In this present study, ZnONPs were rapidly fabricated using GW leaves extract as a bioreductant and stabilizing agents. Different characterization techniques were performed to determine the formation of zinc oxide nanoparticles. The progress of synthesis of ZnONPs was detected by color change after the addition of precursor salt to plant extract at 60 ◦C. The color change in solutions (reddish black) signal the biosynthesis of ZnONPs. This color change in the solution is due to surface plasmon resonance (SPR) [42]. To confirm the stable nature of asynthesized ZnONPs, 1 mg/mL solutions of nanoparticles was prepared and sonicated for ~40 min. The turbid colloidal suspension was allowed to remain stable for 48 h, and SPR was observed with varied time interval. The wavelength scale was fixed between 200–700 nm and solution was scanned between this range. The UV spectra showed absorption peak at 398 nm, indicating that colloidal suspension remained stable for 48 h. The reduction in absorption peak was noticed after 60 h which showed settlement of NPs at the bottom. The UV-Vis spectrum for green ZnONPs are presented in Figure 2A,B. The XRD configurations of thermally annealed green GW-ZnONPs are presented in Figure 2C. The XRD analysis has confirmed the crystalline nature of ZnONPs. The resulting Bragg peaks were in accordance with single and pure phase hexagonal zincite with JCPD card no: 00–036–1451. The XRD spectra showed different distinct diffraction peak with 2Ø value of 32.44, 35.13, 37.34, 47.21, 57.51, 62.58, 65.76 and 67.61, which are corresponding to (100), (002), (101), (102), (110), (103), (112) and (201) Bragg's reflections. XRD spectrum obtained indicates the absence of impurities. The presence of these peaks are due to leaves extract containing organic compounds, thus play significant role in reduction of zinc ions and stabilization of resultant ZnONPs [42]. The average size calculated as ~18 nm. The XRD configuration for ZnONPs are consistent with earlier studies using green synthesis protocol [43,44].

**Figure 2.** UV and XRD spectra analysis for biogenic ZnONPs (**A**) UV visible spectra (**B**) Stability of biosynthesized ZnONPs (**C**) XRD spectra of *Geranium wallichianum* mediated ZnONPs (**D**) Size calculation via Scherer approximation.

The Raman and FTIR analyses were performed to determine the vibrational properties of ZnONPs. The distinct major modes of Raman spectra are located at 92.5 cm−1 (E2L), 180.77 cm−1 (2TAM), 313.23 cm−1 (2E2M), 403.04 cm−1 (E1TO), 565.12 cm−1 (E2H + E2L). Our results of Raman spectra of GW mediated ZnONPs are in agreement with previous studies using *S. thea* [44]. The Raman spectroscopy results are shown in Figure 3A. FTIR analysis was done to recognize the major functional group and their possible role in synthesis and stabilization of ZnONPs. The spectrum of leaves extract mediated zinc oxide nanoparticles is presented in Figure 3B. The peak centered at 3273.31 cm−1, 1395.87 cm−1, 1262.46 cm−1, 1060.47 cm−1 indicate O-H, C-C, -CH and C = C stretching. The other peaks located at 1565.48 cm−1 correspond to C−C. Peaks at 530 cm−1 signify Zn−O bond vibrations from ZnONPs. There was a shift in the peaks of biogenic ZnONPs which suggests that different functional groups of GW leaf extract are involved in synthesis of ZnONPs and prevent agglomeration [45]. The surface morphology of the biogenic ZnONPs was explored using SEM. The SEM images of biogenic ZnONPs are shown in Figure 4A–C. The smaller the size of nanoparticles, the larger the surface area and stronger the activity will be. Thus, ZnONPs have shown significant biological applications. Further, an insight into the morphology and size detail of ZnONPs was revealed by TEM. TEM image in (Figure 4D) shows that ZnONPs are hexagonal in shape with average size of ~18 nm which are consistent with

calculation from XRD. Smaller size NPs with larger surface area have shown numerous applications especially in the field of medicine, chemo, and electrochemistry [46,47]. EDX spectra analysis revealed the surface chemical composition of biogenic ZnONPs. The EDX results have shown that all the ionic zinc was resulted into synthesis of ZnONPs leaving no ionic zinc peak. The EDX peaks showed that both Zn and oxygen exist in test samples while no other elements were observed in the EDX spectrum. The absence of other elements confirms the purity of biosynthesized ZnONPs. The EDX spectrum is shown in Figure 5A. The EDX pattern clearly shows that reduction of Zn salt with GW leaves extract yielding in crystalline ZnONPs. The size distributions, PDI and ζ-potentials (ZP) of thermally annealed ZnONPs were detected by DLS analyses. The results indicated larger particles aggregate of 98.26 nm. The ZP and PDI of ZnONPs were −8.53 mV and 0.232 (Figure 5B,C). The details about zeta size and ZP are given in Table 1. Our DLS results are similar to the previous report of ZnONPs using *Ixora coccinea* [48]. DLS is performed to confirm the size of NPs in colloid suspension in the range of nano and submicron, and its ZP measurement is based on particles movement under electric field.

**Figure 3.** (**A**) Raman spectra of the ZnONPs biosynthesized using zinc nitrate hexahydrate as precursor (**B**) FTIR spectra of ZnONPs.

**Figure 4.** SEM and TEM images of *Geranium wallichianum* mediated ZnONPs using zinc nitrate as a precursor (**A**–**C**) HR-SEM images (**D**) TEM image.

**Figure 5.** (**A**) Elemental composition using EDX (**B**) Size distribution of *Geranium wallichianum* mediated ZnONPs (**C**) Zeta potential measurement of ZnONPs.

**Table 1.** Zeta potential measurements of the ZnONPs.

#### *3.2. Bio-Potentials of Biogenic GW-ZnONPs*

#### 3.2.1. Metabolic Activity of GW-ZnONPs against HepG2 Cells

Cancer is a fatal disease, a major cause of deaths around the globe, and is continuously increasing in cause of death by an estimated ~21 million by the year 2030 [30,49,50]. Among the numerous types of cancers, liver cancer is presently the second deadliest cancer in males and sixth in female causes (~745,517) deaths. The different risk factors related are viral infection, extensive alcohol use, and toxin exposures (aflatoxin) [51]. The cytotoxicity potential of the synthesized zinc oxide nanoparticles against liver cancer cells (HepG2) was evaluated using MTT cytotoxicity assay. The key results obtained by MTT cytotoxicity assay in HepG2 cells treated with various doses of ZnONPs ranging from 7.8125–1000 μg/mL for 48 h are summarized in Figure 6A. Our results of ZnONPs have determined strong reduction in the metabolic activity of HepG2 cancer cells. The metabolic activity was reducing continuously with increase in ZnONPs concentrations. The highest inhibition potential (~71% mortality) was achieved at 1000 μg/mL and cytotoxicity potency was decreasing with a decrease in concentration. The reduction in metabolic activity has shown that ZnONPs might have potential anticancer activity. The IC50 value recorded for GW mediated ZnONPs against HepG2 cell lines was 39.26 μg/mL. The cytotoxic effects induced by GW-NPs at lower concentrations could be due to the plant components attached to the ZnONPs. The results obtained from this study are also very well supported with various evidences for the cytotoxic effect of green ZnONPs using *Rhamnus virgata* leaf extract against the liver cancer HepG2 cell line in vitro [52,53].

#### 3.2.2. Antileishmanial Potential of ZnONPs

Leishmania is a tropical disease with an excessive epidemiological diversity caused by at least 20 *Leishmania* species and is transferred by the bite of female sandflies [54]. Leishmanial parasites have wide distribution range in ~100 countries around the world. The drugs present in commercial market for the treatment of leishmaniasis are generally toxic, less potent and expensive. As for example, Antimonials designed as a potential candidate for leishmaniasis therapy has lost its potential against Leishmanial parasites and has developed resistance against it. Thus, pharmaceutical industries are working hard in this direction to develop novel drugs which will be more effective, less toxic, and cost effective for the treatment. Significant research studies have been performed to design MNPs for the treatment of Leishmania. Various NPs have been used in diverse studies to determine cytotoxic potentials against *L*. parasites [34]. However, the biosynthesized ZnONPs are poorly studied for the treatment of *L. tropica*.

In the present report, antileishmanial potentials of ZnONPs was evaluated using *L. tropica* (KMH23) Figure 6B. The *L. tropica* parasites were exposed to various doses (1–200 μg/mL) of ZnONPs for 72 h. The antileishmanial potentials of ZnONPs were increasing with an increase in concentrations of ZnONPs and determined significant antileishmanial potentials against *L. tropica* promastigotes with IC50: 15.60 μg/mL. Similarly, ZnONPs also shown significant potential against *L. tropica* amastigotes with IC50: 34.5 μg/mL which are consistent with earlier studies of biosynthesized ZnONPs [34,53]. The dose-dependent nature and low IC50 value signify that these ZnONPs can be utilized in drug delivery for the treatment of leishmaniasis.

**Figure 6.** Cytotoxicity assays. The data in all figures represents the mean of three replicates (**A**) Cytotoxicity activities of *Geranium wallichianum* mediated ZnONPs against HepG2 cell line (**B**) Antileishmanial activities of ZnONPs (**C**) Cytotoxicity against brine shrimps.

#### 3.2.3. Antibacterial and Antifungal Activities

The biogenic zinc oxide nanoparticles have also shown significant antibacterial potential against various bacterial strains (BS). For this purpose, different bacterial strains were treated with various doses of ZnONPs ranging from 31.25–1000 μg/mL Figure 7A. The activity was done against different gram positive (*S. aureus* and *B. subtilis*) and gram negative BS (*P. aeruginosa*, *K. pneumoniae*, *E. coli*). Most BS were found susceptible where ZnONPs have shown potential results by inhibiting them. *B. subtilis* was reported to be the most susceptible strain with MIC score of 7.8 μg/mL while *K. pneumoniae*

was found to be the least susceptible strain with an MIC value of 125 μg/mL. Oxytetracycline was taken as positive control while treating BS to different doses of ZnONPs. No single dose has shown stronger potential than positive control. Overall, our ZnONPs have shown potential antibacterial activities against different BS which are in line with the previous reports of biosynthesized nanoparticles [28,55]. The increased antibacterial potential of ZnONPs is due to the bioactive functional groups attached on the surface of NPs. In a nutshell, ZnONPs concluded concentration dependent response against different strains of bacteria. Some other studies have discussed the antibacterial potentials of ZnONPs and shown that ROS generation is the core mechanisms that give antimicrobial potentials to NPs. Further, membrane damage (membrane protein damage) due to NPs absorption on surface result in bacterial cell damaging. Similarly, surface defect in the symmetry of NPs is responsible for bacteria inhibition and causes injury to cells [56]. Besides, different functional groups attached from GW leaves extract result in capped ZnONPs, which play an important role in the bacterial inhibition.

**Figure 7.** Antibacterial and antifungal assays. The data in all figures represents the mean of three replicates (**A**) Antibacterial potential of biogenic ZnONPs (**B**) Antifungal potential of biogenic ZnONPs.

Furthermore, fungicidal potential of ZnONPs was evaluated using various fungal strains (FS). Before activity was performed different concentration of ZnONPs were prepared ranging from 31.25–1000 μg/mL. The drug amp-B was employed as positive control to compare inhibition potential of ZnONPs. Various FS were used; *Fusarium solani*, *M. racemosus*, *A. niger*, *A. flavus* and *C. albicans* (Figure 7B). Substantial work has been performed on antibacterial potentials of ZnONPs while limited fungicidal potentials have been reported for biogenic ZnONPs. Our current study for the first reported

the fungicidal activity of GW-ZnONPs. For this purpose, different FS were treated with various concentrations of ZnONPs (31.25–1000 μg/mL) to determine their antifungal potentials. Generally, a dose dependent inhibition response was reported for ZnONPs where *A. flavus* was the least susceptible fungal strain (MIC: 250 μg/mL while *A. niger* and *M. racemosus* were the most susceptible strain with MIC: 31.25 μg/mL. Among the various FS, *M. racemosus*, *A. niger* and *F. solani* were inhibited at all doses. Besides, ROS, previous studies claim that interaction of ZnONPs with fungal hypea and spores leads to inhibition of their growth. Significant concentration mediated fungicidal assays are reported in previous studies using different fungal strains [34] and are consistent with our present GW-ZnONPs study. MIC values for various bacteria and fungus strain are presented in Table 2.

**Table 2.** MICs values of different bacterial and fungal strains.

#### 3.2.4. Enzyme Inhibition Potentials of ZnONPs

Figure 8A shows protein kinase (PK) enzyme inhibition potential of ZnONPs. These enzymes play significant role in phosphorylation of important amino acids; serine-threonine, tyrosine residues, regulate important processes inside cells like metabolism, apoptosis, proliferations, and differentiation. Deregulated phosphorylation of amino acids can result into genetic abnormalities and result in the development of cancer. Therefore, any substance with potentials to inhibit PK enzymes are of great attention in the field of cancer research [57]. PK phosphorylation has played an important role in the formation of hyphae in *Streptomyces* fungal strain and same mechanism is used to evaluate the PK inhibition potentials and is utilized to study medicinal compounds for determining PK inhibition [5]. The PK inhibition activity was done via disc diffusion method using different doses of ZnONPs ranging from 31.25−1000 μg/mL. The surfactin was taken as a positive control. Different ZOI were observed at different doses of ZnONPs, and the highest ZOI was measured as 15 mm at 1000 μg/mL, which shows important protein kinase inhibition potency of ZnONPs. A concentration-dependent activity is reported for ZnONPs. No single dose has shown stronger potential than the positive control. Therefore, a potential signal transduction inhibitor is identified in the form of nanoscaled ZnO that can be further exploited for anti-infective and anticancer properties. Because of the protein kinase inhibition property, one can pre-deduce that biosynthesized ZnONPs may play an important role in cancer therapeutics. Our results of PK inhibition are in line with the previous findings [53].

**Figure 8.** (**A**) Inhibition potential of *Geranium wallichianum* mediated ZnONPs against protein kinase (**B**) Inhibition potential against alpha amylase (**C**) Antioxidant potential of ZnONPs (**D**) Biocompatibility potential of ZnONPs against human RBCs and macrophages.

Besides PK inhibition assay, alpha amylase (AA) inhibition potency of ZnONPs was determined using various concentrations (31.25–1000 μg/mL) of ZnONPs. The AA play significant role by converting carbohydrates into glucose [57], therefore, blocking or by inhibiting the activity of alpha amylase can prevent the level of glucose formation; thus, it may provide new insights into a nano level treatment of diabetes [58]. In our study, biogenic ZnONPs were explored for their AA inhibition activity and have determined significant potential by the inhibition of AA. The highest inhibition rate was observed 57% at 1000 μg/mL, while AA inhibition potentials was slowly decreasing with a decrease in concentration of ZnONPs. Figure 8B shows the biological inhibition potential of ZnONPs against AA. The results of AA activity are in agreement with the previous findings [44,53].

