol for 30 min, and dehydrated in 100% ethanol. After dehydration, brains were placed in xylene and cover slipped using resin medium. The neurons were quantified using image software (Image J 1.45 s), with three sections from three rats for each group.

#### *2.12. Statistical Analysis*

The data are presented as mean ± SD in triplicate from at least using GraphPad Prism 7.0 software (GraphPad Software, San Diego, CA, USA). *p* < 0.05 was considered statistically significant by LSD test. The results were analyzed by ANOVA and the EC50 value was calculated using the Probit Analysis by SPSS 20.0 software (IBM, Armonk, NY, USA).

#### **3. Results**

#### *3.1. Isolation and Purification of Black Chokeberry Anthocyanins by SMB*

Anthocyanins from black chokeberry were extracted and preliminarily purified by the XAD-7HP macroporous resin column. Four-section SMB was employed to separate the anthocyanins crude extract. After approximately 20 switching times, a cyclic steady state was reached. As shown in Table 1, the optimal parameters of flow rate were QF = 0.42 mL/min, QE = 1.5 mL/min, QR = 6.5 mL/min, and QD = 4.5 mL/min. The HPLC-PDA chromatograms of feed, raffinate and extract by running H are presented in Figure 2A–C. Anthocyanins and impurities are separated in column 3 to 6 (Figure 1). The purity of the SMB ACN was estimated to be 68% to 85%. These results in run A, B and G indicated that a switching time of 141 s maximally purified the anthocyanins. At a certain switching time, the slight decrease of flow rate in the feed, extract and raffinate zone increased the purity comparing run B to C, E to G and D to H. These results confirmed that the performance of SMB was mainly associated with the switching time and flow rate of feed, extract and raffinate [30].

**Table 1.** Operation parameters and separation performance of the SMB.

In order to analyze the anthocyanins crude extract and SMB CAN, HPLC-PDA was used to quantify and UPLC-QTOF-MS to identify the anthocyanins. An HPLC chromatogram of the crude extract showed four peaks (Figure 3A). The λmax value of UV-VIS spectra was 516 nm for cyanidin. The SMB ACN mainly showed two peaks by HPLC-PDA (Figure 3B) which were identified by UPLC-QTOF-MS (Figure 3C,D). Peak 1 of cyanidin 3-*O*-galactoside (m/z 449.1089/287.0560) appeared as a principal peak, and peak 2 was identified as a cyanidin 3-*O*-arabinoside by detection of the respective parent and product ion pairs (m/z 419.0975/287.0557). The four compounds of crude extract were cyanidin 3-*O*-galactoside (72.9% of total anthocyanins), cyanidin 3-*O*-glucoside (2.7%), cyanidin 3-*O*-arabinoside (21.0%), and cyanidin 3-*O*-xyloside (3.4%) (Table 2), which is in accordance with the Oszmianski's results [31]. The mass of total anthocyanins in the crude extract was

2.29 ± 0.19 g/100 g of dry extract, and the mass of total anthocyanins in the SMB ACN was 61.02 ± 6.46 g/100 g of the fraction rich in dry anthocyanins.

**Figure 2.** HPLC-PDA chromatogram of the feed (**A**), the raffinate (**B**), and the extract (**C**) during the SMB process monitored at 278 nm.

**Figure 3.** (**A**) HPLC-PDA chromatogram and UV-VIS scanning spectra of the black chokeberry anthocyanins crude extract at 520 nm and peak identities (1: cyanidin 3-*O*-galactoside; 2: cyanidin 3-*O*-glucoside; 3: cyanidin 3-*O*-arabinoside; 4: cyanidin 3-*O*-xyloside); (**B**) HPLC-PDA chromatogram of the SMB ACN at 520 nm and peak identities (1: cyanidin 3-*O*-galactoside; 2: cyanidin 3-*O*-arabinoside); The UPLC-QTOF-MS mass spectra and structure of cyanidin 3-*O*-galactoside (**C**) and cyanidin 3-*O*-arabinoside (**D**) of the SMB ACN peak 1 and peak 2.

**Table 2.** HPLC quantification and UPLC-QTOF-MS identification of anthocyanins in Black chokeberry crude extract and SMB ACN.

