*Article* **Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from** *Trapa bispinosa* **Roxb.**

**Shijie Ye 1, Dongjie Yin 1, Xiaoyan Sun 1, Qinyi Chen 1, Ting Min 2, Hongxun Wang <sup>1</sup> and Limei Wang 1,\***


**\*** Correspondence: wanglimeiyx@whpu.edu.cn; Tel.: +86-27-8395-6793

**Abstract:** *Trapa bispinosa* Roxb. is an economical crop for medicine and food. Its roots, stems, leaves, and pulp have medicinal applications, and its shell is rich in active ingredients and is considered to have a high medicinal value. One of the main functional components of the *Trapa bispinosa* Roxb. shell is 1-galloyl-beta-D-glucose (βG), which can be used in medical treatment and is also an essential substrate for synthesizing the anticancer drug beta-penta-o-Galloyl-glucosen (PGG). Furthermore, gallate 1-beta-glucosyltransferase (EC 2.4.1.136) has been found to catalyze gallic acid (GA) and uridine diphosphate glucose (UDPG) to synthesize βG. In our previous study, significant differences in βG content were observed in different tissues of *Trapa bispinosa* Roxb. In this study, *Trapa bispinosa* Roxb. was used to clone 1500 bp of the UGGT gene, which was named TbUGGT, to encode 499 amino acids. According to the specificity of the endogenous expression of foreign genes in *Escherichia coli*, the adaptation codon of the cloned original genes was optimized for improved expression. Bioinformatic and phylogenetic tree analyses revealed the high homology of TbUGGT with squalene synthases from other plants. The TbUGGT gene was constructed into a PET-28a expression vector and then transferred into *Escherichia coli* Transsetta (DE3) for expression. The recombinant protein had a molecular weight of 55 kDa and was detected using SDS-PAGE. The proteins were purified using multiple fermentation cultures to simulate the intracellular environment, and a substrate was added for in vitro reaction. After the enzymatic reaction, the levels of βG in the product were analyzed using HPLC and LC-MS, indicating the catalytic activity of TbUGGT. The cloning and functional analysis of TbUGGT may lay the foundation for further study on the complete synthesis of βG in *E. coli*.

**Keywords:** *Trapa bispinosa* Roxb.; TbUGGT; molecular cloning; expression analysis

### **1. Introduction**

*Trapa bispinosa* Roxb. is an annual herbaceous floating plant belonging to Myrtle's *Trapa bispinosa* family and is mainly distributed in tropical and temperate regions [1,2]. Ripe *Trapa bispinosa* Roxb. has a hard deep red shell and creamy white flesh with a sweet taste. It can be used to treat common diseases, such as gastric ulcers, esophageal cancer, and dysentery [3]. The fruit hulls are rich in phenols and flavonoids and have been extensively studied. The extracts from *Trapa bispinosa* Roxb. shell contain many phenolic compounds, such as gallic acid, caffeic acid, naringin, and 1,2,3,4,6-pentagalacyl-β-D-glucose [4–6]. These phenolic compounds have specific physiological antioxidant, anti-inflammatory, and anticancer properties [7–10]. *Trapa bispinosa* Roxb. chestnut is native to Europe and Asia but is only cultivated in China and India [11]. The plant exists in most water bodies in China but is considered one of the aquatic specialties of the Hubei Province and has high economic value.

The compound 1-galloyl-beta-D-glucose (βG) exists in plants such as oak leaves, *Trapa bispinosa* Roxb., and pomegranate and possesses a variety of pharmacological activi-

**Citation:** Ye, S.; Yin, D.; Sun, X.; Chen, Q.; Min, T.; Wang, H.; Wang, L. Molecular Cloning, Expression, and Functional Analysis of Glycosyltransferase (TbUGGT) Gene from *Trapa bispinosa* Roxb. *Molecules* **2022**, *27*, 8374. https://doi.org/ 10.3390/molecules27238374

Academic Editors: Giovanni Ribaudo and Lucia Panzella

Received: 13 October 2022 Accepted: 22 November 2022 Published: 30 November 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 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 (https:// creativecommons.org/licenses/by/ 4.0/).

ties. Studies have shown that βG is a noncytotoxic and selective AKR1B1 inhibitor of aldose reductase, which can protect against oxidative stress and treat secondary complications of diabetes [12]. In a previous study, the protective properties of βG and its mitochondrial antioxidative mechanism reduced the effects of oxidative stress in glaucoma [13]. In Raw 267.4 macrophages, βG prevented LPS-induced activation of JNK and p38 and lowered ROS levels [14]. APRE-19 cells pretreated with βG demonstrated decreased apoptosis induced by retinal microglia [15]. Furthermore, βG inhibited the activation of the NLRP3 and TLR4/NF-κB pathways and decreased the expression of pro-inflammatory cytokines, protecting against LPS-induced sepsis in mice and reducing organ toxicity [16]. The substance has a wide range of applications in clinical practice, but βG is primarily produced via plant extraction, which does not yield high quantities. Therefore, chemical synthesis methods are also being developed but are in opposition to the green concept of modern production. At the same time, biosynthesis has become a popular method for material acquisition. In this study, we aim to develop a βG production method using biosynthesis to meet the medical application demand.

Glycosyltransferase (EC 2.4.x.y) is an enzyme that can transfer activated glycol groups to other small-molecule compounds to complete glycosylation reactions [17]. UGT is a soluble enzyme in plants, with UDPG being the leading sugar donor [18]. According to the different substrate small molecules, the UGT family can be divided into UGGT with gallic acid as the substrate and UDP-glucose with flavonoids as the substrate, such as flavonoid 3-glucosyltransferase (UFGT) [19], etc. UGGT plays an essential role in catalyzing the formation of βG in plant tannin biosynthesis.

U-glutamyl transpeptidase (UGGT) catalyzes the synthesis of βG from gallic acid (GA) and uridine diphosphate glucose (UDPG) [20]. In some higher plants, GA is synthesized via the shikimic acid pathway and converts 3-dehydroshikimic acid to 3,5-dehydroshikimic acid through aroDE enzymes. In addition, 3,5-dehydroshikimic acid can be spontaneously converted to GA via enolization [21–23]. Figure 1 displays the chemical reaction scheme for βG synthesis.

**Figure 1.** Biosynthesis of βG in some higher plants.

Our previous research study identified this substance in *Trapa bispinosa* Roxb., with varying amounts in different tissues [24]. So far, most of the studies on *Trapa bispinosa* Roxb. have focused on the separation and pharmacological effects of active monomer compounds. However, the specific biosynthetic mechanism of the active components of *Trapa bispinosa* Roxb. has rarely been explored. Currently, the secondary metabolites identified in *Trapa bispinosa* Roxb. contain multiple types of galacyl glucose, using the biosynthetic βG of galacyl glucose as the substrate. However, the gene for its synthesis has not been reported.

The TbUGGT gene sequence was obtained with gene annotation and screening using transcriptomics technology. After codon optimization, the recombinant expression vector was constructed and expressed in *Escherichia coli,* and the enzyme activity was determined. This study lays the foundation for future research on the complete synthesis of βG in *Escherichia coli*.

#### **2. Results**

#### *2.1. TbUGGT Gene Cloning and Sequence Analysis*

The βG biosynthesis pathway starts from phosphoenolpyruvate and D-erythritose 4-phosphate (Figure 2a). Combined with the transcriptome data of *Trapa bispinosa* Roxb., the Unigene expression belonging to this pathway detected using RNA-Seq was analyzed and displayed with a heat map (Figure 2b). A total of 44 Unigenes belonging to this pathway were identified using the transcriptome, which was involved in four genes of the pathway, namely, 3-deoxy-D-Arabino-Heptulosonate 7-phosphate synthase (EC 2.5.1.54), 3-dehydroquinate synthase (EC4.2.3.4), 3-Dehydroquinate dehydratase/Shikimate dehydrogenase (EC 4.2.1.10/EC 1.1.1.25), and gallate 1-beta-glucosyltransferase (EC 2.4.1.136). The primary glycosyltransferase gene TbUGGT (CL7060.4) was obtained.

**Figure 2.** βG biosynthesis pathway (**a**) and the expression of related genes (**b**).

The *Trapa bispinosa* Roxb. transferase gene TbUGGT was amplified with PCR using specific primers and the preferred codon optimization of *Escherichia coli*. The gene had a total length of 1500 bp, encoding 499 amino acids. The molecular weight of the protein sequence predicted using ProtParam was about 55.8 kDa and was an unstable hydrophilic protein.

qRT-PCR was used to detect the expression pattern of TbUGGT and identify the expression of the TbUGGT gene in different tissues of *Trapa bispinosa* Roxb., namely, shell (FR), leaf (LR), stem (ST), and root (RT) (Figure 3). Apparent differences in transcription levels were observed in different parts, the highest being shell (FR) expression, and the root (RT) expression being the second. In contrast, the stem (ST) and leaf (LR) yielded relatively low expressions.

According to the conservative structural domain analysis (Figure S1 Supplementary Material), CD Search predicted that the TbUGGT protein belonged to the glycosyltransferase\_GTB-Type (PLN02555) superfamily with a domain range of 1aa–473aa (Figure S1a). ScanProsite predicted that the protein belonged to the UDP Glycosyltransferases superfamily with a domain range of 343aa–386aa (Figure S1b), while Pfam predicted that the protein belonged to the UDPGT family. The domain range was 238aa–423aa (Figure S1c). ProtScale indicated that the TbUGGT protein was hydrophilic (Figure S2). The signal peptide prediction showed no signal peptides in this protein, and the probability of amino acids in each point appearing outside the membrane was close to 1, demonstrating a low probability to appear in the transmembrane region. Therefore, this protein was not a membrane or secreted protein (Figure S3).

**Figure 3.** Differential expression of TbUGGT:TbUGGT in different growth sites, including shell (FR), leaf (LR), stem (ST), and root (RT).

#### *2.2. Structure and Phylogenetic Analyses*

SOPMA showed that the secondary structure of the TbUGGT protein sequence contained α helices (blue), extended chains (red), β rotations (green), and random curls (purple), accounting for 40.48%, 14.23%, 4.21%, and 41.08%, respectively (Figure S4). The protein structure of TbUGGT was predicted using AlphaFold2, in which the model pLDDT was as high as 91.8. (Figure 4a). pLDDT ≥ 90 means that the residue has very high model confidence, which the model can use for later molecular docking analyses. The homology modeling structure was analyzed using PyMOL software (Figure 4b). The docking results between the protein model and the substrate GA molecule showed a binding energy of −6.3. A smaller binding energy indicated a tighter binding between the receptor and the ligand. Visualization revealed that the binding sites were mainly concentrated in Glu at position 139, in Ile at position 143, in Cys at position 145, and in Lys at position 218.

**Figure 4.** AlphaFold2 predicted TbUGGT protein structure model (**a**) and PyMOL software molecular docking results (**b**).

In the homology analysis of the TbUGGT protein sequence, the compared species included *Punica granatum*, *Syzygium oleosum*, *Eucalyptus Grandis*, *Corymbia Citriodora* subsp. Variegata, *Eucalyptus Camaldulensis*, *Rhodamnia argentea*, *Juglans regia*, *Carya illinoinensis*, and *Vitis Vinifera*. The results showed a similarity of 87.60% between the protein and the compared sequence (Figure 5). About 44 amino acid residues in the blue underlined part of Figure 5 correspond to the conservative domain PSPG of glycosyltransferase [25], which is the binding region of glycosyl donors, suggesting that the cloned gene was the UDP glycosyltransferase gene.

**Figure 5.** Comparison of TbUGGT amino acid sequences obtained from GenBank. The species, protein names, and GenBank accession number of the aligned sequences are as follows: *Corymbia Citriodora* subsp. variegata (KAF8013769.1); *Eucalyptustus grandis* (XP 01 0028410.1); *Rhodamniania argentea* (XP 030523235.1); *Syzygiumium oleosum* (XP 030445800.1); *Punicaica granatum* (XP 031382117.1); *Eucalyptus Camaldulensis* (BBB21213.1); *Vitis Vinifera* (XP 002285379.1); *Caryaya illinoinensis* (KAG7977068.1); *Juglansins regia* (XP 018827666.1).

MEGA was used to discuss the phylogenetic relationship between TbUGGT protein sequences and the corresponding proteins in different species. The UGT amino acid sequences of 20 plants were downloaded from the GenBank database for a cluster analysis (Figure 6). Higher scores indicated a closer relationship (the maximum score was 100). The closest relationship occurred between *Trapa bispinosa* Roxb. and pomegranate.

**Figure 6.** Phylogenetic tree of TbUGGT from various species. • *Trapa bispinosa* Roxb.\*: The target gene TbUGGT in this study. The species, protein names, and GenBank accession number are *Eucalyptus Grandis* (XP010028410.1), *Corymbia Citriodora* subsp. Variegata (KAF8013769.1), *Vitis amurensis* (CZS70601.1), *Syzygium oleosum* (XP030445800.1), *Punica granatum* (XP031382117.1), *Eucalyptus Camaldulensis* (BBB21213.1), *Jatropha curcas* (KDP45909.1), *Ricinus communis* (XP002518668.1), *Hevea brasiliensis* (XP021664419.1), *Manihot esculenta* (XP 021619116.1), *Vitis Riparia* (XP 034681626.1), *Vitis labrusca* (ABH03018.1), *Vitis Vinifera* (XP002285379.1), *Vitis quinquangularis* (ASR73556.1), *Rhodamnia argentea* (XP030523235.1), *Cephalotus follicularis* (GAV61182.1), *Canarium album* (QZM06937.1), *Mangifera indica* (XP044479746.1), and *Pistacia vera* (XP031284307.1).

#### *2.3. Prokaryotic Expression of TbUGGT*

To obtain the recombinant expression strain, the recombinant plasmid PET-28a-Tbuggt was transformed into the *Escherichia coli* BL21(DE3) expression strain after colony PCR identification. IPTG was used as the inducer to induce fusion protein expression, and the bands were verified using SDS-PAGE electrophoresis (Figure 7a). Compared with the blank control group, specific protein bands of about 55 kDa (theoretically predicted value of 59.6 kDa) appeared in the experimental group, as indicated by the arrow in the figure.

Furthermore, the recombinant protein was purified with mass culture to eliminate the interference of other proteins, and the bacteria and the bacterial liquid were detected using SDS-PAGE (Figure 7b). The target protein band appeared in the bacterial lane at around 55 kDa, while the protein band did not appear in the bacterial liquid lane, indicating that the protein was expressed in *Escherichia coli*. However, the expressed proteins were mainly concentrated in the bacterial solution, and most of them existed in the form of inclusion bodies, which were broken to release the proteins. SDS-PAGE was used to detect the protein before and after purification (Figure 7b), and the target protein bands appeared at about 55 kDa, indicating that the protein was successfully expressed and purified in *Escherichia coli*.

**Figure 7.** SDS-PAGE results of whole bacterial protein of recombinant strain containing pET-28a-TbUGGT (**a**) and SDS-PAGE results of purified protein (**b**). (**a**) Lane M, protein marker; Lane C1, non-induced whole bacterial protein containing recombinant plasmid; Lane C2, non-induced whole bacterial protein containing empty vector; Lane C3, whole bacterial protein containing recombinant plasmid after induction; Lane C4, whole bacterial protein containing empty carrier after induction. (**b**) Lane M, protein marker; Lane CL1, supernatant before bacterial fragmentation; Lane CL2, purified protein concentrate.

#### *2.4. Determination of Enzyme Activity of TbUGGT Protein In Vitro*

The standard substances of βG, GA, and UDPG were detected under unified-liquidphase conditions. The liquid-phase detection results (Figure 8) showed that the retention times of the three reference substances were 2.487 min for GA, 7.662 min for UDPG, and 6.662 min for βG. The experimental group showed a signal peak at 6.700, with a retention time similar to that of standard βG, indicating the successful production of βG. In order to confirm that the produced substance was indeed βG, LC-MS was used to verify the material composition of the sample and the blank control (Figure 9). The results showed contrast peaks at 7.28 min~7.90 min. There were βG characteristic ion fragments in the mass spectrum at 7.52 min *m*/*z* = 331.06760. The molecular formula was C13H15O10, and the molecular formula of βG is C13H15O10, which aligned with the negative ion scanning situation.

**Figure 8.** *Cont*.

**Figure 8.** Determination of recombinant TbUGGT enzyme activity using HPLC. (**a**) UDPG(-1 ) and GA(-<sup>2</sup> ) standard; (**b**) βG(-3 ) standard; (**c**) experimental group.

Therefore, it is speculated that the recombinant TbUGGT protein has some enzymatic activity and can catalyze the reaction between GA and UDPG to generate βG. However, the product peak area was small, and the conversion rate was low. Subsequent experiments may consider expanding the culture or increasing the amount of enzyme reaction to increase the yield.

**Figure 9.** *Cont*.

**Figure 9.** Determination of recombinant TbUGGT enzyme activity using LC-MS. (**a**) blank control group; (**b**) sample experiment group (**c**) βG mass spectrometry results.

#### **3. Discussion**

As an alien species, *Trapa bispinosa* Roxb. has been domesticated and cultivated in China [26]. Wuhan, China, is one of the cultivation bases of *Trapa bispinosa*, as the climate and environment are suitable for the growth and development of the plant [27]. Presently, research on *Trapa bispinosa* Roxb. focuses on the extraction of active ingredients [28,29], starch materials [30–32], pharmacological activity [33,34], etc. βG is one of the main active components of riboflavin and has significant medicinal value. Moreover, βG is the primary substrate of PGG, which has anti-cancer properties and has been extensively studied. However, the synthesis mechanism of *Trapa bispinosa* Roxb. remains unelucidated. Therefore, a series of experiments on βG biosynthesis were performed.

Many biochemical reactions are associated with glycosylation, and glycosyltransferase (GA) plays an essential role in plant growth and development, hormone balance, and toxic substance removal through glycosylation [35,36]. Meanwhile, a variety of glycosylation donors are involved in the glycosylation reaction. As a UDP-glycosylation donor-dependent enzyme, UGTs can selectively catalyze the site-directed glycosylation modification of natural and non-natural compounds. These are widely used in research

and discovery of new drugs [37,38]. Here, the full-length gene of TbUGGT was amplified using a high-fidelity enzyme, and its amino acid sequence was compared with the glycosyltransferase of other species. The UGT family is highly conserved in different plant species [39]. The conserved functional domain allows the genes to maintain a certain similarity in catalytic potency. Currently, the UGT crystal structure has been obtained mainly in plants, such as cassava [40], Saffron [41], *Arabidopsis thaliana* [42,43], etc. These UGTs only recognize UDPG as a sugar donor [44]. In subsequent experiments in this study, TbUGGT selectively catalyzed UDPG and GA as substrates to generate βG, confirming the results of the functional analysis.

