Comprehensive characterization of hemicelluloses obtained from Gleditsia sinensis Lam. pods and the application of moderately degraded hemicelluloses in galactomannan film.

The Gleditsia sinensis Lam. pods (GSP) are consistently discarded as waste after saponin extraction due to a lack of industrial or high-value utilization. Herein, the hemicelluloses were extracted from two varieties of GSP and subjected to comprehensive characterization. The molar mass of DMSO-soluble hemicelluloses (53.3-66.0 kDa) was higher compared to that of alkali-soluble ones (24.9-32.6 kDa). The presence of minimal acetyl substitution (3.85-4.49 %) on xylan was unequivocally confirmed. NMR spectroscopic analysis indicated that the hemicelluloses in GSP predominantly consist of a 1,4-ß-ᴅ-Xyl backbone with arabinose substituents at O-3 and 4-O-methyl-α-ᴅ-GlcA substituents at O-2 of the xylose residues. p-Coumaric acid substitution also occurred on the 1,4-ß-ᴅ-Xyl backbone. Hydrothermal treatment significantly reduced the hemicelluloses' relative molar mass and produced 7-10 % xylo-oligosaccharides. Furthermore, the moderately degraded hemicelluloses exhibited significantly enhanced biological activity. Finally, the incorporation of the moderately degraded hemicelluloses imparted the galactomannan film with exceptional antioxidant properties (81.1 % DPPH scavenging activity), while negligibly affecting its transparency. Our study's findings will contribute to a comprehensive understanding of the structural and biochemical properties of hemicellulose in waste G. sinensis pods, thereby facilitating their enhanced utilization in industrial applications.

Waterlogging in soil restricts the growth of Gleditsia sinensis seedlings and inhibits the accumulation of lignans and phenolic acids in thorns.

Gleditsia sinensis, commonly known as Chinese Zaojiao, has important economic value and medicinal compounds in its fruits and thorns, making it widely cultivated artificially in China. However, the available literature on the impact of waterlogging on the growth of G. sinensis seedlings and the accumulation of metabolite compounds in its thorns is limited. To address this knowledge gap, G. sinensis seedlings were planted in soil supplemented with pindstrup substrate, which enhances the water-holding capacity of the soil. The analyses of morphological traits and nutrient elements in one-year-old G. sinensis seedlings grown naturally under ambient conditions and metabolite accumulation in its thorns were conducted. The results showed that the waterlogged soil significantly diminished the height, fresh weight, and dry weight of seedling roots and stems (P < 0.05). Furthermore, waterlogging hindered the uptake of iron (Fe) and manganese (Mn), as well as the transport of potassium (K). The identified metabolites within the thorns were categorized into 16 distinct groups. Relative to the control soil, fatty acids and derivatives were the most down-regulated metabolites in the waterlogged soil, accounting for 40.58% of the total metabolites, followed by lignans (38.71%), phenolic acids (34.48%), saccharides and alcohols (34.15%), steroids (16.67%), alkaloids (12.24%), flavonoids (9.28%), and glycerophospholipids (7.41%). Conversely, nucleotides and derivatives experienced the greatest up-regulation in the waterlogged soil, accounting for 50.00% of the total metabolites. In conclusion, waterlogging negatively impacted the growth of G. sinensis seedlings and inhibited the accumulation of metabolites. Hence, when considering the accumulation of secondary metabolites such as lignans and phenolic acids, appropriate management of soil moisture levels should be taken into account.

Gleditsiae fructus regulates osteoclastogenesis by inhibiting the c­Fos/NFATc1 pathway and alleviating bone loss in an ovariectomy model.

