Elsevier

Industrial Crops and Products

Volume 130, April 2019, Pages 615-626
Industrial Crops and Products

Enzymolysis-ultrasonic assisted extraction of flavanoid from Cyclocarya paliurus (Batal) Iljinskaja:HPLC profile, antimicrobial and antioxidant activity

https://doi.org/10.1016/j.indcrop.2019.01.027Get rights and content

Highlights

  • A suitable and efficient method for the extraction of Cyclocarya paliurus flavonoids was determined.

  • New chemical compositions and their contents of Cyclocarya paliurus flavonoids were identified.

  • Antimicrobial activity of Cyclocarya paliurus flavonoids was studied.

Abstract

This study aimed to describe Enzymolysis-ultrasonic assisted extraction of flavanoid from Cyclocarya paliurus (Batal) Iljinskaja: HPLC profile, antimicrobial and antioxidant activity. An enzymolysis-ultrasonic assisted extraction (EUAE) method was developed and optimized for the extraction of Cyclocarya paliurus flavonoids in liquid form (CPF). The key factor influencing the extraction yield of Cyclocarya paliurus flavonoids was identified by two-level Plackett-Burman Design (PBD) with eight factors. Plackett-Burman Design determined the following four key factors as significant for the extraction yield of Cyclocarya paliurus flavonoids viz. Enzymatic temperature, enzymatic pH, complex enzyme concentration and ultrasonic power. The Box-Behnken Design (BBD) was subsequently applied to optimize the four key factors identified from Box-Behnken Design. Chemical composition of Cyclocarya paliurus flavonoids was investigated by high performance liquid chromatography (HPLC). Antimicrobial activity was carried out with the disc diffusion method. Antioxidant activities were investigated by DPPH assays, O2· assays, ABTS+ assays and the reducing power. The optimal extraction conditions were as follow: enzymatic temperature of 50.71 °C, enzymatic pH of 5.08, complex enzyme concentration of 3.23% and ultrasonic power of 108.03 W. Under the optimal extraction conditions, the extraction yield of Cyclocarya paliurus flavonoids was 34.24 ± 0.32 mg/g, which was well-matched with the predicted value (34.47 mg/g). Isoquercetin, quercetin-3-O-α-l-rhamnoside, kaempferol-3-O-α-l-rhamnoside, quercetin and kaempferol were identified from Cyclocarya paliurus flavonoids with the content of 2.37%, 1.94%, 15.77%, 4.86% and 2.64%, respectively. The inhibition zones diameters of Cyclocarya paliurus flavonoids at the concentration of 80 μg/mL against Staphylococcus aureus, Salmonella and Escherichia coli were 21.5 ± 0.45, 17.5 ± 0.35 and 13.5 ± 0.25 mm, respectively. Results also showed a dose dependent scavenging activity as evidenced by IC50 values for superoxide (0.152 mg/mL) and ABTS+ (0.185 mg/mL) radicals. These results indicated that Cyclocarya paliurus flavonoids could be used as antimicrobial and antioxidant agents applying in pharmaceutical, functional foods and natural cosmetics.

Introduction

Cyclocarya paliurus (Batal.) Iljinskaja (C. paliurus), commonly known as “sweet tea tree”, is also called Cash cow and Maliu due to the round fruit and green leaf. It is widely distributed at 420–2500 m elevation in the mountainous regions of Jiangxi, Zhejiang, Jiangsu, Anhui, Fujian, Taiwan, Hubei, Sichuan provinces (Fang et al., 2011). C. paliurus is the sole species in its genus protected by local governments (Xie et al., 2015a,b). The leaves of C. paliurus were authorized as new food raw material by National Health and Family Planning Commission of China in 2013 (Xie et al., 2016).

In recent years, C. paliurus has gained increasing attention due to the wide range of biological activities, such as anti-hypertensive activity (Wang et al., 2013), anti-hyperlipidemic activity (Ma et al., 2015; Yang et al., 2016), hepatoprotective activity (Yang et al., 2018), anti-inflammatory activity (Jiang et al., 2014), immunomodulatory activity (Xiong et al., 2018) and antioxidant activity (Wang et al., 2015). These activities were attributed to the main active ingredients from C. paliurus leaves, such as flavonoids, polysaccharides, triterpenoids, steroids, saponins and phenolic acids compounds (Xiong et al., 2018). Among these compounds, flavonoids were found to be one of the main active compounds in C. paliurus leaves.

