Next Article in Journal
Assessment of Anti-Obesity Potential and Techno-Functional Properties of Bougainvillea spectabilis Willd. Bracts
Previous Article in Journal
Residual Characteristics of Atrazine and Its Metabolites in the Liaoning Province of China
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Separations
Volume 9
Issue 12
10.3390/separations9120398
separations-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Comparative Study of the Phytochemical Profiles of the Rhizomes of Cultivated and Wild-Grown Polygonatum sibiricum

by
Zhibin Pan
1,
Weiqing Cheng
1,
Zhibin Liu
2,
Weibin Wu
1,
Bin Yang
3 and
Junhan Lin
1,*
1
Fujian Vocational College of Bioengineering, Fuzhou 350007, China
2
Institute of Food Science & Technology, Fuzhou University, Fuzhou 350108, China
3
Nanwuyi Professional Cooperative of Chinese Herbal Medicine Planting, Shaowu 354000, China
*
Author to whom correspondence should be addressed.
Separations 2022, 9(12), 398; https://doi.org/10.3390/separations9120398
Submission received: 17 October 2022 / Revised: 25 November 2022 / Accepted: 25 November 2022 / Published: 30 November 2022
(This article belongs to the Section Analysis of Natural Products and Pharmaceuticals)
Browse Figures
Versions Notes

Abstract

:
The rhizome of Polygonatum sibiricum is a traditional Chinese medicine material and also a popular functional food consumed in China. Due to the increasing demand and overexploitation, the use of the cultivated plant is growing rapidly. However, the difference in phytochemical profile and health benefit between the cultivated and wild-grown P. sibiricum has not been revealed yet. The objectives of this study are to compare the phytochemical profiles of two types of P. sibiricum, i.e., the cultivated and the wild-grown types, by using UHPLC-Q-Orbitrap-MS based untargeted metabolomics approach. We tentatively identified 190 phytochemicals belonging to alkaloids, flavonoids, phenolic acids, and terpenoids from both two types of samples. In general, there is distinctive difference in phytochemical profiles between these two types of samples. Specifically, 33 phytochemicals showed significant differences. Of these phytochemicals, 22 compounds, such as laetanine, p-coumaroyl-beta-D-glucose, geniposide, medicagenic acid, were significantly higher in cultivated type; 11 compounds, such as vicenin-2, kaempferol 7-neohesperidoside, vanillic acid, and obacunone, were significantly higher in wild-grown type samples. This study will expand our knowledge regarding the cultivated of P. sibiricum and facilitate its further application in pharmaceutical and food industries.

1. Introduction

Polygonatum sibiricum belongs to the family Liliaceae, which has been used as traditional medicine and foodstuff in China for over 2000 years and is documented in the prestigious pharmaceutical classic “Compendium of Materia Medica” [1]. P. sibiricum is also authorized as “medicine food homology” by China’s Ministry of Health, which means that it combines the function of food and medicine [2]. In addition to its nutritional value, P. sibiricum also has a function in the prevention and treatment of various diseases, and many other healthcare effects. It has been extensively reported that the dried rhizomes of P. sibiricum possess a broad range of pharmacological activities, such as antioxidant [3], anti-microbial [4], anti-inflammatory [5], anti-atherosclerotic [6], anti-diabetic [7], anti-hyperglycemic [8], and anti-cancer activities [9]. Moreover, the wild resources of P. sibiricum are commonly recognized to possess better pharmacological activities. However, due to increasing demand and overexploitation, the wild resources of P. sibiricum have been largely depleted. Therefore, cultivated P. sibiricum has been gaining increasing attention. Nowadays, P. sibiricum is being cultivated. A stable yield of P. sibiricum therefore can be obtained.
Numerous modern pharmacological studies have demonstrated that the main active components of genus of Polygonatum are phenolics, steroid saponins, alkaloids, and polysaccharides [10,11,12,13]. For instance, Chen et al. reported the α-glucosidase inhibitory effect of a phenolic glycoside from P. sibiricum [14]. Luo et al. demonstrated the diabetes alleviation and gut microbiota modulation effects of saponin extracts of P. sibiricum [8]. Therefore, the analysis of the chemical constituents of P. sibiricum is of critical importance for the in-depth understanding of the pharmacological activities and quality control of this economically important traditional herbal material. Zhao and Li reviewed the chemical constituents of the genus Polygonatum and reported that steroidal saponins, flavones, alkaloids, lignins, amino acids, and carbohydrates were major components [15]. Our previous phytochemical investigation on the rhizome part of P. sibiricum revealed the presence of phenolics, terpenoids, alkaloids, fatty acids, amino acid derivatives, carbohydrates, and derivatives, etc. [16]. However, regarding the chemical constituents of the cultivated P. sibiricum, to the best of our knowledge, the relevant information is still lacking.
The prevailing practices on the chemical constituents of P. sibiricum mainly focus on the analysis of the constituents with high concentrations, while those compounds in minor concentrations are normally ignored. Such an approach dismisses the pharmacological activities of minor compounds and overlooks the complexity of the chemical constituents of herbal medicines or foods. The development of sophisticated untargeted metabolomic approaches allows the comprehensive profiling of the chemical constituents presented in major as well as in minor quantities in these traditional herbal materials, and their thorough quality assessment. Due to the rapid and high-throughput data generation, untargeted metabolomic approaches, such as the combination of chromatography and high-resolution mass spectrometry and various chemometric tools, have already been employed in the analysis of the chemical constituents of various plant materials [17,18,19]. In addition to LC-MS-based approach, other techniques, such as high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS-NMR), matrix-assisted laser desorption/ionization (MALDI), and ion mobility spectrometry (IMS), have also been applied to profile the metabolites in several plant materials [20,21].
In this study, the metabolic profiles of rhizomes from the cultivated and wild-grown P. sibiricum were analyzed and compared by employing an integrated strategy of combining UHPLC-Q-Orbitrap-MS technique with chemometrics analysis. Furthermore, the most distinguishing phytochemical compounds between these two plant materials were characterized. The results of the current study can provide useful information for the quality control of the cultivated P. sibiricum and improve the understanding of the chemical nature of this traditional herbal material.

