Development of a hyphenated procedure of heat-reflux and ultrasound-assisted extraction followed by RP-HPLC separation for the determination of three flavonoids content in Scutellaria barbata D. Don

doi:10.1016/j.jchromb.2013.09.015

Journal of Chromatography B

Volume 940, 1 December 2013, Pages 126-134

https://doi.org/10.1016/j.jchromb.2013.09.015 Get rights and content

Highlights

•
A heat-reflux extraction hyphenated with ultrasound (HUAE) was used to extract three flavonoids from Scutellaria barbata D. Don.
•
HUAE process provides a superior extraction yield and saves time, energy, raw materials and solvent consumption.
•
Contents of flavonoids in samples from different geographical regions were varied significantly.
•
The HUAE may serve as a very promising green technology for pharmaceutical and food industry.

Abstract

A hyphenated procedure of heat-reflux and ultrasound-assisted extraction (HUAE), and an accurate high-performance liquid chromatographic (HPLC) method were developed for the determination of apigenin, baicalin and luteolin content in Scutellaria barbara D. Don. The suitable HUAE conditions for the extraction of target compounds from the herb were identified as an ultrasonic frequency of 40 kHz, power of 185 W, duty cycle of 75% (intermittent sonication), mean particle size of 0.355 mm, extraction temperature of 50 °C, ratio of solvent to raw material of 12:1 (mL/g), ethanol concentration of 60% (v/v), extraction time of 30 min and three cycles. Compared with a traditional heat-reflux extraction method, the proposed method reduced the extraction time, extraction temperature and solvent consumption. Also, this HUAE method achieved superior apigenin, baicalin and luteolin yields. Furthermore, the developed HUAE–HPLC method was applied successfully for the simultaneous evaluation of three bioactive compounds in five samples of S. barbara D. Don obtained from different geographical regions. These results clearly demonstrated that the combined HUAE–HPLC process is feasible in the future commercialized manufacture of this highly valuable Chinese herbal medicine.

Introduction

Scutellaria barbata D. Don is a well-known traditional Chinese medicinal herb (named “Ban zhi lian” in Chinese) that has gained increased attention due to its usage as an antitumor remedy, including use as a therapy for human breast cancer, prostate cancer, leukemia, hepatoma carcinoma, uterine carcinoma, cervix carcinoma, lung carcinoma, skin cancer, colorectal carcinoma, renal adenocarcinoma, pancreatic carcinoma, nasopharyngeal carcinoma and oral epidermoid carcinoma [1]. Phytochemical investigations indicate that S. barbata D. Don contains a variety of medicinally active components, mainly flavonoids, phenolic acids, triterpene acid and sterol glucosides [2]. Our interest is focused on the flavonoids due to the variety of important biological functions of these compounds, such as antitumor, hepatoprotective, antibacterial, anti-inflammatory and antioxidant activities, and the ability to reduce the risk of cardiovascular diseases [3]. Three flavones, namely apigenin, baicalin and luteolin, were reported to be the major bioactive flavonoids in S. barbata D. Don. The chemical structures of the three major flavones are shown in Fig. 1. The three flavonoids occur widely in food consumed by humans. Furthermore, apigenin [4], baicalin [5] and luteolin [6] have been reported to possess cytostatic and cytotoxic activities against many human cancer cell lines. Because of the extensive toxicity to various types of human cancer cell lines accompanied by little or no toxicity to normal tissues, these compounds are attractive as candidates compounds for development as new anticancer drugs. The three above-mentioned flavonoids are the most important anticancer constituents of S. barbata D. Don that contribute to the pharmacological efficacy of the herb.

The selection of an effective chromatographic method for the determination of target analytes is a key aspect of the qualitative and quantitative analysis of the chemicals present in medical plants. The majority of the literature been surveyed that high performance liquid chromatographic (HPLC) is the most convenient and comprehensive technique for separating flavonoids in raw materials [3], [7]. However, information on the use of HPLC for the determination of apigenin, baicalin and luteolin from S. barbata D. Don has rarely been reported. Therefore, the objective of this study was to develop a simple, precise and accurate analytical method for the simultaneous determination of the three flavonoids in S. barbata D. Don.

