Separation and identification of terpene-conjugated curcuminoids based on liquid chromatography-tandem mass spectrometry and their in vitro anti-inflammatory activities.

Terpene-conjugated curcuminoids are conjugates of curcuminoids and bisabolanes in the rhizomes of Curcuma longa L. The fragmentation pathways of known three terpene-conjugated curcuminoids (bisabolocurcumin-ether, bisabocurcumin, and demethoxybisabolocurcumin ether) and curcumin, demethoxycurcumin, and bisdemethoxycurcumin were investigated using high-performance liquid chromatography-electrospray ionization tandem mass spectrometry in negative mode to rapidly search and discover similar unknown compounds of the acetone fraction of turmeric. Subsequently, compounds 1-3 were founded in the acetone fraction based on molecular weight and above fragmentation pathways (the characteristic fragment ions, the most and second most abundant fragment ions produced in MS2 spectra). Terpecurcumin X (1) and terpecurcumin Y (3) were further separated by liquid chromatography-tandem mass spectrometry guided isolation technique to verify their structures by nuclear magnetic resonance, electrospray ionization high-resolution mass spectroscopy, ultraviolet and visible spectra and infrared spectra. Interestingly, 1 and 3 were new compounds. The results indicate the feasibility and significant advantages of liquid chromatography-tandem mass spectrometry for the rapid discovery and analysis of new constituents in traditional Chinese medicine. In vitro, Terpene-conjugated curcuminoids had better nitric oxide inhibitory activity than the other seven curcuminoids (demethoxycurcumin, bisdemethoxycurcumin, curdione, curcumenone, bisacurone, curcumenol, and germacron).

Isoprenylated Flavonoids and 2-Arylbenzofurans from the Root Bark of Morus alba L. and Their Cytotoxic Activity against HGC27 Cancer Cells.

Three new compounds (1, 11, and 12), together with 32 known ones, were isolated from the root bark of Morus alba L. using various chromatographic methods. The structures of the undescribed compounds were elucidated based on 1D, 2D NMR, and HRESIMS dataanalysis, while the known ones were identified by comparison of their spectroscopic data with those reported in the literature. All the isolates were evaluated for their cytotoxic activities against human gastric cancer HGC27 cells by CCK-8 assay. Among them, compounds 5, 8, 10, and 30 exhibited cytotoxic activities on HGC27 cells with IC50 values of 33.76 ± 2.64 µM, 28.94 ± 0.72 µM, 6.08 ± 0.34 µM, and 10.24 ± 0.89 µM, respectively. Furthermore, compound 10 was confirmed to reduce proliferation ability, increase apoptosis rate, and inhibit cell migration pathway by annexin V/PI double staining experiment, transwell experiment, and Western blot analysis.

New Monoterpene Glycoside Paeoniflorin Derivatives as NO and IL-1ß Inhibitors: Synthesis and Biological Evaluation.

Several monoterpene glycoside compounds were extracted from Paeonia lactiflora Pall. Among them, paeoniflorin, a water-soluble monoterpene glycoside found in the root of Paeonia lactiflora Pall, exhibits excellent antioxidant pharmacological functions. Initially, Sc(CF3SO3)3 was employed as the catalyst for paeoniflorin's dehydration and rearrangement reactions with alcohols. Subsequently, structural modifications were performed on paeoniflorin through a series of responses, including acetylation, deacetylation, and debenzoylation, ultimately yielding 46 monoterpene glycoside derivatives. The potential inhibitory effects on the pro-inflammatory mediators interleukin-1 beta (IL-1ß) and nitric oxide (NO) were assessed in vitro. The results revealed that compounds 29 and 31 demonstrated notable inhibition of NO production, while eight derivatives (3, 8, 18, 20, 21, 29, 34, and 40) displayed substantial inhibitory effects on the secretion of IL-1ß. Computational research was also undertaken to investigate the binding affinity of the ligands with the target proteins. Interactions between the proteins and substrates were elucidated, and corresponding binding energies were calculated accordingly. The findings of this study could provide valuable insights into the design and development of novel anti-inflammatory agents with enhanced pharmacological properties.

Discovery of a Covalent Inhibitor of Pro-Caspase-1 Zymogen Blocking NLRP3 Inflammasome Activation and Pyroptosis.

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1ß production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1-/- mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

Luteolin and its analog luteolin-7-methylether from Leonurus japonicus Houtt suppress aromatase-mediated estrogen biosynthesis to alleviate polycystic ovary syndrome by the inhibition of tumor progression locus 2.

