Quality evaluation of Lycium barbarum (wolfberry) from different regions in China based on polysaccharide structure, yield and bioactivities
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Lycium barbarum (wolfberry) has been widely cultivated in China, particularly in northwest regions. However, the fruit size and taste of L. barbarum from different habitats are quite different. Traditionally, only the fruit of L. barbarum produced in Ningxia province is recorded as an authentic herb, although the detailed mechanism responsible for this remains obscure. Polysaccharides are considered major active ingredients in L. barbarum which is crucial for its quality evaluation.
In this study, we assessed the yield, monosaccharide composition, molecular weight, and conformation of L. barbarum polysaccharides (LBPs) collected from different regions of China. The antioxidant and immune activities of LBPs were also determined as its quality indicator.
Our results showed that the similarity values of monosaccharide composition were larger than 0.926, and the Mw of the two fractions (peaks 1–2) in LBPs were ranging from 1.36 × 106 to 2.01 × 106 (peak 1), and 6.85 × 104 to 10.30 × 104 (peak 2) which indicated that the structure of LBPs were similar. In addition, results showed that there was no significant difference in antioxidant and immune activities of nine LBPs from different regions. However, the yield of LBPs from Qinghai Province (low atmospheric temperature, high altitude) was significantly lower (p < 0.05) than those collected from Xinjiang and Ningxia province.
These data suggested that the L. barbarum produced in Ningxia and Xinjiang maybe more suitable as materials for medicines and functional foods. This study also provides a reference for improving the quality control standard of LBPs.
KeywordsLycium barbarum L. Polysaccharide Quality evaluation Antioxidative activity Immunomodulatory activity
Lycium barbarum L., a well-known Chinese herb, is one of the medicinal and food homologous traditional Chinese medicines. L. barbarum products have been classified as nutraceutical foods or dietary supplements and have become popular in East Asia, Europe, and North America . Generally, the polysaccharide is the main bioactive ingredient in L. barbarum, which exerts various effects, including immunomodulation, antioxidant, anti-hypertension, and anticancer activities . In China, L. barbarum has been widely cultivated, particularly, in northwest regions, such as Qinghai, Xinjiang, Ningxia, and Gansu. However, the fruit size and taste of L. barbarum from different habitats are considerably different. Traditionally, only the fruit of L. barbarum produced in Ningxia is recorded as an authentic (Daodi) herb in China. Whether differences in the structure and activity of L. barbarum polysaccharides (LBPs) from typical regions exist is crucial for its quality evaluation and related product development.
Unlike small-molecule compounds, polysaccharides are difficult to evaluate only by their content determination or a specific spectrum. In the 2015 edition of Chinese Pharmacopoeia and a few studies , the polysaccharide quality of L. barbarum was evaluated only by determination of the content of total sugars using the phenol–sulfuric acid method with glucose as a reference standard. Indeed, this method is not appropriate for quality evaluation of LBPs due to its poor specificity and inability to reflect the structural characteristics of LBPs . Structural features, including sugar compositions, molecular weight, and conformation are all required for comprehensive description of the polysaccharide products. In recent studies, multiple fingerprints of LBPs have been established . Researcher also compared the LBPs obtained from different locations by molecular weight (Mw) determination . The results in these studies showed that the similarity of those polysaccharides in L. barbarum collected from different regions of China was high. Moreover, antioxidant and immune activity of LBPs have been studied in some researches. But these reports focused on the antioxidant and immune activity of different separated fractions in LBPs [7, 8]. Xie and others evaluated and compared the effects of eight LBPs from different producing areas on macrophage function . In our study, the quality of LBPs from different producing areas was comprehensively evaluated by structure, yield and activity.
In this study, nine LBPs samples of the identical varieties from different locations were extracted and analyzed to determine their structural properties, including the monosaccharide composition, Mw and conformation. The similarity of LBPs from different regions was assessed using statistical and similarity analyses with professional software. Polysaccharides are the major bioactive compounds in L. barbarum by report, which have possessed the immunological adjuvant effect and antioxidant activity. To obtain deep insights into the quality of L. barbarum polysaccharides, their antioxidant and immune activity were also detected as a quality indicator. The antioxidant activities of LBPs were evaluated by hydroxyl and ABTS radicals scavenging assays. In addition, immune activity were evaluated by detect the effect of LBPs on NO, TNF-α, IL-6 production of macrophage. The results could serve as a scientific basis to further evaluate the quality of L. barbarum and will provide a reference for improving the quality control standard of LBPs production.
