**Development of Newly Synthesized Chromone Derivatives with High Tumor Specificity against Human Oral Squamous Cell Carcinoma**

**Yoshiaki Sugita 1,\*, Koichi Takao 1, Yoshihiro Uesawa 2,\*, Junko Nagai 2, Yosuke Iijima 3, Motohiko Sano 4 and Hiroshi Sakagami 5,\***

Received: 3 August 2020; Accepted: 24 August 2020; Published: 26 August 2020

**Abstract:** Since many anticancer drugs show severe adverse effects such as mucositis, peripheral neurotoxicity, and extravasation, it was crucial to explore new compounds with much reduced adverse effects. Comprehensive investigation with human malignant and nonmalignant cells demonstrated that derivatives of chromone, back-bone structure of flavonoid, showed much higher tumor specificity as compared with three major polyphenols in the natural kingdom, such as lignin-carbohydrate complex, tannin, and flavonoid. A total 291 newly synthesized compounds of 17 groups (consisting of 12 chromones, 2 esters, and 3 amides) gave a wide range of the intensity of tumor specificity, possibly reflecting the fitness for the optimal 3D structure and electric state. Among them, 7-methoxy-3-[(1*E*)-2-phenylethenyl]-4*H*-1-benzopyran-4-one (compound **22**), which belongs to 3-styrylchromones, showed the highest tumor specificity. **22** induced subG1 and G2 + M cell population in human oral squamous cell carcinoma cell line, with much less keratinocyte toxicity as compared with doxorubicin and 5-FU. However, 12 active compounds selected did not necessarily induce apoptosis and mitotic arrest. This compound can be used as a lead compound to manufacture more active compound.

**Keywords:** chromone; tumor specificity; QSAR analysis; apoptosis; cell cycle analysis

#### **1. Introduction**

This review is composed of four parts. The first part reviews the adverse effect of chemotherapeutic agents. The second part introduces our life-work research of development of chromone derivatives that show comparable anticancer activity and lower keratinocyte toxicity, as compared with anticancer drug. The third part describes the serious problems of neurotoxicity in G2 + M blocker. The fourth part is the summary of our major findings and future direction of chromone research.

#### **2. Adverse E**ff**ects of Anticancer Drugs**

#### *2.1. Oral Mucositis Associated with Anticancer Drug*

Oral mucositis is one of the most frequent adverse events in cancer drug therapy and hematopoietic stem cell transplantation. Oral mucositis is reported to occur in 5–50% of patients receiving standard-dose chemotherapy and 68–98% of high-dose chemotherapy related to hematopoietic stem cell transplantation [1]. Oral mucositis not only lowers the patient's QOL due to pain but also lowers the oral intake, leads to undernutrition and dehydration, and deteriorates the general condition. It also serves as a gateway for bacterial invasion and may trigger systemic infection. Decreased doses and delayed schedules in chemotherapy lead to reduced efficacy and survival rates, but currently there are no established preventive or curative methods for oral mucositis, hindering smooth cancer treatment [2].

#### *2.2. Neurotoxicity of Anticancer Drugs*

Cancer drug therapy has contributed to the improvement of survival rate and QOL by the development of cytocidal anticancer drugs and molecular targeted therapeutic agents, while the adverse effect of cancer drug therapy causes a decrease in QOL, sometimes causing the discontinuation of the drug therapy. Typical side effects include organ disorders such as bone marrow suppression, physical disorders such as nausea and vomiting, and neuropathy represented by paresthesia. Chemotherapy-induced peripheral neuropathy (CIPN) associated with an anticancer agent is not recovered quickly by a drug withdrawal like myelosuppression, and some disorders may remain for the lifetime. CIPN is a serious adverse event that interferes with the continuation of chemotherapy. It was reported that the incidence of CIPN was 68.1% within 1 month after chemotherapy, 60.0% after 3 months, and 30.0% after 6 months in a follow-up study of 4179 patients with colorectal cancer, breast cancer, gynecologic cancer, and multiple myeloma [3]. However, there are a few reports of preventive and therapeutic drugs for CIPN. Platinum, taxane, and vinca alkaloid are known as causative agents of CIPN. Different drugs have different mechanisms that cause peripheral neuropathy. For example, platinum-based cisplatin causes sensorineural deafness in the high range due to acoustic nerve damage. It has been reported that it is cumulative and that symptoms often continue for a long period of time after discontinuation of administration [4].

Oxaliplatin, a platinum drug, has acute and chronic symptoms. Acute symptoms are characterized by paresthesia around the extremities and around the lips and chronic symptoms may persist for months to years [5]. Carboplatin, a platinum drug, causes relatively few neurological symptoms when used at normal doses, and high doses may cause symptoms similar to cisplatin [6]. Paclitaxel, a taxane-based drug, mainly causes paresthesia of the extremities and is correlated with single dose and total dose [7]. Docetaxel, a taxane-based drug, causes sensory and motor disorders but is less frequent than paclitaxel [8]. Vincristine, a vinca alkaloid drug, causes sensory abnormalities in the fingers and movements within a few weeks after the start of treatment and often persists for a long time after the discontinuation of treatment [9]. CIPN pathological findings are classified into axonopathy, neuronopathy, and myelinopathy. Axonopathy is the most common disorder in CIPN. Neuronal cell body is relatively retained due to damage from thick and long axons. Clinically, glove and stocking type sensory deficits often begin at the extremities. Representative agents are microtubule inhibitors, vinca alkaloids, and taxanes. Neuronopathy is mainly cell bodies of lesions, mainly caused by cell death of dorsal root ganglion cells, and secondary damage to axons and myelin sheaths. Clinically, nerve cell bodies with short axons are also damaged, so sensory deficits occur not only on the extremities but also on the trunk and face. Representative agents are platinum agents such as oxaliplatin and cisplatin [10]. The frequency of mucositis and peripheral neuropathy of various anticancer agents was summarized from the interview form of the pharmaceutical company (Table 1).

**Table 1.** Incidence of oral mucositis and peripheral neuropathy induced by anticancer drugs.

N.D. no data reported.

#### *2.3. Chemotherapy Extravasation*

Systemic intravenous chemotherapy can cause multiple emergencies by local and systemic reactions. Drug extravasation is one of the most devastating complications in chemotherapy. Overall incidences of chemotherapy extravasation ranges from 0.01% to 6.5% [11–13], with reports of extravasation occurrence via central venous catheters ranging from 0.3% to 10.3% [14–17]. The exact incidence rate of extravasation varies greatly due to the general lack of reporting and absence of centralized registry of extravasation events. Therefore, no benchmark existed for the incidence of chemotherapy extravasations.

Extravasation is the accidental leakage of cytotoxic chemotherapy drugs that can cause severe tissue damage, tissue necrosis, blistering, or sloughing into the subcutaneous or subdermal tissue at the injection site [18,19]. Extravasated drugs are further classified into the three groups: vesicants, irritants, and nonvesicants/nonirritants, according to their potential for causing damage as (Table 2) [12,13,19,20].

Vesicant drugs have the capability to induce the formation of blisters and/or cause tissue destruction. Vesicant drugs may be subclassified into DNA-binding and non-DNA-binding compounds [20]. DNA-binding compounds are capable of producing more severe tissue damage and mainly include anthracyclines and alkylating agents. Non-DNA-binding compounds are mainly vinca alkaloids and taxanes.

Irritant drugs can cause pain at the injection site or along the vein, with or without an inflammatory reaction. Some of these agents have the potential to cause soft tissue ulcers only if a large amount of concentrated drug solution is inadvertently extravasated. Nonvesicant or nonirritant drugs, if extravasated, rarely produce acute reactions or tissue necrosis.

**Table 2.** Classification of chemotherapeutic drugs according to tissue damage after extravasation.

Tissue damage related to extravasation occurs by different mechanisms [3]. First, the drug is absorbed by local cells in the tissue and binds to critical structures, causing cell death. After the endocytolysis, surrounding cells can also die through the release of the drug from nearby dead cells. The repetitive nature of this process impairs healing and may result in progressive and chronic tissue injury. Second, the drug that does not bind to cellular DNA may metabolize and be cleared, limiting the degree of tissue injury [21]. However, the literature addressing extravasation is limited to animal studies, case reports, and small human studies. Classic randomized studies in humans for the treatment of extravasations are unthinkable because of ethical reasons. On the whole, the highest possible grade

of recommendation of each measure for extravasations would be low. Novel studies are clearly needed to elucidate the mechanism of chemotherapy extravasations.

#### **3. Development of Newly Synthesized Chromone Derivatives with High Tumor Specificity, but Low Keratinocyte Toxicity**

Our strategy to explore new compounds is composed with the following eight steps: search of natural products that shows the highest tumor specificity (Step 1); QSAR analysis of chromone-related compounds (Step 2); investigation of action mechanism (Step 3), identification of target molecules (Step 4), exploration of more activity compounds by prediction, synthesis, and confirmation (Step 5); check for adverse effects (Step 6); in vivo experiments with animals (Step 7); and clinical application (Step 8) (Figure 1).

