**4. HF ms2**

There are three main spectral components in an HRV spectrum named as high frequency (HF), low frequency (LF), and very low frequency (VLF) bands. The HF band represents the power in the frequency range between 0.15 and 0.4 Hz. HF power is generally believed to represent respiration-linked changes in heart rate and is generally accepted as a measure of respiratory sinus arrhythmia (RSA), or the parasympathetic contribution to HRV. RSA refers to the acceleration in heart rate that occurs during inspiration (due to the cardiovascular control center's inhibition of vagal outflow) and the subsequent heart rate deceleration that occurs during expiration, due to vagal restoration [13, 14].

Doheny et al. in 2015 [15], evaluated the possibility of using a non-invasive biomarker that allows early detection of patients at risk of necrotizing enterocolitis (NEC), that is an acute neonatal inflammatory disease that may lead to intestinal necrosis, multi-system failure and death. For that, they used the high frequency (HF) component of heart rate variability. They studied 70 stable preterm infants (gestational age 28-35 week). HF ms2 was 21.5 ± 2.7 ms2 in infants that remained healthy and 3.9 ± 0.81 ms2 in those that later developed stage 2 + NEC (P < 0.001). The cut-off value in the ROC curve was 4.68ms2 , predictive for developing NEC with sensitivity and specificity of 89% and 87%, and positive and negative predictive values of 50% and 98%, respectively. They concluded that HF ms2 may serve as a potential, non-invasive predictive biomarker of NEC-risk in infants.

In 2004, Abramkin et al. [16], studied 188 patients to compare the prognostic value of different noninvasive reflex tests on days 4-11 of myocardial infarction. The age varied from 34 to 75 years, 68% were men, and 93.6% were on beta-blockers, all without heart failure NYHA IV on the day of tests. HF power < 65 ms2 during active standing (OR 28.8, 95% CI 4.1-104.2; p = 0.001, positive predictive value 29.4%) was an independent predictor of sudden cardiac death.

In a meta-analytic study carried out in 2021 by Heimrich et al. [17], the objective was to verify whether the analysis of heart rate variability could indicate decreased parasympathetic tone in patients with Parkinson's disease. A total of 47 studies were evaluated, including 2772 individuals, 1566 of which had Parkinson's Disease (65.0 ± 0.6 years) and 1206 were healthy controls (62.6 ± 1.0 years). Based on 24 studies, it was possible to detect that the FH ms2 was significantly lower in the group of patients with the disease (145.2 ± 41.1 versus 219.4 ± 48.8 ms2; P = 0.002; heterogeneity 91%).

### **5. Objective**

Considering that the heart rate (HR), the root-mean-square of successive differences between adjacent normal RR intervals (RMSSD) and the HF band power in the frequency range between 0.15 and 0.4 Hz (HFms2), can help to differentiate the homeostatic level between individuals with severe impairment and high risk, and healthy individuals, we performed an intensive review of the literature by collecting published data involving the aforementioned variables, in search of a cutoff value for defining homeostatic reference levels and creating an individualized diagnostic coding.

### **6. Method**

Based on research projects linked to FAPESP - Brazil (2017/12529-7) and CNPq -Brazil (308,555/2018-0), studies involving the use of one or more of the *Heart Rate Variability as a Marker of Homeostatic Level DOI: http://dx.doi.org/10.5772/intechopen.102500*

three variables mentioned above were evaluated. In total, it was possible to analyze 164 studies involving the heart rate of individuals with importantly compromised homeostatic level (HL\_ic), 181 studies involving the heart rate of apparently healthy individuals (HL\_ah), 179 studies involving the RMSSD of individuals with HL\_ic, 221 studies involving the RMSSD from HL\_ah subjects, 125 studies involving the HF ms2 of subjects with HL\_ic and 155 studies involving the HF ms2 of HL\_ah subjects. Obviously, there were concurrent studies in certain situations. Due to a large number of references, they are cited separately and available in a *supplementary file*.

### **7. Statistical analysis**

Data were presented as mean and standard deviation, weighted mean, quantities, percentages and correlation coefficients. Comparisons between groups were made by analysis of variance or the Kruskal-Wallis test and its post-tests, according to the indication. Correlation graphs were constructed and Box-Whisker graphs were used for illustration. An alpha error of 5% was accepted, with P values less than or equal to 0.05 being considered significant. The statistical software used was StatsDirect version 3.3.5 (03/22/2021).

### **8. Results**

The total amount of data analyzed was extremely high. **Table 1** below indicates the amounts for each variable under conditions of significantly compromised and apparently healthy homeostasis, as well as the mean and standard deviation values for the age of the group, the mean and standard deviation of the variable, and the weighted mean of the variable.

### **Table 1.**

*Distribution of the number of studies included, amount of data per variable, according to homeostatic level (importantly compromised [HL\_ic] or apparently healthy [HL\_ah].*

As the behavior of heart rate variability is related to age, linear correlation calculations were made between age (predictor) and the variable to be predicted (HR, RMSSD or HFms2 ) in the HL\_ic and HL\_ah groups (**Table 2**; **Figures 1**–**6**).

### **Table 2.**

*Distribution of simple linear regression, correlation coefficients (r and r2 ) and two-sided P-values, by homeostatic condition.*

#### **Figure 1.**

*Correlation graphs (age x heart rate) in the groups of individuals with importantly compromised homeostatic level (HL-ic) and apparently healthy (HL\_ah).*

### **Figure 2.**

*Correlation graphs (age x RMSSD) in the groups of individuals with importantly compromised homeostatic level (HL-ic) and apparently healthy (HL\_ah).*

*Heart Rate Variability as a Marker of Homeostatic Level DOI: http://dx.doi.org/10.5772/intechopen.102500*

### **Figure 3.**

*Correlation graphs (age x HF ms2 ) in the groups of individuals with importantly compromised homeostatic level (HL-ic) and apparently healthy (HL\_ah).*

### **Figure 4.**

*Scattergram (HR in bpm) for the groups of individuals with importantly compromised homeostatic level (HL- ic; red circles) and apparently healthy (HL\_ah; blue squares).*

### **Figure 5.**

*Scattergram (RMSSD in ms) for the groups of individuals with importantly compromised homeostatic level (HLic; red circles) and apparently healthy (HL\_ah; blue squares).*

### **Figure 6.**

*Scattergram (HF in ms2 ) for the groups of individuals with importantly compromised homeostatic level (HL- ic; red circles) and apparently healthy (HL\_ah; blue squares).*

A moderate negative correlation was found between heart rate and age, both in cases with significant homeostatic impairment and in apparently healthy cases. There was also a moderate negative correlation between RMSSD and age, and between HFms2 and age in the apparently healthy group. The fact that there was only a weak negative correlation between HFms2 and age in the group with significant homeostatic impairment and also the absence of correlation between RMSSD and age in this impaired group, was noteworthy. This may suggest that RMSSD is a more effective or sensitive biological marker of homeostasis, revealing changes regardless of age.

It became also relevant to evaluate the data of the three selected variables, in the group composed of individuals named as being from the general population (HL\_gp). Thus, from the global data survey carried out, a number of 10,121,910 were obtained from individuals from the general population, in different age groups. The values of mean, standard deviation, weighted mean, mean age ± standard deviation and number of articles consulted are found in **Table 3** and **Figure 7**.

Comparative statistical analysis between the 3 groups (HL\_ah, HL\_ic and HL\_gp) for the three selected variables, using the Kruskal-Wallis test with post-test Dwass-Steel-Chritchlow-Fligner, showed a non-significant difference between HR HL\_ah versus HR HL\_gp (P = 0.6228); the statistically significant difference between HR HL\_ah versus HR HL\_ic (P < 0.0001); the statistically significant difference between HR HL\_gp versus HR HL\_ic (P < 0.0001).

### **Table 3.**

*Data and values were obtained in the assessment of the general population (HL\_gp).*

### **Figure 7.**

*Box-whisker graphs of the distributions of values for heart rate (A), RMSSD (B) and HF ms2 (C) variables, by the homeostatic level group.*

Regarding the variable RMSSD, there was a statistically significant difference between RMSSD HL\_ah versus RMSSD HL\_gp (P < 0.0001); the statistically significant difference between RMSSD HL\_ah versus RMSSD HL\_ic (P < 0.0001); the statistically significant difference between RMSSD HL\_gp versus RMSSD HL\_ic (P < 0.0001).

