A breakthrough in phytochemical profiling: ultra-sensitive surface-enhanced Raman spectroscopy platform for detecting bioactive components in medicinal and edible plants.

A perennial challenge in harnessing the rich biological activity of medicinal and edible plants is the accurate identification and sensitive detection of their active compounds. In this study, an innovative, ultra-sensitive detection platform for plant chemical profiling is created using surface-enhanced Raman spectroscopy (SERS) technology. The platform uses silver nanoparticles as the enhancing substrate, excess sodium borohydride prevents substrate oxidation, and methanol enables the tested molecules to be better adsorbed onto the silver nanoparticles. Subsequently, nanoparticle aggregation to form stable "hot spots" is induced by Ca2+, and the Raman signal of the target molecule is strongly enhanced. At the same time, deuterated methanol was used as the internal standard for quantitative determination. The method has excellent reproducibility, RSD ≤ 1.79%, and the enhancement factor of this method for the detection of active ingredients in the medicinal plant Coptis chinensis was 1.24 × 109, with detection limits as low as 3 fM. The platform successfully compared the alkaloid distribution in different parts of Coptis chinensis: root > leaf > stem, and the difference in content between different batches of Coptis chinensis decoction was successfully evaluated. The analytical technology adopted by the platform can speed up the determination of Coptis chinensis and reduce the cost of analysis, not only making better use of these valuable resources but also promoting development and innovation in the food and pharmaceutical industries. This study provides a new method for the development, evaluation, and comprehensive utilization of both medicinal and edible plants. It is expected that this method will be extended to the modern rapid detection of other medicinal and edible plants and will provide technical support for the vigorous development of the medicinal and edible plants industry.

Notoginsenoside R1, An Active Compound from Panax notoginseng, Inhibits Hepatic Stellate Cell Activation and Liver Fibrosis via MAPK Signaling Pathway.

Activation of the hepatic stellate cell is implicated in pathological vascularization during development of liver fibrosis. MAPK signaling is involved in the activation of hepatic stellate cell. Oxidative stress and inflammation are also involved in the pathogenesis of liver fibrosis. Notoginsenoside R1 is an effective saponin isolated from the roots of Panax notoginseng (Burk) F. H. Chen and exerts anti-oxidant, anti-inflammatory and anti-fibrotic roles in various diseases. However, the role of Notoginsenoside R1 in liver fibrosis has not been investigated yet. First, a rat model with liver fibrosis was established through oral gavage administration with carbon tetrachloride. Data from hematoxylin and eosin (H&E) and Masson's trichrome stainings showed that carbon tetrachloride induced severe hepatic damages, including inflammatory cell infiltration, lipid droplets deposition in hepatocytes and liver centrilobular necrosis. Meanwhile, the rats were also intraperitoneal injected with different concentrations of Notoginsenoside R1. Results demonstrated that Notoginsenoside R1 treatment suppressed the pathological changes in the livers with enhanced levels of ALB and TP, and reduced levels of ALP, AST and ALT. Second, Notoginsenoside R1 also significantly attenuated carbon tetrachloride-induced decrease in PPAR-[Formula: see text] and increase in Coll-a1, [Formula: see text]-SMA and TIMP1 in liver tissues ([Formula: see text][Formula: see text] 0.001). Third, the decrease in GSH, SOD and GST and increase in MDA, IL-1[Formula: see text], IL-6 and TNF-[Formula: see text] induced by carbon tetrachloride were markedly restored by Notoginsenoside R1 ([Formula: see text][Formula: see text] 0.001). Lastly, Notoginsenoside R1 counteracted with the promotive effects of carbon tetrachloride on levels of proteins involved in MAPK signaling, including phosphorylated p65 (p-p65), p-ERK, p-JNK and p-p38. In conclusion, Notoginsenoside R1 suppressed the activation of hepatic stellate cells and exerted anti- oxidant and anti-inflammatory to attenuate carbon tetrachloride-induced liver fibrosis through inactivation of NF-[Formula: see text]B and MAPK signaling.