The extraction of flavonoids from plant tissues has been accomplished by traditional extraction processes, such as Soxhlet and heat-reflux extraction (HRE) [3]. These procedures, however, have distinct drawbacks, such as the consumption of large volumes of solvent and amounts of energy, low yields and lengthy extraction procedures that can result in the loss or degradation of target compounds. Owing to problems related to traditional extraction techniques, interest is growing in the development of simpler, faster and more efficient methods for the extraction of bioactive compounds from natural matrices. Recently, ultrasound-assisted extraction (UAE) has been widely employed in the extraction of target compounds from different materials [3], [8], [9], [10], owing to its facilitated mass transport of solvent from the continuous phase into plant cells. In addition, the energy consumption of UAE could be effectively decreased by employing a lower processing temperature and shorter extraction times.

In comparison with other extraction techniques such as microwave-assisted extraction and supercritical fluid extraction, the equipment of UAE is simpler and economically cheaper. Also, the UAE extracts had better quality when compared with conventional extraction [3], [11]. Therefore, UAE has been recognized for potential industrial application in the phyto-pharmaceutical extraction industry for a wide range of herbal extracts.

Hyphenation and/or combination of different extraction techniques in one single process for sample preparation is one of the most recent strategies in food, chemical and pharmaceutical industries [12]. There already were several attempts to apply hyphenated extraction methods to extract the target compounds from various raw materials [3], [12], [13], [14], [15]. Potential exists for combining ultrasound as an adjunct with the other extraction procedures to enhance the extraction efficiency [13]. Sometimes UAE can be effectively combined with other conventional techniques, which can be produced a significant increasing extraction yields and reduction in extraction time at a lower operational condition than under a conventional extraction method [16]. More recently, large scale UAE designs were proposed for stirred tank systems with temperature control [17]. However, no work has been reported in the literature on using the hyphenated system for the extraction of apigenin, baicalin and luteolin from S. barbata D. Don.

The main objective of this study was to develop a hyphenated procedure of heat-reflux and ultrasound-assisted extraction (HUAE) for the preparation of extracts from S. barbata D. Don and to compare these extracts with those obtained from a conventional solvent extraction technique. The concentrations of target compounds were determined by high performance liquid chromatography (HPLC). It is anticipated that these data will provide the theoretical basis for the design and scale-up of the HUAE process for the extraction of apigenin, baicalin and luteolin from S. barbata D. Don.

Section snippets

Chemicals and reagents

The reference compound, apigenin, baicalin and luteolin were purchased as HPLC reference standards from the Acros Organics (Morris Plains, NJ, USA), Aldrich Chemical Co. (Milwaukee, WI, USA) and Fluka Co. Ltd. (Schnelldorf, Germany), respectively. Methanol, ethanol, acetone, acetonitrile, ethyl acetate, n-hexane and phosphoric acid were bought from Merck Co. (Darmstadt, Germany). Deionized water was prepared using a Milli-Q reverse osmosis unit from Millipore (Bedford, MA, USA).

Plant materials

Three batches of

Chromatographic results

The detection wavelength of 280 nm was first selected by checking the absorption maxima of the three standard compounds dissolved in ethanol with a U-300 UV/visible spectrophotometer (Fig. 2). On the basis of the absorption maxima of the three compounds found from their UV spectra using the diode-array detector, the monitoring wavelength was set at 280 nm. The wavelength was chosen to improve the sensitivity and selectivity for the quantitative analysis of baicalin, luteolin and apigenin and

