This study aimed to investigate the composition of three major stilbenes (mulberroside A, oxyresveratrol, and resveratrol) in different portions of mulberries collected in different seasons and their change molds during growth by high-performance liquid chromatography. Mulberroside A levels were the highest in the bark and roots of
Stilbenic compounds (mulberroside A, oxyresveratrol, and resveratrol) (Figure
Structures of compounds
Acetonitrile was of HPLC grade (Tedia, USA). Methanol and alcohol were of analytical grade from Tianjin Ke-Miou Reagent Company (Tianjin, China). Ultrapure water was purified by Milli-Q system (Millipore, Bedford, MA, USA). Formic acid was of analytical grade from Tianjin Fuyu Reagent Company (Tianjin, China). Mulberroside A (123-100311), oxyresveratrol (2009031102), and resveratrol (111535-200502) were from Tianjin Kuiqing Reagent Company (Tianjin, China), Mingyuan Reagent Company (Tianjin, China), and Meidi Reagent Company (Zhejiang, China) and were all proved to be above 98% by HPLC analysis. All samples of mulberries were gathered from the Hunan Institute of Sericulture (Changsha, China) and authenticated by Professor Yan XP of the Hunan Institute of Sericulture (Changsha, China).
An Agilent 1100 liquid chromatography system (Agilent Technologies Deutschland, Waldbronn, Germany), armed with a quaternary solvent delivery system and ultraviolet detector, was used. All analyses were performed with a Hypersil BDS C18 column (200 mm × 4.6 mm, 5
Reference compounds
Linear relation between peak area and concentration (
Compound | Regression equationa |
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Linear range ( |
LODc (ng mL−1) | LOQd (ng mL−1) |
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0.9999 | 0.64–404.40 | 2.60 | 8.67 |
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0.9999 | 0.71–444.00 | 2.10 | 7.00 |
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1 | 0.72–448.00 | 0.95 | 3.17 |
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cLOD: limit of detection.
dLOQ: limit of quantification.
Randomly selected samples of mulberry were first air-dried, milled into powder, dried at room temperature until constant weight, and then passed through a 40-mesh sieve, followed by ultrasonic extraction with 25 mL (for 0.5 g) of 60% methanol for 40 min. After that, the solvent was again added to the resultant mixture to make it equal to the original weight prior to the ultrasonic extraction, followed by filtering the supernatant through a 0.45
Methods to optimize the extraction conditions, calibration graphs, limit of detection (LOD) and quantification (LOQ), and method of validation and application were adapted from the previously reported systems [
In the preliminary study, we found that, compared with other methods, ultrasonic extraction was more effective with less interference. Different concentrations of methanol and ethanol were tested for their efficiency as a solvent. As 60% of methanol as a solvent produces the highest yields for all constituents, it was chosen in the current study. The impact of the length of the extraction time on the efficiency of extraction was evaluated as well. Tested with 60% methanol for 10, 20, 40, and 60 mins, respectively, powdered samples extracted the highest amount of constituents when treated for 40 mins. When column temperature was maintained at 40°C instead of 20 or 30°C, optimized separation was achieved. Various mobile phase compositions were also tested. Results show that water/acetonitrile mixture, not methanol/water mixture, can obtain satisfactory resolution. Addition of acid (0.5% formic acid, 0.5% acetic acid, and 1.0% formic acid) in the mobile phase improves resolution and reduces the peak tailing of the target compounds, with the best results obtained when using acetonitrile/water mixture with 1.0% formic acid. The previously mentioned optimized conditions were used in the current study. According to the absorption maxima of three standards on the ultraviolet spectrum, with three-dimensional chromatograms of HPLC-DAD detection, the wavelength of 320 nm was used in the study.
The concentration of the compounds was determined by external standard method. Linear regression analyses for each compound were conducted by plotting the peak area versus concentration. The calibration curve for each compound was composed of six points representing six different concentrations in triplicate. The results are shown in Table
The LOD and LOQ for each compound under the chromatographic conditions were obtained by measuring the amount of analytical background (Table
The repeatability of the method was tested by intra- and interday variability. Six replicate samples were extracted and analyzed within one day to determine the intraday variability, and the same sample was used on six independent days to obtain the interday variability. The quantity of each ingredient in the sample was determined from its corresponding calibration curve. The relative standard deviation (RSD) obtained by six replicated injections of the solution was taken as a measure of method repeatability. As shown in Table
Intra- and interday repeatability and recovery of the three major stilbenes in mulberry.
Compound | Repeatability ( |
Recovery ( |
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Intraday |
R.S.D. (%) | Interday |
R.S.D. (%) | Mean |
R.S.D. |
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1.1 |
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1.3 | 103.5 | 2.4 |
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1.1 |
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1.7 | 102.4 | 1.9 |
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1.5 |
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2.3 | 100.0 | 2.3 |
bCalculated as detected amount/added amount × 100%. Data were means of six experiments.
cR.S.D. (%) = (S.D./mean) × 100.
The recovery test was done by spiking a solution containing known quantities of the standard and known amounts of powdered mulberry samples, mixed prior to extraction. The standard solutions with their concentration levels in the middle part of the calibration curve and six fortified samples were applied. The recovery rate of the method was 100.0–103.5%, with RSD less than 2.5%, suggesting that the method is accurate (Table
Stability of the solutions of the samples was tested by comparing sample solutions that were kept at room temperature with the standard solutions every 2 h within 24 h, and we found that the sample solutions were stable within 12 h (
All three stilbenes in mulberries were evaluated by a developed analytical method as described in what follows. Peaks in the achieved chromatograms were recognized by comparing the retention times and ultraviolet spectra with those of standard solutions. Representative chromatograms are shown in Figure
HPLC chromatograms of standard mixture (a), leaves of
The complete summary of the results of mulberroside A, oxyresveratrol, and resveratrol from different portions of
Contents of mulberroside A, oxyresveratrol, and resveratrol in the different portions of
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Mulberroside A | Oxyresveratrol | Resveratrol | Mulberroside A | Oxyresveratrol | Resveratrol | Mulberroside A | Oxyresveratrol | Resveratrol | |
Leaves | nd | nd | nd | nd |
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nd | nd |
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nd |
Stem |
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Bark |
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Roots |
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nd: not detected.
Contents of mulberroside A, oxyresveratrol, and resveratrol in the different portions and different seasons of
Sample | Mulberroside A ( |
Oxyresveratrol ( |
Resveratrol ( | |||||||||
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March | June | September | December | March | June | September | December | March | June | September | December | |
Leaves | nd | nd | nd | nd |
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nd |
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nd |
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Stem |
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Bark |
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Roots |
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Pith |
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Tuber |
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nd: not detected.
Contents of components of
Sample | Mulberroside A |
Oxyresveratrol |
Resveratrol |
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Leaves (7th day) | nd |
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Leaves (9th day) | nd |
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Leaves (11st day) | nd |
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Leaves (13rd day) | nd |
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Leaves (15th day) | nd |
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Leaves (17th day) | nd |
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Leaves (20th day) | nd |
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Roots (7th day) |
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Roots (9th day) |
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Roots (11st day) |
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Roots (13rd day) |
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Roots (15th day) |
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Roots (17th day) |
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Roots (20th day) |
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nd: not detected.
In this study, we found that mulberroside A was richest in bark and roots in September, oxyresveratrol was richest in roots and stem in September as well, and resveratrol was very low in all. Mulberroside A levels were the highest in the bark and roots of
Although they have been found in mulberry wood [
Glycosylation of polyphenolic compounds is a common feature in plants, which can enhance the stability of compounds [
The major role of stilbenes in a number of plant families, such as peanut, mulberry, and grapevine, is working as phytoalexins [
The formation of stilbene phytoalexins involves the phenylalanine/polymalonate route (Figure
Biosynthesis of resveratrol via the phenylalanine/polymalonate pathway. Phenylalanine ammonia lyase (PAL); tyrosine ammonia lyase (TAL); cinnamate-4-hydroxylase (C4H); 4-coenzyme A ligase (4CL); stilbene synthase (STS). Adapted from Jeandet et al. [
In order to get new insight about stilbene biotransformation, we examined three major stilbenes in leaves and roots of seedlings of mulberry. This is the first time, to the best of our knowledge, that the amount of mulberroside A, oxyresveratrol, and resveratrol in
The levels of the stilbenes vary in different parts of varieties of mulberries collected in different seasons and in the seedlings of
The authors thank the National Key Technology R&D Program (2012BAD36B07), Hunan Provincial S&T Project (2010FJ1010-3, 2012FJ4291), the project of 12th—five of Hunan Province in Key Disciplines of Pharmacy (2011) no. 76, and the Research Program of the Education Department of Hunan Province (10A091) for financial support of this work. The authors sincerely thank Professor RAO LQ. (School of Bioscience & Biotechnology, Hunan Agricultural University) for his kind help.