Kuijieling decoction suppresses NLRP3-Mediated pyroptosis to alleviate inflammation and experimental colitis in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Ulcerative colitis (UC) is an autoimmune disease. Although the mortality rate of UC is not very high, it has a considerable morbidity rate and an unsatisfactory cure rate. Without effective treatment, UC is likely to develop into colon cancer. Kuijieling (KJL) is an effective empirical formula to treat UC in the clinical setting, and it has been proven to have curative effects against UC. AIM OF THE STUDY: In a previous study, we demonstrated that KJL could suppress NOD-like receptor protein 3 (NLRP3) to reduce inflammatory cytokines and alleviate UC. In this study, we investigated the mechanism of KJL in more detail, from the perspective of pyroptosis. MATERIALS AND METHODS: We established a dextran sulfate sodium-induced UC mouse model and RAW264.7 cells to measure different indicators with different experimental methods. The efficiency of KJL was evaluated by measuring the length and unit weight of mouse colons, and assessment of pathological injury was performed using HE staining. We detected different expression levels of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), caspase-1, gasdermin-D C-terminal domain (GSDMD-C), gasdermin-D N-terminal domain (GSDMD-N), IL-1ß, and IL-18 in colon tissues and cells using RT-qPCR and western blotting. Immunohistochemistry was used for tissues and immunofluorescence for cells to confirm protein expression. IL-1ß and IL-18 were measured with enzyme-linked immunosorbent assay in serum, tissue, and cell culture supernatant. MiR-223 was detected using RT-qPCR. RESULTS: After administration of KJL suspension, colon damage in KJL groups was milder than in model groups. ASC, caspase-1, IL-1ß, and IL-18 mRNA levels in colon tissue were decreased to different degrees in the KJL groups. Protein expression of NLRP3, caspase-1, GSDMD-N, IL-1ß, and IL-18 in vivo decreased significantly in the KJL groups. In addition, Mir-223 level decreased in colon tissue of the KJL groups. In vitro, NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 levels decreased to varying degrees, at both mRNA and protein levels. Mir-223 was lower in the KJL groups than in the model group. Furthermore, KJL was shown to regulate the level of miR-223, which returned to normal after its expression was inhibited or promoted, and the levels of associated indicators also returned to normal after transfection. CONCLUSIONS: KJL is able to inhibit pyroptosis to alleviate UC, but these suppression effects were not mediated through miR-223 regulation.

Yinchenhao decoction attenuates obstructive jaundice-induced liver injury and hepatocyte apoptosis by suppressing protein kinase RNA-like endoplasmic reticulum kinase-induced pathway.

BACKGROUND: Chronic biliary obstruction results in ischemia and hypoxia of hepatocytes, and leads to apoptosis. Apoptosis is very important in regulating the homeostasis of the hepatobiliary system. Endoplasmic reticulum (ER) stress is one of the signaling pathways that induce apoptosis. Moreover, the protein kinase RNA-like endoplasmic reticulum kinase (PERK)-induced apoptotic pathway is the main way; but its role in liver injury remains unclear. Yinchenhao decoction (YCHD) is a traditional Chinese medicine formula that alleviates liver injury and apoptosis, yet its mechanism is unknown. We undertook this study to investigate the effects of YCHD on the expression of ER stress proteins and hepatocyte apoptosis in rats with obstructive jaundice (OJ). AIM: To investigate whether YCHD can attenuate OJ-induced liver injury and hepatocyte apoptosis by inhibiting the PERK-CCAAT/enhancer-binding protein homologous protein (CHOP)-growth arrest and DNA damage-inducible protein 34 (GADD34) pathway and B cell lymphoma/leukemia-2 related X protein (Bax)/B cell lymphoma/leukemia-2 (Bcl-2) ratio. METHODS: For in vivo experiments, 30 rats were divided into three groups: control group, OJ model group, and YCHD-treated group. Blood was collected to detect the indicators of liver function, and liver tissues were used for histological analysis. For in vitro experiments, 30 rats were divided into three groups: G1, G2, and G3. The rats in group G1 had their bile duct exposed without ligation, the rats in group G2 underwent total bile duct ligation, and the rats in group G3 were given a gavage of YCHD. According to the serum pharmacology, serum was extracted and centrifuged from the rat blood to cultivate the BRL-3A cells. Terminal deoxynucleotidyl transferase mediated dUTP nick end-labelling (TUNEL) assay was used to detect BRL-3A hepatocyte apoptosis. Alanine aminotransferase (ALT) and aspartate transaminase (AST) levels in the medium were detected. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) analyses were used to detect protein and gene expression levels of PERK, CHOP, GADD34, Bax, and Bcl-2 in the liver tissues and BRL-3A cells. RESULTS: Biochemical assays and haematoxylin and eosin staining suggested severe liver function injury and liver tissue structure damage in the OJ model group. The TUNEL assay showed that massive BRL-3A rat hepatocyte apoptosis was induced by OJ. Elevated ALT and AST levels in the medium also demonstrated that hepatocytes could be destroyed by OJ. Western blot or qRT-PCR analyses showed that the protein and mRNA expression levels of PERK, CHOP, and GADD34 were significantly increased both in the rat liver tissue and BRL-3A rat hepatocytes by OJ. The Bax and Bcl-2 levels were increased, and the Bax/Bcl-2 ratio was also increased. When YCHD was used, the PERK, CHOP, GADD34, and Bax levels quickly decreased, while the Bcl-2 levels increased, and the Bax/Bcl-2 ratio decreased. CONCLUSION: OJ-induced liver injury and hepatocyte apoptosis are associated with the activation of the PERK-CHOP-GADD34 pathway and increased Bax/Bcl-2 ratio. YCHD can attenuate these changes.