Conclusions

A hyphenated procedure of heat-reflux and ultrasound-assisted extraction (HUAE) was used to extract apigenin, baicalin and luteolin from S. barbata D. Don and was compared to HRE as a control. The effects of seven experimental factors, including duty cycle of ultrasound exposure, particle size, extraction temperature, extraction time, ratio of solvent to raw material, ethanol concentration and number of extraction cycles, on the HUAE recoveries of the three flavonoids were studied. The extracts

Acknowledgments

We gratefully acknowledge the financial support of the National Science Council of Taiwan and Kaohsiung Medical University, Taiwan (NSC90-2316-B-037-003 & KMU-M097014). We greatly thank Chuang Song Zong Pharmaceutical Co., Ltd (Kaohsiung, Taiwan) for kindly providing and authenticating the plant materials used in this research. The authors would like to thank Miss Hsiang-Hung Wei (National Hsinchu University of Education) for the editorial assistance. Finally, the authors would like to

References (40)

X. Hu et al.
Anal. Chim. Acta
(2008)
Y.C. Yang et al.
Ind. Crops Prod.
(2013)
A. Özmen et al.
Phytomedicine
(2010)
M. Li-Weber
Cancer Treat. Rev.
(2009)
X. Tang et al.
Free Radical Biol. Med.
(2011)
P. Wang et al.
J. Chromatogr. B Anal. Technol. Biomed. Life Sci.
(2010)
T. Xia et al.
J. Food Eng.
(2006)
M.A. Rostagno et al.
Anal. Chem.
(2010)
B. Klejdus et al.
J. Chromatogr. A
(2010)
Y.C. Yang et al.
J. Supercrit. Fluids
(2013)

Y.C. Yang et al.

Ind. Crops Prod.

(2013)

C. Da Porto et al.

Ultrason. Sonochem.

(2009)

K. Vilkhu et al.

Innovative Food Sci. Emerg. Technol.

(2008)

L.H. Santos et al.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

(2013)

H.F. Zhang et al.

Innovative Food Sci. Emerg. Technol.

(2009)

G. Romanik et al.

J. Biochem. Biophys. Methods

(2007)

R. Sun et al.

Ultrason. Sonochem.

(2002)

S. Hemwimol et al.

Ultrason. Sonochem.

(2006)

M.A. Rostagno et al.

J. Chromatogr. A

(2003)

M.K. Khan et al.

Food Chem.

(2010)

Cited by (31)