Medical and economic developments have allowed the human lifespan to extend and, as a result, the elderly population has increased worldwide. Osteoporosis is a common geriatric disease that has no symptoms and even a small impact can cause fractures in patients, leading to a serious deterioration in the quality of life. Osteoporosis treatment typically involves bisphosphonates and selective estrogen receptor modulators. However, these treatments are known to cause severe side effects, such as mandibular osteonecrosis and breast cancer, if used for an extended period of time. Therefore, it is essential to develop therapeutic agents from natural products that have fewer side effects. Gleditsiae fructus (GF) is a dried or immature fruit of Gleditsia sinensis Lam. and is composed of various triterpenoid saponins. The anti­inflammatory effect of GF has been confirmed in various diseases, and since the anti­inflammatory effect plays a major role in inhibiting osteoclast differentiation, GF was expected to be effective in osteoclast differentiation and menopausal osteoporosis; however, to the best of our knowledge, it has not yet been studied. Therefore, the present study was designed to examine the effect of GF on osteoclastogenesis and to investigate the mechanism underlying inhibition of osteoclast differentiation. The effects of GF on osteoclastogenesis were determined in vitro by tartrate­resistant acid phosphatase (TRAP) staining, pit formation assays, filamentous actin (F­actin) ring formation assays, western blotting and reverse transcription­quantitative PCR analyses. Furthermore, the administration of GF to an animal model exhibiting menopausal osteoporosis allowed for the analysis of alterations in the bone microstructure of the femur using micro­CT. Additionally, assessments of femoral tissue and serum were conducted. The present study revealed that the administration of GF resulted in a reduction in osteoclast levels, F­actin rings, TRAP activity and pit area. Furthermore, GF showed a dose­dependent suppression of nuclear factor of activated T­cells cytoplasmic, c­Fos and other osteoclastogenesis­related markers.

Fluorescence probe technique for determining the hydrophobic interactions and critical aggregation concentrations of Gleditsia microphylla gum, circular Gleditsia sinensis gum, and tara gum.

Circular Gleditsia sinensis gum, Gleditsia microphylla gum, and tara gum are galactomannans (GMs) with similar mannose/galactose (M/G) molar ratios, which complicates the characterization of physicochemical properties using conventional methods. Herein, the hydrophobic interactions and critical aggregation concentrations (CACs) of the GMs were compared using a fluorescence probe technique, in which the I1/I3 ratio of pyrene indicated polarity changes. With increasing GM concentration, the I1/I3 ratio decreased slightly in dilute solutions below the CAC but decreased sharply in semidilute solutions above the CAC, indicating that the GMs formed hydrophobic domains. However, increases in temperature destroyed the hydrophobic microdomains and increased the CACs. Higher concentrations of salts (SO42-, Cl-, SCN-, and Al3+) promoted hydrophobic microdomain formation, and the CACs in Na2SO4 and NaSCN solutions were lower than those in pure water. Hydrophobic microdomain formation also occurred upon Cu2+ complexation. Although urea addition promoted hydrophobic microdomain formation in dilute solutions, the microdomains were destroyed in semidilute solutions and the CACs increased. The formation or destruction of hydrophobic microdomains depended on the molecular weight, M/G ratio and galactose distribution of GMs. Therefore, the fluorescent probe technique enables the characterization of hydrophobic interactions in GM solutions, which can provide valuable insight into molecular chain conformations.

Enzymatically mediated Gleditsia sinensis galactomannan based hydrogel inspired by wound healing process.

The self-healing property based on metal-ligand physical coordination is particularly interesting in bio-hydrogel science due to its allowance for multiple local healing events to process. As the most abundant renewable green resource in nature, Gleditsia sinensis galactomannan has great potential as a starting material for functional materials. In this study, the biocompatible Gleditsia sinensis galactomannan and cellulose were firstly chemically modified and then taken as the main constituent for constructing the metal-ligand coordination through an enzyme-regulated strategy. The hydrogel could quickly gelatinize in the surrounding environment, corresponding to the violent exothermic phenomenon, and exhibit extraordinary self-healing behavior. The molecular dynamics simulation of the hydrogel confirmed the more stable coordinated configuration from Fe(III)-chelates than Fe(II)-chelates. The morphology, mechanical property, antibacterial, and cytotoxicity of the prepared hydrogel were also studied. Our results indicated that galactomannan hydrogel based on the metal-ligand networks could balance the kinetic stability and intrinsic healability through the enzyme-induced route, which provide a new perspective in the field of biomaterial applications.

A new flavonoid from the thorn of Gleditsia sinensis Lam.

A new flavonoid, (2'''E,6'''S)-4''-(6-hydroxy-2,6-dimethylocta-2,7-dienoyl)-vitexin (1), and five known compounds (2-6) were isolated from the thorn of Gleditsia sinensis Lam. Their structures were determined by comprehensive and comparative spectroscopic analysis of NMR and MS data. The absolute configuration of the new compound was deduced by analysis of the experimental and calculated 13C NMR data. The protective effects against lipopolysaccharide (LPS)-induced apoptosis in normal rat kidney tubule epithelioid (NRK 52e) cells of the isolated compounds were investigated in vitro, whose results showed that compound 1 showed significant protective effect with the EC50 value of 3.0 µM.

