Abstract

   Portulaca oleracea L., known as the “vegetable for long life,” is an
   annual succulent herb that is widely distributed worldwide. Many
   clinical and experimental studies have demonstrated that purslane seed
   (MCXZ) can be used as an adjunctive and alternative therapy for the
   treatment of diabetes mellitus (DM). However, the underlying active
   constituents and pharmacological mechanisms through which MCXZ exerts
   effects in DM remain unclear. In the present study, we confirmed that
   MCXZ treatment resulted in hypoglycemic activity, lowering the fasting
   blood glucose and glycated hemoglobin levels in streptozotocin-induced
   diabetic mice. Then, ultra-high-pressure liquid chromatography coupled
   with linear ion trap-Orbitrap tandem mass spectrometry was used to
   systematically analyze the chemical profile of MCXZ, resulting in the
   identification of 84 constituents, including 31 organic acids and nine
   flavonoids. Finally, the Integrative Pharmacology-based Research
   Platform of Traditional Chinese Medicine was employed to analyze the
   key active components of MCXZ and the molecular mechanisms through
   which these components acted in DM. Ten key active compounds were
   identified based on the topological importance of their corresponding
   putative targets within the known DM-associated therapeutic target
   network of known MCXZ putative targets. Functionally, these candidate
   targets play critical anti-hyperlipidemia, anti-hyperglycemia, immunity
   regulation, and inflammatory roles involving DM-related pathways, such
   as the vascular endothelial growth factor (VEGF) signaling pathway and
   Fc gamma R-mediated phagocytosis, which indicated that MCXZ exhibited
   anti-diabetic activity through multi-faced actions.

   Keywords: purslane seed, diabetes mellitus, UHPLC-LTQ-orbitrap, TCMIP
   v20, molecular network

Introduction

   Diabetes mellitus (DM) represents a major public health issue, causing
   serious economic burdens for both developed and developing countries
   ([40]Papatheodorou et al., 2018). The International Diabetes Federation
   (IDF) reported that approximately 463 million individuals had diabetes
   worldwide in 2019, including 116 million patients in China, which was
   ranked first in the world ([41]International Diabetes Federation (IDF),
   2019). Persistent hyperglycemia and long-term metabolic disorders may
   lead to the development of nephropathy, retinopathy, neuropathy, and
   cardiovascular disease ([42]Zhang H. et al., 2018). Currently, the
   drugs used to treat DM include biguanide, sulfonylureas, α-glycosidase
   inhibitors, benzoin acid, and derivative secretagogues, most of which
   aim to control blood glucose levels and must be used long-term. Gradual
   increases in the required doses of these drugs can lead to liver and
   kidney dysfunction, which can be associated with various complications,
   in addition to those resulting from the disease process ([43]Moukette
   et al., 2017). Therefore, the development of safer, more effective
   drugs, especially those derived from natural products, which can
   provide improved management for blood glucose and diabetes-associated
   complications, has long been the focus of DM studies.

   Traditional Chinese medicine (TCM) is practiced as a form of holistic
   and personalized medicine and has been shown to effectively lower blood
   glucose levels, control diabetic complications, and cause fewer
   side-effects than western medicines, based on syndrome differentiation
   and treatments ([44]Zhang, 2014) that are multi-component,
   multi-pattern, and multi-target. As a result, increasing research has
   focused on the TCM-based treatment of DM. In TCM theory, DM belongs to
   the category of “Xiao-Ke-Zheng,” which was first recorded in the
   classical medical text “Huangdi Neijing” over a thousand years ago.
   Purslane is an annual succulent herb best known as the “vegetable for
   long life” and is distributed throughout diverse geographical
   environments worldwide. Purslane seed (MCXZ) has been used as both food
   and medicine for thousands of years in China ([45]Aberoumand, 2009).
   Clinically, MCXZ, as an adjuvant combined with other treatments, has
   been shown to alleviate DM symptoms, including reduced inflammation and
   improved liver function ([46]El-Sayed, 2011; [47]Dehghan et al., 2016).
   Chemically, a wide variety of compounds have been identified in MCXZ,
   including flavonoids, polysaccharides, fatty acids, proteins,
   glutathione, antioxidants, and vitamins ([48]El-Sayed, 2011).
   Pharmacologically, MCXZ has been associated with various biological
   activities, including hypoglycemic (A. [49]Mohamed et al., 2019),
   hypocholesterolemic ([50]Movahedian et al., 2007), anti-oxidative
   ([51]Guo et al., 2016), diuretic, antipyretic, analgesic, and
   anti-inflammatory ([52]Daniel, 2006) processes. Currently, MCXZ is used
   as an adjuvant treatment for DM to improve glucose tolerance, lipid
   metabolism disorders, liver functions, and insulin sensitivity and
   reduce hyperinsulinemia ([53]Mohanapriya et al., 2006; [54]El-Sayed,
   2011). However, the potential active components and molecular
   mechanisms through which MCXZ acts and that may be applied to the
   direct treatment of DM remain unclear, which limits the clinical
   applications of MCXZ.