ETHNOPHARMACOLOGICAL RELEVANCE: Leonurus japonicus Houtt (L. japonicus, Chinese motherwort), known as Yi Mu Cao which means "good for women", has long been widely used in China and other Asian countries to alleviate gynecological disorders, often characterized by estrogen dysregulation. It has been used for the treatment of polycystic ovary syndrome (PCOS), a common endocrine disorder in women but the underlying mechanism remains unknown. AIM OF THE STUDY: The present study was designed to investigate the effect and mechanism of flavonoid luteolin and its analog luteolin-7-methylether contained in L. japonicus on aromatase, a rate-limiting enzyme that catalyzes the conversion of androgens to estrogens and a drug target to induce ovulation in PCOS patients. MATERIALS AND METHODS: Estrogen biosynthesis in human ovarian granulosa cells was examined using ELISA. Western blots were used to explore the signaling pathways in the regulation of aromatase expression. Transcriptomic analysis was conducted to elucidate the potential mechanisms of action of compounds. Finally, animal models were used to assess the therapeutic potential of these compounds in PCOS. RESULTS: Luteolin potently inhibited estrogen biosynthesis in human ovarian granulosa cells stimulated by follicle-stimulating hormone. This effect was achieved by decreasing cAMP response element-binding protein (CREB)-mediated expression of aromatase. Mechanistically, luteolin and luteolin-7-methylether targeted tumor progression locus 2 (TPL2) to suppress mitogen-activated protein kinase 3/6 (MKK3/6)-p38 MAPK-CREB pathway signaling. Transcriptional analysis showed that these compounds regulated the expression of different genes, with the MAPK signaling pathway being the most significantly affected. Furthermore, luteolin and luteolin-7-methylether effectively alleviated the symptoms of PCOS in mice. CONCLUSIONS: This study demonstrates a previously unrecognized role of TPL2 in estrogen biosynthesis and suggests that luteolin and luteolin-7-methylether have potential as novel therapeutic agents for the treatment of PCOS. The results provide a foundation for further development of these compounds as effective and safe therapies for women with PCOS.

A small molecule inhibitor of caspase-1 inhibits NLRP3 inflammasome activation and pyroptosis to alleviate gouty inflammation.

Caspase-1 is an integral regulator of innate immunity, which plays a key role in inflammasome activation and the release of pro-inflammatory cytokines. The development of novel non-peptidic small molecule caspase-1 inhibitors is an important strategy for antagonizing excessively activated caspase-1 induced by inflammatory diseases, including gouty arthritis. In the present study, we identified 63 caspase-1 inhibitors, with different structures and potencies, from bioactive compound libraries. Among them, NSC697923 potently inhibited the enzymatic activity of caspase-1, with an IC50 value of 1.737 µM. This compound adopted a favorable conformation in the active pocket of caspase-1. Furthermore, NSC697923 potently decreased mature interleukin (IL)-1ß secretion in macrophages stimulated by lipopolysaccharide plus nigericin, ATP, and monosodium urate crystal. NSC697923 also inhibited NLRP3 protein expression by suppressing the NF-κB signaling pathway and the interaction between receptor interacting protein-2 (RIP2) and pro-caspase-1, thereby blocking the priming of the NLRP3 inflammasome. In addition, NSC697923 significantly inhibited caspase-1 mediated gasdermin D cleavage and pyroptosis in macrophages. In an animal model of gouty arthritis, NSC697923 effectively inhibited joint swelling, IL-1ß release, and NLRP3 inflammasome activation. Our results indicate that NSC697923 can effectively suppress NLRP3 inflammasome activation by inhibiting caspase-1, thus warranting further investigation as a potential therapeutic for treating NLRP3 inflammasome-related diseases.

Inhibition of Phosphodiesterase 5 Promotes the Aromatase-Mediated Estrogen Biosynthesis in Osteoblastic Cells by Activation of cGMP/PKG/SHP2 Pathway.

Mechanical stimulation induces bone growth and remodeling by the secondary messenger, cyclic guanosine 3', 5'-monophosphate (cGMP), in osteoblasts. However, the role of cGMP in the regulation of estrogen biosynthesis, whose deficiency is a major cause of osteoporosis, remains unclear. Here, we found that the prenylated flavonoids, 3-O-methoxymethyl-7-O-benzylicaritin (13), 7-O-benzylicaritin (14), and 4'-O-methyl-8-isopentylkaempferol (15), which were synthesized using icariin analogs, promoted estrogen biosynthesis in osteoblastic UMR106 cells, with calculated EC50 values of 1.53, 3.45, and 10.57 µM, respectively. 14 and 15 increased the expression level of the bone specific promoter I.4-driven aromatase, the only enzyme that catalyzes estrogen formation by using androgens as substrates, in osteoblastic cells. 14 inhibited phosphodiesterase 5 (PDE5), stimulated intracellular cGMP level and promoted osteoblast cell differentiation. Inhibition of cGMP dependent-protein kinase G (PKG) abolished the stimulatory effect of 14 on estrogen biosynthesis and osteoblast cell differentiation. Further, PKG activation by 14 stimulated the activity of SHP2 (Src homology 2 domain-containing tyrosine phosphatase 2), thereby activating Src and ERK (extracellular signal-regulated kinase) signaling and increasing ERK-dependent aromatase expression in osteoblasts. Our findings reveal a previously unknown role of cGMP in the regulation of estrogen biosynthesis in the bone. These results support the further development of 14 as a PKG-activating drug to mimic the anabolic effects of mechanical stimulation of bone in the treatment of osteoporosis.

