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A study on the photoisomerization of phenylpropanoids and the differences in their radical scavenging activity using in-situ NMR spectroscopy and on-line radical scavenging activity analysis

Applied Biological Chemistry volume 67, Article number: 69 (2024) Cite this article

Abstract

Phenylpropanoids are naturally occurring secondary metabolites that exhibit various biological activities such as ultra-violet (UV) light protection and reactive-oxygen species (ROS) scavenging. In this study, we utilized a light-emitting diode (LED) based in-situ UV irradiation nuclear magnetic resonance (NMR) technique to monitor the photoisomerization reactions of these phenylpropanoids under UV irradiation in real-time. Through this approach, we measured the photochemical reaction rates and photostationary state (PSS) ratios of these molecules and observed distinct reaction rate and PSS ratio information depending on the variation of substituent groups in each phenylpropanoid molecule. We also evaluated the radical scavenging activity (RSA) for each photochemical product through diphenyl-1-picrylhydrazyl radical (DPPH) assay and 2,2’-azino-bis(3-ethylbenzenthiazoline-6-sulphonic acid) (ABTS) assay. We found that the photoisomerization product of caffeic acid can increase both DPPH and ABTS radical scavenging activities, and confirmed the enhanced ABTS radical scavenging ability of caffeic acid cis-isomer based on the online high-pressure liquid chromatography (HPLC)-ABTS analysis and the PSS ratio information of each isomer.

Introduction

Phenylpropanoids, naturally occurring secondary metabolites, are known to exhibit various physiological activities in plants. These compounds are involved in structural support, plant hormone biosynthesis, and signal transduction within plants [1]. The high UV absorption capacity and antioxidant properties of these compounds, attributed to their structural characteristics, protect plants from direct damage caused by UV radiation and cellular damage induced by reactive oxygen species (ROS) generated from UV absorption [2,3,4]. Photo-switches are molecules that can undergo in structural changes and chemical properties upon absorption of light energy [5]. Azobenzene, for example, which induces cis/trans isomerization through photoisomerization reactions, is a well-known photo-switch compound [6]. These molecules can undergo various structural isomerization reactions upon absorbing specific wavelengths of light. Many phenylpropanoid compounds have been reported to induce cis/trans isomerization of intramolecular double bonds through UV absorption at specific wavelengths [7, 8]. Furthermore, studies have also reported on the altered physiological activities of the corresponding isomeric molecules [7, 9].

The bond dissociation enthalpy (BDE) value of a compound can be used as an important indicator to predict the activity of antioxidants, and lower BDE values may imply stronger antioxidant activity [10, 11]. The BDE value is highly correlated with the molecular structure, and changes in the substituents and stereochemistry of the compound can also lead to variations in the BDE value [12]. There have been some reports on predicting the antioxidant activities of geometric isomers of phenylpropanoid derivatives. In these studies, quantum chemical calculations have suggested distinct BDE value differences among the structures of different isomers [13, 14]. To the best of our knowledge, however, it appears that until recently, systematic studies on the characteristics of photoisomerization reactions and the effects on antioxidant activity caused by each isomerization reaction have not been conducted for phenylpropanoid compounds.

For the photo-switch molecules, monitoring of photochemical profiling could be performed with aid of conventional HPLC-based analyses using time-course sampling measurements [15, 16]. However, this approach may pose difficulties in monitoring fast photochemical reactions and requires repetitive measurements. Moreover, for the DPPH and ABTS assays widely used to evaluate the antioxidant activity of compounds [17], a physical isolation of each photoreaction isomer is necessary. But, some photo-switch molecules exhibit very fast reversible reactions [18], so as to the dead time of the aforementioned measurement methods may pose challenges in evaluating the structure and activity of each isomer.