#### 3.2.5. Antioxidant Activities of ZnONPs

Figure 8C indicates antioxidant potentials of ZnONPs. The antioxidant activities were evaluated in concentration ranging from 1–200 μg/mL. Maximum score for TAC of ZnONPs in terms of AA equivalent/milligrams was reported as 52.43% at 200 μg/mL. TAC assay is mainly used to evaluate the scavenging effect of tested chemicals towards reactive oxygen species (ROS). In the present study, water extract of GW leaves extract was utilized in the reduction and stabilizations of metal ions. It can be concluded from our antioxidant activities, that several phenolic compounds available in the GW leaves extract scavenge ROS which are attached on the surface of ZnONPs.

To further explore the antioxidants species coated on the surface of biogenic nanoparticles, TRP assay was determined. This activity was done to study the reductones that play an important role in the antioxidant potential by providing H-atoms and causing damage to free radical chains [59]. The biosynthesized ZnONPs showed potential antioxidant activity. The reducing power of ZnONPs was decreasing with decrease in concentrations of ZnONPs. The maximum TRP (55%) was observed at its highest concentration of 200 μg/mL. Significant DPPH radicals scavenging activity (71.36%) was observed for ZnONPs at 200 μg/mL. From data presented in Figure 8C, it can be concluded that

numerous antioxidants compound may be responsible in reduction and stabilization of ZnONPs via GW leaf extracts. Our antioxidants result of GW-mediated ZnONPs are consistent with the previous studies of ZnONPs via *S. thea* and *F. indica* [34,44]. The variations and disagreement compare to other studies may be due to various important factors like experiment condition, method of nanoparticles fabrication, plant, plant part used, and nanoparticle size, etc.

#### 3.2.6. Biocompatibility Potential Assays

The biocompatibility and toxicological effect of zinc oxide nanoparticles were determined using human macrophages and RBCs. Biological substance with hemolytic activity of greater than 5% are known as hemolytic, between 2–5% are slightly hemolytic, while less than 2% is non-hemolytic [60]. If a given nanoparticle is hemolytic, it will rupture red blood cells, which further result in hemoglobin release. To confirm the bio-safe nature, hemolysis assay was conducted using human RBCs. The RBCs were exposed to various concentrations of ZnONPs in a concentration ranging from 200–1 μg/mL. The data obtained have shown that the synthesized are non-hemolytic at lower concentration (2 μg/mL), slightly hemolytic at 5 to 50 μg/mL, while hemolytic at concentrations of >50 μg/mL. These results are in agreement to previous reports of *S. thea* mediated ZnONPs [44]. The IC50 value of ZnONPs against human red blood cells was recorded is 491 μg/mL. Our research study confirmed that biosynthesized ZnONPs are non-hemolytic and are considered biocompatible in low concentrations.

The biocompatibility assay was further confirmed by using human macrophages. For this purpose, human macrophages were seeded in 96-well plate and were cultured in RPMI media for 24 h for cells attachment. Further, cells were treated with various concentrations of ZnONPs (200–1 μg/mL). MTT cell viability assay was done to perform biocompatible nature of ZnONPs. The macrophage responded to ZnONPs treatment in a dose dependent manner. The results indicated that ZnONPs at 200 μg/mL inhibit growth of the macrophages by ~32% which determine the bio-safe behavior of biogenic ZnONPs. Normally, macrophages have established mechanisms to deal with ROS produce from external source. According to research studies, ROS are non-toxic to both RBCs and macrophages at a lower concentration unless concentration increases beyond that limit which will be considered toxic for RBCs and macrophages [61]. The IC50 value for ZnONPs was calculated >353.7 μg/mL. The results of biocompatibility assays of ZnONPs are presented in Figure 8D.

#### **4. Conclusions and Future Perspectives**

In summary, a simple, safe, ecofriendly, and one-step process was used for the biofabrication of ZnONPs utilizing GW *leaves* extract without utilizing any chemical reagents or surfactants. Different characterization techniques were performed. TEM analysis showed that ZnONPs was of ~18 nm with a hexagonal shape. Furthermore, different in vitro biological activities of ZnONPs were performed. The bio-potentials of ZnONPs were investigated against different pathogenic microbial strains and confirmed that ZnONPs have shown significant antimicrobial potential. The ZnONPs displayed strong anticancer and antileishmaniasis activities. Furthermore, moderate antioxidant and enzymes inhibition activities have been investigated. The biosafe nature of ZnONPs was confirmed using human RBCs and macrophages. Based on the above findings, we can say that green synthesis is the way forward and new frontier for designing nanomedicine and can be utilized in different theranostic applications for the treatment of different diseases. In addition, different in vivo studies are encouraged on toxicity aspects in different animal models, and once their biocompatibility and bio-safe nature is confirmed, only then can these NPs can be utilized in clinical applications. Further studies are encouraged on the mechanistic and synthesis aspects of the ZnONPs by using different medicinal plant materials.

**Author Contributions:** Conceptualization, B.A.A., J.I., R.A., L.Z., S.K., C.W., T.M., and J.-T.C.; writing—original draft preparation, B.A.A., J.I., and R.A.; writing—review and editing, L.Z., S.K., C.W., T.M., and J.-T.C. All authors have read and agreed to the published version of the manuscript.

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

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

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*

### **Combined Use of Deep Eutectic Solvents, Macroporous Resins, and Preparative Liquid Chromatography for the Isolation and Purification of Flavonoids and 20-Hydroxyecdysone from** *Chenopodium quinoa* **Willd**

**Jia Zeng 1,2,**†**, Xianchao Shang 1,2,**†**, Peng Zhang 1, Hongwei Wang 3,\* , Yanlong Gu 4,\* and Jia-Neng Tan 1,\***

Received: 16 September 2019; Accepted: 21 November 2019; Published: 25 November 2019 -

**Abstract:** Deep eutectic solvents (DESs) were used in combination with macroporous resins to isolate and purify flavonoids and 20-hydroxyecdysone from *Chenopodium quinoa* Willd by preparative high-performance liquid chromatography (HPLC). The extraction performances of six DESs and the adsorption/desorption performances of five resins (AB-8, D101, HPD 400, HPD 600, and NKA-9) were investigated using the total flavonoid and 20-hydroxyecdysone extraction yields as the evaluation criteria, and the best-performing DES (choline chloride/urea, DES-6) and macroporous resin (D101) were further employed for phytochemical extraction and DES removal, respectively. The purified extract was subjected to preparative HPLC, and the five collected fractions were purified in a successive round of preparative HPLC to isolate three flavonoids and 20-hydroxyecdysone, which were identified by spectroscopic techniques. The use of a DES in this study significantly facilitated the preparative-scale isolation and purification of polar phytochemicals from complex plant systems.

**Keywords:** DESs; preparative-scale purification; flavonoids; 20-hydroxyecdysone; *Chenopodium quinoa*

#### **1. Introduction**

Deep eutectic solvents (DESs) exhibit the advantages of negligible volatility, adjustable viscosity, high solubility, preparation simplicity, eco-friendliness, and biodegradability, and have therefore attracted much attention [1–6]. Typically, DESs are homogeneous solutions of quaternary ammonium salts, which act as hydrogen-bond acceptors (HBAs) and engage in strong intermolecular hydrogen-bonding interactions with different hydrogen-bond donors. Among the diverse HBAs, cheap, biodegradable, and non-toxic choline chloride (ChCl) is most commonly used, [7] and ChCl-based DESs have been widely applied as reaction media in the fields of organic synthesis, biomass refinery, polymerization, and materials science [8–13]. Moreover, DESs can be used to extract phytochemicals, such as flavonoids, phenolic acids, κ-carrageenan, anthocyanins, and saponins, from various types of natural sources [14–19]. DES extraction technologies, however, have all been restricted to the

analysis of active constituents, which could only be detected by HPLC, HPLC-MS/MS, or some other analysis instruments using standard chemicals. Until now, there have been almost no studies on the preparative-scale separation and purification of monomer compounds that are not available on-hand, particularly for plant samples, based on DES extraction. Thus, introducing DESs into the area of phytochemistry will provide an interesting approach for researchers.

*Chenopodium quinoa* Willd. (quinoa) is one of the oldest cultivated plants in the Andean region and is well known as a functional food and nutraceutical source rich in essential amino acids, unsaturated fats, phytoecdysteroids, flavonoids, phenolic acids, betalains, and saponins, which can allegedly reduce the risk of cardiovascular disease, cancer, osteoporosis, and diabetes [20–31]. For example, flavonoids are commonly used to treat and prevent diabetes and obesity [32,33], while 20-hydroxyecdysone is believed to exhibit antioxidant, antidiabetic, anti-obesity, antihypertensive, anticancer, and anti-inflammatory properties [34,35]. Therefore, the isolation and characterization of phytochemicals from *C. quinoa* is of high practical significance.

The extraction of phytochemicals from *C. quinoa* has gained significant attention [28–30]. For example, Dini et al. isolated and characterized phenolic constituents (e.g., kaempferol, quercetin (QE), and vanillic acid glycosides) from quinoa seeds [29] by partitioning the methanolic seed extract between *n*-butanol and water. The *n*-butanol phase was subjected to defatting with chloroform (CHC13), and the obtained residue was loaded onto a Sephadex LH-20 column and subjected to droplet counter-current chromatography assisted by thin-layer chromatography to isolate several individual glycosides. In a report by Ho et al., where a series of ecdysteroids were isolated from *C. quinoa* [30], a suspension of the ethanolic (95 vol% aqueous ethanol) extract of ground seeds in water was sequentially treated with hexane, ethyl acetate, and *n*-butanol. Ecdysteroids, including 20-hydroxyecdysone, were separated from the *n*-butanol fraction by the combined use of Diaion HP-20 gel, silica gel, and RP-18 reverse-phase column chromatography. Notably, the above-mentioned organic solvent-based purification procedures are not efficient due to low extraction efficiencies and long extraction times. Therefore, the development of more efficient alternatives for the practical extraction of phytochemicals from *C. quinoa* is required.

Herein, we achieved the separation and purification of flavonoids and 20-hydroxyecdysone from *C. quinoa* by using DESs as extracting solvents in conjunction with the use of macroporous resins, employing preparative high-performance liquid chromatography (HPLC) (Figure 1). The extraction performances of six DESs were investigated and compared with some selected organic solvents. The best DES was thereafter employed in some downstream experiments. A mixture consisting of the DES and quinoa extract was separated using a macroporous resin. Then, the extract was subjected to preparative HPLC for the isolation and purification of monomer compounds, which were further identified by spectroscopic techniques based on comparison with previously reported data.

**Figure 1.** Schematic for routine isolation and purification of flavonoids and 20-hydroxyecdysone using the developed technique.

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

#### *2.1. Chemicals, Reagents, and Instruments*

ChCl, aminoethyl alcohol, D-glucose, glycerol, oxalic acid, lactic acid, urea, methanol-*d*4, QE, 20-hydroxyecdysone, sodium nitrite, and aluminium trichloride (AlCl3) were purchased from Aladdin Industrial Co., Ltd. (Shanghai, China). HPLC-grade methanol and acetonitrile (ACN) were purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). Macroporous resins (D101, HPD 600, NKA-9, HPD 400, and AB-8) and analytical-grade methanol were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). Deionized water was obtained using a Unique-R20 purification system (Xiamen RSJ Scientific Instruments Co., Ltd., Xiamen, China).

Nuclear magnetic resonance (NMR) spectra were recorded on an Agilent DD2 spectrometer (500 MHz for 1H and 125 MHz for 13C NMR; Agilent, CA, USA). High-resolution electrospray ionization–tandem mass spectrometry (HRESI-MS) was performed using an LTQ Orbitrap XL spectrometer (Thermo Scientific, MA, USA).

#### *2.2. Determination of Total Flavonoid (TF) and 20-Hydroxyecdysone Contents*

TF content was determined using a method obtained by the modification of a previously reported method [36]. A 96-well plate was sequentially charged with the concentrate (50 μL), 0.066 M sodium nitrite solution (100 μL), and 10% AlCl3 solution (*w*/*v*, 15 μL) and incubated at room temperature for 6 min. Thereafter, 0.5 M aqueous sodium hydroxide (NaOH, 100 μL) was added to terminate the reaction, and the absorbance of the reaction mixture at 510 nm was measured by a microplate reader using QE as a standard. TF content was expressed as milligrams of QE per gram of dry quinoa material.

20-Hydroxyecdysone content was quantified by ultra-performance liquid chromatography–triple quadrupole tandem mass spectrometry (UPLC-QqQ-MS/MS) using an Acquity UPLC system (Waters Corp., MA, USA) coupled with a TSQ Quantum triple quadrupole tandem mass spectrometer (Thermo Scientific, CA, USA). The mobile phase, comprising ACN (eluent A) and 0.1 vol% aqueous formic acid (eluent B), was supplied at a flow rate of 0.3 mL/min. The following gradient program was used: 0–2 min, 5–10 vol% ACN; 2–4 min, 10–20 vol% ACN; 4–6 min, 20–30 vol% ACN; 6–8 min, 30 vol% ACN; 8–10 min, 30–5 vol% ACN. The injection volume and column temperature were 10 μL and 25 ◦C, respectively.

Low-resolution mass spectrometric detection was carried out in the selected reaction-monitoring mode using an electrospray ionization (ESI) interface. The optimized parameters corresponded to a vaporizer temperature of 450 ◦C, corona discharge voltage of 4.0 kV, capillary temperature of 225 ◦C, sheath nitrogen gas pressure of 17 psi, auxiliary nitrogen gas pressure of 5 psi, and collision gas pressure

of 1.5 mTorr. The Xcalibur software was used to control the LC-MS system and analyze the collected data. The mass spectrometric analysis of 20-hydroxyecdysone was initially performed over the full scan range in a negative-ion mode, and a pseudomolecular ion at *m*/*z* 479.176 (*t*R = 4.65 min) as well as four product fragment ions at *m*/*z* 319.1 (collision energy = 27 eV), 301.1 (collision energy = 33 eV), 159.0 (collision energy = 27 eV), and 83.0 (collision energy = 37 eV) were detected. The fragmentation patterns of 20-hydroxyecdysone were compared with those previously reported in the literature [37].

#### *2.3. Preparation and Selection of DESs*

The ChCl/urea DES was prepared using a modification of a previously reported method [12] (Table 1). Briefly, a glass vial was charged with ChCl and urea, and the obtained mixture was stirred at 280 rpm at 80 ◦C for 2 h. The obtained ChCl/urea DES (clear stable liquid) was mixed with water (7/3, *v*/*v*), and the solution was used for further experiments. Other DESs were prepared in a similar way.