In this study, during the purification and expression of the TbUGGT protein, most proteins existed in the form of inclusion bodies, as predicted with ProtScale and signal peptide. The protein was a non-membrane and non-secretory protein. As reported in the literature, inclusion body proteins could not be inactivated after ultrasound [45]. Here, the TbUGGT protein was extracted and purified by referring to the particular extraction method of the *Escherichia coli* inclusion body [46]. *Escherichia coli* was used as host bacteria for heterologous expression, producing inclusion bodies and inhibiting protein expression, but its activity remained unaffected. The low content of late catalytic might have been related to the particular processing mode of proteins in *Escherichia coli*, resulting in the protein not being wholly purified [47,48]. The specific reasons need to be investigated in further studies.

The qRT-PCR results demonstrated that the expression levels in the shell and root were higher than those in the other two parts, which may have been related to the influence of phenolic tannins on plant growth. The higher shell expression may have been attributed to the accumulation of plant secondary metabolites in fruits. Studies have shown that the presence of binary phenol or polyphenol may inhibit the activity of indole acetate oxidase, reduce the degree of auxin oxidation, and promote plant growth [49]. Specific concentrations of plant endogenous phenols can enhance their rooting ability. As *Trapa bispinosa* Roxb. is a floating aquatic plant with many roots, the generation of polyphenols is essential to meet its rooting needs. Nevertheless, further research is required to elucidate the specific promoting mechanism.

#### **4. Materials and Methods**

#### *4.1. Plant Materials*

In this study, the plant materials of *Trapa bispinosa* Roxb. were collected in Jiangxia District, Wuhan City, Hubei Province (114.10◦ E, 30.27◦ N). After cleaning, the samples were treated with liquid nitrogen and immediately stored at −80 ◦C.

#### *4.2. RNA Extraction and TbUGGT Enzyme Gene Cloning*

RNA was extracted using The Plant Total RNA Isolation Kit (ENOVA BIO, Wuhan, China) The purity and concentration of RNA were determined using an ultra-micro spectrophotometer (MD2000D) and agarose gel electrophoresis (0.8% agarose). Single-strand cDNA was synthesized with PrimeScript IV 1st Strand cDNA Synthesis Mix (Takara Bio, Beijing, China). Specific primers were based on the TbUGGT sequence information obtained from *Trapa bispinosa* Roxb. transcriptome sequencing and designed using PremierX [24], as shown in Table 1. The TbUGGT enzyme gene was amplified using PCR with PrimeSTAR Max DNA Polymerase (Takara Bio, Beijing, China), and an OMEGA PCR purification kit was used to purify the amplified product. The size and quality of PCR products were determined with agarose gel electrophoresis (0.8% agarose).


**Table 1.** Primer sequences.

#### *4.3. TbUGGT Sequence Analysis and Phylogenetic Prediction*

The physicochemical properties of the protein encoded by the TbUGGT gene were analyzed using ProtParam. Subsequently, conserved protein domains, their families, and functional sites were analyzed with CD Search, ScanProsite, and Pfam. Furthermore, ProtScale was used to analyze protein hydrophilicity, while TMHMM was used to predict the protein transmembrane helical region, and the protein signal peptide was predicted with SignalP. SOPMA was used to predict the protein's secondary structure, and AlphaFold2 was used to construct the protein's three-dimensional structure model [50]. The PDB file of the 3D structural model of the protein was downloaded, and the ligand molecules were downloaded from PubChem. Pymol-2.3.4 and AutoDockTools software applications were used to process the ligands and protein molecules, and Vina software was used for molecular docking. For visualization, the docking file was uploaded to Plip after PyMOL processing. The amino acid sequences of the encoded protein were compared using BLAST, and the homology was analyzed using DNAMAN. Mega-x was used to build the phylogenetic tree. Online website addresses are displayed in Table 2.

**Table 2.** Bioinformatic analysis tools.


#### *4.4. TbUGGT Prokaryotic Expression*

#### (1) Vector construction and small-scale expression

The base sequence of the TbUGGT gene was optimized (GenScript Biotech Corp, Nanjing, China) according to the codon preference of *Escherichia coli*. Specific primers were designed, and restriction sites (EcoRI and NotI) were added, as shown in Table 1. The target gene was constructed in the PET-28a vector and transferred to DH5α. The plasmid was then extracted and sent for nucleic acid sequencing (Sangon Biotech (Shanghai) Co., Ltd., Shanghai, China). The corresponding colonies on the plate were carefully selected and inoculated into a kanamycin medium. The colonies were cultured overnight, and the plasmids were extracted and stored in glycerobacteria. The PET-28a recombinant vector was transformed into *Escherichia coli* BL21 (DE3), and colony PCR verification was performed using T7 primers, as shown in Table 2. BL21 was cultured in Luria-Bertani (LB) with kanamycin until the OD600 value reached about 0.6, and isopropyl -β-d-thiogalactoside

(IPTG) was added at the final concentration of 0.5 mM to induce TbUGGT protein expression. Finally, 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed to detect fusion protein expression.

#### (2) Expression and purification of large amounts of protein

Single colonies containing recombinant plasmids were selected and inoculated into a 10 mL LB liquid medium with corresponding resistance. After overnight culture in a 37 ◦C shaker, the colonies were transferred to 1 L of LB liquid medium and cultured until OD600 reached about 0.6 (duration of 2–3 h). The bacterial control solution was collected, and IPTG was added at a final concentration of 0.5 mM. After shaking the culture at 28 ◦C for 5 h, bacterial precipitates were collected using centrifugation (6000 RPM, 4 ◦C, 10 min), and the precipitates were cleaned twice with PBS to remove the residual medium. The bacterial precipitates were then collected and stored at −20 ◦C for future use.

The fusion protein was purified according to the instructions of the His-Tag Protein Purification Kit (Beyotime Biotechnology, Haimen, China). Subsequently, four milliliters of non-denatured lysate was added per gram of bacterial precipitate and complete suspension. The bacteria supernatant was collected using centrifugation. The BeyoGoldTM His-tag packaging column was prepared, and the upper cleaning column was loaded and washed 5 times with 1 mL of washing liquid; then, 0.5 mL of eluent was used ten times. The eluate of each tube was detected using SDS-PAGE electrophoresis, and the eluate that met the requirements was combined. The eluent was concentrated using an ultrafiltration tube, and SDS-PAGE was performed to detect 10 μL of the concentrated protein. The remaining concentrated solution was stored at −80 ◦C.

#### *4.5. Enzyme Activity Detection of TbUGGT Protein*

The total enzymatic reaction system was 100 μL, and 0.5 mM 3,4,5-trihydroxy benzoic acid (GA) and 2.5 mM uridine diphosphate glucose (UDPG) were added. Furthermore, 100 mM MES buffer containing 0.1% β-mercaptoethanol was used to provide a buffer environment. Next, the purified enzyme solution was added to the experimental group, while the enzyme solution was not added to the control group. After 3 h of reaction at 30 ◦C, methanol was added to terminate the reaction, and HPLC and LC-MS were used for detection. The liquid-phase conditions and methods are described below.

HPLC: chromatographic column, Agilent C18 column; mobile phase, 1% acetic acid water (A) and acetonitrile (B); injection volume, 20 μL; flow rate, 1.0 mL/min; column temperature, 35 ◦C; detection wavelength, 280 nm. Liquid-phase method: 0–10 min, 3–5% B; 10–15 min, 5–50% B; 15–25 min, 50–5% B; 25–30 min, 5–3% B; 30–35 min, 3% B. The liquid-phase diagram of standard βG was compared with the experimental results to confirm product formation.

LC-MS: chromatographic column, Waters ACQUITY C18 column (50 mm × 2.1 mm, 1.7 μm); mobile phase, 0.2% formic acid aqueous solution (A) and acetonitrile (B). Gradient elution: 0~1.5 min, 93% A; 1.5~8 min, 93%~80% A; 8~15 min, 80%~75% A. Volume flow rate, 0.4 mL/min; injection volume, 4 μL; column temperature, 35 ◦C.

Mass spectrometry conditions: negative ion scanning mode (ESI; *m*/*z* 100~1400); capillary voltage, 2.64 Kv; collision voltage, 45 V; drying gas temperature, 350 ◦C; source temperature, 150 ◦C; desolvent gas, N2, 800 L/Hr.

#### *4.6. TbUGGT Expression Pattern*

Four samples, including shell (FR), leaf (LR), stem (ST), and root (FR), were selected from the samples frozen at −80 ◦C, and the total RNA of the four samples was extracted using the Kit method. An ultra-micro spectrophotometer (MD2000D) and agarose gel electrophoresis (0.8% agarose) were used to evaluate the purity and concentration of RNA. Reverse transcription into cDNA was performed using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (Takara Bio, Beijing, China) as the template for qRT-PCR. Table 1 displays the primer sequences (q-TbUGGT-F and q-TbUGGT-R) and reference gene EIF5A (C1168.2-F and C1168.2-R). The reaction systems were prepared according to TB Green®

Premix Ex TaqTM II (Takara Bio, Beijing, China). The data were detected using CFX96TM real-time System (Bio-Rad, Wuhan, China) and analyzed using the 2-ΔΔCt method [51].

#### **5. Conclusions**

In this study, the gene TbUGGT was successfully cloned from *Trapa bispinosa* Roxb. After gene optimization, the nucleic acid and protein sequences were analyzed using bioinformatics and the phylogenetic tree and hosted into *Escherichia coli*. The gene was purified and successfully expressed in *Escherichia coli* BL12 (DE3). The HPLC results showed that TbUGGT could catalyze GA and UDPG to produce βG. This study found the catalytic role of TbUGGT in βG biosynthesis, laying the foundation for subsequent related studies on βG biosynthesis in *Escherichia coli*.

**Supplementary Materials:** The following supporting information can be downloaded at: https:// www.mdpi.com/article/10.3390/molecules27238374/s1, Figure S1: Domain prediction of TbUGGT protein (a–c), Figure S2: Hydrophilicity analysis of TbUGGT (a), Figure S3: Signal peptide prediction (a) and transmembrane prediction (b) of TbUGGT, Figure S4: Secondary structure prediction of TbUGGT.

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

**Funding:** This research was supported by Nature Science Foundation of Hubei Province in China (2022CFB429) and Primary Research & Development Plan of Hubei Province (2022BBA0023).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

**Sample Availability:** Samples of *Trapa bispinosa* Roxb., vectors and strains carrying the TbUGGT gene, fermentation products of TbUGGT protein purification are available from the authors.

#### **References**


## *Article* **Synthesis and In Vitro Anticancer Evaluation of Flavone—1,2,3-Triazole Hybrids**

**Alexandra Németh-Rieder 1, Péter Keglevich 1,\*, Attila Hunyadi 2, Ahmed Dhahir Latif 3,4, István Zupkó <sup>3</sup> and László Hazai <sup>1</sup>**


**Abstract:** Hybrid compounds of flavones, namely chrysin and kaempferol, and substituted 1,2,3-triazole derivatives, were synthesized by click reaction of the intermediate *O*-propargyl derivatives. 4-Fluoro- and 4-nitrobenzyl-1,2,3-triazole-containing hybrid molecules were prepared. The mono- and bis-coupled hybrids were investigated on 60 cell lines of 9 common cancer types (NCI60) in vitro as antitumor agents. Some of them proved to have a significant antiproliferative effect.

**Keywords:** flavones; chrysin; kaempferol; hybrids; 1,2,3-triazole; anticancer activity

#### **1. Introduction**

Cancer treatment is one of the most important medical challenges. Permanent research is in progress to produce more effective and less toxic derivatives. One of the exciting and promising directions of this research is the synthesis of antitumor hybrid molecules [1,2]. The concept of molecular hybridization is to incorporate two or more pharmacophores into one molecule with covalent bonds, increasing the chance of effectiveness and improving the drug kinetic properties of the resulting hybrid compared to the corresponding fixed-dose drug combination. It should be noted that rigid distance imposed by the structure of the compound between potentially active parts of the hybrid may prevent biological efficiency.

During our previous work, numerous new molecules exerting a significant antiproliferative effect have been developed in this field. Various hybrids of *Vinca* alkaloids [3] were synthesized, coupling with amino acid esters [4,5], steroids [6], flavones (e.g., **3**, chrysin) [7], phosphorus derivatives [8], amines [5], and compounds containing the known pharmacophore 1,2,3-triazole (**2**) [5]. Recently our work was extended to the synthesis of new aminochrysin derivatives coupled with different aromatics [9].

Several flavonoids with antitumor activity are known in the literature [10,11]. Flavones containing a 2-phenylchromen-4-one (**1**) backbone, and 1,2,3-triazole derivatives keep attracting much research interest, and many 1,2,3-triazole-containing hybrids are known as effective anticancer agents [12–15]. During the last decade, numerous biologically active flavone—1,2,3-triazole hybrids have been synthesized [16–18], for example, **5** apigenin-7 methyl ether derivative, which showed promising activity against ovarian cancer (*IC*<sup>50</sup> = 10, 15 and 20 μM for SKOV3, OVCAR-3 and Caov-3 cancer cell lines) (Figure 1) [19].

In this study, the above outlined results inspired us to develop synthetic possibilities for the preparation of flavone—1,2,3-triazole hybrids.

**Citation:** Németh-Rieder, A.; Keglevich, P.; Hunyadi, A.; Latif, A.D.; Zupkó, I.; Hazai, L. Synthesis and In Vitro Anticancer Evaluation of Flavone—1,2,3-Triazole Hybrids. *Molecules* **2023**, *28*, 626. https:// doi.org/10.3390/molecules28020626

Academic Editor: Giovanni Ribaudo

Received: 15 December 2022 Revised: 4 January 2023 Accepted: 4 January 2023 Published: 7 January 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** The structure of 2-phenylchromen-4-one (**1**), 1,2,3-triazole (**2**), chrysin (**3**), kaempferol (**4**), and an anticancer flavone—1,2,3-triazole hybrid (**5**).

#### **2. Results and Discussion**

In the course of elaborating the synthetic design, chrysin (5,7-dihydroxyflavone) (**3**) and kaempferol (3,4 ,5,7-tetrahydroxyflavone) (**4**) were chosen (Figure 1) to couple with 1,2,3-triazole derivatives. Some chrysin—1,2,3-triazole hybrids prepared by a different way, were previously reported as antibacterial agents [20].

#### *2.1. Coupling Components*

Chrysin (**3**) is among the best-known flavones. It is abundant in nature and present in many edible plants and honey [21]. It has an anticancer effect through inducing apoptosis and autophagy [21,22]. Chrysin (**3**) seems to be suitable for use alone and/or in combination with other chemotherapeutic agents [21]. Kaempferol (**4**) and its derivatives are also found in many plants. They can prevent coronary heart disease and inflammatory problems, and they also show antiproliferative effects and may induce apoptosis [23].

It is known that 1,2,3-Triazole derivatives have been widely used as a pharmacophore in hybrids. In addition to the advantageous physico-chemical properties of this moiety, it is also known to exert various biological effects [24,25]. 1,2,3-Triazole derivatives are characterized by stability, the ability to form hydrogen bonds (increasing their water solubility), and weak basicity (they are not protonated at physiological pH). Moreover, 1,2,3-triazole derivatives have fungicidal, antibacterial, antituberculosis, and anticancer effects [26,27]. The well-known click reaction is used for the preparation of 1,2,3-triazole derivatives, as one of the tools of modern organic synthetic methods based on structure-activity relationships, preferably the *N*1-(4-fluoro- and 4-nitrobenyzl)-1,2,3-triazole derivatives [24,28].

#### *2.2. Chemistry*

Chrysin (**3**) reacted with an equimolar quantity of propargyl bromide (PPGBr) in dimethylformamide in the presence of cesium carbonate at room temperature (Scheme 1), resulting in the 7-substituted product (**6**) (known as an intermediate of antibacterial derivative prepared by a method different from ours [20]). The reason for the regioselectivity is that the proton of the 5-hydroxyl group forms an intramolecular H-bond with the neighboring oxo group. Certainly, with an excess of propargyl bromide (5 equivalent), exclusively the 5,7-disubstituted derivative (**7**) proved to be the product, as expected. Others also synthesized this compound using gold(I) complexes without reporting any preparative and characterization details [29].

The next reaction step was the click reaction (Scheme 2) using 4-fluoro- and 4-nitrobenzyl azide prepared in situ from the corresponding benzyl bromides with sodium azide in DMF at room temperature [30].

The reaction was carried out in the presence of copper(I) iodide, triphenylphosphine, and *N*,*N*-diisopropylethylamine, and resulted in known hybrids **8** and **9**, respectively. These two hybrids were prepared previously with another method, however, only their antibacterial effect has been investigated [20]. Bis(propargyl) derivative **7** was also treated with the same reaction conditions and gave the bis-hybrids **10** and **11**. Avoiding the difficult isolation from the triphenylphosphine oxide formed, the latter click reaction was successfully achieved also with further reagents, namely with copper sulfate pentahydrate and sodium L-ascorbate in a two-phase mixture.

**Scheme 1.** The reaction between chrysin (**3**) and propargyl bromide (PPGBr).

**Scheme 2.** The synthesis of chrysin hybrids (**8**–**11**) containing one or two 1,2,3-triazole units.

The second flavone building block, selected for the synthesis of hybrids, was kaempferol (**4**). The alkylation with propargyl bromide was investigated with different bases and in different solvents (Scheme 3). Using cesium carbonate or potassium carbonate as a base in dimethylformamide compounds **12** and **13** were isolated. However, in acetone solution compound **14** was obtained.

**Scheme 3.** The reaction between kaempferol (**4**) and propargyl bromide.

Derivative **13** was the compound isolated in the relatively largest quantity and was chosen for the click reaction (Scheme 4). Investigating both the reaction conditions resulted in the isolation of the bis-hybrid **15**.

**Scheme 4.** The synthesis of a kaempferol hybrid (**15**) containing two 1,2,3-triazole units.

#### *2.3. Biological Evaluation*

The in vitro antiproliferative activities of chrysin (**3**) and the synthesized compounds (**8**–**11**, **15**) were examined against 60 human tumor cell lines according to the given protocols of NCI (USA) [31–35]. The results are summarized in Table 1. The percentages of growth show the amount of living cancer cells compared to a reference. The negative numbers indicate a significant decrease in the cell number. Since derivatives **8** and **10** had shown remarkable antiproliferative activity on several cancer cell lines during the one-dose test, they were subjected to a five-dose screening. The *GI*<sup>50</sup> (50% growth inhibition) values are also given in Table 1.

**Table 1.** Antiproliferative activities of chrysin (**3**), hybrids **8**–**11** and **15** against 60 human cancer cell lines in vitro. In connection with GPR values, the negative numbers causing cell death are highlighted in bold. Values where *GI*<sup>50</sup> < 10 μM are highlighted in bold, too.



It can be seen from Table 1 that no antiproliferative effect was shown by chrysin (3) and compounds **9** and **11**. Hybrids 8 and 10 cause cell death on several cell lines of different types of cancer and show inhibition effect also on some cases. Despite the relatively limited structural diversity of our compounds, the above results revealed some interesting structure-activity relationships. We found that (i) the bis-hybrid compounds also exert considerable antiproliferative effect and (ii) replacement of the fluorine atom by a nitro group reduces the bioactivity. The kaempferol-triazole hybrid (15) gave rather modest results.

