Introduction

Polysaccharides, widely distributed in plants, microorganisms, and animals, are considered as important biological response modifiers. They play important roles in cell–cell communication, cell adhesion, and molecular recognition in the immune system [1]. Plant polysaccharides are widely recognized as excellent candidates for health and therapeutics. In recent years, many polysaccharides derived from natural plants have been demonstrated to have bioactivities, such as antioxidant, hypolipidemic, immunomodulatory, and anticancer activities [2–5]. The bioactivities of polysaccharides are highly dependent on their unique structures. These structural features include monosaccharide composition, molecular weight, glycosidic bonds, degrees of branching, and tertiary conformation [6–8]. For example, many studies have shown that the anticancer activity and immunomodulatory activity of polysaccharides were related to the (1→3)-β-glucan and (1→6)-glucan in molecule [9, 10]. Low and moderate molecular weight polysaccharides had stronger antioxidant activities [11–13]. Therefore, searching novel polysaccharides with beneficial bioactivities from plant resources has become a hot research topic.

Prunella vulgaris L., known as ‘self-heal,’ is a perennial herb commonly cultured in China, Korea, Japan, and Europe [14]. P. vulgaris, called ‘Xiakucao’ in Chinese, has been used as a health food or traditional Chinese medicine. It has been popularly used for the treatments of jaundice, hepatitis, tuberculosis, mammitis, diabetes mellitus, and hypertension [15]. It is also used to be popular beverages (such as herbal tea) in southern China. In Europe, it was used as food and tea. Phytochemical studies have shown that P. vulgaris contains many kinds of bioactive chemicals, primarily polysaccharides, terpenoids, flavonoids, and saponin [16]. Previous studies have demonstrated that polysaccharides isolated from P. vulgaris have anti-HIV activity and immunomodulatory effects [17, 18].

Extraction is a very important stage in the isolation, identification, and use of bioactive materials. Hot water extraction has been widely used to extract plant polysaccharides. However, the conventional hot water extraction requires a long time, high temperature, and extended extraction times [19]. Therefore, it is essential and desirable to find out an economical and more efficient extraction method. Recently, some novel extraction techniques have been developed for the extraction of nutraceuticals from plants, including ultrasonic extraction, microwave-assisted extraction, supercritical fluid extraction, and solvent extraction. Among these, ultrasonic extraction is an inexpensive, simple, and efficient alternative to conventional extraction methods [20]. Extraction enhancement by ultrasound is mainly attributed to the propagation of ultrasound pressure waves and resulting cavitation forces. Ultrasound offers a mechanical effect causing the cell wall rupture and improving the release of intracellular substances into the solvent [21, 22].

However, there have been no reports on the ultrasonic extraction of polysaccharides from P. vulgaris and evaluation of their antioxidant and antiproliferative activities. Therefore, in this work, an ultrasonic method was employed to extract polysaccharide from P. vulgaris. The extraction conditions were optimized using response surface methodology. The chemical composition and structural characteristics of Prunella vulgaris polysaccharides (PVP) were investigated. In addition, the antioxidant and antiproliferative activities of PVP were evaluated.

Materials and methods

Plant material

The fruit clusters of P. vulgaris (7.7 × 50 mm of a fruit cluster, harvested in Hubei Province, P. R. China) were obtained from Qingping medicinal material market (Guangzhou, China). The fruit clusters were dried by oven and ground into powder by a cutting mill (FW100, Taisite Instrument Co., Ltd, Tianjin, China) and stored at room temperature in a desiccator for further use.

Chemicals and reagents

Fluorescein disodium salt, (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), monosaccharide standards, 2,2ʹ-azobis(2-amidinopropane) (ABAP), dichlorofluorescein diacetate (DCFH-DA), and bovine serum albumin (BSA) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). Trifluoroacetic acid (TFA), galacturonic acid, and glucuronic acid standards were purchased from Aladdin Chemistry Company (Shanghai, China). l-Glutamine was purchased from Guangzhou Feibo Biotechnology Co., Ltd (Guangdong, China). Dulbecco’s modified Eagle’s medium (DMEM), phosphate-buffered saline (PBS, pH 7.4), Hank’s balanced salt solution (HBSS), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco (Long Shang Industry Park, Beijing, China). All other chemicals used were of analytical grade.

Ultrasonic extraction of polysaccharides from P. vulgaris

The dried P. vulgaris power (300 g) was pretreated twice with 1 L of 95 % (v/v) ethanol at 70 °C for 4 h to de-color and remove soluble materials and dried at 50 °C for 24 h.

The dried residues (4.00 g) were weighed and extracted with 100 mL of distilled water. The extraction process was done using an ultrasonic cleaner (KQ-300DE, Kunshan Ultrasonic Instruments Co., Ltd, China), with different ultrasonic power, temperature, and time. The setting temperature was controlled using a circulating bath. The supernatants were collected by centrifugation (DL-5C, Anting Scientific Instrument Co., Shanghai, China). After two rounds of above extraction, the supernatants were combined and concentrated to 25 mL under reduced pressure at 55 °C. The proteins in the extract were removed using the Sevag reagent (chloroform: n-butyl alcohol = 4:1, v/v) [23]. This process was repeated five times. The resulting solution was adjusted using 95 % ethanol to a concentration of 75 % (v/v) ethanol and kept overnight at 4 °C. It was then separated by centrifugation. The precipitates were freeze-dried at −50 °C to obtain crude polysaccharides. The content of polysaccharides was determined by the phenol–sulfuric acid method [24]. The polysaccharide yield (%) is calculated as follows:

$$ {\text{Yield}}\,(\%) = \frac{\text{Weight of dried crude polysaccharide (g)}}{{\text{Weight of }}{P. \, vulgaris}{\text{ on dry basis (g)}}} \times 100$$
(1)

Experiment design

The software Design Expert (Trial Version 7.1.3, State-Ease Inc., Minneapolis, MN, USA) was used for generating the experimental design, data analysis, and model building. A central composite design was employed for optimization. Ultrasonic power (A), extraction time (B), and temperature (C) were set as independent variables, while the extraction yield of PVP was the dependent variable.

The variables and their levels are shown in Table 1. Experiment was randomized to maximize the effects of unexplained variability in the observed response. The nonlinear computer-generated quadratic model used to fit the data is given as follows:

$$Y\text{ = }\beta_{\text{0}} + \sum\limits_{{j\text{ - }1}}^{K} {\beta_{j} } X_{j} \sum\limits_{{j\text{ - }1}}^{K} {\upbeta_{ij} X_{j}^{2} + \sum {\sum {\mathop {\beta ij}\limits_{{i\text{ < }j}} X_{i} X_{j} } } }$$
(2)

where Y is the response variable (yield of polysaccharides); β 0, β j , β jj , and β ij are the regression coefficients for the intercept, linear, square, and interaction terms; X i and X j are the independent coded variables.

Table 1 Central composite design and the response for the extraction yield of PVP
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Preliminary purification of PVP

The crude PVP was obtained under the optimal extraction condition. The extract was collected and concentrated to approximately 1/4 of its initial volume under reduced pressure at 55 °C. The proteins in the extract were removed using the Sevage method. This process was repeated five times. Phenolic compounds and anthocyanins were removed using macroporous resin D101 (Anhui Sanxing Resin Technology Co., Ltd, China) at a ratio of resin/solution 1:2 (v/v) at 35 °C for 2 h. The resulting solution was adjusted to a concentration of 75 % (v/v) ethanol using 95 % ethanol and kept overnight at 4 °C. Then, the precipitate was collected by centrifugation, washed with anhydrous ethanol twice, and finally lyophilized to obtain PVP. The carbohydrate content was determined by the phenol–sulfuric acid method with glucose as the standard. The protein content was determined by the method of Lowry with BSA as the standard [25].

Determination of molecular weight

The molecular weight distribution of PVP was measured using high-performance gel permeation chromatography (HPGPC) instrument (Agilent Co., USA) equipped with an Agilent 1260 Refractive Index Detector [26]. The TSK-GEL columns (Tosoh Co., Ltd, Tokyo, Japan) in series of Guard column (PWXL 6.0 × 40 mm), G-5000 PWXL (7.8 × 300 mm i.d., 10 μm), and G-3000 PWXL (7.8 × 300 mm i.d., 5 μm) were used, eluted with 0.02 M phosphate buffer (pH 6.0) at a flow rate of 0.6 mL/min. The column temperature was kept at 35 ± 0.1 °C. The injection volume was 20 μL. The molecular weight of the sample was determined according to the equation of elution volume and the logarithm of the molecular weight of pullulan standards (Showa Group, Japan).

Fourier transform infrared spectroscopy (FI-IR)

The FT-IR prolife of PVP was determined to the KBr-disk method as described previously [26]. The spectrum was obtained with a resolution of 2 cm−1 between the frequency range of 400–4,000 cm−1.

Chemical composition analysis

The monosaccharide composition was determined gas chromatography (GC) as previously described [7]. Glucose, galactose, mannose, xylose, fucose, rhamnose, and arabinose were used as the monosaccharide standards. Acetyl inositol was used as the internal standard. Five milligrams samples were hydrolyzed with 2 M TFA (4 mL) at 105 °C for 6 h in a sealed tube. Excess TFA was removed by evaporation under reduced pressure. The residue was re-dissolved in 2 mL of methanol and transferred to a glass tube. The solution was taken to dryness with nitrogen. Hydroxylamine hydrochloride (10 mg), inositol hexaacetate (1 mg), and pyridine (2.0 mL) were added in the tube. The sealed tube was immersed in a thermostatic water bath (±1 °C) at 90 °C for 30 min. Then, 2.0 mL of acetic anhydride was added in. The mixture was kept at 90 °C for 30 min. The acetylated derivatives were filtered through 0.22 μm of nylon membrane. GC was performed on a gas chromatography/mass spectrometer (Trace DSQ II, Thermo Fisher Scientific, Waltham) equipped with a TR-5MS capillary column (30 m × 0.25 mm × 0.25 μm). The temperature program was set as follows: the initial temperature of the column was 100 °C and held for 2 min and then increased to 280 °C at 5 °C/min and held for 5 min. The flow rate was 1 mL/min. The injection temperature was 250 °C. The ion source of the mass spectrometer was set at 280 °C. The injection volume was 1 µL and the split ratio was 10:1.

Galacturonic acid and glucuronic acid contents were measured using a Dionex ion chromatography ICS 5000 (Sunnyvale, CA, USA) with a CarboPac PA1 analytic column (4 × 250 mm). The PVP (5 mg) was hydrolyzed with 2 M TFA (4 mL) at 105 °C for 6 h in a sealed tube. Excess TFA was removed by evaporation under reduced pressure at 45 °C. The residue was re-dissolved in distilled water (10 mL) and filtered through 0.22 μm of nylon membrane. The experiment was measured under the following operation conditions: mobile phase 100 mM NaOH (18 min), 100 mM NaOH and 170 mM CH3COONa (7 min), and 100 mM NaOH-170 mM (20 min); flow rate 1.0 mL/min; and column temperature 30 °C. The injection volume was 20 µL.

Periodate oxidation–Smith degradation

The sample was measured using the method [8] with some modifications. PVP (20 mg) was dissolved in 12.5 mL of distilled water, and 12.5 mL of NaIO4 (30 mM) was then added. The solution was thoroughly mixed and kept in the dark at room temperature, and 0.1 mL of aliquots were withdrawn at 6–72 h intervals, diluted to 250 mL with distilled water, and measured using a UV visible spectrophotometer at 223 nm, until the optical density value became stable. Glycol (1.5 mL) was added to stop period ate oxidation. The oxidation solution (2 mL) was titrated by NaOH solution (0.01 M) to calculate the production of formic acid. The rest was extensively dialyzed against distilled water for 48 h. The residue was concentrated to a volume of 10 mL and reduced with NaBH4 (70 mg) in the dark for 24 h, neutralized to pH 6.0–7.0 with 50 % acetic acid, dialyzed as described above, and concentrated to dryness. The residues were hydrolyzed with 2 M trifluoroacetic acid (4 mL) in a sealed glass tube at 105 °C for 6 h. Excess trifluoroacetic acid was removed by evaporation under reduced pressure. The residue was dissolved in 4 mL of methanol and evaporated to dryness for three times. The residue was re-dissolved in 2 mL of methanol and transferred to a glass tube. The solution was taken to dryness with nitrogen. Acetylation was carried out with hydroxylamine hydrochloride (10 mg) and pyridine (1.0 mL) in the sealed tube at 90 °C for 30 min. Then, acetic anhydride (1.0 mL) was added for another 30 min. The acetate derivate was analyzed using an Agilent GC 6890T system with a HP-1701 column (30 m × 0.25 mm i.d., 0.25 µm) and a flame ionization detector. The temperature program was set as follows: the initial temperature of the column was 180 °C, then increased to 220 °C at 2 °C/min and held for 1 min, and elevating to 250 °C at 5 °C/min and held for 1 min. The injection temperature was 220 °C. The ion source of the mass spectrometer was set at 270 °C. The injection volume was 1 µL. Monosaccharide standard, glycol, glycerol, and erythrite were used as standards.

NMR spectroscopy

NMR analysis was carried out on a Bruker 600 MHz (Bruker Corp., Fallanden, Switzerland). PVP (30 mg) was dissolved in D2O (0.6 mL). 1H NMR, and 13C NMR spectra were obtained at 25 °C. Acetone was used as an internal standard for the 13C spectrum. All chemical shifts were expressed in ppm.

DPPH radical scavenging activity

The total antioxidant activity was determined as described by Brand-Williams [27] with some modifications. Briefly, the PVP was accurately weighed and dissolved in distilled water to obtain different concentrations (0.05–2.4 mg/mL). Sample solution (1 μL) was added to 3 mL of 75 μM DPPH methanol solution. After vortexing, the mixture was kept in the dark at room temperature for 30 min. The absorbance was measured at 517 nm. The control was carried out with water instead of polysaccharides solution. Methanol was used as the blank. The ascorbic acid was used as a positive standard. The scavenging activity of DPPH radical scavenging was expressed as:

$${\text{Scavenging activity }}\left( \% \right) \, = \left( {1 - \frac{{A_{s} - A_{c} }}{A}} \right) \times 100$$
(3)

where A s is the absorbance of the reaction solution, A c is the absorbance of the solution including 1 mL of sample and 3 mL of methanol, and A is the absorbance of the solution including 3 mL of DPPH solution and 1 mL of methanol.

Oxygen radical absorbance capacity (ORAC)

The total antioxidant activity was determined using ORAC assay described by Huang et al. [28] and modified by Song et al. [29]. Sample solution was prepared with 75 mM phosphate buffer (pH 7.4). The 20 μL of sample or 20 μL of Trolox standard (range 6.25–50 μM) was added in a well of black-walled 96-well plates (Corning Scientific, Corning, NY), and then, 200 μL of fluorescein sodium salt (0.0956 μM) was added to each well. The mixture was automatically shaken and preincubated for 15 min at 37 °C. Finally, 20 μL of ABAP (119.4 mM) in buffer was added. The fluorescence intensity was measured using a Varioskan Flash multimode reader (Thermo Fisher Scientific Inc., Waltham, MA) at excitation of 485 nm and emission of 538 nm for 70 cycles every 2 min. The buffer was used as blank. All the tested samples including Trolox and blank were detected using the same 96-well microtitre plate. ORAC value was expressed as mean μmol Trolox equivalent (TE) per g of dry weight (DW) for triplicate data.

Cellular antioxidant activity

CAA of polysaccharides

The CAA was determined using the protocol described previously [29]. Briefly, HepG2 cells were seeded at a density of 6 × 104 cells/well on a 96-well microplate in 100 µL of growth medium/well. Twenty-four hours after incubation, the growth medium was removed, and the wells were washed with 100 µL of PBS. Triplicate wells were treated for 2 h with 100 µL of medium containing different concentration of PVP (range 15.6–500 µg/mL) plus 25 µM DCFH-DA. The media were removed, the cells washed with 100 µL of PBS, and 100 µL of 600 µM ABAP in oxidant treatment medium (HBSS with 10 mM Hepes). The control wells containing cells were treated with DCFH-DA and ABAP. The blank cells contain cells were treated with DCFH-DA and no ABAP. The 96-well microplate was placed in a Varioskan Flash spectral scan multimode plate reader (Thermo Labsystems) at 37 °C. Emission wavelength at 538 nm was measured after at 485 nm every 5 min for 1 h.

Quantification of CAA

After the subtraction of the blank and the initial fluorescence values, the area under the fluorescence versus time curve was integrated to calculate the CAA value at each concentration of polysaccharide as follows:

$${\text{CAA unit}} = 100 - \left( {\smallint {\text{SA}}/\smallint {\text{CA}}} \right) \, \times { 1}00 \,$$
(4)

where ∫SA is the integrated area under the sample fluorescence versus time curve, and ∫CA is the integrated area from the control curve. The median effective dose (EC50) was determined for the sample from the median effect plot of log (∫a/∫u) versus log (dose), where ∫a is the fraction affected (CAA unit), and ∫u is the fraction unaffected (1-CAA unit) by the treatment. The EC50 values were expressed as the mean ± SD using triplicate data sets obtained from the same experiment. EC50 values were converted to CAA values, which are expressed as micromoles of quercetin equivalents (QE) per g of DW.

Antiproliferative activity assay

Cell culture

The human cell lines including human hepatoma HepG2, human breast cancer MCF-7, and gastric carcinoma SGC-7901 were obtained from Medical College of Sun Yat-Sen University. The normal liver L02 cell line was purchased from Nanjing KeyGEN Biotech. Co., Ltd (Nanjing, P.R. China). The cell lines in the experiments were cultured in DMEM supplemented with 10 % FBS, 100 µg/mL streptomycin, and 100 units/mL penicillin at 37 °C in a 5 % CO2 humidified atmosphere cultivator.

MTT assay

The antiproliferative activities of polysaccharides were measured by the method of MTT colorimetry [30] with some modifications. Briefly, HepG2, MCF-7, SGC-7901, and L02 cells were seeded at a density of 4 × 103/well on a 96-well microplate in 100 µL of culture medium/well. The cells were incubated for 24 h at 37 °C. After the cells had attached to the wells, the growth medium was removed. Then, medium (100 µL) with different concentrations of PVP was added to each well. Control group without PVP was conducted in parallel. After 48 h of incubation at 37 °C, 20 µL of MTT (5 mg/mL) was pipetted into each well. Then, dimethyl sulfoxide (100 µL) was added after incubation for 4 h. The plates were placed on a table oscillator for 10 min, and the absorbance was measured at 570 nm using the Varioskan Flash spectral scan multimode plate reader. The antiproliferative activity was calculated according to the following formula:

$${\text{Antiproliferative activity }}( \%) = \left( {1 - \frac{{A_{\text{s}} - A_{\text{b}} }}{{A_{\text{c}} - A_{\text{b}} }}} \right) \times 100\,\%$$
(5)

where A c is the absorbance of the control; A s is the absorbance of sample; and A b is the absorbance of the blank.

Statistical analysis

Data were analyzed using SPSS (SPSS Inc., Chicago, IL, USA) and presented as mean ± SD with triplicates. Significance was determined at p < 0.05 by analysis of variance (ANOVA) followed by Duncan’s least significant test.

Results and discussion

Model fitting

The experimental conditions and the results of yield of the PVP are shown in Table 1. There was a considerable variation on the yield of PVP within the range of the extraction conditions. By using the software of Design Expert, a final second-order polynomial equation in terms of coded values was obtained as follows:

$$\begin{aligned} Y & = 1.60 - 0.012 \times A + 0.13 \times B + 0.23 \times C + 0.059 \times A \times B - 0.025 \times A \times C - 0.025 \times B \times C \\ {\kern 1pt} & \quad - 0.079 \times A^{2} \times B + 0.17 \times C^{2} \\ \end{aligned}$$
(6)

where Y is the yield of PVP, and A, B, and C are the coded variables for ultrasonic power, time, and temperature, respectively. As shown in Table 2, the P value for the model was highly significant, which indicated that the fitness of the model was significant, while the lack-of-fit value indicated that lack of fit was not significant relative to the pure error. In addition, the correlation coefficient expressed the quality of fit of the resultant polynomial model. Coefficient of determination (R 2) is defined to be the ratio of the explained variation to the total variation and is a measurement of the degree of fitness [13]. A small value of R 2 indicated a poor relevance of the dependent variables in the model [31]. By analysis of variance, the R 2 value was determined to be 0.9544, which proved that the regression model showed a good fit to the true behavior of the system. The coefficient of variation (CV) indicates the degree of precision with which the treatments are compared. Usually, the higher the value of the CV, the lower is the reliability of the experiment. The CV value was analyzed to be 4.41 %. All of these results suggest that the model was adequate for predicting outcomes within the range of the independent variables used.

Table 2 Analysis of variance for the response surface quadratic model for the yield of PVP
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Validation of the model

From the response surface analysis, when the quality of P. vulgaris was 4.0 g, the optimal condition for the ultrasonic extraction of PVP was ultrasonic power 211.6 W, time 50.1 min, and temperature 70.0 °C. Under these conditions, the maximum response value of yield (2.03 %) was predicted by the model. To validate the adequacy of the model, verification experiments were carried out under the optimal condition (selected ultrasonic power 210 W, time 50 min, and temperature 70 °C). The yield of PVP was 2.11 %. There was no significant difference between the predicted value and experimental value (P > 0.05). The accuracy of the model was validated with triplicate experiments under the optimal reaction condition. The result suggested that the model was accurate and adequate for the prediction of PVP extraction within the range of conditions studied. Moreover, the yield of polysaccharide obtained by ultrasonic extraction was about 1.5-fold higher than that of polysaccharide obtained by hot water extraction (100 °C, 2 h) (data were not shown).

Chemical composition of PVP

The total carbohydrate content of PVP was 87.5 %, and the protein content was 11.4 % (Table 1). Ultraviolet scanning showed PVP was free of nucleic acid.

Molecular weight distribution

The molecular weight distribution of PVP was determined using HPGPC. As shown in Fig. 1, four main peaks at 18.59, 27.12, 31.19, and 33.68 min were detected by refractive index measurements. The equation of the standard curve was LogMw = 40.791 − 6.3165 V + 0.3813 V2 − 0.008 V3 (where Mw represents the molecular weight, while V represents elution volume) with a correlation coefficient of 0.9999. According to the calibration curve with standard dextrans, the weight-average molecular weight (Mw) of PVP was obtained. The molecular weights of PVP consisted of four fractions: 4,720 kDa (56.1 %), 31.8 kDa (3.02 %), 4.87 kDa (11.6 %), and 0.69 kDa (29.2 %).

Fig. 1
figure 1

Molecular weight distribution of PVP

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Infrared spectrum

The IR spectrum of PVP is shown in Fig. 2a. The absorption peaks at 3,427, 2,939, and 1,421 cm−1 were the stretching of the O–H, C–H, and carboxyl C–O groups [26]. The absorption band at 1,633 cm−1 was due to the bound water. The band in the region of 1,254 cm−1 was assigned to O–H deformation vibrations. The stretching peaks at 902, 896, and 895 cm−1 were indicative of possible β-glycosidic linkages between the sugars. The absorption peak at 1,740 cm−1 indicated the presence of uronic acids [32]. As shown in Fig. 2b, ion chromatography analysis showed that the content of galacturonic acid and glucuronic acid in PVP was 6.26 and 0.37 %, respectively (Table 3).

Fig. 2
figure 2

IR spectrum of PVP (a); ion chromatography of uronic acids in PVP (b)

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Table 3 Chemical composition and structural characteristics of PVP
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Monosaccharide composition

PVP was hydrolyzed by TFA into individual monosaccharide, which was further acetylated for GC analysis as shown in Fig. 3. According to the retention time of the monosaccharide standards, the monosaccharide types of PVP were determined. The content of monosaccharide is calculated by each peak area. As shown in Table 3, PVP was mainly composed of mannose, arabinose, xylose, and galactose with molar percentages of 11.0, 28.2, 38.5, and 16.5 %, respectively. It indicated that mannose, arabinose, xylose, and galactose were the predominant monosaccharides and constructed the backbone for P. vulgaris polysaccharides. It also contained rhamnose (2.8 %) and glucose (3.0 %).

Fig. 3
figure 3

Gas chromatography of monosaccharide standards and PVP: a monosaccharide standards, rhamnose (17.41 min), arabinose (17.94 min), fucose (18.10 min), xylose (18.27 min), mannose (22.43 min), glucose (22.66 min), galactose (23.14 min), and internal standard (24.75 min). b Monosaccharide composition of PVP

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The glycosidic linkage of PVP

To exam the position of glycosidic linkages in PVP, it was subjected to periodate oxidation, Smith degradation, and GC analysis. The results of periodate oxidation showed that 1 mol of hexose residue consumed 1.041 mol of HIO4 to generate 0.480 mol of formic acid, indicating the 1→ or 1→6 glycosidic linkages accounted for 48.0 % and 1→2 or 1→4 glycosidic linkages accounted for 8.1 % of all linkages in the molecule. The (1→3)-linked monosaccharides could not be oxidized by HIO4, indicating the 1→3 glycosidic linkages accounted for 43.9 % of all linkages in PVP. The periodate-oxidized and Smith-degraded products were further analyzed by GC. The six types of monosaccharides observed from GC indicated that some of the linkages were (1→3)-linked, (1→3,6)-linked, (1→2,3)-linked, (1→3,4)-linked, and (1→2,3,4)-linked glycosidic linkages. The presence of glycerol and erythrite indicated that PVP also contained the (1→2)-linked, (1→6)-linked, (1→2,6)-linked, and (1→4,6)-linked glycosidic linkages [8].

NMR spectra of PVP

The 1H and 13C NMR spectra of PVP are shown in Fig. 4. The 1H NMR spectrum showed six anomeric proton signals at δ 5.21, δ 5.08, δ 5.02, and δ 4.90 ppm (Fig. 4a). This indicated that PVP mainly contained four kinds of monosaccharide residues in both α-type and β-type glycosidic configuration. There was no the proton signals at δ 5.4 ppm, indicating that PVP was composed of pyranose. The proton signals from C-2, C-3, C-4, C-5, and C-6 of sugar rings concentrated in the region of δ 3.3–4.0 ppm. For the 13C NMR spectrum of PVP, it was difficult to assign all signals without any ambiguities due to the overlaps of signals (Fig. 4b). The six anomeric carbon signals were at 103.84, 103.02, 102.85, 102.19, 101.33, and 100.19 ppm, which suggested that the main configuration of polysaccharide was β-glucan. The strong resonance signal at 82.98 ppm in the range of 78–85 ppm indicated that C-2, C-3, or C-4 positions of sugar units were substituted. The several resonance signals in the region at δ 60–75 ppm indicated that C-6 positions of sugar units were substituted. The result demonstrated that PVP contained 1→2, 1→3, 1→4, and 1→6 glycosidic linkages [33].

Fig. 4
figure 4

1H (a) and 13C (b) NMR spectrum of PVP

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DPPH radical scavenging activity

The DPPH, a relatively stable nitrogen-centered free radical, has been widely used to evaluate free radical scavenging activity. The samples as antioxidants are conceived to their electron- or hydrogen-donating ability [34]. The DPPH changes from purple to yellow when reduced by hydrogen donation. In this study, the DPPH radical scavenging activities of the PVP at different concentrations were measured. As shown in Table 4, PVP exhibited strong scavenging activity on DPPH radical in a concentration-dependent manner. The highest DPPH radical scavenging activity of PVP was 81.70 % at the concentration of 2.4 mg/mL. The polysaccharide (247 kDa) from Isodon lophanthoides was found to exhibit obvious antioxidant activity against DPPH radicals with IC50 value of 211.93 µg/mL [21]. The polysaccharide CPP-1 (1,167 kDa) from Cyclocarya paliurus exhibited significant scavenging effects on DPPH radicals with EC50 values of 52.3 µg/mL [35]. Ma et al. [13] reported that ultrasonic degradation of high molecular weight polysaccharides from Ganoderma lucidum could enhance its DPPH radical scavenging activities. It was widely recognized that the antioxidant activities of polysaccharides were attributed to their hydrogen donation power to the free radicals [21, 36]. In this study, PVP showed a good DPPH radical scavenging activity, which was probably due to its good hydrogen-donating ability.

Table 4 DPPH radical scavenging activity of the PVP
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ORAC

The total antioxidant activity, measured by ORAC, of PVP is expressed as µmol TE/g DW basis. In term of ORAC assay, the peroxyl radical will cause the decrease in fluorescence emission by reacting with fluorescence probe. The antioxidants can prevent the chain reaction between peroxyl radicals and fluorescein by donating their labile hydrogen atoms to peroxyl radicals [28]. In this study, the ORAC value of the PVP was 245.3 ± 40.2 µmol TE/g, indicating that PVP had a strong antioxidant activity. The antioxidant activities of the polysaccharides might be related to their content of proteins and uronic acid, monosaccharide compositions, molecular weights, and types of glycosidic [7, 12]. You et al. [11] found that a moderate molecular weight (306 kDa) polysaccharide from Lentinus edodes showed relatively high antioxidant activity. Zhou et al. [37] reported that lower molecular weights of sulfated polysaccharides have stronger antioxidant capacity. Jiang et al. [12] reported that the Cyclina sinensis polysaccharides with higher contents of protein, uronic acid, and more complicated monosaccharide compositions showed stronger antioxidant. PVP had strong antioxidant activity, which might be associated with its molecular weight and structural characteristics. However, the antioxidant mechanism of glucan is not fully understood. Glucans with different glycosidic linkages and conformations have different free radical scavenging activities. The most pronounced hypothesis is that free radicals can abstract hydrogen atoms at all ring C–H bonds of aldoses, uronic acids, and other sites on carbohydrate [38]. Therefore, the strong total antioxidant activity of PVP was mainly attributed to its active hydroxyl groups of monosaccharides and uronic acids in the polysaccharides.

Cellular antioxidant activity

The cellular antioxidant activity of PVP was measured using the CAA assay. Cellular antioxidant activity assay has been considered as a more biological relevant assay than the common chemical antioxidant activity assays in the measurement of antioxidant activities of food, phytochemicals, and dietary supplements. In term of CAA assay, the formation of dichlorofluorescein by ABAP-generated peroxyl radicals in human hepatocarcinoma HepG2 cells can be retarded in the presence of antioxidants. The decrease in cellular fluorescence when compared to the control cells indicates the antioxidant capacity of the samples [39]. Until today, there have been no reports on the use of the CAA assay on polysaccharides from Prunella vulgaris. The analysis of the CAA assay showed that the IC50 values of the PVP and QE were 373 µg/mL and 4.25 µmol/L, respectively. The CAA value of PVP was 11.4 ± 1.2 µmol QE/g DW, indicating that PVP showed a good antioxidant activity within cells.

Antiproliferative activity

The HepG2, SGC-7901, MCF-7, and L02 cells were chosen to investigate the cytotoxicity of PVP. As shown in Table 5, it could be concluded that PVP exhibited stronger antiproliferative activities on the selected cancer cells than the normal cells. The cytotoxicities to HepG2, SGC-7901, and MCF-7 cells increased in a dose-dependent manner within the tested concentration ranges. At the concentration of 2.0 mg/mL, the levels of growth-inhibitory effects on HepG2, SGC-7901, and MCF-7 cells were 51.2, 35.2, and 31.8 %, respectively. However, the L02 cell viability was above 90 % at the concentrations tested, indicating that PVP had no cytotoxicity against normal liver L02 cell [40]. It has been reported that the antitumor activity of polysaccharide might depend on its monosaccharide composition, molecular weight, structure of the polymeric backbone, and content of protein [13, 41]. Recent studies have demonstrated that (1→3)-β-glucans had strong anticancer effects [10, 42]. In this study, PVP had strong antiproliferative activities against cancer cells, which might be due to the existence of (1→3)-glucans in PVP. However, the mechanisms of polysaccharides against cancer cells are complex and not fully elucidated. Xu et al. [41] demonstrated that polysaccharides with higher antioxidant activities had higher antitumor activities. Choi et al. [14] reported that the antimetastatic effect of aqueous extract isolated from P. vulgaris is mediated through the suppression of MMP-9 expression by the inhibition of NF-kB via ERK1/2 signaling pathway. Bao et al. [43] reported that the antitumor activity of a polysaccharide from Artemisia argyi might be associated with its immunomodulatory effects. However, the mechanism for the antiproliferative activity of polysaccharide from P. vulgaris is still not fully understood and needs further study.

Table 5 Antiproliferative activity of PVP
Full size table

Conclusions

The response surface methodology was proved to be useful for the optimization of ultrasonic extraction of polysaccharides from P. vulgaris. The maximum yield of polysaccharides was obtained at ultrasonic power 211.6 W, time 50.1 min, and temperature 70.0 °C. In order to facilitate the operation, extraction conditions were adjusted as: ultrasonic power 210 W, time 50 min, and temperature 70 °C. Using this condition, the experimental yield was not significantly different from the predicted value. PVP comprised of rhamnose (2.8 %), arabinose (28.2 %), xylose (38.5 %), mannose (11.0 %), glucose (3.0 %), and galactose (16.5 %). PVP contained 1→ and 1→6 glycosidic linkages (48.0 %), 1→2 and 1→4 glycosidic linkages (8.1 %), and 1→3 glycosidic linkages (43.9 %). PVP exhibited good antioxidant and antiproliferative activities, which indicated that P. vulgaris has a potential application to promote health.