5-O-methyldihydroquercetin and cilicicone B isolated from Spina Gleditsiae ameliorate lipopolysaccharide-induced acute kidney injury in mice by inhibiting inflammation and oxidative stress via the TLR4/MyD88/TRIF/NLRP3 signaling pathway.

OBJECTIVE: Inflammation and oxidative stress are the major mechanisms implicated in lipopolysaccharide (LPS)-induced AKI. Spina Gleditsiae is a traditional Chinese anti-inflammatory medicine, from which a large number of flavonoids, such as 5-O-methyldihydroquercetin (GS1) and cilicicone B (GS2), were isolated in the present study. Here, we examined the reno-protective effects and potential underlying mechanisms of GS1 and GS2 in mice with LPS-induced AKI. METHODS: We analyzed renal function; the serum metabolic profile, inflammatory cytokine levels, peripheral white blood cell count, renal cell apoptosis, renal oxidant and antioxidant levels, and renal expression levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), TIR-domain-containing adapter-inducing interferon-ß (TRIF), nuclear factor-ĸB (NF-ĸB), interferon regulatory factor 3 (IRF3), NOD-, LRR- and pyrin domain-containing 3 (NLRP3, inflammasome), cleaved caspase-1, and interleukin 1 receptor type I (IL-1R1) in mice with LPS-induced AKI. RESULTS: GS1 and GS2 improved renal function and significantly reduced the levels of inflammatory cytokines and oxidative stress markers. In addition, PCA score scatter plots suggest that the GS1 and GS2 groups were clustered with the control group, indicating that these compounds contributed to the recovery of mice with AKI toward the normal condition. Moreover, GS1 and GS2 inhibited the expression of TLR4, MyD88, TRIF, p-NF-ĸB, p-IRF3, NLRP3, cleaved caspase-1, and IL-1R1. CONCLUSION: The reno-protective effects of GS1 and GS2 are mediated via the MyD88/TRIF and NLRP3 pathways to reduce inflammation and oxidative stress through TLR4 signaling.

Physicochemical characterization of Gleditsia triacanthos galactomannan during deposition and maturation.

Gleditsia triacanthos polysaccharide, known as galactomannan, has not been exploited as a new functional material even though it possesses industrial potential in food and biomedicine. Galactomannans were recovered from the endosperm of seeds (15 weeks to 25 weeks after flowering) for deposition and maturation analysis. These galactomannans were characterized by using Nuclear magnetic resonance (NMR), X-ray diffraction (XRD), and monosaccharide composition analysis (particularly the mannose to galactose ratio) and molecular weight, solubility, and rheological measurements. The ratio of the three parts in mature seeds was as follows: endosperm (36.67%), hull (34.41%), and embryo (28.92%). The M/G ratio increased from 2.53 to 3.24 between 15 and 23 weeks and then decreased to 3.16 in 25 weeks, consistent with the trends of rheology and solubility. The molecular weight (1.28 × 106 g/mol) and intrinsic viscosity (882.53 mL/g) reached the maximum at 23 weeks and then decreased. Additionally, NMR and XRD showed that the M/G ratio did not change the basic chemical structure but caused slight changes in crystallinity. The purpose of the study was to reveal the changes in galactomannan structure, rheology, and solubility during G. triacanthos galactomannan deposition and maturation to facilitate exploration of its potential industrial applications.

Control of biofouling on pearl oysters Pinctada imbricata using wax and Chinese herbs.

Biofouling poses severe challenges to pearl oyster Pinctada imbricata culture in China, and controlling it is both labor- and capital-intensive. The antifouling properties of wax, and wax mixed with Chinese herbs, sprayed onto pearl oyster shell surfaces during peak biofouling seasons were evaluated. Pearl oysters coated with three wax treatments (plain wax, Chinaberry seed extract, Chinese honeylocust fruit extract) and a control (no treatment), were cultured in nets for up to 60 days. Mortality rate, fouling organism and pearl-oyster weights, and shell height are reported for individual oysters on each of six sampling dates. With the exception of oysters submerged for 12 days, all oysters were significantly affected by treatment type and submersion duration. Fouling weight increased more rapidly over time in the control-treatment oysters. Wax-based coatings deterred fouling-organism settlement on oysters for at least 2 months during the intensive fouling season, reducing mortality and not adversely effecting growth.

