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Supercritical CO2 extraction (SC-CO effect 2 on tiliroside in extraction from content)

2021-11-26 11:17:46 | 日記
Supercritical CO2 extraction (SC-CO effect 2 on tiliroside in extraction from content)
Tiliroside is one of the major flavonoids responsible for the wide range of biological activities of Tilia L. So far, several extraction techniques have been reported for the extraction of this compound L from Tilia. In this work, supercritical CO2 extraction was used for this purpose for the first time. Experiments were carried out using supercritical carbon dioxide and 5% and 10% ethanol as solvents, targeting the recovery content of tiliroside at 45-80°C, 100-220 bar pressure and 20-60 min time. The optimal extraction conditions statistically generated to obtain the highest content of tiliroside were determined as follows: pressure 200 bar, temperature 65°C, 5% ethanol concentration for 45-50 min and pressure 220 bar, temperature 65°C, 10% ethanol concentration for 15 min.

Liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) was used to determine the content of tiliroside in the resulting extracts. In addition, total phenolic (TPC) and flavonoid (TFC) contents and antioxidant activity were determined (DPPH - method).

Keywords: SC-CO2 extraction; tiliroside; flavonoids; linden tree extract; LC-ESI-MS/MS
1 Introduction
Tiliroside (3-O-β-D-(6"-p-coumaroyl)-glucopyranoside) is a glycosidic flavonoid ester that is one of the main components of Tilia L. and is responsible for biological properties of the plant, such as: anti - inflammatory [ 1 ], antibacterial [ 2 ] and anticoagulant activity [ 3 ]. In addition, it has shown antiproliferative and anticancer effects [ 4 , 5 , 6 , 7 ]. Due to its wide range of activities, many scientists are interested in tiliroside and therefore are still developing effective analytical methods for its study.

Many techniques have been used for the extraction of polyphenols, including traditional and modern extraction. The most popular classical polyphenol extraction techniques are soxhlet extraction [ 8 ], maceration [ 8 , 9 ] and percolation [ 10 ]. These methods have been used for more than a century, but some drawbacks have made their application very unprofitable due to overconsumption. Energy, time, large amounts of solvents and thermal decomposition of unstable compounds [ 11 , 12 ]. The disadvantages associated with classical extraction techniques have led to the development of new and more efficient techniques such as ultrasound-assisted extraction (UAE), microwave-assisted extraction (MAE), supercritical fluid extraction (SFE), pressurized liquid extraction (PLE), pressurized hot water extraction (PHWE) [ 13 , 14 ] and supercritical carbon dioxide extraction (SC-CO 2 ) [ 14 , 15 , 16 ].

Supercritical carbon dioxide extraction is an environmentally friendly technology. Carbon dioxide (SC-CO 2 ) is the most commonly used supercritical fluid solvent because of its practical advantages such as inertness, non-toxicity, non-flammability, high purity, low cost and the ability to dissolve lipophilic substances [ 17 , 18 ] ]. In addition, it allows extraction at low temperatures and relatively low pressures [ 19]. Carbon dioxide is mainly used for the extraction of non-polar compounds. However, polar compounds, such as phenolic compounds, have a reduced solubility in supercritical carbon dioxide. The use of co-solvents or polar modifiers has been enhanced by the increased solubility and extraction selectivity of the target compounds, thus allowing to function at lower pressures [ 17 , 20 ]. To date, methanol, ethanol, propanol and acetone have been used for this purpose [ 21 , 22 ]. The addition of this polar co-solvent as a small percentage (1-10%) of CO2 increases its extraction range to include more polar compounds [ 22, 23 ].

So far, several extraction techniques have been reported for phenolic compounds from Tilia [ 24 , 25 , 26 , 27 ], but to our knowledge, no supercritical CO2 extraction has been performed. In this work, the effect of supercritical carbon dioxide extraction conditions on the tiliroside content of the obtained extracts of Tilia linden was analyzed for the first time.

The main subject in this study was the evaluation of the potential linden L. flower extract using SC-CO to obtain 2 solvents by adding ethanol, in terms of the source of tiliroside. The variables temperature, pressure and time were also evaluated to assess the extraction rate.