Ultrasound-assisted extraction was used to extract TF and 20-hydroxyecdysone. The extraction capacities of six DESs were evaluated using the extraction yields (*E*y) of TF and 20-hydroxyecdysone. First, 100 mg of quinoa seed powder and 1.0 mL of DES were added to a 10 mL glass tube in sequence. The mixture was placed on a vortex meter, stirred for 2 min, and further ultrasonicated at 50 ◦C for 30 min (25 kHz, 200 W). After the tube cooled down to the room temperature, the mixture was centrifuged at 3000 rpm for 10 min. The supernatant (1 mL) was diluted with methanol (4 mL), and the mixture was passed through a 0.22 μm filter for subsequent analyses. Each extraction was performed in triplicate, and Ey was calculated as follows:

$$E\_y = (\mathbb{C}\_0 \times V\_0) / M\_{0\prime} \tag{1}$$

where *C*0 is the concentration of TF or 20-hydroxyecdysone found in the DES, *V*0 is the volume of the diluted liquid, and *M*0 is the mass of the sample.

**Table 1.** Compositions and physicochemical properties of deep eutectic solvents (DESs) used in this study.

a Determined at 30 ◦C. b Dehydrate. c Not available.

#### *2.4. Response Surface Methodology (RSM)*

The optimal values for three variables in the selected DES, including water content (*a*), extraction temperature (*b*), and solid (quinoa seed)–liquid (DES) ratio (*c*) at three levels (–1, 0, and +1) were determined using RSM based on a Box–Behnken design (BBD). Table S1 (Supplementary Materials) shows the investigated variables and their values in the three-level BBD. The experiment was designed to reveal the effect of each parameter on TF and 20-hydroxyecdysone contents as well as the interactions between the three main factors. The entire study was comprised of 17 separate experiments including 5 center points, each of which was conducted in triplicate.

Statistical comparisons were made using a single factor analysis of variance (ANOVA); *p*-values < 0.05 were considered significant. Data were processed using Design-Expert 8.5 statistical software and the social package for statistical studies (SPSS 19.0).

#### *2.5. Adsorption*/*Desorption Capacities of the Five Employed Resins*

The adsorption/desorption capacities of the five kinds of macroporous resins were investigated using the adsorption/desorption yields of TF and 20-hydroxyecdysone. The DES extract solution was added to a 200 mL flask containing 1.0 g of the pretreated resins. The mixture was shaken at 120 rpm at room temperature for 24 h to reach adsorption equilibrium, and the resins were washed by deionized water and then desorbed with 50 mL of methanol in the flask, which was continually shaken at 120 rpm at room temperature for 24 h. The adsorption/desorption yields of resins were calculated according to the following Equations (2) and (3):

$$D\_{\mathbf{a}} = (C\_0 - C\_{\mathbf{a}})C\_{0\prime} \tag{2}$$

$$D\_{\rm d} = \mathbb{C}\_{\rm d} \times V\_{\rm d} / (\mathbb{C}\_0 - \mathbb{C}\_{\rm a}) \times V\_{0\prime} \tag{3}$$

where *D*a is the adsorption yield at adsorption equilibrium; *D*d is the desorption yield after adsorption equilibrium; *C*0, *C*a, and *C*d represent the content of TF or 20-hydroxyecdysone at initial equilibrium, absorption equilibrium, and desorption equilibrium, respectively; *V*0 and *V*d are the initial sample volume and desorption solution volume (mL), respectively.

#### *2.6. Small- and Preparative-Scale Sample Extraction*

Quinoa seeds, desaponified by soaking in distilled water for 24 h without the presence of foam, were purchased from Xinjing Quinoa Cultivation and Promotion Co., Ltd. (Shanxi, China) [38]. For small-scale extraction, the seeds were ground and 1.0 g of the obtained powder was mixed with aqueous DES (10 mL). The resulting mixture was sonicated (100 W) at 50 ◦C for 30 min in an ultrasonic bath (Scientz SB25-12D, Ningbo Scientz Biotechnology Co., Ltd, Ningbo, China) and then centrifuged at 5000 rpm for 10 min. Small-scale extraction was used in the selection of DESs and macroporous resins as well as the RSM experiments. For preparative-scale extraction, powdered seeds (1.0 kg) were suspended in 10 L of aqueous DES. The extraction process was similar to that of small-scale extraction. The collected supernatants were loaded onto a macroporous resin column (60 mm × 1200 mm), which was sequentially flushed with deionized water (5000 mL) at a flow rate of 15 mL/min (to remove the DES) and methanol (2000 mL) at a flow rate of 20 mL/min under reduced pressure. The methanolic extract was concentrated, and the concentrate was stored at 4 ◦C for further use.

#### *2.7. Analytical and Preparative HPLC*

For the first separation, analytical HPLC of quinoa extract was performed on a Waters 2489 chromatography system (Waters, MA, USA) equipped with a C18 separation column (5 μm, 4.6 × 150 mm) using ACN and water for gradient elution at a flow rate of 1.0 mL/min. UV detection was performed at 210 nm. The gradient program was as follows: 0–5 min, 8 vol% ACN; 5–7.5 min, 8–15 vol% ACN; 7.5–10 min, 15–30 vol% ACN; 10–15 min, 30–50 vol% ACN; 15–17.5 min, 50–60 vol% ACN; 17.5–30 min, 60 vol% ACN. The first-round preparative HPLC of quinoa extract was performed on a DAC100 preparative HPLC system (Beijing, China) equipped with a C18 separation column (5 μm, 100 × 250 mm) at a flow rate of 300 mL/min, while the other chromatographic conditions were identical to those used for analytical HPLC. The obtained eluent was separated into five (I–V) fractions.

In the second round of separation, fractions I–V were subjected to analytical and preparative HPLC performed on the Waters 2489 chromatography system equipped with 4.6 × 150 mm (5 μm) and 10 × 250 mm (5 μm) C18 columns, respectively. The flow rates of analytical and preparative separations were set to 1.0 and 6.0 mL/min, respectively. The mobile phase composition and UV detection parameters were identical to those used for the first round of separation. The gradient program was as follows: 0–5 min, 5 vol% ACN; 5–10 min, 5–10 vol% ACN; 10–15 min, 10 vol% ACN; 15–20 min, 10–15 vol% ACN; 20–25 min, 15 vol% ACN; 25–30 min, 15–30 vol% ACN; 30–35 min, 30 vol% ACN; 35–40 min, 30–50 vol% ACN; 40–50 min, 50 vol% ACN.

#### **3. Results and Discussion**

#### *3.1. Preparation and Optimization of DES Systems*

The non-toxicity, biodegradability, and low cost of ChCl make it suitable for combination with a series of hydrogen-bond donors (HBDs) to afford biocompatible and renewable DESs. Herein, six DESs were synthesized by combining ChCl with HBDs belonging to the classes of inexpensive and easily available fine chemicals (aminoethyl alcohol and urea), polyalcohols (glycerol), organic acids (oxalic and lactic acids), and sugars (D-glucose). The composition and physicochemical properties (density, conductivity, and viscosity) of the six DESs are listed in Table 1.

DES extraction performance was evaluated by considering the TF and 20-hydroxyecdysone contents of the corresponding extracts. Figure 2A compares the obtained TF contents, revealing that the value obtained for DES-6 (3.93 ± 0.15 mg/g) exceeded those obtained for DES-4 (3.45 ± 0.09 mg/g), DES-3 (3.31 ± 0.10 mg quercetin equivalents (QE)/g), DES-5 (2.78 ± 0.17 mg QE/g), DES-1 (2.52 ± 0.06 mg QE/g), and DES-2 (2.25 ± 0.13 mg QE/g). Dini et al. [39] determined the TF contents before and after cooking in sweet and bitter quinoa seeds. The samples were extracted in methanol–water (80:20 *v*/*v*). It was found that the bitter quinoa seeds processed higher TF content before cooking (1.39 ± 0.35 mg catechin equivalents (CE)/1.0 g) than the sweet seeds (0.81 ± 0.10 mg CE/1.0 g). After cooking, TF contents of bitter and sweet quinoa seeds decreased significantly to 0.63 ± 0.15 mg CE/g and 0.18 ± 0.07 mg CE/g, respectively. In consideration of the similar molecular weights of QE and CE, the TF contents obtained by Dini et al. were lower than those in our study. Figure 2B shows that similar 20-hydroxyecdysone contents were obtained for DES-3 (551.50 ± 4.51 mg/g), DES-5 (546.33 ± 7.60 mg/g), and DES-2 (504.69 ± 12.05 mg/g), while higher values were obtained for DES-6 (606.76 ± 7.98 mg/g), and significantly lower values were obtained for DES-1 (384.80 ± 12.16 mg/g) and DES-4 (107.65 ± 2.88 mg/g). Lafont et al. [30] determined the 20-hydroxyecdysone contents of quinoa seeds obtained from four producing countries (Bolivia, Chile, Ecuador, and Peru). Seed powder was extracted with 20 mL methanol–water (35:65, *v*/*v*) overnight with magnetic stirring. The obtained 20-hydroxyecdysone contents (0.32–0.42 mg/g) were significantly lower than those obtained after extraction by DES-2, DES-3, DES-5, and DES-6. In addition, the 20-hydroxyecdysone content of the DES-6 extract was also much higher than the values previously reported by Raskin et al. (0.18–0.49 mg/g) [28]. It is possible that the weakly alkaline HBD (urea) possessed stronger hydrogen-bond interactions with phenolic hydroxyls and alcoholic hydroxyls in the flavonoids or alcoholic hydroxyls in 20-hydroxyecdysone than the neutral (D-glucose and glycerol), strong alkaline (aminoethyl alcohol), and weakly acidic (oxalic acid and lactic acid) HBDs, contributing to the enhanced extraction yields. Based on the above results, DES-6 (ChCl/urea) was used in subsequent experiments.

**Figure 2.** (**A**) Total flavonoid (TF) and (**B**) 20-hydroxyecdysone extraction efficiencies of the employed DESs. TF extraction efficiency was expressed as milligrams QE equivalents per gram of quinoa sample.

#### *3.2. BBD Experimental Design*

Three values, including water content in the DES (variable *a*), extraction temperature (variable *b*), and solid–liquid ratio (variable *c*), were used as independent variables to efficiently optimize the extraction yields of TF and 20-hydroxyecdysone from *C. quinoa* in the RSM experiments. The extraction yields were considered as related responses in order to evaluate the efficiency of the extraction procedure. The experiments were performed in a random order to avoid systematic error. The results, including the coded variables and related responses, are presented in Table 2. A second-order polynomial equation was applied to express the proposed model after multiple regression analysis of the experimental data. The regression model equations for the responses and variables in terms of the coded levels are as follows:

$$Y\_{\rm TF} = 3.94 + 0.48a + 0.017b - 0.25c \cdot 0.097ab - 0.028ac + 0.20bc - 0.86a^2 - 0.41b^2 - 0.34c^2,\tag{4}$$

$$Y\_{\text{23-Hyythoxyczyczłysowa}} = 0.50 + 0.033a - 0.017b - 0.025c - 0.037ab - 0.020ac - 0.046bc - 0.068a^2 - 0.026b^2 - 0.12c^2,\tag{5}$$

where *a* is the DES water content, *b* is the extraction temperature, and *c* is the solid–liquid ratio.

Variances of the ANOVA regression model equations of TF and 20-hydroxyecdysone are shown in Tables S2 and S3 (Supplementary Materials). The coefficients (*R*2) of the variables of response were 0.9553 and 0.9501 for TF and 20-hydroxyecdysone, respectively. The *F*-values for the lack-of-fit model were all non-significant, which supported our assumption that the models were sufficient to accurately represent the experimental data.

A response surface plot of the model was used for graphically interpreting the significant effects of interactions among the three variables on the contents of TF and 20-hydroxyecdysone (Figure 3). Results showed that the extraction yields of TF and 20-hydroxyecdysone were apparently related to the main variable. In the model, the concentration of water (%) in the DES solution (variable *a*) and solid–liquid ratio (variable *c*) exhibited statistically significant effects (*p* < 0.05) on the extraction yields of TF and 20-hydroxyecdysone. In contrast, extraction temperature (variable *b*) showed a non-significant effect (*p* > 0.05) on the contents.

**Table 2.** Response surface optimization experiments using DES-6 extraction of investigated variables.

Based on RSM results, TF was extracted from *C. quinoa* using the optimum conditions (DES water content, 32.4%; extraction temperature, 50.7 ◦C; and solid–liquid ratio, 97.2 mg/mL). The optimal

extraction conditions for 20-hydroxyecdysone extraction from *C. quinoa*, as well as the related maximal response values (Table 3), were established by applying a simple procedure (DES water content, 32.6%; extraction temperature, 47.8 ◦C; and solid–liquid ratio, 99.7 mg/mL). A verification experiment performed using these conditions gave optimal extraction yields of 4.23 and 0.51 mg/g for TF and 20-hydroxyecdysone, respectively. In order to simplify the operation, the experimental conditions with a DES water content of 30.0%, an extraction temperature of 50.0 ◦C, and a solid–liquid ratio of 100.0 mg/mL were selected for DES extraction (10 L). Under this condition, the extraction yields of TF and 20-hydroxyecdysone were 4.11 and 0.50 mg/g, respectively, which were close to the predicted values.

**Figure 3.** Response surface plots of the models for (**A**,**B**, and **C**) TF and (**D**, **E**, and **F**) 20-hydroxyecdysone contents.

**Table 3.** Optimal conditions of the variables that maximize the response values using response surface methodology.

#### *3.3. Macroporous Resin Selection*

Macroporous resins exhibit the advantages of easy recyclability, low cost, high adsorption/ desorption capacity, and suitability for large-scale production, and have therefore been widely used for the enrichment and purification of phytochemicals extracted from complex plant systems. Here, we used TF and 20-hydroxyecdysone contents to evaluate the performances of five macroporous resins (AB-8, D101, HPD 400, HPD 600, and NKA-9) for removing DESs from extracts prior to preparative HPLC (Figure 4A,B). The D101 resin had the highest TF adsorption capacity (81.2%), which was approximately two-fold higher than that of the HPD 400 resin (38.9%) (Figure 4A). The adsorption capacities of AB-8 (73.4%) and HPD 400 (69.3%) were similar to each other and higher than that of NKA-9 (63.6%). The TF desorption capacities of all the tested resins exceeded 70%. In particular, D101 (89.7%) and AB-8 (79.2%) had similar desorption capacities that exceeded those of HPD 400 (75.4%), HPD 600 (73.4%), and NKA-9 (70.2%). Moreover, D101 exhibited the highest 20-hydroxyecdysone adsorption/desorption capacities among the five tested resins (Figure 4B). Therefore, D101 was selected for implementing the next studies.

**Figure 4.** (**A**) TF and (**B**) 20-hydroxyecdysone adsorption/desorption capacities of the five employed resins.

#### *3.4. Preparative-Scale Isolation and Purification of Monomer Compounds*

Eluate concentrates obtained at the DES removal stage were sequentially subjected to analytical and preparative HPLC, and the corresponding chromatograms (Figure 5) showed that the resolution of preparative HPLC was similar to that of analytical HPLC. Fractions I (242 mg), II (253 mg), III (206 mg), IV (988 mg), and V (265 mg) separated from 9.2 g of the concentrate were further evaluated by analytical HPLC. The corresponding chromatograms, shown in Figure 6A, revealed that the compounds obtained after the first separation round were not sufficiently pure. Therefore, a second round of preparative HPLC (Figure 6B) was performed using a C18 column with a smaller inner diameter (5 μm, 10 × 250 mm), yielding compound **1** (48.7 mg), compound **2** (47.9 mg), compound **3** (39.9 mg), and compound **4** (230.6 mg) from fractions I–IV, respectively. No compound was isolated from fraction V.