**Figure 1.** Strategy for exploring new anticancer drugs using chromone backbone structure.

#### *3.1. Why We Focused on the Chromones*

For the quantification of anticancer activity of text samples, it was necessary to establish the in vitro assay method (Figure 2) [22], using human malignant and nonmalignant cells: four human oral squamous cell carcinoma (OSCC) cell lines (Ca9-22, HSC-2, HSC-3, and HSC-4), three human normal oral mesenchymal cells (gingival fibroblast HGF, periodontal ligament fibroblast HPLF, and pulp cell HPC), and two human normal oral epithelial cells (human oral keratinocyte HOK and primary human gingival epithelial cells HGEP).

**Figure 2.** In vitro assay system for the measurement of tumor specificity.

Tumor specificity (TS) was defined as the ratio of the mean of CC50 against normal cells to that against OSCC cells). When mesenchymal or epithelial cells were used, TSM and TSE could be obtained, respectively. TSE can be used as an index for neurotoxicity. (Figure 1). It would be the most ideal if we could use human epithelial cells as target cells. However, most of anticancer drugs show potent cytotoxicity against epithelial cells (as described later). Therefore, we used TSM value, rather than TSE at the first stage of random screening. Using this method, we found that three major polyphenols, i.e., lignin–carbohydrate complexes, tannins, and flavonoids, showed much lower TSM in comparison to popular chemotherapeutic antitumor drugs (Table 3). On the other hand, the derivatives of chromone, the backbone structure of various flavonoids such as flavonoid, flavone, flavanone, and isoflavone (Figure 3) showed much higher TSM than the majority of polyphenols [22]. These findings encouraged us to explore more active chromone derivatives.

**Table 3.** Tumor specificity (TS) of polyphenols.

1 Cited from [22].

**Figure 3.** Chromone is a backbone structure of some flavonoids.

#### *3.2. Synthesis of Chromones, Esters, and Amides*

We have focused on the following three groups of compounds (Figure 4):

• **Chromone derivatives:**

**having intact chromone ring:** 3-styrylchromones (**A**), 2-styrylchromones (**B**), 2-(*N*-cyclicamino)chromones (**C**), 3-(*N*-cyclicamino)chromones (**D**), 2-azolylchromones (**E**), 3-benzylidenechromones (**F**), pyrano[4,3-*b*]chromones (**G**), furo[2,3-*b*]chromones (**H**). **having chromen ring:** 3-styrylchromenes (**I**) and 3-flavens (**J**) (unpublished). **having cleaved chromone ring:** aurones (**K**) and chalcones (**L**).

**Figure 4.** Structure of chromones (**A**~**L**), esters (**M**,**N**), and amides (**O**~**Q**).

As for chromone derivatives, 3-styrylchromones (**A**) were synthesized by Knoevenagel condensation of the corresponding 3-formylchromones with various phenylacetic acid derivatives [23] (Figure 5).

Here, 2-styrylchromones (**B**) were synthesized by base-catalyzed condensation of the corresponding 2-methylchromones with selected benzaldehyde derivatives [24].

Then, 2-(*N*-cyclicamino)chromones (**C**) were synthesized by the nucleophilic substitution reaction of 2-triazolylchromone derivatives, derived from 3-iodochromones and triazole, with the cyclic secondary amines such as piperidine and piperazine derivatives [25].

Then, 3-(*N*-cyclicamino)chromones (**D**) were synthesized by the condensation of 2,3-epoxychromone derivatives with the cyclic secondary amines [25].

Then, 2-azolylchromones (**E**) were synthesized by the conjugated addition reaction of 3-iodochromone derivatives with various azoles [26].

Next, 3-benzylidenechromones (**F**) were synthesized by base-catalyzed condensation of the corresponding 4-chromanone with substituted benzaldehyde derivatives [27].

Pyrano[4,3*-b*]chromones (**G**) were synthesized by the cycloaddition reaction of 3-formylchromones with selected enol ethers [28].

Furo[2,3-*b*]chromones (**H**) were synthesized by the ring expansion-cycloaddition reaction of methanochromanones with aldehydes or ketones [29].

Basically, 3-styrylchromenes (**I**) were synthesized by Horner-Wadsworth-Emmons reaction of the corresponding 2*H*-chromene-3-carbaldehydes with commercially available diethyl benzylphosphonate derivatives [30]. Additionally, 3-flavens (**J**) were synthesized by reductive intramolecular cycloaddition reaction of 2-hydroxychalcone derivatives [31]. Aurones (**K**) were synthesized by base-catalyzed condensation of 3(2*H*)-benzofuranones with selected benzaldehyde derivatives [32]. Chalcones (**L**) were synthesized by base-catalyzed condensation of the corresponding acetophenones with various benzaldehyde derivatives [31] (Figure 5).

As for esters and amides, cinnamic acid phenethyl esters (**M**) were synthesized by the condensation of cinnamic acid and its analogs, such as caffeic acid, ferulic acid, and *p*-coumaric acid, with the corresponding phenethyl alcohols. In addition, phenylpropanoid amides (**O**) were synthesized by the condensation of the corresponding cinnamic acid derivatives with various biogenic amines.

Piperic acid esters (**N**) were synthesized by the condensation of piperic acid with the corresponding alcohols. In addition, piperic acid amides (**P**) were synthesized by the condensation of the acid chloride of piperic acid with various amines. Piperic acid was prepared by alkaline hydrolysis of piperine.

Oleoylamides (**Q**) were synthesized by the condensation of oleoyl chloride, derived from oleic acid and oxalyl chloride, with the various corresponding biogenic amines (Figure 6).

**Figure 5.** Synthesis of chromone derivatives.

**Figure 6.** Synthesis of esters and amides.

#### *3.3. Tumor Specificity of Chromones, Esters, and Amides*

We investigated a total 291 compounds from 17 different groups (**A~Q**) of their cytotoxicity (assessed by CC50) against four human OSCC (Ca9-22, HSC-2, HSC-3, and HSC-4) and three human normal mesenchymal cells (HGF, HPLF, and HPC), and then their tumor specificity (assessed by TSM, calculated as describe in Figure 2, and potency-selectivity expression (PSE)) [33–52]. PSE, that reflects both tumor specificity and cytotoxicity against tumor cells, was calculated by dividing the TSM by CC50 for tumor cells, and then multiplying by 100. All these values are listed in Supplementary Table S1. This demonstrated that only limited numbers of compounds show higher tumor specificity, although their structures are very similar with each other. It is possible that such highly tumor-specific compounds show the optimal 3D structure, since the tumor specificity of chromone compounds shows the tight correlation with chemical descriptors that reflect the 3-D structure (Table 4) [33,35,37–53].

The most active compounds in each group are shown in Figure 7. Their cytotoxicity against human four OSCC cell lines, and three human normal oral mesenchymal (HGF, HPLF, and HPC), two human epithelial cells (HOK and HGEP), and tumor specificity (TSM (determined with OSCC vs. human normal mesenchymal cells), TSE) (determined with OSCC vs. human normal epithelial cells) are shown in Table 5.

**Figure 7.** The most active compounds in each group, line-upped in the decreasing order of potency.

**Table 5.** Tumor specificity and keratinocyte toxicity of chromones and anticancer drugs.

Further, 7-methoxy-3-[(1*E*)-2-phenylethenyl]-4*H*-1-benzopyran-4-one (compound **22**) showed the highest TS value (TSM = 301.1), followed by 2-[(1*E*)-2-(3,4-dimethoxy)ethenyl]-4*H*-1-benzopyran-4-one (compound **40**) (TSM = 89.1) > 2-[(1*E*)-2-(4-methoxyphenyl)ethenyl]-4*H*-1-benzopyran-4-one (compound **34**) (TSM = 84.1) > (*E*)-3-(4-Hydroxystyryl)-6-methoxy-4*H*-chromen-4-one (compound **11**) (TSM = 69.0) > 7-methoxy-2-(4-morpholinyl)-4*H*-1-benzopyran-4-one (compound **62**) (TSM = 63.4) > (*E*)-3-(4-cholorostyryl)-7-methoxy-2*H*-chromene (compound **182**) (TSM = 59.9) > (3*E*)-2,3-dihydro-3-[(3,4-dihydroxyphenyl)methylene]-7-methoxy-4*H*-1-benzopyran-4-one (TSM = 52.2) (compound **136**). It is noted that these compounds showed comparable TS values of doxorubicin (DXR) and much higher TS value than 5-FU. It was unexpected that DXR and 5-FU showed potent toxicity against human epithelial cells such as human oral keratinocyte (HOK) and human progenitor of human gingival epithelial cells (HGEP) (c/a and d/a in Table 5). We have reported previously that DXR induced apoptosis (characterized by the loss of cell surface microvilli, chromatin condensation, nuclear fragmentation, and caspase-3 activation) in these keratinocytes [54]. On the other hand, compounds **11**, **22**, **34**, **62,** and **69** showed much lower keratinocyte toxicity (Table 5).

