Orcinol gentiobioside inhibits RANKL-induced osteoclastogenesis by promoting apoptosis and suppressing autophagy via the JNK1 signaling.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteoporosis (OP) is a metabolic disorder characterized by disrupted osteoclastic bone resorption and osteoblastic bone formation. Curculigo orchioides Gaertn has a long history of application in traditional Chinese and Indian medicine for treating OP. Orcinol gentiobioside (OGB) is a principal active constituent derived from Curculigo orchioides Gaertn and has been shown to have anti-OP activity. However, the therapeutic efficacy and mechanism of OGB in modulating osteoclastic bone resorption remain undefined. AIM OF THE STUDY: To evaluate the effect of OGB on the formation, differentiation and function of osteoclasts derived from bone marrow macrophages (BMMs), and further elucidate the underlying action mechanism of OGB in OP. MATERIALS AND METHODS: Osteoclasts derived from BMMs were utilized to evaluate the effect of OGB on osteoclast formation, differentiation and bone resorption. Tartrate-resistant acid phosphatase (TRAP) staining and activity assays were conducted to denote the activity of osteoclasts. Osteoclast-related genes and proteins were determined by RT-PCR and Western blotting assays. The formation of the F-actin ring was observed by confocal laser microscopy, and bone resorption pits were observed by inverted microscopy. The target of OGB in osteoclasts was predicted by using molecular docking and further verified by Cellular Thermal Shift Assay (CETSA) and reversal effects of the target activator. The apoptosis of osteoclasts was analyzed by flow cytometry, and autophagic flux in osteoclasts was determined by confocal laser microscopy. RESULTS: OGB inhibited osteoclast formation and differentiation, osteoclast-related genes and proteins expression, F-actin ring formation, and bone resorption activity. Molecular docking and CETSA analysis demonstrated that OGB exhibited good affinity for c-Jun N-terminal Kinase 1 (JNK1). In addition, OGB induced apoptosis and inhibited autophagy in osteoclasts, and the JNK agonist anisomycin reversed the increase in apoptosis and inhibition of autophagy induced by OGB in osteoclasts. CONCLUSION: OGB inhibited osteoclastogenesis by promoting apoptosis and diminishing autophagy via JNK1 signaling.

A rapid and sensitive ultra-performance liquid chromatography-tandem mass spectrometry method for determination of phytohormones in the medicinal plant saffron.

Saffron (Crocus sativus L.) is a valuable Chinese herb with high medicinal value. Saffron pistils are used as medicine, so increasing the number of flowers can increase the yield. Plant hormones have essential roles in the growth and development of saffron, as well as the response to biotic and abiotic stresses (especially in floral initiation), which may directly affect the number of flowers. Quantitative analysis of plant hormones provides a basis for more efficient research on their synthesis, transportation, metabolism, and action. However, starch (which interferes with extraction) is present in high levels, and hormone levels are extremely low, in saffron corms, thereby hampering accurate determination of plant-hormone levels in saffron. Herein, we screened an efficient and convenient pre-treatment method for plant materials containing abundant amounts of starch. Also, we proposed an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method for the quantification of abscisic acid (ABA) and auxin (IAA). Then, the method was applied for the detection of hormone-content differences between flowering and non-flowering top buds, as well as between lateral and top buds. Our method showed high sensitivity, reproducibility, and reliability. Specifically, good linearity in the range 2-100 ng ml-1 was achieved in the determination of ABA and IAA, and the correlation coefficient (R2) was >0.9982. The relative standard deviation was 2.956-14.51% (intraday) and 9.57-18.99% (interday), and the recovery range was 89.04-101.1% (n = 9). The matrix effect was 80.38-90.50% (n = 3). The method was thoroughly assessed employing various "green" chemistry evaluation tools: Blue Applicability Grade Index (BAGI), Complementary Green Analytical Procedure Index (Complex GAPI) and Red Green Blue 12 Algorithm (RGB12). These tools revealed the good greenness, analytical performance, applicability, and overall sustainability alignment of our method. Quantitative results showed that, compared with saffron with a flowering phenotype cultivated at 25 °C, the contents of IAA and ABA in the terminal buds of saffron cultivated at 16 °C decreased significantly. When cultivated at 25 °C, the IAA and ABA contents in the terminal buds of saffron were 1.54- and 4.84-times higher than those in the lateral buds, respectively. A simple, rapid, and accurate UPLC-MS/MS method was established to determine IAA and ABA contents. Using this method, a connection between the contents of IAA and ABA and the flowering phenotype was observed in the quantification results. Our data lay a foundation for studying the flowering mechanism of saffron.