**Figure 5.** Analytical (**A**) and preparative-scale (**B**) HPLC-UV chromatograms of quinoa extracts.

**Figure 6.** Analytical (**A**) and preparative-scale (**B**) HPLC-UV chromatograms of fractions I–IV.

#### *3.5. Identification of Isolated Compounds*

Compounds **1**–**4** were identified based on their HRESI-MS, 1H NMR, 13C NMR, and 2D NMR spectra (Table 4).

Compound **1**. HRESI-MS: found *m*/*z* 755.2048 [M-H]−, calcd. for C33H40O20 *m*/*z* 756.2113. 1H NMR (500 MHz, CD3OD): δ (ppm) 7.69 (1H, d, *J* = 2.2 Hz), 7.56 (1H, dd, *J* = 8.5, 2.2 Hz), 6.87 (1H, d, *J* = 8.5 Hz), 6.36 (1H, d, *J* = 2.1 Hz), 6.19 (1H, d, *J* = 2.2 Hz), 5.66 (1H, d, *J* = 7.8 Hz), 5.59 (1H, d, *J* = 7.7 Hz), 5.21 (1H, d, *J* = 1.4 Hz), 4.54 (1H, d, *J* = 1.5 Hz), 1.18 (3H, d, *J* = 6.5 Hz), and 0.95 (3H, d, *J* = 6.5 Hz). 13C NMR (125 MHz, CD3OD): δ (ppm) 178.1, 164.3, 161.7, 156.9, 156.9, 148.2, 144.4, 133.1, 121.9, 121.6, 115.9, 114.7, 104.5, 100.4, 99.6, 98.3, 98.3, 93.2, 76.0, 74.2, 73.8, 72.6, 72.5, 71.0, 70.9, 70.8, 70.6, 69.4, 68.4, 68.3, 65.3, 16.6, 16.0. A comparison with previously reported data [29] allowed compound **1** to be identified as quercetin-3-*O*-(2,6-di-α-L-rhamnopyranosyl)-β-D-galactopyranoside (Figures S4–S6).

Compound **2**. HRESI-MS: found *m*/*z* 741.1875 [M-H]−, calcd. for C32H38O20 *m*/*z* 742.1956. 1H NMR (500 MHz, CD3OD): δ (ppm) 7.71 (1H, d, *J* = 2.1 Hz), 7.62 (1H, d, *J* = 8.4, 2.1 Hz), 6.86 (1H, d, *J* = 8.4 Hz), 6.37 (1H, d, *J* = 1.9 Hz), 6.17 (1H, d, *J* = 1.9 Hz), 5.40 (1H, d, *J* = 7.9 Hz), 4.52 (1H, d, *J* = 1.5 Hz), 5.46 (1H, d, *J* = 1.2 Hz), 4.06 (1H, d, *J* = 1.2 Hz), 3.99 (1H, dd, *J* = 7.9, 9.4 Hz), 3.79 (1H, m), 3.78 (1H, d, *J* = 3.4 Hz), 3.71 (1H, m), 3.70 (1H, dd, *J* = 12.6, 4.7 Hz), 3.61 (2H, s), 3.57 (1H, dd, *J* = 3.4, 1.5 Hz), 3.55 (1H, d, *J* = 3.5 Hz), 3.51 (1H, dq, *J* = 9.4, 6.2 Hz), 3.42 (1H, dd, *J* = 12.6, 6.8 Hz), 3.27 (1H, t, *J* = 9.4 Hz), 1.18 (3H, d, *J* = 6.2 Hz). 13C NMR (125 MHz, CD3OD): δ (ppm) 178.0, 164.3, 161.3, 157.0, 156.9, 148.3, 144.4, 133.5, 121.8,121.8, 115.9, 114.7, 109.5, 104.3, 100.6, 100.3, 98.3, 93.2, 79.4, 76.6, 74.6, 74.2, 73.4, 73.3, 72.6, 71.8, 70.8, 69.3, 68.3, 65.5, 64.8, 16.5. The 1H and 13C NMR spectra of compound **2** were similar to those of quercetin-3-*O*-β-D-apiofuranosyl-(1→2)-*O*-[α-L-rhamnopyranosyl-(1→6)]-β-Dgalactopyranoside-3 ,4 -dimethyl ether reported by Dini et al. [29] and compound **2** was concluded to be the demethoxylated version of the above species, namely quercetin-3-*O*-β-D-apiofuranosyl- (1→2)-*O*-[α-L-rhamnopyranosyl-(1→6)]-β-D-galactopyranoside. As the NMR data of compound **2** have not been frequently reported, this compound was fully characterized by 1H, 13C, heteronuclear singular quantum correlation (HSQC), heteronuclear multiple bond correlation (HMBC), and 1H-1H correlated spectroscopy (COSY) NMR techniques, and the obtained results are shown in Table S4 (Figures S7–S14).

Compound **3**. HRESI-MS: found *m*/*z* 739.2099 [M-H]−, calcd. for C33H40O19 *m*/*z* 740.2164. 1H NMR (500 MHz, CD3OD): δ (ppm) 8.06 (2H, d, *J* = 8.9 Hz), 6.89 (2H, d, *J* = 8.9 Hz), 6.37 (1H, d, *J* = 1.7 Hz), 6.17 (1H, d, *J* = 1.5 Hz), 5.60 (1H, d, *J* = 7.6 Hz), 5.21 (1 H, d, *J* = 1.5 Hz), 4.52 (1H, d, *J* = 1.5 Hz), 1.17 (3H, d, *J* = 6.5 Hz) and 0.98 (3H, d, *J* = 6.5 Hz). 13C NMR (125 MHz, CD3OD): δ (ppm) 178.1, 164.3, 161.7, 159.9, 157.2, 157.0, 133.0, 130.8, 130.8, 121.6, 114.8, 114.8, 104.6, 101.1, 100.4, 99.4, 98.4, 93.3, 76.1, 74.3, 73.8, 72.6, 72.5, 71.0, 70.9, 70.9, 70.7, 69.3, 68.4, 68.3, 65.7, 16.5, 16.1. Comparison with previously reported data [29] allowed compound **3** to be identified as kaempferol-3-*O*-(2,6-di-α-Lrhamnopyranosyl)-β-D-galactopyranoside (Figures S15–S17).

Compound **4**. HRESI-MS: found *m*/*z* 479.3021 [M-H]−, calcd. for C27H44O7 *m*/*z* 480.3087. 1H NMR (500 MHz, CD3OD): δ (ppm) 5.80 (1H, d, *J* = 2.8 Hz), 3.95 (1H, br s), 3.83 (1H, br s), 1.20 (3H, s), 1.19 (3H, s), 1.18 (3H, s), 0.96 (3H, s), and 0.89 (3H, s). 13C NMR (125 MHz, CD3OD): δ (ppm) 205.0, 166.6, 120.7, 83.8, 77.0, 76.5, 69.9, 67.3, 67.1, 50.4, 49.1, 41.0, 37.8, 35.9, 33.7, 31.4, 31.1, 30.4, 28.3, 27.5, 25.9, 23.0, 20.1, 20.1, 19.6, 16.6. Apart from the missing carbon signal at 47.0 ppm, which was believed to overlap with the solvent peak, the spectral data were in accordance with those reported for 20-hydroxyecdysone [30]. Thus, compound **4** was identified as 20-hydroxyecdysone (Figures S18–S20).

**Table 4.** Structures of the isolated compounds.

#### **4. Conclusions**

A technique combining DES-assisted extraction, macroporous resin-facilitated DES removal, and preparative HPLC was developed for the preparative-scale isolation and purification of flavonoids and 20-hydroxyecdysone from *C. quinoa* Willd. Specifically, three flavonoids and 20-hydroxyecdysone were successfully separated and purified, and their structures were identified by HRESI-MS, 1D NMR, and 2D NMR. No saponins were isolated, as the employed *C. quinoa* sample had been subjected to desaponification. DESs were concluded to be appropriate for the extraction of polar compounds, such as glycosides, or other OH-group-rich compounds. The developed method is expected to be well suited for the preparative-scale separation and purification of minor phytochemicals of interest from complex plant systems. Further work will focus on the application of our method to the purification of triterpenoid saponins.

#### **Supplementary Materials:** The following are available online at http://www.mdpi.com/2218-273X/9/12/776/s1.

**Author Contributions:** J.-N.T., H.W. and Y.G. conceived the experiments and revised the manuscript. J.Z., X.S., and P.Z. collected plant materials. J.Z., and X.S. performed the experiments and analyzed the data. J.-N.T., H.W., and Y.G. wrote the manuscript. All authors have read and approved the final version of the manuscript.

**Funding:** This research was funded by the Fundamental Research Funds for Central Non-Profit Scientific Institution<PHONE_NUMBER>010), Agricultural Science and Technology Innovation Program (ASTIP-TRIC05), Shandong Provincial Natural Science Foundation, China (ZR2016BQ43, ZR2019BB061), and National Natural Science Foundation of China (21703284).

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

#### **References**

#### **Availability of Isolated Substances:** Not available.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

### *Article* **Clerodane Diterpene Ameliorates Inflammatory Bowel Disease and Potentiates Cell Apoptosis of Colorectal Cancer**

**Jia-Huei Zheng 1, Shian-Ren Lin 1 , Feng-Jen Tseng 1,2, May-Jywan Tsai 3, Sheng-I Lue 1,4, Yi-Chen Chia 5, Mindar Woon 6, Yaw-Syan Fu 7,8 and Ching-Feng Weng 7,8,\***

Received: 25 October 2019; Accepted: 20 November 2019; Published: 21 November 2019 -

**Abstract:** Inflammatory bowel disease (IBD) is general term for ulcerative colitis and Crohn's disease, which is chronic intestinal and colorectal inflammation caused by microbial infiltration or immunocyte attack. IBD is not curable, and is highly susceptible to develop into colorectal cancer. Finding agents to alleviate these symptoms, as well as any progression of IBD, is a critical effort. This study evaluates the anti-inflammation and anti-tumor activity of 16-hydroxycleroda-3,13-dien-15,16-olide (HCD) in in vivo and in vitro assays. The result of an IBD mouse model induced using intraperitoneal chemical azoxymethane (AOM)/dextran sodium sulfate (DSS) injection showed that intraperitoneal HCD adminstration could ameliorate the inflammatory symptoms of IBD mice. In the in vitro assay, cytotoxic characteristics and retained signaling pathways of HCD treatment were analyzed by MTT assay, cell cycle analysis, and Western blotting. From cell viability determination, the IC50 of HCD in Caco-2 was significantly lower in 2.30 μM at 48 h when compared to 5-fluorouracil (5-FU) (66.79 μM). By cell cycle and Western blotting analysis, the cell death characteristics of HCD treatment in Caco-2 exhibited the involvement of extrinsic and intrinsic pathways in cell death, for which intrinsic apoptosis was predominantly activated via the reduction in growth factor signaling. These potential treatments against colon cancer demonstrate that HCD could provide a promising adjuvant as an alternative medicine in combating colorectal cancer and IBD.

**Keywords:** colorectal cancer; diterpenes; inflammatory bowel diseases; *Polyalthia longifolia*; herbal medicine

#### **1.  In addition, CRC is the third leading cause of cancer deaths for new cases, and second for estimated deaths in both genders in Taiwan [1]. In the majority of cases, it is a consequence of the progressive accumulation of genetic and epigenetic alterations that leads the transformation and progression of normal colorectal mucosa to adenoma and eventually carcinoma, progressing CRC [1]. When analyzed, the incidence of CRC includes 72% colon cancer and 28% rectum cancer [2]. Surgery is mostly the first choice for all stages of CRC treatment, while for stage IV of CRC, recurrent CRC, or liver cancer metastasis, chemotherapy is the main alternative strategy to treat CRC [3]. Clinically, chemotherapeutic drugs, including 5-fluorouracil (5-FU), oxaliplatin, and capecitabine, are commonly used to treat CRC; however, these typically contribute to several adverse effects, such as fatigue, nausea, bone marrow toxicity, immunosuppression, and easy bleeding. Moreover, chemotherapy is a conventional treatment for late-stage and recurrent colorectal cancer, bit the cancer cells frequently become drug-resistant after treatment [3], and the unpredictability of adverse or side effects ordinarily restricts the administration of an accurate dose. As incidences of adverse results are observed, a new discovery of more efficacious and less toxic agents against CRC is of great urgency.

Noticeably, the risk factors of CRC are complicated, and include excessive alcohol use, obesity, hereditary conditions, and long-standing inflammatory bowel disease (IBD) [3]. IBD is comprised of two conditions: ulcerative colitis (UC; ICD-10-CM code K51.90) and Crohn's disease (CD; ICD-10-CM code K50.90), which are caused by uncontrolled gastrointestinal (GI) inflammation or bacterial infection that subsequently result in fever, edema, intestinal fibrosis, and ulcers [4]. Global prevalence rates for IBD exceed 0.3%, which is mostly attributed to human-development-index countries—e.g., the United States, Canada, Germany, Norway, and Australia—where incidence rates are twice as high as those of Asian and African countries [5]. Moreover, the incidence rates of IBD where it occurs in the industrialized countries of Asia, Africa, and South America were between 4% (Taiwan) and 11.1% (Brazil) from the 1990s to 2010 [5]. Therefore, IBD becomes a burden in Western and industrialized societies.

Within an inflamed GI tract, large numbers of immunocytes, including macrophages, TH cells, neutrophils, and natural killer (NK) cells, are attracted and secrete various forms of cytokines, such as tumor necrosis factor alpha (TNF-α), interferon-γ (IFNγ), and interleukin (IL)-6. When the immunocytes are accumulated, cytokine secretions lead to reductions in incidences of inflammation, and finally potentiate carcinogenesis [6]. Furthermore, one study has illustrated the connection between IBD and the Wnt/β-catenin signaling pathways, which might trigger colon cancer progression and incidences of sporadic colon cancer [7]. Interestingly, vitamin D deficiency, genetic susceptibility, disturbance of the microbiome, and psychological status have recently been confirmed as risk factors for IBD [8,9]. Previous evidence has shown that chronic inflammation may cause GI tumorigenesis, which is familiar and lethal worldwide. Even the known risk factors of IBD that have been broadly explored, as well as the causes, complications, and medications of IBD, including CRC, are still unraveling [10]. Nonetheless, the treatment of IBD remains unsolved and needs to be explored. According to the inquiry of new drug discovery, 46% of new drugs are of natural origin, e.g., derivatives, precursors, or mimetic molecules [11]. This fact exemplifies the continuing research and development of new drugs from natural products, especially medicinal plants, to meet patient demand, as the prospect of exploring new compounds and alternative treatments gains an immediate importance.