#### *3.4. Mechanism of Action*

Compounds **11** and **22** in 3-styrylchromones (**A**), **34** and **40** in 2-styrylchromones (**B**), **95** in 2-azolylchromones (**E**), **228** in chalcones (**L**), and **237** in cinnamic acid phenethyl esters (**M**) induced apoptosis [caspase-3 activation (assessed by western blot analysis) and subG1 cell accumulation (assessed by cell sorter analysis)] in human OSCC cell lines. On the other hand, compounds **62** in 2-(*N*-cyclicamino)chromones (**C**), **95** in 3-(*N*-cyclicamino)chromones (**D**), **107** in 2-azolylchromones (**E**), **154** in pyrano[4,3-*b*]chromones (**G**), and **168** in furo[2,3-*b*]chromones (**H**) did not induce apoptosis (Table 6). Compounds **22**, **34**, **40,** and **107** also induced G2 + M cell accumulation, but only the first 3 compounds induced apoptosis. This indicated that the induction of G2 + M accumulation itself does not guarantee the induction of apoptosis.

**Table 6.** The most active compounds in each group do not necessarily induce apoptosis in human oral squamous cell carcinoma (OSCC) cell line.

*3.5. Other Biological Actions of Chromones, Esters, and Amides*

We searched other biological activities of chromones, esters, and amides (Supplementary Table S2). Table 7 listed up the most potent compounds that showed biological activity higher than positive controls. Compounds **10, 12**, **15**, **124, 136**, **229**, **231**, **237**, **258,** and **261** scavenged the DPPH radical, more potently than ascorbic acid, a well-known antioxidant [23], suggesting its antioxidant action.

**Table 7.** Oher biological activities of chromones, esters, and amides.

Compounds **10**, **14**, **15**, **18**, **131**, **132**, **136**, **266,** and **267** inhibited α-glucosidase (EC <IP_ADDRESS>) that is responsible in breaking down starch and disaccharides to glucose, more potently than acarbose. This suggest their possible antihyperglycemic effect.

Compounds **10**, **15**, **124**, and **136** show both α-glucosidase inhibitory and antioxidant actions, suggestion that they can be lead compounds for manufacturing as antidiabetic drugs.

Compounds **38**, **39**, **87**, **89**, **153**, **173**, **177,** and **236** inhibited monoamine oxidase (MAO-B) more effectively than pargyline, an irreversible selective MAO-B inhibitor drug. This suggests their application to treat the Parkinson's disease and Alzheimer's disease [24,27,28,30,55]. Halogen-containing compounds show more potent inhibitory activity. All compounds showed higher MAO-B-specific inhibition than positive controls and, therefore, were not likely to exert adverse effects due to MAO-A inhibition. Furthermore, they show reversible inhibition and thus were much convenient for the sudden interruption of treatment, as compared with irreversible inhibitors.

Compounds **230** and **235** inhibited the butyrylcholinesterase (BChE) more potently than neostigmine, suggesting that they may serve as lead compounds for the development of novel BChE inhibitors and candidate lead compounds for the prevention or treatment of Alzheimer's disease [55].

We found that all compounds tested showed no anti-HIV activity (SI < 1), in contrast to popular anti-HIV substances (dextran sulfate, curdlan sulfate, azidothymidine, 2 ,3 -dideoxycytidine, azidothymidine, and 2 ,3 -dideoxycytidine) (SI = 53–2512) (Supplementary Table S3).

#### **4. Serious Problems of Neurotoxicity in G2** + **M Blocker**

We found that highly tumor-specific 3-styrylchromone derivatives [7-methoxy-3-[(1*E*)-2 -phenylethenyl]-4*H*-1-benzopyran-4-one (compound **22**) and 3-[(1*E*)-2-(4-hydroxyphenyl)ethenyl]- 7-methoxy-4*H*-1-benzopyran-4-one (compound **29**)] (TSM = 301 and 182, respectively) (Supplementary Table S1) induced subG1 and G2 + M arrest [35]. We also have recently reported that several G2/M blockers such as taxanes paclitaxel (Taxol®, the first microtubule stabilizing agent [57]) and docetaxel, show very high TSM values (>7267 and >86,122, respectively) [58]. Marinho et al. reported recently that 4 -methoxy-2-styrylchromone induced mitotic arrest in human tumor (human Caucasian breast adenocarcinoma MCF-7 and human lung adenocarcinoma NCI-H460) cell lines, in a similar fashion to paclitaxel [59]. Soo et al. reported that cudraflavone C (Cud C), a naturally occurring flavonol, induced apoptosis (caspase activation) in colorectal cancer cells (CRC) and tumor-selective cytotoxicity by targeting the PI3K-AKT pathway [60].

However, many reports, including ours, demonstrated that microtubule-targeted agents have potent neurotoxicity, adversely affecting the quality of life of patients on a long-term basis [61–64]. Iijima et al. recently reported that carboplatin (CBDCA) was highly neurotoxic (TSN = 0.11 (3.2/27.9)), calculated using the data of Table 2 in Ref. [64]. It is urgent to investigate the neurotoxicity, extravasation as well as stomatitis of chromone derivatives, esters, and amides.

#### **5. Conclusions and Future Direction**

We found that:

It is crucial to identify the target molecules (Step 4 in Figure 1). To accomplish this, 13C-labeled compound **22** will be prepared, using 2-hydroxyacetophenone derivatives and 13C-dimethylformamide, or using 13C-iodomethane as methylation agent, and then the differential incorporation of 13C into malignant and nonmalignant cells will be investigated, with LC-MS. Compound **22**, labeled with fluorescence dye (Cy3, CY5, Cy7), will be tested to detect the intracellular uptake and distribution into organelles, using confocal laser microscopy. Binding of cellular protein to and elution from chromone-attached beads may be useful to identify the binding proteins.

In order to explore more potent chromone derivatives, the following three steps will be repeated: (i) prediction by QSAR of the best fit substituents that yield the highest TSM and TSE, (ii) synthesis of compounds introduced with such predicted substituents, and (iii) confirmation of antitumor potential (Step 5). However, it is important to eliminate the compounds that show potent keratinocyte toxicity, neurotoxicity, and extravasation (Step 6), before animal experiment (Step 6) and clinical application (Step 7).

The present study demonstrated that only selected compounds that have the optimal 3D structure show the highest tumor specificity, whereas most of other analogs that have similar structure show much less tumor specificity (Supplementary Table S1). This suggests the presence of binding components or receptors for chromones. It remains to be investigated whether compounds **22** and **40** may interact with estrogen receptors, since these compounds have structural similarity with isoflavones (such as daidzein and genistein) and to some degree with tamoxifen, which have been used for the treatment of oral squamous cell carcinoma that express estrogen receptors [65–67]. In addition, it seems that the "para" like substitution is favorable, possibly because it mimics the structure of estrogen. It is highly probable that different groups of chromone-related compounds have different anticancer mechanisms depending on their structure (Table 6).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2305-6320/7/9/50/s1, Table S1: Tumor specificity of 291 compounds; Table S2: Radical scavenging and monoamine oxidase inhibitory and cholinesterase inhibitory activities of chromones, esters, and amides; Table S3: Test for anti-HIV activity of chromones, esters, and amides.

**Author Contributions:** Conceptualization was done by H.S., Y.U. and Y.S., Y.I., M.S. wrote the sections of adverse effects of anticancer drugs. Y.S. and K.T. wrote the section of chemical synthesis and drew the structures of chromones, esters, and amides and inhibitor assay. Y.U. and J.N. wrote the section of QSAR analysis. H.S. wrote the section of tumor specificity, apoptosis, and cell cycle assay and other parts of the text. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by KAKENHI from the Japan Society for the Promotion of Science (JSPS): Sakagami H, 16K11519, and 20K09885.

**Acknowledgments:** We would like to thank Okudaira (Teikyo University School of Medicine) and Bando (Meikai University School of Dentistry) for technical support.