In recent studies, we have proposed a novel method which enables a monitoring of photoisomerization reactions in real-time using the in-situ UV irradiation NMR technique [19]. This method can afford non-invasive real-time evaluation of the rate of photoisomerization reactions and the exact corresponding isomer ratios. Additionally, the on-line HPLC-ABTS technique has been widely used due to its advantage of providing intuitive antioxidant activity corresponding to each molecular chromatogram peak, without requiring physical separation of individual compounds [20, 21]. Therefore, the utilization of the aforementioned methods will allow an accurate measurement of isomer formation in each photo-switch molecule under light irradiation, and based on this, a radical scavenging activity (RSA) of the individual isomers can be easily evaluated as well.

To this end, we employed the in-situ UV irradiation NMR technique to evaluate the kinetics of photochemical reactions of various phenylpropanoid molecules in real-time and investigated the influence of substituent effects and isomers on photoisomerization reactions. Furthermore, using the online HPLC-ABTS technique, we assessed the differences in antioxidant activity of each phenylpropanoid compound based on the isomerization reaction kinetics data.

Materials and methods

Chemicals and materials

We used eleven trans-phenylpropanoids as standard compounds as following. Cinnamic acid 1, m-coumaric acid 3, and sinapinic acid 7, (TCI, Tokyo, Japan). p-Coumaric acid 2, and o-coumaric acid 4, (Wako, Osaka, Japan). Caffeic acid 5, (Daejung, Korea). Ferulic acid 6, neochlorogenic acid, 9, 4-O-caffeoylquinic acid, 10, and chlorogenic acid 11. (Sigma-Aldrich, MO, USA). Rosmarinic acid, 8, (Biosynth, Compton, UK). For in-situ UV irradiation NMR analysis, deuterium oxide (D2O), dimethylsulfoxide-d6 (DMSO-d6) and methanol-d4 were used. (Eurisotop, Saint Aubin, France). To evaluate the RSA of each compound, 1,1-Diphenyl-2-picrylhydrazyl radical (DPPH), 2,2’-Amino-di(3-ethylbenzthiazoline sulfonate) (ABTS) and Potassium persulfate for ABTS were used (Sigma-Aldrich, MO, USA). As a control in RSA assay, ascorbic acid (Daejung, Korea) was used. For chromatographic analysis, HPLC-grade acetonitrile, water, methanol and formic acid were used (Fisher Scientific, PA, USA).

in-situ UV irradiation NMR spectroscopy

All NMR spectra were obtained using a 500 MHz Bruker Avance NEO spectrometer equipped with a 5 mm CPP BBO probe (Bruker BioSpin, Germany) at 298 K. A fiber-coupled LED, FC-LED (Prizmatix, Israel), was used as the UV 365 nm light source. The pulse sequence shown in Fig. 1b was employed for the acquisition of 1D in situ UV irradiation NMR arrays. The t2 time-domain points were set to 65,536 complex points. Each array was acquired with a single scan using a 90° pulse. The total delay, which is the sum of the acquisition time (Acq.) and the delay between each array (d1), was set to 20 s unless otherwise specified. The number of arrays was set according to the values indicated in the main text. The total acquisition time for a 1D in-situ UV irradiation NMR array per compound was 60 min. In case of DMSO-d6 used as NMR solvent, to alleviate the signal overlap problem during the identification and quantification of each isomer generated by the photochemical reaction on NMR spectra, 5% (v/v) of deuterium oxide was added to the reaction solvent to remove the hydroxy signals through the deuterium exchange effect. Except for compounds 8–11 (25 mM), all other compounds were measured at a concentration of 10 mM.

Fig. 1
figure 1

(a) Schematic illustration of an acquisition system for in-situ UV irradiation NMR spectroscopy (left) and UV irradiation method using optic fiber ending in NMR tube (right). (b) NMR pulse sequence used in this study (left), cis/trans photoisomerization of phenylpropanoids (middle), and schematic diagram of pseudo-2D NMR data acquisition for real-time monitoring of photochemical reactions (right); adapted from Antioxidants 2022, 11, 2206

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NMR data processing

After acquiring the 1D in-situ UV irradiation NMR data arrays, in order to generate photoisomerization profiling data a custom processing script based on Python 3.8 was employed. The script utilized the nmrglue [22] module for python to handle the input and output of NMR data.

