Identification and bioactivity evaluation of miliusanes from Miliusa sinensis.

Miliusanes are a class of anticancer lead molecules belonging to meroterpenoids with an 18-carbon skeleton isolated from Miliusa plants. A phytochemical study of the plant M. sinensis was carried out to discover new miliusanes with diverse structural features in order to better understand their structure-activity relationship. As a result, 20 compounds including 12 new ones (7-14 and 17-20) belonging to two sub-classes of miliusanes were isolated and identified from the twigs and leaves of this plant. Their structures, including absolute configurations, were determined by spectroscopic analyses and electronic circular dichroism. The absolute stereochemistry of miliusane structures has also been confirmed for the first time through the single crystal X-ray diffraction analysis of miliusol (1). Bioactivity evaluation showed that some of the miliusane isolates potently inhibit cell growth of several human derived cancer cell lines with IC50 values ranging from 0.52 to 23.5 µM. Compound 11 demonstrated more potent cytotoxic activity than the known miliusol (1) in stomach cancer cells though its structure contains an unconjugated 1, 4-diketone system, which added a new structure-activity feature to miliusanes. The preliminary mechanism of action studies revealed that they could be a class of dual cell migration inhibitor and senescence inducer.

Transcranial direct current stimulation treated by multilead brain reflex instrument accelerates neural functional recovery in a rat model of stroke.

PURPOSE: Clinical research suggests that transcranial direct current stimulation (tDCS) at bilateral supraorbital foramen and inferior orbital rim and nose intersections may facilitate rehabilitation after stroke. However, the underlying neurobiological mechanisms of tDCS remain poorly understood, impeding its clinical application. Here, we investigated the effect of tDCS applied after stroke on neural cells. MATERIALS AND METHODS: Middle cerebral arterial occlusion (MCAO) reperfusion was induced in rats. Animals with comparable infarcts were randomly divided into MCAO group and MCAO + tDCS group. Recovery of neurological function was assessed behaviorally by modified neurological severity score (mNSS). Ischemic tissue damage verified histologically by TTC and HE staining. Immunohistochemical staining, real-time qPCR, and western blot were applied to determine the changes of neural cells in ischemic brains. RESULTS: The results reveal that tDCS treated by multilead brain reflex instrument can promote the recovery of neurological function, remarkably reduce cerebral infarct volume, promote brain tissue rehabilitation, and can effectively inhibit astrocytosis and enhance neuronal survival and synaptic function in ischemic brains. CONCULSIONS: Our study suggests that tDCS treated by multilead brain reflex instrument could be prospectively developed into a clinical treatment modality.

Hydroxypropyl-ß-cyclodextrin for Delivery of Baicalin via Inclusion Complexation by Supercritical Fluid Encapsulation.

Over the years, various methods have been developed to enhance the solubility of insoluble drugs; however, most of these methods are time-consuming and labor intensive or involve the use of toxic materials. A method that can safely and effectively enhance the solubility of insoluble drugs is lacking. This study adopted baicalin as an insoluble drug model, and used hydroxypropyl-ß-cyclodextrin for the delivery of baicalin via the inclusion complexation by supercritical fluid encapsulation. Different parameters for the complex preparation as well as the physicochemical properties of the complex have been investigated. Our results showed that when compared to the conventional solution mixing approach, supercritical fluid encapsulation enables a more precise control of the properties of the complex, and gives higher loading and encapsulation efficiency. It is anticipated that our reported method can be useful in enhancing the preparation efficiency of inclusion complexes, and can expand the application potential of insoluble herbal ingredients in treatment development and pharmaceutical formulation.

Indole Alkaloids from the Leaves of Nauclea officinalis.

Three new indole alkaloids, named naucleamide G (1), and nauclealomide B and C (5 and 6), were isolated from the n-BuOH-soluble fraction of an EtOH extract of the leaves of Nauclea officinalis, together with three known alkaloids, paratunamide C (2), paratunamide D (3) and paratunamide A (4). The structures with absolute configurations of the new compounds were identified on the basis of 1D and 2D NMR, HRESIMS, acid hydrolysis and quantum chemical circular dichroism (CD) calculation. According to the structures of isolated indole alkaloids, their plausible biosynthetic pathway was deduced.

Acaricidal activity of DHEMH, derived from patchouli oil, against house dust mite, Dermatophagoides farinae.