In the comparative analysis of the variable HF ms2, there was a statistically significant difference between RMSSD HL\_ah versus RMSSD HL\_gp (P < 0.0001); statistically significant difference between RMSSD HL\_ah versus RMSSD HL\_ic (P < 0.0001); statistically significant difference between RMSSD HL\_gp versus RMSSD HL\_ic (P = 0.0002).

Therefore, it is concluded that data from the so-called general population are not suitable to be considered as a normal condition and this must be taken into account when this group is used as a control group.

Finally, based on the weighted average of the results in **Table 1**, on the scatter plots involving the group of individuals with significant homeostatic impairment and the group of apparently healthy individuals, we propose a classification model for the individual homeostatic level. This classificatory model is a three-level, three-stage alphanumeric coding, designed as follows:

Level A: Heart Rate (bpm)

Stage A1: Heart Rate less than 70 bpm Stage A2: Heart Rate between 70 and 85 bpm Stage A3: Heart Rate above 85 bpm Level B: RMSSD (ms) Stage B1: RMSSD above 32 milliseconds. Stage B2: RMSSD between 32 and 28 milliseconds. Stage B3: RMSSD less than 28 milliseconds.

Level C: HF ms2

Stage C1: HF ms2 above 468 ms2

Stage C2: HF ms2 between 468 and 156 ms2 .

Stage C3: HF ms2 less than 156 ms2 .

Thus, a totally healthy individual, with an excellent Homeostatic Level and, therefore, with very low risk, would receive the A1B1C1 classification. An individual with a

**Figure 8.** *Set of possibilities in the alphanumeric classification of the individual homeostatic level (Created by the authors).*

high basal heart rate, a very low RMSSD value and a very low HF power value would be classified as A3B3C3 indicating high severity, low homeostatic level and, therefore, at high risk. Several intermediate combinations would be possible characterizing the current state of each case. The figure below illustrates the full set of possibilities (**Figure 8**).

In conclusion, the present analytical study, based on an extensive amount of data published in the literature (more than 10.5 million values), referring to three recognized variables of heart rate variability markers of the level of homeostasis, allowed us to define cut-off levels indicative of apparently healthy or with important homeostatic compromise. It was possible to conclude that values obtained in the general population are not equivalent to normal values, a fact that must be considered when this group is used as a control. It was also possible, to elaborate a very simple alphanumeric classification with practical applicability in the characterization of the individual homeostatic level.


*Candida albicans***: A Major Fungal Pathogen of Humans**

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This is a reprint of articles from the Special Issue published online in the open access journal *Pathogens* (ISSN 2076-0817) (available at: https://www.mdpi.com/journal/pathogens/special issues/Pathogen Candida albicans).

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**2. Plant cryopreservation methods**

In the last three decades a number of different cryopreservation protocols, such as classical slow-cooling, vitrification, droplet vitrification, encapsulation/dehydration and encapsulation/vitrification protocols have been developed and utilised for germplasm storage (Reed, 2008). The choice of cryopreservation method to attain the highest survival rates is largely dependent on the plant species and tissue type that is being cryostored.

#### **2.1 Slow cooling or controlled rate cooling**

This technique involves the simple dehydration of plant material before cryogenic storage in LN. This is can be done by slow cooling of the plant tissue to a temperature of approximately -40°C (Reinhoud et al., 2000). This forces the formation of extracellular ice ahead of intracellular ice, thus causing an outflow of water from the cells due to the resulting osmotic imbalance and, consequently, dehydration. Dehydration can also be brought about by incubation of tissue material on media containing a relatively high concentration of an osmoregulant, commonly sucrose, although other compounds can also be used (Panis et al., 2002). Usually water concentrations must be decreased to between 10% and 20% of the fresh weight for optimal cryogenic survival (Engelmann, 2004). This has the aforementioned effects of reducing the extent of ice crystal formation due to the reduced water concentration and assisting in the achievement of the vitrified state of water as a result of the increased solute concentration. These techniques do not necessarily make use of cryoprotective agents (CPAs), however they can be used in conjunction with them to further improve dehydration (Reinhoud et al., 2000), though these agents can be toxic to plant cells at high concentrations (Arakawa et al., 1990). Rapid re-warming rates are used after cryogenic storage to prevent ice crystal formation during thawing (Reinhoud et al., 2000). This approach can result in extreme rates of dehydration, which can cause cell volume reductions that are potentially lethal (Day et al., 2008). It has been suggested that slow-cooling is only suitable for non-organised tissues, as sufficient dehydration is more difficult to achieve in tissues with complex structures due to the different rates of water movement between and within plant cells with different characteristics (Gonzalez-Arnao et al., 2008).


2.3.3. Identification of OCT2 808G>T

Candidate SNP (808G>T) in the *OCT2* gene was genotyped using pyrosequencing analysis. Each primer was prepared according to the variation of allele. Forward primer of 5′ -CGGAGAACAGT GGGGATTTTTTAC-3′ , reverse primer of 5′ -CACGTAATTCCTTCCGTCTGAAGA-3′ , and sequencing primer of 5′ -GGTGGTTGCAGTTCACA-3′ were used for *OCT2* 808G>T genotype. Forward primer has a 5′ biotin triethylene glycol label necessary for post PCR processing. PCR was performed in 20 µL reaction mixture including 1 µL extracted DNA, 1 µL of 10 pmol each primer (forward and reverse), and 17 µL autoclaved distilled water. The PCR program comprised an initial denaturation at 94 ◦C for 5 min followed by 45 cycles of denaturation at 94 ◦C for 20 s, annealing at 52.1 ◦C for 30 s, and extension at 72 ◦C for 20 s. A final extension step was performed at 72 ◦C for 5 min. The biotinylated PCR products were immobilized on streptavidin-coated Sepharose beads (Amersham Biosciences, Uppsala, Sweden). A total of 37 µL of binding buffer (10 mM Tris HCl, 2 M sodium chloride, 1 mM EDTA, 0.1% Tween 20, pH 7.6; Pyrosequencing AB, Uppsala, Sweden), 3 µL of streptavidin-coated Sepharose beads, and 20 µL of water were added to 20 µL PCR product; then, the solution was vigorously shaken for 10 min at room temperature. A 96 pin magnetic tool (Pyrosequencing AB, Uppsala, Sweden) was used to transfer up

to 96 samples at a time to solutions as follows. The beads with bound template were first transferred to 70% ethanol solution and 0.2 N sodium hydroxide solution, then to 1X washing buffer (Pyrosequencing AB, Uppsala, Sweden), and finally into a solution of 1X annealing buffer (20 mM Tris acetate, 2 mM magnesium acetate, pH 7.6), including the appropriate sequencing primer of 10 pmol. Lastly, this mixture was heated for 1 min to 95 ◦C and then cooled to 50 ◦C and incubated at room temperature for at least 5 min to bind the sequencing primer to the template. After template preparation, a 96-well plate including the samples was loaded into the instrument (PSQ 96MA; Pyrosequencing AB, Uppsala, Sweden) along with the optimal reagents (Pyro Gold; Biotage AB, Uppsala, Sweden). This instrument sequences the templates by dispensing reagents and deoxynucleotide triphosphates in a user-defined order, achieving real-time sequencing by synthesis in an automated fashion. This is achieved by creating and monitoring an enzyme cascade initiated by nucleotide incorporation that produces light emission. Pyrosequencing data were obtained by using Peak Height Determination Software v2.1 (Pyrosequencing AB, Uppsala, Sweden).

#### 2.3.4. Identification of PEPT1 1287G>C (in Exon 16)

Candidate SNP (1287G>C) in the *PEPT1* gene were genotyped by using PCR-RFLP. Each primer was prepared according to the variation of allele. Forward primer of 5′ -CCCTTGTCAGGGTTAAGATGA-3′ and reverse primer of 5′ -GCTTCTCTAAATCCTATTATAACAGGG-3′ were used for *PEPT1* 1287G>C genotypes. PCR was performed in 20 µL reaction mixture including 1 µL extracted DNA, 1 µL of 10 pmol each primer (forward and reverse), and 17 µL autoclaved distilled water. The PCR program comprised of an initial denaturation at 95 ◦C for 5 min followed by 35 cycles of denaturation at 95 ◦C for 20 s, annealing for at 54.5 ◦C 30 s, and an extension at 72 ◦C for 20 s. The final extension step was performed at 72 ◦C for 5 min. DNA fragments amplified by PCR were reacted at 37 ◦C for 1 h with restriction enzyme *Sau961*, which can recognize and cleave specific sequences. The digested fragments were separated by electrophoresis in 2.5% agarose gel, and were visualized under ultraviolet light after staining the gel with ethidium bromide for 30 min.