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

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

## *Article* **Modulation of Th1**/**Th2 Cytokine Balance by Quercetin In Vitro**

### **Yoshihito Tanaka 1, Atsuko Furuta 2, Kazuhito Asano 3,\* and Hitome Kobayashi 1**

Received: 29 June 2020; Accepted: 28 July 2020; Published: 30 July 2020

**Abstract: Background:** Allergic rhinitis (AR) is well known to be an IgE-mediated chronic inflammatory disease in the nasal wall, which is primarily mediated by Th2-type cytokines such as IL-4, IL-5, and IL-13. Although quercetin is also accepted to attenuate the development of allergic diseases such as AR, the influence of quercetin on Th2-type cytokine production is not well understood. The present study was designed to examine whether quercetin could attenuate the development of AR via the modulation of Th2-type cytokine production using an in vitro cell culture technique. **Methods:** Human peripheral-blood CD4+ T cells (1 × 106 cells/mL) were cultured with 10.0 ng/mL IL-4 in the presence or absence of quercetin. The levels of IL-5, IL-13, and INF-γ in 24 h culture supernatants were examined by ELISA. The influence of quercetin on the phosphorylation of transcription factors NF-κB and STAT6, and mRNA expression for cytokines were also examined by ELISA and RT-PCR, respectively. **Results:** Treatment of cells with quercetin at more than 5.0 μM inhibited the production of IL-5 and IL-13 from CD4+ T cells induced by IL-4 stimulation through the suppression of transcription factor activation and cytokine mRNA expression. On the other hand, quercetin at more than 5.0 μM abrogated the inhibitory action of IL-4 on INF-γ production from CD4+ T cells in vitro. **Conclusions:** The immunomodulatory effects of quercetin, especially on cytokine production, may be responsible, in part, for the mode of therapeutic action of quercetin on allergic diseases, including AR.

**Keywords:** allergic rhinitis; quercetin; human CD4+ T cells; Th1/Th2 cytokine balance; modulation; in vitro

#### **1. Introduction**

Allergic rhinitis (AR) is well accepted to be a chronic inflammatory IgE-mediated disorder of the nasal wall and is characterized by multiple symptoms such as sneezing, itching, and nasal congestion, among others [1,2]. AR is also accepted to be divided into two different phases of allergic reaction: an initial sensitization phase in which allergen exposure results in IgE formation, and subsequent clinical disease after repeated antigen exposure [3]. The clinical reaction is further subdivided into early- and late-phase responses [1,2]. The development of these responses is orchestrated by Th2-type helper T cells via the production of several types of cytokines and chemokines, which are responsible for the migration and activation of inflammatory cells [1,2].

 Consequently, it is desirable to develop safe and effective therapeutic agents for AR. Quercetin is well known to be one of the most abundant dietary flavonoids, found in various vegetables such as onions, broccoli, tomatoes, etc. [6]. For many years, quercetin has been studied for its possible health benefits, and it has been revealed that quercetin attenuates oxidative stress responses through the suppression of free-radical generation [6,7]; increases in the production of thioredoxin [8] and glutathione [9,10]; quercetin–glutathione conjugate formation [11]; and upregulation of glutamate–cysteine ligases [11], which are important endogenous antioxidants [8–11]. In regard to allergic immune responses, quercetin has been reported to inhibit the production of both inflammatory cytokines and chemokines such as IL-5, eotaxin, and RANTES (regulated on activation normal T cell expressed and secreted) from eosinophils and mast cells after immunological stimulation in vitro and in vivo [12–15]. It has also been reported that quercetin inhibits the secretion of harmful chemical mediators, including histamine, leukotrienes, major basic protein, and eosinophil cationic protein from mast cells and eosinophils in vitro and in vivo [14,15]. Furthermore, the influence of quercetin on the production of T-cell cytokines was investigated using an asthmatic mouse model, and it was reported that quercetin could reduce the increased levels of IL-4, and increased IFN-γ levels in bronchoalveolar lavage fluid after antigenic challenge via the modulation of T-box protein expressed in T cells *(T-bet)* and *GATA-3* gene expression, resulting in significant attenuation of all asthmatic reactions [16,17]. Although these reports strongly suggest that quercetin is a good candidate as a supplement for modulation of allergic diseases, including AR, the mechanisms of the therapeutic action of quercetin on allergic responses is not fully understood. There is much evidence that IL-4, one of the Th2-type T-cell cytokines, is a key player in immune modulation of allergic responses and plays essential roles in the development of pathological changes in allergic diseases [2,18]. Although it has been reported that quercetin can inhibit the ability of human peripheral-blood mononuclear cells to spontaneously produce IL-4, but not IFN-γ in vitro via inhibition of cytokine mRNA expression [19], the precise mechanisms of quercetin' on cytokine production are not well understood. In the present study, therefore, we examined the influence of quercetin on IL-4-mediated immune responses by examining the secretion of cytokines from CD4+ T cells in vitro.

#### **2. Materials and Methods**

#### *2.1. Reagents*

Quercetin was obtained from Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA) as a preservative-free pure powder. It was dissolved in dimethyl sulfoxide at a concentration of 10.0 mM and was then diluted with RPMI-1640 medium (Sigma-Aldrich Co., Ltd.) supplemented with 10% heat-inactivated bovine serum (RPMI-FBS; Sigma-Aldrich Co., Ltd.) at appropriate concentrations for experiments. It was then sterilized by passing it through 0.2 μm filters, and stored at 4 ◦C until use. Recombinant human IL-4 was purchased from R & D Systems, Inc. (Minneapolis, MN, USA) as a preservative-free pure powder. IL-4 was also dissolved in RPMI-FBS, sterilized with 0.2 μm filters and stored at 4 ◦C until use. mRNA isolation kits were purchased from Milteny Biotec (Bergisch Gladbach, Germany). The reagents used for cDNA synthesis and the real-time reverse-transcription polymerase chain reaction (RT-PCR) kit were obtained from Invitrogen Corp. (Carlsbad, CA, USA) and Applied Biosystems (Foster City, CA, USA), respectively.

#### *2.2. Preparation of CD4*+ *T Cells*

Heparinized human venous blood was obtained from five healthy subjects (all male, 41.0 ± 10.1 years) after obtaining their written informed consent, which was approved by the Ethics Committee of Showa University (Approved No. 190613; Date of approval: 1 June 2019). Peripheral-blood mononuclear cells (PBMCs) were then obtained after centrifugation (1000× *g* for 30 min) of blood with lymphocyte separation medium (Organon Technica, Durham, NJ, USA). CD4+ T cells were purified from PBMCs using a magnetic cell separator (Milteny Biotec, Bergisch Gladbach, Germany) as described previously [20]. The cells were suspended in RPMI-FBS at a concentration of 1 × 106 cells/mL.

#### *Medicines* **2020**, *7*, 46

The cell purity was more than 95%, as judged using a flow cytometer (FACScan; Becton Dickinson, San Jose, CA, USA).

#### *2.3. Cell Culture*

CD4+ T cells (1 × 106 cells/mL) were introduced into each well of 24 well culture plates in triplicate, where each well contained 10.0 ng/mL of IL-4 and various concentrations of quercetin in a final volume of 2.0 mL [20]. The supernatants were collected 24 h later and stored at −40 ◦C until needed for assays for the levels of cytokines. To prepare cells for examining transcription factor activation and mRNA expression, CD4+ T cells were cultured in a similar manner for 1 and 4 h, respectively [20]. In all experiments, quercetin treatment was started 1 h before IL-4 stimulation.

#### *2.4. Assay for Cytokines*

The levels of IL-5, IL-13, and IFN-γ in culture supernatants were measured in duplicate with human cytokine ELISA kits (R & D) according to the manufacturer's instructions. The sensitivity of the ELISA kits for IL-5, IL-13, and IFN-γ was 3.0 pg/mL, 32.0 pg/mL, and 8.0 pg/mL, respectively.

#### *2.5. Assay for Transcription Factor Activities*

NF-κB and STAT6 activity in cultured cells were examined using ELISA test kits (Active Mortif Co., Ltd., Carlsbad, Calif, USA) following the manufacturer's recommended procedures.

#### *2.6. Assay for mRNA Expression*

Poly A+ mRNA was extracted from cells with oligo(dT)-coated magnetic micro beads (Milteny Biotec, Bergisch Gladbach, Germany). mRNA samples (1.0 μg) were reverse-transcribed to cDNA using a Superscript cDNA synthesis kit (Invitrogen Corp., Carlsbad, CA, USA). Polymerase chain reaction (PCR) was then conducted using a GeneAmp 5700 Sequence Detection System (Applied Biosystems, Forster City, CA, USA). The PCR mixture consisted of 2.0 μL of sample cDNA solution (100 ng/μL), 25.0 μL of SYBR-Green Mastermix (Applied Biosystems), 0.3 μL of both sense and antisense primers, and distilled water to give a final volume of 50.0 μL. The reaction was conducted as follows: 4 min at 94 ◦C, followed by 40 cycles of 4 min at 95 ◦C, 1 min at 60 ◦C, and 1 min at 70 ◦C [20]. GAPDH was amplified as an internal control. mRNA levels for IL-5 and IL-13 were calculated by using the comparative parameter threshold cycle and normalized to GAPDH. The nucleotide sequences of the primers were as follows: for IL-5, 5 -GCTTCTGCATTTGAGTTTGCTAGCT-3 (sense) and 5 -TGGCCGTCAATGTATTTCTTTATTAAG-3 (antisense); for IL-13, 5 -CCACGGTCATTGCTCTC AGGCTGGACTG-3 (sense) and 5 -CCTTGTGCGGGCAGAATCCGCTCA-3 (antisense) [20]; and for GAPDH, 5 -TGCACCACCAACTGCTTAGC-3 (sense) and 5 -GGCATGGACTGTGGTCATGAG-3 (antisense) [7].