*Polyalthia longifolia var.* pendula Linn. (family Annonaceae) is an ornamental tree originally distributed in India, Sri Lanka, and Pakistan, which also contains numerous biological functions, as presented in the literature [12]. By exploring bioactive components, a clerodane diterpene 16-hydroxycleroda-3,13-dien-15,16-olide (HCD; PubChem ID 124820) has been extensively identified [13]. In previous reports, HCD has been shown to have numerous medicinal values as an anti-inflammation, anti-cancer, anti-fungal, anti-diabetic, and anti-bacterial agent [14]. In our previous studies, HCD performed as an executor to induce autophagy in glioma cells and oral squamous cell carcinoma cells, which consequently triggered cancer cell death [15,16]. Moreover, HCD can induce anoikis and reduce cell proliferation via the regulation of both intracellular growth and focal adhesion signaling in renal carcinoma cells [17,18]. In addition to acting as an anti-tumor agent, HCD could also play a supplementary role in the cytotoxicity of tamoxifen-treated breast cancer via the modulation

of the Bax/Bcl-2 ratio, which is directly expressed at cells undergoing apoptosis [19]. Recently, our studies have demonstrated the therapeutic potential of HCD against various types of cancers [19]. Nevertheless, the therapeutic potency of HCD in treating GI inflammation, e.g., IBD and colorectal cancer, has not been clarified. The aim of this study was to determine the dampening effect of HCD on IBD treatment and anticancer activity. In this work, two platforms containing an azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced colitis IBD model (*in vivo*) and colorectal carcinoma cells Caco-2 (*in vitro*) were employed to evaluate the treated efficacy of HCD. Furthermore, the detailed mechanisms of HCD on anti-colorectal cancer were also investigated.

#### **2. Experimental Section**

#### *2.1. Study Design*

The main focus of this study was to evaluate the potential of HCD against IBD and colon cancer. The experimental design was divided into two parts: IBD induction and colon cancer with HCD treatment. In the IBD model, mice were induced by azoxymethane (AOM)/dextran sodium sulfate (DSS). HCD was intraperitoneally (i.p.) injected, and subsequently GI inflammation was observed. In the colon cancer part, Caco-2 cells were used as the sole platform for the determination of cytotoxicity and cell death characteristics, and the underlying mechanisms of colon cancer cytotoxicity in HCD-treated cells, including cell cycle signaling, growth factor signaling, and inflammatory signaling, were under investigation.

#### *2.2. Chemicals*

HCD was kindly provided by Professor Yi-Chen Chia (Department of Food Science and Technology, Tajen University, Taiwan). Isolation and identification of HCD has been described in the previous literature [20]. The reagents and mediums for cell culture were purchased from Thermo-Fisher (Waltham, MA, United States). General chemicals were obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). The antibodies used in this study are listed in Table 1; these were purchased from Genetex International (Hsinchu, Taiwan), Cell Signaling Technology (Danvers, MA, United States), and Merck Millipore (Burlington, MA, United States).

**Table 1.** Antibodies used in this study.

+: Same as above.

**Table 1.** *Cont.*

### *2.3. In Vivo Test*

#### 2.3.1. Animal Source and Care

Eight to ten-week-old C57BL/6 male mice were purchased from BioLASCO (Taipei, Taiwan) and kept in controlled environmental conditions (22 ± 2 ◦C, 55% ± 10% humidity, 12/12 h light/dark cycle). The animals were fed a commercial diet and water ad libitum. Mice experimental protocols were used according to the "Guide for the Care and Use of Laboratory Animals" of National Dong-Hwa University, approved by the National Dong-Hwa University Animal Ethics Committee (approval number 001/2016).

#### 2.3.2. Inflammatory Bowel Disease Model Induction and HCD Treatment

The in vivo IBD model was induced by the injection of AOM and DSS, following a previous study with slight modification [21]. At day 0, mice were weighed and i.p. injected with 10 mg/kg B. wt. of AOM. The next day, the mice were freely supplied with 2% DSS solution for an additional seven days, and regular water for a further seven days. This induction cycle was repeated once. The induction of IBD was verified by checking the histological change of the colon after mice were sacrificed at day 35.

Once IBD induction was confirmed, 15 mg/kg B. wt. of 5-FU, as well as 1.6 and 6.4 mg/kg B. wt. of HCD were i.p. injected once every three days until day 65. Mice were sacrificed by CO2 anesthesia, and their colons were collected for slicing in the literature [16].

#### *2.4. In Vitro Test*

#### 2.4.1. Cell Culture

Human colorectal carcinoma cell lines Caco-2 and HT-29 were obtained from the American Type Culture Collection (ATCC, Manassas, MA, United States). Caco-2 and HT-29 cells were cultured with Dulbecco's modified Eagle medium (DMEM) or RPMI-1640, with 20% fetal bovine serum (FBS) and 1% penicillin/streptomycin (PS) supplementation, respectively. The environmental conditions were 37 ◦C and 5% CO2, and the cultured medium was renewed once every two days. Once cells reached 80% confluence, cells were detached by 0.25% trypsin/EDTA for further experiment. All experiments were carried out within 20 passes, with concern for uniformity and reproducibility.

#### 2.4.2. Cytotoxicity Assay

The cytotoxic effect of HCD was measured by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide; MDBio Inc., Taipei City, Taiwan) assay, as previously described in the literature [16]. Briefly, 7 × 103 cells per well of two cells were inoculated in 96-well plates before incubating with 0.5, 2.0, 5.0, and 10.0 μM of HCD and 1, 10, 50, and 100 μM of 5-FU (a positive control) for 36 and 48 h, respectively. The optical density (OD) at 570 nm was measured after incubation with MTT solution for 4 h, and was solubilized in DMSO. Cytotoxicity was presented by cell viability, which was the ratio of OD570 between treatment and untreated control (0 μM).

#### 2.4.3. Cell Cycle Analysis

The protocol of cell cycle analysis followed a previous study, with slight modification [16]. In brief, 7 × 104 cells per well of Caco-2 cells were seeded into 12-well plates. Cells were incubated with 0.5, 2.0, and 5.0 μM of HCD for 36 and 48 h, respectively. Treated cells were fixed with 70% freeze ethanol and stained with propidium iodide (PI) at 37 ◦C for 1 h. The fluorescent intensity of PI within cells was detected by a CytomicsTM FC 500 flow cytometer (Beckman-Coulter, Brea, CA, United States). Data from 104 cells in each sample were collected, and the different cell cycles were analyzed.

#### 2.4.4. Western Blotting

A total of 2.5 × 105 cells/well of Caco-2 cells were seeded into a six-well plate and incubated until 80% confluence. Cells were treated with 0.5, 1.0, and 2.0 μM of HCD for 24 and 36 h, respectively. After incubation, cells were homogenized, and the desired protein levels were analyzed according to the protocol described in a previous study [16]. The chemiluminometric intensity of each protein was normalized with GAPDH's chemiluminometric intensity. The protein level change was represented by the ratio of normalized chemiluminometric intensity between treated and untreated groups.

#### *2.5. Statistical Analysis*

Data were expressed as mean ± SD from at least three independent experiments. The results were analyzed by one-way analysis of variance (ANOVA) with the Dunnett test. The significant difference (*p* < 0.05) was labelled "\*" on the histogram produced by GraphPad Prism Ver 7.0 (GraphPad Software, La Jolla, CA, United States). The IC50 of the dose-dependent cytotoxicity was determined using non-linear regression embedded in GraphPad Prism, and the chosen model was the highest R2 value.

#### **3. Results**

#### *3.1. Histological Change of Intestine Tissue after AOM*/*DSS Induction and HCD Treatment*

To generate the IBD mouse model, mice were chemically induced by AOM/DSS. After AOM/DSS induction, enlarged lymph nodes, lymphocyte infiltration, irregular and shorter villi, and thicker muscle mucous and muscle layers were observed in the intestines of mice, which consequently confirmed that mice were successfully induced with IBD after the AOM/DSS given (Figures 1B and 2B), compared to the control without induction (Figures 1A and 2A). In the next experiment, IBD-induced mice were employed to evaluate the amelioration efficacy of 5-FU and HCD on the histopathological signs of IBD. The tissue section showed that the lymphocytes were less or not infiltrated into the lamina propria layer after treatment with 5-FU and HCD (Figure 1C–E). The arranged villi in neat rows were found in an induced group as a positive control (AOM/DSS alone), and this feature was not observed in the 5-FU and HCD-treated groups. Additionally, the lymph nodes were reduced after treating with 5-FU and HCD (Figure 2C–E). These histological changes elicited that HCD could reduce IBD symptoms. The next experiments were performed to evaluate the efficacy of HCD on colorectal cancer cells.

#### *3.2. Cytotoxicity E*ff*ects of HCD and 5-Fluorouracil on Colorectal Cancer Cells*

To check cytotoxicity of HCD against colorectal cancer cells, cells were treated with various concentrations of HCD and 5-FU (conventional chemotherapeutic agent, as a positive control), respectively. When compared to the untreated control (0 μM), cell viability in HCD-treated groups was significantly decreased (Figure 3A). The IC50 values of HCD in Caco-2 cells were 4.10 μM (36 h) and 2.32 μM (48 h), which were lower in 5-FU (100 μM for 36 h; 66.79 μM for 48 h) (Figure 3B). To further validate the potential of HCD for colon cancer treatment, another colon cancer cell, HT-29, was treated with various concentrations of HCD. The results showed that a dose-dependent decrease of cell viability was also observed in HCD-treated HT-29 cells. The IC50 values of HCD against HT-29 were 10.18 μM (36 h) and 1.39 μM (48 h), and were higher than those of Caco-2 (Figure 3C). According to these results, we confirmed that the cytotoxicity of HCD in colorectal cancer cells (Caco-2 and HT-29) had a higher potential than 5-FU. Therefore, the subsequent experiments were focused on the investigation of underlying mechanisms in HCD against Caco-2 cells.

**Figure 1.** Histological appearances of the longitudinal section in the intestine of mice. Mice treated with (**A**) control and (**B**) azoxymethane (AOM)/dextran sodium sulfate (DSS) induction, as well as AOM/DSS induction followed by (**C**) 15 mg/kg B.wt of 5-fluorouracil (5-FU), (**D**) 1.6 mg/kg B.wt, and (**E**) 6.4 mg/kg B.wt of 16-hydroxycleroda-3,13-dien-15,16-olide (HCD) treatment (*n* = 5 in each group), were sacrificed, and the longitudinal section of tissues was stained with hematoxylin and eosin. The yellow asterisk and triangles indicate lymph node and lymphocyte infiltration, respectively, in the intestine. Magnification = ×100.

**Figure 2.** Histological changes of intestinal lymph nodes in HCD-treated mice with inflammatory bowel disease (IBD). The intestinal lymph nodes of (**A**) the control, (**B**) mice with AOM/DSS induction, and mice with AOM/DSS induction followed by (**C**) 15 mg/kg B.wt of 5-FU, (**D**) 1.6 mg/kg B.wt, and (**E**) 6.4 mg/kg B.wt of HCD treatment (*n* = 5 in each group) were stained and photographed. The yellow asterisk indicates the lymph node in the intestine. Magnification = ×40.

**Figure 3.** Cell viability of Caco-2 and HT-29 cells after HCD and 5-FU treatments. HCD and 5-FU were used to treat with (**A**,**B**) Caco-2 and (**C**,**D**) HT-29 cells for 36 and 48 h, respectively. The cell viability was determined by MTT assay. In addition, the cell viability was calculated according the comparison of control group (0 μM). Data (*n* = 3) were represented as mean ± SD. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, as compared with the untreated control (0 μM).

#### *3.3. Characteristics of HCD-Induced Cell Death*

To identify features of HCD-induced cell death, Caco-2 cells were treated with various concentrations of HCD, and intracellular DNA content was checked using flow cytometry coupling with PI-staining. After 36 h and 48 h of HCD treatment, the sub-G1 and G0/G1 cell cycle exhibited significant change in a dose-dependent fashion (Figure 4A), which could be caused by increasing the sub-G1 ratio, referred to as the apoptotic population. Interestingly, the significant decrease of the G2/M phase was found only at 5 μM HCD (Figure 4B). Concurrently, analyzing apoptotic markers, the increase of cleaved caspase-3, -8, -9, and PARP specified that apoptotic cell death appeared at 24 h and 36 h of treatments, respectively (Figure 5). These results clearly illustrate that HCD-caused cell death in Caco-2 cells was dominate in apoptosis. Moreover, by observation of the dynamics of apoptotic markers, intrinsic apoptotic inducer caspase-3 expression was enhanced at the first 24 h, and then cleaved at the following 12 h (Figure 5A). Conversely, Bcl-2, the apoptotic inhibitor, had no significant change until 36 h of treatment (Figure 5B). Additionally, the extrinsic apoptotic markers, cleaved caspase-8 and -9, also had significantly higher expression at either 24 h or 36 h of treatment (Figure 5). The data confirmed that both intrinsic and extrinsic apoptotic signaling pathways were involved in HCD-induced apoptosis, and intrinsic apoptosis might be prior to the extrinsic event. The following experiments would be carried out to determine whether the dynamic change of growth signaling pathway is affected by HCD.

**Figure 4.** Cell cycle change after HCD treatment. Caco-2 cells were stained by propidium iodide after (**A**) 36 h and (**B**) 48 h of HCD treatment, and further analyzed by fluorescent intensity by flow cytometry. Data (*n* = 3) were represented as mean ± SD. \* *p* < 0.05; \*\* *p* < 0.01; \*\*\* *p* < 0.001, as compared with the untreated control (0 μM).

**Figure 5.** Dynamic change of apoptotic markers after HCD treatment. The protein levels of PARP, caspase-3, caspase-8, and caspase-9 were measured after (**A**) 24 h and (**B**) 36 h of HCD treatment. The protein levels were represented as the ratio of GAPDH-normalized chemiluminometric intensity between the untreated control (0 μM) and treatment. Data (*n* = 3) were represented as mean ± SD. \* *p* < 0.05 as compared with the untreated control (0 μM).

#### *3.4. Growth Signal Reduced by HCD Triggered Caco-2 Cell Apoptosis*

Notably, the intrinsic apoptotic pathway is regulated by the balance of growth signal and anti-growth signal. The subsequent experiments were applied to measure the signals of PI3K/Akt for growth, p53/p21 for anti-growth, and cyclin D1/PCNA for cell division after HCD treatments. Western blot data indicated that the protein levels of Akt and cyclin D1 were significantly decreased in 2 μM of HCD treatment after the first 24 h, and a significant decrease of the cyclin D1 level at 1 μM of HCD treatment was observed (Figure 6A). Subsequently, the protein levels of p53 and p21 were up-regulated and PCNA was down-regulated at 36 h (Figure 6B). This result hinted that HCD-induced apoptosis could be the reducing result of growth signaling via Akt mediation. The downregulation of growth signaling caused the elevation of p53/p21 protein expression, and consequently turned down cyclin D1 expression, which was the key for overcoming the G1/S checkpoint. However, upstream of Akt, the protein levels of PI3K were not associated with the mediation of Akt (Figure 6), which meant

that Akt might be reduced by other signaling pathways. The next section was performed to test the characteristics of the inflammatory pathway within HCD-induced apoptosis.