2. Materials and Methods

2.1. Chemicals

LC-MS grade reagents of formic acid, methanol, and acetonitrile were obtained from CNW Technologies, Inc. (Düsseldorf, Germany). The internal standard compound 2-chlorophenylalanine was obtained from HC Biotech (Shanghai, China). Ultrapure water was prepared through a Millipore Alpha-Q water purification system (Millipore, Billerica, MA, USA).

2.2. Plant Materials

Two types of P. sibiricum rhizomes, including the cultivated type (C) and the wild-grown type (W)–five samples in each type–were collected for analysis. The cultivated plants were cultured with NPK (ammonium, phosphate, and potassium) compound fertilizer (15-15-15, 37.5~45.0 g/m2). The fertilizer application was repeated every six months. The wild-grown plants were grown in the wild field in Shaowu city (Fujian, China). Their basic information, such as weight, length, yield, and polysaccharide content, are summarized in Supplementary Materials Table S1.

2.3. Sample Extraction

After the collection of the fresh rhizomes from the cultivated and wild-grown P. sibiricum, all the plant materials were cut into small pieces and freeze-dried. The dried rhizomes were then crushed into powder. Approximately 1 g of powder samples were taken and extracted with five times the volume of extraction solution. The extraction solution was 25% methanol/water containing 1 μg/mL 2-chlorophenylalanine as internal standard. The metabolites of all rhizome samples were thoroughly extracted with the assistance of sonication for 1 h in an ice-water bath. After filtration and centrifugation (13,800× g, 15 min, 4 °C), an aliquot of 300 μL supernatant was taken for further untargeted metabolomic analysis. Additionally, a quality control (QC) sample, which was the mixture of equal amount of all samples, was prepared.

2.4. Ultra-High Performance Liquid Chromatography Analysis

Chromatographic analysis of the P. sibiricum extracts was performed using an Agilent ultra-high-performance liquid 1290 UPLC system (Agilent, Santa Clara, CA, USA). A BEH C18 column (150 mm × 2.1 mm, 1.7 μm; Waters, Milford, MA, USA) with guard column (5 mm × 2.1 mm, 1.7 μm; Waters, Milford, MA, USA) was used to perform the chromatographic separation. The mobile phases consisted of water with 0.1% (v/v) formic acid (solvent A) and acetonitrile with 0.1% (v/v) formic acid (solvent B). The following gradient was utilized: 0–3.5 min, 95–85% solvent A; 3.5–6 min, 85–70% solvent A; 6–6.5 min, 70% solvent A; 6.5–12 min, 70–30% solvent A; 12–12.5 min, 30% solvent A; 12.5–18 min, 30–0% solvent A; 18–25 min, 0% solvent A; 25–26 min, 0–95% solvent A; 26–30 min, 95% solvent A., which was eluted at a flow rate of 400 μL/min. The volume of samples injection was at 5 μL. The QC sample was injected once at the beginning, after 10 sample injections, and at the end of the run to obtain three sets of data to monitor the stability of instrument.

2.5. Mass Spectrometry Analysis

After chromatographic separation, mass spectrometry was performed using an Q Exactive Focus Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) equipped with heated-ESI-II (HESI-II), an ion source was employed to obtain the MS and MS/MS data. Two analytical sequences (positive and negative modes) were executed. The following HESI-II parameters were utilized: the positive and negative spray voltage were 4.0 kV and 3.6 kV, respectively; nitrogen sheath gas was set at a flow rate of 45 arbitrary units (AU); nitrogen auxiliary gas was set at a flow rate of 15 AU; capillary temperature was set at 400 °C; the acquisition scan range 100–1500 m/z; full MS scan resolution at 70,000 full width at half maximum (FWHM) and data-dependent MS/MS scan resolution at 17,500 FWHM were used. The accuracy of mass spectrometer was adjusted by using an external calibration based on the manufacturer’s guidelines. XCalibur software (version 4.0, Thermo Fisher Scientific, Bremen, Germany) was used for data acquisition.

2.6. Data Processing and Multivariate Statistical Analysis

Raw data of all samples acquired from LC-MS were converted to the mzXML format by using msConvert software (Version 3, ProteoWizard, Palo Alto, CA, USA) and then processed by using XCMS package in R software (Version 3.6.1, R Core Team, New Zealand) for peak extraction, peak alignment, and peak integration. Data processing was performed in both positive ionization mode and negative ionization mode. For multivariate statistical analysis, the output data from XCMS was further processed by R software. The overall differences in the metabolic profile among all collected samples were analyzed by principal component analysis (PCA) and hierarchical cluster analysis (HCA). For identification of the metabolites in the samples, the detected ion features from the UHPLC-Q-Orbitrap-MS were qualitatively analyzed based on the in-house metabolite database (Shanghai Biotree biotech Co., Ltd., Shanghai, China) and the public database, such as HMDB, METLIN, and M/Z cloud. Furthermore, in order to filter the differential metabolites between the two types of plant materials, a volcano plot analysis was performed.

3. Results

3.1. Method Validation

The reliability and reproducibility of the analytical method used in the current study was evaluated by measuring and comparing the outcomes of the three injections of QC. The base peak chromatograms (BPCs) of the QC sample and the extracted ion chromatograms (EICs) of the internal standard of 2-chlorophenylalanine in the QC sample in both positive and negative ionization modes were shown in Figures S1 and S2. The BPCs of the three injections and the EICs of 2-chlorophenylalanine were well overlapped, indicating the satisfying stability of the analytical method used in this study. Furthermore, low relative standard deviations of the intensities of the internal standard were detected between the three injections of the QC sample (8.1% and 3.4% for positive and negative ionization mode, respectively), suggesting good signal stability. These results suggested that the established untargeted metabolomic method was reliable and suitable for sample analysis.

