Network pharmacology and bioinformatics to identify the molecular mechanisms of Gleditsiae Spina against colorectal cancer.

Objective: In this study, network pharmacology, bioinformatics and molecular docking were used to explore the active phytochemicals, hub genes, and potential molecular mechanisms of Gleditsiae Spina in treating of colorectal cancer.. Methods: The targets of Gleditsiae Spina, and targets related to CRC were derived from databases. We identified the hub genes for Gleditsiae Spina anti-colorectal cancer following the protein-protein-interaction (PPI) network. Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the hub genes from a macro perspective. Finally, we verified the hub genes by molecular docking, GEPIA, HPA, and starBase database. Results: We identified nine active phytochemicals and 36 intersection targets. The GO enrichment analysis results showed that Gleditsiae Spina may be involved in gene targets affecting multiple biological processes, including response to radiation, response to ionizing radiation, cyclin-dependent protein kinase holoenzyme complex, serine/threonine protein kinase complex, cyclin-dependent protein serine/threonine kinase regulator activity and protein kinase regulator activity. KEGG enrichment analysis results indicated that the P53 signaling pathway, IL-17 signaling pathway, Toll-like receptor signaling pathway, PI3K-Akt signaling pathway, and JAK-STAT signaling pathway were mainly related to the effect of Gleditsiae Spina on colorectal cancer. Molecular docking analysis suggested that the active phytochemicals of Gleditsiae Spina could combine well with hub genes (PTGS1, PIK3CG, CCND1, CXCL8 and ADRB2). Conclusion: This study provides clues for further study of anti-CRC phytochemicals as well as their mechanisms of provides a basis for their development model.

[Content determination of ten flavonoids and alkaloids in Gleditsiae Sinensis Fructus, Gleditsiae Fructus Abnormalis, and Gleditsiae Spina].

To study the quality control of three traditional Chinese medicines derived from Gleditsia sinensis [Gleditsiae Sinensis Fructus(GSF), Gleditsiae Fructus Abnormalis(GFA), and Gleditsiae Spina(GS)], this paper established a multiple reaction monitoring(MRM) approach based on ultra-high performance liquid chromatography-triple quadrupole-linear ion-trap mass spectrometry(UHPLC-Q-Trap-MS). Using an ACQUITY UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 µm), gradient elution was performed at 40 ℃ with water containing 0.1% formic acid-acetonitrile as the mobile phase running at 0.3 mL·min~(-1), and the separation and content determination of ten chemical constituents(e.g., saikachinoside A, locustoside A, orientin, taxifolin, vitexin, isoquercitrin, luteolin, quercitrin, quercetin, and apigenin) in GSF, GFA, and GS were enabled within 31 min. The established method could quickly and efficiently determine the content of ten chemical constituents in GSF, GFA, and GS. All constituents showed good linearity(r>0.995), and the average recovery rate was 94.09%-110.9%. The results showed that, the content of two alkaloids in GSF(2.03-834.75 µg·g~(-1)) was higher than that in GFA(0.03-10.41 µg·g~(-1)) and GS(0.04-13.66 µg·g~(-1)), while the content of eight flavonoids in GS(0.54-2.38 mg·g~(-1)) was higher than that in GSF(0.08-0.29 mg·g~(-1)) and GFA(0.15-0.32 mg·g~(-1)). These results provide references for the quality control of G. sinensis-derived TCMs.

Content determination of ten flavonoids and alkaloids in Gleditsiae Sinensis Fructus, Gleditsiae Fructus Abnormalis, and Gleditsiae Spina / 中国中药杂志