   In the current study, an integrative pharmacology approach was used to
   investigate the active constituents and the underlying pharmacological
   mechanisms through which MCXZ acts during the treatment of DM. This
   study combined high-throughput chemical analysis, target prediction,
   and network construction and analysis, which was performed by following
   a three-step analytical process ([55]Figure 1). 1) Chemical information
   databases, including the Encyclopedia of Traditional Chinese Medicine
   (ETCM) and other electronic databases, were searched for the
   constituents of purslane. 2) Ultra-high-pressure liquid chromatography
   coupled with linear ion trap-Orbitrap tandem mass spectrometry
   (UHPLC-LTQ-Orbitrap) was performed to rapidly characterize the
   preliminary chemical profile of MCXZ. 3) The TCMIP V2.0 platform was
   utilized to predict MCXZ putative targets, construct a drug
   target-disease-gene network based on predicted interactions among MCXZ
   putative targets and known therapeutic targets associated with
   DM-related diseases, and identify potential active constituents
   correlated with the candidate MCXZ targets during the treatment of DM.

FIGURE 1.

   [56]FIGURE 1
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   Schematic diagram for revealing the active constituents and the
   underlying pharmacological mechanisms of MCXZ in the treatment of DM by
   combining high-throughput technology and integrative pharmacology
   method.

Materials and Methods

Chemicals and Materials

   Mass spectrometry-grade methanol, acetonitrile, and formic acid were
   acquired from Fisher Scientific Co. (Loughborough, United Kingdom).
   Purified water was prepared by a Milli-Q system (Millipore, Billerica,
   MA, United States). Other reagents used in the experiment were of
   analytical grade. MCXZ was supplied by Guangzhou Zhongda Pharmaceutical
   Development Co., Ltd. (Batch NO. 180201; Guangzhou, China). This drug
   was identified as a dry, mature seed from Portulaca oleracea by the
   pharmacist Yan Jin (research assistant at the China Academy of Chinese
   Medical Sciences).

Animals and Experimental Design

   Healthy specific-pathogen-free (SPF)-grade male Balb/c mice (body
   weight: 18.0–21.0 g) were purchased from the Department of Experimental
   Animal Science, Department of Medicine, Peking University (Beijing,
   China). The project identification code was 20160010. All animal
   experiments were approved by the Committee on Animal Care and Use of
   the Institute of Chinese Materia Medica, China Academy of Chinese
   Medical Sciences. Before the experiment began, all animals were placed
   in a standard laboratory environment, during which they were provided
   free access to food and water. The experiment did not begin until the
   animals had adapted to the environment for 3 days.

   Dried MCXZ was ground into a powder with 80 mesh by a high-speed
   multi-function grinder (JP-500C, Yongkang Jiupin Industry and Trade
   Co., Ltd.). Before administration, the powder was dissolved in normal
   saline containing 0.5% carboxymethyl cellulose sodium to prepare 54.17,
   108.33, and 216.67 mg/ml suspensions ([58]Liu, 2018).