3.2. General Comparison of the Metabolic Profiles

A UHPLC-Q-Orbitrap-MS-based untargeted metabolomic approach was used to define a comprehensive profile of the constituents from both the types of plant. The representative BPCs in both positive and negative ionization modes of the two sample types have been depicted in Figure 1. The differences in metabolic profile between them were evident as observed in both ionization modes.
Next, the XCMS package in R software was used to perform the chromatographic peak detection, alignment, filtration, and extraction, which generated two data matrixes consisting of 7867 and 5986 ion features in positive and negative ionization modes, respectively. Subsequently, the resultant ion features were used as the variables for further multivariate statistical analysis. PCA was carried out first to observe the distribution of the data of all samples under reduced dimensionality. As demonstrated in Figure 2A,B, the score plots of PCA in both ionization modes demonstrated that all samples, except for an outlier of C4, were clearly separated into two clusters, which was consistent with the types of samples. Then, in order to classify the samples with similar metabolic profile, a hierarchical cluster analysis (HCA) was conducted, and the result was illustrated in Figure 3. A clear grouping pattern, consistent with their types, was observed in both dendrograms representing the results derived from the two ionization modes. Therefore, the results of PCA and HCA suggested that the general metabolic profile of the rhizomes of cultivated plants was different from that of the wild-grown type.

3.3. Identification of the Phytochemicals

The detected ion features were putatively identified by their accurate mass and fragmentation data in an established in-house database and multiple public metabolites databases with a mass tolerance of 5 ppm. In this study, a total of 98 and 92 phytochemical compounds in the positive and negative ionization modes in LC-MS were tentatively identified as alkaloids (22), flavonoids (33), phenolic acids (34), and terpenoids (101). The detailed information including their compound names, composite score, molecular formula, class, accurate mass, retention time, and the integrated peak areas in all samples was provided in Table S2. Additionally, as shown in Figure S3, we used heatmaps to directly demonstrate the variations in the contents of these tentatively identified phytochemicals. Furthermore, the differentiating phytochemicals between the two sample types were visualized in a volcano plot, as shown in Figure 4. In the volcano plot, the negative logarithm to base 10 of p-value was used as the vertical axis, and the logarithm to base 2 of fold change between cultivated and wild-grown samples was used as the horizontal axis. Each point in the volcano plot represented a detected phytochemical. Phytochemicals with a fold change ≥2.0 or ≤0.5 and p-value < 0.05 were considered as differentially altered compounds, and thus colored as red (significantly higher in cultivated samples, fold change ≥2.0 and p-value < 0.05) or green (significantly higher in wild-grown samples, fold change ≤0.5 and p-value < 0.05). The results are illustrated in Figure 4. It can be seen that 22 phytochemicals were significantly higher in cultivated samples, of which 11 were acquired from the positive ionization mode and 11 were acquired from the negative ionization mode. In addition, 11 phytochemicals were significantly higher in wild-grown samples, of which five were acquired from the positive ionization mode and six were acquired from the negative ionization mode.
Detained information for these 33 differentiating phytochemicals is listed in Table S2. The variations in the contents for these differentiating phytochemicals and the clustering pattern of the two groups based on the differentiating phytochemicals were depicted in Figure 5. The color squares changed from light blue to dark blue, indicating the increasing amount of the phytochemicals.
A recent study profiled the chemical constituents of seeds of four Polygonatum species, including P. sibiricum, Polygonatum cyrtonema, Polygonatum kingianum, and Polygonatum macropodium [22]. In this study, 185 flavonoids, 127 lipids, 105 phenolic acids, and 36 steroids were tentatively identified by using the UPLC-QTOF-MS/MS system-based untargeted metabolomics approach [22]. Recently, Wang et al. also compared the metabolic profiles of three Polygonatum species, including P. sibiricum, P. cyrtonema, and P. kingianum, by using UPLC-Q-TOF-MS/MS, and found that adenosine, sucrose, and pyroglutamic acid could be used to distinguish the three Polygonatum species [23]. It has been reviewed by Zhao and Li [15] that the major chemical constituents of the genus Polygonatum are steroidal saponins, flavones, alkaloids, lignins, amino acids, and carbohydrates. Furthermore, it was summarized that steroidal saponins, flavonoids, lectin, and polysaccharides were the main bioactive compounds in this genus [15]. Due to the potential bioactivities of the phytochemical compounds, and for the purpose of the identification of the distinguishing metabolites between cultivated and wild-grown type of P. sibiricum, this study mainly focused on the identified 33 differentially altered phytochemical compounds with a fold change ≥2.0 or ≤0.5 and p-value < 0.05 between the two types of plant materials. These phytochemicals include four alkaloids, six flavonoids, seven phenolic acids, and 16 terpenoids, as listed in Table S2. The compound identification of several specific phytochemicals and their comparison between the two types of plant materials are presented below.

3.4. Differentially Altered Alkaloids

Alkaloids are plant secondary metabolites containing cyclic structures with at least one basic nitrogen atom [24]. Several alkaloids exhibit significant therapeutic effects, such as the antitumor agent taxol from Taxus, the antiarrhythmic quinidine from Cinchona, the antiasthma efedrina from Ephedra, and the antibiosis berberine from Coptis [25]. Alkaloids are among the most important active components in natural herbs. In this study, a total of 22 alkaloids were identified across all samples. Among them, four alkaloids, including nicotinic acid, 3-formylindole, laetanine, and calcium pantothenate, were found to be differentially abundant in the rhizomes of cultivated and wild-grown types of P. sibiricum. Taken laetanine as an example–its representative EIC and fragmentation spectrum were shown in Figure 6A1,A2. It is obvious that significant higher laetanine were detected in the cultivated samples. Laetanine, a noraporphine alkaloid, has been reported to possess antiplasmodial activity [26].

