Abstract
Eucalyptus is a fast-growing and high-yield tree species producing approximately one third of the timber of the world. Eucalyptus leaves are a by-product of timber with comparable biomass, and are largely unused. Eucalyptus leaves are rich in polyphenols, of which oenothein B is the most abundant. In this study, the authors developed an ultrasonic-based method for extracting oenothein B from Eucalyptus leaves. The ethanol concentration was proven to be a key determinant for the extraction efficiency and quality of oenothein B. Extracting with an ethanol concentration greater than 20% resulted in the altered chemical structure of oenothein B. The optimized conditions for Oenothein B extraction from Eucalyptus leaves used 10% ethanol and a 1 to 50 (g/mL) material to liquid ratio for 1 h under 40 kHz ultrasonic at a temperature of 87 °C. The highest extraction yield obtained was 12.4%. The oenothein B extract showed the capability of reactive oxygen scavenging. The accumulation pattern of oenothein B during the developmental processes of Eucalyptus leaves was detected using the developed method, and the rapid-accumulation period of oenothein B was determined, which will facilitate the utilization of Eucalyptus leaf resources.
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An Optimized Method for Extracting Oenothein B from Eucalyptus Leaves
Yating Deng,a Ziyi Tan,a Xiaoqi Dang,a Rongjie Mao,a Siyu Long,a Wei Li,b and Meng Li a,*
Eucalyptus is a fast-growing and high-yield tree species producing approximately one third of the timber of the world. Eucalyptus leaves are a by-product of timber with comparable biomass, and are largely unused. Eucalyptus leaves are rich in polyphenols, of which oenothein B is the most abundant. In this study, the authors developed an ultrasonic-based method for extracting oenothein B from Eucalyptus leaves. The ethanol concentration was proven to be a key determinant for the extraction efficiency and quality of oenothein B. Extracting with an ethanol concentration greater than 20% resulted in the altered chemical structure of oenothein B. The optimized conditions for Oenothein B extraction from Eucalyptus leaves used 10% ethanol and a 1 to 50 (g/mL) material to liquid ratio for 1 h under 40 kHz ultrasonic at a temperature of 87 °C. The highest extraction yield obtained was 12.4%. The oenothein B extract showed the capability of reactive oxygen scavenging. The accumulation pattern of oenothein B during the developmental processes of Eucalyptus leaves was detected using the developed method, and the rapid-accumulation period of oenothein B was determined, which will facilitate the utilization of Eucalyptus leaf resources.
DOI: 10.15376/biores.17.4.6094-6107
Keywords: Oenothein B (OeB); Eucalyptus leaves; Ultrasonic extraction; Ethanol; Antioxidant
Contact information: a: Central South University of Forestry and Technology, Changsha, China; b: Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Shenzhen 518071 China; Corresponding author: limeng0422@foxmail.com
INTRODUCTION
Eucalypt is the general name of the species of genus Angophora, Corymbia, and Eucalyptus of the Myrtaceae family, containing a total of 945 species and varieties (Xie 2019). Eucalyptus, as one of the representative fast-growing tree species in the world, has the following characteristics: fast growth rate and short rotation period. It is widely used in the following industries: papermaking, furniture, and wood-based artificial plate, etc. (Qi 2006; Ding et al. 2021). Eucalyptus is a deciduous tree species. In addition to timber production, considerable biomass is also produced in Eucalyptus leaves every year. Looking at the Guangxi province of China as an example, the total output of Eucalyptus timber was greater than 30 million m3, and the total production of Eucalyptus leaves was approximately 2500 tons in 2020. However, these biological resources are not only unused, but they also consume additional costs in waste disposal. Eucalyptus leaves contain a large number of secondary metabolites with bioactivities, which are important resources to be developed and utilized (Mu 2021). Therefore, the development and utilization of Eucalyptus leaves has great potential and is also an important supplement to the Eucalyptus industry.