Data presented in fresh fruit weight (FW) and dried extract weight (DW). ND: not detected.

The SMB continuously purified the anthocyanins, and efficiently enriched the two main anthocyanins and eliminated the impurity. A similar observation was made by Wang, who reported the use of a two-step simulated moving bed chromatographic process to purify the EGCG from tea polyphenol [32]. It was suggested that the SMB was a highly efficient way to separate and purify the secondary metabolites from a natural extract. Due to the instability of secondary metabolites like anthocyanins and the complexity of the plant phenols, the extraction process of monomer was extremely difficult. Therefore, these results indicated that SMB may be a feasible and effective strategy to isolate and purify the anthocyanins from black chokeberry.

#### *3.2. The Free Radical Scavenging Abilities of Black Chokeberry Anthocyanin Extracts*

To evaluate the SMB purification process, we tested the DPPH and ABTS free radical scavenging abilities of the black chokeberry anthocyanins crude extract and the SMB purified anthocyanin extract. As shown in Figure 4, the EC50 values of anthocyanins crude extract were significantly higher than Trolox, Cyn-3-gal and SMB ACN of the free radical scavenging abilities (*p* < 0.001). The DPPH• and ABTS•+ scavenging of crude extract (54.18 ± 19.59 g/L and 4.21 ± 1.50 g/L) were 65- and 30-fold more than SMB ACN (0.83 ± 0.20 g/L and 0.14 ± 0.06 g/L). The lower EC50 of SMB ACN indicated a higher antioxidant activity. Moreover, the ABTS radical scavenging ability of SMB ACN was similar to Cyn-3-gal and Trolox (*p* > 0.05). These results suggest that the SMB process increased the free radical scavenging ability of the anthocyanin extract.

**Figure 4.** (**A**) DPPH free radical scavenging activity of Trolox, cyanidin 3-*O*-galactoside (Cyn-3-gal), anthocyanins crude extract and SMB ACN; (**B**) ABTS free radical scavenging activity of Trolox, Cyn-3-gal, anthocyanins crude extract and SMB ACN. Date represent mean ± SD (*n* = 6) were expressed by the EC50 inhibition of the free radical scavenging and the **\*\*\*** *p* < 0.001 by LSD test of one-way ANOVA.

The results showed that the ABTS free radical scavenging abilities of anthocyanins extracts were higher than those of DPPH; notably, cyanidin 3-*O*-galactoside had a high free radical scavenging ability due to its hydroxyl group. It may be that the anthocyanin extracts neutralized the DPPH free radicals mainly by hydrogen atom transfer and the ABTS•+ by a fast, non-selective electron transferring process. Therefore, the application of the SMB chromatography purified and enrichened the anthocyanins and improved the antioxidant ability than crude extract. Deneva et al. found that anthocyanins were the second biggest contributor to antioxidant activity after the proanthocyanidins among black chokeberry polyphenols [3]. Moreover, cyanidin-3-*O*-glucoside could regulate cellular antioxidant defense induced by Aβ1–40 in SH-SY5Y cells through the Nrf2 signaling pathway [33]. The purified anthocyanins may scavenge the free radicals and inhibit the Aβ1–40 neurotoxicity. Therefore, the next section of this study is concerned with SMB ACN, investigating its neuroprotective activity against amyloid-β induced in rats.

#### *3.3. The Protection of Anthocyanins on Memory Impairment in Aβ-Induced Toxicity Rats*

The Morris water maze (MWM) is a method to assess spatial learning and memory in rats. The escape latency time of all groups had a decreasing tendency over four days

(Figure 5A). The results showed that the SMB ACN treatment group presented a less steep learning curve between days 1 and 2, which might indicate that anthocyanins had increased the short-term memory. The sham group showed a more stable spatial learning rate than the Aβ+SMB ACN group. In the probe trial, the time spent in the targeted quadrant of the sham group was shorter compared to the Aβ group impaired by Aβ1–40, which indicated that the Aβ group showed lower long-tern learning and memory ability (Figure 5B). The rats given the anthocyanins extract achieved more platform crossings in comparison with the Aβ-induced group, and showed a nonsignificant difference with the sham group (Figure 5C). The results indicated that anthocyanin group had a significantly higher spatial learning ability than the Aβ-induced group (*p* < 0.05). Interestingly, the sham group achieved higher scores than the Aβ group, although this was not significant, and may have been due to intervariability among the rats. As shown in Figure 5D, the Aβ + SMB ACN group allayed behavioral deficits compared to the Aβ group regarding the swim path. The behavioral test results showed that the purified anthocyanins extract alleviated the damage induced by amyloid-β to spatial learning and memory.