The two promising compounds (**8** and **10**) were tested for their antiproliferative activity on further two human cervical cancer cell lines HeLa and SiHa (Table 2). Interestingly, the monohybrid derivative 8 was active only against HeLa cells, and SiHa cells were relatively resistant to it. The bis-hybrid derivative 10 was more potent and similarly active against both cell lines, with a sub-micromolar IC50 value against HeLa. Both derivatives exhibited higher activity than the reference agent cisplatin against HeLa cells.

**Table 2.** In vitro antiproliferative activity of compounds **8** and **10** against human cervical cancer cell lines. Compounds were tested in the concentration range of 0.1–30 μM in 2 biological replicates, 5 parallel measurements each. *IC*<sup>50</sup> values and their 95% confidence intervals (C.I.) are presented. Value where *IC*<sup>50</sup> < 1 μM is highlighted in bold. Cisplatin was included as a reference agent.


The results obtained in this paper are encouraging for the future optimization of the derivatives. We want to emphasize that this study may be the starting point for more detailed synthetic and anticancer research.

#### **3. Materials and Methods**

#### *3.1. General Materials and Methods*

All chemicals were purchased from Sigma-Aldrich (Budapest, Hungary) and were used as received. Melting points were measured on a VEB Analytik Dresden PHMK-77/1328 apparatus (Dresden, Germany) and are uncorrected. IR spectra were recorded on Zeiss IR 75 and 80 instruments (Thornwood, NY, USA). NMR measurements were performed on a Bruker Avance III HDX 500 MHz NMR spectrometer equipped with a 1H{13C/15N} 5 mm TCI CryoProbe (Bruker Corporation, Billerica, MA, USA). 1H And 13C chemical shifts are given on the delta scale as parts per million (ppm) relative to tetramethyl silane. One-dimensional 1H, and 13C spectra and two-dimensional 1H–1H COSY, 1H–1H NOESY, 1H–13C HSQC, and 1H–13C HMBC spectra were acquired using pulse sequences included in the standard spectrometer software package (Bruker TopSpin 3.5, Bruker Corporation). ESI-HRMS and MS-MS analyses were performed on a Thermo Velos Pro Orbitrap Elite (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was ESI, operated in positive ion mode. The protonated molecular ion peaks were fragmented by CID (collision-induced dissociation) at a normalized collision energy of 35–65%. For the CID experiment, helium was used as the collision gas. The samples were dissolved in methanol. EI-HRMS analyses were performed on a Thermo Q Exactive GC Orbitrap (Thermo Fisher Scientific, Bremen, Germany) system. The ionization method was EI and operated in positive ion mode. Electron energy was 70 eV and the source temperature was set at 250 ◦C. Data acquisition and analysis were accomplished with Xcalibur software version 4.0 (Thermo Fisher Scientific). TLC was carried out using DC-Alufolien Kieselgel 60 F254 (Merck, Budapest, Hungary) plates. Preparative TLC analyses were performed on silica gel 60 PF254+366 (Merck) glass plates.

#### *3.2. Chemistry*

#### 3.2.1. 7-(*O*-Propargyl)chrysin (**6**)

Chrysin (**3**) (330 mg, 1.3 mmol) and cesium carbonate (426 g, 1.3 mmol) were dissolved in DMF (15 mL), the solution was stirred at 10 min, then propargyl bromide (0.142 mL, 1.3 mmol) was added in 80% toluene solution. After stirring at room temperature for 18.5 hrs, the reaction mixture was evaporated to dryness, and the residue was dissolved in dichloromethane (60 mL). Next, water (60 mL) was added, and the pH was adjusted to 1 with 2*M* hydrochloric acid solution. The water phase was extracted with dichloromethane (2 × 30 mL), then the combined organic phase was washed with water (60 mL) and saturated sodium chloride solution (60 mL). The organic phase after drying with magnesium sulfate was evaporated to dryness, and the crude product was purified with preparative TLC (dichloromethane-methanol = 40:1) to give 274 mg (72%) of compound **6** as a yellow solid. M.p.:180–182 ◦C. TLC (dichloromethane-methanol = 30:1); *Rf* = 0.83. IR (KBr) 3284, 1663, 1624, 1540, 1331, 1155, 767 cm<sup>−</sup>1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 3.69 (t; *J* = 2.4 Hz; 1H; C(7)-OCH2C≡CH); 4.97 (d; *J* = 2.4 Hz; 2H; C(7)-OCH2); 6.48 (d; *J* = 2.3 Hz; 1H; H-6); 6.88 (d; *J* = 2.3 Hz; 1H; H-8); 7.07 (s; 1H; H-3); 7.58–7.62 (m; 2H; H-3 , H-5 ); 7.62–7.66 (m; 1H; H-4 ); 8.09–8.13 (m; 2H; H-2 , H-6 ); 12.84 (s; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO*d*6) *δ* (ppm) 56.2 (C(7)-OCH2); 78.3 (C(7)-OCH2C≡CH); 79.0 (C(7)-OCH2C≡CH); 93.7 (C-8); 98.6 (C-6); 105.2 (C-10); 105.4 (C-3); 126.4 (C-2 , C-6 ); 129.1 (C-3 , C-5 ); 130.5 (C-1 ); 132.1 (C-4 ); 157.1 (C-9); 161.1 (C-5); 163.1 (C-7); 163.5 (C-2). 182.1 (C-4). ESI-HRMS:M + H = 293.08086 (delta = 0.08 ppm; C18H13O4). HR-ESI-MS-MS (CID = 55%; rel. int. %): 269(5); 265(100); 251(56); 247(4); 239(6); 223(10).

#### 3.2.2. 5,7-Bis(*O*-propargyl)chrysin (**7**)

Chrysin (**3**) (500 mg, 1.97 mmol) and cesium carbonate (3.2 g, 9.84 mmol) were dissolved in dimethylformamide (20 mL), the solution was stirred at 10 min, then propargyl bromide (1.1 mL, 9.84 mmol) was added in 80% toluene solution. After stirring at room temperature for 45 min, the reaction mixture was evaporated to dryness, and the residue was dissolved in dichloromethane (40 mL). Next, water (40 mL) was added, and the pH was adjusted to 1 with 2*M* hydrochloric acid solution. The water phase was extracted with dichloromethane (3 × 20 mL), then the combined organic phase was washed with water (2 × 20 mL) and saturated sodium chloride solution (20 mL). The organic phase after drying with magnesium sulfate was evaporated to dryness and 620 mg (95%) pure product (**7**) was obtained. M.p.: 204–206 ◦C. TLC (dichloromethane-methanol = 30:1); *Rf* = 0.33. IR (KBr) 3214, 1635, 1597, 1450, 1343, 1164, 833 cm<sup>−</sup>1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 3.63 (t; *J* = 2.4 Hz; 1H; C(5)-OCH2C≡CH); 3.70 (t; *J* = 2.4 Hz; 1H; C(7)-OCH2C≡CH); 4.93 (d; *J* = 2.4 Hz; 2H; C(5)-OCH2); 4.98 (d; *J* = 2.4 Hz; 2H; C(7)-OCH2); 6.67 (d; *J* = 2.3 Hz; 1H; H-6); 6.82 (s; 1H; H-3); 7.00 (d; *J* = 2.3 Hz; 1H; H-8); 7.54–7.62 (m; 3H; H-3 , H-4 , H-5 ); 8.01–8.09 (m; 2H; H-2 , H-6 ). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 56.2 (C(7)-OCH2); 56.4 (C(5)-OCH2); 78.3 (C(7)-OCH2C≡CH); 78.6 (C(5)-OCH2C≡CH); 78.8 (C(5)-OCH2C≡CH); 79.0 (C(7)-OCH2C≡CH); 95.0 (C-8); 98.8 (C-6); 108.2 (C-3); 109.1 (C-10); 125.9 (C-2 , C-6 ); 129.0 (C-3 , C-5 ); 130.7 (C-1 ); 131.4 (C-4 ); 157.8 (C-5); 158.8 (C-9); 159.7 (C-2); 161.2 (C-7); 175.4 (C-4). ESI-HRMS: M + H = 331.09618 (delta = −0.9 ppm; C21H15O4). HR-ESI-MS-MS (CID = 35%; rel. int. %): 313(6); 303(89); 292(47); 289(16); 275(13); 265(13); 251(100); 185(36); 157(10).

3.2.3. Click Reaction of 7-(*O*-Propargyl)chrysin (**6**) with 4-Fluorobenzyl Azide; Preparation of **8**

To 7-*O*-propargyl chrysin (**6**) (48 mg, 0.164 mmol) was added 4-fluorobenzyl azide (25 mg, 0.164 mmol) in toluene solution (4 mL) prepared in situ [30], triphenylphosphine (9 mg, 0.0328 mmol), copper(I) iodide (4 mg, 0.0164 mmol) and 0.09 mL (0.492 mmol) diisopropylethylamine. After reflux for 2 h, the reaction mixture was diluted with toluene (25 mL), then the mixture was washed with water (30 mL). After washing the water phase with toluene (10 mL), the combined organic phase after drying with magnesium sulfate was evaporated to dryness. The preparative TLC (dichloromethane-methanol = 40:1) of the crude product resulted in 5 mg (7%) pure product (**8**). M.p.:163–165 ◦C. M.p. *lit*.: 190-191oC [20]. TLC (dichloromethane-methanol = 40:1); *Rf*=0.34. IR (KBr) 3424, 1660, 1614, 1558, 1161, 766, 541 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 5.29 (s; 2H; H2-1 ); 5.62 (s; 2H; H2-7 ); 6.50 (d; *J* = 2.2 Hz; 1H; H-6); 6.96 (d; *J* = 2.2 Hz; 1H; H-8); 7.06 (s; 1H; H-3); 7.19–7.24 (m; 2H; H-10 , H-12 ); 7.39–7.44 (m; 2H; H-9 , H-13 ); 7.58–7.66 (m; 3H; C(2)-Ph: 2x H*meta*, H*para*); 8.09–8.12 (m; 2H; C(2)-Ph: 2x H*ortho*); 8.35 (s; 1H; H-6 ); 12.8 (br; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 52.0 (C-7 ); 61.7 (C-1 ); 93.5 (C-8); 98.6 (C-6); 105.0 (C-10); 105.3 (C-3); 115.5 (d; <sup>2</sup>*J*CF = 21.6 Hz; C-10 , C-12 ); 124.9 (C-6 ); 126.4 (C(2)-Ph: C*ortho*); 129.1 (C(2)-Ph: C*meta*); 130.3 (d; <sup>3</sup>*J*CF = 8.5 Hz; C-9 , C-13 ); 130.5 (C(2)-Ph: Cipso); 132.0–132.1 (m; C-8 , C(2)-Ph: C*para*); 142.0 (C-2 ); 157.2 (C-9); 161.1 (C-5); 161.8 (d; <sup>1</sup>*J*CF = 244.2 Hz; C-11 ); 163.5 (C-2); 163.9 (C-7); 182.0 (C-4). ESI-HRMS: M+H = 444.13547 (delta = 0.13 ppm; C25H19O4N3F). HR-ESI-MS-MS (CID = 45%; rel. int. %): 416(100); 363(32); 307(24); 293(12); 291(60); 267(26); 255(47).

3.2.4. Click Reaction of 7-(*O*-Propargyl)chrysin (**6**) with 4-Nitrobenzyl Azide; Preparation of **9**

To 7-*O*-propargyl chrysin (**6**) (48 mg, 0.164 mmol) was added 4-nitrobenzyl azide (29 mg, 0.164 mmol) in toluene solution (4 mL) prepared in situ [30], triphenylphosphine (9 mg, 0.0328 mmol), copper(I) iodide (4 mg, 0.0164 mmol) and 0.09 mL (0.492 mmol) diisopropylethylamine. After reflux for 4 h, the reaction mixture was diluted with toluene (25 mL), then the mixture was washed with water (30 mL). After washing the water phase with toluene (10 mL), the combined organic phase after drying with magnesium sulfate was evaporated to dryness. The residue was dissolved in dichloromethane and after filtration, the filtrate was evaporated to dryness, then 24 mg (31%) product (**9**) was obtained. M.p.: 219–221 ◦C. M.p*. lit*.: 187–188 ◦C [20]. TLC (dichloromethane-methanol = 40:1); *Rf* = 0.45. IR (KBr) 809; 1155; 1349; 1524; 1617; 1656; 3083 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 5.32 (s; 2H; H2-1 ); 5.83 (s; 2H; H2-7 ); 6.51 (d; *J* = 2.1 Hz; 1H; H-6); 6.97 (d; *J* = 2.1 Hz; 1H; H-8); 7.07 (s; 1H; H-3); 7.53–7.58 (m; 2H; H-9 , H-13 ); 7.58–7.67 (m; 3H; C(2)-Ph: 2x H*meta*, H*para*); 8.07–8.13 (m; 2H; C(2)-Ph: 2x H*ortho*); 8.22–8.27 (m; 2H; H-10 , H-12 ); 8.43 (s; 1H; H-6 ); 12.83 (s; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 51.9 (C-7 ); 61.7 (C-1 ); 93.6 (C-8); 98.7 (C-6); 105.1 (C-10); 105.4 (C-3); 123.9 (C-10 , C-12 ); 125.4 (C-6 ); 126.4 (C(2)-Ph: C*ortho*); 129.0 (C-9 , C-13 ); 129.1 (C(2)-Ph: C*meta*); 130.5 (C(2)-Ph: Cipso); 132.1 (C(2)-Ph: C*para*); 142.2 (C-2 ); 143.2 (C-8 ); 147.2 (C-11 ); 157.2 (C-9); 161.1 (C-5); 163.5 (C-2); 163.9 (C-7); 182.0 (C-4). ESI-HRMS: M+H=471.12976 (delta = −0.3 ppm; C25H19O6N4). HR-ESI-MS-MS (CID=35%; rel. int. %): 443(25); 425(12); 307(18); 291(26); 255(100); 189(2).

3.2.5. Click Reaction of 5,7-Bis(*O*-propargyl)chrysin (**7**) with 4-Fluorobenzyl Azide; Preparation of **10**

(a) To 5,7-bis(*O*-propargyl) chrysin (**7**) (44 mg, 0.133 mmol) was added 4-fluorobenzyl azide (40 mg, 0.265 mmol) in toluene solution (6 mL) prepared in situ [30], triphenylphosphine (14 mg, 0.0532 mmol), copper(I) iodide (5 mg, 0.0265 mmol) and 0.14 mL (0.798 mmol) diisopropylethylamine. After reflux for 5 hrs, the reaction mixture was diluted with toluene (25 mL), and the mixture was washed with water (30 mL), then the water phase was washed with toluene (10 mL). The combined organic phase was dried with magnesium sulfate and the precipitated product (**10**) (32 mg, 38%) could be separated with filtration.

(b) To 5,7-bis(*O*-propargyl) chrysin (**7**) (44 mg, 0.133 mmol) was added 4-fluorobenzyl azide (40 mg, 0.266 mmol) in dichloromethane solution (4.5 mL) prepared in situ [30], copper(II) sulfate pentahydrate (56 mg, 0.222 mmol), sodium L-ascorbate (88 mg, 0.443 mmol) and water (4.5 mL). After 18 hrs of intensive stirring at room temperature, the reaction mixture was diluted with water (18 mL) and extracted with dichloromethane (2 × 20 mL). The combined organic phase was washed with saturated sodium chloride solution (50 mL), and after drying with magnesium sulfate the solution was evaporated. The residue was separated with preparative TLC (dichloromethane-methanol = 15:1) and 27 mg (32%) product (**10**) was obtained (Figure 2). Mp.: 229–231 ◦C. TLC (dichloromethanemethanol = 20:1); *Rf* = 0.30. IR (KBr) 1642, 1605, 1511, 1352, 1225, 1159 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 5.23 (s; 2H; H2-1"); 5.31 (s; 2H; H2-1 ); 5.63 (s; 2H; H2-7 ); 5.64 (s; 2H; H2-7"); 6.76 (s; 1H; H-3); 6.80 (d; *J* = 2.2 Hz; 1H; H-6); 7.07 (d; *J* = 2.2 Hz; 1H; H-8); 7.18–7.24 (m; 4H; H-10 , H-12 , H-10", H-12"); 7.38–7.42 (m; 2H; H-9", H-13"); 7.40–7.45 (m; 2H; H-9 , H-13 ); 7.54–7.61 (m; 3H; C(2)-Ph: 2x H*meta*, H*para*); 8.02–8.06 (m; 2H; C(2)-Ph: 2x H*ortho*); 8.31 (s; 1H; H-6 '); 8.37 (s; 1H; H-6 ). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 51.9 (C-7"); 52.0 (C-7 ); 61.6 (C-1 ); 62.6 (C-1 '); 94.8 (C-8); 98.4 (C-6); 108.2 (C-3); 108.8 (C-10); 115.4–115.6 (m; C-10 , C-12 , C-10", C-12 '); 124.5 (C-6"); 124.9 (C-6 ); 125.8 (C(2)-Ph: C*ortho*); 129.0 (C(2)-Ph: C*meta*); 130.2 (d; <sup>3</sup>*J*CF = 8.5 Hz; C-9", C-13"); 130.3 (d; <sup>3</sup>*J*CF = 8.5 Hz; C-9 , C-13 ); 130.7 (C(2)-Ph: Cipso); 131.4 (C(2)-Ph: C*para*); 132.1–132.2 (m; C-8 , C-8"); 142.1 (C-2 ); 142.9 (C-2 '); 158.7 (C-5); 159.0 (C-9); 159.6 (C-2); 161.78 (d; <sup>1</sup>*J*CF = 244 Hz), 161.81 (d; <sup>1</sup>*J*CF = 244 Hz): C-11 , C-11"; 162.2 (C-7); 175.4 (C-4). ESI-HRMS: M + H = 633.20636 (delta = 1.14 ppm; C35H27O4N6F2). HR-ESI-MS-MS (CID = 35%; rel. int. %): 605(43); 588(5); 577(6); 552(8); 498(8); 496(8); 456(5); 452(26); 444(48); 424(5); 399(6).

**Figure 2.** The skeleton numbering of compound **10** used for NMR assignment.

3.2.6. Click Reaction of 5,7-Bis(*O*-propargyl)chrysin (**7**) with 4-Nitrobenzyl Azide; Preparation of **11**

(a) To 5,7-bis(propargyl) chrysin (**7**) (44 mg, 0.133 mmol) was added 4-nitrobenzyl azide (47 mg, 0.266 mmol) in toluene solution (6 mL) prepared in situ [30], triphenylphosphine (14 mg, 0.0532 mmol), copper(I) iodide (5 mg, 0.0265 mmol) and 0.14 mL (0.798 mmol) diisopropylethylamine. After reflux for 4 hrs, the reaction mixture was diluted with toluene (25 mL), and the mixture was washed with water (30 mL), then the water phase was washed with toluene (10 mL). The combined organic phase was dried with magnesium sulfate, and the precipitated crude product could be separated with filtration. After preparative TLC (dichloromethane-methanol = 15:1) of the crude product, 9 mg (10%) pure product (**11**) was obtained.