Eucalyptus leaves are rich in acyl phenols and derivatives, flavonoids, and tannins (Cao et al. 2016a). Among them, oenothein B (OeB) has been identified as one of the most abundant secondary metabolites accumulated in Eucalyptus leaves (Bazylko et al. 2007; Schepetkin et al. 2009; Yoshida et al. 2018). Oenothein B is an ellagic acid tannin composed of two tellimagrandin I units, which form a macrocyclic dimer through binding with glucose and gallic acyl groups via ester bonds and glycoside bonds (Fig. 1) (Santos et al. 2014; Li et al. 2020).
Fig. 1. The chemical structure of OeB
Oenothein B has been reported as having high antioxidant activity, and it considerably reduces the content of reactive oxygen species (ROS) by inhibiting the activities of hyaluronidase and peroxidase in vivo (Toth et al. 2009). In addition to antioxidant activity, OeB also shows an inhibitory effect on a variety of tumor cells. For example, OeB can inactivate the ROS induced PI3K/Akt/NF-κB pathway to restrict the cells in the G1 stage, thus it effectively inhibits the proliferation of lung cancer cell A549 (Pei et al. 2018). The growth of breast cancer cell lines MCF7, MDA-MB-468, and MDA-MB-231 was inhibited by OeB in low concentration and ceased in high concentration (Maruška et al. 2017). Oenothein B also shows antiviral and anti-inflammatory activity; thus, it has been used for the therapy of benign prostatic hyperplasia, urethritis, gastric ulcers, and other diseases (Kadam et al. 2018; Dacrema et al. 2020; Esposito et al. 2021). For example, OeB extracted from Oenothera glazioviana effectively inhibited the invasion of the hepatitis C virus (HCV) (Satoru et al. 2019). In addition, OeB plays a role in neuroprotection and body immunity improvement, which might function through activating the extracellular signal regulated kinase 2 in the brain or enhancing the production of human interferon γ (IFNγ) in T cells (Amakura et al. 2009; Kiss et al. 2011; Ramstead et al. 2012; Okuyama et al. 2021). Taken together, OeB is a plant secondary metabolite with a variety of biological activities, and the high accumulation of OeB in Eucalyptus leaves shows great potential. At present, plant extracts with high content of OeB are mainly used for clinical research and treatment of diseases, for example, the extract of Epilobium angustifolium L. (Esposito et al. 2021; Maruška et al. 2017). Although OeB extract is effective in treating diseases, almost none of the purified OeB was used. It can be seen that it is necessary to separate OeB.
To better use Eucalyptus resources, in this study, the authors developed an ultrasonic-based, simple and low-cost method to extract OeB from Eucalyptus leaves. The effects of various parameters, including the solvent concentration, temperature, material to liquid ratio, and extraction time, on the extraction efficiency and product quality were analysed and optimized.
EXPERIMENTAL
Materials
The experimental materials were collected from the clonal forest of Eucalyptus urophylla × E. grandis grown in the Southern Seedling Base of the China Eucalyptus Research Centre, Zhanjiang City, Guangdong Province. The fresh and healthy leaves of 3-year-old Eucalyptus urophylla × E. grandis with CEPT 1774 were selected, dried at a temperature of 60 °C for 4 h, and then stored in a -70 °C refrigerator for future use. The leaves of CEPT 1774 grow at an annual average temperature of 23.2 °C, annual sunshine hours of 1714 to 2038 h, an annual total solar radiation of 102 to 118 Kcal/cm2, and an annual average rainfall of 1417 to 1802 mm.
Chromatographic Conditions
The chromatographic column was a Diamonsil C18 (2) column (5 µm 250 mm × 4.6 mm). The organic phase was methanol, the aqueous phase was 0.1% formic acid, the column temperature was 30 °C, the injection volume was 10 µL, the flow rate was 1 mL/min, the detection wavelength was 270 nm, the gradient elution program was 0 min to 35 min, the methanol was 10% to 50%, 0.1% formic acid was 90% to 50%; 30 min to 35 min, methanol was 10%, and 0.1% formic acid was 90% (Cao et al. 2016b). The sample was detected via a U3000 ultra-high performance liquid chromatograph (Thermo Fisher, Waltham, MA, USA).