#### *2.4. Determination of Plasma Tiropramide Concentrations*

Plasma concentrations of tiropramide were determined using a validated column-switching semi-micro high-performance liquid chromatography (HPLC) method based on a previous study [12].

#### 2.4.1. Chromatographic Conditions

The analytical system consisted of the Nanospace SI-2 series (Shiseido, Tokyo, Japan) with an ultraviolet-visible (UV–VIS) detector 3002, two 3001 pumps, a 3014 column oven, a high pressure six-way switching valve 3012, a 3010 degasser, and a 3023 autosampler. The system operation and signal processing were operated by Syscon (Shiseido, Tokyo, Japan). The columns used in this on-line extraction system include an analytical column (Capcell Pak C18 UG120, 150 × 1.5 mm I.D. Shiseido), a pre-column (Capcell Pak MF Ph-1, 10 × 4 mm I.D. 5 µm Shiseido), and an enrichment column (Capcell Pak C18 UG120, 35 × 2 mm I.D. Shiseido). The mobile phase for primary separation of tiropramide in the pre-column and concentration in the enrichment column was phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v) with a flow rate of 0.5 mL/min. The mobile phase for analytical column was phosphoric acid–phosphate buffer (50 mM, pH 7.0)-acetonitrile (0.04/59.96/40, v/v/v) with a flow rate of 0.1 mL/min. All the columns were maintained at 25–30 ◦C. The quantification was performed at 230 nm wavelength. The peak with the retention time of tiropramide was verified using a photodiode array detector (2017 Diode Array, Shiseido, Tokyo, Japan).

#### 2.4.2. Analytical Procedures

The performance of column-switching semi-micro HPLC consists of three steps, as follows: sample loading and primary separation, enrichment of analyte fraction, and chromatographic separation. When the column-switching valve was at the precolumn inlet position, an aliquot of filtered (by 0.22 µm, Millex-GV syringe filter unit, Millipore, Burlington, MA, USA) plasma sample (80 µL) was loaded to the pre-column, and a primary separation of tiropramide from plasma proteins was conducted by using phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v). Subsequently, the valve was switched to the enrichment column inlet position, and the tiropramide fraction was eluted from the pre-column and concentrated in the enrichment column by phosphate buffer (50 mM, pH 7.0)-acetonitrile (88/12, v/v). Afterwards, the valve was switched to analytical column inlet position, and tiropramide was finally isolated and quantified by phosphoric acid–phosphate buffer (50 mM, pH 7.0)-acetonitrile (0.04/59.96/40, v/v/v).


**2. Putative neuroplastic mechanisms of pharmacotherapy**

Pharmacotherapy constitutes first-line treatment modality for majority of psychiatric disorders, and various theories exist as to how drug-induced neurochemical changes reverse different psychiatric symptoms. The posited purely neurotransmitter-based mechanism of action postulates either increased or reduced synaptic concentration of a target neurotransmitter that is implicated in a given disorder. This model is challenged by disjunction in the timescale of the onset of neurochemical versus therapeutic effects, wherein the potentiation or attenuation of neurotransmitter function often occurs within hours of administration and the clinical improvement is typically seen days or weeks after [3]. In quest of new rapidacting agents, contemporary approaches to understanding of drug action focus on the role of adaptive neuroplastic processes that correlate in time with the onset of clinical improvement, hence are hypothesized to represent a more direct treatment target.


**Preface**

In recent years, the biomedical engineering field has seen remarkable advancements, focusing mainly on developing novel solutions for enhancing tissue regeneration or improving therapeutic outcomes. One of the key components of the latest developments is polymers (natural, synthetic, or blended formulations) due to their indisputable wide range of properties and functionalities that transform them into ideal materials for designing drug delivery systems or scaffolds for tissue engineering. This reprint, devoted to the topic of "Polymer Materials for Drug Delivery and Tissue Engineering", gathers a multidisciplinary collection of articles that merge the knowledge and expertise of worldwide researchers that present their latest research in the field of polymer-based materials. This reprint also brings together articles that report original research on the design and synthesis of novel drug delivery systems and scaffolds or that provide insights into polymer interaction. Polymers are valuable tools for the development of drug delivery systems and biomimetic scaffolds due to their tailorable design, versatility, attractive physiochemical properties, and excellent biocompatibility. With ongoing advancements in polymer science and personalized medicine, polymers hold great promise in revolutionizing the current landscape of the biomedical field, providing a real opportunity for designing personalized biomedical products that can be easily adjusted to meet patients' needs.

This reprint, entitled "Polymer Materials for Drug Delivery and Tissue Engineering", is a tribute to all the dedicated scientists engaged in the field of polymer materials for drug delivery and tissue engineering applications. The editors express their gratitude to all the contributing authors of this Special Issue as their insightful research and commitment to advancing the field have not only elevated the quality of our Special Issue but also contributed significantly to the broader scientific community. Also, we acknowledge all the members of the *Polymers* Editorial Office for their indispensable support and invaluable work in the development of this Special Issue.

> **Ariana Hudita and Bianca Gˇalˇat¸eanu** *Editors*


Dietary Antioxidants in the Chemoprevention of Prostate Cancer

*Dwayne Tucker, Melisa Anderson, Fabian Miller, Kurt Vaz, Lennox Anderson-Jackson and Donovan McGrowder*

## **Abstract**

Prostate cancer is the second most common cancer and the fifth leading cause of cancer death. The incidence of prostate cancer is rising due to increased screening and awareness, and there is epidemiological evidence suggesting an interaction among biological and environmental risk factors in the development and progression of prostate cancer. Vegetables and fruits provide a wide range of antioxidants and phytochemicals that have been demonstrated to have a negative, positive, or no association with prostate cancer risk. Therefore, it is evident that the effect of dietary antioxidants on risk of prostate cancer remains undecided and inconclusive. The main focus of this review was to examine recent and past literature of the chemoprotective properties of five major groups of phytochemicals against prostate cancer development including both *in vivo* and *in vitro* findings.

**Keywords:** antioxidants, prostate, cancer, risk, association

#### **1. Introduction**

Among men worldwide, prostate cancer is the second most common cancer and the fifth leading cause of cancer death, with an estimated recorded amount of 1.3 million cases and 359,000 deaths in 2018 [1]. The incidence of prostate cancer is rising due to increased awareness and screening, and it is estimated that 42% of prostate cancer cases occur in men over 50 years old [2]. There is epidemiological proof that suggests an interaction among several known biological and environmental risk factors in the development and progression of prostate cancer [3]. These include age, race, family history, genetic risk, socioeconomic status, and modifiable risk factors such as physical activity, obesity, and possibly dietary factors [4].

Oxidative stress defined as an imbalance between prooxidant and antioxidant processes, and interference of the oxidation-reduction circuitry is one of the many proposed underlying mechanisms of prostate carcinogenesis [5, 6]. There is increasing epidemiological data that diet plays a key role in the biology and tumorigenesis of prostate cancer, and higher intake of the main phytochemical-containing diets lowers the risk of the disease [7]. Vegetables and fruits provide a wide range of phytochemicals and antioxidants that have been demonstrated to have a positive effect on decreasing the incidence or averting the occurrence of prostate cancer [8]. Several of these antioxidants may attenuate prostate cancer development, given that oxidative stress from reactive oxygen species and loss of antioxidant enzymes may contribute to genomic instability prior to prostate cancer [9].

This paper will review information in the literature on the relationship between nutrients with antioxidant properties from the diet, and the risk of prostate cancer.

## **2. Method of article selection**

A literature search was conducted for all English language literature published before December 2018. The search was conducted using the electronic databases, including PubMed, Embase, Web of Science, and Cochrane Library. The search strategy included keywords such as prostate cancer, epidemiology, incidence, mortality, risk factor, selenium, vitamin E, vitamin C, carotenoids, and polyphenols.