A modified strategy to improve the dissolution of flavonoids from Artemisiae Argyi Folium using ultrasonic-assisted enzyme-deep eutectic solvent system
2023, Journal of Chromatography A
In this study, enzyme-deep eutectic solvent-assisted ultrasonic extraction technique (EnDUE) was developed for the efficient dissolution of flavonoids from Artemisiae Argyi Folium. The extraction results of Artemisiae Argyi Folium flavonoids (quercetin, luteolin, and isorhamnetin) were used as indicators to investigate the influencing factors through single factor experiment, Placket-burman design, and Box-behnken design, so as to obtain satisfactory yields. After systematic optimization, the optimal conditions for extraction of the target flavonoids were: Choline chloride/1,4-butanediol with a water content of 25%, cellulase+pectinase with a concentration of 1.6%, solid-liquid ratio of 1/32 g/mL, pH of 4.2, ultrasonic frequency of 80 kHz, ultrasonic power of 160 W, ultrasonic temperature of 40 °C, and ultrasonic time of 25 min, respectively, which derived a total yield of 8.06 ± 0.29 mg/g. Compared with the reference techniques, the proposed EnDUE technique showed significant advantages in the yield and extraction efficiency of flavonoids. In addition, after preliminary purification, the Artemisiae Argyi Folium flavonoids showed good antioxidant activity. Deep eutectic solvent (DES) can degrade the cell wall components and increase the action site of enzyme, and enzyme can promote the penetration of DES into the cell wall matrix, which is mutually beneficial to the dissolution of intracellular components. Therefore, the extraction technique proposed in this work (EnDUE) greatly promotes the dissolution of flavonoids from Artemisiae Argyi Folium, and provides theoretical support for the further application of plant flavonoids.
Validation of a greener procedure for the extraction of triterpenic acids from Hedyotis corymbosa
2021, Microchemical Journal
A hyphenated procedure (HUAE) of ultrasound-assisted extraction (UAE) and heat-reflux extraction (HRE) was developed and validated to extract oleanolic acid (OA) and ursolic acid (UA) from Hedyotis corymbosa. High-performance liquid chromatography and ultraperformance liquid chromatography/mass spectrometry were utilized to analyze the extracts. The HUAE procedure not only provided higher yields but also demanded less organic solvent and time at a lower temperature than HRE and UAE alone. Furthermore, the kinetic data of UA obtained from HUAE and HRE are excellently fitted by the second-order kinetic model, indicating that the fitting parameters obtained by HUAE, such as the extraction rate constant, initial extraction rate and equilibrium concentration, were higher than those obtained by HRE. The activation energies derived by HUAE and HRE of UA from H. corymbosa were 9.62 and 11.88 kJ/mol, respectively, showing that the energy barrier required for HUAE was significantly lower than that for HRE. Derivation from Fick's second law indicated that the mass transfer, diffusion coefficients and equilibrium constant acquired from the HUAE of UA were significantly greater than those obtained from the HRE, which further confirmed the superior efficiency of the HUAE procedure. The obtained thermodynamic properties, such as the enthalpy, entropy, and Gibbs free energy, obviously demonstrated that the HUAE and HRE of UA from H. corymbosa were endothermic and irreversible processes; the rate-controlling step was the internal mass transfer.
Separation and quantification of bioactive flavonoids from Scutellaria barbata using a green procedure
2019, Food and Bioproducts Processing
Six bioactive flavonoids, including apigenin, baicalein, baicalin, luteolin, naringenin and wogonin, were extracted from a herbal tea plant, Scutellaria barbata D. Don, using a hyphenated procedure (HUAE) combining heat-reflux extraction (HRE) and ultrasonic-assisted extraction (UAE); the flavonoids were analyzed by high-performance liquid chromatography–mass spectrometry (HPLC/MS). HUAE had the highest flavonoid yield, which was 8.1–9.2% and 18.3–21.1% higher than those of UAE and HRE, respectively, while using shorter extraction times (less than 45 and 165 min) and less extraction solvent (less than 100 and 220 mL). The enhancement of the extraction efficiency obtained by HUAE was mainly due to the combined cavitation, mechanical and thermal effects caused