UV spectroscopy

The UV spectrum was obtained using a UV/Vis spectrometer, Lambda 35 (PerkinElmer, MA, USA), with a quartz cell. The analytical sample was dissolved in HPLC plus grade methanol (Sigma-Aldrich, MO, USA) at a concentration of 0.05 mM. The spectrum range was set from 210 to 450 nm, and the data interval was 1.0 nm.

UV-lamp based photoisomerization reaction

To minimize solvent evaporation due to the heat generated by the light source during irradiation and the resulting change in solute concentration, the photochemical reaction was carried out in DMSO-d6. Each compound was dissolved in a DMSO-d6: D2O (95:5, v/v) solvent mixture at a concentration of 5 mM. 400 µL of each sample was transferred to a well in a 48-well plate. All samples were then irradiated with UV light for 40 min using a custom-built UV lamp with a wavelength of 370 nm and intensity of light was measured using a TM-223 UVAB Light Meter (TENMARS, Thailand) and the light intensity was 11 mW/cm2. The resulting photochemical reaction products were used to evaluate the radical scavenging activity. UV irradiation onto the microplate was performed under the same conditions as for the UV intensity measurements above.

DPPH radical scavenging assay

The DPPH radical scavenging assay was performed using the method reported by Wang et al. [23] with some modifications. Stock solution of 0.2 mM DPPH in methanol was prepared. Analytical samples were diluted in methanol. 100 µL of each sample and 150 µL of DPPH stock solution was mixed into the 96-well plate. The reaction plate was wrapped in aluminum foil and kept at room temperature for 60 min in darkness. The absorbance was measured at 517 nm with microplate reader. Ascorbic acid was used as the positive control, and the capability to scavenge the DPPH radical was calculated using the following equation:

Radical scavenging activity (RSA, %) = [(1 – absorbance of sample / absorbance of blank) × 100] at 517 nm.

The results are expressed as the means with standard deviation (SD) for experiments conducted in three times.

ABTS radical scavenging assay

The ABTS radical scavenging assay was performed by the Nazir et al. method [24] with modifications. For the ABTS radical reagent, equal quantity of 7 mM ABTS was added to 2.45 mM of potassium persulfate was prepared in water. This stock was incubated 16 h in darkness at room temperature for radical stabilization. Before using this solution, it was diluted with methanol to get an absorbance of 0.8 at 734 nm. 20 µL of each sample and 180 µL of working solution mixture were placed in 96-well plate for 30 min in the dark. The absorbance was measured at 734 nm with microplate reader. Ascorbic acid was used as the positive control. The ABTS radical scavenging activity of each sample was calculated as the percent inhibition according to the following equation:

Radical scavenging activity (RSA, %) = [(1 – absorbance of sample / absorbance of blank) × 100] at 734 nm.

All sample were tested triplicates.

On-line HPLC-ABTS analysis

The HPLC-ABTS system consisted of Agilent 1260 series HPLC-DAD system (Agilent Technologies, CA, USA), fitted with an additional pump to supply the ABTS radical solution. The ABTS radical reagent was prepared from stock using the same method as off-line ABTS assay and diluted 20-fold in HPLC grade methanol. Analytical samples were diluted in methanol to a concentration of 0.5 mM and 10 µL was injected into the on-line HPLC-ABTS system. For the stationary phase, YMC Triart C18 (4.6 × 150 mm, 3 μm) column was used and the column temperature was maintained at 30 °C. Mobile phase was acetonitrile (A) and water (B); flow rate was kept at 0.7 mL/min. The elution gradient was carried out as follows: 90% B for 0–3 min; 90 − 10% B for 3–23 min; 10% B for 23–28 min; 10–90% B for 28–30 min. The ABTS radical solution was supplied with a flow rate of 0.3 mL/min. The chromatograms were recorded at 254 nm as positive peak and the visible detector was set at 734 nm to measure the decrease of ABTS radicals as negative peak. The data were analyzed by Chemstation software (Agilent Technologies, CA, USA).