3.5. Differentially Altered Flavonoids

Flavonoids are a large group of naturally occurring phenylchromones. It is well known that flavonoids are powerful reducing agents or free radical quenchers in various systems [27]. Flavonoids can also act as antimicrobial defensive compounds [28]. A flavonoid-rich diet is commonly recommended due to the functionality of flavonoids in the reduction of risks of multiple acute and chronic diseases, including cardiovascular disease, cancer, and diabetes. The underlying mechanisms at the molecular level may associate with the management of oxidative stress [29]. Flavonoids are widely distributed in plants in glycoside-bound and free aglycone forms. The genus Polygonatum is rich in flavonoids [3]. It has been reviewed by Tao that a total of 54 flavonoids belonging to six structurally different subtypes presented in eight species of genus Polygonatum. In this study, we also identified 33 flavonoids from the rhizomes of cultivated and wild-grown types of P. sibiricum. Of these identified flavonoids, one flavonoid aglycone (genistein) and five flavonoid glycosides, including kaempferol-glucorhamnoside, kaempferol-neohesperidoside, apigenin-C-glucoside, vicenin-2 (apigenin 6,8-di-C-glucoside), and naringin, were found to be distinguishingly abundant in the rhizomes of cultivated and wild-grown types of P. sibiricum. Figure 6B1,B2 demonstrated the representative EIC and fragmentation spectrum of vicenin-2. This compound gave an [M-H]− ion at m/z 593.15057, indicating a molecular formula of C27H30O15 (Δm/z = −1.05 ppm). The data-dependent MS/MS fragmentation yielded five main fragment ions with m/z values of 353.06427; 383.07532; 473.10889; 297.07657; 325.06989. The fragmentation pattern fits the description of vicenin-2 as documented in the database of HMDB. Therefore, this compound was tentatively identified as vicenin-2, a 6,8-di-C-glucoside of apigenin. This compound has been reported to exist in many plants, such as Urtica circularis [30], Artemisia capillaris [31], and Citrus aurantium [32]. It has also been documented that vicenin-2 exerts various health beneficial effects, including antioxidant, anti-hepatotoxic, trypanocidal, antispasmodic, and anti-nociceptive activities, as well as the improvement of functional gastrointestinal discomfort [31]. Moreover, many studies have claimed that vicenin-2 is the main responsible compound in some herbs for their anti-inflammatory activity [30] and angiotensin-converting enzyme inhibitory activity [33]. This study is the first report indicating the existence of vicenin-2 in P. sibiricum. Comparing the abundance of vicenin-2 in the rhizomes of cultivated and wild-grown types of P. sibiricum, a much higher amount of vicenin-2 was detected in the wild-grown plant.

3.6. Differentially Altered Phenolic Acids

Phenolic acids, including hydroxybenzoic acids and hydroxycinnamic acids, can be found in many plants. This group of phenolic compounds are well known for their diverse biological activities and commonly work as bioactive molecules regularly used in therapeutics, cosmetics, and food industries [34]. In this study, two hydroxybenzoic acids and their derivatives, including vanillic acid and sibiricose A3, and five hydroxycinnamic acids, including ferulic acid, methyl-cinnamic acid, methyl 4-hydroxy-methoxycinnamate, arillatose B, and p-coumaroyl-beta-D-glucose, were found to be differentially abundant in the rhizomes of cultivated and wild-grown types of P. sibiricum. Figure 6C1,C2 demonstrated the representative EIC and fragmentation spectrum of p-coumaroyl-beta-D-glucose, respectively. This compound gave an [M-H]− ion at m/z 325.09323, indicating a molecular formula of C15H18O8 (Δm/z = 1.04 ppm). The data-dependent MS/MS fragmentation yielded three main fragment ions with m/z values of 163.03929; 119.05024; 145.02943. This fragmentation pattern fits the description of vicenin as documented in the database of HMDB. Therefore, this compound was tentatively identified as p-coumaroyl-beta-D-glucose. This compound is found in many plants, like strawberries, jostaberries, and blackcurrants. In this study, a higher abundance of p-coumaroyl-beta-D-glucose was detected in the cultivated P. sibiricum than that in the wild-grown samples.