Kuijieling-Containing Serum Regulates Th17 and Treg Cell Differentiation by Inhibiting STAT3 Signaling In Vitro.

OBJECT: To investigate the effect of Kuijieling (KJL) on the balance between T helper 17 (Th17) and regulatory T (Treg) cells in peripheral blood mononuclear cells (PBMC) in vitro and explore the underlying mechanism. MATERIALS AND METHODS: PBMCs isolated from rats were stimulated with transforming growth factor-ß, interleukin (IL)-6, and IL-23 to induce the imbalance of Th17 and Treg cells and were treated with 10, 5, or 2.5% KJL-containing serum. The proportion of Th17 or Treg cells in CD4+ T cells was analyzed by flow cytometry, the concentrations of IL-17, IL-21, and IL-10 were assayed by ELISA, mRNA expressions of retinoic acid-related orphan receptor γt (RORγt), forkhead box protein 3 (Foxp3), and signal transducer and activator of transcription 3 (STAT3) were quantified by PCR, and phosphorylated STAT3 (p-STAT3) was analyzed by flow cytometry. RESULTS: KJL-containing serum decreased the proportion of Th17 cells and increased the proportion of Treg cells in CD4+ T cells, decreased the concentration of IL-17 and IL-21, enhanced the level of IL-10 in the cell culture supernatant, promoted the expression of Foxp3, and inhibited the levels of RORγt, STAT3, and p-STAT3. CONCLUSION: KJL suppresses the STAT3 pathway to remedy the imbalance between Th17 and Treg cells.

Labdanes and megastigmanes from Vitex negundo var. heterophylla.

The aromatic plants of Vitex negundo var. heterophylla are not only herb medicine but also a functional food and an industrial crop. Its leaves can be used as a functional food for improving human's health, but the previous studies mainly focused on the volatile constituents, lignans, and iridoids. Our research led to the isolation of four new terpenoids (1-4), together with fifteen known compounds including seven flavonoids (9-15), two jasmonates (7-8) and six terpenoids (5-6, 16-19) from the leaves. Among all these compounds, 1, 2, 11, and 19 exhibited strong inhibitory activity against NO production in lipopolysaccharide (LPS)-induced RAW264.7 macrophage. The anti-inflammatory mechanism of the most active compound (2) is related to the inhibition of iNOS and COX-2, and the suppression of NF-κB pathway. Therefore, terpenoids and flavonoids from the leaves of Vitex negundo var. heterophylla might be used as potential anti-inflammatory candidates for developing medicine or value-added functional food.

HBD Inhibits the Development of Colitis-Associated Cancer in Mice via the IL-6R/STAT3 Signaling Pathway.

Colitis-associated cancer (CAC) is a malignant disease of the colon that is caused by recurrent episodes of chronic intestinal inflammation. Huangqi Baizhu decoction (HBD) is a classic prescription comprised of Radix Astragali and Rhizoma Atractylodis, which are usually used to treat digestive conditions, such as peptic ulcers, colitis, or colorectal carcinoma in clinics. HBD is well known for "tonifying qi and spleen" based on the theories of traditional Chinese medicine, and has the preponderant effect of alleviating chronic intestinal mucosa damage associated with disease. However, the underlying mechanism behind this is still unknown. In the current study, we employed the AOM/DSS mouse model to analyze the effects of HBD on the development of inflammation in colonic carcinoma. The in vivo study showed that HBD could significantly reduce the mortality of mice and control the incidence and size of colonic tumors by inhibiting the IL-6/STAT3 signaling pathway. In vitro, Astragaloside and Atractylenolide (CAA), the main components of HBD, inhibited the proliferation of HCT-116 cells as determined by an MTT assay. Furthermore, CAA notably suppressed the protein expression of IL-6R, STAT3, Survivin, and Cyclin D1 induced by IL-6 in HCT-116 and RAW264.7 cells. These results suggested that HBD exhibits anti-inflammatory and anti-proliferative effects, inhibiting the development of CAC in mice.