Materials and methods
Chemical composition of LBPs in nine samples
Protein content (%)
Total carbohydrate content (%)
Mean ± SD (n = 3)
67.4 ± 3.21
4.0 ± 0.32
Mean ± SD (n = 3)
61.9 ± 5.80
1.7 ± 0.07*
Mean ± SD (n = 3)
62.5 ± 4.05
4.5 ± 0.31
d-Mannose (Man), d-ribose (Rib), l-rhamnose (Rha), d-glucuronic acid (GlcA), d-galacturonic acid (GalA), d-glucose (Glc), d-galactose (Gal), d-xylose (Xyl), and l-arabinose (Ara) were obtained from the National Institute for Food and Drug Control (China). Bovine serum albumin (BSA) was manufactured by Thermo (USA). 1-Phenyl-3-methyl-5-pyrazolone (PMP), 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydroxyl (DPPH) were purchased from Sigma-Aldrich Trading (St. Louis, MO, USA). Other reagents and chemicals were of analytical grade and obtained from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China).
Preparation of LBPs
Analytical methods for composition determination
Neutral sugar levels were determined by the phenol–sulfuric acid method as d-glucose equivalents. The sulfuric acid–carbazole method was adapted to measure the uronic acid content with d-galacturonic acid used as a standard. The protein content was determined by the Bradford method using bovine serum albumin (BSA) as a standard .
Determination of molecular weight and polydispersity index
The Mw and polydispersity (Mw/Mn) of LBPs were measured using HPSEC-MALLS-RID methods. Each sample of 20 mg was dissolved in 2 mL of the mobile phase and then filtered through a 0.45-µm membrane. The Mw of LBPs were established by a HPLC system (Shimadzu Company, Japan), equipped with a refractive index detector (RID), combined with a multi-angle laser light scattering detector (MALLS, DAWN HELEOS, Wyatt Technology Co., Santa Barbara, CA, USA). After optimizing the chromatographic conditions, multiple size exclusion columns Shodex SB806 (300 mm × 7.8 mm, i.d.), Shodex SB805 (300 mm × 7.8 mm, i.d.), and Shodex SB804 (300 mm × 7.5 mm, i.d.) were used to obtain a good separation efficiency. The mobile phase was 0.1% NaNO3 aqueous solution applied at a flow rate of 0.5 mL/min. An injection volume of 100 µL was used, and each sample was run for 80 min, and a temperature maintained at 40 °C.
Monosaccharide composition analysis
The composition analysis of polysaccharide is an important step to control the quality and obtain basic information. Briefly, each of the LBPs (1 mg/mL) was hydrolyzed by trifluoroacetic acid (TFA, 4 mol/L) at 120 °C for 4 h, followed by complexing with PMP (0.5 mol/L) . Sample solution (20 μL) was injected and analyzed by HPLC-PAD using a ZORBAX Eclipse XDB-C18 column (250 × 4.6 mm, 5 μm, Agilent, USA) and UV detection at 250 nm. The flow rate of the mobile phase consisting of acetonitrile and 0.125 mol/L KH2PO4 (v/v = 16:84, pH 6.9) was 1.0 mL/min.
Antioxidative activity study
Hydroxyl radical scavenging activity
Total antioxidant capacity
RAW264.7 macrophage proliferation
RAW264.7 macrophage cells in an RPMI-1640 medium containing 10% FBS were plated in a 96-well microplate (1 × 104 cells/well, 100-µL volume; ATCC). The cells were incubated with 100 µL of the LBPs at different concentrations (50, 100 and 200 µg/mL) in triplicate. The cell cultures were kept in humidified atmosphere containing 5% CO2 at 37 °C for 72 h. Then, the WST-1 solution (20 µL) was added to the wells and the solution was further incubated for 4 h at 37 °C. The optical density was measured at 450 nm using a microplate reader (EL-800; BioTek Instruments, Winooski, VT, USA). The absorbance (A) was translated into a macrophage proliferation ratio (%) = At/Ac 100, where At and Ac are the absorbance of the test group and control group, respectively.
Reverse transcription-polymerase chain reaction (RT-PCR)
RAW264.7 cells in logarithmic growth phase were seeded in 6-well plates with a cell density of 1 × 105/mL. After 12 h of culture, the culture medium was replaced with the Dulbecco’s modified Eagle’s medium (DMEM) containing NX1, QH1, XJ1 at the concentrations of 50, 100 and 200 µg/mL, respectively, or 1 μg/mL LPS. After incubation at 37 °C for 18 h. The TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract the total RNA from RAW264.7 cells according to the manufacturer’s protocol. Reverse transcriptase-generated complementary DNA encoding iNOS, IL-6 and TNF-α genes were amplified by polymerase chain reaction using specific primers. The nucleotide sequences of the primers were as follows: iNOS, 5′-TGCCACGGACGAGACGGATAC-3′(forward) and 5′-CCATTGCACAACTCTTTTCTCA-3′(reverse); IL-6,5′-CTCCCAACAGACCTGTCTATAC-3′(forward) and 5′-CCATTGCACAACTCTTTTCTCA-3′(reverse); TNF-α,5′-ATGTCTCAGCCTCTTCTCATTC-3′(forward) and 5′-GCTTCTCACTCGAATTTTGAGA-3′(reverse); GAPDH,5′-TGGCCTTCCGTGTTCCTAC-3′(forward) and 5′-GAGTTGCTGTTGAAGTCGCA-3′(reverse). Each sample was tested three times and the average of the three values was used for calculation. In a parallel experiment, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control.
Each assay was performed in triplicate. The data are expressed as mean ± standard deviation (SD). Statistical differences between the different groups were assessed by Student’s t-test. The similarities in the HPLC chromatograms were evaluated by the software “Similarity Evaluation System for Chromatographic Fingerprint of traditional Chinese medicine (TCM)” published by China Pharmacopoeia Committee (Version 2012A).
Results and discussion
Chemical composition of LBPs
The contents of protein, total carbohydrate, and yield of LBPs, collected from different sites are summarized in Table 1. Each assay was performed in triplicate, the data are expressed as mean ± SD.
The L. barbarum fruits collected from Qinghai (average altitude 2740 m, annual average temperature 5.0 °C) had significantly bigger sizes than those from Ningxia (average altitude 1180 m, annual average temperature 10.2 °C) and Xinjiang (average altitude 750 m, annual average temperature 9.5 °C). The results of the determination of 100-grain weight, one of the evaluation indexes utilized in the commercial grade standard for L. barbarum evaluation in China were also in agreement with observed appearance characteristics, i.e., the size of the fruits. The average 100-grain weight of three bathes L. barbarum fruits from Qinghai (25.59 g) was obviously higher (nearly twice higher) than that of the sample from Ningxia (13.32 g) and Xinjiang (14.29 g). Previous studies revealed that low atmospheric temperature, high altitude, and long sunshine time exerted favorable effects on the appearance characteristics of L. barbarum fruits. This conclusion is consistent with our observations. In addition, the L. barbarum fruits from Qinghai had the biggest grains, but the yield of LBPs from them was lower than others, which indicated that the yield of LBPs did not increase with the increase of the size of the grain.
Molecular weight and conformation of LBPs
Molecular weight and polydispersity index of LBPs from nine samples
Mw × 106 Da
Mw × 104 Da
1.37 (± 1.4%)
8.83 (± 1.6%)
1.40 (± 1.2%)
8.68 (± 1.5%)
1.39 (± 1.3%)
8.30 (± 1.6%)
1.99 (± 1.2%)
10.21 (± 1.7%)
1.92 (± 1.0%)
10.30 (± 1.4%)
2.01 (± 1.0%)
9.36 (± 1.0%)
1.46 (± 0.8%)
6.85 (± 2.7%)
1.57 (± 0.8%)
6.97 (± 1.6%)
1.36 (± 1.8%)
7.35 (± 2.6%)
The results showed that the Mw of the two polysaccharide fractions (peaks 1–2) in LBPs collected from Ningxia, Qinghai, and Xinjiang were similar, ranging from 1.36 × 106 to 2.01 × 106 (peak 1), and 6.85 × 104 to 10.30 × 104 (peak 2), respectively. These findings were in accordance with the ones of previous studies showing that the molecular weights of polysaccharides in LBPs were within the range from 1.0 × 104 to 2.3 × 106 Da . Furthermore, the polydispersity index of the two polysaccharide fractions (peaks 1 and 2) in LBPs were from 1.56 to 2.59 and from 1.19 to 1.48, respectively, suggesting that molecular the weight distribution of each polysaccharide fraction in L. barbarum was relatively narrow.
The polymer conformation of polysaccharides, another important parameter reflecting structure can be determined by HPSEC-MALLS . The conformation, denoted as α, can be examined by determining both Mw and size (rms radius, rg) at each elution volume, independently, then, plotting log(Rg) as a function of log(Mw). The resulting slopes reveal whether the molecule’s conformation is approximated by a sphere (slope of about 0.33), random coil (slope of 0.5–0.6) or rod (slope of 1.0) . Of much greater interest was the comparison in molecular conformation, shown in Table 2. The conformation plot, α ranging from 0.189 to 0.252, suggested that the natural LBPs have a compact, sphere-like structure. It also be observed that the conformation of LBPs from nine samples showed high similarity which further demonstrate the consistency of similarity of polysaccharide structure in L. barbarum.
Monosaccharide analysis and HPLC-PAD fingerprints
Monosaccharide composition of LBPs
Mean ± SD (n = 9)
3.9 ± 0.37
3.5 ± 0.44
3.7 ± 0.34
1.8 ± 0.19
24.9 ± 1.84
10.2 ± 0.76
19.3 ± 1.23
2.5 ± 0.26
30.2 ± 1.18
With the development of modern analytical technology, extensive exploratory work has been conducted on the fingerprints of polysaccharides from herbs [23, 24]. These studies on polysaccharide fingerprint profiling have often been performed by HPLC. Similarity analysis (SA) have been usually employed to evaluate chromatographic fingerprinting data . In our study, the standard fingerprints were established and the similarity of fingerprints were calculated by the software “Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (TCM)”. Nine characteristic peaks in fingerprints are displayed in Fig. 3b. The mixed standard sample of nine types of monosaccharide in Fig. 3c was used for qualitative analysis of monosaccharide peaks of samples in the same chromatographic conditions. The results showed that all the similarity values of each of the LBPs with the standard fingerprint were larger than 0.926. The values had highly similar fingerprint characteristics of LBPs although the samples were from different geographical regions. It also implied the standard fingerprint was representative enough that could be used to control and evaluate the quality of L. barbarum.
Antioxidant activities of LBPS from different locations
Our findings indicate that all LBPs from different regions had effective scavenging activities, which may be associated with the structure of LBPs. As mentioned in previous reports [27, 28], polysaccharides is characterized by high contents of galacturonic acid, a high degree of branching and favorable linkage usually showed a strong free radical scavenging activity. Interestingly, LBPs just has these structural features which together contribute to their predominant antioxidant activity. Results also showed that there was no significant difference in antioxidant capacity of nine LBPs towards hydroxyl and ABTS radicals determination.
Immunomodulatory activities of LBPS from different locations
Comparison of RAW264.7 cell viability
Effect of LBPs from different locations on RAW264.7 TNF-α, IL-6 and NO production
Regulation of macrophage function by polysaccharides has been reported in diverse ways including promoting macrophage activation, inducing the secretion of TNF-α, IL-6 and NO, and enhancing phagocytosis and antigen presenting ability [29, 30]. Thus, the effects of LBPs from different regions induced the secretion of NO, IL-6 and TNF-α of macrophage were evaluated and compared in this study. The results showed that RAW264.7 cells treated with NX1, QH1, XJ1 or LPS resulted in significantly increased the mRNA transcription levels of iNOS, IL-6 and TNF-α (Fig. 5b).
Our results showed that the structural properties including monosaccharide composition, Mw and conformation of LBPs from different areas were similar, which is consistent with the results obtained from their antioxidant and immune activities. It may implied that the species is a more important factor affecting the structure and bioactivities of LBPs than the origin. However, the average yield of LBPs from different producing areas showed great differences: the LBPs from Qinghai were significantly lower (p < 0.05) than those from Xinjiang and Ningxia, which may indicate that the L. barbarum produced in Ningxia and Xinjiang maybe more suitable as materials for medicines and functional foods.
This study also provides a reference for improving the quality control standard of LBPs. According to our research results, in consideration of structural similarity of LBPs from different producing areas, the yield of LBPs should be used as a key index to evaluate the quality of L. barbarum. First, the HPLC-PAD typical fingerprint of the monosaccharide composition and Mw of peaks can be determined to identify the LBPs and the related products from polysaccharides of other herbs. Then the yield of LBPs can be used as an important index to evaluate the quality of L. barbarum. On the basis of the existing standards for the determination of LBPs content, the identification tests for the determination of monosaccharide composition and Mw should also be developed. Nevertheless, conducting a large-scale study, including the analysis of larger numbers of samples from Ningxia, Xinjiang, and Qinghai, is required to further confirm the present results.
All persons who meet authorship criteria are listed as authors, and all authors certify that they have participated sufficiently in the work to take public responsibility for the content, including participation in the concept, design, analysis, writing, or revision of the manuscript. Furthermore, each author certifies that this material or similar material has not been and will not be submitted to or published in any other publication. The specific contributions made by each author have been listed as follows: YW: conception and design of study, acquisition of data and drafting the manuscript; HJ: acquisition of data, analysis and interpretation of data; XD, SY: revising the manuscript critically for important intellectual content; SM: conception and design of study; JN: conception and design of study, analysis and interpretation of data. All authors read and approved the final manuscript.
This study was supported by Grants from “12th Five-Year Plan,” and “Creation of Major New Drugs,” as well as “The Platform of Safety Testing Technology and Standards of Traditional Chinese Medicine” (NO. 2014ZX09304307-002) from the Important Program of Ministry of Science and Technology of the People’s Republic of China.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing interests.
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