3.7. Differentially Altered Terpenoids

Terpenoids are the largest and structurally most diverse class of plant secondary metabolites. They are responsible for the fragrance, taste, and pigment of many plants [35]. Moreover, many terpenoids have substantial pharmacological bioactivity, such as antioxidant activity, anti-malarial activity, anti-inflammatory effect, anti-cancer property, and anti-ulcer activity [36]. Therefore, in order to further our understanding of the pharmacological application potential of herbal materials such as P. sibiricum, it is of great interest to characterize their terpenoid profiles. In this study, a total of 101 terpenoids were identified across all samples. When comparing the abundance of these terpenoids in the rhizomes of cultivated and wild-grown types of P. sibiricum, 16 terpenoids were found to be distinguishingly different.
Iridoids, a type of monoterpenoids, comprise a large group of compounds in the general form of cyclopentanopyran, prevalently occurred in a wide range of plants, such as Scrophulariaceae, Pyrolaceae, Oleaceae, Labiatae, Rubiaceae, and Gentianaceae [37]. Iridoids most frequently exist in plants as glycosides, commonly bound to glucose. Iridoids have been regarded as defense chemicals against herbivores and pathogens. Additionally, various pharmacological effects, such as neuroprotective, hepatoprotective, anti-inflammatory, hypoglycemic, hypolipidemic, and antitumor activities, have been reported [37]. Due to their importance in pharmaceutical applications, iridoids have attracting more and more attentions. In this study, six iridoid glycosides, including daphylloside, asperuloside acid, geniposide, harpagide, loganoside, and 7-deoxyloganetate hexioside were identified as the distinguishing iridoids in the rhizomes of cultivated and wild-grown types of P. sibiricum. Taking geniposide as an example, as shown in Figure 6D1, this compound produced an [M-H]− ion with m/z value of 387.12978, indicating the molecular formula of this compound was C17H24O10 (Δm/z = 0.28 ppm). The m/z values of the main MS/MS fragmentation were 225.07722, 166.06348, 341.10785, and 151.04004 (Figure 6D2). The fragmentation pattern fits the description of geniposide as documented in the database of HMDB. The most abundant ion fragment peak 225.07722 represents the loss of sugar moiety. Therefore, this compound was tentatively identified as geniposide. This compound is an iridoid monoterpenes containing a glycosyl moiety linked to the iridoid skeleton. It has been found in over 40 species of plants, most of which are herbal medicine materials [38,39]. Zhou et al. have systematically reviewed the diverse pharmacological activities and potential medicinal benefits of geniposide, which include neuroprotective, antidiabetic, hepatoprotective, anti-inflammatory, analgesic, antidepressant-like, cardioprotective, antioxidant, immune-regulatory, antithrombotic, and antitumoral effects [40]. In this study, significant higher abundance of geniposide was shown in the rhizomes of cultivated P. sibiricum than that in the wild-grown plants.
Steroidal saponins are another group of important terpenoids in plant. This group of compounds are characterized as triterpenoids linked to one or more carbohydrate chains. It has been reported that steroidal saponins are widely distributed in the genus Polygonatum, such as P. sibiricum [41], P. kingianum [42,43], P. odoratum [44], and P. zanlanscianense [43]. Numerous studies have clearly documented the pharmacological activities of steroidal saponins. Some of them showed promising anti-microbial, anti-inflammatory, anti-thrombotic, and hypocholesterolemic effects [45]. More importantly, steroidal saponins are used as substrates in the production of steroid hormones and drugs [46]. In this study, four steroidal saponins, including lithocholic acid, digitonin, medicagenic acid, and obacunone, were found to be differentially altered in the rhizomes of cultivated plant as compared to wild-grown type of P. sibiricum. As shown in Figure 6E1, the molecular formula of this compound was C30H46O6, as established by the [M-H]− peak at m/z 501.32217 (Δm/z = 0.01 ppm). The MS/MS spectrum of this compound showed four major fragment ions, including 457.33429, 439.32462, 73.02901, and 353.24725 (Figure 6E2), which is similar to the compound medicagenic acid as documented in database of HMDB. Medicagenic acid, also known as medicagenate, has been reported to possess antimycotic activities. In this study, a significantly higher abundance of medicagenic acid was detected in the rhizomes of cultivated P. sibiricum than that in the wild-grown type.

4. Conclusions

In this study, a UHPLC-Q-Orbitrap-MS-based untargeted metabolomic approach was employed to reveal the phytochemical variation of two types of P. sibiricum rhizomes, namely the cultivated and wild-grown types. A total of 190 phytochemicals, belonging to alkaloids, flavonoids, phenolic acids, and terpenoids, were tentatively identified. As demonstrated in PCA and HCA, distinctive differences in phytochemical profiles between the two sample types were present. Specifically, of these identified phytochemicals, 33 compounds showed significant differences. Although it is commonly believed that the wild-grown type P. sibiricum rhizomes possesses better pharmacological activities, the higher content of multiple phytochemicals, such as laetanine, p-coumaroyl-beta-D-glucose, geniposide, medicagenic acid, in the cultivated plant suggests the cultivation may also have potent application potentials in medicine and functional foods. Regarding the underlying mechanism for the large disparities between the two sample types, it is as of yet unknown but may relate to the different cultivation conditions. It is speculated that some of the differentiating phytochemicals are caused by the differences in gene and protein expression. Further transcriptome and proteome studies to explore the differentiating gene and protein are warranted. In addition, in vitro and in vivo studies are also necessary to further compare the health benefits of these two types of plant materials.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/separations9120398/s1. Table S1: The basic information of the cultivated and wild-grown types P. sibiricum samples. Table S2: The tentatively identified phytochemicals acquired from positive and negative ionization modes. Figure S1: The base peak chromatograms of QC samples in both positive and negative ionization modes. Figure S2: The extracted ion chromatograms of the internal standard of 2-chlorophenylalanine in QC samples in positive and negative ionization modes. Figure S3: Heatmaps and hierarchical cluster analysis of the tentatively identified phytochemicals.

Author Contributions

Z.P.: writing—original draft, performed experiments and sorted the data. Z.P., J.L. and Z.L.: conceptualization, analyzed the validation data. W.C.: data curation. Z.P., W.C., W.W. and B.Y.: performed experiments and analyzed the data. Z.L. and J.L.: writing—review and editing, conceptualization, supervision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the biomedical health care innovation team of Fujian Vocational College of Bioengineering (2020TD01); the central government guide local special projects for science and technology development from Science and Technology Department of Fujian Province (2020L3018); the middle-aged and youth project of Education Department of Fujian Province (JAT201225) and the key program of Fujian Vocational College of Bioengineering (2020ZD02).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data will be provided upon request.

Conflicts of Interest

All the authors declare that they have no conflict of interest.