#### *2.7. Statistical Analysis*

Statistical analyses were performed with ANOVA followed by Dunnett's multiple-comparison test. Values of *p* < 0.05 were considered statistically significant.

#### **3. Results**

#### *3.1. Influence of Quercetin on the Production of T-Cell Cytokines*

The first set of experiments was undertaken to examine whether quercetin could suppress the production of Th-2-type cytokines IL-5 and IL-13, by CD4+ T cells after IL-4 stimulation. CD4+ T cells (1 × 106 cells/mL) were cultured with 10.0 ng/mL IL-4 in the presence of 1.0 to 10.0 μM quercetin for 24 h. The levels of IL-5 and IL-13 in culture supernatants were measured by ELISA. Treatment of cells with quercetin at lower than 2.5 μM did not inhibit IL-5 production: IL-5 levels in experimental culture supernatants were similar (not significant) to those that received IL-4 stimulation alone (Figure 1a). On the other hand, higher concentrations of quercetin (more than 5.0 μM) caused significant suppression of IL-5 production, which was increased by IL-4 stimulation (Figure 1a). We then examined the influence of quercetin on IL-13 production by CD4+ T cells after IL-4 stimulation. Quercetin suppressed IL-13 production as it did IL-5 production (Figure 1b). The minimum concentration of quercetin that caused significant suppression of IL-13 production was 5.0 μM (Figure 1b). We finally examined the influence of quercetin on Th-1-type cytokine production using IFN-γ. Stimulation of cells with IL-4 significantly decreased IFN-γ levels in culture supernatants (Figure 2). Although addition of quercetin at less than 2.5 μM did not inhibit the suppressive activity of IL-4 on IFN-γ production, quercetin at more than 5.0 μM suppressed the downregulation of IFN-γ production induced by IL-4 stimulation (Figure 2).

**Figure 1.** Influence of quercetin on Th2-type cytokine production from human peripheral-blood CD4+ T cells in vitro. CD4+ T cells (1 × 106 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. Cytokine levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects. (**a**): IL-5; (**b**): IL-13; \* *p* > 0.05 versus IL-4 alone; \*\* *p* < 0.05 versus IL-4 alone.

**Figure 2.** Influence of quercetin on interferon (IFN)-γ production from human peripheral-blood CD4+ T cells in vitro. CD4+ T cells (1 × 106 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 24 h. IFN-γ levels in culture supernatants were examined by ELISA. The results were expressed as the mean pg/mL ± SE of five subjects.

#### *3.2. Influence of Quercetin on Transcription Factor Activation and Cytokine mRNA Expression*

The final set of experiments was carried out to examine the possible mechanisms by which quercetin could inhibit Th2-type cytokine production from CD4+ T cells after IL-4 stimulation. CD4+ T cells were stimulated with IL-4 in the presence of 1.0 to 10.0 μM quercetin. Activation of transcription factors NF-κB and STAT6 in 1 h cultured cells was examined by ELISA. As shown in Figure 3a, lower concentrations (1.0 and 2.5 μM) of quercetin did not affect NF-κB activation, which was increased by IL-4 stimulation. However, treatment of cells with higher concentrations (5.0 to 10.0 μM) of quercetin significantly inhibited IL-4–induced NF-κB activation. We then examined the influence of quercetin on STAT6 activation after IL-4 stimulation. The data presented in Figure 3b clearly showed that quercetin inhibited STAT6 activation, as was the case for NF-κB. The minimum concentration of quercetin that caused significant suppression was 5.0 μM. The final experiments in this section were performed to examine the influence of quercetin on Th2-type cytokine mRNA expression in 4 h cultured cells by real-time RT-PCR (Figure 4). Addition of quercetin at 2.5 μM did not suppress mRNA expression for either IL-5 or IL-13, but mRNA expression, which was increased by IL-4 stimulation, was significantly inhibited when cells were treated with quercetin at more than 5.0 μM.

**Figure 3.** Influence of quercetin on transcription factor activation in CD4+ T cells in vitro. CD4+ T cells (1 × 106 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 1 h. Activation of transcription factors NF-κB (**a**) and STAT6 (**b**) was assessed by ELISA. The results were expressed as the mean OD at 450 nm ± SE of five subjects. \* *p* > 0.05 versus IL-4 alone; \*\* *p* < 0.05 versus IL-4 alone.

**Figure 4.** Influence of quercetin on mRNA expression for Th2-type cytokines in vitro. CD4+ T cells (1 × 106 cells/mL) were stimulated with 10.0 ng/mL IL-4 in the presence of various concentrations of quercetin for 4 h. mRNA expression for IL-5 (**a**) and IL-13 (**b**) was examined by real-time RT-PCR. The results were expressed as the mean cytokine/GAPDH ± SE of five subjects. \* *p* > 0.05 versus IL-4 alone; \*\* *p* < 0.05 versus IL-4 alone.

#### **4. Discussion**

Quercetin, a natural compound belonging to the flavonol subgroup, has been shown to favorably modify the clinical conditions of allergic diseases, including AR, through the inhibition of inflammatory cell (e.g., mast cells and eosinophils) activation [12–15]. It has also been reported that quercetin exerts suppressive effects on the production of neuropeptides, which are responsible for the development of AR symptoms [21]. Although it is established that Th2-type T cells play a key role in triggering the allergic inflammatory responses in AR [2,18], the influence of quercetin on Th2-type T-cell functions is not clearly defined. The present study, therefore, was undertaken to examine the influence of quercetin on Th2-type T-cell functions by examining Th2-type cytokine production.

The present results clearly showed that quercetin inhibited the ability of CD4+ T cells to produce IL-5 and IL-13 after IL-4 stimulation through inhibition of the activation of transcription factors NF-κB and STAT6, and inhibition of cytokine mRNA expression. It is also showed that quercetin abrogated the suppressive activity of IL-4 on INF-γ production by CD4+ T cells. The minimum concentration of quercetin that caused significant modulation of cytokine production was 5.0 μM. After oral administration of quercetin at 1200 mg, which is a standard recommended dosage, plasma levels of quercetin gradually increase and peak at 12 μM [22,23], which is a much higher level than that which caused modulation of cytokine production by CD4+ T cells after IL-4 stimulation in vitro in this study. From these reports, the findings of the present in vitro study may reflect the biological function of quercetin in vivo.

AR is well known to consist of type I hypersensitivity allergic responses in nasal membranes against several types of aeroallergens [1,2]. It is also accepted that type I allergic responses consist of two different phases [2,3]. The sensitization phase comprises IgE formation against specific allergens based on the Th2-type immune system. In the triggering phase, allergic symptoms are triggered by to secretion of several kinds of chemical mediators from mast cells and eosinophils after re-exposure to the same allergen [2,3] These two phases are orchestrated by T cells, especially Th2-type helper T cells, through the secretion of several cytokines [1,2]. Among Th2-type cytokines, the first important cytokine is IL-4, which promotes the special production of IgE from resting B cells [2,18]. IL-3 and IL-5 are other important Th2-type cytokines, and have been shown to enhance the proliferation and differentiation of mast cells and eosinophils from their precursors [24]. IL-13 is a pleiotropic cytokine produced by activated Th2-type T cells [25]. It has a wide variety of effects on Th2-dominated inflammatory disorders, such as enhancement of IgE production and vascular cell adhesion molecule 1 expression, which increases the migration of inflammatory cells into the site of inflammation [26]. It has also been reported that IL-13 as well as IL-5 can activate and inhibit the apoptosis of eosinophils [25]. On the other hand, IFN-γ, the principal Th1-type effector cytokine, initiates and maintains Th1-type immune responses, which dampen diseases promoted by Th2-type immune responses through the inhibition of Th2-tpe T-cell recruitment/differentiation, induction of apoptosis in eosinophils, and blockage of IgE isotype switch in B cells, among other actions [27]. From these reports, the present results strongly suggest that the beneficial immunomodulatory effects of quercetin may comprise, in part, the therapeutic mode of action of quercetin on allergic diseases, including AR.