To study the quality control of three traditional Chinese medicines derived from Gleditsia sinensis [Gleditsiae Sinensis Fructus(GSF), Gleditsiae Fructus Abnormalis(GFA), and Gleditsiae Spina(GS)], this paper established a multiple reaction monitoring(MRM) approach based on ultra-high performance liquid chromatography-triple quadrupole-linear ion-trap mass spectrometry(UHPLC-Q-Trap-MS). Using an ACQUITY UPLC BEH C_(18) column(2.1 mm × 100 mm, 1.7 μm), gradient elution was performed at 40 ℃ with water containing 0.1% formic acid-acetonitrile as the mobile phase running at 0.3 mL·min~(-1), and the separation and content determination of ten chemical constituents(e.g., saikachinoside A, locustoside A, orientin, taxifolin, vitexin, isoquercitrin, luteolin, quercitrin, quercetin, and apigenin) in GSF, GFA, and GS were enabled within 31 min. The established method could quickly and efficiently determine the content of ten chemical constituents in GSF, GFA, and GS. All constituents showed good linearity(r>0.995), and the average recovery rate was 94.09%-110.9%. The results showed that, the content of two alkaloids in GSF(2.03-834.75 μg·g~(-1)) was higher than that in GFA(0.03-10.41 μg·g~(-1)) and GS(0.04-13.66 μg·g~(-1)), while the content of eight flavonoids in GS(0.54-2.38 mg·g~(-1)) was higher than that in GSF(0.08-0.29 mg·g~(-1)) and GFA(0.15-0.32 mg·g~(-1)). These results provide references for the quality control of G. sinensis-derived TCMs.

Profiling of widely targeted metabolomics for the identification of chemical composition in epidermis, xylem and pith of Gleditsiae Spina.

Gleditsiae Spina, the thorn of Gleditsia sinensis Lam., has a long history of being used as a traditional medicine in East Asian countries. However, only a few biologically active substances have been identified from it. In this study, the epidermis, xylem and pith of Gleditsiae Spina, respectively Gs-E, Gs-X and Gs-P, were studied. We used a widely targeted metabolomics method to investigate the chemical composition of Gs-E, Gs-X and Gs-P. A total of 728 putative metabolites were identified from Gleditsiae Spina, including 211 primary metabolites and 517 secondary metabolites. These primary and secondary metabolites could be categorized into more than 10 different classes. Flavonoids, phenolic acids, lipids, amino acids and derivatives, and organic acids constituted the main metabolite groups. Multivariate statistical analysis showed that the Gs-E, Gs-X and Gs-P samples could be clearly separated. Differential accumulated metabolite (DAM) analysis revealed that more than half of the DAMs exhibited the highest relative concentrations in Gs-E, and most of the DAMs showed the lowest relative concentrations in Gs-X. Moreover, 11 common differential primary metabolites and 79 common differential secondary metabolites were detected in all comparison groups. These results further our understanding of chemical composition and metabolite accumulation of Gleditsiae Spina.

[Quality standards of Gleditsiae Spina and its decoction pieces].

In view of the current inadequate standards for Gleditsiae Spina in the Chinese Pharmacopoeia, this study put forward some new items of the quality standards of Gleditsiae Spina. Thin-layer chromatography(TLC) was performed for identification with the reference substance of taxifolin and the reference material of Gleditsiae Spina as the control. According to the general principles of the Chinese Pharmacopoeia(2020 edition, Vol. 4), the moisture, total ash content, and alcohol-soluble extract of medicinal materials and decoction pieces of Gleditsiae Spina were determined. The content determination method for medicinal materials and decoction pieces of Gleditsiae Spina was established using high-performance liquid chromatography(HPLC), with taxifolin as the quality control index. Based on the determination results of 30 batches of samples of Gleditsiae Spina from different habitats, the draft quality standards of Gleditsiae Spina were developed, which provided suggestions for the revision of the quality standards of Gleditsiae Spina in the Chinese Pharmacopoeia.

Quality standards of Gleditsiae Spina and its decoction pieces / 中国中药杂志

In view of the current inadequate standards for Gleditsiae Spina in the Chinese Pharmacopoeia, this study put forward some new items of the quality standards of Gleditsiae Spina. Thin-layer chromatography(TLC) was performed for identification with the reference substance of taxifolin and the reference material of Gleditsiae Spina as the control. According to the general principles of the Chinese Pharmacopoeia(2020 edition, Vol. 4), the moisture, total ash content, and alcohol-soluble extract of medicinal materials and decoction pieces of Gleditsiae Spina were determined. The content determination method for medicinal materials and decoction pieces of Gleditsiae Spina was established using high-performance liquid chromatography(HPLC), with taxifolin as the quality control index. Based on the determination results of 30 batches of samples of Gleditsiae Spina from different habitats, the draft quality standards of Gleditsiae Spina were developed, which provided suggestions for the revision of the quality standards of Gleditsiae Spina in the Chinese Pharmacopoeia.