Triacanthine exerts antitumor effects on bladder cancer in vitro and in vivo.

BACKGROUND: Numerous studies have focused on solvent extracts from locust trees (Gleditsia spp.), which contain diverse bioactive components including saponins, flavonoids, and alkaloids. However, because of the undefined nature of such phytochemicals, their clinical application as chemotherapeutic agents has often been limited. PURPOSE: This study aimed to evaluate the anti-oncogenic activity of triacanthine, an alkaloid obtained from Gleditsia triacanthos L. STUDY DESIGN: The anti-oncogenicity of triacanthine in vitro was evaluated via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, cell-counting kit-8 assay (CCK-8 assay), flow cytometry, imunoblot, migration and invasion assays, zymography, and electrophoretic mobility shift assay in the human bladder carcinoma cell line EJ. The in vivo efficacy of triacanthine was evaluated via oral administration to EJ-xenografted BALB/c nude mice. To identify the side effects of triacanthine, cisplatin was also administered and an acute toxicity test was performed. RESULTS: Triacanthine significantly inhibited EJ cell proliferation (IC50 600 µM). Flow cytometry analysis revealed that cells were arrested in the G1 phase, and apoptotic cells accumulated in sub-G1 phase in a dose-dependent manner. Triacanthine inhibited the G1-S transition by deterring complex formation between cyclin-dependent kinases and cyclins, thereby up-regulating cell cycle inhibitors p21WAF1 and p27KIP1. In addition, triacanthine induced a caspase-dependent extrinsic pathway of apoptosis and autophagy. Early responsive kinases, extracellular signal-regulated kinase (ERK) and Janus kinase (JNK) were up-regulated by triacanthine. Triacanthine-mediated inhibition of the migratory and invasive potential of EJ cells was attributed to reduction of matrix metalloproteinase (MMP)-9 due to suppression of binding activities of the transcription factors activator protein (AP)-1, specificity protein (Sp)-1, and nuclear factor (NF)-κB. In an in vivo study, triacanthine significantly limited growth of xenografted tumors. Interestingly, while cisplatin resulted in significant weight loss after a 5-mg/kg dose, triacanthine did not cause weight loss, behavioral abnormalities, altered biochemical parameters, or tissue staining. A single oral dose acute-toxicity test (triacanthine 2,000 mg/kg) produced no adverse cytotoxic effects via blood biochemical tests and tissue-organ staining. CONCLUSION: To our knowledge, this is the first systematic evaluation of the anti-oncogenic activity of triacanthine. Therefore, we believe that our findings may guide the development of novel chemotherapeutic agents for bladder cancers.

Comparison and characterization of galactomannan at different developmental stages of Gleditsia sinensis Lam.

Pods from a Gleditsia sinensis Lam. tree were collected and the galactomannan content and other properties were determined at their different developmental stages. In green and immature seed, galactomannan was substituted to a great extent with a mannose to galactose (M/G) ratio of 2.4 from crude polysaccharides. During late galactomannan deposition, it was substituted to a lower extent and this ratio increased rapidly, reaching a M/G ratio of 3.1. Average molecular weight (Mw) of the extracted polysaccharides first increased, reached the maximum (1.19 × 106) at 17 weeks after flowering (WAF), and then decreased. These changes might result from primary galactomannan biosynthesis and from galactose removal by α-galactosidase in the endosperm. The solubility of crude polysaccharides decreased with increased M/G ratio and maximum solubility was more than 89% that collected at 13 WAF. Rheological properties showed that apparent viscosity was largely influenced by the molecular weight and M/G ratio of galactomannans.

Anticancer Effects of Gleditsia sinensis Extract in Rats Transplanted With Hepatocellular Carcinoma Cells.

The thorns of Gleditsia sinensis have been historically used in Chinese medicine and are considered one of the fundamental therapeutic herbs. Its anticancer effects are currently being explored. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and still requires the development of new drugs with higher efficiency. By using a rat HCC model implanted with cancerous Walker-256 cells, the therapeutic effects of G. sinensis extract (GSE) were assessed, as well as its regulatory effects on miRNAs. GSE significantly restored liver morphology and dramatically induced cell apoptosis in HCC rats. In addition, miR-21/181b/183 was upregulated in the HCC liver, and the elevation of these miRNAs could be alleviated by both GSE and sorafenib. PTEN/TIMP3/PDCD4 downregulation was consistent with the targets of miR-21/181b/183 in the HCC liver, and the alteration of these target genes was restored by both GSE and sorafenib. TIMP3 effects on MMP-2/9 expression were also determined. Our present findings indicate the potential of GSE in HCC treatment, and expand the understanding of miRNA-related mechanisms in the anticancer effects of GSE.