Traditional extracting methods (heating, boiling, and refluxing) usually need too much time and a massive of solvent, and even may cause a loss of flavonoids because of hydrolysis, ionization and oxidation during extraction (Li et al., 2005). Subsequently, ultrasonic-assisted extraction (UAE) was extensively used as the alternative extraction method for flavonoids, the reason being that super agitation allows effective mass transfer between immiscible phases (Liao et al., 2015). In the recent years, another extraction method termed enzyme-assisted extraction (EAE) began to be available. In this extraction process, enzymes can effectively catalyze the degradation of cell walls to cause the release of flavonoids contained inside plant cells (Zhu et al., 2014). Although both of these two methods can significantly improve the extraction yield of target compounds, single method usually requires long extraction time or high energy but low extraction efficacy. Compared with the traditional extraction method, EUAE method has the advantages of time saving, energy saving and high extraction yield. Therefore, UAE coupled with EAE could be an effective method for flavonoids extraction. To the best of our knowledge, the combination of UAE and EAE used in extraction of CPF have not been reported yet. Basically, there are a number of factors affecting extraction efficiency and yield of target compound. The methodology of Plackett-Burman is a powerful and useful tool in searching for the key factors rapidly from a multivariable system (Aa et al., 2014; Dayana and Bakthavatsalam, 2016).

Previously, literatures have demonstrated that CPF exhibited a variety of biological activities, such as free radical-scavenging, diabetes mellitus prevention and antihyperlipidemia (Li et al., 2011; Ma et al., 2015; Xie et al., 2015a,b). Fang et al. (2017) showed that flavonoids from Ilex pubescens presented neuroprotective effect against focal cerebral ischemia/reperfusion injury in rats. Chen et al. (2017a,Chen et al., 2017b demonstrated that Oliv. leaf extract (COE) exhibited significant ameliorative effects on regulating glucose and lipid metabolism in high-glucose-fat diet-fed and streptozotocin-induced diabetic model mice. Jiang et al. (2017) found that flavonoids from sea buckthorn inhibited the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages through the MAPK and NF-κB pathways. In the past decades, flavonoids have played an important role in treating diabetes, obesity and coronary heart disease (Zhang et al., 2010). As a consequence of increasing demands for natural products, CPF are applied to pharmaceutical industries, functional foods and natural cosmetics (Brahmia et al., 2016; Kaya et al., 2016).

The aims of the present study were to investigate Enzymolysis-ultrasonic assisted extraction of flavanoid from Cyclocarya paliurus (Batal) Iljinskaja: HPLC profile, antimicrobial and antioxidant activity. In the present study, single factor experiments were used to establish suitable ranges of multiple extraction variables. These ranges were screened for the key factors influencing the yield of CPF using two-level PBD. After screening the key factors with PBD, a Three-level, four-variable BBD was sequentially employed to optimize EUAE conditions. Nowadays, structural characteristics of CPF have gained increasing attention due to their unique properties, many chemical compositions were identified, such as quercetin-3-O-β-d-glucuronide, quercetin, kaempferol-3-O-β-d-glucuronide, kaempferol-7-O-α-l-rhamnoside and kaempferol (Xie et al., 2015a,b; Xie et al., 2018). However, none of previous studies had focused on antimicrobial activity of CPF. Health-promoting agents were exploited to investigate antioxidant activities, including DPPH radical-scavenging assay, superoxide radical-scavenging assay, ABTS+ radical-scavenging assay and the reducing power assay. High performance liquid chromatography provided a detailed description of the possible functional chemical compositions of CPF.

Section snippets

Plant materials

The leaves of C. paliurus were collected from Xiushui County, Jiangxi Province, China. It was authenticated by Prof. Qingfeng Zhang in Jiangxi Agricultural University of Key Laboratory of Natural Products Research and Development, China. A voucher specimen (No.: KLFSE 36) was deposited at the Key Laboratory of Food Science and Engineering, Jiangxi Agricultural University, China. The leaves of C. paliurus were air-dried and pulverized in a mill before extraction (Xie et al., 2013).

Chemicals and reference compounds

Cellulase (30

Single factor experimental analysis of EUAE

To evaluate the effects of various EUAE conditions on extraction yield of CPF, single factor experiments were carried out. The fixed parameters were set as the follows: enzymatic time of 1.5 h, enzymatic temperature of 50 °C, enzymatic pH of 4.0, complex enzyme concentration of 3%, ultrasonic power of 90 W, ultrasonic time of 30 min, ethanol concentration of 50% and liquid-solid ratio of 15 mL/g.

Conclusion

In this study, the optimal enzymolysis-ultrasonic assisted extraction conditions using combination of Plackett-Burman design and Box-Behnken design were as follows: enzymatic temperature of 50.71 °C, enzymatic pH of 5.08, complex enzyme concentration of 3.23%, and ultrasonic power of 108.03 W. Under the optimal extraction conditions, the extraction yield of CPF was 34.24 ± 0.32 mg/g, which was close with the predicted value (34.47 mg/g) of BBD model. Five chemical compositions were identified

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

The authors gratefully acknowledge the financial supports by National Natural Science Foundation of China (Nos. 31560459, 31160319), Jiangxi Provincial Academic and Technical Leaders Program (20182BCB22003), the Science Funds of Educational Commission of Jiangxi Province, China (Nos. KJLD13027, GJJ13281) and the Graduate Innovative Special Fund Projects of Jiangxi Province, China (No. YC2015-S176).

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