The authors include many interventional and observational studies that have reported findings of dietary antioxidants, prostate cancer incidence, and progression. The majority of these studies focused on vitamins E and C, carotenoids, specifically beta- and alpha-carotene and lycopene, phenols including tea and coffee, and the flavonoids, as well as selenium.

#### **3. Vitamin E and prostate cancer**

Vitamin E is a potent lipid-soluble antioxidant, which is well recognized for safeguarding the body against free radical-mediated peroxidative damage. It is a naturally occurring essential vitamin mainly found in foods such as nuts, oils, fruits, and vegetables and is available as a dietary supplement. Vitamin E scavenges highly reactive free radical species such as hydroxyls, superoxides, lipid peroxyls, hydroperoxyls, and nitrogen radicals; and prevents lipid peroxidation related to carcinogen-induced DNA damage [10].

It is known that a deficient antioxidant defense system can result in oxidative stress. As such, increased levels of reactive oxygen species over time may have an etiological role in the development of malignancies such as prostate cancer [11]. Vitamin E may therefore be considered as adjuvant therapy for the prevention of prostate cancer [12]. However, despite emerging evidence supporting vitamin E as a powerful antioxidant, its effect on prostate cancer risk remains poorly understood.

Two categories of vitamin E compounds exist: tocopherols (α, β, γ, and δ-Toc) and tocotrienols (α, β, γ, and δ-T3) [12]. Despite structural differences between both categories, tocopherols and tocotrienols each have sufficient antioxidant properties [12].

#### **3.1 Alpha-, gamma-, and delta-tocopherols**

Alpha-tocopherol accounts for the most abundant and active isoform of vitamin E in human tissues and is the most widely used in dietary supplements [10]. Alphatocopherol terminates free radical chain reactions by transferring hydrogen protons to free radicals yielding nonradical products [13]. Fairly stable alpha-tocopheroxyl radicals are generated, which do not react with polyunsaturated fatty acids but with each other or couples with other free radicals to form nonradical products [13]. The generation of nonradical products by vitamin E may therefore provide a protective effect against free radical-mediated cell membrane damage and consequently reduces mutagenesis and carcinogenesis.

A number of studies have reported findings on vitamin E supplementation (alpha-, gamma-, and delta-tocopherols) and risk of prostate cancer [14–18].

**221**

*Dietary Antioxidants in the Chemoprevention of Prostate Cancer*

Notably, in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC), daily supplementation of alpha-tocopherol (50 mg) reduced the risk of prostate cancer [17] and moderate dose decreased posttrial mortality [15]. However, a follow-up of the Physicians Health Study II, a large-scale randomized trial, suggested that vitamin E supplementation had no immediate or long-term effect on the incidence of prostate cancer (HR 0.99; 95% CI: 0.89–1.10) [14]. Conversely, findings from the large-scale selenium and vitamin E cancer prevention trial (SELECT) demonstrated that the risk of prostate cancer was significantly increased with dietary vitamin E supplementation containing alpha-tocopherol [16]. However, it was found that the incidence of prostate cancer did not increase in men who received combination therapy of vitamin E and selenium [19]. As such, it can be speculated that there may be a synergistic effect between both antioxidants which attenuates prostate cancer risk [19]. The increased risk of the disease associated with vitamin E therapy could be attributed to the disturbance of the normal physiological balance of vitamin E isomers by the high dosage of alpha-tocopherol, which may result in depletion of other important isomers such as gamma-tocopherol [20]. Studies have supported that gamma-tocopherol may have more superior chemopreventive effects than alpha-tocopherol, considering its stronger antiinflammatory and antinitrative effects [12]. However, it is important to note that analysis of 15 prospective studies involving data for prostate cancer cases and controls and using risk estimation by multivariable-adjusted conditional logistic regression found that gamma-tocopherol was not associated with risk of aggressive prostate cancer, and the latter was inversely associated with alpha-tocopherol [21]. As such, it was suggested that the protective effect against prostate cancer may be lost with impaired balance of vitamin E isomers [20]. Findings from the SELECT trial were later recapitulated, as alpha-tocopherol was found to upregulate prostate cancer cell proliferation in the early stages of the disease [22]. It was found that premalignant rather than benign or malignant prostate cells had increased proliferation in response to vitamin E [22]. These data indicate that the effect of vitamin E antioxidant activity may be dependent on the stage of the prostate cells in the tumor development process [22]. Conversely, it was later found that combination therapy of delta-tocotrienol and gamma-tocopherol was efficacious in inhibiting the proliferation of prostate cancer cells by apoptosis and cell cycle arrest in the G1

A recent study conducted on mice revealed that delta-tocopherol and not alphatocopherol blocks the activation of the Akt pathway which drives tumorigenesis, inhibiting the survival of prostate cancer cells [23]. Another study which supports the chemopreventative activity of delta-tocopherol is that of Wang et al. which reported a novel mechanism by which this antioxidant inhibits prostate cancer cell growth by the attenuation of EGF/IGF-induced activation of Akt on T308 [24]. In examining the efficacy of other tocopherol, gamma-tocopherol (0.3% in diet) supplementation was found to significantly reduce the development of mouse prostatic intraepithelial neoplasia lesions and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced elevation of nitrotyrosine, 8-oxo-deoxyguanosine, p-Akt,

There is supporting evidence that gamma-tocopherol significantly inhibits the growth of human prostate PC-3 tumor cell line by decreasing progression into the S-phase, upregulation of transglutaminase 2 and downregulation of (TG2), and downregulation of cyclin D1 and cyclin E levels [26]. These findings suggest that different isoforms of vitamin E may differ in their influence on prostate cancer risk and that alpha-tocopherol supplementation alone may increase the risk of the disease. It was reported that the association between vitamin E and prostate cancer risk may be linked to genetic variation in genes that regulate antioxidant and vitamin E

*DOI: http://dx.doi.org/10.5772/intechopen.85770*

and G2/M phases of the cell cycle [12].

Ki-67 and COX-2, and the loss of Nrf2 and PTEN [25].

#### *Dietary Antioxidants in the Chemoprevention of Prostate Cancer DOI: http://dx.doi.org/10.5772/intechopen.85770*

*Antioxidants*

**2. Method of article selection**

coffee, and the flavonoids, as well as selenium.

**3. Vitamin E and prostate cancer**

carcinogen-induced DNA damage [10].

**3.1 Alpha-, gamma-, and delta-tocopherols**

reduces mutagenesis and carcinogenesis.

oxidative stress from reactive oxygen species and loss of antioxidant enzymes may

This paper will review information in the literature on the relationship between nutrients with antioxidant properties from the diet, and the risk of prostate cancer.

A literature search was conducted for all English language literature published before December 2018. The search was conducted using the electronic databases, including PubMed, Embase, Web of Science, and Cochrane Library. The search strategy included keywords such as prostate cancer, epidemiology, incidence, mortality, risk factor, selenium, vitamin E, vitamin C, carotenoids, and polyphenols. The authors include many interventional and observational studies that have reported findings of dietary antioxidants, prostate cancer incidence, and progression. The majority of these studies focused on vitamins E and C, carotenoids, specifically beta- and alpha-carotene and lycopene, phenols including tea and

Vitamin E is a potent lipid-soluble antioxidant, which is well recognized for safeguarding the body against free radical-mediated peroxidative damage. It is a naturally occurring essential vitamin mainly found in foods such as nuts, oils, fruits, and vegetables and is available as a dietary supplement. Vitamin E scavenges highly reactive free radical species such as hydroxyls, superoxides, lipid peroxyls, hydroperoxyls, and nitrogen radicals; and prevents lipid peroxidation related to

It is known that a deficient antioxidant defense system can result in oxidative stress. As such, increased levels of reactive oxygen species over time may have an etiological role in the development of malignancies such as prostate cancer [11]. Vitamin E may therefore be considered as adjuvant therapy for the prevention of prostate cancer [12]. However, despite emerging evidence supporting vitamin E as a powerful antioxidant, its effect on prostate cancer risk remains poorly understood. Two categories of vitamin E compounds exist: tocopherols (α, β, γ, and δ-Toc) and tocotrienols (α, β, γ, and δ-T3) [12]. Despite structural differences between both categories, tocopherols and tocotrienols each have sufficient antioxidant

Alpha-tocopherol accounts for the most abundant and active isoform of vitamin E in human tissues and is the most widely used in dietary supplements [10]. Alphatocopherol terminates free radical chain reactions by transferring hydrogen protons to free radicals yielding nonradical products [13]. Fairly stable alpha-tocopheroxyl radicals are generated, which do not react with polyunsaturated fatty acids but with each other or couples with other free radicals to form nonradical products [13]. The generation of nonradical products by vitamin E may therefore provide a protective effect against free radical-mediated cell membrane damage and consequently

A number of studies have reported findings on vitamin E supplementation (alpha-, gamma-, and delta-tocopherols) and risk of prostate cancer [14–18].

contribute to genomic instability prior to prostate cancer [9].