   Streptozotocin (STZ, 60 mg/kg, dissolved in 0.1 M sodium citrate
   buffer, pH 4.5) was injected intraperitoneally, continuously for 5 days
   to induce DM in mice ([59]Goodarzi et al., 2019). To establish the
   normal control group, 12 healthy mice were injected intraperitoneally
   with an equal volume of sodium citrate buffer. One week after the last
   injections, the mice were fasted for 5 h, after which blood was
   collected from the tail vein, and the fasting blood glucose (FBG)
   levels were measured using a blood glucose monitor (GT-1980. Aikelai
   Medical Electronics (Pinghu) Co., Ltd.). Mice with FBG levels greater
   than 11.1 mmol·L^−1 were considered to be successful DM model mice. All
   successfully modeled DM mice were randomly divided into five groups
   according to body weight and FBG: Model (STZ) group, Met (metformin
   hydrochloride) group, and MCXZ low-, medium-, and high-dose groups (n =
   12 for each group). The dose of metformin hydrochloride used was
   130 mg/kg body weight (BW)/day; the low, medium, and high doses of MCXZ
   powder were 812.5, 1,625, and 3,250 mg/kg BW/day, respectively. The
   normal control group and model group were administered an equivalent
   volume of 0.9% NaCl. All groups were treated through intragastric
   administration for four consecutive weeks.

   FBG (fasted for 5 h) was detected every 2 weeks for 4 weeks. At the end
   of the experiment, the mice were fasted for more than 12 h, and then
   eye blood samples were collected. Blood samples were collected in blank
   sterile tubes and allowed to coagulate at room temperature for 1 h.
   Then, whole blood was centrifuged at 3,500 rpm for 15 min. The serum
   was separated and stored at −80°C for further studies.

Measurement of Glycated Hemoglobin (HbAlc)

   A specific enzyme-linked immunosorbent assay (ELISA) kit (Cusabio,
   batch number: M03033575) was used to quantify HbAlc from serum samples.
   This assay employs the competitive inhibition enzyme immunoassay
   technique. The experiment was performed according to the manufacturer’s
   instructions.

Histopathological Evaluation of Liver and Kidney Tissues

   The liver and kidney tissues were removed and immersed in 4% formalin
   for 72 h at 4°C. To analyze the histopathological changes that occurred
   in the liver and kidney, sections from paraffin-embedded tissues were
   stained with hematoxylin and eosin and captured under a light
   microscope (Olympus, BX51, Japan).

Chemical Information Database for the Compounds Found in Purslane

   The chemical compound database information associated with purslane
   primarily included chemical name, molecular formula, molecular weight,
   structural formula, and other information. The chemical components
   associated with purslane were collected from existing databases, using
   “Portulaca oleracea” as the keyword. These databases included the
   Encyclopedia of Traditional Chinese Medicine (ETCM,
   [60]http://www.nrc.ac.cn:9090/ETCM/), which contains information about
   a total of 7,274 herbal ingredients ([61]Xu et al., 2019). Other
   resources included electronic databases such as PubMed
   ([62]https://pubmed.ncbi.nlm.nih.gov/, update on 2019) and CNKI (China
   Journals of Full-text database; [63]https://www.cnki.net/, update on
   2019). Detailed information regarding the identified compounds in
   purslane is presented in [64]Supplementary Table S1.

Preparation of Sample Solutions

   MCXZ was ground into a powder with 40 mesh, and 1 g of powder was
   accurately weighed. The powder was dissolved in 10 ml 70% methanol and
   submitted to ultrasonic extraction for 40 min. The extracts were
   centrifuged for 12 min (at 12,000 rpm), and the supernatant was
   separated. The sample solution was subjected to 0.22-mm nylon membrane
   filtration and analyzed directly by UHPLC-LTQ-Orbitrap.