**Figure 5.** SMB ACN treatment alleviated Aβ induced spatial learning deficits. (**A**) The escape latency during the spatial acquisition trials of Sham, Aβ and Aβ + SMB ACN group; (**B**) The time spent in the target quadrant of the spatial probe test; (**C**) The number of platform crossing of the spatial probe test; (**D**) The swim path of the spatial acquisition trial. The results were expressed by mean ± SD (*n* = 12) and the **\*** *p* < 0.05, **\*\*** *p* < 0.01 by LSD test of one-way ANOVA.

Previous reports have shown that anthocyanin galactosides are better maintained in the intestines compared to glucosides, while arabinosides or xylosides showed negligible losses [34]. Notably, cyanidin 3-*O*-galactoside and cyanidin 3-*O*-arabinoside crossed the blood-brain barrier into the cortex and hippocampus [16]. Moreover, the neuroprotective ability of black chokeberry has been reported; additionally, anthocyanins have been associated with antiaging [7]. Anthocyanins were shown to regulate the cell cycle and senescence by regulating the expression of the DNA damage signaling pathway and antioxidant enzymes in neuronal cells [7,11]. Our results support the hypothesis that the administration of SMB ACN ameliorates learning and memory impairment by Aβ-induced severe behavioral dysfunction.

#### *3.4. The Neuroprotective Effect of SMB Anthocyanins Extract in Rat Brain*

To further elucidate the neuroprotective effect of anthocyanins, we examined the hippocampus. The Nissl staining stains the nuclei of all cells, as well as clumps of material surrounding the nuclei of neurons. Neuronal loss in the brain, especially in the CA1 region of the hippocampus [35] and basal forebrain, is one of the most important pathological hallmarks of Alzheimer's disease [36]. The pyramidal cells in the CA1 and CA3 regions of the hippocampus in the brain modulates memory and emotions [37].

The results showed that the survival of neuron cells in the CA1 and CA3 of the sham group was greater than in the Aβ1–40 treated group (*p* < 0.05) (Figure 6). The neurons in the Aβ group were disorganized and deformed with deep stain nuclei due to the neurotoxicity of Aβ. The results of the sham group indicated that the injection into the ventricle of the brain did not damage the hippocampus region. The neurons and kernel in the visual field of the Aβ + SMB ACN group were clearer and more intact than the Aβ group, and the numbers of pyramidal cells in the CA1 and CA3 were significantly increased (*p* < 0.05). The neurons in the Aβ1–40 treated group were damaged with extensive degenerative changes including sparse cell arrangements, swollen cell bodies, loss of integrity, shrunken cytoplasm and oval or triangular nucleus [38,39]. These results confirmed that Aβ1–40 can induce cellular loss and disorganization of the pyramidal cells, and indicated that the treatment of anthocyanins can reverse these changes to alleviate the loss of memory.

**Figure 6.** Representative images of Nissl staining in the CA1 and CA3 regions of the hippocampus of sham group (**A**,**B**), Aβ group (**C**,**D**), and Aβ + SMB ACN group (**E**,**F**). (**G**) The site of intracerebral ventricle injection in rat brain (Black) and the sites of the section in CA1 and CA3 region of the hippocampus (**H**). The number indicated the surviving neurons in CA1 (**I**) and CA3 (**J**), \* *p* < 0.05 and \*\* *p* < 0.01. Stained sections of each group were viewed at 200× magnification. Scale bar = 100 μm.