A new neolignan from the thorns of Gleditsia japonica var. delavayi.

A new neolignan, 5-(3″-acetoxypropyl)-2-(4'-hydroxy-3'-methoxyphenyl)-7-methoxy-3-methylbenzofuran (1) along with nine analogues were isolated from the thorns of Gleditsia japonica var. delavayi by solvent extraction and repeated column chromatography. Their structures were elucidated by means of extensive spectroscopic analysis including 1D, 2D-NMR techniques and HR-ESIMS. All the isolates were reported for the first time from this species.

Anti-breast cancer triterpenoid saponins from the thorns of Gleditsia sinensis.

One new triterpenoid saponin (1), as well as six known ones (2-7), were isolated from the ethanol extract of the thorns of Gleditsia sinensis. Their structures were elucidated by extensive spectroscopic analysis in conjunction with chemical evidence. Cytotoxic activity of compounds 1-6 was evaluated against human breast cancer MCF 7 cells in vitro by the MTT method. Our results revealed moderate activities for compounds 1-6 with IC50 values of 18.43, 30.47, 18.46, 10.02, 30.76, and 17.32 µM, respectively. Furthermore, compounds 1, 3, 4, and 6 induced apoptosis in MCF 7 cell, with 1 and 6 causing late apoptosis of MCF 7 cells, while 3 and 4 acting oppositely.

Identification of potential genes involved in triterpenoid saponins biosynthesis in Gleditsia sinensis by transcriptome and metabolome analyses.

Gleditsia sinensis is widely used as a medicinal plant in Asia, especially in China. Triterpenes, alkaloids, and sterols were isolated from Gleditsia species. Among them, triterpenoid saponins are very important metabolites owing to their various pharmacological activities. However, the triterpenoid saponin biosynthesis pathway has not been well characterized. In the present study, we performed de novo transcriptome assembly for 14.3 Gbps of clean reads sequenced from nine tissues of G. sinensis. The results showed that 81,511 unique transcripts (unitranscripts) (47,855 unigenes) were constructed, of which 31,717 unigenes were annotated with Gene Ontology and EC numbers by Blast2GO against the NCBI-nr protein database. We also analyzed the metabolite contents in the same nine tissues by LS-MS/MS, and saponins including gleditsioside I were found in fruit at higher levels. Many of the genes with tissue-specific expression in fruit are involved in the flavonoid biosynthesis pathway, and many of those have UDP-glucosyltransferase (UGT) activity. We constructed a saponin biosynthesis pathway and identified two key enzyme families in the triterpenoid saponin biosynthesis pathway, cytochrome P450 and UDP-glucosyltransferase, that are encoded by 37 unigenes and 77 unigenes, respectively. CYP72A, CYP716A, and CYP88D, which are known as key enzymes for saponin biosynthesis, were also identified among the P450s. Our results provide insight into the secondary metabolite biosynthesis and serve as important resources for future research and cultivation of G. sinensis.

[Research on morphological characteristics change of quercetin and polyphenols of accumulation dynamic of spines of Gleditsia sinensis].