Although the present results clearly showed a favorable modification of quercetin on IL-4-mediated Th1/Th2 cytokine balance, the precise mechanism(s) by which quercetin modulates cytokine balance after IL-4 stimulation is not fully understood. IL-4 exerts its biological functions by binding to a high-affinity receptor, IL-4 receptor α chain (IL-4Rα), on the cell surface [28,29], and this complex then induces the activation of the tyrosine kinases, Janus kinase 1 and 3, which cause the phosphorylation of the transcription factor STAT6, which is essential for cytokine production from Th2-type T cells [28,29]. These reports may suggest that the immunomodulatory effect of quercetin on cytokine production is partially dependent on its suppressive activity on the STAT6 signal pathway. This speculation may be supported by the observation that treatment of CD4+ cells with quercetin at more than 5.0 μM inhibited STAT6 phosphorylation after IL-4 stimulation. In addition to IL-4Rα, IL-4 binds with the common γ chain and induces phosphorylation of NF-κB, which is responsible for cytokine mRNA

expression [30,31]. From these reports, there is another possibility that quercetin inhibits the NF-κB signal pathway and results in suppression of Th2-type cytokine production from CD4+ T cells after IL-4 stimulation. This speculation may be supported by the present observation showing the suppressive activity of quercetin at more than 5.0 μM on NF-κB activation induced by IL-4 stimulation.

Activation of Janus kinase 1 and 3 and STAT6 phosphorylation require an increase in intracellular Ca2+ levels [32]. Quercetin has been reported to be able to inhibit an increase in intracellular free Ca2+ levels in human mast cells after inflammatory stimulation in vitro [33]. Quercetin has also been reported to inhibit the phosphorylation of several types of tyrosine kinases, which are responsible for transcription factor activation [34,35]. On the basis of these reports, quercetin might inhibit the phosphorylation of tyrosine kinases through the inhibition of an increase in Ca2+ levels in CD4+ cells after IL-4 stimulation, resulting in suppression of Th2-type cytokine production. Further experiments are required to clarify this point.

#### **5. Conclusions**

The present results strongly suggest that quercetin modulates IL-4-mediated immune responses, especially Th1/Th2 cytokine balance, and results in attenuation of the development of allergic immune responses.

**Author Contributions:** Cell culture, assays for cytokines and for mRNA expression: Y.T.; assay for transcription factor activation: A.F.; statistical analysis of the data and drawing figures: H.K.; conceptualization, study design, and manuscript writing: K.A. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** All the authors have no conflict of interest in this study.

#### **References**

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).


**Theoretical and Economic Evaluation of Low-Cost Deep Eutectic Solvents for E**ff**ective Biogas Upgrading to Bio-Methane**

#### **Edyta Słupek, Patrycja Mako´s \* and Jacek G ˛ebicki**

Department of Process Engineering and Chemical Technology, Faculty of Chemistry, Gdansk University of Technology, G. Narutowicza St. 11/12, 80-233 Gdansk, Poland<EMAIL_ADDRESS>(E.S<EMAIL_ADDRESS>(J.G.)

**\*** Correspondence<EMAIL_ADDRESS>Tel.: +48-508997100

Received: 29 May 2020; Accepted: 28 June 2020; Published: 1 July 2020

**Abstract:** This paper presents the theoretical screening of 23 low-cost deep eutectic solvents (DESs) as absorbents for effective removal of the main impurities from biogas streams using a conductor-like screening model for real solvents (COSMO-RS). Based on thermodynamic parameters, i.e., the activity coefficient, excess enthalpy, and Henry's constant, two DESs composed of choline chloride: urea in a 1:2 molar ratio (ChCl:U 1:2), and choline chloride: oxalic acid in a 1:2 molar ratio (ChCl:OA 1:2) were selected as the most effective absorbents. The σ-profile and σ-potential were used in order to explain the mechanism of the absorptive removal of CO2, H2S, and siloxanes from a biogas stream. In addition, an economic analysis was prepared to demonstrate the competitiveness of new DESs in the sorbents market. The unit cost of 1 m3 of pure bio-methane was estimated to be in the range of 0.35–0.37 EUR, which is comparable to currently used technologies.

**Keywords:** biogas; deep eutectic solvents; upgrading; absorption; COSMO-RS; economic analysis


**2.2 Organic encapsulants**

The organic encapsulants may be developed in two structural scales, such as nano or micro. They include polymers and surfactants that are being introduced to develop PWTCs. It is worth-noting that the surfactants are normally polymers, but precisely, they are thermo-plastic elastomers consisting of co-polymers blocks. The potential polymeric encapsulants include low-density polyethylene (LDPE), highdensity polyethylene (HDPE), melamine-formaldehyde (MF), polypropylene and polyacrylate, etc., while surfactant-based encapsulants are styrene-b-(ethyleneco-butylene)-b-styrene (SEBS) tri-block copolymer, styrene–butadiene–styrene (SBS), olefin block copolymer (OBC) and polystyrene, among others. However, the main challenge with organic encapsulants is that their thermal conductivity is approximately similar to that of paraffin waxes [25]. Therefore, organic encapsulants are only SSs. The need of TCEs with encapsulated PWTCs definitely results in a triplex thermal composite, implying that the latent heat has to be further sacrificed. Such a design challenge can only be resolved by recommending the encapsulated PWTCs for those applications where thermal energy storage is moderately acquired but fast charging/discharging is not a primary objective, for example, in flame retardancy and thermal management of buildings, fabrics and food packages. The fabrication of encapsulated PWTCs is based on dry or wet-physical methods;

however, the fabrication of the encapsulants is usually enabled by the chemical reactions which are complex due to the inclusion of several preparation steps and process conditions [26]. The little variance in process conditions can result in broken capsules (shells), posing the liquid drainage threats for PWTCs. Until recently, encapsulated PWTCs have been successfully fabricated. For example, duplex encapsulated PWTCs have been presented comprising of paraffin wax (melting temperature of 52–54 C, latent heat of 182.6 Jg−1) as a core and MF as shell, as shown in **Figure 9** [27]. With 10 g of paraffin wax and 61.6% encapsulation efficiency, the latent of encapsulated PWTC is 107.4 Jg−1.

It is thus obvious that the encapsulation process covers the several steps at various process constraints to create the final PWTCs. Normally, the morphology is spherical and it is filled with paraffin wax, as shown in Figure, therefore encapsulation efficiency (how much core material can be surrounded and uptaken by the shell of encapsulant) is the fundamental parameter to be emphasized for encapsulated PWTCs. In another example of triplex PWTC is presented where HDPE has been employed as an encapsulant for paraffin wax (melting temperature of 54–56°C and latent heat of 199 jg−1), and copper metal foam has been introduced enhancing the thermal conductivity of HDPE/paraffin wax from 0.72 Wm−1 K−1 to 2.14 Wm−1 K−1 and reducing the latent heat as minimum as 151.6 jg−1. The PWTCs also

#### **Figure 10.**

*Thermal stability of paraffin wax and PWTCs via TGA [30].*

provided favorable shape-stability, allowing 2.3% mass loss of paraffin wax after 50 thermal cycles [28]. In a word, the stronger the encapsulant shell is, the more effective is the shape-stability. Generally, several other examples are also found where carbonic thermal reinforcements have been introduced to accelerate thermal response of encapsulated PWTCs, such as EG [25, 26].

Apart from the salient merits of providing shape-stabilization, organic encapsulants are thermally stable too [29], imparting the adequate thermal stability to PWTCs. For example, SEBS has been employed to encapsulate paraffin wax (melting temperature of 52–54°C and latent heat of 176.6 jg−1) in different percent contents (5–20%), and 20% of SEBS enhances the thermal decomposition temperature of PWTC to around 221.4°C, as demonstrated in **Figure 10** [30].

## **3. Bottlenecks, recommendations and design standards for PWTCs**

The fabrication of PWTCs is simple until investigated recently; nonetheless thermo-physical bottlenecks of the paraffin waxes still exist. For example, their inert features do not allow chemical reactions, so the utilization of chemical methods for fabrication of PWTCs can be overlooked. Thus, physical methods are only viable options for the fabrication of PWTCs. However, physical methods can also be wet or dry, for example, mixing the melted paraffin waxes with thermal reinforcement is the dry-physical method, while dissolving the paraffin wax into a solvent and then mix with thermal reinforcement is the wet-physical method or encapsulating the paraffin wax in the capsules is also the wet-physical method. Meanwhile, the shape-stabilization can be achieved via both methods, but thermal conductivity improvements have abundantly been reported based on dry-physical methods. Besides, thermal conductivity of PWTCs prepared via dry-physical methods is not sufficiently high, leaving a wide research roam and urging to put rigorous efforts into this serious matter. However, the PWTCs with increased thermal conductivity suffer from another challenge which consists of a short and uprising isothermal zone, indicating the reduced duration of temperature-control capacity. The uprising isothermal zone dictates that the temperature-control capacity of PWTC is not perfectly constant, but nearly constant and fast. A case-study describing this standpoint can be seen above in **Figure 7**.