Results

Real-time monitoring of photoisomerization of phenylpropanoids using in-situ UV irradiation NMR technique

To evaluate the photoisomerization reactions of various naturally occurring phenylpropanoid compounds using an in-situ UV irradiation NMR spectroscopic technique (Fig. 1), we selected a total of eleven compounds, including seven phenylpropanoids (1–7) based on their phenyl ring functional groups, and four caffeoyl derivatives (8–11) (Fig. 2).

Fig. 2
figure 2

Structures of phenylpropanoid derivatives. Each number indicates a compound number used in this study. Cinnamic acid 1, p-coumaric acid 2, m-coumaric acid 3, o-coumaric acid 4, caffeic acid 5, ferulic acid 6, sinapinic acid 7, rosmarinic acid 8, neochlorogenic acid 9, 4-O-caffeoylquinic acid 10, chlorogenic acid 11

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The kinetic reaction profile and isomer ratio of photoisomerization for each phenylpropanoid molecule was monitored using an in-situ UV irradiation NMR spectroscopic technique (Fig. 1). A series of 1H NMR spectrum was acquired in 20 s intervals upon UV irradiation for 40 min. Besides, in order to monitor a reversible thermal isomerization, 1H NMR data also measured in every 20 s under a dark condition after an interruption of the light irradiation. Then, we calculated an area of unsaturated alpha carbonyl 1H NMR signals of each isomer under 365 nm UV irradiation.

We monitored the photoisomerization reactions of phenylpropanoid derivatives by dividing them into three groups according to their chemical group characteristics: (1) cinnamic acid and its monohydroxy derivatives (Fig. 3a), (2) cinnamic acid derivatives with two or more hydroxy or methoxy groups (Fig. 3b), and (3) caffeoyl group derivatives (Fig. 3c). As shown in Fig. 3b and c, all the compounds in this group reached photoisomerization reaction equilibrium within 10 min of UV irradiation, with cis-isomer ratios ranging from 40 to 48%, and monitoring of the thermal reverse reaction under dark conditions for 20 min after interruption of UV irradiation exhibited only changes in cis-isomer ratio within 1% (Table 1).

Fig. 3
figure 3

In-situ UV irradiation NMR photoisomerization profile of phenylpropanoids upon UV (365 nm) irradiation in DMSO-d6 solution. Profiling data of (a) cinnamic acid and its monohydroxy derivatives (1–4), (b) two or more hydroxy or methoxy cinnamic acid derivatives (5–7), and (c) caffeoyl group derivatives (8–11). Solid and dotted lines denote relative NMR signal intensity of trans and cis isomers of phenylpropanoids over acquisition time under UV irradiation, respectively. Each NMR signal intensity, in arbitrary units, were normalized. Each colored number in figures represents the compound number. NMR measurements were performed under UV irradiation between 0 and 40 min, and after 40 min under dark conditions

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Table 1 The ratios of cis-isomers of phenylpropanoids after UV irradiation and 1 h post-irradiation
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However, there were distinct photoisomerization reaction profiles depending on the presence or absence of the hydroxy group in the phenyl ring of cinnamic acid and the substitution position (Fig. 3a). For cinnamic acid 1, and m-coumaric acid 3, none of or only very small isomerization reactions were observed, respectively. In case of the p-coumaric acid 2, its isomerization rate was relatively slow, and the resulted ratio of the cis isomer was only about 21%. For o-coumaric acid 4, its reaction equilibrium was reached faster than for other molecules in this group (about 20 min) and, interestingly, the cis-isomer ratio reached about 68% which is highest among tested compounds. We also measured the ratio of cis-isomers 1 h after interruption of UV irradiation. Except for chlorogenic acid 11, all showed an exhibited a rise of less than 1% of increase in the trans isomer by thermally reversible isomerization (Table 1). Through this, it was verified the stability of each isomer ratio within the timescale of subsequent in vitro activity and on-line HPLC-ABTS activity evaluation.