Influence of improved behavioral inhibition on decreased cue-induced craving in heroin use disorder: A preliminary intermittent theta burst stimulation study.

BACKGROUND: Impaired behavioral inhibition is a critical factor in drug addiction and relapse. Repetitive transcranial magnetic stimulation (rTMS) reduces the craving of heroin-addicted individuals for drug-related cues. However, it is unclear whether this technique also improves impaired behavioral inhibition and how improved behavioral inhibition affects craving. OBJECTIVE: The intermittent theta-burst stimulation (iTBS) has been recently shown to be non-inferior relative to rTMS for depression. Here, we aim to investigate the effect of iTBS on heroin-addicted individuals' behavioral inhibition and cue-induced craving and the relationship between the alteration of behavioral inhibition and craving. METHOD: 42 of 56 initially recruited individuals with the heroin-use disorder in the abstinent-course treatment were randomized to undergo active or sham iTBS to the left dorsolateral prefrontal cortex and received three daily iTBS treatments for 10 consecutive days. We measured participants' performance during a two-choice oddball task (80% standard and 20% deviant trials) and heroin-related cue-induced craving before and immediately after treatment. RESULTS: The group that received active iTBS showed significantly improved two-choice oddball task performance after 10 days of intervention compared to both pre-intervention and the group who received sham iTBS. Similarly, a significant reduction in cue-induced craving was observed after following the intervention in the active iTBS group but not the sham iTBS group. The moderation model indicated that iTBS categories play a significant moderating role in the relationship between accuracy cost changing and altered cue-induced craving. CONCLUSIONS: The iTBS treatment protocol positively affects behavioral inhibition in patients with heroin addiction. Improvements in behavioral inhibition can substantially reduce craving.

Screening of Key Proteins Affecting Floral Initiation of Saffron Under Cold Stress Using iTRAQ-Based Proteomics.

BACKGROUND: Saffron crocus (Crocus sativus) is an expensive and valuable species that presents preventive and curative effects. This study aimed to screen the key proteins affecting the floral initiation of saffron under cold stress and thus increasing yield by regulating the temperature. RESULTS: Protein expression profiles in flowering and non-flowering saffron buds were established using isobaric tags for relative or absolute quantitation (iTRAQ). A total of 5,624 proteins were identified, and 201 differentially abundant protein species (DAPs) were further obtained between the flowering and non-flowering groups. The most important functions of the upregulated DAPs were "sucrose metabolic process," "lipid transport," "glutathione metabolic process," and "gene silencing by RNA." Downregulated DAPs were significantly enriched in "starch biosynthetic process" and several oxidative stress response pathways. Three new flower-related proteins, CsFLK, CseIF4a, and CsHUA1, were identified in this study. The following eight key genes were validated by real-time qPCR in flowering and non-flowering top buds from five different growth phases: floral induction- and floral organ development-related genes CsFLK, CseIF4A, CsHUA1, and CsGSTU7; sucrose synthase activity-related genes CsSUS1 and CsSUS2; and starch synthase activity-related genes CsGBSS1 and CsPU1. These findings demonstrate the important roles played by sucrose/starch biosynthesis pathways in floral development at the mRNA level. During normal floral organ development, the sucrose contents in the top buds of saffron increased, and the starch contents decreased. In contrast, non-flowering buds showed significantly decreased sucrose contents under cold stress and no significant changes in starch contents compared with those in the dormancy stage. CONCLUSION: In this report, the protein profiles of saffron under cold stress and a normal environment were revealed for the first time by iTRAQ. A possible "reactive oxygen species-antioxidant system-starch/sugar interconversion flowering pathway" was established to explain the phenomenon that saffron does not bloom due to low temperature treatment.

Single-molecule real-time transcript sequencing identified flowering regulatory genes in Crocus sativus.