2. materials and methods
2.1 Plant materials
Tiliae Inflorescentia (Tiliae flos) was purchased from Kawon Herb Company (Gostyń, Poland). According to the information provided by the manufacturer, linden flowers were harvested from Tilia cordata Mill and/or Tilia platyphyllos L. in June and July 2015. The plant material was dried in air and made into powder according to the Polish Pharmacopoeia IX.

2.2 Extraction method
The plant material (about 10 g) was extracted using a multipurpose pilot plant for supercritical fluid extraction manufactured by Waters (Milford, MA, USA) at a flow rate of 10.33 ml/min for CO 2 and 0.49 ml/min for ethanol and 13.65 ml/min for CO 2 and 0.97 ml/min for ethanol for 5% EtOH. /The following ranges were studied; extraction pressures between 100 and 220 bar, temperatures between 45°C and 80°C, and extraction times of 20, 40 and 60 minutes. The extracts are marked with the symbols "Ex. 1-15/5%" for 5% ethanol and "Ex. 1-15/10%" for 10% ethanol. Extraction details are given in Tables 1 and 2.
All extracts obtained were filtered, vacuum evaporated to dryness, and lyophilized in a Free Zone 1 unit (Labconco, Kansas City, KS, USA). The residue was weighed and redissolved in ethanol to obtain a st°Ck solution of suitable concentration.

To prepare samples for LC-MS analysis of flavonoid sapogenins, SPE extraction was used. For this purpose, a portion of the dissolved extract was passed through a °Ctadecyl SPE microcolumn (500 mg, JT Baker Inc., Philipsburg, USA) in methanol (10 ml) and water (10 ml). After loading the sample, individual columns were washed with 5 ml of 100% methanol. All solutions obtained were evaporated to dryness under vacuum and redissolved in a suitable solvent to obtain samples for LC MS/MS analysis.

2.3 Total phenol and flavonoid content (TPC and TFC)
All spectrophotometric measurements were performed using a 96-well clear microplate (Nunclon. Nunc Roskilde, Denmark) and an Infinite Pro 200F ELISA (Tecan Group Ltd., Männedorf, Switzerland).

The total phenolic content (TPC) of the extracts was determined by the Folin-Ci°Calteu method with some modifications [ 28 ]. The absorbance was measured at 680 nm after 20 min of incubation. The total phenolic content was calculated from the calibration curve and the results were expressed as μg of gallic acid equivalent per gram of dry extract.

Total flavonoid content (TFC) analysis was performed using a modified Lamaison and Carnet method [ 29 , 30 ]. The absorbance was read at 430 nm after 30 min of incubation with a blank solution containing methanol instead of the test sample. The total flavonoid content was calculated from the calibration curve and the results were expressed as μg quercetin/g dry extract.

2.4 Antioxidant activity
The DPPH radical scavenging capacity of each extract (1 g per ml of dry plant material) was determined by the 2,2-diphenyl-1-picrylhydrazyl (DPPH - ) assay [ 28 , 31 ].

The absorbance was measured at 517 nm after 30 min of incubation. dPPH - solution was used as a control. the rate of reduction of dPPH - was calculated by the following equation.

where A Ç is the absorbance of the control and A minus is the absorbance of the extract.

2.5 LC-ESI-MS/MS analysis
The contents of tiliroside and flavonoid glycosides were determined by reversed-phase high performance liquid chromatography and electrospray ionization mass spectrometry (LC-ESI-MS/MS). For this purpose, an Agilent 1200 series HPLC system (Agilent Technologies, USA) equipped with a binary gradient solvent pump, degasser, autosampler and column oven was used, which was connected to a 3200 QTRAP mass spectrometer (AB Sciex, USA).

Tiliroside was separated on a Zorbax SB-C18 column (2.1 x 50 mm, 1.8 μm particle size; Agilent Technologies, USA) at 25°C using a 5 μl injection. The solvents used were water (A) containing 0.1% HCOOH and acetonitrile (B). The flow rate was 200 μl/min with the following gradients: 0-0.5 min 10% B; 1-1.5 min 50% B; 2.5-3.5 min 80% B; 4-5.5 min 7-% B; 5-8% 90% B.