Mass Spectroscopy (MS) Conditions
The column was a Waters Acquity UPLC BEH C18 column (1.7 µm x 2.1 mm × 50 mm). The organic phase was acetonitrile, the aqueous phase was 0.1% formic acid, the injection volume was 10 µL, the flow rate was 0.4 mL/min, the ion source was ESI, the scanning mode was multiple reaction detection (MRM), and the samples were detected via a 6500 Q-TOF instrument (Agilent Technologies, Santa Clara, CA ).
Determination of the Oenothein B (OeB) Extraction Yield
Sample preparation
Approximately 10 mg of Eucalyptus leaf powder was used for liquid phase detection, with different ethanol concentrations, extraction times (min), solid to liquid ratios (g/mL), extraction temperatures (°C), and ultrasonic cleaning at 40 kHz. It was then extracted for 1 h, centrifuged at 4000 r/min for 10 min, the supernatant was removed and passed through a 0.22-μm filter, and then stored at a temperature of 4 °C for subsequent detection.
Preparation of the standard curve
The OeB standard product was provided by the School of Food Science of South China Agricultural University. The standard curve was drawn with the concentration of OeB standard solution (mg/mL) as the X-axis and the peak area (mAu*min) as the Y-axis (linear range 0.0125 to 1 mg/mL).
Calculation
The OeB extraction yield was calculated according to Eq. 1,
where c is the concentration of OeB (mg/mL), v is the volume of the extract (mL), and m is the mass of Eucalyptus leaves powder (mg).
Orthogonal experimental design and analysis of variance
On the basis of a single factor experiment, the ethanol concentration (A), extraction temperature (B), and solid-liquid ratio (C) were selected as the factors to be investigated, and a L9 (34) orthogonal experiment was designed to study the extraction process of Eucalyptus OeB (as shown in Table 1).
Table 1. Factor and Level of the Orthogonal Test
Assessment of Antioxidant Activity
First, the sample stock solution was diluted 8-fold to a concentration of 312.5 μg/mL, and then sequentially halved to 5 sample working solutions of different concentrations, and the scavenging ability of these samples against 1,1-diphenyl-2-picrylhydrazyl (DPPH)/2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) radicals was determined according to Eq. 2,
where AS is the absorbance value of the sample tube to be tested, and AC is the absorbance value of the blank control tube (Li et al. 2021; Zhang 2021).
Statistical analysis
One-way ANOVA was performed on the mean of the experimental data using SPSS ver. 25.0 for Windows (SPSS Inc., Chicago, IL, USA) using the honestly significant difference test of LSD, Duncan’s test, and Dunnett’s test. The level of significance was set to a p-value less than 0.05.
RESULTS AND DISCUSSION
Effect of Ethanol Concentration on Extraction Efficiency of Oenothein B (OeB)
According to its chemical structure, OeB is easily soluble in polar solvents. The authors compared the extraction results among different polar solvents including water, methanol, ethanol, and isopropyl alcohol. The results showed that OeB has the highest solubility in water, while ethanol can improve the purity of the extracting product (data not shown). In addition, the change of the OeB extraction rate was compared under the conditions of no ultrasound (6.2% ±0.04) and ultrasound (8.7% ± 0.03), and the results showed that ultrasound significantly ameliorated the extraction yield of OeB (Fig. S1). The ethanol concentration was reported to greatly affect the extraction amount. For example, the extraction amount of ginsenosides reached the highest (74.3 mg/g) with 55% ethanol. However, the extraction amount was greatly decreased when lower or higher ethanol concentration were used (Yuan et al. 2021). Therefore, different ethanol concentrations were adopted for the OeB extraction. The extracts obtained with ethanol concentration less than 20% showed a single absorption peak, while the products showed a double absorption peak when extracted at 20% or higher ethanol concentrations. The extracts showed a mixed absorption peak when the ethanol concentration reached 50%, which indicated that a high concentration of ethanol introduced impurities or changed the chemical structure of OeB (as shown in Fig. 2).
Fig. 2. Extraction effect of OeB with different ethanol concentrations (standard solution dissolved in 10% ethanol)
After treating OeB with low-concentration sulfuric acid (0.05 mol/L), the macrocyclic structure of OeB was destroyed, and two isomers with m/z of 1591 formed (Hatano et al. 1990). To further reveal the effects of the ethanol concentration on OeB, the authors performed time of flight mass spectrometry (TOF MS) analysis on the extracts obtained at different ethanol concentrations (10%, 30%, and 60%).