Photoisomerization efficiency depending on the UV absorption properties

We measured UV absorption spectra in methanol solvent for a group of single hydroxy-substituted cinnamic acid derivatives (1–4) that exhibit distinct differences in their intermolecular photoisomerization profiles.

Prior to analysis of UV spectra, we acquired 1H NMR spectra (before/after UV irradiation) of compound 1–4 in methanol-d4 in order to verify the equivalence of photochemical reaction trends in different solvent conditions (Fig. 4). It exhibited similar isomerization trend compared to UV irradiation results in DMSO-d6; Compounds 2 and 4 showed UV induced cis-isomer NMR signals, whereas 1 and 3 showed not cis-isomer signals at all. This allowed us to see similar photoisomerization trends in the methanol solvent system as in the DMSO. In UV absorption spectra of 1–4, as shown in Fig. 5a and b, both 2 and 4 exhibited UV absorption up to around 350 nm, and considering the bandwidth, it was confirmed that absorption of UV light in the 365 nm wavelength range is possible. Compound 1 showed an absorption peak at 275 nm and only displayed an absorption spectrum up to the early 300 nm wavelength range, confirming the difficulty of absorption by the light source used in the experiment (Fig. 5c). On the other hand, 3, which has a hydroxy group substituted at the meta position, showed a similar spectrum pattern to 4, but the ratio of absorption peaks around 275 and 330 nm was observed to be relatively lower for the 330 nm absorption peak compared to 3 (Fig. 5d).

Fig. 4
figure 4

Comparison of the enlarged aromatic signal region of 1H NMR spectra: (a) p-coumaric acid 2 and (b) o-coumaric acid 4, (c) cinnamic acid 1, and (d) m-coumaric acid 3, before (upper, black) and after (lower, red) UV (365 nm) irradiation in methanol solution. Asterisks in (a) and (b) denote NMR signals corresponding to their corresponding cis-isomers

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Fig. 5
figure 5

UV absorption spectra of (a) p-coumaric acid 2 (b) o-coumaric acid 4, (c) cinnamic acid 1, and (d) m-coumaric acid 3. Each compound was dissolved in 0.5 mM concentration in methanol, respectively

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Comparison of the difference of radical scavenging activities by photoisomerization

The DPPH and ABTS RSA evaluation on each trans isomers and their isomeric mixture through UV irradiation enabled a comparison of the RSA based on the cis/trans isomeric forms of each phenylpropanoid structure (Table 2). For this, the photoisomerization reactions were carried out using the same solvent system as NMR measurement within a microplate using a 370 nm UV lamp instead of in-situ UV irradiation NMR method. Then, the equivalence of cis isomer formation and its ratio between the in-situ UV-NMR and UV-lamp method was verified through the comparison of each 1H NMR data, respectively (See the supplementary document).

Table 2 Evaluation of the DPPH and ABTS radical scavenging activities of phenylpropanoids upon UV exposure
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As shown in Table 2, monohydroxy substituted cinnamic acid derivatives (2 and 4) exhibited relatively low DPPH RSA compared to the rest of the compounds both before and after the UV reaction. Furthermore, except for caffeic acid 5, it showed no significant difference in the DPPH RSA before and after UV irradiation among tested compounds. As shown in Table 2; Fig. 6a, in the case of 5, the trans isomer (before UV irradiation) exhibited an RSA of approximately 74%, and after the formation of cis isomer by UV irradiation, an increase of approximately 17% in RSA (91%) was observed. In the ABTS RSA evaluation, similar DPPH results were measured. The remaining eight cis/trans isomeric mixture samples, except for 5, showed no significant change in ABTS RSA after UV irradiation or exhibited a decrease in RSA of about 10%, as in the case of sinapinic acid 7. However, in the case of 5, a two-fold increase in RSA was observed, reaching approximately 83% (Table 2; Fig. 6b).