**220**

properties [12].

Notably, in the Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC), daily supplementation of alpha-tocopherol (50 mg) reduced the risk of prostate cancer [17] and moderate dose decreased posttrial mortality [15]. However, a follow-up of the Physicians Health Study II, a large-scale randomized trial, suggested that vitamin E supplementation had no immediate or long-term effect on the incidence of prostate cancer (HR 0.99; 95% CI: 0.89–1.10) [14]. Conversely, findings from the large-scale selenium and vitamin E cancer prevention trial (SELECT) demonstrated that the risk of prostate cancer was significantly increased with dietary vitamin E supplementation containing alpha-tocopherol [16]. However, it was found that the incidence of prostate cancer did not increase in men who received combination therapy of vitamin E and selenium [19]. As such, it can be speculated that there may be a synergistic effect between both antioxidants which attenuates prostate cancer risk [19]. The increased risk of the disease associated with vitamin E therapy could be attributed to the disturbance of the normal physiological balance of vitamin E isomers by the high dosage of alpha-tocopherol, which may result in depletion of other important isomers such as gamma-tocopherol [20].

Studies have supported that gamma-tocopherol may have more superior chemopreventive effects than alpha-tocopherol, considering its stronger antiinflammatory and antinitrative effects [12]. However, it is important to note that analysis of 15 prospective studies involving data for prostate cancer cases and controls and using risk estimation by multivariable-adjusted conditional logistic regression found that gamma-tocopherol was not associated with risk of aggressive prostate cancer, and the latter was inversely associated with alpha-tocopherol [21]. As such, it was suggested that the protective effect against prostate cancer may be lost with impaired balance of vitamin E isomers [20]. Findings from the SELECT trial were later recapitulated, as alpha-tocopherol was found to upregulate prostate cancer cell proliferation in the early stages of the disease [22]. It was found that premalignant rather than benign or malignant prostate cells had increased proliferation in response to vitamin E [22]. These data indicate that the effect of vitamin E antioxidant activity may be dependent on the stage of the prostate cells in the tumor development process [22]. Conversely, it was later found that combination therapy of delta-tocotrienol and gamma-tocopherol was efficacious in inhibiting the proliferation of prostate cancer cells by apoptosis and cell cycle arrest in the G1 and G2/M phases of the cell cycle [12].

A recent study conducted on mice revealed that delta-tocopherol and not alphatocopherol blocks the activation of the Akt pathway which drives tumorigenesis, inhibiting the survival of prostate cancer cells [23]. Another study which supports the chemopreventative activity of delta-tocopherol is that of Wang et al. which reported a novel mechanism by which this antioxidant inhibits prostate cancer cell growth by the attenuation of EGF/IGF-induced activation of Akt on T308 [24]. In examining the efficacy of other tocopherol, gamma-tocopherol (0.3% in diet) supplementation was found to significantly reduce the development of mouse prostatic intraepithelial neoplasia lesions and 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine-induced elevation of nitrotyrosine, 8-oxo-deoxyguanosine, p-Akt, Ki-67 and COX-2, and the loss of Nrf2 and PTEN [25].

There is supporting evidence that gamma-tocopherol significantly inhibits the growth of human prostate PC-3 tumor cell line by decreasing progression into the S-phase, upregulation of transglutaminase 2 and downregulation of (TG2), and downregulation of cyclin D1 and cyclin E levels [26]. These findings suggest that different isoforms of vitamin E may differ in their influence on prostate cancer risk and that alpha-tocopherol supplementation alone may increase the risk of the disease.

It was reported that the association between vitamin E and prostate cancer risk may be linked to genetic variation in genes that regulate antioxidant and vitamin E metabolism [18, 27]. Furthermore, it was found that genetic variation in SOD genes responsible for detoxifying superoxide free radicals and protecting cells from oxidative stress may be associated with an increased risk of high-grade prostate cancer and disease recurrence [18]. Similarly, it was shown that single nucleotide polymorphisms (SNPS) in genes associated with vitamin E metabolism such as SEC14L2, SOD1, and TTPA may influence an individual's response to vitamin E supplementation and associated prostate cancer risk [28]. As such, inherited genotypes may confer prostate cancer risk.

It is therefore anticipated that clinical trials will be undertaken with vitamin E isomers combination therapy for further assessment of prostate cancer risk. It may be useful to conduct more studies including isomers other than alpha-tocopherol. Men with a strong family history of prostate cancer should undergo genetic testing, to identify antioxidant gene mutations that may be implicated in prostate cancer.


**Author details**

Kadarkarai Kirupa\*, R. Rajashri, Kamali Raman, Aishwarya Balaji, Pavithra Elango, Swetha Karupaiah and Leelavathy Gopalakrishnan DR. M.G.R. Education and Research Institute, India

\*Address all correspondence to<EMAIL_ADDRESS>
© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


**Purification E**ff**ect of Sequential Constructed Wetland for the Polluted Water in Urban River**

#### **Xueyuan Bai 1, Xianfang Zhu 2, Haibo Jiang 1, Zhongqiang Wang 1, Chunguang He 1,\*, Lianxi Sheng 1,\* and Jie Zhuang 3**

Received: 19 February 2020; Accepted: 3 April 2020; Published: 8 April 2020

**Abstract:** Constructed wetlands can play an active role in improving the water quality of urban rivers. In this study, a sequential series system of the floating-bed constructed wetland (FBCW), horizontal subsurface flow constructed wetland (HSFCW), and surface flow constructed wetland (SFCW) were constructed for the urban river treatment in the cold regions of North China, which gave full play to the combined advantages. In the Yitong River, the designed capacity and the hydraulic loading of the system was 100 m3/d and 0.10 m3/m2d, respectively. The hydraulic retention time was approximately 72 h. The monitoring results, from April to October in 2016, showed the multiple wetland ecosystem could effectively remove chemical oxygen demand (COD), ammonia nitrogen (NH4 +-N), total nitrogen (TN), total phosphate (TP), and suspended solids (SS) at average removal rates of 74.79%, 80.90%, 71.12%, 78.44%, and 91.90%, respectively. The removal rate of SS in floating-bed wetland was the largest among all the indicators (80.24%), which could prevent the block of sub-surface flow wetland effectively. The sub-surface flow wetland could remove the NH4-N, TN, and TP effectively, and the contribution rates were 79.20%, 64.64%, and 81.71%, respectively. The surface flow wetland could further purify the TN and the removal rate of TN could reach 23%. The total investment of this ecological engineering was \$12,000. The construction cost and the operation cost were \$120 and \$0.02 per ton of polluted water, which was about 1/3 to 1/5 and 1/6 to 1/3 of the conventional sewage treatment, respectively. The results of this study provide a technical demonstration of the restoration of polluted water in urban rivers in northern China.