This research shows that purified anthocyanins by SMB exhibit excellent antioxidative activity against oxidative stress and prevent amyloid-β induced neurotoxicity in the brain. However, we still need to investigate the mechanism of antioxidative ability in vivo and the bioavailability of anthocyanins and their metabolites to determine the efficacy of such a

treatment. The cell signaling pathway of anthocyanins regulating the cellular antioxidant system and alleviating the Aβ neurotoxicity still needs to be examined. We used UPLC-QTOF-MS to analyze a black chokeberry anthocyanins crude extract and found many secondary metabolites, such as polyphenols, that have antioxidant activities. In a future study, the neuroprotective ability of black chokeberry may found to be associated with the ability of these secondary metabolite compounds to protect neuronal cells against oxidative stress.

#### **4. Conclusions**

In this study, the anthocyanins from black chokeberry (*Aronia melanocarpa*) were isolated and purified by XAD-7HP macroporous resin and SMB chromatography, yielding a purity of 85%. The main purified anthocyanins (cyanidin 3-*O*-galactoside and cyanidin 3-*O*-arabinoside) had a strong antioxidant ability and ameliorated learning and memory impairment among Aβ-induced neurotoxicity rats. Taken together, these results demonstrate that simulated moving bed chromatography could be a feasible and effective strategy to separate highly bioactive anthocyanins from black chokeberry. The present study was an attempt to establish a method to isolate secondary metabolites from natural plants to exploit their neuroprotective abilities.

**Author Contributions:** Conceptualization, L.M. and J.L.; methodology, H.W., H.C., and H.T.; software, H.W. and X.Z.; validation, X.Z. and C.R.; investigation, H.W., H.C., and H.T.; data curation, L.M. and J.L.; writing—original draft preparation, H.W. and X.Z.; writing—review and editing, H.W., H.C., C.R. and J.L.; visualization, H.W.; supervision, L.M. and J.L.; project administration, J.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work was funded by the National Natural Science Foundation of China, grant number 31571840.

**Institutional Review Board Statement:** The study was approved and conducted according to the Animal Ethical and Welfare Committee (Approval No. IRM-DWLL-2017095).

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data are not publicly available because the research is still ongoing.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

*Article*


**4. Concluding Remarks and Future Prospects**

Seaweeds remain largely untapped reservoirs of natural bioactive molecules [10]. In fact, more than 11,300 species of seaweeds are reported on Algabase, of which only 42 species were surveyed on category 4 (studies of extracts enriched in isolated groups or classes of complex lipids) and category 5 (studies of isolated complex lipid molecular species), most of them within the Ochrophyta phylum. This reveals that the bioprospecting potential of seaweed lipids remains largely untapped.

Complex lipids from seaweeds are emerging as bioactive molecules with hidden potential; however, their exploitation is far from being optimized and their action mechanisms are still poorly understood. This figure is likely to change as more seaweeds have their bioactive complex lipids characterized and more mechanism-oriented studies are performed.

To date, not only do most studies lack a systematic research approach, but most of the lipid bioactivities already identified refer to total lipid extracts. Indeed, only a few studies have achieved molecular isolation and characterization of bioactive lipids. Interestingly, complex lipids isolated from seaweed species with reported bioactivity have been classified mainly as GLs species. This systematic analysis pinpoints the promising results of naturally occurring GLs in seaweeds, with emphasis to their antitumor and antiinflammatory potential. The advances of emerging food/feed, nutraceutical, cosmeceutical, pharmaceutical, and complementary medicine research fields [91–93], as well as biological and experimental sciences, will contribute to boost structural characterization of complex lipids and to link lipid structure and bioactivity through different mechanisms of action.

Regardless of their polyphyletic nature, it is unquestionable that seaweeds as a whole, remain an important reservoir of lipid phytochemicals. Despite the low abundance of these biomolecules in seaweeds, they remain largely uncharacterized and unexplored. Complex lipids from seaweeds offer an unmatched chemical diversity and structural complexity when compared to terrestrial phytochemicals. It seems that seaweeds species or genera feature unique lipidomes, which likely enhances the potential number of target applications. Lipidomic characterization strategies using high-resolution apparatus, such as mass spectrometry, can be paramount to unleash the true potential of these biomolecules. The species-specific lipidome for each seaweed could be applied to the production of target bioactive lipids. Otherwise, isolated bioactive complex lipids can be used as a largescale synthesis model. While some of their natural chemotherapy diversity has already been studied, resulting in open access and proprietary compound libraries, there is still a multitude of lipids from algal origin that have hardly been characterized. The potential of these biomolecules to develop new products and processes is certainly far from being exhausted. It is expected that the bioprospecting of seaweed extracts enriched in active lipids for the formulation of high-end products can foster the added value of seaweed biomass production.