by the ultrasonic energy. It was further found that the experimental data obtained for the total extract of S. barbata via the HUAE and HRE procedures can achieve excellent correlation using the second-order rate model. The second-order rate constant obtained by the HUAE correlation decreased with increasing temperature, which was completely opposite to that of the HRE procedure. However, the remaining two related parameters (the equilibrium concentration of analytes and the initial extraction rate), whether obtained through HUAE or HRE procedures, increased with increasing temperature. In addition, this study also relied on the evaluation of the extraction activation energy and thermodynamic parameters to explain and demonstrate the superiority of the HUAE procedure for the extraction of S. barbata.
Effect of emergent non-thermal extraction technologies on bioactive individual compounds profile from different plant materials
2019, Food Research International
Citation Excerpt :
Bioactive compounds have been extracted by UAE from different plant materials such as Stevia rebaudiana Bertoni (Barba, Grimi, & Vorobiev, 2015; Šic Žlabur et al., 2015), ginseng root (Lu, Yin, & Yu, 2017b; Wu, Lin, & Chau, F.-T., 2001), Hemerocallis citrina baroni (Yang, Geng, Lu, & Fan, 2016), Harpagophytum procumbens (Baghdikian et al., 2016), yarrow (Bashi, Mortazavi, Rezaei, Rajaei, & Karimkhani, 2012), Piper auritum and Porophyllum ruderale (Conde-Hernández & Guerrero-Beltrán, 2014), Withania somnifera (Dhanani, Shah, Gajbhiye, & Kumar, 2017), Salvia officinalis L. (Glisic, Ristic, & Skala, 2011; Zeković et al., 2017), Mentha piperita L., Origanum majorana L. and Chamomilla recutita L. (Hossain et al., 2012; Kowalski, Kowalska, Jamroz, Nawrocka, & Metyk, 2015), Thymus serpyllum L. (Jovanović et al., 2017; Tuzimski, 2010), Aloe vera (Jawade & Chavan, 2013), Datura innoxia Mill. ( Oniszczuk, Waksmundzka-Hajnos, Gadzikowska, Podgórski, & Oniszczuk, 2013), Equisetum arvense L. (Oniszczuk et al., 2014), Rosmarinus officinalis (Paniwnyk, Cai, Albu, Mason, & Cole, 2009), Artemisia selengensis Turcz (Peng, Jia, Wang, Zhu, & Chen, 2010), Berberis koreana bark (Rodríguez-Rojo, Visentin, Maestri, & Cocero, 2012), Achillea biebersteinii and Achillea wilhelmsii, (Salarbashi et al., 2014), Gymnema sylvestre (Sheoran, Panda, Admane, Panda, & Wajid, 2015), Angelica dahurica (Wang, Mei, Wang, Liu, & Chen, 2011), Artemisia annua L. (Wang et al., 2011), Scutellaria barbata D. Don (Wei, Yang, Chiu, & Hong, 2013), Hedyotis diffusa and Hedyotis corymbosa (Wei & Yang, 2015), Euryale ferox Salisb. ( Wu et al., 2014), Ligusticum chuanxiong and Hort Angelica (Yang, Chen, Lee, & Wang, 2008), Rabdosia rubescens (Yang & Wei, 2015; Yang, Wei, & Huang, 2012) and Ginseng Radix, Astragali Radix and Dendrobii Officinalis Caulis (Yip et al., 2016) (Table 3).
Extraction is the first step for isolation and purification of interesting bioactive compounds, by mixing of the plant material with an adequate solvent. Those bioactive compounds are, usually, secondary metabolites, such as phenolic acids and flavonoids which are present in closed insoluble structures, making its extraction a challenge. There are many different traditional extraction methods, such as Soxhlet, heat reflux, and maceration. Nevertheless, due to several disadvantages, they are being replaced by new methods, using emergent technologies, such as high hydrostatic pressure, ultrasounds, pulsed electric fields, and supercritical fluids. The use of novel technologies allows enhancing mass transfer rates, increasing cell permeability as well as increasing secondary metabolite diffusion, leading to higher extraction yields, fewer impurities on the final extract, extractions at room temperature with thermo-sensitive structures preservation, use of different non-organic solvents, low energy consumption, short operation time, and have no significant or lower effect on the structure of bioactive compounds. This paper aims to review the effect of the main emergent extraction technologies (high hydrostatic pressure, pulsed electric fields, ultrasounds, and supercritical fluid assisted) on the individual profile of bioactive compounds from plant material.
Development and characterization of a green procedure for apigenin extraction from Scutellaria barbata D. Don
2018, Food Chemistry
Citation Excerpt :