**Keywords:** polluted urban river; sequential constructed wetlands; purification effect; water restoration; Yitong River

#### **1. Introduction**

In recent decades, with the rapid development of urban economies and urbanization, urban rivers all over the world have been facing the threat of pollution. Japan, the United States, and some European countries began to implement water purification of urban rivers in the 1950s and 1960s [1]. However, the research and application of further technology was only carried out in the past 20 to 30 years [2,3]. At present, river water purification technologies can be categorized into physical, chemical, biological, and ecological methods. Physical methods include aeration [4] and sediment dredging [5–7]. Chemical methods include chemical precipitation [8] and the application of chemical

algaecide [9]. Biological methods include bioremediation [10], biofilms [11,12], contact oxidation [13–15], and membrane bioreactor technology [16,17]. Ecological methods include ecological ponds [18,19], plant purification treatment [20,21], ecological floating beds [22–24], and constructed wetlands [25–27]. Among these methods, aeration is the easiest, fastest, most effective, and most widely applied. It has been applied to the restoration of the Emscher River in Germany, the Homewood Canal in the United States, and the Thames River in the United Kingdom [28]. However, the operation and maintenance costs are high. Sediment dredging can remove endogenous pollution sources and is also a widely applied method to improve the water environment. There are problems associated with this method, such as the large amount of construction work, challenges to processing the sludge, and the additional pollution created by improper dredging [29,30]. Physical and chemical methods are often used only as secondary approaches to mediate pollution or to treat emergent water pollution. Although biological methods effectively clean up pollution and have low energy consumption requirements and small environmental impacts, they also have issues such as time-consuming processes to cultivate microorganisms and purification that is subject to restrictions from external conditions such as the temperature and water flow. Biofilm technology requires large-scale construction and involves land use issues. Constructed wetlands, on the other hand, use physical, chemical, and biological synergies in an ecosystem to efficiently remove pollutants by simulating the natural environment [31]. The operating costs are low, and they are easy to maintain and manage without secondary pollution. They also offer ecological benefits: they have been demonstrated to be an economical and efficient sewage treatment and management method [32], and they have become a preferred ecological method to improve the water quality of rivers in cities around the world. Therefore, the development of optimized functions of constructed wetlands that suit different goals and requirements has been a focal area of research. However, the ecological ponds occupy a large area of land, which is easily affected by environmental conditions, and the removal effect of nitrogen and phosphorus is not stable. The plant purification treatment and ecological floating beds only rely on aquatic plants to absorb nitrogen, phosphorus, and organic matter; thus, the capacity is limited. The constructed wetlands have disadvantages for purifying water because of the large area and low hydraulic load for the surface flow constructed wetland (SFCW); moreover, the horizontal subsurface flow constructed wetland (HSFCW) has a poor resistance to shock loads and is easily blocked. Therefore, the river water purification is a complex system engineering, and needs the joint application of various ecological technologies. The Yitong River is the mother river of Changchun, a capital city in northeast China. To create a water landscape for the city, multiple dams have been constructed to reserve water on the river section within Changchun. As a result, the flow rate has become slow, the dissolved oxygen (DO) level has decreased sharply, the river's self-purification capacity has been lost, and the water quality has begun to deteriorate. Eutrophication is especially severe in seasons with high temperatures. This situation has brought hidden risks to the water environment and the ecological security of the areas of Changchun near the lower reaches of the river, which severely affects the surrounding ecological environment and the daily life of nearby residents and restricts the sustainable development of the urban waters.

In this paper, we built a sequential combination system of the floating-bed constructed wetland (FBCW) + horizontal subsurface flow constructed wetland (HSFCW) + surface flow constructed wetland (SFCW) to purify the polluted water of Yitong River, using the favorable terrain of the park on the shore. This system was continuously monitored for one year and the results were analyzed. They can provide necessary data support for the popularization and application of the technology.

#### **2. Method**

#### *2.1. Study Area*

The study area is along the Yitong River in the city of Changchun, Jilin Province, northeast China (Figure 1, 43◦50 32" N, 125◦21 32" E), which has a temperate continental monsoon climate zone. The annual rainfall in the study area is 600 to 700 mm. The average annual temperature is 4.6 ◦C. The maximum temperature in the summer is 40 ◦C. The annual freezing period is 5 months.

**Figure 1.** Location and overview of the study area.

The water quality was monitored in the study area from April to October 2014. Water quality data are shown as averages ± standard deviations (Table 1). The average chemical oxygen demand (COD) and ammonia nitrogen (NH4 +-N) concentrations were 57.65 mg/L and 5.60 mg/L, respectively, which were both beyond the lower limits of Class V water in the Environmental Quality Standards for Surface Water (GB3838-2002) [33]. Therefore, the water quality was classified as worse than Class V.

**Table 1.** Results of water quality monitoring in the study area of the Yitong River.

COD—chemical oxygen demand; NH4 +-N—ammonia nitrogen; TN—total nitrogen; TP—total phosphate; SS—suspended solids; DO—dissolved oxygen.

#### *2.2. Ecological Engineering Design*

The engineering process is shown in Figure 2. The wetland system was composed of FBCW, HSFCW, and SFCW in sequence. Firstly, the water of the Yitong River was introduced into FBCW by gravity from the upstream rubber. The FBCW mainly realized the pre-treatment function, introducing the adsorption carrier and suspended biological filler, which attached a large number of microbial flora, and formed a collaborative purification system of aquatic plants, porous substrate, and biofilm. This is to improve the degradation efficiency of organics, nitrogen, and phosphorus, reduce the pollution load for follow-up HSFCW, and improve the impact resistance of the system. Additionally, the FBCW can remove suspended solids (SS) effectively and reduce the risk of clogging of the HSFCW. The FBCW used the terrain elevation difference to design a multi-level water drop for reaeration, and created favorable conditions of the denitrification for HSFCW. The HSFCW was the core of the system, and formed the aerobic-anoxic-anaerobic environment, which contributed to the smooth progress of nitrification and denitrification, and thus, could effectively promote the removal of nitrogen in sewage. Falling water and reoxygenation by FBCW improved the concentration of DO, which was conducive to the transformation of NH4 +-N and improved the nitrification reaction capacity in the front end of HSFCW. However, in the middle and back end of HSFCW, the anoxic and anaerobic environment, which for the DO was lower, could promote the denitrification process. Then, the effluent from the HSFCW entered the SFCW. The SFCW was rich in microtopography, which could improve the DO, further removed the pollutants of nitrogen and phosphorus, and provided a safety guarantee for the effluent reaching the standard. The effluent of SFCW flowed back to Yitong River. The system has played an effective collaborative role of FBCW, HSFCW, and SFCW, which reduced the risk of wetland blocking and improved the antipollution load capacity and operation stability. At last, the system improved the efficiency of pollutant purification.

**Figure 2.** Processing flow and configuration.

The designed water volume was 100 m3/d. According to the water quality monitoring results in 2014, the design of influent water was COD ≤ 70 mg/L, NH4 +-N ≤ 8.0 mg/L, SS ≤ 60 mg/L. Therefore, the design of effluent water was COD ≤ 40 mg/L, NH4 +-N ≤ 2.0 mg/L, SS ≤ 6.0 mg/L. The total surface hydraulic loading was approximately 0.10 m3/m2d. The total area of the wetlands was 1000 m2. The hydraulic retention time was 3 d. Among them, the surface hydraulic loading of FBCW, HSFCW, and SFCW was 0.42 m3/m2d, 0.50 m3/m2d, and 0.18 m3/m2d, respectively. The area of FBCW, HSFCW, and SFCW was 240 m2, 200 m2, and 560 m2, respectively. The hydraulic retention time of FBCW, HSFCW, and SFCW was 0.83 d, 1.17 d, and 1.0 d, respectively.

The FBCW comprised 25 floating beds, with 3.00 × 2.00 m per floating bed. The total area of floating beds was 150 m2, and a coverage rate was 62.5% in FBCW. The top of the floating beds was set up with 3000 planters with diameters of 10cm and heights of 30 cm (20 planters/m2). They were filled with absorbent volcanic rocks as the substrate to a height of 25 cm. Two water spinach plants were planted in every planter. AquaMats (Hangzhou Zijing Envrionmental Engineering CO.,LTD., Hangzhou, China), a biofiltration media, were hung 20 cm apart from one another under the floating beds. The height of the media was 30 cm.

The depth of the HSFCW was 1.2 m. The SSFCW was filled with natural volcanic rocks with a particle size of 10–20 cm and a filling height of 1.0 m. *Lythrum salicaria L.*, a wetland plant, was planted in a density of 25 clumps/m2 and three plants/clump. The distribution zone and the catchment zone were established front and after end in the wetland to ensure the even distribution of water in the system and to maximize the wetland efficiency.

The designed depth of SFCW was 0.1 m to 0.8 m. The SFCW was comprised of shallow waters, deep pools, sandbars, and ecological islands, with *Lythrum salicaria L.*, *Iris pseudacorus L.*, *Salix integra*, and other wetland plants. It was also supplemented by ecological bag revetment and slope bank with vegetation.