Fig. 3. Total ions chromatogram (left) and mass spectrometry (right) detection of OeB extracts with different ethanol concentrations. The OeB standard product dissolved in: 10% ethanol (a); OeB extracted by 10% ethanol (b); OeB extracted by 30% ethanol (c); and OeB extracted by 60% ethanol (d) (Note: The abscissa is time in total ions chromatogram, and the numbers shown in the mass spectrometry are molecular weights)
The structure of OeB is an asymmetric and difficult-to-separate macrocyclic structure, which is difficult to analyse (Bazylko et al. 2007). Therefore, the comparison of the components was based on the molecular weight difference among these extracts. The results demonstrated that a differential component with a molecular weight of 907 was detected in the 30% ethanol extracts, while three differential components with molecular weights of 797, 798, and 959, were detected in the 60% ethanol extracts, and there was no difference was detected between the 10% ethanol extracts and standard sample (as shown in Fig. 3). These results suggested that a high concentration of ethanol might alter the chemical structure of OeB, thus reducing the purity of the OeB extracts. The threshold for the ethanol concentration to ensure the extraction quality of OeB is 20%. Afterward, the authors assessed the extraction yield of OeB at 5%, 10%, and 15% ethanol. The results showed that the extraction yield of OeB reached the highest value (11.1%) when 10% concentration of ethanol was adopted (Fig. 4a). Therefore, 10% was determined as the optimal ethanol concentration for further study.
Fig. 4. Effect of different factors on the OeB extraction yield: the effect of the ethanol concentration on the extraction of OeB (a); the effect of time on the extraction of OeB (b); the effect of the temperature on the extraction of OeB (c); and the effect of the material to liquid ratio on the extraction of OeB (d)
Effect of the Temperature, Material to Liquid Ratio, and Extraction Time on the Extraction Efficiency of Oenothein B (OeB)
The ultrasonic extraction method is widely used because of its simplicity, high efficiency, and low amount of harm. Some studies have used ultrasonic-assisted extraction to extract polyphenols from Lvmaofeng tea. Compared to the extraction yield obtained by conventional water extraction (10.9%), the extraction yield of ultrasonic alcohol extraction was considerably improved (14.5%) (Wang et al. 2021).
Temperature is an important parameter for OeB extraction. Therefore, the authors set the extraction temperature range from 50 to 94 °C. The extraction yield of OeB increased as the temperature increased from 50 to 87 °C, while no major increase was seen when the extraction temperature was greater than 87 °C (Fig. 4b). For the material to liquid ratio, when it ranged from 1 to 10 to 1 to 40, the extraction yield of OeB gradually increased, while no major improvement was observed when the material to liquid ratio was greater than 1 to 40 (Fig. 4c). In addition, the extraction yield of OeB increased as the extraction time was extended, but it did not further increase after 1 h (Fig. 4d).
Optimized Method for OeB Extraction Determined Through Orthogonal Analysis
According to the existing results, the ethanol concentration, temperature, and material to liquid ratio are critical factors for determining the extraction efficiency of OeB. Therefore, the authors performed an orthogonal test on these parameters (Table 1). The factor priority order for OeB extraction is the ethanol concentration, followed by the material to liquid ratio and temperature. Under the conditions of the predicted optimum combination (A2B2C3), the actual extraction yield of OeB from Eucalyptus leaves equaled 12.4% (Table 2). The ANOVA analysis confirmed that the ethanol concentration was the most critical factor for extraction efficiency of OeB (Table 3). Using deionized water, the aerial part of willow leaf was extracted with a solid to liquid ratio of 1 to 100 (g/mL), and an extraction yield of OeB was approximately 7.3% as reported in Granica et al. 2012. Compared with this, the OeB extraction effect of this study was further improved.
Table 2. Orthogonal Test for Extraction
Table 3. The ANOVA Analysis Results of Variance
The Antioxidant Activity of the OeB Extracts
Plants, vegetables, and fruits are rich in polyphenols, which block their own excessive free radical responses to combat adverse environments (Sharma et al. 2019). Phenolic compounds can form coupling compounds with 2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) and 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radicals, and their antioxidant activity can be determined based on the degree of decolorization (Gulcin İ 2020; Ilyasov et al. 2020).