To study morphological characteristics change of the development process and quercetin and polyphenols of accumulation dynamic of the spines of Gleditsia sinensis, measure and compare morphological indexes using Vernier caliper, ruler and balance, calibrate and analysis quercetin and polyphenols content using HPLC and colorimetric method. The spines of G. sinensis development process is divided into formation period (the beginning of August to the beginning of November), dormant period (the beginning of November to the end of March in the following year), germination period (the end of March to the middle of April), fast growth period (the middle of April to the middle of August), browning period (the middle of August to the end of August) and mature period (the beginning of September to the end of December). Formation period the spines primordium divides and forms the scale bubs; dormant period the scale bubs are in a dormant state; germination period the bubs scales fall off, spines primordium began to development; fast growth period rapid growth to maximum; browning period browning from the tip and browns until the whole becomes brown; mature period The early stage of maturity is full of luster, gradually the color deepened and the luster faded. The accumulation of quercetin was gradually decreasing after increasing. The total polyphenol accumulation was significantly decreased and then gradually increased, decreased finally. The content of quercetin was increased from 0.000 4%-0.002 6%, and the polyphenol content decreased from 0.761 9%-0.049 1% and then slowly increased to 0.286 9% in the fast growth period.The quercetin continuous increase to 0.004 3% and total polyphenol increased to 0.421 6% in the browning period. In the mature period, the quercetin content significantly decreased after reaching 0.009 6% in September, and the polyphenols content decreased after reaching 0.723 5% in October. Using principal component analysis results: September first, October 2nd, November 3rd. The morphological characteristics change of the development process and quercetin and polyphenols accumulation were determined. The development process is divided into six periods, the best harvest time is the early stage of mature period. Provide theoretical support for the utilization of the spines of G. sinensis and cultivation techniques of high yield.

Effective lactic acid production from waste paper using Streptococcus thermophilus at low enzyme loading assisted by Gleditsia saponin.

Waste paper has considerable potential as a raw material for lactic acid (LA) production due to high cellulose content, abundance and low cost. In this study, four kinds of waste papers were used for LA production through simultaneous saccharification and fermentation (SSF) by Streptococcus thermophilus. The SSF of office paper achieved the highest LA concentration (39.71 g/L), while the highest LA yield was observed for magazine (99.56%), followed by office paper (82.85%). High LA concentration is unfavorable to total LA conversion because of product inhibition. However, the addition of Gleditsia saponin (GS) could obtain both high yield and high concentration of LA at a low enzyme loading, indicating that product inhibition could be moderated. A lactic acid yield of 86.30% was obtained from office paper at an enzyme loading of 9 FPU/g-cellulose with GS, which was higher than that of without GS at a higher loading of 18 FPU/g-cellulose.

Potential of near infrared spectroscopy and pattern recognition for rapid discrimination and quantification of Gleditsia sinensis thorn powder with adulterants.

The Gleditsia sinensis Lam thorn (GST) is a classical traditional Chinese medical herb, which is of high medical and economic value. GST could be easily adulterated with branch of Rosa multiflora thunb (BRM) and Rosa rugosa thumb (BRR), because of their similar appearances and much lower cost for these adulterants. In this study Fourier transform near-infrared spectroscopy (FT-NIR) combined with chemical pattern recognition techniques was explored for the first time to discriminate and quantify of cheaper materials (BRM and BRR) in GST. The Savitzkye-Golay (SG) smoothing, vector normalization (VN), min max normalization (MMN), first derivative (1 st D) and second derivative (2nd D) methods were used to pre-process the raw FT-NIR spectra. Successive projections algorithm was adopted to select the characteristic variables and linear discriminate analysis (LDA), support vector machine (SVM), as while as back propagation neural network (BPNN) algorithms were applied to construct the identification models. Results showed that BPNN models performance best compared with LDA and SVM models for it could reach 100% accuracy for identifying authentic GST, and GST adulterated with BRM and BRR based on the spectral region of 6500-5500 cm-1 combined with 1 st D pre-processing. In addition, the BRM and BRR content in adulterated GST were determined by partial least squares (PLS) regression. The correlation coefficient of prediction (rp), root mean square error of prediction (RMSEP) and bias for the prediction by PLS regression model were 0.9972, 1.969% and 0.3198 for BRM, 0.9972, 1.879% and 0.05408 for BRR, respectively. These results suggest that the combination of NIR spectroscopy and chemometric methods offers a simple, fast and reliable method for classification and quantification in the quality control of the tradition Chinese medicine herb of GST.

Sulfated Purine Alkaloid Glycosides from the Pupal Case Built by the Bruchid Beetle Bruchidius dorsalis Inside the Seed of Gleditsia japonica.

Three new sulfated isoguanine alkaloid glycosides, designated as saikachinoside A monosulfate (1), saikachinoside A disulfate (2), and locustoside B disulfate (3), have been isolated from the pupal case of the wild bruchid seed beetle Bruchidius dorsalis (Chrysomelidae, Bruchinae) infesting the seed of Gleditsia japonica Miq. (Fabaceae). Their structures were determined by spectroscopic methods and the inhibitory activity of 2 and 3 against acid phosphatase was evaluated.

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.