LC System

   Sample analysis was performed using the Thermo Accela UHPLC system
   (Thermo Fisher Scientific, San Jose, California, United States).
   Chromatographic separation was performed on a maintained reverse-phase
   column Waters HSS T3-C18 (2.1 × 100 mm, 1.8 µm). The mobile phase was a
   mixture of methanol (A) and 0.1% formic acid in water (B). The
   following elution gradient was used: 0–5 min, 3%–10% A; 5–25 min,
   10%–40% A; 25–35 min, 40%–60% A; 35–45 min, 60%–80% A; 45–50 min,
   80%–95% A; 50–60 min, 95% A. The flow rate was set to 0.3 ml/min, and
   the injection volume was 1 µL.

Mass Spectrometry and Data Processing

   For LC-ESI-MS^n experiments, the samples were detected in the positive
   and negative ion modes by electrospray ionization (ESI) source and were
   scanned in one-stage and multi-stage modes separately. The parameters
   for the ESI source were as follows: ion source voltage, 3.5 kV;
   capillary temperature, 350°C; sheath and auxiliary gas pressure, 0.24
   and 0.07 MPa, respectively; ion source temperature, 350°C. The sheath
   and auxiliary gas was nitrogen in both cases. The mass axis of MS was
   calibrated using an external standard method (the mass error was less
   than 5 ppm); mass calibration positive ion selection: 74.09643,
   3.06037, 195.08465, 262.63612, 524.26496, and 1,022.00341; negative ion
   selection: 230.10174, 249.15299, and 407.28030. The MS^1 was fully
   scanned and acquired in the range of 50–1,500 m/z, with a resolution of
   30,000. The MS^2 uses a data-dependent scan (DDS). The three peaks with
   the highest abundance were selected for collision-induced dissociation
   to obtain MS^2 data.

   Mass Frontier 6.0 (Thermo Fisher Scientific) software and Xcalibur 2.1
   (Thermo Fisher Scientific) software were employed for data analysis.
   The accuracy error threshold was fixed at 10 ppm.

Prediction of Putative MCXZ Targets

   According to the results of MCXZ component recognition, the
   corresponding targets were obtained through target prediction and
   functional analyses of TCM (including prescriptions) using the
   Integrative Pharmacology-based Research Platform of Traditional Chinese
   Medicine (TCMIP V2.0 [65]http://www.tcmip.cn/TCMIP/index.php) ([66]Xu
   H. Y. et al., 2017). The principle underlying target prediction is the
   use of MedChem Studio (version 3.0) software to search DrugBank for the
   structural similarities between the two-dimensional structures of
   chemical components and the certified drug (Approved), followed by
   scoring the similarity using the Tanimoto coefficient. When the
   similarity score was ≥0.8 (moderate-high similarity), the potential
   targets for the MCXZ chemical components were obtained.

Prediction of Known Therapeutic Genes Acting on DM

   The candidate therapeutic genes associated with DM were collected from
   the TCMIP V2.0 database using “Diabetes Mellitus” as the keyword. The
   platform integrates HPO, OMIM, TTD, Drugbank, DisGeNET, ORPHANET, and
   other drug, biological, and symptom databases.

Protein–Protein Interaction Data

   Protein–protein interactions (PPIs) were obtained by importing putative
   MCXZ targets and DM-related genes into the STRING database
   ([67]http://string-db.org/, version 11.0). To ensure the accuracy of
   the results, the species was set to “Homo sapiens,” and the confidence
   was set to 0.4.

Network Analysis and Visualization

   To scientifically explain the complex relationships between putative
   MCXZ targets and known DM-related genes and to identify key nodes,
   Cytoscape software (version 3.7.1, Boston, MA, United States) was used
   to create an interaction network between identified components,
   putative targets, and known DM-related genes. This complex network
   analysis method includes data integration, analysis, and visualization.
   The Network Analyzer in Cytoscape software was then used to calculate
   the three topological parameters of each node gene, including “degree,”
   “betweenness,” and “closeness.” The core nodes of the interaction
   network between MCXZ and DM-related targets were obtained by selecting
   those targets with degree values greater than 2-fold the median value
   and the key core target network through which MCXZ acts on DM was
   acquired by selecting nodes that meet all three topological parameters
   simultaneously. These three topological parameters are typically used
   to evaluate the topological importance of nodes in molecular
   interaction networks. The higher the center of a node, the more
   important that the node was to the network ([68]Mao et al., 2019).