#### *2.3. Data Collection and Analysis*

The water quality was continuously monitored from April to October 2016. Samples were taken from 9:00–11:00 on the 17th of each month. Sampling staff used clean plastic samplers to take water samples at the inflow and outflow of each wetland along the direction of the flow. The sampling points are shown in Figure 2: sampling point A (system inflow), sampling point B (outflow of the FBCW), sampling point C (outflow of the HSFCW), and sampling point D (outflow of SFCW). Three samples of 500 mL water were taken in clean plastic sampling bottles at each sampling point as duplicate samples. A water quality analysis of the samples was conducted within 24 h. The main monitoring indicators included pH, SS, DO, COD, NH4 +-N, total nitrogen (TN) and total phosphate (TP). The methods were based on the Water and Wastewater Monitoring and Analysis Method (Fourth Edition) [34].

#### **3. Results**

The system was operated from April to June in 2016. The effectiveness of the removal of various pollutants gradually stabilized, and the water quality of the system improved significantly. Monitoring data showed that the SS concentration of inflow fluctuated between 46.38 and 89.26 mg/L (the mean was 64.84 mg/L) while the system was operating. The SS concentration of the outflow was 4.35–5.68 mg/L (the mean was 4.90 mg/L). The removal rate of SS was 87.86%–94.80% (the mean was 91.90%). From June to September, the root system of the plants in the floating-bed wetland continued to grow. After the formation of a stable biofilm on the hanging biofiltration media, the filtration by the plant roots and the interception and adsorption of the biofiltration media became prominent. The maximum removal rate of SS was 94.80%. As seen in Figure 3, the average removal rate of SS by the FBCW was 80.24%. The removal of SS in the HSFCW increased by 9.45% on average compared to the outflow of the FBCW. The removal of SS in the SFCW increased by 2.21% on average compared to the outflow of HSFCW. These results indicated that the removal of SS was mainly completed in the FBCW. The HSFCW and the SFCW enhanced the removal of SS.

During the operation of the system, the DO concentration of the inflow was basically maintained at 1.43–1.86 mg/L. The average DO concentration of the system inflow was 1.59 mg/L. The DO concentration of the outflow was 3.86–5.58 mg/L. The average DO concentration of the outflow was 4.79 mg/L. The DO concentration of the outflow met the water quality standard of Class IV water in the Environmental Quality Standards for Surface Water (GB3838-2002) [33]. As seen in Figure 4, oxygenation by the waterfalls in the FBCW significantly increased the DO concentration of the outflow. The consumption of the HSFCW brought the DO concentration back down to a lower level at the outflow of the HSFCW. Then, the effect of the SFCW gradually improved the DO concentration of the system outflow. The DO concentration in general showed a pattern of first increasing, then decreasing, and increasing again. The DO concentration of the outflow increased by an average of 2.03 times. The DO concentration of the outflow reached a maximum of 5.58 mg/L. The increase in DO was mainly

attributed to the waterfalls in the FBCW. The terrain changes in the SFCW, such as deep ponds, shallow waters, jumps, and waterfalls, adjusted the form and speed of the flow.

**Figure 3.** Concentration of SS in the inflow and outflow and the removal rate.

**Figure 4.** DO concentration of the inflow and the increase rate.

During the operation of the system, the COD concentration of the inflow fluctuated between 62.35 and 83.48 mg/L. The average COD concentration of the system inflow was 71.57 mg/L. The average COD concentration of the outflow was 20.57 mg/L. The COD removal rate of the system was 38.14%–82.05%, and the average removal rate was 74.79%. As seen in Figure 5, the COD removal rate was the lowest in April at 38.14%. It rose to 61.29% in May. With the exception of April, May, and October, when the system just started and reached the end of operation, the COD removal rate could reach more than 80%. The COD concentration of the outflow in June to September met the water quality standard of Class III water in the Environmental Quality Standards for Surface Water (GB3838-2002) [33]. In this system, the contribution of the HSFCW to the COD removal was close to 54.42%. The floating-bed wetland contributed approximately 34.56% to the COD removal. The contribution of the SFCW was only approximately 11.02% because the COD removal by the HSFCW was mainly achieved through microbial degradation and adsorption by plants and filtration media. Compared to the COD removal rate in the HSFCW, the COD removal rate in the FBCW was lower due to the shorter retention time. Additionally, the SFCW was at the downstream end of the system where the COD concentration of

the inflow was lower. In addition, the SFCW had a limited ability to remove pollutants thus, its COD removal rate was not high.

**Figure 5.** COD concentration of the inflow and outflow and the removal rate.

During the operation of the system, the NH4 +-N concentration of the inflow was 5.65–8.26 mg/L. The average NH4 +-N concentration of the inflow was 6.98 mg/L. The average NH4 +-N concentration of the outflow was 1.25–4.66 mg/L, and the average removal rate was 80.90%. As seen in Figure 6, excluding the initial stage in April and May and the late operation season in October, the average NH4 +-N concentration of the outflow from June to September was 1.35 mg/L. The removal rate of NH4 +-N was higher than 78%. The NH4 +-N concentration of the outflow met the water quality standard of Class IV water in the Environmental Quality Standards for Surface Water (GB3838-2002) [33]. The removal of NH4 +-N in the entire wetland system was largely achieved in the HSFCW, which contributed to 79.20% in the system purification.

**Figure 6.** Concentration of NH4 +-Nin the inflow and outflow and the removal rate.

During the operation of the system, the concentration of TN in the inflow was 6.43–8.89 mg/L. The average TN concentration of the system inflow was 7.73 mg/L. The TN concentration in the outflow was 1.75–6.28 mg/L, and the average removal rate was 71.12%. As seen in Figure 7, in April, the TN concentration in the outflow was 6.28 mg/L, and the removal rate was only 19.00%. In May, the TN

concentration in the outflow was 4.16 mg/L, and the removal rate was approximately 35%. From June to September, the average TN concentration of the outflow was 1.83 mg/L, and the removal rate reached approximately 76.75% and was stable. In October, the TN concentration in the outflow was 2.64 mg/L, and the removal rate decreased. The TN removal rate by the FBCW, the HSFCW, and the SFCW was 12.35%, 64.66%, and 23.00%, respectively

**Figure 7.** Concentration of TN in the inflow and outflow and the removal rate.

During the operation of the system, the concentration of TP in the inflow was 1.48–1.69 mg/L. The average TP concentration in the inflow was 1.56 mg/L. The TP concentration in the outflow was 0.32–0.38 mg/L. The average TP concentration in the outflow was 0.35 mg/L. As seen in Figure 8, the removal rate of TP reached an average of 78.39%. The TP concentration in the outflow met the water quality standard of Class V water in the Environmental Quality Standards for Surface Water (GB3838-2002) [33]. The system had a strong removal capacity for TP, which was mainly attributed to the HSFCW. The removal of TP by the FBCW and the SFCW was not significant, with an average removal rate of 12.86% and 5.43%, respectively. This might be related to the form of phosphorus and the mechanism and method of removal. It is generally believed that the removal of phosphorus in artificial wetlands is mainly achieved by adsorption to the substrates. Therefore, it is possible that the substrate structures of the HSFCW played a major role in the adsorption and retention of phosphorus. The FBCW and SFCW only removed a small portion of the suspended phosphorus.