This study characterized the acaricidal activity of constituents of patchouli oil extracted from (Pogostemon cablin (Blanco) BENTH) against the house dust mite, Dermatophagoides farinae. A new compound, 2-(1,3-dihydroxy-but-2-enylidene)-6-methyl-3-oxo-heptanoic acid (DHEMH), was isolated from patchouli oil and characterized by (1)H-NMR, (13)C-NMR, LC-MS and elemental analysis (EA). This active component was identified as the hydrolysate of pogostone. Fifteen other constituents found in patchouli oil were also identified by GC-MS, including patchouli alcohol and pogostone. LD(50) studies carried out over 24 h using contact toxicity tests identified DHEMH as the most toxic compound to D. farinae (2.04 µg/cm(2)), followed by patchouli oil (6.11 µg/cm(2)), benzyl benzoate (BP) (9.31 µg/cm(2)) and dibutyl phthalate (DBP) (58.52 µg/cm(2)). In vapor phase toxicity tests, all of these compounds were more effective in closed than open containers, indicating that the most efficient mode of delivery for these compounds is the vapor phase. These results indicate that DHEMH and patchouli oil merit further study as potential agents for the control of D. farinae.

Advanced research on acteoside for chemistry and bioactivities.

Acteoside is one kind of phenylethanoid glycoside, which has shown a lot of biological activities. This article reviewed the study progress of acteoside, such as distribution, preparation, identification, and bioactivities.

Plant-derived isoquinoline alkaloids that target ergosterol biosynthesis discovered by using a novel antifungal screening tool.

The ergosterol pathway is a prime antifungal target as it is required for fungal survival, yet is not involved in human homeostasis. Methods to study the ergosterol pathway, however, are often time-consuming. The minimum inhibitory concentration (MIC) assay is a simple research tool that determines the lowest concentration at which a novel antimicrobial is active in vitro with limited scope to determine the mechanism of action for a drug. In this study, we show that by adding hydrogen peroxide, an oxidative stressor, or glutathione (GSH), an antioxidant, to modify a commonly performed MIC assay allowed us to screen selectively for new antifungal drugs that target ergosterol biosynthesis in fungi. A human pathogen and dermatophyte, Microsporum gypseum, was used as a test organism. When exposed to ergosterol targeting drugs, the hydrogen peroxide treatment significantly decreased fungal survival by reducing ergosterol in the cell wall, whereas GSH increased survival of M. gypseum. Further, by performing a series of experiments with M. gypseum and Trichophyton rubrum, it was determined that the oxidative stress from hydrogen peroxide causes cell death at different developmental stages based on fungal species. These findings allow us to describe a simple, high-throughput method for simultaneously screening new antifungal drugs for activity and effects on the ergosterol pathway. By using this tool, two isoquinoline alkaloids were discovered to be potent inhibitors of ergosterol biosynthesis in vitro by reducing the amount of ergosterol without affecting the expression of 1,3-ß-glucan. Both compounds also significantly reduced the severity of acanthosis, hyperkeratosis, spongiosis and dermal edema in vivo.

Acaricidal activities of traditional Chinese medicine against the house dust mite, Dermatophagoides farinae.

OBJECTIVE: This paper assessed the potential of traditional Chinese medicine (TCM) preparations for using as environmentally acceptable and alternative commercial acaricides. METHODS: 22 kinds of TCM, which contained abundant essential oils and showed insecticidal effects, were collected. Samples extracted with petroleum ether, ethyl acetate and methanol were tested against house dust mites Dermatophagoides farinae and their toxicity assessed. RESULTS: The results showed that 3 TCM of Cinnamonum cassia, Eugenia caryophyllata and Pogostemon cablin have higher activity, and the parallel tests showed that the petroleum ether extract had higher activities (0.0046 mg/cm2, 0.005 mg/cm2 and 0.006 mg/cm2 respectively, 24 h, LD50) than the extracts of ethyl acetate and methanol. The acaricidal activity of the ethyl acetate extracts from C. cassia, P. cablin and Asarum sieboldii (0.00144 mg/cm2, 0.00347 mg/cm2 and 0.05521 mg/cm2 respectively, 24 h, LD50) were almost comparable to that of benzyl benzoate and dibutyl phthalate. However, the methanolic extracts of were less effective. CONCLUSIONS. This study shows the use of extracts with petroleum ether of C. cassia, P. cablin and E. caryophyllata as eco-friendly biodegradable agents for the control of the house dust mite.

Anti-inflammatory properties of uvaol on DSS-induced colitis and LPS-stimulated macrophages.