Fig. 5. Scavenging activity of OeB extract against DPPH and ABTS free radical: the scavenging extent of OeB extract on DPPH free radical (a); comparison of ABTS free radical scavenging extent between Vc and the 312.5 μg/mL concentration sample (b); the scavenging extent of OeB extract on DPPH free radical (c); and the comparison of ABTS free radical scavenging extent between Vc and the 312.5 μg/mL concentration sample (d) (Note: 1/8 = 312.5 μg/mL; 1/16 = 156.3 μg/mL; 1/32 = 78.1 μg/mL; 1/64 = 39.1 μg/mL; 1/128 = 19.5 μg/mL; 1/256 = 9.8 μg/mL; 1/512 = 4.9 μg/mL; and Vc = 8.0 μg/mL)
Antioxidant activity is a basic attribute of OeB; thus the authors measured the antioxidant activity of the OeB extracts obtained through this optimized method. The scavenging percentage on the free radicals of OeB extracts were 74.3% ± 0.1% and 83.6% ± 0.1%, indicated by DPPH and ABTS, respectively (as shown in Fig. 5a and 5c). The activity of the scavenging free radicals of OeB is comparable to that of Vc (as shown in Fig. 5b and 5d). Onion polyphenols were extracted with 75% ethanol and when the concentration of onion polyphenols was 0.3 mg/mL, the scavenging rate of the DPPH free radicals reached 86.2% (Cao et al. 2020). Therefore, it was suggested that the extract obtained by this method retained antioxidant activity of OeB.
The Accumulation Pattern of OeB in Eucalyptus Leaves During Development
Eucalyptus leaves at 8 different developmental stages were collected to detect the OeB content using the optimized conditions (as shown in Fig. 6a). The OeB content increased along with leaf development. The OeB content dramatically increased at stages T1 through T3, stabilized in mature Eucalyptus leaves during the T4 through T7 stages, and decreased in aged Eucalyptus leaves at the stage T8 (as shown in Fig. 6b). The pattern of OeB accumulation in Eucalyptus leaves indicated that molecular regulation of OeB biosynthesis is initiated at T1.
Fig. 6. Eucalyptus leaves with different leaf ages and OeB content
CONCLUSIONS
- Ethanol concentration is a key factor affecting oenothein B (OeB) extraction yield of Eucalyptus leaves. Low concentration (< 20%) is conductive to the dissolution of Eucalyptus OeB.
- The optimized conditions for OeB extraction from Eucalyptus leaves were determined to be as follows: 10% ethanol, a 1 to 50 (g/mL) material to liquid ratio, 40 kHz ultrasonic treatment, and a temperature of 87 °C for 1 h.
- The 8 times diluted Eucalyptus OeB reached 74.3% and 83.6% of DPPH and ABTS free radicals, respectively, showing strong antioxidant ability.
- The OeB content dramatically increased at early growth period (TI~T3), stabilized in mature Eucalyptus leaves (T4~T7), and decreased in aged Eucalyptus leaves (T8).
ACKNOWLEDGMENTS
This work was financially supported by the Key Research Projects of Hunan Education Department (Grant No. 20A521), the Natural Science Foundation of Hunan Province (Grant No. 2021JJ31144), and the Innovation Project of Forestry Science and Technology in Hunan Province (Grant No. XLK201984).
Author Contributions
Meng Li: guided the experimental; Yating Deng: wrote the article, methodology, and investigation; Ziyi Tan, Xiaoqi Dang, Rongjie Mao, and Siyu Long: processed the Eucalyptus raw material; and Wei Li: provided the OeB standard products. All authors have read and agreed to the published version of the manuscript.
Conflict of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Data Availability Statement
Data sharing not applicable to this article as no datasets were generated or analysed during the current study.
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Article submitted: July 1, 2022; Peer review completed: August 21, 2022; Revisions accepted: September 6, 2022; Published: September 14, 2022.
DOI: 10.15376/biores.17.4.6094-6107