A new lignan from Gleditsiae spina / 药学学报

The chemical constituents from ethyl acetate extract of Gleditsiae spina were isolated and purified by various chromatographic methods such as MCI gel CHP-20, ODS, Sephadex LH-20, silica gel and semi-preparative HPLC. Seven lignans were isolated and identified by spectroscopic data analyses as (7R,8S,7'E,7''S,8''R)-buddlenol P (1), (+)-syringaresinol (2), (+)-isolariciresinol (3), (7S,8R)-cedrusin (4), (7S,8R)-4,9,9'-trihydroxy-3,3'-dimethoxy-7,8-dihydrobenzofuran-1'-propylneolignan (5), 3',4-O-dimethylcedrusin (6), balanophonin (7). Among them, compound 1 is a new lignan, compounds 2-7 are isolated from the Gleditsia L. for the first time. MTT method was used to investigate the effect of compounds 2-7 on LPS-induced injury of NRK-52e cells. As a result, compounds 2, 3 and 7 exhibit protective effects against LPS-induced damage to NRK-52e cells.

HPLC-MS/MS determination of flavonoids in Gleditsiae Spina for its quality assessment.

Gleditsiae Spina, the thorn of Gleditsia sinensis Lam., has been used as an anti-inflammatory, anti-tumor, and anti-bacterial traditional medicine for hundreds of years in China. This study used high-performance liquid chromatography and tandem mass spectrometry combined with chemometric methods to allow the fast and accurate identification and quantification of the flavonoids compounds in Gleditsiae Spina, and created reliable criteria for accurate identification of Gleditsiae Spina and its adulterants. This research provides good evidence for the classification and quality evaluation of Gleditsiae Spina. Firstly, eight flavonoids compounds were detected and identified on the basis of their mass spectra, fragment characteristics, and comparison with published data. Then the mass spectroscopic fragmentation pathways of these compounds were determined and, in addition rutin, isoquercitrin, and quercitrin were detected in Gleditsiae Spina for the first time. The quantification was performed on a triple quadrupole tandem mass spectrometer in multi-reaction monitoring mode, and the baseline separation of the eight bioactive flavonoids components was achieved within 13 min. Furthermore, the proposed method was successfully applied for simultaneous quantitative determination of the eight Gleditsiae Spina compounds and adulterants obtained from different sources in China. Then, we built a classification model which showed a high level of accuracy predicting 100% of the samples, correctly.

Chemical constituents from ethyl acetate extract of Gleditsiae Spina / 中草药

Objective To investigate the chemical constituents from the ethyl acetate extract of Gleditsiae Spina. Methods The chemical constituents were isolated and identified by chromatography on silica gel, Toyopearl HW-40C, Sephadex LH-20, MCI Gel CHP-20, ODS, and RP-HPLC. Their structures were elucidated on the basis of physicochemical properties and spectral analyses. Results Eighteen compounds were isolated from the ethyl acetate extract of Gleditsiae Spina, and identified as scopoletin (1), (-)-(7R,8S)-erythro-guaiacylglycerol (2), 5-methoxy-guaiacylglycerol (3), (-)-(7R,8R)-threo-guaiacylglycerol (4), 3,4’- dihydroxypropiophenone (5), dihydroferulic acid (6), p-hydroxybenzoic acid (7), indole-3-carbaldehyde (8), protocatechuic acid methyl (9), protocatechuic aldehyde (10), syringic acid (11), 2-guaiacylpropane-1,3-diol (12), (2R*,3R*,4S*)-2,3-diguaiacyl-4- hydroxyl tetrahydrofuran (13), 3-(2-oxopropyl)-3-hydroxy-indolin-2-one (14), juglanin D (15), C-veratroylglycol (16), 3-(4-hydroxyl-3-methoxyphenyl)-propan-1,2-diol (17), and (-)-syringaresinol (18). Conclusion Eighteen compounds are isolated from this plant for the first time.