Thus, either maximum or minimum, threshold limit of the thermal reinforcements is very important in PWTCs, which is however not standardized yet. Thermal reinforcements always demand the equal replacement of base material in PWTCs, signifying that the equivalent reduction in latent heat storage capability [12]. In such a situation, the design parameters of PWTCs should be adjusted according to the target applications. For example, thermal management of photovoltaic panel is required to be done through PWTC. In this case, the liquid drainage of the PWTC may not be considered a design parameter of the primary importance because the mechanical enclosure can assist in controlling the liquid drainage, meaning that the form-stability of PWTC can be neglected. However, the heat accumulation at the interface of PV and the hot-spots in the PWTC body are altogether supposed to be the primary design parameters. Therefore, thermal reinforcements for such applications should solely be the TCEs that can help fabricate the duplex PWTCs. Counter-institutively, Another case can be discussed regarding the thermal management of buildings where PWTCs should capable of ensuring prolonged temperature-control capacity as well as adequate shape-stability. So, thermal reinforcements for such applications should solely be the SSs, but the SSs needs to conform the property prerequisites of the building ingredients like cement, sand, and clay, etc., which altogether leads to the triplex PWTCs. In between these two scenarios, special applications such as thermal management of satellites, robots, and astronauts can be put forth, where the total quantity of PWTC emerges is another design parameter together with consideration of temperature-control capacity and shape-stabilization.

PWTCs can also suffer from the challenge of saturation energy storage limit which is dependent on the total thermal energy storage (sensible heat + latent heat)

**Figure 11.** *Proposal of design standards for PWTCs.*

#### *Paraffin Wax-Based Thermal Composites DOI: http://dx.doi.org/10.5772/intechopen.97195*

of paraffin wax. This challenge is expected to emerge in all kinds of applications. Therefore, it is strongly recommended to evaluate the total saturation duration that can anticipate saturation energy storage limit. Accordingly, the periodic heat regeneration of PWTCs, i.e., extracting the stored thermal energy, becomes essential to keep the whole thermal management system fresh for the coming cycles. The possible methods for periodic heat regeneration encompass the natural convection or forced convection achieved either via air or water for which deep design considerations of the whole thermal management systems are required.

Among thermal reinforcements, EG can act both as TCE and SS [29, 31], pointing out that duplex PTWCs can be fabricated for thermal management with high charging/discharging rates. Therefore, EG is declared to be the most effective carbonic thermal reinforcement.

Although the design standardization of the optimal PWTCs is complicated, the parameters as-proposed in **Figure 11** may serve as the preliminary design principles.


**5. The role of institutions in fostering entrepreneurship**

Interactions between institutions and the business environment are also fundamental for economic and social development. The difficulties that emerge in these partnerships might inhibit entrepreneurs' desire to invest, which would be detrimental to both parties because it decreases the productivity of the businesses and consequently reduces the level of public sector funding in these circumstances, new institutional

#### *Entrepreneurship in Emerging Economies: The Role of Innovation and Institutions DOI: http://dx.doi.org/10.5772/intechopen.109893*

strategies are required to address the interchange issues that stimulate economic growth and significantly affect the nature of economic development. A phenomenon that we can refer to as Institutional Economics is produced as a consequence of the role that institutions play in the development of entrepreneurship [25]. This phenomenon suggests a particular method for examining theoretical and real-world economic issues in relation to social and institutional developments. With this method, the connections between economic and non-economic processes can be better understood [26]. The functioning of phenomena including economic agent behavior, supply and demand laws, market mechanism activity, and transaction management is based on widely accepted institutions [27]. These institutions' principal purpose is to establish standards or basic rules that control behavior and influence how entrepreneurs interact with one another in the entrepreneurial ecosystem. Institutions are essentially the civic or organizational norms that facilitate interaction between entrepreneurs by helping them establish reasonable expectations that one can have toward the other.

At the same time, plays the role of controlled models of human interactions, and the term *institution* refers to both formal rules and their application characteristics as well as informal limitations. This idea also outlines a certain way that social and political connections are organized in accordance with the laws that have been developed in various domains of endeavor. Thus, it is possible to distinguish between two notions of institutions: the first refers to the idea of an institution as a set of policies guiding people's behavior and the second is the idea of an institution as a distinct entity [28]. In some circumstances, it is reasonably obvious to distinguish between institutions as a set of regulations and institutions as actual entities or organizations. This category includes businesses, labor unions, and political parties. Guidelines for how these entities should be represented are on the one hand, and the organizations that have been designated as such are on the other. The institutions that govern transactions specify whose preferences matter and how these preferences will be taken into account during the process of production, distribution, and consumption is actually the rules that govern economic interactions and relationships. By creating a solid framework for how people interact with the economy, institutions serve as a primary tool for increasing human behavior predictability and reducing uncertainty. As a result, the term institution can be defined as a system of laws that can decrease the uncertainty that is frequently linked to human conduct while initiating, launching, developing, and completing transactions [29, 30]. The analysis that tracks the linkages with various types of institutions is referred to as "the function of institutions in the impact that entrepreneurship has on economic development" [31].

The requirement for entrepreneurial education is the first aspect that stands out when considering ties with academic and research institutes. Modern civilization places higher demands on the educational system, resulting in a social structure that did not exist before. These changes are primarily brought about by the substantial developments of the last few decades and the demand for proactive, independent individuals who can integrate into society and participate in its ongoing development. In this context, educational institutions at all levels must adopt particular twenty-first-century tools and methods to provide the ideal educational environment and foster creativity, innovation, and the capacity for a global perspective [32]. The capacity to develop leadership and interpersonal skills through an entrepreneurial mindset is a significant asset that is gaining a growing amount of attention. The entrepreneurial spirit and innovation emerge in new approaches to learning, living, and working. For the incorporation of entrepreneurship and innovation [33], transdisciplinary approaches, and interactive teaching methods in education, different

models, architectures, and standards are expected. Undoubtedly, current systems must be evaluated, and the worldwide educational system needs to be fundamentally reformed. In order to provide the economic environment with a workforce that is educated and capable of fostering economic development, changes in the educational system are required at all levels [34]. These improvements should focus on the process of continuous learning as well as the essential connections and interactions between levels (secondary, high school, higher, and continuing education). On the other hand, time is critical to bear in mind this relationship's vulnerability, which is the formation of institutional commitment. Institutions of higher learning must alter their curricula to support the development of twenty-first-century competencies [35]. In this regard, institutional commitment is essential, along with clear strategies and realistic action plans. However, at moment, there is no concrete application of any discussions relating to entrepreneurship education in terms that students may use. Academic institutions must turn the debates' conclusions into practice, which involves providing students and instructors with the appropriate motivation and support. At this time, governments everywhere must act to narrow the widening competency gaps. At the highest political levels, there must be a consistent and unequivocal commitment to this. Both the strategic framework within which schools and universities can work to implement programs and activities inside their institutions, as well as a clear signal of support for entrepreneurship, should be conveyed through policies. Better action and coordination are required at the national, regional, and local levels to achieve objectives [36]. A key component of developing policies and implementing programs is including stakeholders from academia, industry, the NGO sector, and institutions.

#### **5.1 Learning organizations and innovation systems**

In a knowledge-based economy, companies search for connections to foster interactive learning among companies and outside partners and networks search for methods to offer complementary assets. Through these connections, businesses can access cutting-edge research findings, obtain vital technology components, reduce the costs and risks of innovation, and pool resources for production, marketing, and distribution [37]. Companies choose the tasks they will carry out on their own, collaboratively with other enterprises, with universities or research organizations, and with government support as they create new goods and processes. Thus, innovation is the outcome of various interactions between institutions and actors, which collectively make up an innovation system [38]. The interactions within these systems have an impact on how innovatively successful businesses perform, which in turn has an impact on the economy. These innovation systems are made up of the flows and relationships between emerging industry, government, and academia, as well as the advancement of science and technology. Therefore, one factor influencing prosperity is the system's ability for knowledge distribution, or its capacity to guarantee innovators' timely access to relevant knowledge resources.

In a knowledge-based economy, knowledge serves as the primary resource. The level of knowledge and information incorporation in economic activity is now so high that it results in rather significant structural and qualitative changes in how the economy functions, altering the foundation of competitive advantage. The value of knowledge has increased for all economic system participants as a result of the growing competitiveness of the global knowledge economy and our willingness to disseminate knowledge [39]. This has significant ramifications for company goals, governmental policies, as well as the institutions and processes in place to control economic behaviors.

