cyclodextrin inclusion complexes: Selective inclusion, enhancement of antioxidant activity and thermal stability

Industrial Crops and Products 95 (2017) 60–65

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Chimonanthus praecox extract/cyclodextrin inclusion complexes: Selective inclusion, enhancement of antioxidant activity and thermal stability Shu Zhang, Hongyang Zhang, Zhizhen Xu, Mengqi Wu, Wei Xia ∗ , Wenqing Zhang ∗ Shanghai Key Laboratory of Functional Materials Chemistry, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China

a r t i c l e

i n f o

Article history: Received 22 May 2016 Received in revised form 5 September 2016 Accepted 13 September 2016 Keywords: Chimonanthus praecox Cyclodextrin Inclusion complex Selective inclusion Antioxidant activity Thermal stability

a b s t r a c t In this research, the inclusion complexes of Chimonanthus Praecox extract (CPE) with cyclodextrins (CDs) were prepared. The samples before and after encapsulation were analyzed by UHPLC-QTOF-MS and the variation in the contents of each identified bioactive compound were visualized in the heat maps. It was found that ␤-CD have selective inclusion capacity to flavonoids. Moreover, encapsulation with CDs could significantly improve the antioxidant activity and thermal stability of CPE, enabling application of Chimonanthus Praecox extract as natural antioxidants and/or food additive especially when expected to be thermally processed. Therefore, encapsulation with CDs was a promising way in further application of bioactive compounds in plants. Additionally, this study gives new insight into the inclusion behavior between the complicated guests and different hosts, which provided specific guidance on the choice of bioactive guests with appropriate hosts. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Chimonanthus praecox, famous for its fragrant flowers, is a significant industrial crop native to China, Japan, and some regions of Europe and America (Wang et al., 2011; Kozomara et al., 2008). The species received much attention in folk medicine that is used as treatments for coughs and rheumatic arthritis. Nowadays, the products of Chimonanthus praecox such as fresh cut flower and volatile oil have been widely used in ornamental and cosmetic industries. Especially, in our previous study (Zhang et al., 2016), comprehensive phytochemical profile of Chimonanthus praecox extracts (CPE) has been studied and many bioactive compounds were identified. It is well known that plant bioactive compounds, gaining growing interest recently, have shown the health benefits with antioxidative, anticarcinogenic, antihypertensive, and so on (Belˇscˇ ak-Cvitanovic´ et al., 2011). However, the use of these compounds is restricted since they have limited water solubility and low stability against environmental factors such as temperature, pH and light (Pinho et al., 2014). To circumvent these drawbacks,

∗ Corresponding authors. E-mail addresses: [email protected] (W. Xia), [email protected] (W. Zhang). http://dx.doi.org/10.1016/j.indcrop.2016.09.033 0926-6690/© 2016 Elsevier B.V. All rights reserved.

encapsulation with cyclodextrins has been proven to be a promising approach (Liu et al., 2016). Cyclodextrins (CDs) are a family of cyclic oligosaccharides with six, seven, or eight D-glucose units linked by ␣-1,4-glucose bonds, which are called ␣, ␤, or ␥-CDs. They possess a toroidal shape with the primary hydroxyl groups at the narrow side and the secondary hydroxyl groups at the wide side. CDs show several advantages compared to other ring molecules: they are water-soluble, nontoxic, commercially available compounds with low price and can be functionalized by a wide variety of synthetic methods. Most importantly, CDs, as widely used supramolecular macrocyclic host molecules with a hydrophobic cavity, are known to form inclusion complexes with a great deal of inorganic/organic/biological guest molecules and ions in both aqueous solution and the solid state (Chen and Liu, 2010), which play an important role in supramolecular chemistry, analytical science, food industry, and so forth. Among them, ␤-CDs are being widely used in these fields due to its suitable cavity size, and ␥-CDs are more appropriate to moderate or larger size guest molecules in some cases, while ␣-CDs are limited to complex with some small molecules or long alkyl chains (Simoes et al., 2015). Therefore, the goal of this work was to encapsulate CPE with ␤CD and ␥-CD; analyze and visualize the changes in content of each identified chemical compound in CPE before and after encapsulation; determine the selective inclusion characters to flavonoids;

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and investigate the enhancement of antioxidant activity and thermal stability of CPE. To the best of our knowledge, there is no previous study on the protection of bioactive compounds in Chimonanthus Praecox. In addition, results here may contribute to a further application of Chimonanthus praecox in the production of functional bioactive components.