Fig. 6
figure 6

Comparison of the radical scavenging activity (RSA) with and without UV irradiation for selected phenylpropanoids. The sample and control concentration were 40 µM and 20 µM for the DPPH and ABTS assays, respectively. Results of (a) DPPH and (b) ABTS radical scavenging activities for compounds 2, 5 and 8. The vertical axis represents the calculated RSA values (see the Methods section in details). Each number on the horizontal axis indicates the compound number. For each compound number, the gray bar represents the RSA of the compound without UV irradiation, while the orange bar represents the RSA with UV irradiation. Ascorbic acid (black bar) was used as a control

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Next, to evaluate the individual RSA of trans and cis isomers of 5, without a separation process of each isomer, simultaneously, we employed an on-line HPLC-ABTS analysis method. As shown in Fig. 7a, the peak separation of each trans and cis isomer was observed on HPLC chromatograms. And the conformation of each isomer peak was confirmed by comparing the standard UV spectrum of 5 (Fig. 7b) with the UV spectra of each HPLC peak (Fig. 7c and d). This enabled the comparison of ABTS RSA for each isomer of 5 by calculation of an area under the curve (AUC) of the absorbance difference at 734 nm wavelength. As a result, the ratio of AUC corresponding to the trans and cis isomers was calculated to be approximately 37:63. According to the actual molar ratio of the trans and cis isomers in the UV irradiated sample, as determined by 1H NMR spectral analysis, it was 53:47. Thus, the ABTS absorbance difference per molar equivalent of trans and cis isomer was calculated to be 1:1.51.

Fig. 7
figure 7

On-line HPLC-ABTS analysis of the photoisomerization reaction sample of caffeic acid 9. (a) Comparison of 254 nm (top, black) and 734 nm (bottom, red) HPLC chromatograms; y-axis: absorbance (arbitrary units), x-axis: analysis time. Each area under the curve (AUC) value was calculated by integrating of the peak in 734 nm chromatogram, respectively. Comparison of the UV absorption spectra of 9 (b) and peak 1 (c) and peak 2 (d) in the chromatogram

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Discussion

Many photo-switches exhibit different isomer conversion ratios depending on reaction conditions [25,26,27], and the accurate evaluation of these ratios in isomer mixtures presents obstacles due to the thermal reverse reaction [28, 29], which impedes the physical separation of each pure isomer. In this study, by employing the in-situ UV irradiation NMR technique, it was possible to accurately measure the ratio of photo-isomers in mixtures by evaluating the formation of each isomer upon irradiation, and their reverse reaction under dark conditions as well. This enabled the accurate acquisition of information about isomer ratios for subsequent evaluations of radical scavenging activity.

The photoisomerization kinetic profiling data (Fig. 3), except for the cinnamic acid and coumaric acid derivatives, 1–4, (Fig. 3a), the rest of compounds exhibited similar photochemical reaction equilibrium states under 365 nm UV irradiation (Fig. 3b and c). Meanwhile, from the ratio of equilibrium in the first-order kinetic reaction, the ratio of reaction rates between the forward and reverse reactions can be inferred, confirming that all these derivative molecules exhibit similar forward and reverse photoisomerization reaction constants under reaction condition used in this study. The compounds 1–4 in Fig. 3a, however, showed significant differences in the rate of photochemical reaction and the equilibrium constant ratio of reactants within the group and compared to the molecules in the other groups (Fig. 3b and c).