*Entrepreneurship in Emerging Economies: The Role of Innovation and Institutions DOI: http://dx.doi.org/10.5772/intechopen.109893*

A system of consumption and production based on intellectual capital is known as the new economy. It often makes up a sizable portion of all economic activity in developed nations. In the knowledge-based economy, intangible assets, such as the value of employees' knowledge or intellectual capital, can account for a sizeable portion of a company's worth [40]. However, businesses are not authorized to incorporate these assets on their balance sheets in accordance with generally accepted accounting policies. The global economy has transitioned to a knowledge-based economy in the digital era, bringing with it the best practices from each country's economic growth [41]. Additionally, knowledge-based development elements produce an interconnected global economy where information sources, such as human expertise, are critical drivers of economic growth and are regarded as significant economic assets.

#### **6. Conclusions**

Economic development benefits remarkably from entrepreneurship. The approach considered to be the most important is innovation, which includes creating new goods and procedures, finding untapped sources of supply, coming up with creative ways to manage a business, as well as leveraging on new markets. The main contribution of analyses in innovation-driven countries is a broader knowledge of the causes and effects of innovation in developing countries, as well as of the institutions and policies that promote or inhibit innovation. Three important conclusions that may be drawn from this chapter are as follows:

• Finally, the institutional and political environment does have a significant impact on how innovative an entrepreneur is. This aspect explains why entrepreneurs with remarkably comparable characteristics may exhibit divergent outcomes in terms of their innovative performance. To encourage innovation, a solid organizational framework and an efficient innovation infrastructure are needed.

The necessity for both direct and indirect government support for innovation is essential for sustainable development in the twenty-first-century entrepreneurial ecosystem. The business environment, grants for research and development, risk and venture capital assurance, maximizing the potential of migrant workers and the diaspora, enhancing technical and managerial education, expanding infrastructure, and active public partnerships are just a few illustrations of the several different manners this can be accomplished. By fostering competition, an adverse climate can often encourage innovative behavior, and entrepreneurs themselves can serve as catalysts for political and institutional change.

Given the current and upcoming challenges facing global development, entrepreneurial innovation is going to become increasingly important. These include ongoing socioeconomic inequalities as well as the rising susceptibility of nations to outside shocks such as financial crises, calamities, and threats like environmental issues.

Innovative entrepreneurship is fundamental to overcoming these obstacles and seizing the opportunities they offer, and the idea itself ought to be expanded. In particular, increasing roles for social entrepreneurship, public entrepreneurship, institutional entrepreneurship, and even sovereign non-state entrepreneurship are examined.

To better comprehend the innovative contributions of various business types and forms of entrepreneurship in countries with various levels of development, it is important to comprehend the current challenges:

The current chapter has improved our understanding of how different forms of entrepreneurship prosper in different environments and at different stages of development. However, we definitely must conduct additional research and analysis and look for the most appropriate patterns for different configurations and developmental stages.

It is still difficult to create institutions, groups, and regulations that successfully encourage both invention and entrepreneurship. The fact that developing nations usually lack the capacity and resources to conduct significant, evidence-based research makes this process increasingly challenging. Future research will concentrate on how to react to the junction of the fields of entrepreneurship, development economics, and innovation studies in order to support this argument.

Several dynamic influences, such technology advancement, economic fluctuations, or demographic shifts, have reshaped societies all over the world and provided new challenges as well as opportunities to business ecosystems. Governments, public and private organizations, as well as economic actors, are gradually becoming more conscious of the significance of entrepreneurship, which is seen as a multifaceted

#### *Entrepreneurship in Emerging Economies: The Role of Innovation and Institutions DOI: http://dx.doi.org/10.5772/intechopen.109893*

phenomenon and one of the leading drivers of economic development on all stages, from the local to the global level.

Scientific research on entrepreneurship and economic development became more prominent in the second decade of the twenty-first century, alongside technological innovation. Both concepts have established themselves as significant research topics for economists and researchers around the world who, in the context of globalization, have worked to develop new indicators that could quantify the impacts of the two beliefs. Additionally, there is increasing interest in the nexus between entrepreneurship and economic development as a method of explaining changes in economic performance throughout time. Entrepreneurship, a common component of human activity, is critical to the growth of the economy. Today, entrepreneurship is widely recognized as an important element of economic progress on a global scale by professionals and academics everywhere.

However, entrepreneurship has both positive and negative effects on economic growth. Economic development and entrepreneurship are now closely linked in today's modern global economy, and economic progress can emerge from the creation of a critical mass of effective policies and initiatives. This transition requires the implementation of an adapted strategy and governance, which is particularly difficult for large corporations. These conglomerates will be under pressure to advocate for the establishment of a separate sector for artificial intelligence and spin-offs. To avoid the imbalance between businesses that can apply AI compared to those that cannot, it is necessary to take into consideration and leverage the fact that the overwhelming majority of AI investors are actually the leading players in the technology economy.

A significant increase in productivity results from the economic utilization of artificial intelligence. As a technical innovation, it contributes to both the inputs—consumer goods or services—and the outputs—internal processes like management, logistics, or customer service. A substantial loss of competitiveness could result from the inability to adapt to these technologies. However, it is important to consider the risks associated with economic dependency, sovereignty, and the potential for increased inequality. It is imperative to remove the possibility of turning businesses into consumers of solutions created offshore given the current structure of the digital economy. The platforming and value-capturing effects that are now characteristic of the digital economy could then be further improved by the development of artificial intelligence. Few actors in the economic arena have the data and computational resources that this development requires. The ability to interpret these technologies' significance and contents also need to be preserved due to their ability to be used as decision-supporting or even decision-making tools. This is important because the dissemination of artificial intelligence-derived technologies has the potential to cause significant social and territorial inequality, particularly because of the concentration of wealth in some areas.

The global business environment is evolving due to the digital revolution, which has changed methods and approaches for creating economic ecosystems as well as processes and systems. To be competitive in the context of global competitiveness, companies worldwide must take advantage of this new environment. The establishment of start-ups and the transformation of existing businesses through the development of new digital technologies are generally understood as examples of the digitization of entrepreneurship. These activities are perceived as a pillar of innovation, prosperity, and employment generation in many economies. As we observe a variety of global initiatives aimed at stimulating the acceleration of entrepreneurial activity, a holistic and integrated approach is required. A nation's capacity for digital entrepreneurship mainly relies on digital entrepreneurial behavior, culture, and strategies, as well as an

innovation ecosystem where governments, industries, businesses, educational institutions, and NGOs operate around each other. Entrepreneurship thrives in ecosystems where all stakeholders take an active role. Education and practice in particular should be more integrated to guarantee that future abilities meet employment prospects. Students should be given the chance to experience and practice entrepreneurship education, and the academic environment should be encouraged to connect with the solid business community to incorporate these abilities into the learning process. It is important to raise awareness and increase access to good practice models in order to motivate youngsters to reach their full potential. In this regard, it is essential to broaden partnerships among interested parties.

To ensure sustainable economic development, the strategy's actions must be focused on: fostering cooperation and relationships between institutions and the entrepreneurial ecosystem; establishing business-friendly governance by developing a dialog strategy between the factors involved (public institutions and the business environment); strengthening entrepreneurial capacities; and supporting, encouraging, and promoting local businesses by training providers. The institutions' purpose is to improve the business environment by strengthening the potential of SMEs and their role in economic growth, building confidence between the business community and the government, and overall fostering a more supportive business environment.

The qualities of the entrepreneurial ecosystem that have been emphasized in this chapter's examination of the function of institutions lead us to assume that this relationship is significant because it is a mechanism through which various participants from the public sector, the business community, and non-governmental sector cooperate to enhance the conditions for economic growth and to generate employment. In order to improve the quality of life for everyone in the community, dynamic entrepreneurial culture is built, maintained, and tangible resources are generated for the community and the business environment. The amount of time and money required to establish a strategy and a public-private partnership toward economic growth that is under the control of the community largely depends on the current condition in addition to the expertise and competence of the actors and institutions involved.

Underestimating the institutional framework is the cause of the inadequate performance of businesses in the sector since institutions' primary purpose in society is to eliminate uncertainty by creating a stable foundation for human interactions. Therefore, the slow growth of the entrepreneurial ecosystem has been primarily caused by the absence of sufficient forms of social and political relationship organization. In order to obtain an agreement on these concerns, it is fundamental that the interested parties, the entrepreneurs, are thoroughly consulted in a participatory process when formulating policies for the expansion of entrepreneurship as well as when creating institutions. In contrast to when these structures are imposed by external authorities, the establishment of local institutions that are based on the needs of the economic environment and that would benefit from improved collaboration with the locals can lead to significantly greater performances and reduced costs.

*Entrepreneurship in Emerging Economies: The Role of Innovation and Institutions DOI: http://dx.doi.org/10.5772/intechopen.109893*