2. Materials and methods 2.1. Reagents and chemicals ␤-Cyclodextrin, ␥-Cyclodextrin, Formic acid, Rutin trihydrate, Quercetin, Quercitrin, Kaempferol, Hyperoside, Quercitrin, and 2,2-Diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (Shanghai, China). UHPLC grade acetonitrile was purchased from Fisher Scientific (Shanghai, China). Other reagents were purchased from Shanghai Chemical Reagent Co., Ltd. (Shanghai, China).

2.2. Preparation of plant materials Plant materials (Chimonanthus praecox flowers) were collected from Research Base of Chimonanthus praecox, Shanghai, China in January 2016. Identification were supervised by Dr. Du Yongqin (Research Institute of Chimonanthus praecox) and deposited in Shanghai Key Laboratory of Functional Materials Chemistry, East China University of Science and Technology, Shanghai, China. The petal samples were then dried at 40 ◦ C and sieved through a 40mesh sieve. 5 g of powdered samples were mixed with 25-fold methanol and sonicated three times for 1 h. The Chimonanthus praecox extracts (CPE) were then dried by an R-201 rotary evaporator (Senco, Shanghai, China) at 40 ◦ C and stored in the dark at −20 ◦ C for further use.

2.3. Preparation of inclusion complexes In the encapsulation experiment, two inclusion complexes with ␤-CD and ␥-CD were prepared according to the method described by Ding (Ding et al., 2013) and Kalogeropoulos (Kalogeropoulos et al., 2010) with some modifications. The CPE (100 mg) was dissolved into 5 mL methanol, and suspended in 50 mL aqueous solution containing 375 mg ␤-CD (molar ratio based on the hypothesis that the extract was quercetin, Mr of quercetin and ␤-CD ≈ 1:3). Then the solution was stirred for 5 h in the dark at 60 ◦ C and subsequently filtered through 0.45 mm PVDF filter (Anaqua chemical supply, Houston, USA). After this process, the water soluble filtrate was frozen and freeze-dried. The obtained CPE/␤-CD inclusion complex powder was stored under nitrogen at −20 ◦ C until use. The CPE/␥-CD inclusion complex was prepared with the same method mentioned above, except that the same CPE methanol solution was suspended in 50 mL aqueous solution containing 429 mg ␥-CD (molar ratio based on the hypothesis that the extract was quercetin, Mr of quercetin and ␥-CD ≈ 1:3). 2.4. Fourier transform infrared spectroscopy FT-TR spectra were obtained in the frequency range between 4000 and 500 cm−1 using a Nicolet 6700 FT-IR spectrophotometer. Each sample was ground with spectroscopic grade potassium bromide (KBr) powder and then pressed into a 1 mm pellet. The IR spectra of inclusion complexes were analyzed and compared with the spectra of CDs, CPE alone and their physical mixtures.