References

  1. Cheng, X.; Ji, H.; Cheng, C.; Sun, Y.; Cheng, H.; Wang, D.; Pan, Y.; Liu, X. Comprehensive determination of the processing level of rhizome of Polygonatum sibiricum by macroscopic, micromorphological, and microscopic characterizations. Microsc. Res. Tech. 2022, 85, 2669–2678. [Google Scholar] [CrossRef] [PubMed]
  2. Hou, Y.; Jiang, J.-G. Origin and concept of medicine food homology and its application in modern functional foods. Food Funct. 2013, 4, 1727–1741. [Google Scholar] [CrossRef] [PubMed]
  3. Debnath, T.; Park, S.R.; Jo, J.E.; Lim, B.O. Antioxidant and anti-inflammatory activity of Polygonatum sibiricum rhizome extracts. Asian Pac. J. Trop. Dis. 2013, 3, 308–313. [Google Scholar] [CrossRef]
  4. Li, Y.-L. Isolation and identification of endophytic fungi from Polygonatum sibiricum in Mountain Tai and study on their antimicrobial activity. Chin. Tradit. Herb. Drugs 2013, 44, 1490–1494. [Google Scholar]
  5. Zhao, H.; Wang, Q.-L.; Hou, S.-B.; Chen, G. Chemical constituents from the rhizomes of Polygonatum sibiricum Red. and anti-inflammatory activity in RAW264. 7 macrophage cells. Nat. Prod. Res. 2019, 33, 2359–2362. [Google Scholar] [CrossRef] [PubMed]
  6. Yang, J.-X.; Wu, S.; Huang, X.-L.; Hu, X.-Q.; Zhang, Y. Hypolipidemic activity and antiatherosclerotic effect of polysaccharide of Polygonatum sibiricum in rabbit model and related cellular mechanisms. Evid.-Based Complement. Altern. Med. 2015, 2015, 391065. [Google Scholar] [CrossRef] [Green Version]
  7. Chai, Y.; Luo, J.; Bao, Y. Effects of Polygonatum sibiricum saponin on hyperglycemia, gut microbiota composition and metabolic profiles in type 2 diabetes mice. Biomed. Pharmacother. 2021, 143, 112155. [Google Scholar] [CrossRef]
  8. Luo, J.; Chai, Y.; Zhao, M.; Guo, Q.; Bao, Y. Hypoglycemic effects and modulation of gut microbiota of diabetic mice by saponin from Polygonatum sibiricum. Food Funct. 2020, 11, 4327–4338. [Google Scholar] [CrossRef]
  9. Long, T.; Liu, Z.; Shang, J.; Zhou, X.; Yu, S.; Tian, H.; Bao, Y. Polygonatum sibiricum polysaccharides play anti-cancer effect through TLR4-MAPK/NF-κB signaling pathways. Int. J. Biol. Macromol. 2018, 111, 813–821. [Google Scholar] [CrossRef]
  10. Zhao, H.; Hou, D.; Sun, T.; Lin, W. Quantitative Determination of Total Flavonoids from Polygonatum sibiricum by Spectrophotometry. In Proceedings of the IOP Conference Series: Materials Science and Engineering, Wuhan, China, 10–12 October 2019; p. 022126. [Google Scholar]
  11. Hu, C.-Y.; Xu, D.-P.; Wu, Y.-M.; Ou, S.-Y. Triterpenoid saponins from the rhizome of Polygonatum sibiricum. J. Asian Nat. Prod. Res. 2010, 12, 801–808. [Google Scholar] [CrossRef]
  12. Tang, C.; Yu, Y.-M.; Qi, Q.-L.; Wu, X.-D.; Wang, J.; Tang, S.-A. Steroidal saponins from the rhizome of Polygonatum sibiricum. J. Asian Nat. Prod. Res. 2019, 21, 197–206. [Google Scholar] [CrossRef] [PubMed]
  13. Sun, L.-R.; Li, X.; Wang, S.-X. Two new alkaloids from the rhizome of Polygonatum sibiricum. J. Asian Nat. Prod. Res. 2005, 7, 127–130. [Google Scholar] [CrossRef] [PubMed]
  14. Chen, Z.-Y.; Liu, Y.; Zhang, Y.-Q.; Huang, J.-H.; Shao, J.-H.; Zhu, J.-Z.; Zhao, C.-C. A New Phenolic Glycoside from Polygonatum sibiricum and its α-Glucosidase Inhibitory Activity. Chem. Nat. Compd. 2021, 57, 50–52. [Google Scholar] [CrossRef]
  15. Zhao, X.; Li, J. Chemical constituents of the genus Polygonatum and their role in medicinal treatment. Nat. Prod. Commun. 2015, 10, 683–688. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  16. Luo, G.; Lin, J.; Cheng, W.; Liu, Z.; Yu, T.; Yang, B. UHPLC-Q-Orbitrap-MS-Based Metabolomics Reveals Chemical Variations of Two Types of Rhizomes of Polygonatum sibiricum. Molecules 2022, 27, 4685. [Google Scholar] [CrossRef]
  17. Xie, H.; Wang, H.; Chen, B.; Lou, J.; Wang, H.; Xiong, Y.; Hu, Y.; Xu, X.; Jing, Q.; Jiang, M. Untargeted metabolomics analysis to unveil the chemical markers for the differentiation among three Gleditsia sinensis-derived herbal medicines by ultra-high performance liquid chromatography/quadrupole time-of-flight mass spectrometry. Arab. J. Chem. 2022, 15, 103762. [Google Scholar] [CrossRef]
  18. Liu, Z.; Bruins, M.E.; de Bruijn, W.J.; Vincken, J.-P. A comparison of the phenolic composition of old and young tea leaves reveals a decrease in flavanols and phenolic acids and an increase in flavonols upon tea leaf maturation. J. Food Compos. Anal. 2020, 86, 103385. [Google Scholar] [CrossRef]
  19. Meng, X.; Zhu, B.; Liu, Y.; Fang, L.; Yin, B.; Sun, Y.; Ma, M.; Huang, Y.; Zhu, Y.; Zhang, Y. Unique Biomarker Characteristics in Gestational Diabetes Mellitus Identified by LC-MS-Based Metabolic Profiling. J. Diabetes Res. 2021, 2021, 6689414. [Google Scholar] [CrossRef]
  20. Augustijn, D.; de Groot, H.J.; Alia, A. HR-MAS NMR applications in plant metabolomics. Molecules 2021, 26, 931. [Google Scholar] [CrossRef]