BACKGROUND: Apocynum venetum leaves are used as a kind of phytomedicine and the main ingredient in some traditional Chinese medicine products for the relief of colitis. To understand the bioactive constituents of A. venetum L., we did a phytochemistry study and investigated anti-Inflammatory effects of compounds and explored the underlying mechanisms. METHODS: We isolated compounds from ethanol extract of A. venetum L. leaf and detected the most effective compound by NO inhibition assay. We investigated anti-Inflammatory effects on dextran sulfate sodium (DSS)-induced colitis mice and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. The disease activity index was determined by scores of body weight loss, diarrhea and rectal bleeding; histological damage was analyzed by H&E staining; macrophages change in the colon were analyzed by immunohistochemistry (IHC); myeloperoxidase activity was measured by myeloperoxidase assay kits; levels of proinflammatory cytokines were determined by qPCR and ELISA; protein production such as COX-2, iNOS, STAT3 and ERK1/2 were determined by western blotting. RESULTS: We isolated uvaol from ethanol extract of A. venetum L. leaf and found uvaol has excellent potential of inhibiting NO production. We further found uvaol could attenuate disease activity index (DAI), colon shortening, colon injury, and colonic myeloperoxidase activity in DSS-induced colitis mice. Moreover, uvaol significantly reduces mRNA expression and production of pro-inflammatory cytokines (TNF-α, IL-6, IL-1ß, and MCP-1) and infiltration of macrophages in colonic tissues of colitis mice. Studies on LPS challenged murine macrophage RAW246.7 cells also revealed that uvaol reduces mRNA expression and production of pro-inflammatory cytokines and mediators. Mechanically, uvaol inhibits the pro-inflammatory ERK/STAT3 axis in both inflamed colonic tissues and macrophages. CONCLUSIONS: A. venetum leaf contains uvaol and uvaol has potent anti-inflammatory effects on DSS-induced experimental colitis and LPS-stimulated RAW264.7 macrophage cells. These results suggest uvaol is a prospective anti-inflammatory agent for colonic inflammation.

Antiviral constituents against respiratory viruses from Mikania micrantha.

Phytochemical investigation of the dried aerial parts of Mikania micrantha led to the isolation of a new sesquiterpene, 3beta-acetoxy-1,10-epoxy-4-germacrene-12,8;15,6-diolide (1), along with six known constituents: 1,10-epoxy-4-germacrene-12,8;15,6-diolide (2), dihydromikanolide (3), potassium mikanin 3-sulfate (4), mikanin (5), alpinetin (6), and ergosta-7,22-dien-3beta-ol (7). Their structures were elucidated by spectroscopic methods, and the molecular structures and stereochemistry of sesquiterpene lactones 1-3 were revealed by single-crystal X-ray analysis. Compound 2 showed moderate activity against respiratory syncytial virus (IC(50) = 37.4 uM) and parainfluenza type 3 virus (IC(50) = 37.4 uM) with a therapeutic index (TI) of 16.0 for both compounds. Compound 4, the main component of M. micrantha, exhibited inhibitory activity against parainfluenza type 3 virus with IC(50) (19.7 uM) and TI (24.0) values comparable to those of ribavirin, serving as a positive control.

Indigo Naturalis Suppresses Colonic Oxidative Stress and Th1/Th17 Responses of DSS-Induced Colitis in Mice.

Indigo naturalis (also known as Qing-dai, or QD), a traditional Chinese medicine, has been widely used as an anticolitis regimen in the clinical practice of Chinese medicine. However, the precise mechanisms behind its efficacy remain unknown. We investigated the protective effects and associated molecular mechanisms of QD in DSS-induced colitis in mice. We found that QD administration attenuated DSS-induced colon shortening, tissue damage, and the disease activity index during the onset of colitis. Moreover, QD administration significantly suppressed colonic MPO activity and increased the activities of colonic T-SOD, CAT, and GSH-Px, as well the expression of p-AMPK and Nrf-2 in colon tissues of colitic mice. In addition, QD was capable of reducing the colonic Th1 and Th17 cell cytokines, the frequencies of Th1 and Th17 cells, and the phosphorylation of p-STAT1 and p-STAT3 in the mesenteric lymph nodes of colitic mice. An in vitro assay showed that QD significantly suppressed the differentiation of Th1 and Th17 cells. These findings suggest that QD has the potential to alleviate experimental colitis by suppressing colonic oxidative stress and restraining colonic Th1/Th17 responses, which are associated with activating AMPK/Nrf-2 signals and inhibiting STAT1/STAT3 signals, respectively. These findings also support QD as an effective regimen in the treatment of IBD.

TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts.