The difference in the amount of photon absorption and the number of molecules transitioning to the excited state during the photochemical reaction can cause these differences in reaction rates, which can be attributed to the difference in the absorption spectra of each compound at the 365 nm wavelength used in the study. The bathochromic shift of the UV absorption spectra of these compounds due to di- or tri-hydroxy substitution effects shows absorption peaks around the 330 nm, which allows for relatively strong photon absorption from the light source compared to 1–4, explaining the difference in reaction rates. Notably, in the case of 1, no cis isomer formation was observed under 365 nm light irradiation. This result appears to be simply due to the lack of 365 nm wavelength light absorption by cinnamic acid. It can be expected that the isomerization reaction of cinnamic acid could be induced through irradiation with shorter wavelengths of light. This can be inferred from previous reports showing the formation of cis isomers of cinnamic acid under extensive UV irradiation [30]. In the case of 2–4, significant differences were observed in terms of reaction rate and equilibrium constant, depending on the position of the hydroxy group substitution.

The difference in the presence or absence of the photoisomerization reaction upon UV irradiation in 3 and 4 was presumed to be due to differences in the excited state of each molecule and the photoisomerization reaction mechanism, apart from light absorption. Zhou et al. [31] reported results on the increased trans → cis photoisomerization efficiency of ortho-substituted arylacrylate compounds; it was mentioned that the ortho-substituted trans compound exhibited a relatively longer triplet state lifetime compared to the meta- and para-substituted compound as well. Considering the relationship between photochemical reaction efficiency and triplet state lifetime [32], the interpretation of the high photoisomerization reaction efficiency of ortho-substituted molecules becomes possible. Thus, given the structural similarity between phenylpropanoid derivatives and arylacrylate compounds, the relatively higher trans → cis conversion rate of ortho-hydroxy substituted phenylpropanoids in this study is considered to be consistent with the results of the aforementioned study [31].

In the DPPH and ABTS assay results, the RSA were proportional to the number of hydroxyl substitution groups on the phenyl ring (mono hydroxy vs. di- or tri-hydroxy) showing a good agreement with the results of DPPH RSA for cinnamic acid derivatives in previous reports [33]. In a theoretical study, in addition, the high negative correlation coefficient between the BDE values of the catechol group in cinnamic acid derivatives using a quantum chemical calculation was reported as well [34]. Thus, it seems the presence and increasing number of catechol groups within these molecules are the most important factors in the expression of radical scavenging activity, and the strongest DPPH RSA of rosmarinic acid 8, which has two catechol groups within the molecule, also supported this correlation. In the ABTS results, although the difference between mono-hydroxy cinnamic derivatives and other groups was not as distinct as in DPPH, the results still showed a similar trend between the phenyl group substitution structure and the radical scavenging ability, as observed in the DPPH results. As mentioned in previous studies [34], while DPPH has a high correlation with the BDE value of the catechol substituent, ABTS RSA is known to be influenced by various factors such as pH, reaction time, and secondary antioxidant reactions caused by ABTS radical reactants [35].

Interestingly, in the DPPH and ABTS assay results for cis/trans isomer mixtures, the compound 5 showed an increase of 17% and 43% in DPPH and ABTS RSA values, respectively, between the samples before and after UV irradiation while rest of compounds do not exhibit significance difference in RSA in both assays. But, in vitro assay results for the isomer mixtures still have limitations in interpreting whether the cause of this increase in RSA activity, especially in the ABTS assay, is due to the effect of the cis isomer itself or the synergistic effect of the trans and cis isomer molecules or the formation of complexes of each isomer’s radical reaction products.

On the other hand, it was reported that a lower BDE calculation value for the 4-hydroxy group in the cis isomer of 5 than that of trans isomer [36], thus it also supports the interpretation of the higher RSA results for the cis isomer in this experiment. Furthermore, it showed that the AUC value of the ABTS absorbance peak for the cis isomer compound was approximately 1.7 times higher than the corresponding AUC value for the trans compound in on-line HPLC-ABTS chromatogram. Since each trans and cis isomer exists in nearly equal ratios in the reaction sample as calculated by 1 H NMR analysis, we concluded that the cis isomer possesses higher ABTS radical scavenging ability per equivalent than the trans isomer of 5. It should be note that compounds 8–11, which possess the same caffeoyl group as 5, did not show a significant change in radical scavenging ability due to the cis isomers. Recent DPPH and ABTS activity evaluation results for the cis isomers of seco-iridoid molecules containing the p-coumaroyl group reported a decrease in radical scavenging ability with an increase in the proportion of cis isomers [37]. Based on these results, the RSA of phenylpropanoids suggests that not only the catechol substituent effect but also various complex factors in terms of chemical structure, such as other substituent groups and the cis/trans isomer structure, can influence their radical scavenging ability.

In this study, we exploited in-situ UV irradiation NMR spectroscopy to characterize the UV irradiation-induced photoisomerization reactions of naturally occurring phenylpropanoid derivatives. We monitored the generation of cis-isomer by the photoisomerization reaction depending on the phenyl substituent group, and in particular identified a very high cis-isomerization ratio due to the effect of the ortho substituent on the hydroxy group. Furthermore, their DPPH and ABTS assay data suggested an increase in radical scavenging activity as a result of the generation of cis-caffeic acid, and on-line HPLC-ABTS analysis confirmed the higher ABTS radical scavenging activity of the cis-caffeic acid compared to the its trans-isomer. This work provides structural insights into the UV irradiation-induced isomerization and its effect to radical scavenging activity of phenylpropanoid molecules, which are known to be natural antioxidants, and will be widely used in the study of naturally occurring antioxidants.

Data availability

Availability of data and materials All data generated or analyzed during this study are included in this published article.

Abbreviations

ABTS:

2,2’-Azino-Bis (3-Ethylbenzenthiazoline-6-Sulphonic Acid)

AUC:

Area Under the Curve

BDE:

Bond Dissociation Enthalpy

DPPH:

2,2-Diphenyl-1-Picrylhydrazyl Radical

HPLC:

High Performance Liquid Chromatography

LED:

Light-Emitting Diode

NMR:

Nuclear Magnetic Resonance

PSS:

Photostationary State

ROS:

Reactive Oxygen Species

RSA:

Radical Scavenging Activity

UV:

Ultra-Violet

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Funding

This work was supported by the Korea Institute of Science & Technology—Research Program 2E33301.

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Authors and Affiliations

  1. KIST Gangneung Institute of Natural Products, Innovative Enterprise Cooperation Center, Gangneung, 25451, Korea

    Sangah Park & Byung-Hun Um

  2. Department of Chemistry, Gangneung-Wonju National University, Gangneung, 25457, Korea

    Sangah Park

  3. KIST Gangneung Institute of Natural Products, Natural Product Informatics Research Center, Gangneung, 25451, Korea

    Hojin Kim, Miyeon Bang & Jin Wook Cha

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  1. Sangah Park
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  2. Hojin Kim
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  3. Miyeon Bang
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  4. Byung-Hun Um
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  5. Jin Wook Cha
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Contributions

JWC conceived and conceptualized study; JWC and SP designed experiment; HK and MB prepared material and performed formal analysis; HK, MB and SP performed in vitro assay and spectroscopic analysis; Funding acquisition was provided from JWC and BHU. SP and JWC writing original draft of manuscript, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

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Correspondence to Jin Wook Cha.

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Park, S., Kim, H., Bang, M. et al. A study on the photoisomerization of phenylpropanoids and the differences in their radical scavenging activity using in-situ NMR spectroscopy and on-line radical scavenging activity analysis. Appl Biol Chem 67, 69 (2024). https://doi.org/10.1186/s13765-024-00925-3

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