Pathway Enrichment Analysis

   To elucidate the biological functions of putative MCXZ targets, the
   targets were introduced into DAVID ([69]https://david-d.ncifcrf.gov/,
   version 6.7), and pathway enrichment analysis was conducted on targets
   within the network using the Kyoto Encyclopedia of Genes and Genomes
   database (KEGG, [70]http://www.genome.jp/kegg/). Relevant pathways with
   p-values < 0.05 were selected as significant pathways.

Quantitative Real-Time Reverse Transcriptase-Polymerase Chain Reaction
(qRT-PCR)

   Total RNA was isolated from pancreatic tissue using RNAiso Plus
   (TaKaRa, Tokyo, Japan). The PCR reaction procedures were performed as
   follows. Stage 1: Pre-denaturation, one cycle at 95°C for 5s. Stage 2:
   PCR reaction, 40 cycles at 95°C for 10s and 60°C for 30s. Stage 3: 1
   cycle heating from 60°C–95°C, at 0.05°C/s. The relative expression
   levels of vascular endothelial growth factor (VEGF), erb-b2 receptor
   tyrosine kinase 2 (ErbB2), androgen receptor (AR), and protein kinase B
   (Akt1) were calculated using the 2^−ΔΔCt method. β-actin (ACTB) was
   used as the internal control. All quantitative real-time reverse
   transcriptase-polymerase chain reaction (qRT-PCR) experiments were
   repeated three times. The primer sequences used in this study were as
   follows: VEGF-F, 5′-CCT GGG AAA TGT GCC TGT GA-3′ and VEGF-R, 5′-ATT
   CGC ACA CGG TCT GT-3’; ErbB2-F, 5′-ATT GGC TCT CAT TCA CCG CA-3′ and
   ErbB2-R, 5′-CCA AGC CCT CAA GAC CAC AT-3’; Akt1-F, 5′-GAT AAC GGA CTT
   CGG GCT GT-3′ and Akt1-R, 5′-CGG CCA CAC ATC TCG TA-3’; androgen
   receptor (AR)-F, 5′-GCC CGA ATG CAA AGG TCT TC-3′ and AR-R, 5′-CCC AGA
   GCT ACC TGC TTC AC-3’; ACTB-F, 5′-AGG GAA ATC GTG CGT GAC AT-3′ and
   ACTB-R, 5′-AAC CGC TCG TTG CCA ATA GT-3’.

Statistical Analysis

   All data were analyzed by SPSS 25.0 software (SPSS Inc., Chicago, IL,
   United States). Data were expressed as the mean ± standard error of the
   mean (SEM). The results were presented using GraphPad Prism 7.0
   software (GraphPad Software, San Diego, CA, United States). Significant
   differences between normally distributed gene expression data were
   determined by one-way analysis of variance (ANOVA). The FBG and HbA1c
   data, which were not normally distributed, were analyzed using the
   nonparametric Kruskal-Wallis test. p < 0.05 was considered significant.

Results

Effects of MCXZ on FBG and HbAlc Levels in DM Model Mice

   As shown in [71]Figure 2A, the FBG concentrations were significantly
   increased in diabetic model mice (model group) compared with those in
   normal mice (control group), whereas the MCXZ and Met groups showed
   significantly reduced FBG concentrations compared with that in the
   model group.

FIGURE 2.

   [72]FIGURE 2
   [73]Open in a new tab

   [74]MCXZ alleviates the symptoms of the mice with DM. (A) [75]MCXZ
   reduce the FBG of the mice with DM. (B) [76]MCXZ downregulated the
   level of HbA1c in the serum of mice with DM. [77]MCXZ-L, [78]MCXZ low
   dose group 812.5 mg/kg; [79]MCXZ-M, [80]MCXZ middle dose group
   1625 mg/kg; [81]MCXZ-H, [82]MCXZ high dose group 3250 mg/kg. Data are
   mean ± SD.***p < 0.001, **p < 0.01 vs. Control; ^#### p < 0.0001, ^## p
   < 0.01, ^# p < 0.05 vs. the modle group; n = 8–12 animals per group.