Under this scope it will be possible to put forward innovative processes for the production of farmed seaweeds biomass under controlled conditions, as these will allow to target new markets and consumers under a circular and sustainable blue bioeconomy framework.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/10 .3390/md19120686/s1, Table S1: Eligible studies distributed by title, published year, doi (when applicable), seaweeds species, genus, phylum, bioactivity reported, and category where they were inserted.

**Author Contributions:** Conceptualization, D.L. and M.R.D.; methodology, D.L.; validation, M.R.D., F.R., M.C.L., A.I.L., R.C. and M.R.D.; formal analysis, D.L.; data curation, D.L. and F.R.; writing original draft preparation, D.L.; writing—review and editing, F.R., M.C.L., A.I.L., R.C. and M.R.D.; supervision, A.I.L., R.C. and M.R.D. All authors have read and agreed to the published version of the manuscript.

**Funding:** The authors are grateful to Fundação para a Ciência e a Tecnologia (FCT, Portugal), European Union, QREN, POPH, FEDER, and COMPETE by funding CESAM (UIDP/50017/2020 + UIDB/50017/2020), and LAQV/REQUIMTE (UIDP/50006/2020 + UIDB/50006/2020). Thanks

to the project Omics 4 Algae: Lipidomic tools for chemical phenotyping, traceability, and valorization of seaweeds from aquaculture as a sustainable source of high added-value compounds (POCI-01-0145-FEDER-030962), funded by Centro2020, through FEDER and PT2020.Diana Lopes (SFRH/BD/119027/2016) is grateful to FCT, Programa Operacional do Capital Humano (POCH) and European Union through European Social Fund (FSE) for her grant. FCT is also thanked for the Scientific Employment Stimulus 2017, with a Junior Researcher contract to Felisa Rey (CEECIND/00580/2017), and an Assistant Researcher contract to Miguel Leal (CEECIND/01618/2020). This is a contribution of the Marine Lipidomics Laboratory.

**Conflicts of Interest:** The authors declare no conflict of interest.


**3.1. Anatomy of the retina**

The retina is considered to be a part of the *central nervous system* (CNS). This is the only part of the CNS that can be observed noninvasively. It is a light-sensitive layer of cells located at the back of the eye with a thickness of 0.2–0.4 mm. It is responsible for sensing the light rays that hit it through the pupil and an eye lens that turns and inverts the image. The retina is a complex structure with several layers of neurons linked by synapses (**Figure 10**). The only neurons that react directly to light are *photoreceptors*. These are divided into two main types: *cones* and *rods*. In adults, the retina covers approximately 72% of the inner eye. The entire surface of the retina contains about 7 million cones and 75–150 million rods. This would compare the eye to a 157 MP camera. Rods are used to detect light and are capable of responding to the impact of one to two photons by providing black and white vision. Cones are used to detect colors and are divided into three types depending on which base color they are sensitive to (red, green, blue), but these are less sensitive to light intensity. In these cells, there is a phenomenon called *transduction* where the cascade of chemical and electrical phenomena changes into electrical impulses. These are then transmitted through the optic nerve to the central nervous system.

We can observe the two most distinctive points on an eye's retina. It is a *blind spot* (or an optical disc) and a *macula* (*yellow spot*). A blind spot is the point where the optic nerve enters the

**Figure 10.** Structure of the retina [11].

semi-automatic scanning to automatic, but the error rate of these systems is still relatively

• Images of the iris may be of poor quality, resulting in errors in registration, verification, or

• The iris can change over time, especially due to various illnesses. Changing the iris is possible in cataract surgery and illnesses such as *nystagmus* (shaking eyes) or *aniridia* (a completely missing iris). For some blind people, the iris may not be visible at all due to clouding

• The individual parts of the iris are related to the various internal organs of the human body, resulting in the possibility of misusing the scanned pattern to determine the health

Recognition by the retina is another option offered by the eye. Perhaps, the most complicated part of the entire retinal identification procedure is to obtain a good-quality eye image. Here, it is possible to inspire the principles of medical devices for the examination of an eye. It is also necessary to understand the function of the retina for human vision, its location, and the elements contained therein, according to which biometric identification can be carried out.