Withanolides and aromatic glycosides isolated from Nicandra physaloides and their anti-inflammatory activity.

Two new withanolides (1-2) together with five known ones (3-7), and three known aromatic glycosides (8-10) were isolated from the dried stems and leaves of Nicandra physaloides, an edible and medicinal plant. Their structures were identified by extensive spectroscopic analyses or comparison with literature data. The absolute configuration of 2 was assigned via X-ray crystallography. Compound 1 with a spiroketal moiety is relatively unusual in withanolides. Aromatic glycosides (8-10) showed potent inhibitory activity against LPS-induced nitric oxide production in RAW 264.7 macrophages, with IC50 values from 4.69 to 16.12 µM.

Kuijieling regulates the differentiation of Treg and Th17 cells to ameliorate experimental colitis in rats.

BACKGROUND: Regulatory T (Treg) cells and T helper 17 (Th17) cells play crucial roles in ulcerative colitis (UC). Kuijieling (KJL) is an effective Chinese medicine formula for treating UC in clinic. Kuijieling has shown remedy effect on the imbalance between Treg and Th17 cells. This study aimed to further reveal the exact underlying mechanism of how Kuijieling regulates the differentiation of Treg and Th17 cells in the treatment of UC. METHODS: Colitis was induced by trinitrobenzene sulfonic acid in rats and treated by KJL. Pathological injury was evaluated by HE staining and pathological score. Transforming growth factor-ß1 (TGF-ß1), interleukin(IL)-2, IL-6, IL-10, IL-17, IL-23 and IL-21 in plasma were assayed by ELISA. Forkhead box P3 (Foxp3), signal transducer and activator of transcription (STAT) 5 expressed in colon mucosa were measured by western blot. Immunohistochemistry was employed for quantifying retinoic acid-related orphan receptor γt (RORγt) and STAT3 in colon. RT-PCR was used to analyze the expression of IL-2, IL-17, IL-23, IL-21 mRNA in colon. RESULTS: After the administration of KJL, pathological injury in colon mucosa was reduced and histological score was decreased, transforming growth factor-ß1 (TGF-ß1), interleukin(IL)-2, IL-10 in blood and Foxp3, STAT5, IL-2 in colon increased significantly, IL-6, IL-23, IL-17, IL-21 in blood and RORγt, STAT3, IL-23, IL-17, IL-21 in colon decreased. Our result showed that KJL regulates the related cytokines and transcription factors to promote Treg cells and suppress Th17 cells. CONCLUSION: KJL restores the balance between Treg and Th17 cells through regulating the differentiation of them, therefore contributes to the treatment of UC.

Global protein expression analysis of molecular markers of DS-1-47, a component of implantation-promoting traditional chinese medicine.

This study investigated the molecular markers of DS-1-47, a component of an implantation- promoting traditional Chinese medicine consisting of Astragalus mongholicus, Atractylodes macrocephala, Scutellaria baicalensis and Dipsacales, in an attempt to clarify the molecular mechanism and action targets of DS-1-47. Controlled ovarian stimulation (COS) method was used to establish the implantation dysfunction models of mice. Animals were divided into normal pregnant group, COS model group and DS-1-47 group. Laser capture microdissection-double dimensional electrophoresis-mass spectrum (LCM-DE-MS) was used to analyze the uterine protein molecules that were possibly involved in the promotion of implantation. Twenty-three proteins in DS-1-47 group were significantly changed as compared to those in COS model group, with 7 proteins down-regulated and 16 proteins up-regulated. Except for some constituent proteins, the down-regulated proteins included collagen α-1 (VI) chain, keratin 7, keratin 14, myosin regulatory light chain 12B, myosin light polypeptide 9, heat shock protein ß-7, and C-U-editing enzyme APOBEC-2; the up-regulated proteins included apolipoprotein A-I, calcium regulated protein-3, proliferating cell nuclear antigen, L-xylulose reductase, and calcium binding protein. These 23 proteins that were regulated by DS-1-47 represented a broad diversity of molecule functions. The down-regulated proteins were associated with stress and immune response, and those up-regulated proteins were related to proliferation. It was suggested that these proteins were important in regulating the uterine environment for the blastocyst implantation. By identification of DS-1-47 markers, proteomic analysis coupled with functional assays is demonstrated to be a promising approach to better understand the molecular mechanism of traditional Chinese medicine.