Optimal values for source parameters are: capillary temperature 500°C, gas curtain 30 psi, negative ionization mode source voltage -4500 V. Nitrogen is used as the gas curtain and collision gas. The data obtained were processed using Analyst 1.5 software from AB Sciex.

Multiple reaction monitoring (MRM) was used for the quantitative analysis of tiliroside and flavonoid sapogenins. The calibration curves obtained in MRM mode were used for the quantification of the analytes. The identified compounds were quantified based on peak areas and comparison with the calibration curves of the corresponding standards. The linear ranges of the calibration curves were specified and the limits of detection (LOD) and quantification (LOQ) of tiliroside and flavonoid sapogenins were determined.
2.6 Statistical analysis
The results are expressed as the mean ± standard deviation (SD) of three replicates. In addition, the Pearson correlation coefficients between the two components (i.e., total phenolic and flavonoid content) and antioxidant activity were determined.

Calculations and surface plot 3W were performed in STATISTICA 10.0 (StatSoft).

3. results and discussion
Our study aimed to discover the optimal conditions for supercritical CO2 extraction for the maximum increase of tiliroside content from the extracted flowers of Tilia spp.

Although the main drawback of supercritical CO2 extraction is the expensive equipment and analytical process, the possibility of using lower temperatures during the extraction avoids thermal degradation of unstable compounds and makes the method attractive. In addition, the lack of light and oxygen protects the extract from oxidation and loss of biological activity [ 17 , 33 , 34 ].

Due to the non-polar nature of supercritical carbon dioxide, the addition of 5% and 10% ethanol has been used as a polar co-solvent to modify the polarity and increase the solubility.

Thirty different linden extracts were studied. The effect of a range of solvents, temperature, pressure and time on the tiliroside content of the extracts was investigated. The results are expressed as the mean ± standard deviation (SD) of three replicates. The extraction rate was calculated as the percentage of dry extract obtained from 1 g of raw material.

As a fast, simple and reliable analytical tool, tiliroside was determined by LC-ESI-MS/MS method.

The total phenolic content (TPC) and flavonoid content (TFC) of SC-CO 2 extracts and their antioxidant activity measured by DPPH -method were also determined.
Statistical analysis is an efficient way to collect and summarize data. In chemical studies, it is used to summarize (descriptive statistics) experimental data and variance (standard deviation, standard error of the mean, confidence interval or range) and to perform hypothesis testing. Statistics are also very helpful in developing experimental designs and drawing appropriate inferences from the collected data [ 35 ].

Experimental conventional optimization (one method at a time) was performed in order to obtain the maximum amount of tiliroside and other flavonoids in the SC-CO 2 extract of Bodhi tree. In the present study, the independent variables were: extraction time, pressure and temperature (Tables 1 and 2). Another effective variable was the amount of co-solvent (ethanol) obtained by screening experiments, such as 5% and 10% ethanol. The total yield of the extract, the amount of tiliroside, the total phenolic content (TPC), the total flavonoid content (TFC) and additionally the antioxidant activity were the response variables (dependent variables).

The obtained responses are shown in Tables 5 and 6. The average extraction rates obtained using different ethanol concentrations were comparable (1.16% and 1.19% for 5% and 10% ethanol concentrations, respectively). However, the extracts containing 10% ethanol showed a significant increase in tiliroside content, TPC and TFC, especially the tiliroside content increased by about 10-fold.

The tiliroside content at 5% ethanol concentration ranged between 2.121 ± 0.195 µg per gram of dry extract and 9.268 ± 0.145 µg per gram of dry extract. 3/5% and Ex. 13/5%, respectively. So far, for 10% ethanol, the lowest content of tiliroside was 3.742 ± 0.289 μg/g Ex. dry extract. 8/10% and the highest content of tiliroside was 309.033 ± 5.50 μg per g of dry extract. 11/10%. Therefore, the use of higher concentrations of ethanol in the extraction process slightly increased the extraction efficiency and strongly affected the tiliroside content.

Based on the surface plot 3W model, the optimal extraction conditions using 5% and 10% ethanol concentrations can be determined. Figures 1, 2 and 3 relate to 5% ethanol concentrations. The analysis of all extracts (Figure 1) showed that the extraction rate of tiliroside increased with increasing temperature and extraction time. Using a higher pressure
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