The retina is considered to be a part of the *central nervous system* (CNS). This is the only part of the CNS that can be observed noninvasively. It is a light-sensitive layer of cells located at the back of the eye with a thickness of 0.2–0.4 mm. It is responsible for sensing the light rays that hit it through the pupil and an eye lens that turns and inverts the image. The retina is a complex structure with several layers of neurons linked by synapses (**Figure 10**). The only neurons that react directly to light are *photoreceptors*. These are divided into two main types: *cones* and *rods*. In adults, the retina covers approximately 72% of the inner eye. The entire surface of the retina contains about 7 million cones and 75–150 million rods. This would compare the eye to a 157 MP camera. Rods are used to detect light and are capable of responding to the impact of one to two photons by providing black and white vision. Cones are used to detect colors and are divided into three types depending on which base color they are sensitive to (red, green, blue), but these are less sensitive to light intensity. In these cells, there is a phenomenon called *transduction* where the cascade of chemical and electrical phenomena changes into electrical impulses. These are then transmitted through the optic nerve to the

We can observe the two most distinctive points on an eye's retina. It is a *blind spot* (or an optical disc) and a *macula* (*yellow spot*). A blind spot is the point where the optic nerve enters the

of the person. This alternative medicine area is called *iridology* [10].

high.

identification.

16 Machine Learning and Biometrics

of the eyes.

**3. Recognition by retina**

**3.1. Anatomy of the retina**

central nervous system.

• Relatively high cost for high-performance systems.

eye, has a size of about 3 mm2 , and lacks all receptors. So, if the image falls into the blind spot, it will not be visible to a person. The brain often "guesses" how the image should look in order to fill this place. The existence of a blind spot can be tested as shown in **Figure 11**. If we close the left eye and observe the cross, then the black circle disappears at a certain distance from the image. This is precisely the moment when this image lands on a blind spot [5].

On the other hand, the *macula* (*yellow spot*) is referred to as the sharpest vision area, has a diameter of about 5 mm, and the cones predominate it (it is less sensitive to light). This area has the highest concentration of light-sensitive cells whose density is decreasing toward the edges. The center of the macula is *fovea*, which is the term describing receptor concentration and visual acuity. Our direct view is reflected in this area. Interestingly enough, the macula (yellow spot) is not really yellow, but slightly redder than the surrounding area. This attribute, however, was given by the fact that yellow appears after the death of an individual.

The retina is nourished by the *choroid*, which is a layer located between the retina and the sclera. It contains blood vessels and a pigment absorbing excess light. **Figure 12** shows how the retina is richly interwoven with nourishing vessels and nerves. It shows a similar apparatus to the brain, where the structure and venous tangle remain unchanged throughout life. The retina has two main sources of blood supply—the retinal artery and vessels. Larger blood flow to the retina is through the blood vessel that nourishes its outer layer with photoreceptors. Another blood supply is provided by the retinal artery, which primarily nourishes the inside of the retina. This artery usually has four major branches.

The retina located inside the eye is well protected from external influences. During life, the vessel pattern does not change and is therefore suitable for biometric purposes.

(bleeding or blood clots) appear in the retina; however, as mentioned earlier, potential damage (e.g., perforation or retinal detachment) or retinal disease is such a matter to go to ophthalmologists. Since our research group works with medical doctors, we process images or video sequences in which we look for pathological manifestations. At the present time, we focus on detecting and delimiting the exudates/druses and hemorrhages in the image, automatically detecting the position of the macula and blind spot. These are the reference points by which we determine the location of pathological findings. The worst area is the part called the fovea centralis, where the sharpest vision is located. Once this area is damaged, it has a very significant impact on our sight. An example of the detection of pathological findings is shown in **Figure 13**. We also deal with colleagues by detecting the quality of blood flow in the retina. There is still much to do in all areas of imaging and video processing for medical purposes, as input data are very different. For the time being (and probably still remaining so

Recognition of Eye Characteristics

19

http://dx.doi.org/10.5772/intechopen.76026

Every part of the human body can be affected by a disease, whether it's curable or not. An incurable disease will be understood as a disability that cannot be surgically or otherwise removed without the biometric information (e.g., amputation) disappearing. The curable disease is removable with minimal consequences (e.g., inflammation, cuts). The retina can be affected by both types of these diseases. These diseases can significantly affect the course of recognition. If a disease disrupts the structure of the retina, it may cause erroneous evaluation

*Macular degeneration* is a disease that occurs in 90% of cases with age, also known as agedrelated macular degeneration (ARMD). In the remaining percentage, macular degeneration occurs in children or young people in the form of *Best's macular degeneration* or *Stargardt's*

In macular degeneration, the area of the retina, which forms at the center of the field of vision, is violated (**Figure 14**). As a result, a major disturbance of the central field of vision arises. In the center, the patient sees a gray shadow down to a black spot. The peripheral vision of the

**Figure 13.** Hemorrhage (left), detection of suspected areas (center), and highlighted hemorrhage (right).

for a long time), the best diagnostic tool is a medical doctor.

*disease*. These diseases arise on the basis of inheritance.

or a complete rejection of the pattern.

*3.2.1. Macular degeneration*

**Figure 11.** Blind spot testing.

**Figure 12.** A snapshot of the retina taken by the fundus camera.

The retina acquires an image similar to a camera. The beam passing through the pupil appears in the focus of the lens on the retina, much like film. In medical practice, specialized optical devices are used for the visual examination of the retina.

#### **3.2. Eye diseases**

In the field of ophthalmology, the iris is not very interesting because when we neglect the extreme and very rare cases of a disease (e.g., irrigation or perforation of the iris, irritation of the iris), pigment changes occur often, which is not the result of a disease and has no effect on human health. The main focus is on ophthalmology in regard to examining the retina of the eye, of course taking into account the overall health of the eye (e.g., cataracts or increased intraocular pressure). In the retina, there is a relatively large line of diseases and damage that interest medical doctors, but they are detailed in an encyclopedia of ophthalmology with hundreds of pages (e.g., [12] (1638 pages) or [13] (2731 pages)). The large group is diabetes and age-related macular degeneration (ARMD). Occasionally, exudates/druses or hemorrhages (bleeding or blood clots) appear in the retina; however, as mentioned earlier, potential damage (e.g., perforation or retinal detachment) or retinal disease is such a matter to go to ophthalmologists. Since our research group works with medical doctors, we process images or video sequences in which we look for pathological manifestations. At the present time, we focus on detecting and delimiting the exudates/druses and hemorrhages in the image, automatically detecting the position of the macula and blind spot. These are the reference points by which we determine the location of pathological findings. The worst area is the part called the fovea centralis, where the sharpest vision is located. Once this area is damaged, it has a very significant impact on our sight. An example of the detection of pathological findings is shown in **Figure 13**. We also deal with colleagues by detecting the quality of blood flow in the retina. There is still much to do in all areas of imaging and video processing for medical purposes, as input data are very different. For the time being (and probably still remaining so for a long time), the best diagnostic tool is a medical doctor.

Every part of the human body can be affected by a disease, whether it's curable or not. An incurable disease will be understood as a disability that cannot be surgically or otherwise removed without the biometric information (e.g., amputation) disappearing. The curable disease is removable with minimal consequences (e.g., inflammation, cuts). The retina can be affected by both types of these diseases. These diseases can significantly affect the course of recognition. If a disease disrupts the structure of the retina, it may cause erroneous evaluation or a complete rejection of the pattern.


**4.10. GLUT9** *(SLC2A9)*

**4.7. Class II family members**

26 Glucose Homeostasis

**4.8. GLUT5** *(SLC2A5)*

into the portal vein (Figure 3B).

*Mouse Models of GLUT5 deficiency*

*4.8.1. GLUT5 physiology*

tension.

**4.9. GLUT7** *(SLC2A7)*

to two proteins that only differ in their N-terminal region.

With GLUT5 being identified in 1990 and established as a fructose transporter initial transport studies for GLUT7 and 9 indicated that those transporters might also transport fructose. A specific feature for class II transporters is that cytochalasin B as a classical GLUT inhibitor does not block glucose transport. Furthermore, all isoforms do not show an affinity for 2-DG and galactose. Unique for GLUT9 and GLUT11 is the alternative splicing /promoter usage, where either two or three different mRNA's are transcribed, respectively. In case of GLUT9 this leads

Human GLUT5 was initially cloned from an intestinal epithelial cell line. GLUT5 is considered as the prototypic fructose transporter – when expressed in *Xenopus laevis* oocytes the human protein transports fructose with a Km of 6 mM without any noticeable glucose transport activity. However, fructose transport is not inhibited by cytochalasin B, phlore‐ tin or phlorizin. Besides fructose, the rat GLUT5 transports glucose, an activity that can be blocked by cytochalasin B. In humans, rats and mice, GLUT5 is primarily expressed in the jejunal region of the small intestine. Lower levels of the protein are expressed in the kidney, the brain, skeletal muscle, and adipose tissue. GLUT5 mediates fructose absorption in the jejunum at the apical, and potentially, at the basolateral membrane, of the epithelial cells

GLUT5 deficiency is associated with reduced fructose absorption when animals are challenged by a high fructose diet. While wildtype mice upon high fructose feeding display an enhanced salt absorption in their jejuna and develop systemic hypertension, GLUT5 knockout mice do not show fructose stimulated salt absorption. Instead, the animals display impaired nutrient absorption that is accompanied by hypotension. Absence of GLUT5 leads to a massive dilatation of the cecum and colon, consistent with severe malabsorption. On a normal chow diet, GLUT5 deficient mice have normal blood pressure and display normal weight gain. The phenotype of GLUT5 deficient mice demonstrates that this isoform is essential for fructose absorption by the intestine and thereby fundamentally involved in fructose induced hyper‐

The human GLUT7 was cloned from an intestinal cDNA library using a PCR-based strategy. GLUT7 is primarily expressed in the small intestine and colon, although mRNA has been detected in the testes and prostate as well. The protein has been localized to the apical membrane of the small intestine and colon. GLUT7 shows a rather high affinity for glucose and fructose (*K*m for glucose=0.3mM) while galactose, 2-DG, and xylose are not transported. Sugar transport by GLUT7 is not inhibited by cytochalasin B or phloretin. Sequence alignments Human GLUT9 cDNA was isolated by PCR amplification from a human kidney cDNA library on the basis of sequence information from ESTs and from its genomic sequence. GLUT9 mRNA is detected almost exclusively in the kidney and liver and at low levels in the small intestine, placenta, lung and leucocytes. GLUT9 is localized to the insulin-secreting β-cells of human and mouse islets, where downregulation of the protein by siRNA in rat and mouse insulinoma cells leads to a reduced glucose-stimulated insulin secretion. In humans as well as in mice alternative splicing/or promoter usage results in two proteins, GLUT9a and GLUT9b, which only differ in their N-terminal region. While human and mouse GLUT9b are mainly expressed in the kidney, placenta and liver, GLUT9a shows a broader tissue distribution. The different N-termini of human GLUT9a and b determine basolateral versus apical sorting in polarized cells *in vitro*, respectively. In mouse kidney, GLUT9 is localized to apical as well as basolateral membranes of distal convoluted tubules. Initial characterization of GLUT9 determined a rather low affinity for 2-DG. However, a high affinity transport has been reported for glucose and fructose with Km values of 0.6 mM and 0.4 mM, respectively. More recently, GLUT9 has been identified as a high-affinity uric acid transporter with a Km of 0.9 mM and 0.6 mM for the human and mouse protein, respectively. Although transport of glucose and fructose has not been observed by some investigators, GLUT9 is thought to exchange glucose or fructose for urate. The classical inhibitor cytochalasin B does not block GLUT9 function. GLUT9 has been established to represent a major regulator of urate homeostasis in dogs, mice, and men.

#### *4.10.1. GLUT9 physiology*