**Figure 6.** Dynamics of cell growth related signaling pathway after HCD treatment. Caco-2 cells were treated with HCD and the changes in PI3K, Akt, p53, p21, cyclin D1, and PCNA were measured at (**A**) 24 h and (**B**) 36 h. The protein levels were represented as the ratio of GAPDH-normalized chemiluminometric intensity between the untreated control (0 μM) and treatment. Data (*n* = 3) were represented as mean ± SD. \* *p* < 0.05 as compared with the untreated control (0 μM).

#### *3.5. Inflammation-Suppressing E*ff*ect of HCD in Caco-2 Cells*

From the literature, inflammation has been proven as a promoting cause of colorectal cancer [22], and inflammatory signaling could crosstalk with Wnt/β-catenin, as well as being involved in colorectal cancer growth [23]. Thus, the subsequent experiment examined the regulation of inflammatory-related proteins, including COX-2, NF-κB, and β-catenin. At 24 h of HCD treatment, the p50 subunit of NF-κB showed a significantly decreasing manner (Figure 7A). The decrease of p50 was diminished, whereas β-catenin was up-regulated at 36 h (Figure 7B). This result refutes that NF-κB and β-catenin are involved in HCD-mediated apoptosis; however, the underlying mechanisms were not fully interpreted. Of note, inflammatory signaling was proven by the significant characteristics of IBD pathological progression, and then the subsequent experiment will be employed to evaluate whether oral gavage HCD may ameliorate IBD symptom in an AOM/DSS- induced mouse model by the mediation of inflammatory signals.

**Figure 7.** Inflammation-related protein changes after HCD treatments. NF-κB, β-catenin, and Cox-2 were determined by Western blotting at (**A**) 24 h and (**B**) 36 h HCD-treated Caco-2 cells. The chemiluminescent intensity of each protein was normalized with GAPDH and represented as the protein levels. Data (*n* = 3) were represented as mean ± SD. \* *p* < 0.05 as compared with the untreated control (0 μM).

#### **4. Discussion**

In chemical-induced IBD and colitis-associated cancer research, this study broadened the application of HCD in medical use, and could provide a new approach for IBD treatments. These experimental results conferred the anticancer effect of HCD against colon cancer, which led to intrinsic and extrinsic regulation for apoptotic cell death by down-regulating Akt-mediated growth signaling. Moreover, the anti-inflammation fashion of HCD in colon cancer might be one reason for the down-regulation of Akt, and is postulated for the IBD curing effect in vivo.

Usually, the conventional therapies for IBD can be grouped into the two following approaches: anti-inflammation, *e.g.*, corticosteroids, mesalazine, and cyclosporine; and anti-microbial, including ornidazole and rifaximin [24]. Ordinarily, these drugs are useful for treating mild to moderate IBD. However, side effects, such as drug resistance to antibiotics and opportunistic infection, are still of concern [25]. Likewise, about one-third of chronic IBD patients have failed responses to corticosteroid medication, which are valid for acute IBD [25]. Hence, natural components are potentially considered to be the new strategies or approaches for curing IBD. Five types of phenylpropanoids and four types of lignan glycosides—phytochemicals originated from a warm-season perennial legume, *Lespedeza cuneata*—were synthesized, and these compounds could ameliorate UC [26]. Curcumin, the primary active compound of turmeric, alleviates CD and UC by inhibiting NF-κB activity [27]. Macrophage infiltration into the intestines could also be impaired by α-eleostearic acid isolated from *Momordica charantia* [28]. After AOM/DSS induction, polypus and enlargement lymph nodes were found in the colon by histopathological examinations in our study (data not shown). Additionally, the observations of irregular villi arrangement and lymphocyte infiltration into the lamina propia layer in this study are typical characteristics of IBD. The IBD mice treated with HCD had neat rows of villi and lymph nodes that were not enlarged or infiltrated into lamina propia layer, which indicates that HCD could relieve the symptoms of IBD.

In indigenous medication, *P. longifolia* is an antipyretic drug used in past decades [12]. Under modern chemical and medical research, various antimicrobial and anti-inflammatory compounds are purified and identified, such as 16α-hydroxy-cleroda-3,13(14)-Z-diene-15,16-olide, (3S,4R)-3,4,5-trihydroxy pentanoic acid-1,4-lactone and 16-Oxo-cleroda-3,13E-dien-15-oic acid [12]. HCD has been demonstrated to alleviate lipopolysaccharide-induced microglia inflammation via

reducing iNOS, COX-2, and NF-κB gene expression [13]. In addition, HCD repressed COX-2 protein expression in Caco-2 and further ameliorated inflammation in AOM/DSS-induced mice. These results imply that HCD represents a novel and potential clinical approach for IBD treatment and CRC chemotherapy.

In our previous study, HCD was demonstrated to be a non-toxic agent to normal cells [16]. When compared to 5-FU, the cytotoxicity of HCD against colorectal cancer was higher than 5-FU (4.10 vs. 100 μM in IC50 at 36 h treatment), suggesting that HCD had higher efficacy and more potent when applied to colon cancer treatments. In the cell cycle analysis of Caco-2 cells treated with HCD, the ratio of the sub-G1 phase was significantly increased, and this increase was associated with an increase of HCD concentrations. This result was accompanied by the analysis of pro-apoptotic markers, such as PARP; caspase-3, -8, and -9; and Bcl-2. During observations from 24 h to 36 h of HCD treatment, the protein levels of pro-apoptotic molecules changed, and these signaling transductions could be precisely determined by caspase-8, caspase-3, Bcl-2, and PARP. Again, the caspase-3 expression level was increased in the first 24 h, which also indicated that intrinsic apoptotic pathways were activated at this time. These results imply that both intrinsic and extrinsic apoptotic signaling pathways were simulanteously activated in Caco-2 cells. This was the first evidence that HCD could induce the apoptosis of cancer cells.

As we know, extrinsic and intrinsic apoptotic signaling pathways exert themselves with different signaling molecules [29]. In general, an extrinsic apoptotic signaling pathway is started from the activation of death receptor (TRAIL receptor or TNF receptor) and terminated at pro-caspase-3 cleavage via caspase-8 activation [29,30]. Different from extrinsic apoptosis, intrinsic apoptotic signaling is mediated by Bcl-2 to cause the loss of mitochondrial membrane potential or growth signaling depletion, which leads to cytochrome c release, pro-caspase-9 cleavage, and consequently, caspase-3 activation [29,31,32]. Therefore, by observing the altered levels of cleavage caspase-8, caspase-9, and Bcl-2, the type of chemical-induced apoptosis could be putatively addressed. Previous studies of HCD-induced cancer cell death were focused on autophagic cell death and intrinsic apoptosis [15,16,33,34]. To the best of our knowledge, this study is the first to show the involvement of HCD-induced extrinsic apoptosis in colorectal cancer. Moreover, the underlined targets of HCD in extrinsic apoptosis still need to be further explored, because this work only focused on the alteration of caspase-8 proteins.

HCD-mediated intrinsic apoptosis was found by down-regulating β-catenin/NF-κB/Akt and activating p53/p21 expression. Previously, HCD had potentiated apoptosis via blocking the PI3K/Akt signaling pathway, promoting Aurora B degradation, and modifying histone-modifying enzymes in leukemia cells [33,34]. Remarkably, the cytotoxicity (cell death) of oral squamous cell carcinoma (OSCC) and glioma cells were also demonstrated in the treatment of HCD through an autophagic manner via Western blotting analysis, without an increase in sub-G1 [15,16]. Moreover, HCD activated the autophagy in lung cancer (A549) via reducing the protein level of mTOR, PI3K/p85, Akt, and Beclin 1, and suppressed apoptosis by lessening cleaved-PARP formation (Chiu et al., 2019, unpublished data). Thereby, one possibility for cell death is that this difference in apoptosis or autophagy was regardless of different types of cancer cells (adenoma, carcinoma, glioma, or neuroblastoma) and p53 (wild or mutant type). The cell death features of HCD induced in Caco-2 cells and other cancer cells imply that the critical point of the apoptosis/autophagy switch needs to be clarified.

The known roles of p53 are in cell cycle regulation, apoptosis induction, DNA repair activation, and aerobic respiratory improvement [35]. When measuring the apoptosis-related protein levels, the p53 protein level showed an increase at 36 h after HCD treatment. In intestine and colon tissues under IBD, p53 was overexpressed by TNF-α induction, and subsequently triggered cell apoptosis [36]. Interestingly, the induction of TNF-α was only observed in mutant p53, but not in wild-type p53 [37]. Over 50% of colitis-induced colorectal cancer and colon neoplasia could be found with the TP53 mutation. This mutation is believed to be the first step of colitis-associated carcinogenesis when compared with sporadic CRC [38]. Of note, p53 in Caco-2 is an aberrant type [39]. HCD caused the

alteration of p53 protein expression in CRC cells, which indicated that HCD might affect p53 protein levels in IBD tissues. Therefore, the effects of HCD on intestinal epithelial cells with wild-type p53 might differ from similar Caco-2 cells.

In addition, as an apoptosis-inducing feature, HCD also possesses inhibiting activity for Wnt/β-catenin in an anti-inflammation manner. Wnt/β-catenin dysregulation has been reported as a key factor for CRC initiation [40]. The Wnt/β-catenin signaling pathway acts as a central regulator in intestine homeostasis and epithelial stem cell proliferation. The Wnt ligand binds to the Frizzled/LRP receptor, and activates a signal cascade to subsequently result in the stabilization of β-catenin. Stable β-catenin can translocate into the nucleus and initiate gene expression of MYC and its downstream target, CCND1 [41]. Impaired activation of the Wnt/β-catenin signaling pathway could cause uncontrolled cell proliferation and finally, elicit colon cells carcinogenesis. Therefore, the Wnt/β-catenin signaling pathway would become the target of CRC prevention, prognosis, and diagnosis. Numerous studies have sought new compounds or herbal medicine for treating CRC. Fermented culture broth of *Antrodia camphorate*, hydnocarpin (a natural lignan), and bark extract of *Mesua ferrea* have been shown to inhibit activity of Wnt/β-catenin in colon cancer cells [42–44]. In this study, the reduction of β-catenin levels after HCD treatment showed the inhibited manner of the Wnt/β-catenin signaling pathway, which indicated a possibility of reducing colon cancer initiation.

#### **5. Conclusions**

This study illustrated anti-colorectal cancer activity from HCD by modifying intrinsic growth signaling and inflammatory modulators, which subsequently triggered both intrinsic and extrinsic signals to induce cell apoptosis. Furthermore, the inflammatory symptoms of AOM/DSS-induced enteritis in the in vivo mouse model were also ameliorated by HCD treatments. This is the first evidence showing the medicinal efficacy of HCD on IBD mice and colon cancer, suggesting that HCD could provide an alternative and complementary regimen for anti-colon cancer and IBD treatments.

**Author Contributions:** Conceptualization, C.-F.W. and Y.-C.C.; methodology, Y.-S.F., S.-R.L., S.-I.L., and C.-F.W.; software, M.-J.T. and F.-J.T.; validation, S.-R.L., F.-J.T., and M.W.; formal analysis, S.-I.L.; investigation, J.-H.Z.; resources, C.-F.W.; data curation, M.-J.T. and Y.-C.C.; writing—original draft preparation, J.-H.Z. and S.-R.L.; writing—review and editing, C.-F.W.; project administration, C.-F.W.; funding acquisition, C.-F.W.

**Funding:** This study was supported by Ministry of Science and Technology (Grant number 104-2320-B-259-001-MY3)

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

#### **References**

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Review*

### **Recent Trends in the Application of Chromatographic Techniques in the Analysis of Luteolin and Its Derivatives**

#### **Aleksandra Maria Juszczak 1 , Marijana Zovko-Konˇci´c 2 and Michał Tomczyk 1,\***

Received: 1 October 2019; Accepted: 8 November 2019; Published: 12 November 2019

**Abstract:** Luteolin is a flavonoid often found in various medicinal plants that exhibits multiple biological effects such as antioxidant, anti-inflammatory and immunomodulatory activity. Commercially available medicinal plants and their preparations containing luteolin are often used in the treatment of hypertension, inflammatory diseases, and even cancer. However, to establish the quality of such preparations, appropriate analytical methods should be used. Therefore, the present paper provides the first comprehensive review of the current analytical methods that were developed and validated for the quantitative determination of luteolin and its *C*- and *O*-derivatives including orientin, isoorientin, luteolin 7-*O*-glucoside and others. It provides a systematic overview of chromatographic analytical techniques including thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC) and counter-current chromatography (CCC), as well as the conditions used in the determination of luteolin and its derivatives in plant material.

**Keywords:** luteolin; hyphenated techniques; chromatography

#### **1. Introduction**

Luteolin (Figure 1) is a yellow dye commonly found in fresh plants. It is a flavonoid of the flavone type that is distributed widely throughout the plant kingdom. Similar to other derivatives of 2-phenylbenzo-γ-pyrone, its basic skeleton has a characteristic C6-C3-C6 system, containing two benzene rings and a bridge with a C2-C3 double carbon bond and an attached oxygen atom [1–4]. Structure-activity studies have demonstrated that the pharmacological effects of luteolin and other flavonoids are strongly related to the presence of hydroxyl groups at the C5, C7, C3' and C4' carbons as well as to the presence of the double bond in the C2-C3 position [3,5]. The presence of the -OH group at position C3' distinguishes luteolin from apigenin, and the lack of this group at the C3 carbon is an element that places luteolin in the flavone group [6].

**Figure 1.** Chemical structure of luteolin.

Luteolin exhibits multiple biological effects such as antioxidant, anti-inflammatory and immunomodulatory activity. Plants rich in luteolin are often used in traditional medicine for treatment of various diseases such as hypertension, inflammatory disorders, and even cancer [7].

Because luteolin bears four hydroxyl groups (at the C5, C7, C3' and C4' positions), many derivatives of luteolin can be created. Various types of functional groups and/or sugar molecules can be attached to those positions, creating a huge number of different but structurally similar molecules. The most common are methyl derivatives, as well as *C*- and -*O*-glycosides [8,9].

Orientin, an 8-*C*-glucoside derivative of luteolin, displays an array of health-related biological properties, such as antioxidant, anti-ageing, antiviral, antibacterial, anti-inflammatory, vasodilatation, cardioprotective, radiation protective, neuroprotective, antidepressant-like, anti-adipogenesis, and antinociceptive effects. It may be found in different medicinal plants such as *Ocimum sanctum* (holy basil), *Phyllostachys nigra* (bamboo leaves), *Passiflora* sp. (passion flower), *Linum usitatissimum* (flax), *Euterpe oleracea* (Acai palm) and many others [10]. Another luteolin derivative, isoorientin (luteolin-6-*C*-glucoside) acts as an antioxidant, photoprotective [11], skin lightening [12], hepatoprotective [13] and anti-inflammatory agent [14]. *O*-glucosides of luteolin also display biological activities. For example, luteolin 7-*O*-glucoside alleviates skin lesions in murine models of atopic dermatitis [15] and protects cells against apoptosis induced by hypoxia/reoxygenation [16].