   Meanwhile, to further examine the effects of MCXZ on DM, HbAlc levels
   were detected using an ELISA kit. HbA1c is currently considered the
   gold standard for glucose monitoring in patients with DM and has been
   increasingly adopted as a criterion for DM diagnosis. HbA1c levels were
   substantially increased in the diabetic model mice (model group)
   compared with those in normal animals (control group). Compared with
   the model group, mice treated with MCXZ showed significantly decreased
   HbA1c levels ([83]Figure 2B). Surprisingly, the hypoglycemic effect
   observed in the low-dose MCXZ group was better than those observed in
   the medium- and high-dose groups.

Effects on Liver and Kidney Tissue Histopathology

   As shown in [84]Figure 3, compared with the control group, the
   structures of the liver and kidney tissues were not significantly
   altered in any of the experimental groups, including the Model, Met,
   MCXZ-L, MCXZ-M, and MCXZ-H groups.

FIGURE 3.

   [85]FIGURE 3
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   The effects of MCXZ on liver and kidney histopathological injury (×
   40).

Identifification of the Primary Compounds Found in MCXZ Using
UHPLC‐LTQ‐Orbitrap

   The systemic characterization of chemical profiles is an important
   precondition for determining the pharmacological mechanisms through
   which TCM agents exert their effects. To perform this characterization
   in MCXZ, the UHPLC-LTQ-Orbitrap method, together with the ETCM
   database, was initially applied for the rapid and high-throughput
   identification of MCXZ compounds (both known and unknown) in the
   present study. The UHPLC-LTQ-Orbitrap method combines efficient
   separation and strong structural characterization abilities to achieve
   the high-resolution acquisition of parent and daughter ion data, both
   quickly and simultaneously, to obtain multi-stage mass spectrometry
   fragment information, which can significantly improve the ability to
   rapidly identify and analyze the chemical components of complex
   systems, such as those used in TCM ([87]Wang et al., 2015). The ETCM
   contains 7,274 herbal ingredients. Any identified molecular formulas
   that are not included in the purslane chemical components database may
   represent either known compounds that have not previously been
   associated with purslane or new compounds; compounds can be searched in
   his database and confirmed against various types of information. The
   total ion chromatograms (TIC) of MCXZ were presented in [88]Figure 4,
   corresponding to the positive and negative signals.

FIGURE 4.

   [89]FIGURE 4
   [90]Open in a new tab

   Total ion chromatogram of MCXZ detected by UHPLC-LTQ-Orbitrap. (A):
   Positive ion detection mode; (B): Negative ion detection mode.

   During the identification process, the compounds were first analyzed
   and identified in positive ion mode, and then further analyzed and
   verified in negative ion mode. Compounds in MCXZ were identified or
   tentatively characterized according to their retention times and MS^n
   data, which are summarized in [91]Table 1. The specific method used
   Xcalibur 2.1 to extract molecular ion peaks from first-order,
   high-resolution, mass spectrometry data, which were then matched with
   the high-precision excimer ions in the purslane chemical compound
   database (the collected compounds were calculated by [M-H]^−, [M +
   CH3COO]^−, [M]^+, [M + H]^+, and [M + Na]^+]. All possible compounds
   were obtained with a mass error of 5.0 ppm. The MS^n information could
   also be compared against the precise relative molecular mass,
   fragmentation patterns, and pathways reported in the related literature
   to identify compounds ([92]Sun et al., 2014; [93]Yu et al., 2016).
   Using the described sample treatment methods and analytical conditions,
   a total of 84 compounds were analyzed and identified in MCXZ using both
   positive and negative ion modes, including 31 organic acids, 22
   alkaloids, nine flavonoids, eight coumarins, et al.

TABLE 1.

   Identification of chemical compounds in MCXZ by UHPLC-LTQ-Orbitrap.
   Peak no tR min Measured mass m/z MS2 Formula Compound Name References