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2.5. Dissociation of inclusion complexes The inclusion complexes were dissociated to determine the guest according to the method reported by Chen (Chen et al., 2006) with some modifications. The inclusion complex (50 mg) was dispersed into methanol (100 mL). Then the dissociation was aided by sonication for 30 min. This process was repeated for six times. After sonication, the suspension liquid was filtered through 0.22 ␮m PTFE filter (Anaqua chemical supply, Houston, USA) and dried, then stored at −20 ◦ C. The obtained guests are defined as ␤-CPE and ␥-CPE for short, respectively. 2.6. UHPLC-ESI-QTOF-MS analysis Analyses were performed by an Agilent 1290 Infinity UHPLC system (Santa Clara, CA, USA). Separation was achieved on an Agilent Extend C18 UHPLC reversed-phase column (2.1 × 50 mm, 1.8 ␮m). The mobile phases consisted of solvent (A) 0.1% formic acid in water (v/v) and solvent (B) acetonitrile. A linear gradient program at a flow rate of 0.25 mL/min was used as follows: 0 min, 5% B; 0–5 min, 5–10% B; 5–13 min, 10–30% B; 13–18 min, 30–95% B. The injection volume was 1 ␮L, and the column temperature was set to 40 ◦ C. The concentrations of samples were 12 mg/mL. The QTOF-MS was operated in positive mode using an Agilent 6530 ultrahigh definition Q-TOF mass spectrometer (Santa Clara, CA, USA), equipped with an electrospray ionization (ESI) source. The optimized MS spectrometric parameters were as followed: gas temperature 350 ◦ C, flow rate 10 min/L, nebulizer 30 psig, VCap 3500 V, and Fragmentor 150 V. 2.7. Determination of total flavonoid contents The total flavonoid contents were determined according to a colorimetric method described by Lin (Lin and Tang, 2007) and modified in our laboratory. Appropriately sample (5 mL in 70% ethanol solution) was reacted with 1 mL NaNO2 solution (5%) for 6 min, followed by reaction with 1 mL Al(NO3 )3 solution (10%) for another 6 min. Then, 10 mL NaOH solution (4%) were added. The absorbance at 510 nm was measured 15 min later on UV2550 Spectrophotometer and compared to that of rutin standards. The total flavonoid contents were expressed in mg rutin equivalent (RE)/g CPE, ␤-CPE and ␥-CPE. 2.8. In vitro antioxidant capacity A modified DPPH radical scavenging assay (Brahmi et al., 2013) was carried out for determining the antioxidant capacity of CPE and inclusion complexes. Briefly, 1.5 mL of samples in methanol or ultrapure water with different concentrations were added to 1.5 mL of DPPH methanolic solution (0.1 mM). The absorbance of the mixture against methanol or ultrapure water as blank were determined at 517 nm after reacting for 30 min in the dark. Methanol or ultrapure water was used as the negative control. The results were given by IC50 value, which was defined as the sample concentration obtaining a 50% scavenging capacity. Thus, the lower the value, the higher antioxidant activity. IC50 were calculated from the graph of the DPPH•-scavenging percentage against the sample concentration and converted to the amount of guest. 2.9. Thermal studies The enhancement in thermal stability of the CPE by inclusion with CDs was evaluated on NETZSCH- STA409PC synthesized thermogravimetry analyzer by thermogravimetric analysis (TGA). Sample (10 mg) measurements were carried out in alumina pans

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Fig. 1. (A) FT-IR spectra of (a) ␤-CD, (b) CPE, (c) physical mixture, (d) CPE/␤-CD inclusion complex, (B) FT-IR spectra of (a) ␥-CD, (b) CPE, (c) physical mixture, (d) CPE/␥-CD inclusion complex.

under nitrogen atmosphere. TGA analysis was studied in the temperature range of room temperature–600 ◦ C with a heating rate of 10 ◦ C/min. 2.10. Statistical analysis Heat maps were created to visualize the changes in content of each identified compound in CPE before and after encapsulation with CDs using MATLAB 2012a (The MathWorks, Inc., Natick, MA). All analyses and determinations were conducted in triplicate. The results were calculated as mean ± SD. Significant differences were determined by one-way ANOVA, followed by Duncan’s multiple range test using SPSS (Version 19, IBM, USA). Differences at p < 0.05 were considered to be significant. 3. Results and discussion 3.1. FT-IR spectroscopy FT-TR was used to obtain more evidences about the formation of the complexes and the functional groups involved in the inclusion process. Fig. 1A shows the IR spectra of CPE/␤-CD inclusion complex compared with the physical mixture, the CPE alone and ␤-CD alone. Fig. 1A-a shows transmittance spectrum of ␤-CD, which is in agreement with literature data (Barba et al., 2015; Ding et al., 2013; Gomes et al., 2014). A characteristic wide-transmittance band around 3400 cm−1 and another around 1400 cm−1 can be ascribed

to hydroxyl groups stretching vibration. A peak at 2923 cm−1 belongs to the asymmetric and symmetric C H stretching. The 1641 cm−1 band reveals H O H bending of water molecules attached to ␤-CD. The peak at 1157 cm−1 indicated C O C and C C stretching and C OH bending. The 1029 and 1080 cm−1 absorption band can be attributed to C C stretching vibrations. The vibrational bands corresponding to the 942 cm−1 region are related to the skeletal vibration involving the ␣-1, 4 linkage of the ␤-cyclodextrin molecule. The peak at 860 cm−1 corresponds to C C H bending and C O and C C stretching, and the 574 cm−1 is due to the skeletal vibration. CPE (Fig. 1A–b) had several characteristic bands different from CD, such as the peak at 2854 cm−1 indicating symmetric CH2 stretching, 1056 cm−1 revealing stretching of the C O C bond and 1740 cm−1 corresponding to C O bond. The FT-IR spectrum of the physical mixture (Fig. 1A–c) manifests all the peaks described above though slight changes in intensity could be observed, indicating that physical mixture was not sufficient to induce inclusion. In the IR spectrum of ␤-CD inclusion complex (Fig. 1A–d), the 944 cm−1 band was significantly enhanced when compared with ␤CD, indicating the framework vibration in the molecule of ␤-CD was disturbed. The peak at 1028 cm−1 was greatly enhanced, showing that bend vibration of O H in the molecule of ␤-CD was increased (Chen et al., 2006). These results were most likely due to the hostguest interaction. Moreover, the intensity of some characteristic peaks in CPE was suppressed in the spectrum of the inclusion complex. It can be observed that the peak at 2854, 1056, and 1740 cm−1 of CPE are not present in inclusion complex, owing to the existence of strong interactions between hosts and guests hampering some vibration modes (Vilanova and Solans, 2015). Then, the absorption around 3400 cm−1 of inclusion complex shows a stronger intensity than ␤-CD alone, which might indicate the hydroxyl groups of flavonoid outside the cavity of ␤-CD (Li et al., 2015). It is also worth noting that although the CPE was yellow and viscous solid, the inclusion complex was pale yellow powder without stickiness, revealing that the inclusion complex was successfully formed. The FT-TR results of ␥-CD, ␥-CD/CPE mixture and CPE/␥-CD inclusion complex (Fig. 1B) were similar to the ones of ␤-CD, showing that CPE/␥-CD inclusion complex was also successfully formed. 3.2. Identification of the compounds To identify the bioactive compounds, the representative sample (mixture of CPE, ␤-CPE and ␥-CPE) was analyzed by UHPLC-ESIQTOF-MS based on the methods established in our previous work (Zhang et al., 2016). The corresponding extract ion chromatograms (EICs) are shown in Fig. S1. The peaks were well separated and 38 compounds were identified. The MS data of each spectrum are shown in Table S1, affording retention times, accurate mass, molecular formulas and scores. The score value (Gómez et al., 2010) is calculated by the software taking into account both the accurate masses and the isotopic distribution. In this research, peaks with scores above a threshold at 85.00 to ensure the accuracy of formulas’ matching results, have to be checked manually to define the compounds responsible. Four major classes of compounds were found, including flavonoids, coumarins, carboxylic acids and alkaloids. 3.3. Selective inclusion characteristics of CDs Selective inclusion characteristics of CDs were represented by comparing the changes in content of each identified compound using UHPLC/MS, and heat maps demonstrating their fold changes were illustrated in Fig. 2. The fold changes were the ion intensities (peak area) of detected peaks’ mean value ratio of in guest to

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Fig. 2. Heat maps of identified compounds in (A) CPE before encapsulation, (B) CPE after encapsulation with ␤-CD and (C) CPE after encapsulation with ␥-CD. Heat maps show the content changes (red = increased contents, green = decreased contents) as a result of selective inclusion. Each cell in the heat maps represents the fold change of a compound, which is the ratio of the content in guest to CPE.

Scheme 1. Parent nucleus of flavonoids.

CPE. About the inclusion capacity, four classes of compounds varied from each other.

3.3.1. Flavonoids with CDs Flavonoids (Scheme 1) are formed by a series of condensation reactions between hydroxycinnamic acid (B-ring and carbon atoms 2, 3 and 4 of the C-ring) and malonyl residues (A-ring), leading to a C6 -C3 -C6 base structure (Cuyckens and Claeys, 2004). There were majority of the publications reporting the encapsulation of flavonoids with CDs in order to improve their water solubility and stability. As stated in the literature (Kim et al., 2009; Pinho et al., 2014), two major binding modes in flavonoid-CD inclusion complexes have been summarized: (1) The B-ring of flavonoids directs toward the secondary rim while A-ring toward the primary rim of CDs; (2) The A-ring orients toward the secondary rim, in opposite to the first mode. About ␤-CPE, each of the identified flavonoids displayed a higher content than in CPE. For example, Epicatechin had a significant 75.7% increase in ␤-CPE. Meanwhile, analogous increasing trends can also be found in most flavonoids of ␥-CPE. For instance, the content of Naringenin increased by 126.3%. However, as mentioned above, not all the flavonoids in ␥-CPE showed the same increase regularity as compared with ␤-CD. The probable reason might be that the cavity of ␥-CD is larger than ␤CD, causing molecules without certain size more liable to dissociate from the ␥-CD inclusion complexes than the ␤-CD. Thus, they were difficult to form stable inclusion complexes with ␥-CD.

3.3.2. Non-flavonoids with CDs In this part, inclusion behavior of non-flavonoids containing coumarins, carboxylic acids and alkaloids was discussed. With regard to coumarins, a large variety of complexes with CDs have been investigated (Dsouza et al., 2011). However, the change trend in content of each compound in CPE was not uniform. It should be attributed to various structures with hydrophilic groups, such as hydroxyl and amino at both ends of coumarin molecules, preventing the access into cavity of CDs by forming hydrogen bond (Dai and Wu, 2000). Referring to carboxylic acids, it has the similar regular with coumarins. It might depend on several factors such as watersolubility of carboxylic acid molecules, the possible antagonistic relationship (Olga et al., 2015) and the above-mentioned reasons in coumarins. Alkaloids were reported to have a range of action in various biological systems such as scavengers of reactive oxygen (Moura et al., 2007). In our previous study (Zhang et al., 2016), a negative correlation was found between antioxidant capacity and total alkaloid contents. The content of identified alkaloid all reduced in ␤-CPE, on the contrary, content of several alkaloid molecules increased in ␥-CPE. The reason might be that parent nucleus of alkaloids in CPE is too large to be encapsulated into ␤-CD. In general, there were two major factors to determine the selective inclusion characteristics of CPE in CDs. The one was the properties of the host and the guest, such as the relative size of the cyclodextrin to the guest, certain key functional groups in the guest, and the thermodynamic interactions between the different components of the system (Del Valle, 2004). The other one was the possible synergistic or antagonistic coencapsulation among guests. In such complex system of CPE, ␤-CD revealed significant selective inclusion characteristics to flavonoid compounds.

3.4. Determination of total flavonoid contents The total flavonoid contents (TFC) of CPE, guests of inclusion complexes were presented in Fig. 3, expressed as mg rutin equivalent (RE)/g extract or guest. The ␤-CPE had the highest TFC (177.62 ± 3.51 mg/g) than the other ones (p < 0.001). This level was significant different from those in CPE (103.02 ± 3.00 mg/g,

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In summary, different cyclodextrins can achieve various effects. For example, ␤-CD could selectively enrich flavonoids; meanwhile, ␥-CD could enhance antioxidant activity in a much higher degree than ␤-CD. Therefore, different CDs can be chosen for diverse purposes.

3.6. Thermal stability studies

Fig. 3. Total flavonoid contents (TFC) of (A) CPE; (B) ␤-CPE and (C) ␥-CPE. Data are the mean of three independent analyses of each representative variety (mean ± SD; ¨ , p < 0.01;*** ¨ , p < 0.001. n = 3).*¨ ¨indicates significant differences at p < 0.05;** ¨ ¨

Fig. 4. Antioxidant activities of (A) CPE; (B) CPE/␤-CD inclusion complex and (C) CPE/␥-CD inclusion complex. Data are the mean of three independent analyses of each representative variety (mean ± SD; n = 3).*¨ ¨indicates significant differences at ¨ , p < 0.01;*** ¨ , p < 0.001. p < 0.05;** ¨ ¨

p < 0.001) and ␥-CPE (70.77 ± 2.30 mg/g, p < 0.001). It should be noticed that the TFC of ␤-CPE raised by 72.4% than CPE. Therefore, ␤-CD indicated enrichment effect on flavonoid, which exactly coincided with the result in heat maps. Flavonoids are polyphenolic compounds present in many herbs, fruits, and vegetables. These compounds have gained attention because of their antioxidant activity and health-promoting effects by reducing the risk of cardiovascular disease, neurodegenerative disorders, and cancer (Zhang et al., 2013). However, the flavonoid contents of CPE was not high enough. Therefore, the enrichment of flavonoids by cyclodextrin could give insights into the further utilization of Chimonanthus Praecox. This strategy could also be used in other industrial crops to prepare products rich in flavonoids. 3.5. In vitro antioxidant capacity The antioxidant capacity of bioactive compounds can be modified in the presence of cyclodextrins (García-Padial et al., 2013). In the DPPH assay (Fig. 4), the antioxidant activities among three samples were statistically different (p < 0.05). ␥-CD/CPE (44.30 ± 0.46 ␮g/mL) stood out from the others. This level was significant higher than CPE (59.28 ± 0.21 ␮g/mL, p < 0.01). ␤-CD/CPE (54.31 ± 2.98 ␮g/mL, p < 0.05) also indicated much higher antioxidant capacity than CPE. The improvement in antioxidant activity might be ascribed to two reasons. On the one hand, the compounds with high antioxidant activities were enriched by cyclodextrin. On the other hand, the redox behavior of certain molecules is changed due to the access of hydrophobic part into the CD cavity and possibly to the stabilization of the radical oxidation product, causing antioxidant capacity enhancement (Celik et al., 2015).

Thermogravimetric analysis (TGA) is an effective method to study physical and chemical properties of material. For CD inclusion complexes, the thermal stability and the evaporation of the guest molecules shift to a higher temperature upon inclusion complexation due to host-guest interactions (Kayaci and Uyar, 2011). Hence, the TGA and derivative TG (DTG) were performed to evaluate for the CPE and inclusion complexes to determine their thermal stability. In the TGA of CPE (Fig. 5A), the weight loss was started at around 100 ◦ C, associated with the decomposition of compounds in CPE, which indicated that CPE have a volatile nature. This thermal degradation process lasted till the end. TGA thermogram of inclusion complexes (Fig. 5 B and 5C)showed two weight losses: the initial weight loss below 100 ◦ C due to dehydration of water strongly bound to CDs (Yousaf et al., 2015), and the major one between 300 ◦ C and 350 ◦ C showing thermal degradation of inclusion complex, respectively. The TGA profiles of the CPE/CDs inclusion complexes revealed that evaporation of CPE in CDs occurred over a much higher temperature range when compared with pure CPE, demonstrating that the thermal stability of CPE was improved as a result of inclusion complexation with CDs. In addition, the temperatures at which maximum rate of weight loss occurred are described by the position of the peaks in the DTG curve. The DTG curve of CPE exhibited several peaks, and the ones at 128 ◦ C and 176 ◦ C with highest intensity owning to the major degradation process of CPE. Comparing with CPE, the DTG peaks of inclusion complexes were around 320 ◦ C, corresponding to the major weight loss between 300 ◦ C and 350 ◦ C revealed in TGA curves. The observation in TGA and DTG curves indicated that the formation of the CPE/CDs inclusion complexes retarded the weight loss of CPE during heating. Therefore, the thermal stability of CPE was improved when they were included by CDs.

4. Conclusion In conclusion, this is the first report on the encapsulation of Chimonanthus praecox extract with CDs and the characterization of inclusion complexes were investigated. The UHPLC-QTOF-MS heat maps demonstrated a comprehensive image of complexation results and showed a selective inclusion capacity to flavonoids in ␤CD based on host-guest interaction. Therefore, encapsulation with CDs is a promising way used in Chimonanthus praecox and even other industrial crops to prepare products rich in flavonoids. Moreover, it may bring new insight into the inclusion behavior between the complicated guests and different hosts, which provided specific guidance on the choice of bioactive guest with a corresponding host. Additionally, the antioxidant activity and thermal stability of CPE were significantly improved by the interaction with CDs. Thus, cyclodextrin complexation technology have good potential to expand the application of industrial crops. The CPE/CDs inclusion complexes can be further used either to fortify foods, especially those expected to be thermally processed by increasing the shelflife or as a good candidate for natural antioxidants in the industries related to pharmaceutical and functional ingredients.

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Fig. 5. TGA and DTG thermograms of (A) CPE, (B) CPE/␤-CD inclusion complex and (C) CPE/␥-CD inclusion complex.

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