An increasing number of herbal preparations on the market contain luteolin and its derivatives, either as single-ingredient products or in mixtures with other phytochemicals, e.g., in form of medicinal plants extracts. To establish the quality of such products, it is important to use appropriate analytical methods. However, there is a lack of quality reviews of the available methods for quantification of luteolin derivatives, and the information on their comparison is lacking. Therefore, the aim of this article is to systematize knowledge and information in the field of chromatographic analytical techniques used for quantification of luteolin and its derivatives. The presented review is the first description of this type and provides a systematic overview of chromatographic analytical techniques including thin layer chromatography (TLC), high performance thin layer chromatography (HPTLC), liquid chromatography (LC), high performance liquid chromatography (HPLC), gas chromatography (GC) and counter-current chromatography (CCC), as well as the conditions used to assess luteolin and its derivatives.

#### **2. Chromatographic Techniques for the Analysis of Luteolin Derivatives**

Chromatography occupies a leading position among other instrumental methods in the analysis of chemical compounds. As a physicochemical method of separation and analysis of mixtures of chemical compounds, it allows detection and identification as well as quantitative determination of the test substance with high accuracy. The coupling of chromatography with other methods of analysis contributes to a more accurate detection and expansion of analytical capabilities, especially for complex mixtures of organic compounds [2,17,18].

Chromatographic techniques are based on the interaction of the mixture components with the mobile and stationary phases of the chromatographic system. This results in the division of the mixture components between the two phases. In addition, the interaction of the mobile and stationary phases is also important in the separation process [17,18]. According to the aggregation state of the mobile phase, chromatographic techniques are divided into gas, liquid and supercritical chromatography. Another criterion for classification of chromatographic techniques is the type of stationary phase. If the stationary phase is a liquid, the chromatography technique is referred to as partition chromatography. In the case of a solid, the technique is referred to as adsorption chromatography. Another example of the classification of chromatographic methods is their division depending on the chromatographic processing method. This classification allows distinguishing column chromatography and planar techniques, which include TLC and paper chromatography [17,19]. Many different chromatographic techniques are used in the analysis of luteolin derivatives. These include TLC, HPTLC, LC, HPLC, GC and CCC [19].

#### *2.1. Thin Layer Chromatography in the Analysis of Luteolin Derivatives*

Thin layer chromatography (TLC) is a rather simple but relatively popular method used in the analysis of flavonoids since 1960 [20]. It is a variation of LC that is carried out on a plane and is therefore referred to as planar chromatography. Despite the dynamic development of other chromatography techniques, TLC has not lost its importance in phytochemical analysis [18]. Its values are still recognized as is the basic tool in the qualitative analysis of natural products, and as such, it is still recommended by most modern Pharmacopoeias. In addition, the TLC technique is currently being improved, and the scope of its application is widening, while the results are becoming comparable to those obtained by GC or HPLC [20–22]. The stages of chromatographic analysis consist of placing the sample on the stationary phase and developing the chromatogram, followed by its visualization. In the final step, qualitative and/or quantitative determinations of the tested components are made [18,22,23]. General guidelines for flavonoid separation on TLC plates are presented in Table 1.

**Table 1.** Recommended combinations of solvents / adsorbents for identification. of different flavonoid types by thin layer chromatography (TLC).

Abbreviations: AcOH, acetic acid; BuOH, butanol; CHCl3, chloroform; EtOAc, ethyl acetate; FA, formic acid; MeOH, methanol; MEK, methyl ethyl ketone; Py, pyridine; To, toluene. a The mobile phase is thoroughly mixed in the separating funnel and the upper phase is used. b The mobile phase is thoroughly mixed in the separating funnel and the excess water is discarded.

The main advantage of TLC is that it is a relatively simple and inexpensive technique that allows for rapid qualitative and quantitative analysis of the tested compounds. Samples analyzed with this method usually do not require pre-treatment, such as purification or concentration. In addition, several dozens of samples can be analyzed simultaneously on one plate. A large amount of the diluted sample can be applied to the stationary phase because the solvent evaporates during the application to the plate. Furthermore, due to the evaporation of the solvent phase after the development of the chromatogram, the detection method does not depend on the type of mobile phase used for separation. In TLC, it is possible not only to compare the analyzed components with the standards, but also to differentiate between substances bearing specific functional groups using the appropriate reagents for detection [18].

Thin layer chromatography and column chromatography (CC) are interchangeable techniques that may be combined, which significantly reduces costs and analysis time [22]. To achieve this, the same adsorbents are ideally used for both TLC and CC [18,22,24]. Nevertheless, other solvent systems may also be used. Elution in CC may be carried out in one mobile phase, or its composition may be changed during chromatography (mobile phase gradient), thereby increasing the elution force. In this case, the TLC mobile phase should be changed accordingly [18,24].

The type and quality of the stationary phase greatly affect the separation of mixture components. Thus, the selection of an appropriate adsorbent is very important. However, most TLC analyses are carried out in a normal phase system where hydrophilic (polar) adsorbents are used [18,23,24]. Reversed phase systems with lipophilic (non-polar) stationary phases are common and have little significance in the analysis of flavonoids [20]. Currently, the most commonly used stationary phase for the analysis of flavonoids is silica gel [20]. However, the use polyamide coated chromatography plates, in both normal and reversed phase systems, is not uncommon [24].

Detection of flavonoids on TLC plates is most often conducted under ultraviolet (UV) light at wavelengths of 254 or 366 nm. Luteolin derivatives also display fluorescence, which can be enhanced using the appropriate derivatization reagents, e.g., with the so-called NP/PEG reagent. The most frequent procedure consists of spraying the plate with 1% methanolic diphenylboric acid-β-ethylamino ester (natural product reagents, NP), followed by 5% ethanolic polyethylene glycol 4000 (PEG) solution [20]. A densitometer may also be used for the qualitative analysis of the substance. The analysis is performed by comparing the retardation factor (Rf) and absorption spectrum of the test substance and the standard. Analytes can also be identified by extracting the separated substances from the plate. Then, the analysis is carried out using Fourier transform infrared mass spectrometry (MS), UV spectrometry, Raman spectrometry or other techniques [23]. Even though TLC separation of luteolin derivatives (Table 2) can be performed in different types of stationary phases such as a polyamide phase [26,28], it is most frequently performed on silica gel plates that are often coated with a fluorescent indicator [27,29] (F254 plates) for preliminary detection. Such an approach has been used in case of analysis of luteolin 3'-*O*-glucoside, luteolin 6,8-*C*-dihexoside and luteolin 7-*O-*rutinoside in *Phlomis* sp. [27]. However, subsequent analysis with NP/PEG is the standard procedure for TLC analysis of luteolin [26–29], and it is almost always performed regardless of additional types of detection such as detection of flavonoids in *Ligustrum vulgare* with aniline phthalate [26]. Typically, the mobile phase for luteolin derivative separation consists of a mixture of aprotic organic solvents such as ethyl acetate (EtOAc) [26,28] or acetone (Ace) [29] and H2O with a significant amount of formic (FA) and/or acetic acid (AcOH) to avoid tailing of the separated zones [26–29].

Thin layer chromatography is often used as a complementary method to other chromatographic techniques. For example, analysis of the butanol (BuOH) fraction of the methanol (MeOH) extract from the leaves of the common privet (*Ligustrum vulgare*) conducted by Muˇcaji et al. [26] allowed isolation of two luteolin derivatives from the plant. TLC was carried out, among other techniques, on polyamide plates, and the optimal mobile phase was found. TLC was used for analysis of individual fractions obtained in column chromatography with or without acid hydrolysis of compounds. MS detection and nuclear magnetic resonance (NMR) spectra were also used [26]. Furthermore, TLC was used together with high performance liquid chromatography combined with mass spectrometry and pulsed amperometric detection (HPLC-PAD-MS) for analysis of luteolin and other phenolic compounds in *Leontopodium alpinum*. In addition to NP/PEG, UV, infrared (IR) and NMR analyses were used for identification of these compounds [29].

Compared to other chromatographic techniques, HPTLC results in reduced time and costs of analysis and provides much greater efficiency of separation. It is suitable even for the analysis of crude extracts similar to TLC, and a relatively small amount of solvent is used to analyze several samples, making this method environmentally friendly [60]. In the analysis of luteolin derivatives (Table 3), HPTLC silica gel 60 is almost exclusively used as the stationary phase [45,47,49,51], while HPTLC NH2 plates are rarely used, e.g., for separation of flavonoids in some Lamiaceae species such as *Mentha piperita* [53] and *Thymus* sp. [55]. In addition to NP/PEG (e.g., [52,57]), other detection systems may be employed for visualization of luteolin derivatives, such as bis-diazotized sulfanilamide [53] or aqueous solutions of Al3+ ions for flavonoids in *M. piperita* [53], honey [49] or *Thymus* sp. [55]. Similar to TLC, mixtures of organic solvents, H2O, and FA are most often used as the mobile phase [44,45,47–49,51,52].

High performance thin layer chromatography may also be used as a complimentary method to other chromatographic techniques. Chelyn et al. [44] used the HPTLC technique in the analysis of the ethanol (EtOH) extract of *Clinacanthus nutans* leaves. The analysis revealed the presence of, among others, luteolin 8-*C*-glucoside (orientin) and luteolin 6-*C*-glucoside (isoorientin) in the raw material. Their detection was conducted by comparing Rf coefficients using derivatization reagents and 366 nm UV light. In this work, the characteristic fluorescent bands after derivatization provided important clues for the identification of the major flavone present in the samples, while high performance liquid chromatography combined with ultraviolet spectrometry or a diode array detector (HPLC-UV/DAD) technique was employed for the simultaneous detection and quantification of these compounds [44].

**Table 2.** Thin layer chromatography in the analysis of luteolin derivatives.

Abbreviations:FBS, Fastethanolic polyethyleneglycol4000;To,toluene;UV,ultravioletspectroscopy;MS,massspectrometry;NMR,nuclearmagneticresonance;IR,infrared.

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**Table3.**Highperformancethinlayerchromatographyintheanalysisofluteolinderivatives.

**Table 3.** *Cont.* Abbreviations: Ace, acetone; AcOH, acetic acid; AlCl3, aluminium trichloride; Al2(SO4)3, aluminium sulfate; BuOH, butanol; CHCl3, chloroform; DCM, dichloromethane; DI, 1,4-dioxane; DIPE, diisopropyl ether; ESI-MS/MS, electrospray ionization combined with tandem mass spectrometry; EtFo, ethyl formate; EtOAc, ethyl acetate; Et, ether; FA, formic acid; H2SO4, sulfuric acid; Hx, hexane; MeOH, methanol; MEK, methyl ethyl ketone; *n*Hx, *n*-hexane; NP, 1% methanolic diphenylboric acid-β-ethylamino ester - natural product reagents; neurotransmitters; PEG, 5% ethanolic polyethylene glycol 4000; THF, tetrahydrofuran; To, toluene; UV-VIS, ultraviolet-visible spectroscopy; ESI-MS, electrospray ionization combined with mass spectrometry.

#### *2.2. High Performance Liquid Chromatography in the Analysis of Luteolin Derivatives*

Among the many chromatographic methods, adsorptive chromatography, in which the mobile phase is liquid and the stationary phase is solid, is of the greatest practical importance. For example, this method has a much wider application than GC because it allows the analysis of substances in the form of liquids and soluble solids. Furthermore, it is also suitable for analysis of thermolabile substances because it is usually performed at low temperatures, and such samples are not degraded [61]. Due to the long analysis time, high mobile phase consumption and low efficiency, traditional CC is currently used mainly for preparative purposes. However, improved methods, such as HPLC and especially liquid chromatography combined with mass spectrometry (LC-MS), are increasingly used for analysis of natural compounds including luteolin derivatives [62].

High performance liquid chromatography has been performed since 1960. It is a dynamically developing method with a wide range of uses and has been proven to be very useful in phytochemical analysis. The principle of operation consists of pumping the mobile phase from the tank (or tanks) through the stationary phase-filled column. Eluents are previously filtered and degassed. Some systems are additionally equipped with thermostats that regulate the temperature of the column. If the chromatographic system is properly selected and applied, then the individual components are separated and detected. Strengthened signals are transmitted to the computer, where the obtained data are registered and properly processed [19]. The separation is based on competition of the molecules of the eluent and the substance being analyzed for the space on the adsorbent surface in the column [63,64].

When choosing a column, one should be guided by the size of the sample, time of analysis and expected effect of the separation. Although columns with different diameters are available, those with a diameter of 4.6 mm are by far the most common. However, due to better detection of the components separated in smaller diameter columns, these columns are increasingly being used for separation and analysis [19,63,65]. In addition, improved separation can be achieved using a column filled with smaller particles. In addition to 5 μm particles, which are most common, particles of 3 μm in diameter or even smaller can be used. For example (Table 4), this approach was chosen when separating luteolin 2"-*O*-feruloylhexosyl-6-*C*-hexoside and luteolin 6-*C*-glucoside from *Arenaria montana* [66,67] and isoorientin in *Achillea millefolium* [68,69], as well as separating luteolin 6-*C*-hexosyl-8-*C*-pentoside, luteolin 2"-*O*-deoxyhexosyl-6-*C*-glucoside and luteolin 6-*C*-glucoside in *Cymbopogon citratus* [70,71]. Particles less than 2 μm in diameter were used for ultra-performance liquid chromatography (UPLC) analysis of luteolin derivatives and other flavonoids in *Lactuca sativa* [72,73] and date palm (*Phoenix dactylifera*) [74].

Stationary phases with different polarity may be used in HPLC. In the normal phase system, polar column fillings are used, and most often the filling is silica gel. However, silica gel can adsorb water, which leads to the loss of the original separating properties of the column and thus to impaired reproducibility of the obtained results [19,65]. Therefore, the gel is often modified with the aim of enabling better separation of mixture components. This is performed mainly by bonding alkyl chains (or alkyl chains bearing other functional groups) to functional groups on the gel surface. Silica gels optimized in this way are referred to as the associated phase [2,65]. In the so-called reversed phase (RP) system, non-polar associated phases are used. Such systems are especially useful in the analysis of insoluble or poorly water-soluble compounds, as well as in the analysis of polar compounds, provided that a mobile phase with high water content is used. Typically, an octadecylsilane (ODS) phase, composed of 18 carbon atoms (RP-18), is employed. Such fillers can have different properties depending on the silica gel type and/or production method [19,65]. According to the available literature, the analysis of luteolin derivatives (Table 4) was performed exclusively with RP systems, and the RP-18 system was the most frequently used system, as exemplified in the separation of six luteolin derivatives in *Securigera securidaca* [75] or as many as ten derivatives in *Capsicum annuum* [76]. Stationary phases composed of 8 carbon atoms (RP-8) were rarely used. Examples include the separation of flavonoids in *Coriandrum sativum* [77] and *Achillea millefolium* [68,69].