Apigenin is among the major bioactive flavonoids purified from S. barbata D. Don that contribute to plant pharmacological efficacy. Flavonoids have minimal toxicity and have many important pharmacological activities, including antitumor, hepatoprotective, antibacterial, anti-diabetes, anti-inflammatory and antioxidant activities and the ability to reduce the risk of cardiovascular diseases (Wei, Yang, Chiu, & Hong, 2013; Xie et al., 2017; Yang, Wei, Huang, Lee, & Lin, 2013; Yang, Wei, Huang, & Lee, 2013). Several techniques have been used to extract flavonoids from plant matrices, including maceration (Albuquerque et al., 2017), supercritical CO2 extraction (SC-CO2) (Martinez-Correa et al., 2017), heat-reflux extraction (HRE) (Yang, Wei, Huang, et al., 2013; Wei et al., 2013; Bosso, Guaita, & Petrozziello, 2016), ultrasound-assisted extraction (UAE) (Yang, Wei, & Huang, 2012; Albuquerque et al., 2017; Moorthy et al., 2017), microwave extraction (Albuquerque et al., 2017; Xie et al., 2017) and pressurized hot water extraction (Liau, Ponnusamy, Lee, Jong, & Chen, 2017).
This study compared the use of ultrasound-assisted supercritical CO₂ (USC-CO₂) extraction to obtain apigenin-rich extracts from Scutellaria barbata D. Don with that of conventional supercritical CO₂ (SC-CO₂) extraction and heat-reflux extraction (HRE), conducted in parallel. This green procedure yielded 20.1% and 31.6% more apigenin than conventional SC-CO₂ extraction and HRE, respectively. Moreover, the extraction time required by the USC-CO₂ procedure, which used milder conditions, was approximately 1.9 times and 2.4 times shorter than that required by conventional SC-CO₂ extraction and HRE, respectively. Furthermore, the theoretical solubility of apigenin in the supercritical fluid system was obtained from the USC-CO₂ dynamic extraction curves and was in good agreement with the calculated values for the three empirical density-based models. The second-order kinetics model was further applied to evaluate the kinetics of USC-CO₂ extraction. The results demonstrated that the selected model allowed the evaluation of the extraction rate and extent of USC-CO₂ extraction.
Functional components in Scutellaria barbata D. Don with anti-inflammatory activity on RAW 264.7 cells
2018, Journal of Food and Drug Analysis
The objectives of this study were to determine the variety and amount of various functional components in Scutellaria barbata D. Don as well as study their anti-inflammatory activity on RAW 264.7 cells. Both ethanol and ethyl acetate extracts were shown to contain the functional components including phenolics, flavonoids, chlorophylls, and carotenoids, with the former mainly composed of phenolics and flavonoids, and the latter of carotenoids and chlorophylls. Both extracts could significantly inhibit (p < 0.05) the production of lipopolysaccharide-induced nitric oxide, prostaglandin E₂, interlukin-6, and interlukin-1β, as well as the expressions of phosphor extracellular signal-regulated kinase and phosphor-c-Jun N-terminal kinase (p-JNK), but failed to retard tumor necrosis factor-α expression. Both ethanol and ethyl acetate extracts had a dose-dependent anti-inflammatory activity on RAW 264.7 cells. Furthermore, the anti-inflammatory efficiency can be varied for both ethanol and ethyl acetate extracts, which can be attributed to the presence of different varieties and amounts of functional components, as mentioned above. This finding suggested that S. Barbata extract may be used as an anti-inflammatory agent for possible future biomedical application.

View all citing articles on Scopus

View full text

Development of a hyphenated procedure of heat-reflux and ultrasound-assisted extraction followed by RP-HPLC separation for the determination of three flavonoids content in Scutellaria barbata D. Don

Highlights

Abstract

Introduction

Section snippets

Chemicals and reagents

Plant materials

Chromatographic results

Conclusions

Acknowledgments

Anal. Chim. Acta

Ind. Crops Prod.

Phytomedicine

Cancer Treat. Rev.

Free Radical Biol. Med.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

J. Food Eng.

Anal. Chem.

J. Chromatogr. A

J. Supercrit. Fluids

Ind. Crops Prod.

Ultrason. Sonochem.

Innovative Food Sci. Emerg. Technol.

J. Chromatogr. B Anal. Technol. Biomed. Life Sci.

Innovative Food Sci. Emerg. Technol.

J. Biochem. Biophys. Methods

Ultrason. Sonochem.

Ultrason. Sonochem.

J. Chromatogr. A

Food Chem.