HPLC fingerprint analysis and content determination of Gleditsiae Spina / 中草药

Objective: An HPLC method was developed to evaluate the quality of Gleditsiae Spina through three components determination and fingerprint analysis. Methods: The assay was performed on Waters symmetry C18 (250 mm × 4.6 mm, 5 μm) column. The mobile phase consisted of acetonitrile and 0.1% methanoic acid in water with gradient elution by PDA detection at 338 nm. Results: Thirteen batches of Gleditsiae Spina were analyzed with the established method. In the fingerprint, 14 common peaks were marked and three of them were determined. The three compounds showed good linearity (r ≥ 0.999 2) in the range of 0.091 3-5.840 0, 0.176 3-11.280 0, and 0.014 0-0.895 0 mg/mL, respectively. The average recoveries were 98.97%-99.66% with RSD < 2.5%. Conclusion: This method has good repeatability and stability and provides a new method for the quality control of Gleditsiae Spina.

Flavonoids from Gleditsiae Spina and their antitumor activities / 中草药

Objective: To investigate the flavonoids from Gleditsiae Spina (thorns of Gleditsia sinensis) and their antitumor activities. Methods: The chemical constituents in the EtOAc fraction from Gleditsiae Spina were isolated and purified by the chromatography on silica gel, Sephadex LH-20 columns, and semi-preparative HPLC. Their structures were identified by various spectroscopic analyses, and the cytotoxicity of compounds 7-16 were evaluated in vitro against MCF-7 cell lines by SRB method. Results: Sixteen compounds were isolated from the extracts of Gleditsiae Spina and identified as tricin (1), liquiritigenin (2), 7,4'-dihydroxy-5,3'-dimethoxyflavanonol (3), garbanzol (4), 7,3',5'-trihydroxyflavanone (5), 7,4'-dihydroxyflavonol (6), dihydrokaempferol (7), butein (8), (2S)-5,7,3',5'- tetrahydroxyflavanone (9), 7,3',5'-trihydroxy-5-methoxyflavanonol (10), quercetin (11), fustin (12), fisetin (13), leucorobinetinidin (14), thevetiaflavon (15), and isovitexin (16). Compound 8 showed the inhibitory effect against MCF-7 cells with IC50 value of 28.53 μmol/L. Conclusion: Compounds 1-6, 8-10, 14 and 15 are isolated from the plants of Gleditsia L. for the first time, compound 8 shows the significant cytotoxic activity against MCF-7 cells.

Investigation of three coumarin components in thorns of Gleditsia sinensis and their cytotoxic activity to tumor cells / 中草药

Objective: To investigate the chemical constituents from Gleditsiae Spina (the thorns of Gleditsia sinensis). Methods: The compounds were isolated and purified by silica gel, Sephadex LH-20 column chromatographic techniques, and their chemical structures were confirmed on the basis of spectroscopic analysis by the physicochemical properties. Cytotoxic activity using the MTT colorimetry method was performed to measure the inhibitory effect of the compounds on cell proliferation of HepG2, A-549, and EC109. Results: Three coumarins were obtained from the ethyl acetate soluble fraction of the 90% ethanolic extract and their structures were identified as scoparone (1), isoscopoletin (2), and 6-amino-7-methoxycoumarin (3). Conclusion: Compound 3 is a novel coumarin named gledisinmarin A. Compounds 1 and 2 are isolated from this plant for the first time. Compound 2 displays the stronger cytotoxicity against A549 cell with an IC50 value of 34.47 μg/mL, while cisplatin with an IC50 value of 11.50 μg/mL as a positive control.

Flavanonol compounds from thorns of Gleditsie Spina and their cytotoxicity / 中草药

Objective: To study the flavonoids constituents from the thorns of Gleditsia sinensis and their cytotoxicity against tumor cells. Methods: The compounds were isolated by silica gel, Sephadex LH-20 column chromatography, and HPLC techniques. Their structures were elucidated on the basis of spectroscopic analyses. Results: Twelve flavonoids were obtained and identified as (2R,3R)-5,3',4'-trimethoxyl-7-hydroxyl-flavanonol (1), 5,7,3',4'-tetrahydroxyl-flavanonol (2), 5-methoxyl-3',4',7-trihydroxyl-flavanonol (3), dihydrokaempferol (4), epicatechin (5), 5,7,3',5'-tetrahydroxyl-flavanonol (6), fustin (7), (2R,3R)-7,3',5'-trihydroxyl-flavanonol (8), (2R,3R)-5,7,3'-trihydroxyl-4'-methoxyl-flavanonol (9), quercetin (10), 5,7,4'-trihydroxylflavone-8-C-glucopyranose (11), and 2,7-dimethyl-xanthone (12), respectively. Conclusion: Compound 1 is a new component named G. spina flavanonol A, and compounds 3,8,9, and 12 are isolated from the thorns of G. sinensis for the first time. The results of cytotoxicity test show that the dihydroflavonol compound 7 displays the stronger cytotoxicity against HepG2, A549, and EC109 cell strains, while compounds 1 and 3 have the effects on HepG2 and EC109, and compound 2 has the effect on EC109 cancer cells, respectively.