  21. Patel, M.K.; Pandey, S.; Kumar, M.; Haque, M.I.; Pal, S.; Yadav, N.S. Plants metabolome study: Emerging tools and techniques. Plants 2021, 10, 2409. [Google Scholar] [CrossRef]
  22. Qi, J.; Wei, J.; Liao, D.; Ding, Z.; Yao, X.; Sun, P.; Li, X. Untargeted Metabolomics Analysis Revealed the Major Metabolites in the Seeds of four Polygonatum Species. Molecules 2022, 27, 1445. [Google Scholar] [CrossRef] [PubMed]
  23. Wang, S.; Li, W.; Zhang, X.; Li, G.; dong Li, X.; Chang, H.; Niu, J.; Wang, Z. Metabolomics Study of Different Germplasm Resources for Three Polygonatum Species Using UPLC-Q-TOF-MS/MS. Front. Plant Sci. 2022, 13, 826902. [Google Scholar] [CrossRef] [PubMed]
  24. Mondal, A.; Gandhi, A.; Fimognari, C.; Atanasov, A.G.; Bishayee, A. Alkaloids for cancer prevention and therapy: Current progress and future perspectives. Eur. J. Pharmacol. 2019, 858, 172472. [Google Scholar] [CrossRef] [PubMed]
  25. Fang, L.; Liu, Y.; Yang, B.; Wang, X.; Huang, L. Separation of alkaloids from herbs using high-speed counter-current chromatography. J. Sep. Sci. 2011, 34, 2545–2558. [Google Scholar] [CrossRef] [PubMed]
  26. Omar, H.; Fadaeinasab, M.; Taha, H.; Widyawaruyanti, A.; Nafiah, M.A.; Rachmatiah, T. Aporphine alkaloids with in vitro antiplasmodial activity from the leaves of Phoebe tavoyana. J. Asian Nat. Prod. Res. 2020, 22, 52–60. [Google Scholar] [CrossRef] [PubMed]
  27. E Middleton, M.D., Jr. Biological properties of plant flavonoids: An overview. Int. J. Pharmacogn. 1996, 34, 344–348. [Google Scholar] [CrossRef]
  28. Cushnie, T.T.; Lamb, A.J. Antimicrobial activity of flavonoids. Int. J. Antimicrob. Agents 2005, 26, 343–356. [Google Scholar] [CrossRef] [PubMed]
  29. Slimestad, R.; Fossen, T.; Brede, C. Flavonoids and other phenolics in herbs commonly used in Norwegian commercial kitchens. Food Chem. 2020, 309, 125678. [Google Scholar] [CrossRef]
  30. Marrassini, C.; Davicino, R.; Acevedo, C.; Anesini, C.; Gorzalczany, S.; Ferraro, G. Vicenin-2, a potential anti-inflammatory constituent of Urtica circularis. J. Nat. Prod. 2011, 74, 1503–1507. [Google Scholar] [CrossRef]
  31. Islam, M.N.; Ishita, I.J.; Jung, H.A.; Choi, J.S. Vicenin 2 isolated from Artemisia capillaris exhibited potent anti-glycation properties. Food Chem. Toxicol. 2014, 69, 55–62. [Google Scholar] [CrossRef]
  32. Alam, F.; Mohammadin, K.; Shafique, Z.; Amjad, S.T.; Asad, M.H.H.b. Citrus flavonoids as potential therapeutic agents: A review. Phytother. Res. 2022, 36, 1417–1441. [Google Scholar] [CrossRef] [PubMed]
  33. Zhang, Y.-Q.; Luo, J.-G.; Han, C.; Xu, J.-F.; Kong, L.-Y. Bioassay-guided preparative separation of angiotensin-converting enzyme inhibitory C-flavone glycosides from Desmodium styracifolium by recycling complexation high-speed counter-current chromatography. J. Pharm. Biomed. Anal. 2015, 102, 276–281. [Google Scholar] [CrossRef] [PubMed]
  34. Kumar, N.; Goel, N. Phenolic acids: Natural versatile molecules with promising therapeutic applications. Biotechnol. Rep. 2019, 24, e00370. [Google Scholar] [CrossRef] [PubMed]
  35. Wang, G.; Tang, W.; Bidigare, R.R. Terpenoids as therapeutic drugs and pharmaceutical agents. In Natural Products; Springer: Berlin/Heidelberg, Germany, 2005; pp. 197–227. [Google Scholar]
  36. Yang, W.; Chen, X.; Li, Y.; Guo, S.; Wang, Z.; Yu, X. Advances in pharmacological activities of terpenoids. Nat. Prod. Commun. 2020, 15, 1934578X20903555. [Google Scholar] [CrossRef] [Green Version]
  37. Wang, C.; Gong, X.; Bo, A.; Zhang, L.; Zhang, M.; Zang, E.; Zhang, C.; Li, M. Iridoids: Research advances in their phytochemistry, biological activities, and pharmacokinetics. Molecules 2020, 25, 287. [Google Scholar] [CrossRef] [Green Version]
  38. Shan, M.; Yu, S.; Yan, H.; Guo, S.; Xiao, W.; Wang, Z.; Zhang, L.; Ding, A.; Wu, Q.; Li, S.F.Y. A review on the phytochemistry, pharmacology, pharmacokinetics and toxicology of geniposide, a natural product. Molecules 2017, 22, 1689. [Google Scholar] [CrossRef] [Green Version]
  39. Costa, R.; Albergamo, A.; Pellizzeri, V.; Dugo, G. Phytochemical screening by LC-MS and LC-PDA of ethanolic extracts from the fruits of Kigelia africana (Lam.) Benth. Nat. Prod. Res. 2017, 31, 1397–1402. [Google Scholar] [CrossRef]
  40. Zhou, Y.-X.; Zhang, R.-Q.; Rahman, K.; Cao, Z.-X.; Zhang, H.; Peng, C. Diverse pharmacological activities and potential medicinal benefits of geniposide. Evid.-Based Complement. Altern. Med. 2019, 2019, 4925682. [Google Scholar] [CrossRef] [Green Version]
  41. Ahn, M.-J.; Kim, C.Y.; Yoon, K.-D.; Ryu, M.Y.; Cheong, J.H.; Chin, Y.-W.; Kim, J. Steroidal Saponins from the Rhizomes of Polygonatum sibiricum. J. Nat. Prod. 2006, 69, 360–364. [Google Scholar] [CrossRef]