We previously demonstrated that overexpression of tropomyosin receptor kinase A (TrkA) promotes the survival and Schwann cell-like differentiation of bone marrow stromal stem cells in nerve grafts, thereby enhancing the regeneration and functional recovery of the peripheral nerve. In the present study, we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts. Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA, TrkA-shRNA or the respective control. The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect. Then, 8 weeks after surgery, hematoxylin and eosin staining showed that compared with the control groups, the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged, whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group. Western blot assay showed that compared with the control groups, the TrkA overexpressing group had higher expression of the myelin marker, myelin basic protein and the axonal marker neurofilament 200. The TrkA overexpressing group also had higher levels of various signaling molecules, including TrkA, pTrkA (Tyr490), extracellular signal-regulated kinases 1/2 (Erk1/2), pErk1/2 (Thr202/Tyr204), and the anti-apoptotic proteins Bcl-2 and Bcl-xL. In contrast, these proteins were downregulated, while the pro-apoptotic factors Bax and Bad were upregulated, in the TrkA-shRNA group. The levels of the TrkA effectors Akt and pAkt (Ser473) were not different among the groups. These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway. All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University, China in December 2014 (approval No. AEWC-2014-001219).

Neuroprotective Effects of Ginsenoside Rg1 against Hyperphosphorylated Tau-Induced Diabetic Retinal Neurodegeneration via Activation of IRS-1/Akt/GSK3ß Signaling.

We have recently demonstrated that tau hyperphosphorylation causes diabetic synaptic neurodegeneration of retinal ganglion cells (RGCs), which might be the earliest affair during the pathogenesis of diabetic retinopathy (DR). Thus, there is a pressing need to seek therapeutic agents possessing neuroprotective effects against tau hyperphosphorylation in RGCs for arresting the progression of DR. Here, using a well-characterized diabetes model of db/db mouse, we discovered that topical ocular application of 10 mg/kg/day of ginsenoside Rg1 (GRg1), one of the major active ingredients extracted from Panax ginseng and Panax notoginseng, ameliorated hyperphosphorylated tau-triggered RGCs synaptic neurodegeneration in diabetic mice. The neuroprotective effects of GRg1 on diabetic retinae were abrogated when retinal IRS-1 or Akt was suppressed by intravitreal injection with si-IRS-1 or topically coadministered with a specific inhibitor of Akt, respectively. However, selective repression of retinal GSK3ß by intravitreal administration of si-GSK3ß rescued the neuroprotective properties of GRg1 when Akt was inactivated. Therefore, the present study showed for the first time that GRg1 can prevent hyperphosphorylated tau-induced synaptic neurodegeneration of RGCs via activation of IRS-1/Akt/GSK3ß signaling in the early phase of DR. Moreover, our data clarify the potential therapeutic significance of GRg1 for neuroprotective intervention strategies of DR.

[Chemical study on ethyl acetate portion of Ehretia thyrsiflora, boraginaceae species of Kudingcha].

OBJECTIVE: To investigate the chemical constituents of Ehretia thyrsiflora. METHOD: Compounds were isolated by using silica gel, Sephadex LH-20 and RP-C18 chromatography; their structures were elucidated by means of spectral data analysis. RESULT: Seven compounds were isolated and identified as methyl rosmarinate (1), caffeic acid (2), quercetin (3), kampferol (4), kaempferol 3-O-alpha-D-arabinoside (5), quercetin 3-O-alpha-D-arabinoside (6), and p-hydroxy benzoic acid (7). CONCLUSION: All these compounds were isolated from E. thyrsiflora for the first time. Compounds 2-7 were isolated from genus Ehretia for the first time.

In vivo antifungal activity of dipyrithione against Trichophyton rubrum on guinea pig dermatophytosis models.

The treatment of dermatophytosis has improved considerably over the past several decades following the introduction of the oral antifungals such as azoles and amphotericin B. However, these drugs have had limited success because the treated fungi often develop drug resistance, resulting in recurrence when applied in various topical formulations. Thus, there are constant needs for new topical agents that are effective against dermatophytosis. Dipyrithione is an attractive candidate to become an antifungal agent due to its broad spectrum of antimicrobial activities. In this study, we determined that dipyrithione could potently inhibit the growth of Trichophyton rubrum, which is the most common cause of dermatophytosis. The MIC50 value of dipyrithione against T. rubrum was measured as 6.03 µM, as compared with miconazole (MIC50: 1.38 µM). Additionally, the compound caused morphological changes in the fungi, which was examined using the morphological interference assay. The in vivo experiment further revealed that dipyrithione had a healing effect on the skin of guinea pigs infected with T. rubrum. Our studies have demonstrated that dipyrithione had a potent antifungal activity in vitro and in vivo, suggesting that it could be formulated as a potential antifungal lead compound in search for novel therapeutic agents against dermatophytosis.

Notoginsenoside R7 suppresses cervical cancer via PI3K/PTEN/Akt/mTOR signaling.