Chemical constituents of Gleditsiae Spina and their antitumor activity / 中草药

Objective: To investigate the chemical constituents from Gleditsiae Spina (the thorns of Gleditsia sinensis) and their antitumor activitives. Methods: The chemical constituents were isolated and purified by the chromatography on repeated silica gel, Sephadex LH-20 column, semi-preparative HPLC, etc. Their structures were elucidated by NMR and MS spectroscopic data analyses; The cytotoxicity of compounds 1-4 and 6 was evaluated against human liver cancer SK-HEP-1 cells by MTT assay. Results: Twelve compounds were isolated from Gleditsiae Spina, and identified as 2-aminoimidazole (1), 2,3-dihydro-5-(2-formylvinyl)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-3-benzofuranmethanol (2), E-cinnamic acid (3), 3-O-trans-feruloylquinic acid (4), trans-caffeic acid (5), 4-hydroxy-3-methoxybenzamide (6), threo-guaiacylglycerol-β-coniferyl aldehyde ether (7), 5,7-dihydroxy-chromone (8), vanillic acid (9), protocatechuic acid (10), 3-O-caffeoylquinic acid methyl ester (11), and 3-O-caffeoylquinic acid ethyl ester (12); Compound 1 exhibited the potent cytotoxicity against SK-HEP-1 cells with IC50 value of 34.47 μg/mL. Conclusion: All the compounds except compound 5 are isolated from the plants of Gleditsia L. for the first time; Compound 1 shows significant cytotoxic activity against SK-HEP-1 cells.

Prevention and treatment of total flavonoids from Gleditsiae Spina on lung cancer and its mechanisms / 中草药

Objective: To explore the prevention and treatment of total flavonoids from Gleditsiae Spina (TFGS) on lung cancer and its mechanisms. Methods: Mouse Lewis lung cancer (LLC) and embryonic lung fibroblast (L929) cells were treated with different doses of TFGS for 48 h, cell proliferation and adhesion were examined by MTT assay, and gap junctional intercellual communication (GJIC) was measured through scrape loading and dye transfer. The mice were randomly divided into model, quercetin (100 mg/kg, positive control), high-and low-dose (100 and 30 mg/kg) TFGS groups. The mice were ip injected with urethane twice weekly for five weeks to induce lung carcinogenesis and treated once daily for 10 weeks following the first urethane injection. The prevention of TFGS on chemocarcinogenesis was evaluated and the expression of gap junctional protein connexin 43 (Cx43) in lung tissue with tumors was compared by immunohistochemistry. The LLC cells were injected into the lateral axilla and tail vein respectively to establish the LLC sc allograft and experimental lung metastasis. The tumor-inocubating mice were randomly divided into model, doxorubicin (5 mg/kg, positive control), high-and low-dose (same as above) TFGS groups. The mice received the treatment for three weeks following tumor inocubation, and the effects of TFGS on the tumor size, metastasis, and life span were evaluated. Results: TFGS inhibited LLC cell proliferation in a dose dependent manner but had no effect on L929 cell proliferation in vitro. TFGS with a little effect on cell proliferation decreased cell adhesion and promoted GJIC in a dose dependent manner in LLC cells but did not affect the L929 cell adhesion. TFGS was able to prevent carcinogenesis induced by urethane and enhance Cx43 staining in lung region with tumor in immunohistochemistry. Compared with untreated model mice, GJIC reduced the tumor size and metastasis and prolongated life span in a dose dependent manner. Conclusion: TFGS could promote GJIC to prevent and treat tumor and might be a potential antitumor agent.