  42. Xing-Cong, L.; Chong-Ren, Y.; Makoto, I.; Hiromichi, M.; Ryoji, K.; Kazuo, Y. Steroid saponins from Polygonatum kingianum. Phytochemistry 1992, 31, 3559–3563. [Google Scholar] [CrossRef]
  43. Jin, J.-M.; Zhang, Y.-J.; Li, H.-Z.; Yang, C.-R. Cytotoxic steroidal saponins from Polygonatum z anlanscianense. J. Nat. Prod. 2004, 67, 1992–1995. [Google Scholar] [CrossRef]
  44. Wang, D.; Li, D.; Zhu, W.; Zhang, J.; Peng, P. Steroidal saponins from the rhizomes of Polygonatum odoratum. Nat. Prod. Res. 2009, 23, 940–947. [Google Scholar] [CrossRef] [PubMed]
  45. Passos, F.R.S.; Araújo-Filho, H.G.; Monteiro, B.S.; Shanmugam, S.; de Souza Araújo, A.A.; da Silva Almeida, J.R.G.; Thangaraj, P.; Júnior, L.J.Q.; Quintans, J.D.S.S. Anti-inflammatory and modulatory effects of steroidal saponins and sapogenins on cytokines: A review of pre-clinical research. Phytomedicine 2021, 96, 153842. [Google Scholar] [CrossRef] [PubMed]
  46. Güçlü-Üstündağ, Ö.; Mazza, G. Saponins: Properties, applications and processing. Crit. Rev. Food Sci. Nutr. 2007, 47, 231–258. [Google Scholar] [CrossRef] [PubMed]
Separations 09 00398 g001 550
Figure 1. The representative base peak chromatograms of the cultivated and wild-grown P. sibiricum acquired in positive (A) and negative (B) ionization modes.
Figure 1. The representative base peak chromatograms of the cultivated and wild-grown P. sibiricum acquired in positive (A) and negative (B) ionization modes.
Separations 09 00398 g001
Separations 09 00398 g002 550
Figure 2. PCA score plots of the metabolites acquired from positive (A) and negative (B) ionization modes.
Figure 2. PCA score plots of the metabolites acquired from positive (A) and negative (B) ionization modes.
Separations 09 00398 g002
Separations 09 00398 g003 550
Figure 3. HCA dendrograms of the metabolites acquired from positive (A) and negative (B) ionization modes.
Figure 3. HCA dendrograms of the metabolites acquired from positive (A) and negative (B) ionization modes.
Separations 09 00398 g003
Separations 09 00398 g004 550
Figure 4. Volcano plots of the identified phytochemicals of the cultivated and wild-grown P. sibiricum.
Figure 4. Volcano plots of the identified phytochemicals of the cultivated and wild-grown P. sibiricum.
Separations 09 00398 g004
Separations 09 00398 g005 550
Figure 5. Heatmaps and hierarchical cluster analysis of the 33 significantly altered phytochemicals.
Figure 5. Heatmaps and hierarchical cluster analysis of the 33 significantly altered phytochemicals.
Separations 09 00398 g005
Separations 09 00398 g006 550
Figure 6. The extract ion chromatograms and data-dependent MS2 fragmentation spectra of representative phytochemical compounds. The extract ion chromatogram at m/z 314.13865 in positive ionization mode (A1) and its MS2 fragmentation spectrum (A2). The extract ion chromatogram at m/z 593.15057 in negative ionization mode (B1) and its MS2 fragmentation spectrum (B2). The extract ion chromatogram at m/z 325.09323 in negative ionization mode (C1) and its MS2 fragmentation spectrum (C2). The extract ion chromatogram at m/z 387.12978 in negative ionization mode (D1) and its MS2 fragmentation spectrum (D2). The extract ion chromatogram at m/z 501.32217 in negative ionization mode (E1) and its MS2 fragmentation spectrum (E2).
Figure 6. The extract ion chromatograms and data-dependent MS2 fragmentation spectra of representative phytochemical compounds. The extract ion chromatogram at m/z 314.13865 in positive ionization mode (A1) and its MS2 fragmentation spectrum (A2). The extract ion chromatogram at m/z 593.15057 in negative ionization mode (B1) and its MS2 fragmentation spectrum (B2). The extract ion chromatogram at m/z 325.09323 in negative ionization mode (C1) and its MS2 fragmentation spectrum (C2). The extract ion chromatogram at m/z 387.12978 in negative ionization mode (D1) and its MS2 fragmentation spectrum (D2). The extract ion chromatogram at m/z 501.32217 in negative ionization mode (E1) and its MS2 fragmentation spectrum (E2).
Separations 09 00398 g006
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Pan, Z.; Cheng, W.; Liu, Z.; Wu, W.; Yang, B.; Lin, J. Comparative Study of the Phytochemical Profiles of the Rhizomes of Cultivated and Wild-Grown Polygonatum sibiricum. Separations 2022, 9, 398. https://doi.org/10.3390/separations9120398

AMA Style

Pan Z, Cheng W, Liu Z, Wu W, Yang B, Lin J. Comparative Study of the Phytochemical Profiles of the Rhizomes of Cultivated and Wild-Grown Polygonatum sibiricum. Separations. 2022; 9(12):398. https://doi.org/10.3390/separations9120398

Chicago/Turabian Style

Pan, Zhibin, Weiqing Cheng, Zhibin Liu, Weibin Wu, Bin Yang, and Junhan Lin. 2022. "Comparative Study of the Phytochemical Profiles of the Rhizomes of Cultivated and Wild-Grown Polygonatum sibiricum" Separations 9, no. 12: 398. https://doi.org/10.3390/separations9120398

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop