Tiliroside exhibits a wide spectrum of effects on the human body; considering expensive synthesis of tiliroside, linden trees seem to be a good source of this compound. For the first time, 46 various extraction methods were developed to receive tiliroside from
Kaempferol–3-O-
Structure of tiliroside (using the ACD/Chemsketch program, version 12.5 (Advanced Chemistry Development, Inc.)).
Tiliroside exhibits a wide spectrum of effects on the human body. The plant materials containing tiliroside show antithrombotic [
Due to its antioxidant and anti-inflammatory properties, tiliroside has been used in the cosmetic industry. One of the available studies has shown that tiliroside does not show high activity against free radicals but may be useful as a UV B blocker [
In combination with sorbitol, tiliroside was used by Merck in the production of a new cosmetic, RonaCare® Tiliroside. According to the manufacturer, the product is mainly intended for dry skin care, shows anti-inflammatory properties, and shields the skin from aching and itching. The above was confirmed by Carola et al. [
Raw material details are shown in Table
Raw material details.
Plant material | Systematic affiliation | Harvest time | Origin |
---|---|---|---|
|
|
June-July 2015 | KAWON company |
Portions of powdered plant material were extracted using various extraction methods, i.e., maceration, maceration with stirring, extraction under reflux, ultrasound-assisted extraction, and accelerated solvent extraction.
Extracts were marked with symbols denoting the individual extraction methods: MAC: maceration, MACst: maceration with stirring, UR: extraction under reflux, ASE: accelerated solvent extraction, UAE: ultrasound-assisted extraction), and the concentration and symbols of solvents are M: methanol, E: ethanol, W: water, DE: diethyl ether, CH: chloroform); additionally the extraction temperatures were provided in brackets (rt: room temperature, 80: 80°C, etc.). Moreover, the influence of addition of acetic acid (AA) and diethyl ether (DE) was studied, and the samples were marked with symbols denoting the compound and its concentration, e.g., MACst-E70% + AA0.1% means the extract obtained by maceration with stirring using 70% ethanol and addition of 0.1% acetic acid.
In the case of maceration, plant material (10 g) was flooded with 100 mL of 70% ethanol, stirred, and put aside for 7 days at room temperature.
Maceration with stirring (10 g of plant material) was conducted at room temperature with the following eluents: water, methanol, and ethanol (50%, 70%, and 100%), 70% and 100% ethanol with addition of acetic acid (0.5, 1, 2, 5, and 10%), 100% ethanol with addition of acetic acid (0.5 and 1%), and 70% ethanol with addition of diethyl ether (5 and 10%). This extraction lasted 2 days, while stirring for 6 hours per day.
The extraction with a boiling solvent under reflux (10 g of plant material) was carried out with 100 mL of 70% ethanol 2 times for 1 hour each (UR-E70%) in duplicate.
One extract was soaked with 9 portions of 100 ml of diethyl ether. Each time, the ether fraction was collected, pooled, evaporated to dryness under vacuum, and lyophilized (UR-DE).
Moreover, the influence of the addition of acetic acid (1%AA) was studied. In this case, 10 g of the plant material was flooded with 100 mL of 70% ethanol with addition of 1% acetic acid. Extraction with a boiling solvent under reflux was carried out 2 times for 1 hour each.
Accelerated solvent extractions (3 times for 10 minutes each) were conducted in the ASE 150 system from Dionex Corporation (Sunnyvale, CA, USA) at 80°C with different methanol and ethanol concentrations (50%, 70%, and 100%) and chloroform (100%).
Moreover, the influence of addition of acetic acid (0.1; 0.5; 1; and 5%) was evaluated.
In the ultrasound-assisted extraction, the plant powder (2 g) was extracted twice (15 minutes each time) with 20 ml of methanol and ethanol (50%, 70%, and 100%). Extractions were performed at room temperature (rt) and at 80°C. The influence of addition of acetic acid (0.5 and 1%) was studied.
In all cases, the extracts obtained were filtered, evaporated to dryness under vacuum, and lyophilized in the Free Zone 1 apparatus (Labconco, Kansas City, KS, USA). The residue was weighed and redissolved in the same solvent as the one used for extraction to obtain stock solutions at suitable concentrations.
All samples were prepared in triplicate.
The total phenolic content (TPC) and total flavonoid content (TFC) were determined using 96-well transparent microplates (Nunclon. Nunc. Roskilde, Denmark) and an Infinite Pro 200F microplate reader (Tecan Group Ltd., Männedorf, Switzerland).
Analysis of TPC was carried out using the Folin–Ciocalteu phenol reagent according to the method described by Olech with some modifications [
The total flavonoid content (TFC) was determined according to the modified Lamaison and Carret method [
The antioxidant assay was determined by the DPPH˙ (2,2-diphenyl-1-picrylhydrazyl) method with some modifications [
The antiradical activity was assessed using an improved ABTS+˙ decolourization assay with modifications [
ABTS was dissolved in water to 8.7 mM concentration. ABTS radical cation (ABTS˙+) was produced by allowing 1.25 ml of ABTS stock solution to react with 5 ml of potassium persulfate (12.2 mM) and allowing the mixture to stand in the dark at room temperature for 12–18 h before use. The ABTS˙+ solution was diluted with methanol (1 : 100).
To determine IC50 of samples, the technique with 96-well microplates was used. Aliquots of 180
The results of antioxidant activity were expressed as Trolox equivalent antioxidant capacity (TEAC) (mM of Trolox per g sample) based on their IC50 values.
Tiliroside was determined by a newly developed, simple, and rapid method using liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS).
For this, an Agilent 1200 Series HPLC system (Agilent Technologies, USA) equipped with a binary gradient solvent pump, a degasser, an autosampler, and a column oven connected to a 3200 QTRAP Mass spectrometer (AB Sciex, USA) was used.
The contents of tiliroside were determined. The compound was separated at 25°C on the Zorbax SB-C18 column (2.1 × 50 mm, 1.8-
The 3200 QTRAP MS/MS system was operated using an electrospray ion source in the negative mode. The optimal mass spectrometer parameters were determined experimentally and were as follows: curtain gas 30 psi, capillary temperature 500°C, nebulizer gas 30 psi, and negative ionization mode source voltage −4000 V.
Nitrogen was used as a curtain and collision gas. The data were acquired and processed using Analyst 1.5 software from AB Sciex, USA.
Multiple reaction monitoring (MRM) was used for quantitative analysis of tiliroside. The compound was identified by comparing the retention time and
Summary of optimized parameters for quantitative analysis of tiliroside.
Compound | Retention time (min) | Q1 ( |
Q3 ( |
DPa (V) | EPb (V) | CEPc (V) | CEd (eV) | CXPe (V) |
---|---|---|---|---|---|---|---|---|
Tiliroside | 6.17 | 302.7 | 124.9 | −45 | −3.5 | −18 | −26 | 0 |
284.8 | −45 | −3.5 | −18 | −14 | −4 |
aDP: declustering potential; bEP: entrance potential; cCEP: cell entrance potential; dCE: collision energy; eCXP: collision cell exit potential.
Analytical parameters of LC-MS/MS quantitative method.
Compound | LOD (ng/mL) | LOQ (ng/mL) |
|
Linearity range (ng/mL) |
---|---|---|---|---|
Tiliroside | 0.5 | 2 | 0.9995 | 2–500 |
Validation of the method for precision was measured by using a standard solution containing tiliroside concentrations covering the entire calibration range.
The method was validated for instrumental precision, intraday precision, and interday precision. The instrumental precision was tested by repeated analysis (
Instrumental precision.
Nominal concentration (ng/mL) | Measured concentration ( |
|
---|---|---|
Mean ± SD | % RSD | |
100 | 104.00 ± 2.32 | 2.2 |
The intraday data reflect the precision of the method under the same conditions within one day. Intraday precision was obtained by analyzing six replicates of three tiliroside standard solution concentrations (50, 100, and 500 ng/mL) (Table
Intraday precision data.
Nominal concentration (ng/mL) | Measured concentration ( |
|
---|---|---|
Mean ± SD | % RSD | |
50 | 51.06 ± 1.04 | 2.0 |
100 | 103.00 ± 1.90 | 1.8 |
500 | 499.80 ± 4.79 | 1.0 |
The interday precision was verified by repeating the above procedure at three different days within the period of one week (Table
Interday precision data.
Nominal concentration (ng/mL) | Measured concentration ( |
|
---|---|---|
Mean ± SD | % RSD | |
50 | 51.01 ± 1.06 | 2.1 |
100 | 103.12 ± 2.90 | 1.0 |
500 | 499.97 ± 4.80 | 0.3 |
Results were expressed as mean ± standard deviation (SD) of three replications for each extract tested. Moreover, the relative standard deviation (RSD) for instrumental, interday, and intraday precision was determined.
Calculations, spread chart 3W, surface chart 3W, and median graph were performed in STATISTICA 10.0 (StatSoft).
In our study, the effects of a particular extraction technique, solvent, additives, and temperature on the tiliroside content were evaluated. For this purpose, 46 different
Tiliroside, as mentioned earlier, possesses anti-inflammatory, antioxidant, anticarcinogenic, cytochrome P450 inhibitory, and hepatoprotective activities. Recently, its anti-diabetic effects have also been reported [
Extraction of the plant material is the first and very important step in the analytical process of various studies dealing with plant secondary metabolites [
To date, the extraction process for the highest content of tiliroside from
The extractive yields vary among different solvents, methods, and conditions of extraction. Table
Tiliroside contents in extracts from
Samples | Efficiency of extraction (%)a | Content of tiliroside (mg per g dry extracts) |
---|---|---|
MAC-E70% | 13.7 | 1.530 ± 0.001 |
MACst-H | 17.4 | 0.142 ± 0.005 |
MACst-E50% | 14.6 | 1.105 ± 0.010 |
MACst-E70% | 17.4 | 1.717 ± 0.005 |
MACst-E100% | 5.3 | 1.289 ± 0.002 |
MACst-E70% + AA0.5% | 18.5 | 5.010 ± 0.024 |
MACst-E70% + AA1% | 18.4 | 5.370 ± 0.033 |
MACst-E70% + AA2% | 20.1 | 2.870 ± 0.011 |
MACst-E70% + AA5% | 21.1 | 2.540 ± 0.010 |
MACst-E70% + AA10% | 19.5 | 2.990 ± 0.009 |
MACst-E70% + DE5% | 15.4 | 3.900 ± 0.013 |
MACst-E70% + DE10% | 13.2 | 3.560 ± 0.012 |
MACst-E100% + AA0.5% | 6 | 2.130 ± 0.008 |
MACst-E100% + AA1% | 6 | 2.160 ± 0.006 |
ASE-M100% (80) | 14.8 | 4.980 ± 0.037 |
ASE-M70% (80) | 22.3 | 4.440 ± 0.028 |
ASE-M50% (80) | 22.0 | 1.371 ± 0.013 |
ASE-CH100% (50) | 1.5 | 0.193 ± 0.001 |
ASE-E100% (80) | 3.3 | 6.810 ± 0.014 |
ASE-E70% (80) | 19.6 | 2.980 ± 0.007 |
ASE-E50% (80) | 8.8 | 2.320 ± 0.012 |
ASE-E70% (80) + AA0.1% | 23.0 | 3.200 ± 0.013 |
ASE-E70% (80) + AA0.5% | 18.9 | 5.160 ± 0.017 |
ASE-E70% (80) + AA1% | 26.4 | 7.400 ± 0.019 |
ASE-E70% (80) + AA5% | 27.8 | 1.802 ± 0.004 |
ASE-E100% (80) + AA0.5% | 18.9 | 5.850 ± 0.012 |
ASE-E100% (80) + AA1% | 10.8 | 5.560 ± 0.015 |
UR-E70% | 23.6 | 1.225 ± 0.006 |
UR-DE | 5.6 | 9.842 ± 0.028 |
UR-E70% + AA1% | 18.1 | 1.821 ± 0.010 |
UAE-M100% (rt) | 3.6 | 4.281 ± 0.084 |
UAE-M70% (rt) | 5.1 | 1.358 ± 0.016 |
UAE-M50% (rt) | 6.6 | 0.829 ± 0.004 |
UAE-E100% (rt) | 2.5 | 3.291 ± 0.059 |
UAE-E70% (rt) | 4.0 | 1.728 ± 0.028 |
UAE-E50% (rt) | 4.1 | 1.244 ± 0.019 |
UAE-M100% (80) | 9.4 | 2.116 ± 0.038 |
UAE-M70% (80) | 9.2 | 1.120 ± 0.019 |
UAE-M50% (80) | 5.4 | 1.650 ± 0.054 |
UAE-E100% (80) | 7 | 2.406 ± 0.001 |
UAE-E70% (80) | 7.5 | 1.607 ± 0.007 |
UAE-E50% (80) | 5.8 | 2.072 ± 0.015 |
UAE-E70% (80) + AA0.5% | 8.4 | 1.848 ± 0.004 |
UAE-E70% (80) + AA1% | 6.6 | 3.212 ± 0.021 |
UAE-E100% (80) + AA0.5% | 2.8 | 3.322 ± 0.012 |
UAE-E100% (80) + AA1% | 4 | 3.368 ± 0.057 |
aCalculated as percentage of dry extract obtained from 1 g of raw material.
The liquid chromatography-electrospray ionization-tandem mass spectrometry method has many advantages. It is a simple, rapid, reliable, and effective analytical tool [
First, the method was validated. The correlation coefficient for tiliroside over the concentration range of 2–500 ng·mL−1 was 0.9995. The limit of detection (LOD) and the limit of quantitation (LOQ) at a signal-to-noise ratio of three and ten (S/N = 3 and 10) for tiliroside were 0.5 and 2 ng·mL−1, respectively (Table
Instrumental precision was tested by repeated analysis (
The intraday and interday precisions for tiliroside standard are summarized in Tables
Table
In the case of ultrasound-assisted extraction (Figure
Spread chart 3W of the content of tiliroside vs. type of solvent and solvent concentration for ultrasound-assisted extraction (UAE).
Similar dependencies were observed for accelerated solvent extraction (Figure
Spread chart 3W of the content of tiliroside vs. type of solvent and solvent concentration for accelerated solvent extraction (ASE).
Noteworthy, the choice of an appropriate solvent is of utmost importance along with application of a compatible extraction method [
Moreover, the analysis of all ethanol extracts (Figure
Surface chart 3W of the content of tiliroside vs. addition of acetic acid and ethanol concentration for all extraction methods (smoothing of smallest squares weighted by distance).
70% ethanol with addition of 1% acetic acid was selected as the best solvent for extraction of tiliroside. Figure
Median graph of the content of tiliroside vs. different extraction methods.
Moreover, for MACst-E70% + AA1%, ASE-E70% (80) + AA1%, UAE-E70% (80) + AA1%, and UR-E70% + AA1% samples, spectrophotometric determination of total phenolic content (TPC) and total flavonoid content (TFC) as well as antioxidant activity tests were performed (Table
Total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity by the DPPH˙ method, and free radical scavenging ability (ABTS+˙) in different extracts (70% ethanol with addition of 1% acetic acid) from
Sample | TPC (mg·GA·g−1 of dry extract) | TFC (mg·Q·g−1 of dry extract) | TEAC (mM·Trolox·g−1 dry extract) |
IC50 (mg·mg−1·DPPH˙) |
---|---|---|---|---|
MACst-E70% + AA1% | 239.09 ± 15.66 | 23.70 ± 0.89 | 1.06 ± 0.03 | 0.25 ± 0.01 |
ASE-E70% (80) + AA1% | 296.96 ± 15.66 | 16.89 ± 0.86 | 1.54 ± 0.05 | 0.30 ± 0.02 |
UAE-E70% (80) + AA1% | 238.69 ± 15.46 | 20.58 ± 0.08 | 1.54 ± 0.02 | 0.25 ± 0.01 |
UR-E70% + AA1% | 181.39 ± 2.62 | 14.05 ± 0.71 | 1.17 ± 0.01 | 0.34 ± 0.01 |
The antiradical activity was assayed using a DPPH• method with some modifications [
The tested extracts also contained a high amount of polyphenols and flavonoids and had high antioxidant activity (Table
In conclusion, the results of this study showed that the extracts of
Our study presented a newly developed method for determination of tiliroside using simple, rapid, and reliable liquid chromatography-electrospray ionization-tandem mass spectrometry, appropriate to industrial applications.
The effective methods to receive tiliroside from
Accelerated solvent extraction is an appropriate method for analytical application. However, due to economic conditions, classical maceration with the same solvents also giving a very active extract with high amounts of tiliroside and other phenolic compounds can be recommended for industrial application.
The data used to support the findings of this study are included within the article.
The authors declare that there are no conflicts of interest regarding the publication of this paper.