The selection of an appropriate mobile phase is extremely important for chromatographic separation. The type of analyte, mixture composition, stationary phase and detector used should be taken into account. Mixtures of up to three components are most commonly used. In the reverse system, mixtures of MeOH/H2O or acetonitrile (ACN)/H2O are routinely used. As the amount of organic solvent increases, the retention time for non-polar substances decreases, while the addition of H2O extends their retention times. As a rule of thumb, systems containing ACN/H2O eluents are more efficient than those containing MeOH/H2O [19]. In the normal phase system, however, the base solvent is a non-polar eluent, and its polarity is appropriately modified by the addition of another solvent of higher polarity, e.g., chloroform (CHCl3) [65]. According to the available literature, virtually all mobile phases used for analysis of luteolin derivatives consisted either of ACN/H2O, e.g., [26,66,67,78] or, less often, MeOH/H2O mixtures [72,79–81]. Additionally, a modifier such as FA, e.g., [79,82–84], AcOH, e.g., [44,78,85–87], or trifluoroacetic acid (TFA) [88] was added to avoid peak tailing [89]. Analyses performed without acidic modifiers are rare [90].

In the chromatography process, the elution can be carried out in two ways. The first method is isocratic elution, which involves running with the same composition of mobile phase during the analysis. This means that a constant elution force is maintained throughout the entire separation period. In this case, even a slight change in the composition of the mobile phase may affect the results of the analysis. Isocratic elution is only rarely used for analysis of flavonoids. In the case of luteolin derivatives, this method was used only for analysis of luteolin 7-rhamnosyl- (1-6)-galactoside in *Filago germanica* [91], luteolin 7-rhamnosyl(1-6)galactoside in *Galactites elegans* [90], and various luteolin derivatives in *Apium graveolens* [80]. If the composition of the eluent changes during the division of the mixture, then the gradient elution can be described. The gradient can be linear or specifically programmed. With a change in the composition of the eluent, its elution force increases. For this reason, this method is used particularly for the separation of mixtures composed of substances of different polarity [2,64]. The vast majority of papers describing the separation of luteolin derivatives in plant mixtures use a gradient approach. Examples include the separation of luteolin 8-*C*-glucoside, luteolin 6-*C*-glucoside, luteolin 4'-*O*-glucoside and luteolin 7-*O*-glucoside from *Chrysanthemum trifurcatum* [92], thirteen luteolin derivatives from *Cymbopogon citratus* [70,71] and many others.

By far, the most common method for detection of luteolin derivatives is using diode array detectors (DADs) (also called photodiode array detectors, PDA), which were used in as many as 46 instances (e.g., for the analysis of luteolin derivatives and other flavonoids in *Cymbopogon citratus* [79], *Dianthus versicolor* [88], *Clinacanthus nutans* [44], *Thymus alternans* and others [93]). More often than not, however, DADs were combined with other detectors for additional structure determination or confirmation, with or without prior isolation. Examples of DADs combined with other detection methods for analysis of luteolin derivatives in plants include the use of tandem mass spectrometry (MS/MS) and NMR for *Ligustrum vulgare* [26], diode array detectors combined with electrospray ionization and mass spectrometry (DAD-ESI-MS) for *Phoenix dactylifera* [94] and *Nerium indicum* [95], electrospray ionization combined with tandem mass spectrometry (ESI-MS/MS) for *Achillea moschata* [96], electrospray ionization combined with time of flight mass spectrometry (ESI-TOF-MS) and electrospray ionization combined with ion trap and tandem mass spectrometry (ESI-IT-MS/MS) for *Aloysia citrodora* and many others.

For example, the HPLC-PAD-MS technique was used in the analysis of aerial flowering parts of the Edelweiss alpine region (Leontopodium alpinum). This method allowed the basic separation of almost all components of the L. alpinum extract prepared by exhaustive dichloromethane (DCM) followed by MeOH extraction. In total, 14 compounds have been isolated from the extract, including several luteolin derivatives. The authors used a gradient as the mobile phase as follows: H2O:0.9% FA:0.1% AcOH:1.5% BuOH (A) and ACN:30% MeOH:0.9% FA:0.1% AcOH (B) and MeOH (C). The structure of the isolated components was additionally confirmed using NMR spectroscopy [29].

**Table 4.***Cont.*

**Table 4.** *Cont.*

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**Table 4.** *Cont.*

**Table4.***Cont.*

**Table 4.** *Cont.*

ESI-IT-MS/MS,

ionization combined with tandem mass spectrometry;

ion-mobility

amperometric

detection combined with electrospray ionization and mass spectrometry;

UV/pulsed amperometric

 detection combined with mass spectrometry.

 detection;

 spectrometry

 combined with quadrupole – time of flight mass spectrometry;

PDA-ESI-MS/MS,

 pulsed

amperometric

 detection combined with electrospray ionization and tandem mass spectrometry;

 PDA-MS, pulsed amperometric

 detection combined with mass spectrometry;

 electrospray ionization combined with ion trap and tandem mass spectrometry;

ESI-QTOF-MS,

 electrospray ionization combined with quadrupole – time of flight mass spectrometry;

 ESI-MS, electrospray ionization combined with mass spectrometry;

 MeOH, methanol; MS/MS, tandem mass spectrometry;

 ESI-MS/MS, electrospray IMS-QTOF-MS,

 FA, formic acid;

 PA, phosphoric acid; PAD, pulsed

 PDA-ESI-MS,

 TFA, trifluoroacetic

 pulsed

 acid; UV/PAD-MS,

amperometric

#### *2.3. Liquid Chromatography in the Analysis of Luteolin Derivatives*

LC combined with tandem mass spectrometry (LC-MS/MS) (Table 5) is especially useful in the analysis of multicomponent mixtures, such as herbal extracts because it does not require a large amount of the sample or previous separation. To further reduce the influence of other factors on the analysis, more advanced techniques, involving the combination of more than one detection method, e.g., LC combined with NMR and MS (LC-NMR-MS), are increasingly used [139].

Research on ethanol extract from *Lophatherum gracile* stems and leaves was performed using LC coupled with MS/MS. Gradient elution was performed using 0.3% FA and MeOH. Analysis of the species revealed the presence of, among others, luteolin 7-*O*-β-D-glucoside, and luteolin 6-*C*-glucoside [140].

Another raw material containing luteolin and its derivatives is the cocoa seed (*Theobroma cacao*). In the analysis of the H2O:MeOH extract, the following mobile phase was used: H2O:0.1% FA and ACN:0.1% FA. The elution was carried out in a linear gradient, whereas LC combined with electrospray ionization and tandem mass spectrometry (LC-ESI-MS/MS) coupling allowed the identification of the tested compounds [141].

Lin and Harnly performed a water-methanol analysis of the flower extract of *Chrysanthemum morifolium* and distinguished many compounds, including numerous derivatives of luteolin. In this case, the mobile phase was a mixture of 0.1% FA:H2O and 0.1% FA:ACN, in varying proportions. The qualitative determination of the analyzed substances was based on a comparison of retention times as well as mass and UV/Vis spectra [142].

**Table5.**Liquidchromatographyintheanalysisofluteolinderivatives.

**Table 5.** *Cont.*

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**Table 5.** *Cont.* Abbreviations:AFNH4,arrayelectrosprayspectrometry;DAD, diode array detector; DAD-ESI-MS/MS, diode array detector combined with electrospray ionization and tandem mass spectrometry; ESI, electrospray ionization; ESI-MS, electrospray ionization combined with mass spectrometry; FA, formic acid; MeOH, methanol; MS/MS, tandem mass spectrometry; PDA-MS, pulsed amperometric detection combined with mass spectrometry; QTOF, quadrupole time-of-flight; TQMS, triple quadrupole mass spectrometer.

#### *2.4. Gas Chromatography in the Analysis of Luteolin and Its Derivatives*

Gas chromatography (GC) is a technique used to analyze volatile as well as non-volatile compounds after their derivatization. GC is characterized by chromatographic distribution of either a gas mobile phase on a solid adsorbent (gas-solid chromatography) or a liquid on an inert support (gas-liquid chromatography). GC can be hyphenated with various detection techniques such as GC combined with mass spectrometry (GC-MS), GC combined with tandem mass spectrometry (GC-MS/MS) or GC combined with time of flight mass spectrometry (GC-TOF-MS), thus greatly increasing the versatility, sensitivity and accuracy of the method [164,165]. The analyzed substances should be thermally stable, and their boiling (or sublimation) temperature should not exceed 350-400◦C. To achieve this, non-volatile substances are often derivatived. Polar functional groups are transformed into their less polar counterparts, thus increasing the volatility of the prepared compounds. The most common examples include substitution with a trimethylsilyl (TM) group, organic radicals or compounds such as trimethylchlorosilane (TMCS), hexamethyldisilazane (HMDS) or *N*,*O*-bis(trimethylsilyl)-trifluoroacetamide (BSTFA). Such an approach greatly increases the number of possible analytes [2,17]. For example, phenolic groups of flavonoids are often transformed into their less polar trimethylsilyl counterparts allowing for rapid and effective separation of complex mixtures [166].

In adsorption GC, a gas that is chemically inert to the stationary phase as well as the components being analyzed is used as the mobile phase. Most often hydrogen, nitrogen or argon is used. Helium is being used less often due to its higher cost than other gases and the implementation of the principles of chemical safety. The mobile phase must be properly selected for compatibility with the detector used. However, the carrier gas itself does not have a significant influence on the separation effects of the analyzed mixtures [17]. In the process of separation, the method of application of the sample to the chromatography column is very important. The sample should always have as small of a volume and the shortest dosing time as possible. This ensures better separation and narrower bands [17,164].

Gas chromatography uses open-ended columns, i.e., capillary columns and packed columns. Open-ended (OT-open tubular) columns are characterized by much higher efficiency than packed columns; therefore, they are chosen much more often [164]. Capillary columns are particularly useful in the separation of substances with significantly different boiling points. Ideally, the column should have a similar polarity to the analyzed components. However, due to the higher efficiency and greater durability of stationary phases with low polarity, so capillary columns are recommended for chromatographic analyses [17,164].

Another factor that influences the efficiency of the separation of the analyzed sample components, as well as the time of analysis, is temperature. The separation temperature should be selected depending on the stationary phase used and the boiling point of the analytes [17,164].

The analysis of luteolin derivatives has been carried out in accordance with the general procedure used for analysis of other flavonoids (Table 6) [165]. MS was used for detection of volatile derivatives of luteolin, while the use of a flame ionization detector (FID) was described only in two papers [167,168]. Most commonly, helium was used as a carrier gas (e.g., [169–171]), but the use of nitrogen was also recorded [167,168]. The derivatization of luteolin, which is necessary for chromatographic separation, was mostly achieved with BSTFA/TCMS [170–172]. For the analysis of lipophilic luteolin derivatives, such as 7,3',4'-trimethyl-luteolin in *Arnica alpina,* no derivatization was necessary [168].

#### *2.5. Counter-Current Chromatography in the Analysis of Luteolin Derivatives*

Counter-current chromatography (CCC) is a variation of liquid chromatography in which both the stationary and mobile phases are liquid. The separation of the constituents of the mixture is carried out in a system of immiscible liquids that are in equilibrium with each other. The method is simple and rapid, offering the possibility of introducing the raw sample to the column without need for previous clean-up [173]. Counter-current chromatography is mainly used for purification of natural compounds, while its use as an analytical technique is far less common. In CCC, various dividing

techniques can be used, thus distinguishing centrifugal partition chromatography (CPC) and rapid- or high-speed CCC (HSCCC), often referred to as hydrodynamic chromatography [174,175]. High-speed CCC is particularly often encountered in studies involving the separation of flavonoids [176]. Table 7 presents the conditions for the separation of mixtures containing luteolin and its derivatives using rapid counter-current chromatography. Separation of luteolin derivatives from mixtures of different phytochemicals is usually carried out with mixtures of EtOAc with one of alcohol (BuOH, EtOH or AcOH) and H2O. Upon separation, structure determination by NMR is often necessary [173,177]. However, instead of NMR, high resolution mass spectrometry (HRMS) can also be employed for structure confirmation, as evidenced by the use of this technique for differentiation of various luteolin derivatives in *Lippia origanoides* [178].

**Table6.**Gaschromatographyintheanalysisofluteolinanditsderivatives.

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Abbreviations: Py, pyridine, BSTFA, N,O-bis(trimethylsilyl)trifluoroacetamide; TMS, trimethylsily; HMDS, hexamethyldisilazane; TMCS, trimethylchlorosilane; APCI-TOF-MS, atmosphericpressure chemical ionization combined with quadrupole – time of flight mass spectrometry; FID, flame ionization detector; QMS, quadrupole mass spectrometry.

Abbreviations: AcOH, acetic acid; BuOH, butanol; EtOAc, ethyl acetate; EtOH, ethanol; HRMS, high resolution mass spectrometry; Hx, hexane, rpm, revolutions per minute.

#### **3. Conclusions**

The presented comparison of chromatographic methods currently used to determine luteolin and its derivatives provides a systematic summary of the available knowledge. Without a doubt, chromatographic analysis may be successfully employed as an efficient method for both qualitative assessment (fingerprinting) and quantitative determination of luteolin derivatives. In tedious and time-consuming determinations of multi-ingredient plant extracts, including those that contain the most prevalent luteolin derivatives, combinations of large-scale chromatographic techniques with serially aligned detection modalities such as LC-MS/MS or LC/NMR/MS were found to be particularly useful. Such beneficial coupling of chromatography with other analytic techniques expands analytical capabilities while additionally improving the accuracy, sensitivity, and precision of assays. Despite the dominant position of LC in the analysis of natural compounds and the dynamic development of novel chromatographic methods, the TLC/HPTLC has not lost its important place in the phytochemical analysis of luteolin derivatives. The technique is relatively simple and inexpensive while facilitating rapid qualitative and quantitative analysis of test compounds. In addition, it facilitates large quantities of diluted samples being deposited in the stationary phase, allowing for a wider choice of mobile phase carriers. The technique is subject to continuous improvements and its range of applicability is expanding.

Effective chromatographic analysis in the determination of luteolin derivatives requires appropriately selected chromatographic separation conditions. The appropriate choice of stationary phase sorbent may significantly improve test conditions. Most TLC analyses of luteolin derivatives are carried out in normal phase systems featuring a polar (hydrophilic) sorbent phase. In HPLC, separation conditions can be chosen more arbitrarily as the technique allows for the use of stationary phases of varying polarity which is particularly advantageous in the analysis of compounds that are either insoluble or poorly soluble in water. In order to additionally improve the sensitivity of HPLC analyses, one should focus on the column parameters responsible for appropriate separation. Quite often, the fairly successful analyses are used as starting points for further modifications, including the development of preparative-scale analyses.