Tanshinone IIA improves miR-133 expression through MAPK ERK1/2 pathway in hypoxic cardiac myocytes.

Tanshinone IIA is a lipid-soluble pharmacologically active compound extracted from the rhizome of Chinese herb Salvia miltiorrhiza, a well-known traditional Chinese medicine used for the treatment of cardiovascular disorders. Previous studies have identified that tanshinone IIA inhibited overexpression of miR-1 in hypoxic neonatal cardiomyocytes. This study was designed to examine the effects of tanshinone IIA on miR-133 expression under hypoxic condition. Neonatal rat cardiomyocytes were cultured in a hypoxic environment (2% O(2)+93% N(2)+5% CO(2)) at 37°C for 24 hours. MTT, TUNEL assays, and Flow Cytometry (FCM) were performed to identify cell apoptosis. Western blot was used to examine the expression of ERK1/2 and miR-133 level was quantified by Real-time PCR. Our results showed that apoptosis was induced by hypoxia. Typical apoptotic cells were seen by TUNEL assay, and FCM showed an apoptosis rate of 13.32% in hypoxic group. Apoptosis rate in hypoxic cells was reduced significantly by tanshinone IIA. In addition, the expression level of miR-133 was increased in hypoxic cells and further upregulated by tanshinone IIA. The stress-activated protein kinase MAPK ERK1/2 was activated by hypoxia and further increased with tanshinone IIA treatment. The present study demonstrated that tanshinone IIA enhanced cell resistance to hypoxic insult by upregulating miR-133 expression through activating MAPK ERK1/2 in neonatal cardiomyocytes.

[Effect of Kuijieling decoction on proteins expression of IKK-alpha in colonic mucosa of ulcerative colitis model rats].

OBJECTIVE: To explore the changes of protein expression of IKK-alpha as well as the effects of Kuijieling Decoction (KD) on colonic mucosa of ulcerative colitis (UC) model rats. METHODS: UC model rats were induced by TNBS. The rats were randomly divided into six groups: normal control (NC) group, model control (MC) group, Kuijieling low dose (KLD), middle dose (KMD) group, high dose (KHD) group and SASP group. After 10-days' treatment the rats were killed to get their colonic tissues. The positive rate of IKK-alpha expression was detected by immunohistochemical (IHC). RESULTS: The positive rate of IKK-alpha in MC group was significantly higher than that in NC group (P < 0.01). The positive rate of IKK-alpha in KMD group was significantly lower than that in MC group (P < 0.05). The positive rate of IKK-alpha in KHD and SASP group were significantly lower than that in MC group (P < 0.01). CONCLUSION: IKK-alpha may be involved in the pathogenesis of UC, and KD can inhibit positive rate of IKK-alpha in colonic mucosa of UC model rats induced by TNBS. The inhibitory effects of KD on UC may be associated with this.

Effects of Dangshen root extract serum on intracellular free calcium in gastric parietal cells of dissociated rats.

OBJECTIVE: To study the effects of Dangshen root extract on intracellular free calcium concentration [Ca2+] i of parietal cells. METHODS: The Dangshen-containing serum was obtained from rat blood after a continuously five days' feeding with Dangshen root extract of three doses and parietal cells were isolated from Sprague-Dawley rats; [Ca2+] i in single cells was measured by confocal microscope loaded with Fluo3-AM as fluorensent indicator; The change of [Ca2+] i was represented by fluorensent intensity (FI). RESULTS: There were differences in the FI of [Ca2+] i increased by gastrin both between high and control group and between middle and control group (P < 0.05). But no difference was found between low and control groups (P > 0.05). And time to peak of FI, it was not found any difference between any two of the groups. CONCLUSION: Dangshen-containing serum can inhibit the intracellular [Ca2+] i increase induced by gastrin in a dose-dependent manner and it may be one of the mechanisms of its reduction on acid secretion which has a close relation with the formation of ulcerous diseases.