BACKGROUND: Saffron crocus (Crocus sativus) is a valuable spice with medicinal uses in gynaecopathia and nervous system diseases. Identify flowering regulatory genes plays a vital role in increasing flower numbers, thereby resulting in high saffron yield. RESULTS: Two full length transcriptome gene sets of flowering and non-flowering saffron crocus were established separately using the single-molecule real-time (SMRT) sequencing method. A total of sixteen SMRT cells generated 22.85 GB data and 75,351 full-length saffron crocus unigenes on the PacBio RS II panel and further obtained 79,028 SSRs, 72,603 lncRNAs and 25,400 alternative splicing (AS) events. Using an Illumina RNA-seq platform, an additional fifteen corms with different flower numbers were sequenced. Many differential expression unigenes (DEGs) were screened separately between flowering and matched non-flowering top buds with cold treatment (1677), flowering top buds of 20 g corms and non-flowering top buds of 6 g corms (1086), and flowering and matched non-flowering lateral buds (267). A total of 62 putative flower-related genes that played important roles in vernalization (VRNs), gibberellins (G3OX, G2OX), photoperiod (PHYB, TEM1, PIF4), autonomous (FCA) and age (SPLs) pathways were identified and a schematic representation of the flowering gene regulatory network in saffron crocus was reported for the first time. After validation by real-time qPCR in 30 samples, two novel genes, PB.20221.2 (p = 0.004, r = 0.52) and PB.38952.1 (p = 0.023, r = 0.41), showed significantly higher expression levels in flowering plants. Tissue distribution showed specifically high expression in flower organs and time course expression analysis suggested that the transcripts increasingly accumulated during the flower development period. CONCLUSIONS: Full-length transcriptomes of flowering and non-flowering saffron crocus were obtained using a combined NGS short-read and SMRT long-read sequencing approach. This report is the first to describe the flowering gene regulatory network of saffron crocus and establishes a reference full-length transcriptome for future studies on saffron crocus and other Iridaceae plants.

A comprehensive analysis and comparison between vacuum and electric oven drying methods on Chinese saffron (Crocus sativus L.).

The red stigmas of saffron are one of the most expensive spices in the world and serve as a traditional Chinese medicine. More saffron has been cultivated in China, and different drying technologies have been studied. However, a comprehensive and comparative analysis of different drying approaches has not been well studied. In this study, we compared electric oven and vacuum oven drying approaches on saffron. We found saffron was dried quicker under high vacuum drying mode with high temperature and the quicker drying rate provided, the more open microstructural interstices on the saffron surface. Both methods were best fit to Midilli and Kucuk model. Besides, the coloring, aroma and bitterness strength after drying showed the similar results. In sum, our data suggested the optimal drying temperature was 100 °C for 20 min for two evaluated methods, however considering the machine cost, the electric oven drying would be the first choice.

Crocin induces autophagic apoptosis in hepatocellular carcinoma by inhibiting Akt/mTOR activity.

BACKGROUND: Autophagy induction is a common mechanism for antitumor chemicals in induction of cancer cell death. However, the role of autophagy in crocin-induced apoptosis is barely studied in hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The influence of crocin on growth, apoptosis, and autophagy and its mutual relations were analyzed by Cell Counting Kit-8 assay, flow cytometer, EGFP-LC3 puncta analysis, and Western blot in HCC cells. The activities of Akt/mTOR axis and its roles in autophagy regulation were also detected by Western blot in HCC cells treated with crocin. Finally, the roles of Akt/mTOR axis in crocin-induced autophagic apoptosis were analyzed by Western blot and flow cytometer in HCC cells. RESULTS: The results showed that crocin can induce growth inhibition in a does- and time-dependent pattern by apoptosis. Increased LC3 puncta and upregulated LC3-II accumulation was observed as early as at 6 hours in HepG2 and HCCLM3 cells treated with 3 mg/mL crocin. Moreover, apoptosis analysis using flow cytometer and cleaved poly (ADP-ribose) polymerase detection revealed that autophagy initiation was prior to apoptosis activation in HCC cells treated with crocin. When autophagy was blocked with 3-methyladenine, crocin-induced apoptosis was inhibited in HCC cells. Furthermore, crocin treatment constrained the activities of key proteins in Akt/mTOR signaling, such as p-Akt (S473), p-mTOR (S2448), and p-p70S6K (T389), suggesting that crocin could induce autophagic apoptosis in HCC cells in an Akt/mTOR-dependent mechanism. Indeed, when autophagy was suppressed by forced expression of Akt, the crocin-induced apoptosis was also impaired in HCC cells. CONCLUSION: The results suggested that crocin could induce autophagic apoptosis in HCC cells by inhibiting Akt/mTOR activity. This study originally revealed that autophagic apoptosis is a novel cytotoxic function of crocin, which lays the theoretical foundation for clinical application of crocin in HCC.

[Comparison on polysaccharide content and PMP-HPLC fingerprints of polysaccharide in stems and leaves of Dendrobium officinale].