Notoginsenoside R7 was isolated from Panax notoginseng, a plant used commonly in traditional Chinese medicine. We investigated the anti-cancer effects of R7 in HeLa cells in vitro and in vivo, and explored the underlying mechanisms of action. R7 dose-dependently inhibited HeLa cell proliferation and induced apoptosis in vitro, In silico docking-based screening assays showed that R7 can directly bind Akt. Pretreatment with the Akt inhibitor LY294002 synergistically enhanced the R7 anti-proliferation and anti-apoptosis effects in HeLa cells, confirming that R7 acts through the PI3K/Akt pathway. Consistent with the in vitro findings, R7 exerted anti-tumor effects in a mouse xenograft model by targeting PI3K (PTEN) and Akt, activating the pro-apoptotic Bcl-2 family and, subsequently, caspase family members. R7 treatment activated PTEN and downregulated mTOR phosphorylation without affecting mTOR expression, though regulatory-associated protein of mTOR (raptor) expression declined. Our study suggests that R7 is a promising chemotherapeutic agent for the treatment of cervical cancer and other PI3K/PTEN/Akt/mTOR signaling-associated tumors.

Application of microscopic techniques in authentication of herbal tea--Ku-Ding-Cha.

Ku-Ding-Cha, a kind of herbal tea, has been widely used in China for a long time to support cardiovascular health. Confusion arises because in different parts of China different plants are used to produce the commercial teas. It is important to determine the identity of the used species in order to authenticate genuine Ku-Ding-Cha. However, once the plants have been processed, it is difficult to identify them through macroscopic identification; other means are necessary. A comparative study was made on 24 samples of Ku-Ding-Cha, including five standard identified and authenticated plants and 19 commercial samples, by microscopic techniques. The results showed that the shapes of leaf blades, xylem cells, stone cells, and calcium oxalate crystals could be used for the identification of the plants from which the commercial products were made. The method was proven to be quick, handy, simple, and convenient.

Rourinoside and rouremin, antimalarial constituents from Rourea minor.

Bioassay-directed fractionation of the antimalarial active CHCl(3) extract of the dried stems of Rourea minor (Gaertn.) Aubl. (Connaraceae) liana led to isolation of two glycosides, rourinoside (1) and rouremin (2), as well as five known compounds, 1-(26-hydroxyhexacosanoyl)-glycerol (3), 1-O-beta-D-glucopyranosyl-(2S,3R,4E-8Z)-2-N-(2'-hydroxypalmitoyl)-octadecasphinga-4,8-dienine, 9S,12S,13S-trihydroxy-10E-octadecenoic acid, dihydrovomifoliol-9-beta-D-glucopyranoside, and beta-sitosterol glucoside. Compounds 1-3 showed weak in vitro activities against Plasmodium falciparum. Their structures and stereochemistry were elucidated by spectroscopic methods and selected enzyme hydrolysis.

[Determination of two polyacetylenes in Herba Lobeliae Chinensis by HPLC analysis].

OBJECTIVE: To develop an HPLC method for determination of two polyacetylenes, lobetyolin and lobetyolinin, in Herba Lobeliae Chinensis. METHOD: C18 column was used with the mobile phase consisted of acetonitrile and water. Linear gradient elution from 10% to 40% acetonitrile in 25 min was applied, at the flow rate of 1.0 mL x min(-1), the detection wavelength was at 267 nm. RESULT: Lower contents of lobetyolin and lobtyolinin were found in collected samples of Herba Lobeliae Chinensis. The highest amounts of lobetyolin and lobetyolinin were found to be 0.461 and 0.436 mg x g(-1) in a sample procured from Hong Kong. However, there were no lobetyolin and lobetyolinin in some of the samples. CONCLUSION: A simple and effective HPLC method to analyze the two polyacetylenes in Herba Lobeliae Chinensis was established. It could be applied for the quality control of this herb.

Design and fabrication of hydrogel-based nanoparticulate systems for in vivo drug delivery.

Hydrogels are swellable polymer networks which can imbibe a substantial amount of fluids. Compared to bulk hydrogels, hydrogel nanoparticles exhibit added values in drug delivery because they can be internalized into cells to deliver pharmaceutical agents into the cytosol. Their large surface area also allows for multivalent conjugations, making optimization of the nanoparticles towards in vivo and clinical applications possible. These features, together with the tunable properties and biocompatibility of hydrogels, account for the widespread use of hydrogel-based nanoparticulate systems in drug formulation. From an in vivo perspective, this article presents the latest advances in fabrication and engineering of hydrogel nanoparticles, and illuminates future avenues to enhance the potential of hydrogel-based nanoparticulate systems in treatment development and drug administration.