(b) To 5,7-bis(*O*-propargyl) chrysin (**7**) (176 mg, 0.532 mmol) was added 4-nitrobenzyl azide (190 mg, 1.064 mmol) in dichloromethane solution (18 mL) prepared in situ [30], copper(II) sulfate pentahydrate (224 mg, 0.888 mmol), sodium L-ascorbate (352 mg, 1.772 mmol) and water (18 mL). After 16.5 h of intensive stirring at room temperature, the reaction mixture was diluted with water (72 mL) and extracted with dichloromethane (3 × 80 mL). The combined organic phase was washed with saturated sodium chloride solution (200 mL), and after drying with magnesium sulfate the solution was evaporated. The residue was separated with preparative TLC (dichloromethane-methanol = 15:1) and 30 mg (17%) product (**11**) was obtained. M.p. = 182–184 ◦C. TLC (dichloromethane-methanol = 20:1); *Rf* = 0.40. IR (KBr) 805; 1109; 1167; 1348; 1521; 1608; 1644; 3080 cm<sup>−</sup>1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 5.27 (s; 2H; H2-1 '); 5.34 (s; 2H; H2-1 ); 5.83 (s; 2H; H2-7 ); 5.84 (s; 2H; H2-7 '); 6.76 (s; 1H; H-3); 6.82 (d; *J* = 2.3 Hz; 1H; H-6); 7.09 (d; *J* = 2.3 Hz; 1H; H-8); 7.53–7.61 (m; 7H; H-9 , H-13 , H-9 ', H-13 ', C(2)-Ph: 2x H*meta*, H*para*); 8.02–8.06 (m; 2H; C(2)-Ph: 2x H*ortho*);

8.22–8.26 (m; 4H; H-10 , H-12 , H-10 ', H-12 '); 8.39 (s; 1H; H-6 '); 8.45 (s; 1H; H-6 ). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 51.8 (C-7 '); 51.9 (C-7 ); 61.6 (C-1 ); 62.6 (C-1 '); 94.9 (C-8); 98.4 (C-6); 108.2 (C-3); 108.8 (C-10); 123.8 (C-10 , C-12 , C-10 ', C-12 '); 125.0 (C-6 '); 125.4 (C-6 ); 125.8 (C(2)-Ph: C*ortho*); 129.0 (C-9 , C-13 , C-9 ', C-13 ', C(2)-Ph: C*meta*); 130.7 (C(2)-Ph: Cipso); 131.4 (C(2)-Ph: C*para*); 142.2 (C-2 ); 143.1 (C-2 '); 143.2 (C-8 ); 143.3 (C-8 '); 147.2 (C-11 , C-11"); 158.7 (C-5); 159.0 (C-9); 159.6 (C-2); 162.1 (C-7); 175.4 (C-4). ESI-HRMS: M+H = 687.19286 (delta = −2.6 ppm; C35H27O8N8). HR-ESI-MS-MS (CID = 35%; rel. int. %): 659(45); 631(10); 507(84); 471(100); 443(12); 343(3); 291(7).

#### 3.2.7. *O*-Alkylation of Kaempferol (**4**) with Propargyl Bromide

(a) Kaempferol (**4**) (113 mg, 0.393 mmol) and cesium carbonate (129 mg, 0.393 mmol) were dissolved in dimethylformamide (5 mL) and after 10 min stirring propargyl bromide (0.043 mL, 0.393 mmol) was added in 80% toluene solution. The reaction mixture was stirred at room temperature for 2.5 hrs and was evaporated to dryness. The residue was dissolved in dichloromethane (20 mL), then water (20 mL) was added and the pH was adjusted to 1 with 2*N* hydrochloric solution. The water phase was washed with dichloromethane (2 × 10 mL), the combined organic phase was treated with water (20 mL), and then with saturated sodium chloride solution (20 mL). After drying with magnesium sulfate the solution was evaporated to dryness and using preparative TLC (dichloromethane-methanol = 20:1) two products were obtained: 8 mg (6%) of monopropargylated derivative (**12**), and 25 mg (19%) of 3,7-bis(*O*-propargyl) kaempferol (**13**).

(b) Kaempferol (**4**) (1130 mg, 3.93 mmol) and potassium carbonate (543 mg, 3.93 mg) were dissolved in dimethylformamide (15 mL). After 10 min stirring at room temperature propargyl bromide (0.43 mL, 3.93 mmol, in 80% toluene solution) dissolved in dimethylformamide (5 mL) was dropped into the reaction mixture. After 2.5 h further potassium carbonate (272 mg, 1.97 mmol) and propargyl bromide (0.22 mL, 1.97 mmol, in 80% toluene solution) dissolved in dimethylformamide (2 mL) were added. The reaction mixture was stirred for a further 5 hrs at room temperature, evaporated to dryness, and the residue was dissolved in chloroform (200 mL). Next, water (200 mL) was added, and the pH was adjusted to 1 with 2*N* hydrochloric acid. The water phase was extracted with chloroform (2x100 mL), and the combined organic phase was washed with water (200 mL) and with saturated sodium chloride solution (200 mL) and evaporated to dryness. Preparative TLC (dichloromethane-methanol = 20:1) separation of the residue 40 mg (3%) of **12** and 330 mg (23%) of **13** were obtained.

3-(*O*-Propargyl)kaempferol (**12**): M.p. = 180–182 ◦C. TLC (dichloromethane-methanol = 20:1); *Rf* = 0.19. IR (KBr) 821; 1180; 1235; 1557; 1660; 3293 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 3.50 (t; *J* = 2.4 Hz; 1H; C(3)-OCH2C≡CH); 4.89 (d; *J* = 2.4 Hz; 2H; C(3)-OCH2); 6.22 (d; *J* = 2.1 Hz; 1H; H-6); 6.46 (d; *J* = 2.1 Hz; 1H; H-8); 6.90–6.95 (m; 2H; H-3 , H-5 ); 7.98–8.01 (m; 2H; H-2 , H-6 ); 10.28 (br s; 1H; C(4 )-OH); 10.90 (br; 1H; C(7)-OH); 12.55 (s; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 58.8 (C(3)-OCH2); 78.6 (C(3)-OCH2C≡CH); 79.2 (C(3)-OCH2C≡CH); 93.7 (C-8); 98.6 (C-6); 103.8 (C-10); 115.4 (C-3 , C-5 ); 120.4 (C-1 ); 130.4 (C-2 , C-6 ); 134.8 (C-3); 156.25 (C-9); 156.34 (C-2); 160.1 (C-4 ); 161.1 (C-5); 164.2 (C-7); 177.6 (C-4). EI-HRMS: M = 324.06174 (delta = −3.4 ppm; C18H12O6).

3,7-Bis(*O*-propargyl)kaempferol (**13**): M.p. = 185–187 ◦C. TLC (dichloromethanemethanol = 20:1); *Rf* = 0.49. IR (KBr) 1180; 1289; 1332; 1493; 1602; 1662; 3259 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 3.51 (t; *J* = 2.4 Hz; 1H; C(3)-OCH2C≡CH); 3.68 (t; *J* = 2.4 Hz; 1H; C(7)-OCH2C≡CH); 4.91 (d; *J* = 2.4 Hz; 2H; C(3)-OCH2); 4.95 (d; *J* = 2.4 Hz; 2H; C(7)-OCH2); 6.46 (d; *J* = 2.3 Hz; 1H; H-6); 6.82 (d; *J* = 2.3 Hz; 1H; H-8); 6.93–6.97 (m; 2H; H-3 , H-5 ); 8.01–8.05 (m; 2H; H-2 , H-6 ); 10.3–10.4 (br; 1H; C(4 )-OH); 12.55 (s; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 56.2 (C(7)-OCH2); 58.9 (C(3)- OCH2); 78.3 (C(7)-OCH2C≡CH); 78.5 (C(3)-OCH2C≡CH); 79.0 (C(7)-OCH2C≡CH); 79.3 (C(3)-OCH2C≡CH); 93.3 (C-8); 98.4 (C-6); 105.2 (C-10); 115.4 (C-3 , C-5 ); 120.3 (C-1 ); 130.5 (C-2 , C-6 ); 135.1 (C-3); 155.9 (C-9); 156.8 (C-2); 160.3 (C-4 ); 160.8 (C-5); 162.9 (C-7); 177.8 (C-4). EI-HRMS: M = 362.07804 (delta = −1.2 ppm; C21H14O6).

(c) Kaempferol (**4**) (200 mg, 0.699 mmol) and potassium carbonate (106 mg, 0.769 mg) were dissolved in acetone (7 mL). After 10 min stirring at room temperature propargyl bromide (0.076 mL, 0.699 mmol, in 80% toluene solution) dissolved in acetone (3 mL) was dropped into the reaction mixture. After 7 hrs reflux further potassium carbonate (53 mg, 0.35 mmol) and propargyl bromide (0.038 mL, 0.35 mmol, in 80% toluene solution) dissolved in acetone (1.5 mL) were added. The reaction mixture was refluxed for a further 6 hrs, evaporated to dryness, and the residue was dissolved in dichloromethane (40 mL). Next, water (40 mL) was added and the pH was adjusted to 1 with 2*N* hydrochloric acid. The water phase was extracted with dichloromethane (2 × 20 mL), the combined organic phase was washed with water (40 mL) and with saturated sodium chloride solution (40 mL) and evaporated to dryness. After preparative TLC (dichloromethane-methanol = 20:1) separation of the residue, 10 mg (3%) of product **14** was obtained. M.p. = 158–160 ◦C. TLC (dichloromethane-methanol = 20:1); *Rf* = 0.82. IR (KBr) 1174; 1185; 1509; 1605; 1627; 3287 cm−1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 3.47 (t; *J* = 2.4 Hz; 1H; C(3)-OCH2C≡CH); 3.64 (t; *J* = 2.4 Hz; 1H; C(4 )-OCH2C≡CH); 3.65 (t; *J* = 2.4 Hz; 1H; C(5)-OCH2C≡CH); 3.69 (t; *J* = 2.4 Hz; 1H; C(7)-OCH2C≡CH); 4.91 (d; *J* = 2.4 Hz; 2H; C(3)-OCH2); 4.92 (d; *J* = 2.4 Hz; 2H; C(4 )-OCH2); 4.93 (d; *J* = 2.4 Hz; 2H; C(5)-OCH2); 4.97 (d; *J* = 2.4 Hz; 2H; C(7)-OCH2); 6.64 (d; *J* = 2.3 Hz; 1H; H-6); 6.96 (d; *J* = 2.3 Hz; 1H; H-8); 7.15–7.19 (m; 2H; H-3 , H-5 ); 8.07–8.11 (m; 2H; H-2 , H-6 ). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 55.5 (C(4 )-OCH2); 56.2 (C(7)-OCH2); 56.5 (C(5)-OCH2); 58.2 (C(3)-OCH2); 78.2 (C(7)-OCH2C≡CH); 78.5 (C(5)-OCH2C≡CH); 78.6 (C(4 )-OCH2C≡CH); 78.76 (C(4 )-OCH2C≡CH); 78.79 (C(3)-OCH2C≡CH); 78.9 (C(5)-OCH2C≡CH); 79.05 (C(7)- OCH2C≡CH); 79.10 (C(3)-OCH2C≡CH); 94.7 (C-8); 98.4 (C-6); 108.8 (C-10); 114.7 (C-3 , C-5 ); 123.0 (C-1 ); 129.8 (C-2 , C-6 ); 137.4 (C-3); 152.7 (C-2); 157.80 (C-9); 157.82 (C-5); 158.8 (C-4 ); 161.2 (C-7); 171.9 (C-4). EI-HRMS: M = 438.10893 (delta = −2.0 ppm; C27H18O6).

3.2.8. Click Reaction of 3,7-Bis(*O*-propargyl)kaempferol (**13**) with 4-Fluorobenzyl Azide; Preparation of **15**

(a) To 3,7-bis(*O*-propargyl) kaempferol (**13**) (140 mg, 0.387 mmol) was added 4-fluorobenzyl azide (117 mg, 0.773 mmol) in toluene solution (10 mL) prepared in situ [30], triphenylphosphine (41 mg, 0.155 mmol), copper(I) iodide (15 mg, 0.077 mmol), 0.43 mL (2.322 mmol) diisopropylethylamine and 13 mL toluene. After reflux for 5.5 hrs, the reaction mixture was diluted with toluene (70 mL), and the mixture was washed with water (45 mL), then the water phase was washed with toluene (15 mL). The combined organic phase was dried with magnesium sulfate and after preparative TLC (dichloromethanemethanol = 20:1) of the residue, 17 mg (7%) product (**15**) was obtained.

(b) To 3,7-bis(*O*-propargyl) kaempferol (**13**) (193 mg, 0.532 mmol) was added 4 fluorobenzyl azide (161 mg, 1.064 mmol) in dichloromethane solution (18 mL) prepared in situ [30], copper(II) sulfate pentahydrate (222 mg, 0.888 mmol), sodium L-ascorbate (351 mg, 1.77 mmol) and water (18 mL). After 8 h of intensive stirring at room temperature, the reaction mixture was diluted with water (70 mL) and extracted with dichloromethane (3 × 80 mL). The combined organic phase was washed with saturated sodium chloride solution (200 mL), and after drying with magnesium sulfate the solution was evaporated. The residue was separated with preparative TLC (dichloromethane-methanol = 20:1) and 18 mg (5%) product (**15**) was obtained (Figure 3). M.p. = 151–153 ◦C. TLC (dichloromethanemethanol = 20:1); *Rf* = 0.31. IR (KBr) 1172; 1225; 1512; 1587; 1602; 1665; 3139 cm<sup>−</sup>1. 1H NMR (499.9 MHz; DMSO-*d*6) *δ* (ppm) 5.20 (s; 2H; H2-1 '); 5.27 (s; 2H; H2-1 ); 5.51 (s; 2H; H2-7 '); 5.62 (s; 2H; H2-7 ); 6.47 (d; *J* = 2.2 Hz; 1H; H-6); 6.85–6.87 (m; 2H; C(2)-Ar: 2x H*meta*); 6.88 (d; *J* = 2.2 Hz; 1H; H-8); 7.14–7.19 (m; 2H; H-10 ', H-12 '); 7.19–7.25 (m; 4H; H-10 , H-12 , H-9 ', H-13 '); 7.39–7.44 (m; 2H; H-9 , H-13 ); 7.88–7.92 (m; 2H; C(2)-Ar: 2x H*ortho*); 8.14 (s; 1H; H-6 '); 8.34 (s; 1H; H-6 ); 10.30 (br s; 1H; C(2)-Ar: C*para*-OH); 12.68 (s; 1H; C(5)-OH). 13C NMR (125.7 MHz; DMSO-*d*6) *δ* (ppm) 51.7 (C-7 '); 52.0 (C-7 ); 61.7 (C-1 ); 64.1 (C-1 '); 93.0 (C-8); 98.3 (C-6); 105.2 (C-10); 115.3 (C(2)-Ar: C*meta*); 115.4 (d; <sup>2</sup>*J*CF = 21.6 Hz; C-10 ', C-12 '); 115.5 (d; <sup>2</sup>*J*CF = 21.6 Hz; C-10 , C-12 ); 120.3 (C(2)-Ar: Cipso); 124.9 (C-6 ); 125.0 (C-6 '); 129.8 (d; <sup>3</sup>*J*CF = 8.4 Hz; C-9 ', C-13 '); 130.27 (C(2)-Ar: C*ortho*); 130.30 (d; <sup>3</sup>*J*CF = 8.3 Hz; C-9 , C-13 ); 132.1 (d; <sup>4</sup>*J*CF = 3.0 Hz; C-8 , C-8 '); 135.6 (C-3); 142.1 (C-2 ); 142.4 (C-2 '); 156.1 (C-9); 156.5 (C-2); 160.1 (C(2)-Ar: C*para*); 160.9 (C-5); 161.4 (d; <sup>1</sup>*J*CF = 244.2 Hz; C-11 '); 161.7 (d; <sup>1</sup>*J*CF = 244.4 Hz; C-11 ); 163.7 (C-7); 178.0 (C-4). ESI-HRMS: M + H = 665.19497 (delta = −0.7 ppm; C35H27O6N6F2). HR-ESI-MS-MS (CID = 35%; rel. int. %): 637(51); 620(5); 584(8); 530(20); 512(51); 484(26); 476(100); 456(7); 448(51); 431(8); 299(4).

**Figure 3.** The skeleton numbering of compound **15** used for NMR assignment.

#### *3.3. Biological Evaluation*

#### 3.3.1. One-Dose Screen

All compounds were tested initially at a single high dose (10−<sup>5</sup> *M*) in the full NCI60 cell panel [31–35]. The number reported for the one-dose assay is growth relative to the no-drug control and relative to the time zero number of cells. This allowed the detection of both growth inhibition (values between 0 and 100) and lethality (values less than 0). For example, a value of 100 means no growth inhibition. A value of 30 would mean 70% growth inhibition. A value of 0 means no net growth over the course of the experiment. A value of −30 would mean 30% lethality. A value of −100 means all cells are dead.

#### 3.3.2. Five-Dose Screen

Compounds that exhibited significant growth inhibition in the one-dose screen were evaluated against the 60-cell panel at five concentration levels. The human tumor cell lines of the cancer screening panel were grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. Typically, cells were inoculated in 96-well microtiter plates in 100 μL at plating densities ranging from 5000 to 40,000 cells/well, depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates were incubated at 37 ◦C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to the addition of experimental drugs. After 24 h, two plates of each cell line were fixed in situ with trichloroacetic acid (TCA), to represent a measurement of the cell population for each cell line at the time of drug addition (*t*z). Experimental drugs were solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate was thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg ml−<sup>1</sup> gentamicin. Additional four, 10-fold or <sup>1</sup> <sup>2</sup> log serial dilutions were made to provide a total of five drug concentrations plus control. Aliquots of 100 μL of these different drug dilutions were added to the appropriate microtiter wells already containing 100 μL of medium, resulting in the required final drug concentrations.