In order to provide scientific basics for exploitation and sufficient application of Dendrobium officinale leaves resources, the phenol-sulfuric acid method was applied to determine the polysaccharide content. The monosaccharides were derivated by PMP and the derivatives were identified by HPLC-DAD-ESI-MS(n) and the contents of mannose and glucose were determined simultaneously. Similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (2004A) was employed to generate the mean chromatogram and similarity analysis of the samples was carried out. The results demonstrated that polysaccharide content, monosaccharide compositions and composition ratio had an obvious difference between stems and leaves. The polysaccharide content of stems was higher than that of leaves. Monosaccharide composition in leaf was significantly different from that in stem. The polysaccharide from stems was composed of mannose and glucose, however the polysaccharide of leaves was acid heteropolysaccharide and was mainly composed of five monosaccharides, including mannose, galacturonic acid, glucose, galactose and arabinose. The similarity value of the 14 batches was above 0.9, indicating that similarity of fingerprints among different samples was high. The study can provide evidence for expanding the medicinal parts of D. officinale.

[HPLC specific chromatogram of Dendrobium officinale].

OBJECTIVE: To establish the method of specific chromatogram analysis of ether extract of Dendrobium officinale for identification of D. officinale. METHOD: Chromatographic separation was carried out at 30 degrees C on an Ultimate C18 column (4.6 mm x 250 mm, 5 microm) eluted with methanol and water containing 0.2% phosphoric acid in a gradient elution at a flow rate of 1.0 mL x min(-1). The detection wavelength was set at 280 nm. The similarity evaluation system for chromatographic fingerprint of NPC (National Pharmacopoeia Committee) was adopted to specific chromatogram construction. RESULT: The HPLC specific chromatogram of D. officinale was constructed with 6 common specific chromatographic peaks including naringenin as a reference peak. CONCLUSION: The method shows good precision and repeatability of relative retention time. It can be used to identify D. officinale.

[Determination of naringenin in Dendrobium officinale by HPLC].

OBJECTIVE: To explore a characteristic chemical marker of Dendrobium officinale, establish determination method of its content and determine the naringenin content in D. officinale from different sources and growth years. METHOD: The content of naringenin was determined by HPLC. HPLC analysis was made on a XB -C18 (4.6 mm x 250 mm, 5 microm) with methanol and water containing 0.2% phosphoric acid as mobile phase. The detection wavelength was 290 nm. RESULT: The HPLC method showed good linearity within the range of 0.026-0.208 microg (r = 1). The average recovery of naringenin was 96.3% (RSD 1.8%). The naringenin content was the highest in 3 years D. officinale and had some differences from different sources. CONCLUSION: The method is accurate and reliable. It is appropriate for the quantitative determination of naringenin in D. officinale and it's production.

[Comparative studies on scavenging DPPH free radicals activity of flavone C-glycosides from different parts of Dendrobium officinale].

OBJECTIVE: To study the scavenging DPPH free radicals activity of flavone C-glycosides from different parts of Dendrobium officinale. METHOD: The types and contents of flavonoids from different parts of D. officinale were analyzed by TLC and HPLC. The antioxidant effect was tested by scavenging DPPH free radicals activity. RESULT: The stems, leaves and flowers contained the same type of flavone C-A glycosides and 8 common peaks were identified. The content of flavone C-A glycosides was significantly different. The content of flavone C-glycosides in leaves and flowers was higher than that in stems. The flavonoid in roots was less. Stems contained naringenin, which was not identified in root, leave and flower. Both stems and leaves had antioxidant capacity of eliminating DPPH free radicals, of which scavenging DPPH free radicals activity of leaves was better than stems. CONCLUSION: Considering the content of flavonoid and antioxidant activity leave and flower of D. officinale may substitute stems. The study provides a preliminary basis for the development and utilization of leave and flower of D. officinale.

[Experimental studies of hypoglycemic action on total flavone of Ampelopsis grossedentata from Guangxi].

OBJECTIVE: To study hypoglycemic action of total flavone (GXTF) of Ampelopsis grossedentata from Guangxi by observing the effects of GXTF on blood glucose levels in many strain animal models. METHOD: The blood glucose levels in many strain animal models were determined after oral administration, with the models of diabetes induced by alloxan, of hyperglycemic mice induced by epinephrine and glucose, and normal mice. RESULT: GXTF had better therapeutical action on diabetes mice induced by alloxan, and could significantly lowered the blood glucose levels of hyperglycemic mice induced by epinephrine and glucose, but had no significant effects on blood glucose levels of normal mice. Acute toxicity test showed that the maximum oral dosage is 26.0 g.kg-1. CONCLUSION: GXTF has better hypoglycemic effect on many strain animal models and toxicity is vary small.