**Figure 8.** Concentration of TP in the inflow and outflow and the removal rate.

#### **4. Discussion**

Due to the cold winter in northern China, artificial wetlands cannot function properly in the winter. As a result, the wetland system stops operating in November every year. It restarts in March to April the next year when the weather becomes warmer again. When the system first started operating, the low temperature inhibited microbial activities, and the plants were not growing yet. The system mainly relied on substrate adsorption and interception to purify the water, and the efficiency of pollutant removal was low. As shown in Figures 5–7, in April, with an average temperature of 3–12 ◦C, the removal rates of COD and NH4 +-N were less than 40%, and the removal rate of TN was only 19.00%. In May, the removal rates of COD, NH4 +-N and TN increased to 61.29%, 55.92%, and 35.30%, respectively, when the average temperature was 12–23 ◦C and the wetland plants started to grow. Between June to September, the temperature gradually rose and the average temperature was 16–29 ◦C, when the plants grew up. As the operating time increased, the operation of the system gradually became stable and the microbial activities increased significantly. The integrated effect of the wetland plants, substrate, and microorganisms was fully functional, thus, the purification efficiency was best. The both removal rates of COD and NH4 +-N were over 80%, when the TN could exceed 75%. After September, the temperature gradually decreased, and the plants stopped growing and started to wither. When the average temperature was 2–4 ◦C in October, the removal rates of COD, NH4 +-N, and TN decreased to 67.14%, 66.39%, and 66.41%, respectively. The average temperature was −7–2 ◦C in November, and the wetland system progressively stopped running. Existing studies have shown that the nitrogen removal in a wetland is achieved by microbial activities, substrate adsorption, and plant absorption [35,36]. Low temperatures seriously affect the efficiency of water purification in artificial wetlands [37,38], because denitrifying microorganisms in artificial wetlands are active only when the temperature is above 15 °C [39,40]. In addition, during the operation of the system from June to September, despite the poor water quality of the inflow, the wetland system still operated stably. The removal rates of COD and NH4+-N still exceeded 75%. The COD concentration of the outflow was ≤20 mg/L, and the NH4+-N concentration of the outflow was ≤1.5 mg/L. The system showed a good effectiveness for pollutant removal.

The purpose of having a combination of different types of wetlands is to balance the advantages and disadvantages of each type of wetland [41]. Such a design can break through the restrictions imposed by the technical characteristics and environmental conditions of a single wetland type, enhance the system's resistance to loading fluctuations, and improve the efficiency of the water treatment [42]. According to a survey of more than 100 constructed wetlands in the United States, nearly half of them became clogged within five years of operation [43]. It is believed that the accumulation of SS is the main cause of blockage in the constructed wetlands [44]. Studies have also shown that horizontal and vertical flow wetlands can remove more than 90% of organic matter and almost all N and P if effective pretreatment facilities are set up prior to the constructed wetlands or if the wetland area is large enough [45]. Compared to a settling basin or an oxidation pond, using a FBCW as a pretreatment unit for the HSFCW can greatly improve the removal rate of SS and the purification efficiency of COD, nitrogen, and phosphorus. It can reduce the pollution load for the subsequent wetland process, improve the resistance of the loading capacity of the entire wetland system, and reduce the risk of clogging the HSFCW. The HSFCW is the core of the multiple wetlands and the key to effective denitrification in the system. The nitrification and denitrification by the microorganisms are the important processes for denitrification in constructed wetlands [46]. Golterman found that there must be both aerobic and anaerobic conditions in a wetland to achieve the nitrification and denitrification processes and the effective removal of nitrogen [47]. The nitrification process is aerobic. NH4 +-N is mainly removed by nitrification. The DO concentration should normally be maintained above 2.0 mg/L for nitrification. Although the FBCW was not efficient in removing NH4 +-N, the waterfalls designed by using the height differences in the terrain enabled oxygenation. This maintained a higher concentration of DO before water went into the HSFCW and promoted nitrification in the upper stream of the HSFCW and the conversion and removal of NH4 +-N in the HSFCW. Denitrification requires anaerobic environments. The DO level in the middle and lower streams of the HSFCW decreased rapidly. A hypoxic or anaerobic environment was formed, which promoted the process of denitrification and achieved an ideal removal of TN from the system. The removal of TN by a single type of wetland is not effective, because it is difficult to form both aerobic and hypoxic environments [31]. Multiple wetlands can effectively improve the level of DO in the outflow through the FBCW and form an anaerobic environment in the middle and lower streams in the HSFCW, which are conducive to denitrification in the wetland and critical to the effective denitrification of the system. Compared to the HSFCW, the average removal rate of NH4 +-N was much lower in the SFCW in the lower stream of the system because the concentration of NH4 +-N was lower in the inflow. However, the removal rate of TN in the SFCW was significant, accounting for 23% of the total removal of TN in the wetland system. This might be because the SFCW was usually in a saturated state during the operation and had considerable oxygen consumption, which was conducive to denitrification. In addition, the DO concentration going into the SFCW was very low. The multiple changes in the terrain between deep pools and shallow waters helped form a local environment with aerobic-hypoxic-anaerobic microcirculations, which promoted denitrification and the removal of TN. In addition, the seasonal temperature differences are large in northern China, and seasonal changes have a great impact on the effectiveness of constructed wetlands [25]. Multiple constructed wetlands can effectively make up for the disadvantage of a single wetland type [41], prolong the operation time of wetlands, and improve the efficiency of wetland purification.

For improved sewage treatment methods such as cyclic-activated sludge technology, activated charcoal adsorption, biofilms, and contact oxidation, the construction cost is usually \$300–600 per ton of sewage, and the operating cost is approximately \$0.07–0.15 per ton of sewage. Not only are the construction and operation costs high, but the management is also complicated. The construction cost of a constructed wetland system is approximately \$100–180 per ton of sewage. The operating cost is approximately \$0.014–0.056 per ton of sewage. Compared with the traditional biochemical treatment, constructed wetlands have the advantages of low investment, low operating cost, simple operation and management, and high ecological efficiency [48]. Therefore, these systems have great advantages in the restoration of polluted rivers. In this study, the total investment in construction was only \$0.12 million, which was \$120 per ton of water, and was about 1/5–1/3 of the investment in the construction of traditional sewage treatment. The system used gravity flow, no power was needed for operation, and the operating cost only involved regular maintenance. The maintenance costs were covered by the park maintenance budget, and the park created a part-time maintenance job for the wetlands. The part-time staff's wage was 70 dollar/month, and the system maintenance costs were only 0.023 dollar per ton of water, equal to 1/6–1/3 of the traditional sewage treatment. Compared with generic constructed wetland systems, the system described in this paper has a lower operating cost. Currently, the economic factor has become an important parameter for the selection of sewage treatment methods. Compared with operating constructed wetlands and other river water purification technologies in China and other countries, the multiple wetland technology in this study has obvious advantages and practicality.

#### **5. Conclusions**

(1) The one-year continuous monitored results of the sequential wetland system of FBCW, HSFCW, and SFCW showed that the system had a good effect on pollution removal. The system was mature and stable between June and September, and the average removal rates of COD, NH4 +-N, TN, TP, and SS were 74.79%, 80.09%, 71.12%, 78.39%, and 91.90%, respectively. The FBCW had the highest removal rate of SS (80.24%), which could effectively prevent the blocking of HSFCW. The HSFCW contributed 79.20%, 64.64%, and 81.71% to the total removal of NH4 +-N, TN, and TP, respectively. However, the SFCW could further remove TN, which contributed 23.00% to the total TN. The construction cost of the system was only \$120 per ton of water, and the operation cost was only 0.023 dollar per ton of water. The system was an economic and efficient river water purification technology.

(2) The temperature directly affected the operation and treatment of the sequential series system of the constructed wetlands. The system was in a stage of operation and growth in April and May, while the removal efficiency of pollutants was relatively low, and then gradually increases over time. The system was in a stable stage from June to September; thus, the removal efficiency of pollutants was the highest, and both removal rates of COD and NH4 +-N were over 80%, while the TN exceeded 75%. The removal efficiency of pollutants decreased with the temperatures in the October. Then, the system stopped running when the water froze. In a temperate continental monsoon climate zone, the system cannot run stably all year; thus, we should take the measures of heat preservation and regulation of cold-adapted microorganism to prolong the operation time and improve purification efficiency.

(3) The sequential wetland system of FBCW, HSFCW, and SFCW by using the nearshore land on both sides of the river is an effective measure to treatment of polluted river water. The system can give full play to the advantages of different wetland and realize the maximization of the overall function. The system has high pollutant purification efficiency, good operation stability, strong anti-pollution load capacity, lower construction investment and operation cost, and simple management and maintenance. The technology can provide a reference for the restoration of polluted water bodies in urban rivers in temperate continental monsoon climate zone.

(4) The system integrates water purification, ecological restoration, landscape beautification, and entertainment. Based on the concept of "close to nature, multi-functional and sustainable" in the ecological restoration, the research gives a great theory and practice to promote the health of the river environment and the sustainable development of regional ecology economy society.

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

**Funding:** This research was founded by the Foundation of Jilin Scientific and Technological Development Project, grant numbers<PHONE_NUMBER>7JH;<PHONE_NUMBER>8GH); the National Natural Science Foundation of China, grant numbers: 41901116.

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

#### **References**

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