Following drug addition, the plates were incubated at 37 ◦C, 5% CO2, 95% air, and 100% relative humidity for an additional 48 h. For adherent cells, the assay was terminated by the addition of cold TCA. Cells were fixed in situ by the addition of 50 μL of cold 50% (*w*/*v*) TCA, and incubated at 4 ◦C for 60 min. The supernatant was discarded, and the plates were washed with water (5×) and dried in air. Sulforhodamine B (SRB) solution (100 μL) at 0.4% (*w*/*v*) in 1% acetic acid was added to each well, and plates were incubated at room temperature for 10 min. After staining, the unbound dye was removed by washing five times with 1% acetic acid, and the plates were dried in the air. The bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at λ = 515 nm. Using the seven absorbance measurements [time zero (*t*z), control growth (*c*), and test growth in the presence of drug at the five concentration levels (*t*i)], the percentage growth was calculated at each of the drug concentration levels. Growth inhibition (%) was calculated as:

$$\left[\left(t\_{\mathrm{i}} - t\_{\mathrm{z}}\right)/\left(\mathrm{c} - t\_{\mathrm{z}}\right)\right] \times 100\text{, for concentrations where }t\_{\mathrm{i}} \ge t\_{\mathrm{z}}\tag{1}$$

$$\left[\left(t\_{\mathrm{i}} - t\_{\mathrm{z}}\right)/\left(t\_{\mathrm{z}}\right)\right] \times 100\text{, for concentrations where } t\_{\mathrm{i}} < t\_{\mathrm{z}}.\tag{2}$$

Three dose-response parameters were calculated as follows. *GI*<sup>50</sup> (growth inhibition of 50%) was calculated from Equation (3), which is the drug concentration resulting in a 50% reduction in the net protein increase (as measured by SRB staining) in control cells during the drug incubation. The drug concentration resulting in total growth inhibition (TGI) was calculated from Equation (4), where *t*<sup>i</sup> = *t*z. The *LC*<sup>50</sup> indicating a 50% net loss of cells following treatment was calculated from Equation (5):

$$[(t\_{\rm i} - t\_{\rm z})/(c - t\_{\rm z})] \times 100 = 50\tag{3}$$

$$\left[\left(t\_{\rm i} - t\_{\rm x}\right)/\left(c - t\_{\rm x}\right)\right] \times 100 = 0\tag{4}$$

$$[(t\_{\rm i} - t\_{\rm x})/(t\_{\rm x})] \times 100 = -50.\tag{5}$$

3.3.3. Antiproliferative Assay on HeLa and SiHa Cells

Cervical adenocarcinoma (HeLa) and cervical carcinoma (SiHa) cells were obtained from the European Collection of Cell Cultures (Salisbury, UK) and the American Type Tissue Culture Collection (Manassas, VA, USA), respectively. The cells were cultured in Minimum Essential Medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, and 1% penicillin-streptomycin at 37 ◦C in a humidified atmosphere. Media and supplements were purchased from Lonza Group Ltd. (Basel, Switzerland). Cell viability was assessed by the MTT assay as published before [36]. Briefly, the cells were seeded in 96 well plates at 5000 cells/well density. After 24 h, 100 μL of new media containing the test samples was added. After incubation for 72 h, an aliquot of 44 μL of MTT solution (5 mg/mL) was added. After incubation for a further 4 h, the medium was removed by aspiration, the precipitated formazan crystals were dissolved by adding 100 μL of DMSO to each well, and the plates were shaken at 37 ◦C for 1 h. The absorbance was measured at 545 nm with a microplate reader. IC50 values were calculated by fitting sigmoidal dose–response curves by the nonlinear regression model log (inhibitor) vs. normalized response and variable slope fit of GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA). Clinically utilized anticancer agent cisplatin (Ebewe GmbH, Unterach, Austria) was included as a reference molecule.

#### **4. Conclusions**

As a result of the current study, hybrid compounds containing chrysin coupled with substituted 1,2,3-triazole pharmacophores showed significant in vitro anticancer activities on several cell lines of different types of cancer. Moreover, the activity of the bis-conjugated derivatives of chrysin was also considerable. Therefore, it may be a reasonable strategy to prepare further hybrid molecules of flavones with more complex structures to obtain potentially valuable new antitumor leads.

**Author Contributions:** A.N.-R. performed the experiments; P.K. and L.H. conceived and designed the experiments; A.D.L. performed bioactivity testing supervised by I.Z. and A.H.; L.H. wrote the paper. All authors have read and agreed to the published version of the manuscript.

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

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not available.

**Acknowledgments:** Project no. RRF-2.3.1-21-2022-00015 has been implemented with the support provided by the European Union. A.H. acknowledges support from the Ministry of Innovation and Technology of Hungary (TKP2021-EGA-32). We thank Áron Szigetvári, Miklós Dékány and Csaba Szántay Jr., Gedeon Richter Plc., for the NMR and MS measurements and interpretations.

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

**Sample Availability:** Samples of the compounds are not available from the authors.

#### **References**


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## *Review* **Therapeutic Potential of Phenolic Compounds in Medicinal Plants—Natural Health Products for Human Health**

**Wenli Sun \*,† and Mohamad Hesam Shahrajabian †**

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

**\*** Correspondence: sunwenli@caas.cn; Tel.: +86-13-4260-83836

† These authors contributed equally to this work.

**Abstract:** Phenolic compounds and flavonoids are potential substitutes for bioactive agents in pharmaceutical and medicinal sections to promote human health and prevent and cure different diseases. The most common flavonoids found in nature are anthocyanins, flavones, flavanones, flavonols, flavanonols, isoflavones, and other sub-classes. The impacts of plant flavonoids and other phenolics on human health promoting and diseases curing and preventing are antioxidant effects, antibacterial impacts, cardioprotective effects, anticancer impacts, immune system promoting, anti-inflammatory effects, and skin protective effects from UV radiation. This work aims to provide an overview of phenolic compounds and flavonoids as potential and important sources of pharmaceutical and medical application according to recently published studies, as well as some interesting directions for future research. The keyword searches for flavonoids, phenolics, isoflavones, tannins, coumarins, lignans, quinones, xanthones, curcuminoids, stilbenes, cucurmin, phenylethanoids, and secoiridoids medicinal plant were performed by using Web of Science, Scopus, Google scholar, and PubMed. Phenolic acids contain a carboxylic acid group in addition to the basic phenolic structure and are mainly divided into hydroxybenzoic and hydroxycinnamic acids. Hydroxybenzoic acids are based on a C6-C1 skeleton and are often found bound to small organic acids, glycosyl moieties, or cell structural components. Common hydroxybenzoic acids include gallic, syringic, protocatechuic, *p*-hydroxybenzoic, vanillic, gentistic, and salicylic acids. Hydroxycinnamic acids are based on a C6-C3 skeleton and are also often bound to other molecules such as quinic acid and glucose. The main hydroxycinnamic acids are caffeic, *p*-coumaric, ferulic, and sinapic acids.

**Keywords:** phenolics; curcumin; protocatechuic; quinones; stilbenes; curcuminoids

#### **1. Introduction**

Medicinal plants are very important worldwide, both when used alone and as a supplement to traditional medication [1–5]. For many years, humans have employed plants as a source of food, flavoring, and medicines [6–10]. Various parts of medicinal plants such as seeds, leaves, flowers, fruits, stems, and roots are rich sources of bioactive compounds [11–13]. Bioactive compounds should be considered as important dietary supplements [14–19]. Polyphenols are a group of secondary metabolites involved in the hydrogen peroxide scavenging in plant cells [20]. Phenolic compounds are second only to carbohydrates in abundance in higher plants, and they display a great variety of structures, varying from derivatives of simple phenols to complex polymeric materials such as lignin [21–26]. Phenolic compounds are known for their notable potential activity against various human viruses, and phenolic compounds also have immunomodulatory and anti-inflammatory activity [27]. The most abundant phenolic compounds are phenolic monoterpenes (carvacrol and thymol) and diterpenes (carnosol, carnosic acid, and methyl carnosate), hydroxybenzoic acids (*p*-hydroxybenzoic, protocatechuic, gallic, vanillic, catechol, and ellagic), phenylpropanoic acids (*p*-coumaric, caffeic, rosmarinic, chlorogenic, ferulic, cryptochlorogenic, and neochlorogenic), phenylpropenes (eugenol), coumarins

**Citation:** Sun, W.; Shahrajabian, M.H. Therapeutic Potential of Phenolic Compounds in Medicinal Plants—Natural Health Products for Human Health. *Molecules* **2023**, *28*, 1845. https://doi.org/10.3390/ molecules28041845

Academic Editor: Giovanni Ribaudo

Received: 6 January 2023 Revised: 11 February 2023 Accepted: 13 February 2023 Published: 15 February 2023

**Copyright:** © 2023 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 (https:// creativecommons.org/licenses/by/ 4.0/).

(herniarin and coumarin), flavanoes (naringenin, eriocitrin, naringin, and hesperidin), flavones (apigenin, apigetrin, genkwanin, luteolin, luteolin 7-glucuronide, cynaroside, scolymoside, salvigenin, and cirsimaritin), and flavanols (catechin, astragalin, kaempferol, methyl ethers, quercetin, hyperoside, isoquercetin, miquelianin, and rutin) [28,29].

Plant phenolics are considered promising antibiofilm and antifungal agents [30,31]. Diaz et al. [32] also reported that the levels of phenolic and flavonoid compounds were correlated with the anti-inflammatory and antioxidant activities of medicinal plants. Tukun et al. [33] reported that phenolic content is significantly connected to antioxidant activity, and halophytes have high content of nutrients and phenolic metabolites. Some of the most important phenolic compounds recognized from medicinal plants are syringic acid and gallic acid from *Moringa oleifera* [34]; gallic acid, vanillic acid, 4-hydroxybenzoic acid, and syringic acid from *Peganum harmala* [35]; rosmarinic acid from *Rosmarinus officinalis* L. and *Mentha canadensis* L. [36]; vanillin from *Thymus vulgaris* [37]; caffeic acid and *p*-coumaric acid from *Ocimum basilicum* L., *Thymus vulgaris* L., *Salvia officinalis* L., and *Origanum vulgare* L. [36]; piceatannol glucoside, resveratroloside, and piceid from *Polygonum cuspidatum* [38]; trans-rhapontin, cis-rhapontin, and trans-desoxyrhaponticin from *Rheum tanguticum* Maxim. Ex Balf. [39]; herniarin from *Matricaria chamomilla* [40]; kayeassamin I, mammeasin E, and mammeasin E from *Mammea siamensis* [41]; scopoletin, fraxetin, aesculetin, fraxin, and aesculin from *Fraxinus rhynchophylla* [42]; phyllanthin, niranthin, hypophyllanthin, nirtetralin, virgastusin, heliobuphthalmin lactone, and bursehernin from *Phyllanthus amarus* [43]; schisanchinin A, schisanchinin B, schisanchinin C, and schisanchinin D from *Schisandra chinensis* [44]; 7-methyljuglone from *Drosera rotundifolia* [45], rhein, physcion, chrysophanol, emodin, and aloe-emodin from *Rheum palmatum* and *Rheum hotaoense* [46]; curcumin, demethoxycurcumin, and bis-demethoxycurcumin from *Curcuma longa* [47]; luteolin, apigenin, orientin, apigenin-O-glucuronide, and luteolin-O-glycoside from *Origanum majorana* [48]; glycitein, genistein, formononetin, daidzein, prunetin, biochanin A and daidzin, and genistin from *Medicago* spp. [49]; kaempferol 3-O-glucoside and isorhamnetin 3-O-galactoside from *Tephrosia vogelii* [50]; rutin, kaempferol 3-O-rhamnoside, and quercetin 3-O-glucoside from *M. oleifera* [34]; gallocatechin and catechin from *Mentha pulegium* [48]; taxifolin, taxifolin methyl ether, and dihydrokaempferide from *Origanum majorana* [48]; hesperidin, naringenin-O-rhamnoglucoside, and isosakuranetin-O-rutinoside from *Mentha pulegium* [48]; and punicalagin, pedunculagin I, granatin A, ellagic acid, ellagic acid pentoside, ellagic acid glucoside, and punigluconin from *Punica granatum* [51]. Phenolic phytochemicals include flavonoids, flavonols, flavanols, flavanones, flavones, phenolic acids, chalcones, isoflavones, tannins, coumarins, lignans, quinones, xanthones, curcuminoids, stilbenes, cucurmin, phenylethanoids, and several other plant compounds, owing to the hydroxyl group bonded directly to an aromatic hydrocarbon group [52]. The classes of phenolic compounds in plants are shown in Table 1.

**Table 1.** Classes of phenolic compounds in plants [53].


Phenolic acids include two subgroups, i.e., hydroxybenzoic and hydroxycinnamic acids [53]. Hydroxybenzoic acids consist of gallic, *p*-hydroxybenzoic, vanillic, protocatechuic, and syringic acid, which, in common, have the C6-C1 structure [53]. Hydroxycinnamic acids, on the other hand, are aromatic compounds with a three-carbon side chain (C6-C3), with caffeic, *p*-coumaric, ferulic, and sinapic acids being the most common [52]. Gallic acid is present in cloves (*Eugenia caryophyllata* Thunb.), while protocatechuic acid can be found in coriander (*Coriandrum sativum* L.), dill (*Anethum graveolens* L.), and star anise (*Illicium verum* Hook. f.) [54]. Caffeic acid is found among others in parsley (*Petroselinum crispum* L.), ginger (*Zingiber officinale* Rosc.), and sage (*Salvia officinalis* L.), and *p*-coumaric acid is found in oregano (*Origanum vulgare* L.), basil (*Ocimum basilicum* L.), and thyme (*Thymus vulgaris* L.) [54]. Some samples of hydroxybenzoic and hydrozycinnamic acids are presented in Table 2.


**Table 2.** Examples of hydroxybenzoic and hydroxycinnamic acids.

Flavonoids include the largest group of plant phenolics, responsible for over half of the eight thousand naturally occurring phenolic constituents [55,56]. Flavonoids are low molecular weight compounds, including fifteen carbon atoms, arranged in a C6-C3-C6 configuration [53]. The genetic structure of main classes of flavonoids are shown in Table 3.

Phenolic phytochemicals play a variety of protective roles against abiotic stresses, such as UV light, or abiotic stresses, namely predator and pathogen attacks [57]. Phenolic phytochemicals are utilized by humans to treat several ailments including bacterial, protozoal, fungal, and viral infections, inflammation, diabetes, and cancer. Biosynthesis and accumulation of polyphenol and other secondary metabolites in plants is considered as an evolutionary reaction of biochemical pathways under adverse environmental influences, i.e., biotic/abiotic limitations, including increased salinity and drought stress [58–60]. Some of the extraction methodologies of phenolic components from medicinal and aromatic plants are maceration, digestion, infusion, decoction, Soxhlet extraction, percolation, aqueous alcoholic extraction by fermentation, counter-current extraction, ultrasound extraction, supercritical fluid extraction, and phytonics stage. The principle factors shaping the production of phenolic components are the water supplied to plants and the time of stress exposure, and, among the various quantification methods, HPLC and colorimetric tests are the most utilized to quantify the phenolic compounds analyzed [61]. Djeridane et al. [62] reported that the phenolics in medicinal plants provide substantial antioxidant activity. A positive, significant linear connection between antioxidant activity and total phenolic content revealed that phenolic components were the dominant antioxidant constituents in medicinal plants [63,64]. Various groups of tests on phenolics indicated significant mean alterations in radical scavenging activity; tannins demonstrated the strongest activity, while most quinones, isoflavones, and lignans tested revealed the weakest activity [65,66]. The most abundant flavone in *Cytisus multiflorus* is the chrysin derivative, Kaempferol-3-*O*-rutinoside is the major flavonol in *Malva sylvestris*, and Quercetin-3-*O*-rutinoside is the principle flavonol in *Sambucus nigra* [66]. *Nepeta italica* subsp. *cadmea* and *Teucrium sandrasicum* are rich in phenolics, which indicated antioxidant and cytotoxic properties [67]. Through LC-ESI-MS analysis, five phenolic acids (quinic acid, syringic acid, gallic acid, *p*-coumaric acid, and trans-ferulic acid) and five flavonoids (catechin, epicatechin, quercetrin, rutin, and naringenin) were predominant and common in some desert shrubs of Tunisian flora (*Pituranthos tortuosus*, *Ephedra alata*, *Retama raetam*, *Ziziphus lotus*, *Calligonum comosum*, and *Capparis spinosa*) [68].


**Table 3.** Generic structure of major classes of flavonoids.

The main phenolic compounds in Matico (*Piper angustifolium* R.), Guascas (*Galinsoga parviflora*), and Huacatay were chlorogenic acid and hydroxycinnamic acid derivatives [69]. High phenolic and antioxidant activity-containing medicinal plants and species such as Chanca Piedra (*Phyllanthus nirui* L.), Yerba Mate (*Ilex paraguariensis* St-Hil), Zarzaparrilla (*Smilax officinalis*), and Huacatay (*Tagetes minuta*) have the highest anti-hyperglycemiarelevant in vitro α-glucosidase inhibitory activities with no effect on α-amylase [69]. Nineteen phenolic compounds from different groups are used in wound treatment, and the compounds are tyrosol, curcumin, hydroxytyrosol, luteolin, rutin, chrysin, kaempferol, quercetin, icariin, epigallocatechin gallate, morin, silymarin, taxifolin, hesperidin, naringin, puerarin, isoliquiritin, genistein, and daidzein [70–73]. The most important identified phenolics in *Phlomis angustissima* and *Phlomis fruticosa*, medicinal plants from Turkey, by RP-HPLC-DAD were hesperidin, catechin, kaempferol, epicatechin, eupatorin, and epigallocatechin, and chlorogenic, syringic, vanillic, *p*-coumaric, ferulic, and benzoic acids [74]. Quercetin of *Cordia dichotoma* G. Forst. (Lashusa) is the most notable phytoconstituent responsible for the therapeutic efficacy [75]. Vanillic acid, nepetin, verbascoside, and hispidulin, of *Clerodendrum petasites* S. Moore (CP) were chosen as potential phenolic active compounds in Thai traditional medicine for the treatment of different kinds of skin diseases [76–78]. Bouyahya et al. [79] reported that compounds such as terpenoids, alkaloids, flavonoids, phenolic acids, and fatty acids of *Arbutus unedo* L., *Thymus capitatus* managed diabetes by several mechanisms such as enzymatic inhibition, interference with

glucose and lipid metabolism signaling pathways, and the inhibition and the activation of gene expression involved in glucose homeostasis.

*Grewia tenax*, *Terminalia sericea*, *Albizia anthelmintica*, *Corchorus tridens*, and *Lantana camara* are frequently used to treat gastroenteritis and include higher total phenolic and flavonoid contents in Namibia [80–85]. The most important phenolics identified from pomegranate are punicalin, gallic acid, ellagic acid, pyrogallol, salycillic acid, coumaric acid, vanillic acid, sesamin, and caffeic [86], and phenolic compounds have been discovered to have inhibitory effects again α-glucosidase activities [87]. Two new phenolics, leucoxenols A and B, were obtained and identified as major secondary metabolites from the leaves of *Syzygium leucoxylon* [88]. Phenolics are main phytochemicals found in *Cyathea* species, and *Cyathea* has been considered to be a potential source of novel cancer therapeutic compounds [89]. Purified phenolic compounds from the bark of *Acacia nilotica* showed insecticidal potential against *Spodoptera litura*, and they could provide substitutes to synthetic pesticides for controlling various pests [90]. Bellumori et al. [91] reported that the roots of *Acmella oleracea* L. had about twice as many phenols as the aerial parts, and caffeic acid derivatives were the main phenolic compounds in roots and aerial parts. Kaempferol was found as the most abundant phenolic compound in basil leaf extract after using an HPLC-UC method (61.4 mg.kg<sup>−</sup>1) [92]. Apple fruit (*Annona squamosa* L.) has a specific spatial distribution of microbes and phenolics, its peel phenolics contain antimicrobial activity against several Gram-positive bacteria, and its peel phenolics had a growth-promoting effect toward autochthonous yeasts [93–96]. The phenolic contents of *Cyathea dregei* (root and leaves), *Felicia erigeroides* (leaves and stems), *Felicia erigeroides* (leaves and stems), *Hypoxis colchicifolia* (leaves), *Hypoxis colchicifolia* (leaves), and *Senna petersiana* (leaves) have shown high antimicrobial and cyclooxygenase (COX) inhibitory activities [97].