A Comparative Study on 5hmC Targeting Regulation of Neurons in AD Mice by Several Natural Compounds.

A series of studies have confirmed that DNA methylation disorder (5mC/5hmC) is closely related to the occurrence and development of some diseases, such as Alzheimer's disease (AD). This study aims at discovering natural compounds that could adjust and control 5-hydroxymethylcytosine (5hmC) levels and improve Alzheimer's disease (AD) neuronal status. Cordycepin and cordycepic acid were selected as research materials, with resveratrol as positive control. The results of Dot Blot indicated that cordycepin, cordycepic acid, and resveratrol significantly increased the expression level of 5hmC. Combined with qPCR results, it was revealed that cordycepin increased the expression of ten-eleven translocation (TETs) mRNA compared with the abovementioned cordycepic acid and resveratrol. Besides, cordycepin dramatically reduced the transcription level of Apolipoprotein E (ApoE), suggesting that cordycepin might hinder the formation of NFTs (neurofibrillary tangles) and the accumulation of amyloid ß-protein (Aß) in the brain by reducing the expression of ApoE, resulting in affecting the progression of AD. Meanwhile, the immunofluorescence (IF) staining results demonstrated that the percentage of differentiation of SHSY-5Y cells reasonably increased after the treatment of cordycepin and cordycepic acid. Simultaneously, the length of axons and the number of dendritic branches in mouse primary neurons were substantially increased by cordycepin. The screening results illustrated that cordycepin had a positive influence on the level of 5hmC and the morphology of neurons, and most of the effects were better compared to the positive control (resveratrol). It indicated that cordycepin delayed the progression of neurodegenerative diseases such as AD. However, the specific mechanism of action still needs to be further investigated. Our research provided a foundation for further discussion about the influence of cordycepin on AD and a new idea for the pathological study of related diseases.

Comprehension of the unique characteristics of traditional Chinese medicine.

Traditional Chinese Medicine (TCM) has made great contributions to human history, especially in China, as has been recorded for thousands of years. The characteristics of TCM in both theory and practice make it different from Conventional Western Medicine (CWM). The characteristics of TCM include phenomenological, philosophical, and methodological aspects. This paper briefly discusses some historical turning points and innovations in TCM development in China, explores human body enigmas such as the heart, compares different aspects of TCM and CWM, and identifies the unique characteristics of TCM treatment, as compared to other medical systems.

[Exploration and analysis of theories of five phases, eight trigrams and four elements].

The theory of five phases is significantly different from that of eight trigrams of I Ching (Book of Changes): the eight trigrams represent the materials or phenomena in the nature, whereas five phases put "metal" created by human activities into nature, which organically incorporate the human activities and all the things in the nature and promote the formation of holistic concept of heaven and human being. Without the attendance of man, earth can't generate metal, metal can't restrict wood, and metal can't generate water. So, it can be concluded that the five phases is the improved eight trigrams. Together with monism of original qi and the dualism of yin-yang, it contributes to Chinese medicine the scientific world view and methodology to shake off wizards, and is also the root cause for the Chinese medicine being good at resolving complicated problems. The theory of five phases is also significantly different from the theory of four elements in the western world: The five phases create the relationship of the interaction and interrelation among everything on earth and is the embryo and basis of systemic science. The theory of four elements belongs to constructivism and is the sprout of analytical science.

[Historic significance of Hua Tuo's theory of "Six parts" and "Three methods" in cold pathogenic diseases].

In the end of the Eastern Han dynasty, Hua Tuo and Zhang Zhongjing explored deeply and separately the diagnostic and therapeutic principles of cold pathogenic diseases, though they didn't know each other. This was not only because of their excelsior spirit, but also showed the severity of cold pathogenic diseases at that time. The doctors in past dynasties researched and inherited the achievements of Zhongjing's Shang han lun (Treatise on Cold Pathogenic Diseases). The theory of Hua Tuo's "transformation of diseases by six parts" was different from Su wen re lun (Discussion on Heat in Plain Questions). It doesn't put 6 - channel theory equally, but giving a series of rules for diagnosing the syndromes transforming from superficies to interior, from the surface to the bottom, and from upper to lower which is the beginning of wei - qi - ying - xue differential diagnosis and three - jiao differential diagnosis. Hua Tuo treated cold pathogenic disorders by "sweating, emesis, and catharsis" with abundant clinical approaches. Hua Tuo's idea of "rotten stomach with petechiae" was valued by scholars of warm disorders and of clinical significance even today.