The most important techniques for analysis of phenolic compounds and extracts are nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC) with ultraviolet-visible (UV-Vis) or photodiode array (PDA) detector or coupled to mass spectrometry (MS), derivatization (silylation, alkylation, etc.) as well as gas chromatography (GC) or GC-MS analysis, phytochemical screening such as total flavonoid content (TFC), total phenolic content (TPC), etc., and antioxidant potential tests such as 2,2-dipehnyl-1-picrylhydrazyl (DPPH), etc. [97–107]. Solid-liquid extraction (SLE) is one of the main methods for extraction of phenolic compounds, specially syringic acid, catechin, and *p*-coumaric acid, which is simple, well established, and widely used [108]. Ultrasoundassisted extraction (UAE) is often used for extraction of gallic acid and rutin, which is easy to execute, uses inexpensive equipment, and consumes less solvents, and has fast extraction, good extraction yield, and low impacts on the environment [109]. Supercritical fluid extraction (SFE) usually applies for gallic acid, anthocyanin, and protocatechuic acid, which has high selectivity, cheaper and safer solvent, easily controlled extraction conditions, environmental friendliness, low operating temperature, and easy separation of solvent from solutes [110]. Microwave-assisted extraction (MAE) is used for extraction of 3-caffeoylquinic acid, 5-caffeoylquinic acid, and ellagic acid, which has short extraction time and low solvent consumption [111]. Pressurized liquid extraction (PLE) applies for extraction of rutin and quercetin, which consumes fewer organic solvents, has higher probability to avoid organic solvents by using water only, and is fast and efficient [112]. For extraction of proanthocyanidin, naringin, and hesperidin, enzyme-assisted extraction (EAE) is proposed, which is safe and green and does not need complex paraphernalia [113]. Key points about phenolic acids and their derivatives are shown in Table 4. This work aims to provide an overview of phenolic compounds and flavonoids as potential sources of pharmaceutical and medical application from recently published studies, as well as some interesting directions for future research.


**Table 4.** Important points about phenolic acids and their derivatives.

#### **2. The Important Health Benefits of Phenolic Components**

Flavonoids and phenolics are commonly known as the largest phytochemical molecules with antioxidant characteristics [124]. Traditional Chinese medicinal plants that contain phenolic acids and flavonoids have shown high antioxidant activity. *Nepeta italica* subsp. Cadmea and *Teucrium sandrasicum* are rich in phenolic, tannin, and flavonoids content, which showed antioxidant and cytotoxic properties. *Bauhinia variegata* L. contained flavonoid compounds and revealed antioxidant properties against oxidative damage by radical neutralization, iron binding, and decreasing power abilities [125]. The rhizome extracts of *Polygonatum verticillatum* (L.) All. exhibited antioxidant activity, which is connected to the level of phenolic composition [126]. Singh and Yadav [127] have reported that, among medicinal plants, oregano, clove, thyme, and rosemary contain the highest amounts of phenolic compounds. Flavan-3-ol oligomers and monomers were potent antioxidant compounds abundantly identified in *Camellia fangchengensis* [128].

*Bellis perennis* L. was rich in phenolic compounds, and it can be used for wounds, cancer, inflammation, and eye diseases [129]. A total of 27 kinds of phenolic compounds were identified by HPLC-ESI-QTOF-MS/MS, and okra (*Abelmoschus esculentus*) polyphenols exhibited great antioxidant activity in vitro [130]. The *Althaea officinalis* extracts showed stronger antioxidant activity and excellent α-glucosidase, 5-lipoxygenase, and nitric oxide inhibitory properties [131]. *Dendrobium densiflorum* was rich in flavonoid, alkaloid, and antioxidant activity, *Acampe papillosa* was rich in total phenol, total tannin, and total saponin content, and *Coelogyne nitida* exhibited higher antioxidant activity because of its higher quercetin content [132]. Cirak et al. [133] showed that *Achillea arabica* Kotschy is an important source of natural antioxidants. The antioxidant property and bioactive constituents from the fruits of *Aesculus indica* (Wall. Ex Cambess.) Hook, which were quercetin and mandelic acid, were the major bioactive molecules with notable antioxidant properties to decrease oxidative stress caused by reactive oxygen species (ROS) [134]. The phytochemical compounds and biological activity of *Pinus cembra* L. contain higher concentration of total phenolics and flavonoids than that of needle extract, and its bark extract showed better ability as a free radical scavenger [135]. Higher antioxidant activity in normal-tannin lentil seed coats than low-tannin ones was reported; kaempferol tetraglycoside was dominant in low-tannin seed coats, and procyanidins, kaempferol tetraglycoise, and catechin-3-*O*-glucoside in normal-tannin has been found [136]. Zhang et al. [137] also reported that antioxidant activity and prebiotic impacts were positively correlated for oat phenolic compounds. 3,4-dihydroxybenzoic, rutin, vanillic acid, and quercetin were detected from aqueous extracts of *azendjar* and *taamriouth* figs, and a dark peel variety consisted of more phenolics and exerted a higher antioxidant capacity [138]. Although gallic acid was the most important compound in carob (*Ceratonia siliqua* L.) pulp extract, geographic origin strongly influenced the contents of bioactive compounds and antioxidant activities [139].

*Asplenium nidus* L. contained gliricidin 7-*O*-hexoside and quercetin-7-*O*-rutinoside that can fight against three pathogens, i.e., *Proteus vulgaris* Hauser, *Proteus mirabilis* Hauser, and *Pseudomonas aeruginosa* (Schroeter) Migula [140]. Flavones, which were extracted from the root of *Scutellaria baicalensis* Georgi, were proven as potential antibacterial agents against *Propionibacterium acnes*-induced skin inflammation both in in vitro and in vivo models [141]. Kaempferol that was isolated from the *Impatiens balsamina* L. exhibited potential activity to inhibit the growth of *P. acnes* [142]. Phenolics from kernel extract *Mangifera indica* L. also showed anti-acne properties to inhibit the growth of *P. acnes* [143]. Medicinal plants such as *Albizia procera*, *Atalantia monophylla*, *Asclepias curassavica*, *Azima tetracantha*, *Cassia fistula*, *Costus speciosus*, *Cinnamomum verum*, *Nymphaea stellata*, *Osbeckia chinensis*, *Punica granatum*, *Piper argyrophyllum*, *Tinospora cordifolia*, and *Toddalia asiatica* have shown antifungal activity [144]. The strictinin isolated from the leaves of *Camellia sinensis* var. *assamica* (J.W. Mast.) Kitam was a good substitute for antibacterial activities [145]. Phenolic compounds, especially flavonoids, have long been reported as chemopreventive factors in cancer therapy [146–148]. The extract of *Curcuma longa* L. rhizome has been suggested as a promising source of natural active compounds to fight against malignant melanoma due to its potential anticancer property in the B164A5 murine melanoma cell line [149]. Glircidia 7-*O*-hexoside and Quercetin 7-*O*-rutinoside, which were flavonoids isolated from the medicine fern (*Asplenium nidus*), were also proposed as potential chemopreventives against human hepatoma HepG2 and human carcinoma HeLa cells [140]. Quercetin can

induce miR-200b-3p to regulate the mode of self-renewing divisions of the tested pancreatic cancer [150], and a soy isoflavone genistein inhibited the activation of the nuclear factor kappa B (NF-KB) signaling pathway that maintains the balance of cell survival and apoptosis; this soy isoflavone could also take its action to fight against cell growth, apoptosis, and metastasis, including epigenetic modifications in prostate cancer [151]. Curcumin exhibits anticancer impacts towards skin cancers, as this phenolic can influence the cell cycle by acting as a pro-apoptotic agent [152]. Curcumin acts as a non-selective cyclic nucleotide phosphodiesterase (PDE) inhibitor to inhibit melanoma cell proliferation, which is associated with epigenetic integrator UHRF1 [153]. Curcumin inhibited proliferation of the selected cell lines in prostate cancer and induced apoptosis of the cancer cells with a dose-dependent response [154].

The cardioprotective impacts from various kinds of phenolics and flavonoids occurring in medicinal plants have been investigated in many studies [155,156]. Many phenolic and flavonoid compounds have been studied and had reported their cardioprotective properties via different mechanisms including inhibition of ROS generation, apoptosis, mitochondrial dysfunction, NF-KB, p53, and DNA damage both in vitro and in vivo, and clinical studies [157]. Kaempferol, luteolin, rutin, and resveratrol showed their efficacy against doxorubicin-induced cardiotoxicity [158,159]. Isorhamnetin provided a cardioprotective effect against cardiotoxicity of doxorubicin and potentiated the anticancer efficacy of this drug [160]. The total phenolic and flavonoid contents of the aqueous fraction from *Marrubium vulgare* L. have effects on ischemia-reperfusion injury of rat hearts, which proved that the aqueous fraction from *M. vulgare* had cardioprotective potential [156]. Aspalathin and phenylpyruvic acid-2-*O*-β-D-glucoside, two of the major compounds from *Aspalathus linearis* (Burm.f.) R. Dahlgren, were demonstrated as potential protective compounds to protect myocardial infarction caused by chronic hyperglycemia [155]. Puerarin is a potential isoflavone that was reported as an interesting candidate for cardioprotection by protecting myocardium from ischemia and reperfusion damage by means of opening the Ca2+-activated K+ channel and activating the protein kinase C [161]. Quercetin, hesperidin, apigenin, and luteolin were reported as flavonoids containing potential anti-inflammatory impacts [162]. The flavonoids and phenolic compounds of *Phyllanthus acidus* leaves could be correlated with the analgesic, antioxidant, and anti-inflammatory activities [163]. Hydroxytyrosol and quercetin 7-*O*-α-L-rhamnopyranoside exhibited anti-inflammatory activity through lowering the levels of TNF-α, and hydroxytyrosol and caffeic acid showed significant anti-inflammatory activity at 100 μm by reducing the release of NO in LPS-stimulated macrophages comparable to positive control indomethacin [164].

The most important chemical compounds extracted from ethanol of *Cardiospermum halicacabum* were chrysoeriol, kaempferol, apigenin, luteolin, methyl 3,4-dihydroxybenzoate, 4-hydroxybenzoic acid, quercetin, hydroquinone, protocatechuic acid, gallic acid, and indole 3-carboxylic acid, which have shown high anti-inflammatory and antioxidant activities [165]. The most important phenolic components with antiviral effects against COVID-19 were curcumin, Theaflavin-3,3 -digallate, EGCG, Paryriflavonol A, Resveratrol, Quercetin, Luteolin, Scutellarein, Myricetin, and Forsythoside A [166]. In traditional Persian medicinal science, medicinal plants such as *Glycyrrhiza glabra* L., *Rheum palmatum* L., *Punica granatum* L., and *Nigella sativa* L. have been introduced for treating respiratory disorders and infections because of their phenolic compounds [167]. The anti-inflammatory activity of polyphenolic compounds in *Gaillardia grandiflora* Hort. Ex Van Houte and *Gaillardia pulchella* Foug from Egypt were reported [168]. Anti-inflammatory properties of two medicinal plant species, *Bidens engleri* O.E. Schulz from Asteraceae family as well as *Boerhavia erecta* L. from Nyctaginaceae family, were identified and reported in various fractions [169]. *Plantago subulata* has shown anti-inflammatory properties on macrophages and a protective effect against H2O2 injury [170]. Phenolic content changes with aromatic and medicinal plant species and extraction method used [171]. Astilbin, a dihydroflavonol, from *Smilax glabra* Roxb significantly inhibited nitric oxide production, tumor necrosis factor-α (TNF-α), and mRNA expression of inducible nitric oxide synthase in the tested cells [172]. Apigenin is a

main flavone with skin protective impact against UV light; this flavone can be identified in various edible medicinal plants or plants-derived beverages, e.g., beer, red wine, and chamomile tea [173,174]. Quercetin is a flavonol that can be discovered in apple peel, onion skin, and *Hypericum perforatum* L. leaves [175]. Silymarin, a standardized extract of flavonolignans from the milk thistle (*Silybum marianum* (L.) Gaernt.) fruits, consists of silybin, a principle active component [176]. Genistein is a soybean isoflavone that was also reported as photoprotective molecule against photocarcinogenesis by inhibiting UV-induced DNA damage in human skin-equivalent in vitro model [177]. Equol is considered as an isoflavonoid metabolite from isoflavone daidzein or genistein produced by gut microflora [178,179]. Genistein is an obvious example of an interesting choice of a flavonoid phytoestrogen for improving endothelial roles in postmenopausal women with MetS [180]. A chrysin derivative was the most abundant flavone in *Cytisus multiflorus*, quercetin-3-*O*rutinoside was the main flavonol in *Sambucus nigra,* and kaempferol-3-*O*-rutinoside was the main flavonol in *Malva sylvestris* [181]. Biological properties of phenolic compounds are presented in Table 5.

**Table 5.** Biological activities of phenolic compounds.




#### **3. Hydroxybenzoic Acids (Gallic Acid and Protocatechuic Acid)**

Hydroxybenzoic acids (HBAs) are antioxidant phytochemicals found in many medicinal plants and are efficient for prevention of various human diseases [247,248]. Joshi et al. [249] reported that 4-hydroxybenzoic acid (4HBA) is a potential antidiabetic, anticancer, antifungal, antioxidant, and cardioprotective, etc. *Piper garagaranum* C. DC contains prenylated hydroxybenzoic acids, and prenylated hydroxybenzoic acids indicated anti-inflammatory characteristics, as determined in murine macrophage assays [250].

#### *3.1. Gallic Acid*

Gallic acid is one of the most abundant polyphenols identified in nature [251,252]. Behera et al. [253] reported that gallic acid reveals antioxidant or free radical scavengers in adipocyte proliferation. Gallic acid is found in a wide range of natural plants, it is associated with the health of human beings, and it has well-documented anticancer, antibacterial, anti-inflammatory, and antifungal activities [254,255]. Gallic acid in *Emblica officinalis* mediated antidiabetic potential and delineated the upregulation of pAkt, PPAR-γ, and Glut4 through gallic acid-mediated antidiabetic properties, thus providing potent therapy for diabetes [256]. Gallic acid inhibited about 44–57% of the total CaOx crystal formations, and it is a promising agent with antiurolithiatic properties for the treatment and prevention of urinary or kidney stones [257]. Gallic acid supplementation adjusted serum lipid metabolism by decreasing serum triglyceride, fat digestibility, and bacteroidetes/firmicutes ratio [258]. Gallic acid prevents the development and occurrence of gastric precancerous lesions (GPL) by inhibiting the Wnt/β-catenin signaling pathway and then suppressing the epithelial– mesenchymal transition (EMT) process [259]. Gallic acid is a direct thrombin inhibitor with a platelet aggregation inhibitory effect [260]. Gallic acid shows significant binding and disruption of protease structure, and gallic acid has a potential phytotherapeutic effect against fungal protease, which is a notable virulence factor [261]. Gallic acid can boost gut microbiota alterations connected with cardiovascular disease (CVD) and suggests that males suffering from atherosclerosis may benefit from gallic acid supplementation, as this polyphenol partially restored microbiome dysbiosis [262]. Gallic acid could decrease the noxious impacts of diclofenac (DIC) on the antioxidant defense system and renal tissue [263].

#### *3.2. Protocatechuic Acid*

Protocatechuic acid (3,4-dihydroxybenzoic acid) is a natural phenolic acid, and one of the chief metabolites of complex polyphenols [264]. It can be identified in many plants such as bran and grain brown rice, particularly in the scales of onion, plums, grapes, gooseberries, and nuts such as ordinary almonds [265,266]. Da-Costa-Roch et al. [267] and Adedara et al. [268] reported that protocatechuic acid can be found in many medicinal plants, especially *Hibiscus sabdariffa* L. (Hs, roselle; Malvaceae). Protocatechuic acid has different activities such as neuroprotective activities, antiosteoporotic activities, antitumor activities, and the protective effects against hepatotoxic and nephrotoxic activities [269,270]. It has also antibacterial, antiulcer, anti-aging, antidiabetic, anticancer, antiviral, antifibrotic, analgesic, anti-inflammatory, anti-atherosclerotic, and cardiac activity [271,272]. Protocatechuic acid from bitter melon (*Momordica charantia*) alleviates cisplatin-induced oxidative renal damage, which proves it has protective activity against anticancer drug-induced oxidative nephrotoxicity [273]. Protocatechuis acid inhibits Cd-induced neurotoxicity in rats, increases the Nrf2 signaling pathway, and exhibits anti-apoptotic and anti-inflammatory activities [274]. *Veronica montana* has protocatechuic acid as the main phenolic molecule, and it kills bacteria by affecting its cytoplasmic membrane [275].

#### **4. Hydroxycinnamic Acids (***p***-Coumaric Acid, Caffeic Acid, Ferulic Acid, Sinapic Acid)**

Hydroxycinnamic acid derivatives are a notable class of polyphenols found in vegetables, fruits, and medicinal plants, and extensively consumed in human diet [276,277]. Hydroxycinnamic acids significantly contribute to antioxidant capacity [278]. Hydroxycinnamic acids are widely found in plants and their products such as cereals, fruits, coffee, vegetables, etc. [279,280].

#### *4.1. p-Coumaric Acid*

*p*-Coumaric acid is a plant metabolite with antioxidant and anti-inflammatory impacts [281,282]. *p*-Coumaric acid boosts hepatic fatty acid oxidation and fecal lipid excretion, and it affects inflammatory and insulin resistance-related adipokines. *p*-Coumaric acid stimulates electrical factors of biological and model lipid membranes [283].

#### *4.2. Caffeic Acid*

Caffeic acid (3,4-dihydroxycinnamic acid) has been known as an important source of natural antioxidants in different agricultural products [284,285]. It has immense use in cancer treatment [286,287], and it could be known as an important natural antioxidant [288]. Caffeic acid can induce apoptosis in cancer cells through increasing ROS levels and impairing mitochondrial function, and it also benefits from reducing aggressive behavior of tumors via suppressing metastasis [289]. Caffeic acid has anti-inflammatory and antioxidant properties against 6-propyl-thiouracil (PTU)-induced hypothyroidism [290]. Meinhart et al. [291] reported that higher sums of mono-caffeoylquinic acids were found in mulberry, quince, and bilberry, and the dicaffeoylquinic acids sum was higher in granadilla, passion fruit, and kumquat. It is a phenolic compound extensively discovered in commonly consumed foods such as apples, pears, and coffee [292]. The biosynthesis pathway of caffeic acid can be categorized into two modules, (1) L-tyrosine is synthesized from carbon sources via the glycolytic pathway, the pentose phosphate pathway, and the shikimate pathway; (2) caffeic acid is generated by the continuous deamination and hydroxylation of L-tyrosine [293]. Trifan et al. [294] found that caffeic acid oligomers reported in *Symphytum officinale* L. root may contribute to the anti-inflammatory activity for which comfrey preparations are used in traditional medicine. Caffeic acid phenethyl ester extracted from *Rhodiola sacra* could provide health benefits, decreasing the magnitude of the inflammatory process triggered by endotoxin shock and the production of inflammatory mediators [295]. Caffeic acid from the leaves of *Annona coriacea* have shown antidepressant-like impacts, which involve important neurotransmitter systems [296]. Spagnol et al. [297] reported that caffeic acid presented antioxidant activity greater than ascorbic acid and trolox. Caffeic acid regulates lipogenesis-related protein expression in high-fat diet (HFD)-fed mice, alleviates endotoxemia and the proinflammatory response in HFD-fed mice, and attenuates gut microbiota dysbiosis in HFD-fed mice [298]. Caffeic acid decreases oxidative stress levels in the hippocampus and regulates microglial activation in the hippocampus [299].

#### *4.3. Ferulic Acid*

Ferulic acid (4-hydroxy-3-methoxycinnamic acid) is a polyphenol that is widely known for its therapeutic potential, showing anti-aging, anti-inflammatory, and neuroprotective impacts [300,301]. The ferulic acid molecule reveals cis-trans isomerism, with the most abundant form in nature being the trans isomer, and both isomers have proven results in the treatment of several pathologies such as diabetes, cancer, and neurodegenerative and cardiac diseases [302]. Ferulic acid is important for the synthesis of significant chemical molecules such as coniferyl alcohol, di ferulic acid, vanillin, synaptic, and curcumin, as well as for giving the cell wall stiffness [303]. Ferulic acid can be applied as an antioxidant to prevent damage from ultraviolet (UV) radiation and skin carcinogenesis [304]. It is ample in numerous fruits and vegetables, including bananas, eggplant, citrus fruits, and cabbage, as well as in seeds and leaves [305,306]. In Chinese medicinal science, ferulic is normally joined with polysaccharides by covalent bonds in various plant cell walls such as cereal bran and regarded as the main bioactive compound of *Angelica sinensis*, chuanxiong rhizoma, and ferula [307], and it has several biological activities such as anti-apoptosis, anticancer, antioxidant, and anti-inflammatory impacts [308]. Free ferulic acid is related to the natural content of ferulic acid in herbs, and total ferulic acid refers to the sum of free ferulic acid plus the amount of related hydrolyzed components [309,310]. *Angelica sinensis* is a perennial herbaceous species that creates the bioactive metabolite ferulic acid [311,312]. The ferulic compounds of *Salvia officinalis* could be useful as a safe natural source for estrogenic characteristics [313]. Singh et al. [314] indicated that ferulic acid is a phenol derivative from natural sources and applied it as a potential pharmacophore that exerts multiple pharmacological properties such as neuroprotection, Aβ aggregation modulation, antioxidant, and anti-inflammatory. Ferulic acid increases cerebellar functional and histopathological changes induced by diabetes, which can be attributed to its antioxidative effect and its ability to modulate nitric oxide synthase (NOS) isoforms [315]. Ramar et al. [316] showed that

ferulic acid and resveratrol revealed antioxidant as well as antidiabetic effects, consequently modulating liver, kidney, and pancreas damage caused by alloxan-induced diabetes, possibly via inhibition of the proinflammatory factor, NF-KB. Ferulic acid treatment prevents radiation-induced lipid peroxidation and DNA damage and restores antioxidant status and histopathological alterations in experimental animals [317]. Hu et al. [318] found that ferulic acid could alleviate inflammation and oxidative stress. Ferulic acid can inhibit cancer proliferation through various mechanisms, including changing the cancer cell cycle, inducing apoptosis, and regulating proteins involved in cell proliferation [319], and ferulic acid could be used as a potential official adjuvant for breast cancer treatment [320].

#### *4.4. Sinapic Acid*

Sinapic acid, a widely prevalent hydroxycinnamic acid, contains numerous biological activities related to its antioxidant property [321,322]. It protects lysosomes and prevents lysosomal dysfunction [323]. Saeedavi et al. [324] reported that sinapic acid may be a new therapeutic potential to treat allergic asthma through suppressing T-helper 2 immune responses. Sinapic acid phenethyl ester boosts gene expression related to the cholesterol metabolic process [325]. Hu et al. [326] indicated that sinapic acid can be utilized as an effective chemo preventive agent against lung carcinogenesis. It can also alleviate blood glucose levels by improving insulin production in pancreatic β-cells, and it can exhibit an antioxidative impact by suppressing lipid peroxidation and increasing the activity of antioxidant enzymes [327]. Sinapic acid significantly increases caspase-3 activity and inhibits cell invasion, and it has anticancer impacts on prostate cancer cells [328]. Sinapic acid pretreatment mitigates renal impairment and structural injuries through the downregulation of oxidative/nitrosative stress, inflammation, and apoptosis in the kidney [329]. Raish et al. [330] indicated the ability of sinapic acid to restore the antioxidant system and to suppress oxidative stress, pro-inflammatory cytokines, extracellular matrix, and TGF-β, and showed that sinapic acid treatment (10 and 20 mg/kg) significantly ameliorated bleomycin (BML)-induced lung injuries. Singh and Verman [331] revealed that sinapic acid increases streptozotocin (STZ)-induced cognitive impairment by ameliorating oxidative stress and neuro inflammation in the cortex and hippocampus. Sinapic acid can modulate the redox state in high-fat diet (HFD) rats [332].

#### **5. The Health Benefits of Coumarins (Umbelliferone, Esculetin, Scopoletin)**

Coumarins (2*H*-chromen-2-one ring) with the molecular formula C9H6O2 are an important group of natural compounds and are used as additives in both cosmetics and foods [333], and they constituent a notable class of heterocyclic compounds with the characteristic benzo-α-pyrone moiety in its structure [334]. Coumarin has been reported to have antibacterial, anticancer, antioxidant, anti-inflammatory, anticoagulant, and anti-Alzheimer's disease (AD) activities [335,336]. Coumarin derivatives are found naturally as secondary metabolites in more than 150 species of plants and in over 30 plant families such as *Clusiaceae*, *Umbelliferae*, *Guttiferae*, *Rutaceae*, *Oleaceae*, *Fabaceae*, and many more [337]. Seo et al. [338] reported that different coumarins were identified from the roots of *Angelica dahurica* using NMR spectroscopy, and each coumarin revealed remarkable differences in content and inhibitory effect. Kassim et al. [339] indicated that the good antioxidant activity of *Melicope glabra* (Rutaceae) is because of umbelliferone, glabranin, and scopoletin. Coumarin-based compounds extracted from the medicinal plants are shown in Table 6.

**Table 6.** Coumarin-based compounds obtained from the medicinal plants used by various ancient medical systems [340].


#### *5.1. Umbelliferone*

Umbelliferone is a 7-hydroxycoumarin and an isomer of caffeic acid [341], and it has been reported for different pharmacological activities against numerous diseases such as cancer [342]. The plant sources of umbelliferone are *Acacia nilotica*, *Angelica decursiva*, *Aegle marmelos*, *Artemesia tridentata*, *Aster praelatus*, *Balsamocitrus camerunensis*, *Chamomilla recutita*, *Citrus aurantium*, *Cirtus natsudaidai*, *Citrus paradise*, *Coriandrum sativum*, *Diospyros oocarpa*, *Diplostephium foliosissimum*, *Dystaenia takeshimana*, *Edgeworthia chrysantha*, *Edgeworthia gardneri*, *Eriostemon apiculatus*, *Ferula communis*, *Ferula communis*, *Ferula assafoetida*, *Fructus Aurantii*, *Glycyrrhiza glabra*, *Angelica archangelica*, *Haplophyllum villosum*, *Harbouria trachypleura*, *Haplopappus desertzcola*, *Haplophyllum patavinum*, *Hydrangea chinensis*, *Hydrangea macrophylla*, *Hieracium pilosella*, *Ipomoea mauritiana*, *Justicia pectoralis*, *Matricaria recutita*, *Melicope glabra*, *Musa* spp., *Parkinsonia aculeata*, *Peucedanum praeruptorum*, *Picea abies*, *Potentilla evestita*, *Rhododendron lepidotum*, *Platanus acerifolia*, *Selaginella stautoniana*, *Saussurea eopygmaea*, *Stellera chamaejasme*, and *Typha domingensis* [343]. It has been reported to have antioxidant, anti-inflammatory, free radical scavenging, and antihyperglycemic properties [344], and antifungal characteristics [345]. Althunibat et al. [346] reported that umbelliferone prevented isoproterenol cardiotoxicity in rats, and it decreased isoproterenolinduced oxidative stress and inflammation. Kutlu et al. [347] reported that umbelliferone has a strong antioxidant and anti-inflammatory effect on sepsis, and it can be considered as a new treatment for organ dysfunction. Umbelliferone ameliorates atopic dermatitis (AD)-associated symptoms and inflammation via regulation of various signaling pathways, suggesting that umbelliferone might be a potential therapeutic of AD [348]. Umbelliferone downregulates TGF-β1 levels in kidney tissue and it may promote kidney function and ameliorate renal oxidative stress [349]. Mohamed et al. [350] indicated that umbelliferone ameliorated oxidative stress-related hepatotoxicity via its ability to augment cellular antioxidant defenses by activating Nrf2-mediated HO-1 expression. Umbelliferone exhibits anticancer impacts on human oral carcinoma (KB) cell lines, with the increased generation of intracellular reactive oxygen species (ROS) triggering oxidative stress-mediated depolarization of mitochondria [351]. Umbelliferone has gastric protective activity in vivo, and it has antidiarrheal activity in vivo [352].

#### *5.2. Esculetin*

Esculetin (6,7-dihydroxycoumarin), a natural coumarin derived from herbs, has shown different pharmacological activities [353]. Kadakol et al. [354] reported that esculetin, a naturally occurring 6,7-dihydroxy derivative of coumarin, has revealed its potential function in various non-communicable diseases (NCDs) including obesity, diabetes, renal failure, cardiovascular disease, cancer, and neurological disorders. Esculetin reduced both chronic and acute topic skin inflammation, and mitigated inflammation by suppressing infiltration of inflammatory cells [355]. It can be found in many medicinal plants such as *Artemisia capillaris*, *Matricaria chamomilla* L., *Artemisia scoparia*, *Citrus limonia*, *Cortex Fraxini,* and *Ceratostigma willmottianum* [356–358]. Esculetin supplementation could protect against development of non-alcoholic fatty liver in diabetes via regulation of glucose, lipids, and inflammation [359]. The esculetin protects human hepatoma HepG2 cells from hydrogen peroxide-induced oxidative injury, and the production is provided via the induction of protective enzymes as part of an adaptive response mediated by Nrf2 nuclear accumulation [360]. Esculetin prevents progressive renal fibrosis under insulin resistance (IR) and type 2 diabetic nephropathy (T2D) conditions, and it decreases oxidative stress in the kidney under IR and T2D conditions [361]. Esculetin has the ability to suppress tumor growth and metastasis via Axin2 suppression, which can be an attractive therapeutic strategy for the treatment of metastatic colorectal cancer (CRC) [362]. Esculetin treatment decreased neurological defects and improved cognitive impairments in transient bilateral common carotid artery occlusion (tBCCAO)-treated mice, and the mechanism underlying the pharmacological impacts of esculetin involved its action on mitochondrial autophagy and the apoptosis triggered by mitochondrial oxidative stress via mediation of mitochondrial fragmentation during transient cerebral ischaemia and reperfusion injury [363]. Zhang et al. [364] reported that esculetin could be a potential therapeutic drug for the treatment of hepatic fibrosis by inducing stellate cell senescence. Wang et al. [365] indicated that esculetin is safe and reliable, is easy to be absorbed by the body, and can be synthesized in a variety of ways. Esculetin inhibits the pyroptosis of microvascular endothelial cells through the NF-KB/NLFP3 signaling pathway and is expected to be conducive in treating pyroptosisrelated diseases [366]. Esculetin directly binds to hnRNPA1 and decreases the concentration of hnRNPA1 in endometrial cancer cells, and it downregulates the levels of BCL-XL and XIAP expression, resulting in apoptosis and an arrest in proliferation [367]. Esculetin inhibits clear cell renal cell carcinoma growth in a dose- and time-dependent manner, and it induces apoptosis and cell cycle arrest [368]. Esculetin could be used as a dietary therapy for the prevention of alcoholic liver disease, and it can markedly prevent ethanol-induced liver injury in mice [369].

#### *5.3. Scopoletin*

Scopoletin (6-methoxyl-7-hydroxy coumarin) has a phenolic hydroxyl structure and is a member of the coumarin family [370]. It has a long history of use for its medicinal characteristics in traditional Chinese medicine [371]. Scopoletin is one of the main bioactive components of *Convolvulus prostratus* Forssk, known to have a role in acetylcholinesterase inhibitor, antimicrobial, memory enhancer, and antioxidative properties [372]. It is a major component of noni (*Morinda citrifolia* L.), which contributes to the anti-inflammatory, antioxidative, immunomodulatory, and hepatoprotective properties [373]. Scopoletin could be a potential phagocytic enhancer, and it can increase immunity through enhancing macrophage phagocytic capabilities [374]. Scopoletin improved vancomycin-induced renal injury via restoring the antioxidant defense system [375]. Scopoletin reduces non-alcoholic fatty liver disease in high-fat diet-fed mice [376]. It has been reported that scopoletin could exert a positive impact on anti-aging related to autophagy via modulation of p53 in human lung fibroblasts [377].

#### **6. The Health Benefits of Stilbenes (Resveratrol, Piceatannol, Pterostilbene)**

Stilbenes (based on the 1,2-diphenylethylene skeleton) are a group of plant polyphenols with rich structural and bioactive diversity [378]. They originate from plant families such as Vitaceae, Gnetaceae, Leguminaceae, and Dipterocarpaceae, and, structurally, they have a C6-C2-C6 skeleton, normally with two isomeric forms [379,380]. They have wonderful potential for anti-inflammatory, antiviral, anticancer, and antioxidant activities, as well as an application as cosmetic materials, coloring agents, and dietary supplements [381–383]. Wine and grapes are the main dietary source of stilbenes [384]. These compounds are synthetized by plants in response to abiotic or biotic stress situations [385]. Most stilbene compounds reveal antimicrobial properties, acting as phytoalexins in response to pathogen or herbivore attack [386]. Phytochemical phenols of stilbene families indicated good stability at elevated temperatures [387].

#### *6.1. Resveratrol*

Resveratrol (3,5,4 -trihydroxy-trans-stilbene) is a plant polyphenol, extensively popularized during the last decades, owing to its promising beneficial effects on human health [388]. It is a famous non-flavonoid polyphenol, related to the family of stilbenes whose structure consists of two phenolic rings linked by a double bond, which promotes two isomeric conformations: trans- and cis-resveratrol [389,390]. Resveratrol's cis-isomer is unstable, and its trans-isomer contains greater stability, but converts to the cis-isomer under exposure to high pH or UV light [391,392], with heat increasing the degradation process [391]. It exists in many traditional herbs, and in several types of fruits, especially in the muscadine grape, red wine, cranberry, lingonberry, and redcurrant [393], and roots of various plant species including *Polygonum cuspidatum* and rhubarb (*Rheun rhapontiicum*) [394]. It is also useful in common age-related diseases such as cancer, cardiovascular diseases, type 2 diabetes, and neurological conditions, and it has also positive impacts on metabolism and can boost the lifespan of various organisms [395]. Resveratrol supplementation can be considered as an adjuvant therapy for relieving inflammation [396]. It has great potency in treating cardiovascular diseases [397]. Resveratrol attenuates kidney damage in malignant hypertension rats, and it can increase glomerular filtration while decreases proteinuria [398]. It inhibits the release of proinflammatory cytokines and leads to the release of anti-inflammatory cytokines, and it scavenges free radicals and upregulates antioxidant enzymes [399]. Chowdhury et al. [400] indicated that resveratrol treatment indicated beneficial impacts on preventing oxidative stress and fibrosis in the kidneys of high-fat (HF) diet-fed rats, probably by modulating the gene expression of oxidative stress and inflammation-related parameters and enzymes. Resveratrol can downregulate the pro-inflammatory cytokine release decreasing lung injury [401]. Resveratrolcontaining fruits could be a promising substitute for the management of Alzheimer's disease [402]. It can be more effective in cardiotoxicity prevention [403]. *Polygonum cuspidatum* is an important medicinal plant in China and a rich source of resveratrol compounds, which is a secondary metabolite formed in the long-term evolution procedure of plants to increase their response to adverse environments such as pathogens and ultraviolet radiation [404]. As an anticancer parameter, resveratrol promotes apoptosis in hepatocellular carcinoma cells [405]. Bhaskara et al. [406] reported that resveratrol is a potential reducing factor that can prevent carcinogenesis due to its antioxidant abilities, and it acts as an immunomodulatory agent for treating cancer. Resveratrol can exhibit anti-aging activity through a variety of signaling pathways [407]. Resveratrol shows potent anti-rotavirus efficacy in vitro and in vivo, and it blocks viral structural expression and genomic RNA synthesis [408]. Resveratrol oligomers from *Paeonia suffruticosa* indicate neuroprotective effects in vitro and in vivo by regulating cholinergic, antioxidant, and anti-inflammatory pathways, and they may have promising applications in the treatment of Alzheimer's disease [409]. Resveratrol is also involved in neurodegenerative diseases (NDs) with multiple neuroprotective activities [410]. Antimicrobial activity of resveratrol against many bacteria and fungi has been reported, such as antimicrobial activity against Gram-positive bacteria such as *Bacillus cereus*, *Bacillus megaterium*, *Staphylococcus aureus*, *Enterococcus faecalis*, *Enterococcus faecium*, *Mycobacterium tuberculosis*, *Mycobacterium smegmatis*, *Streptococcus pneumoniae*, *Streptococcus pyogenes*, *Propionibacterium acnes*, and *Listeria monocytogenes*; against Gram-negative bacteria such as *Escherichia coli*, *Klebsiella pneumoniae*, *Salmonella enterica serovar Typhimurium*, *Pseudomonas aeruginosa*, *Helicobacter pylori*, *Arcobacter butzleri*, *Arocobacter cryaerophilus*, *Haemophilus ducreyi*, *Neisseria gonorrhoeae*, *Neisseria meningitidis*, *Vibrio cholerae*, *Fuscobacterium nucleatum*, *Campylobacter jejuni*, and *Campylobacter coli*; and against fungi such as *Trichophyton mentagrophytes*, *Trichophyton tonsurans*, *Trichophyton rubrum*, *Epidermophyton floccosum*, *Microsporum gypseum*, *Candida albicans*, *Saccharomyces cerevisiae*, *Botrytis cinerea*, and *Trichosporon beigelii* [411]. Resveratrol has powerful anticancer characteristics in different cancer cells and organs such as pancreatic cancer, colorectal cancer, gastric cancer, esophageal cancer, hepatocellular cancer, oral cancer, and biliary tract cancer [412]. Resveratrol decreases damage to pancreatic tissue via suppression of calcium overload; it suppresses calcium overload and, thereby, decreases trypsinogen activation, oxidative stress, mitochondrial dysfunction, and disorders, and it also reduces damage to other organs such as lung and heart by decreasing microcirculatory dysfunction [413].

#### *6.2. Piceatannol*

Piceatannol (3,4,3 ,5 -tetrahydroxy-trans-stilbene), a natural polyphenolic stilbene, has pleiotropic pharmacological potentials [414]. It can be found in different kinds of fruits and vegetables such as blueberries, grapes, and passion fruit [415]. Piceatannol is a metabolite of resveratrol found in red wine, which prevents cardiac hypertrophy in rat neonatal cardiomyocytes [416]. It has previously been known as an antileukemic principle, which has been shown to be an inhibitor of protein-tyrosine kinase activity [417]. It has been reported that its low water-solubility and bioavailability could limit its application in both food and

pharmaceutical fields [418]. Piceatannol, compared with the renowned resveratrol, is a better anticancer factor and a superior agent with other biological properties [419]. Piceatannol lightened oxidative injury and collagen synthesis in lung tissues during pulmonary fibrosis, and it suppressed the activation and collagen synthesis of TGF-β-induced lung fibroblasts [420]. It appears to be an appropriate nutritional or pharmacological biomolecule that modulates effector T cell functions, namely cytokine production, differentiation, and proliferation [421]. Piceatannol attenuates fat accumulation in steatosis-induced HepF2 cells, it suppressed lipogenesis and fatty acid uptake in steatosis-induced HepG2 hepatocytes, and it suppressed fatty acid-induced oxidative stress [422]. It shows antiaggregation activity, and it increases catalase and glutathione peroxidase activity [423]. It can also be considered as a potential chemotherapeutic factor in the treatment of leukemia, but it may be connected with the risk of multi-drug resistance [424]. Passion fruit seed extract and piceatannol could exert anticancer activity via human glyoxalase I (GLO I) inhibition [425]. Piceatannol is a promising medication for preventing acute liver failure and the mechanisms may be associated to its inhibitory impacts on ER stress, inflammation, and oxidative stress [426]. Piceatannol has a potential inhibitory activity against human glyoxapase I (GLO I), and it inhibits the proliferation of GLO I-dependent human lung cancer [427]. It protects ARPE-19 cells against apoptosis induced by photo-oxidation, and the protective effect of piceatannol is because of the activation of the Nrf2/NQO1 pathway [428]. Piceatannol is a potent enhancer of cisplatin-induced apoptosis, and it reveals the potential for clinical development for the treatment of ovarian cancer [429]. It has been reported that piceatannol significantly decreases the degree of bovine serum albumin (BSA) glycosylation, and this suggests its potential impact on preventing the progression of diabetes mellitus [430].

#### *6.3. Pterostilbene*

Pterostilbene, a dimethyl ester derivative of resveratrol, may act as a cytotoxic and anticancer factor [431]. It primarily exists in blueberries, grapevines, and heartwood of red sandalwood [432,433]. Phenolic resveratrol, pterostilbene has been reported to have antifungal activity against a broad range of important phytopathogenic fungi such as *Leptosphaeria maculans* and *Peronophythora litchii* [434]. It is an anti-inflammatory and antioxidant agent with preventive effects toward skin disorders, and its anticancer impacts include inducing necrosis, apoptosis, and autophagy [435]. It can alleviate hepatic damage and oxidative stress and increase hepatic antioxidant function in piglets [436]. It possesses the abilities of antiproliferation, reversing epithelial to mesenchymal transition (EMT), and suppression of cancer stemness, and it could suppress tumor growth and inhibit the metastasis of tumor cells to livers and lungs with therapeutic safety in BALB/C mice [437].

#### **7. The Important Health Benefits of Lignan (Sesamin)**

Lignans are naturally occurring compounds produced and accumulated in different edible and medicinal plants, which can be subdivided bio-synthetically into neolignan and lignans [438,439]. Lignans, as the notable subgroup of phenylpropanoids, are involved in the plant defense responses to numerous biotic and abiotic stresses [440]. Lignans, with different biological activities, such as antitumor, antibacterial, antioxidant, and antiviral activities, are generally distributed in nature and mostly exist in the xylem of plants [441,442]. The level of lignans varies between plant parts of all species [443].

Sesamin, a major lignan derived from sesame seeds, has several benefits and medicinal characteristics [444]. It exerts various pharmacological impacts, such as prevention of hyperlipidemia, hypertension, and carcinogenesis, as well as anticancer and chemopreventive activity in vitro and in vivo [445,446], and antioxidant and anti-inflammatory characteristics [447,448]. Plants reported to contain sesamin are *Paulownia tomentosa* Staud., *Phyllarthron comorense*, *Justicia simplex*, *Hyptis tomentosa*, *Anacyclus pyrethrum*, *Artemisia absinthium*, *Artemisia gorgonum*, *Chrysanthemum cinerariaefolium*, *C. frutescens*, *C. indicum*, *Diotis maritima*, *Eupatorium ageratina*, *E. ritonia*, *E. fleischmannia*, *Otanthus maritimus*, *Aptosimum*

*spinescens*, *Gmelina arborea* Roxb., *Acanthopanax senticosus*, *A. sessiliflorum*, *Eleutherococcus divaricatus*, *Asarum sieboldii*, *Aristolochia cymbifera*, *Alnus glutinosa*, *Salicomia europaea*, *Austrocedrus chilensis*, *Evodia micrococca*, *Fagara xanthoxyloides*, *Fagara tessmannii*, *Fagara heitzii*, *Micromelum minutum*, *Melicope glabra*, *Spiranthera odoratissima*, *Flindersia pubescens*, *Zanthoxylum naranjillo*, *Zanthoxylum tingoassuiba*, *Zanthoxylum piperitum*, *Zanthoxylum nitidum*, *Zanthoxylum flavum*, *Zanthoxylum alatum* Roxb., *Zanthoxylum bungeanum*, *Ginkgo biloba*, *Machilus glaucescens*, *Ocotea usambarensis*, *Aiouea trinervis* Meisn., *Talauma hodgsonii*, *Magnolia* spp., *Picea abies*, *Macropiper excelsum*, *Piper sarmentosum*, *Sesamum indicum*, *S. radiatum*, *S. mulayanum*, *S. malabaricum*, *S. alatum*, *S. angustifolium*, *S. angolense*, *S. calycinum*, *Anemopsis californica*, *Quercus frainetto* Ten., *Vernicia fordii*, *Jatropha curcas*, *Larrea tridentata*, *Morinda citrifolia*, *Glossostemon bruguieri*, *Ligustrum japonicum*, and *Triclisia sacleuxii* [449]. Sesamin could boost the proliferation and adhesion of intestinal probiotics, leading to modulating gut microbiota, which provided the basis for sesamin as a food-borne functional parameter for improving intestinal health [450]. Sesamin suppressed breast cancer proliferation, and it downregulated programmed death ligand 1 (PD-L1) expression, which is mediated by NF-KB and AKT [451]. Sesamin increased osteoblast differentiation by the increase of type I collagen (COL1A1) and alkaline phosphatase (ALP) gene expression as well as ALP activity [452]. Sesamin ameliorated lead-induced neuroinflammation in rats, and decreased accumulation of lead in blood and neuronal tissues of rats [453]. It ameliorated polymorphonuclear neutrophils infiltration and exudate volume [454]. Majdalawieh et al. [455] reported that sesamin can potentially be utilized as an effectual adjuvant therapeutic agent in ameliorating tumor development and progression, and it could be utilized in the prevention and treatment of different types of cancer. It has been reported that sesamin promoted diabetes-induced neuroinflammation in rats, exhibited neurotrophic supportive action in diabetic rats, and prevented neuronal loss in diabetic rats [456]. Sesamin has a chondroprotective effect through inhibition of proteoglycans (PGs) degradation induced by IL-1beta and inhibition of collagen degradation [457].

#### **8. The Health Benefits of Condensed Tannins or Proanthocyanidins (Procyanidin B1)**

Proanthocyanidins, also known as condensed tannins [458,459], belong to the oldest of plant secondary metabolites, and these constituents are widespread in woody plants, but are also discovered in certain forages, as well as fruits, seeds, nuts, and bark [460,461]. Yu et al. [462] reported that proanthocyanidins were prevalent in lotus seed coats. They can be categorized into three groups according to their component units and the linkages between them: procyanidins, prodelphinidins, and propelargonidins [463]. The biological activity of plant proanthocyanidins is associated with their chemical concentration and structure [464]. Proanthocyanidins from *Pinus thunbergii* mainly included catechin/epicatechin, and they showed significant antioxidant capacity [465]. Proanthocyanidins in tea, black currant, grapes, bilberry, pine bark, cranberry, and peanut skin may lead to a decrease in the oxidative stress (ROS), induce lower iNOS and COX-2 overexpression, then lower inflammation, and, lastly, show activities against diabetes, asthma, neuropathologies, cardiovascular ailments, obesity, and cancer [466]. The precursors of proanthocyanidins are produced by the phenyl propanoid pathway in the cytosol and are converted to the vacuole, where they polymerize to create proanthocyanidins [467]. They have various bioactivities, such as anticancer, antibacterial, and antioxidant [468]. Proanthocyanidins stimulate antioxidant capacity and increase resistance against oxidative stress-induced senescence in fruits after harvest [469].

Procyanidins are associated with the class of natural products known as proanthocyanidins or condensed polyphenols [470]. They have been reported to reveal broad advantages to human health and are applied in the prevention of cancers, diabetes, cardiovascular diseases, etc. [471]. They are structurally diverse constituents and can be divided into monomeric, oligomeric, or polymeric variants associated with degree of polymerization, which plays a role in manifesting various impacts that are associated with human health [471]. The anti-digestion and antioxidant impacts of grape seed procyanidins have

been proven [472]. Procyanidin B1 is also a promising liver cancer antitumor drug [473] (Na et al., 2020). Procyanidins increase the glycometabolism and decrease the secretion of inflammatory factors of postpartum mice with gestational diabetes mellitus (GDM) [474].

#### **9. The Health Benefits of Curcuminoids (Curcumin, Demethoxycurcumin,**

#### **Bisdemethoxycurcumin)**

#### *9.1. Curcuminoids*

Curcuminoids are a group of polyphenol coloring constituents that exist in the plant species *Curcuma*, such as *Curcuma longa*, *C. Wenyujin*, *C. zedoaria*, etc. [475,476]. They are synthesized in turmeric from cinnamic acid precursors obtained via the phenylpropanoid biosynthetic pathway, and there are three different precursors, namely curcuminoids biosynthesis-cinnamic acid, ferulic acid, and coumaric acid [477]. Ramirez-Ahumada et al. [478] reported that curcuminoid synthase activity in turmeric crude protein extracts converts feruloyl-CoA into curcumin. Curcumins are the commercially available component in curcuminoids, as the principle constituents, and the other two, demethoxycurcumin and bisdemethoxycurcumin, as minor components [479,480]. Curcumin and demethoxycurcumin are distinctive because of the phenylmethoxy group [481]. Curcuminoids share important pharmacological characteristics possessed by turmeric, a distinguished curry spice, considered as an important factor in Alzheimer's disease [482]. It has been reported that curcuminoids of turmeric can be considered as a modern medicine for the treatment of knee osteoarthritis [483] as well as a potential anticancer agent [484]. Zhou et al. [485] also reported that turmeric rhizomes exhibit versatile biological activities such as a significant anticancer property. Three curcuminoids, namely curcumin, demethoxycurcumin, and bisdemethoxycurcumin, in turmeric were found and were shown to contain significant synergistic anticancer activities [486]. Curcuminoids rescued neurotoxin-induced inflammatory gene expression and rescued neurotoxin-induced apoptotic gene expression, and individual curcuminoids showed significant function useful for Alzheimer's disease [482].

#### *9.2. Curcumin*

Curcumin (bis-α,β-unsaturated β-diketone), also known as diferuloylmethane, is a hydrophobic polyphenol obtained from the rhizome of the perennial herb genus *Curcuma,* which belongs to the ginger family (Zingiberaceae) and consists of species such as *Curcuma longa*, *Curcuma amada*, *Curcuma aromatic*, *Curcuma zedoaria*, and *Curcuma raktakanta* [487,488]. Curcumins contain different medicinal values such as antioxidant, anti-pulmonary fibrosis, anti-inflammation, antiviral, and chronic obstructive pulmonary disease impacts, and attractively docked with multi-target molecular proteins related to diabetes [489–494]. Curcumin is insoluble in water and easily efficient in organic solvents [495]; the active functional groups of curcumin can be oxidized by electron transfer and hydrogen abstraction [496], and curcumin is more durable in acidic to neutral conditions than in alkaline circumstances [495–497]. Curcumin, as an enzyme inhibitor, has proper structural characteristics including a flexible backbone, hydrophobic nature, and different available hydrogen bond (H-bond) donors and acceptors [498]. Curcumin is stable to heat but is light-sensitive and produces singlet oxygen and other reactive oxygen species (ROS) when exposed to the sun, which is also a photodynamic and photobiological property of curcumin [499]. Curcumin decreases inflammation by inhibiting lipopolysaccharideinduced nuclear factor-KB (NF-KB) p65 translocation and mitogen-activated protein kinase activation in dendritic cells [500]. Curcumin decreases morphine dependence in rats through an inhibitory influence on neuroinflammation and a decline in the expression of μ-opioid receptors in the prefrontal cortex [501]. Curcumin influences synaptic plasticity genes (Arc and Fmr1) to decrease amnesia [502]. Xie et al. [503] reported that curcumin together with photodynamic therapy have been confirmed as effective in many kinds of cancer cells in vitro and animal models. It has been extensively applied in cancer treatment because of its ability to trigger cell death and suppress metastasis [504]. Mahjoob and Stochaj [505] reported that curcumin improves aging-related cellular and

organ dysfunctions. Curcumin can be a promising antifatigue substitute for improving exercise performance [506]. Its derivatives have anti-inflammatory actions for drug repurposing in traumatic brain injury (TBI), but their molecular targets are not clear [507].

#### *9.3. Demethoxycurcumin*

Demethoxycurcumin is one of the principle active compounds of curcuminoids discovered in turmeric powder, which is used as a spice in Asian cooking and traditional medicine [508]. Recent studies reveal that demethoxycurcumin has various biological activities including antioxidant, anti-inflammation, and anticancer activities [509–511]. Lin et al. [512] reported that demethoxycurcumin is the most active constituent against various kinds of breast cancer cell lines and induces apoptosis and autophagy. Demethoxycurcumin, a natural derivative of curcumin, revealed stronger inhibitory activity on nitric oxide and tumor necrosis factor-α production in comparison with curcumin in lipopolysaccharideactivated rat primary microglia [513]. Demethoxycurcumin remitted the inflammation of nucleus pulposus cells without overt cytotoxic impacts [514].

#### *9.4. Bisdemethoxycurcumin*

Bisdemethoxycurcumin is a demethoxy derivative of curcumin and is much more stable than curcumin in physiological media [514–516]. It can scavenge free radicals and control cellular redox balance because of its antioxidant property [517,518], and it has potential anti-allergic effects [519]. Mahattanadul et al. [520] reported that bisdemethoxycurcumin's antiulcer impacts might be because of its characteristics of decreasing gastric acid secretion and increasing the mucosal defensive mechanism via suppression of inducible nitric oxide synthase (iNOS)-mediated inflammation. Bisdemethoxycurcumin inhibits human pancreatic α-amylase (HPA) [521].

#### **10. Conclusions**

Phenolic compounds are one of the most important types of compounds with an important role in growth and reproduction, providing protection against pathogens and predators, and they could be the main determinant of antioxidant potential of foods. Phenolics are a heterogeneous collection of compounds generated as secondary metabolites in plants. Phenolic compounds are aromatic or aliphatic compounds with at least one aromatic ring to which one or more OH groups are connected. They are subdivided into different groups depending on the number of phenolic rings that they possess and the structural elements joined to them. They are naturally occurring compounds present in several foods such as cereals, fruits, vegetables, and beverages. Polyphenols can also be found in dried legumes and chocolate. The distribution of phenolic compounds in plant tissues and cells change considerably according to the type of chemical compound. They also contribute towards the color and sensory characteristics of fruits and vegetables. Different classes of phenolic compounds in plants are simple phenolics, benzoquinones, hydroxybenzoic acids, acetophenones, phenylacetic acids, hydroxycinnamic acids, phenylpropanoids, naphthoquinones, xanthones, stilbenes, anthraquinones, flavonoids, isoflavonoids, lignans, neolignans, biflavonoids, lignins, and condensed tannins. Hydroxybenzoic acids are gallic acid and Protocatechuic acid. Hydroxycinnamic acids are *p*-coumaric acid, caffeic acid, ferulic acid, sinapic acid, and other components such as coumarins (umbelliferone, esculetin, scopoletin, resveratrol, piceatannol, pterostilbene), curcuminoids (curcumin, demethoxycurcumin, bisdemethoxycurcumin), condensed tannins or proanthocyanidins (procyanidin B1), and lignan (sesamin). From a human physiological viewpoint, phenolic compounds are important in defense responses such as antioxidant, anti-aging, antiproliferative, and antiinflammatory. High phenolic activity in many species could prove to be beneficial towards human health if included as part of food designs for a healthy diet.

Flavonoids are the largest group of natural phenolic compounds, and, based on the differences in the pyran ring, flavonoids can be divided into flavones, isoflavones, flavanonols, flavonols, flavanones, flavan-3-ols, and anthocyanidins. They can be subdivided

into different subgroups on the basis of the carbon of the C ring on which the B ring is attached and the degree of unsaturation and oxidation of the C ring. Flavonoids in which the B ring is linked in position 3 of the C ring are called isoflavones. Those in which the B ring is linked in position 4 are called neoflavonoids, while those in which the B ring is linked in position 2 can be further subdivided into several subgroups on the basis of the structural characteristics of the C ring. The most prominent health benefits of phenolic compounds are antioxidant activity, anti-inflammatory properties, antifungal activity, antimicrobial activity, antibacterial properties, anti-coronavirus activities, neuroprotective potential, appropriate for skin health, suitable for wound healing, and anticancer activities. Flavonoids, a group of natural substances with variable phenolic structure, are found in vegetables, fruits, grains, bark, stems, roots, flowers, wine, and tea. Flavonoids are considered as an important constituent in different pharmaceutical, medicinal, nutraceutical, and cosmetic applications. They belong to a class of low-molecular-weight phenolic compounds that are extensively distributed in the plant kingdom. Future research is needed to determine the pharmaceutical benefits of phenolic and flavonoid compounds of medicinal plants, especially traditional Chinese medicinal plants, and to gain a better understanding of these chemical compounds in medicinal plants and herbs. It is also important to increase analytic techniques to allow the collection of more data on excretion and absorption.

**Author Contributions:** W.S., writing—original draft preparation; M.H.S., writing—original draft preparation and editing. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Natural Science Foundation of Beijing, China (Grant No.M21026). This research was also supported by the National Key R&D Program of China (Research grant 2019YFA0904700).

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

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

#### **References**


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