Anti-hepatitis B virus activity and hepatoprotective effect of des(rhamnosyl) verbascoside from Lindernia ruellioides in vitro.

Although clinically approved hepatitis B virus (HBV) polymerase inhibitors (lamivudine-3TC, entecavir, etc.) serve as effective therapeutics, the virus can easily generate resistance to them. Therefore, the treatment of HBV infection remains a public health problem. Numerous studies have shown that natural products have prospective anti-HBV activity. The purpose of this study was to isolate and extract des(rhamnosyl) verbascoside from Lindernia ruellioides (Colsm.) Pennell and explore its anti-HBV and hepatoprotective effects. Anti-HBV activity was evaluated in HepG2.2.15 cells, a human hepatocellular carcinoma cell line with HBV-stable infection, and its protective effect was evaluated in HL-7702 cells, a normal human liver cell line. HepG2.2.15 cells maintained normal growth morphology within the selected concentration range of des(rhamnosyl) verbascoside. It also inhibited the expression of HBV antigens and HBV DNA in a dose- and time-dependent manner in vitro. Further, western blot experiments showed that it could downregulate HBV X protein (HBx) expression in a dose-dependent manner. In the H2 O2 -induced hepatocyte injury model, the cell-survival rate of the HL-7702 cells with the highest drug dose reached 85.25%, which was significantly improved compared with that of the model group. Most of the cells returned to normal morphology, showing polygonal or fusiform structures. Thus, it may be stated that des(rhamnosyl) verbascoside exhibits anti-HBV activity and hepatoprotective effects in vitro and may exert an anti-HBV effect via antigen inhibition, HBV DNA secretion, and HBx protein expression.

Experimental Data Based Machine Learning Classification Models with Predictive Ability to Select in Vitro Active Antiviral and Non-Toxic Essential Oils.

In the last decade essential oils have attracted scientists with a constant increase rate of more than 7% as witnessed by almost 5000 articles. Among the prominent studies essential oils are investigated as antibacterial agents alone or in combination with known drugs. Minor studies involved essential oil inspection as potential anticancer and antiviral natural remedies. In line with the authors previous reports the investigation of an in-house library of extracted essential oils as a potential blocker of HSV-1 infection is reported herein. A subset of essential oils was experimentally tested in an in vitro model of HSV-1 infection and the determined IC50s and CC50s values were used in conjunction with the results obtained by gas-chromatography/mass spectrometry chemical analysis to derive machine learning based classification models trained with the partial least square discriminant analysis algorithm. The internally validated models were thus applied on untested essential oils to assess their effective predictive ability in selecting both active and low toxic samples. Five essential oils were selected among a list of 52 and readily assayed for IC50 and CC50 determination. Interestingly, four out of the five selected samples, compared with the potencies of the training set, returned to be highly active and endowed with low toxicity. In particular, sample CJM1 from Calaminta nepeta was the most potent tested essential oil with the highest selectivity index (IC50 = 0.063 mg/mL, SI > 47.5). In conclusion, it was herein demonstrated how multidisciplinary applications involving machine learning could represent a valuable tool in predicting the bioactivity of complex mixtures and in the near future to enable the design of blended essential oil possibly endowed with higher potency and lower toxicity.

Microwave-Assisted versus Conventional Isolation of Glucosinolate Degradation Products from Lunaria annua L. and Their Cytotoxic Activity.

Glucosinolates (GSLs) from Lunaria annua L. seeds were analyzed qualitatively and quantitatively by their desulfo counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products, isothiocyanates (ITCs), using GC-MS technique. GSL breakdown products were obtained by conventional techniques (hydrodistillation in a Clevenger type apparatus (HD), CH2Cl2 extraction after myrosinase hydrolysis (EXT) for 24 h) as well as by modern techniques, microwave-assisted distillation (MAD) and microwave hydrodiffusion and gravity (MHG). Seven GSLs were identified as follows: isopropyl GSL (1), sec-butyl GSL (2), 5-(methylsulfinyl)pentyl GSL (3), 6-(methylsulfinyl)hexyl GSL (4), 5-(methylsulfanyl)pentyl GSL (5), 6-(methylsulfanyl)hexyl GSL (6), and benzyl GSL (7). Additionally, pent-4-enyl- and hex-5-enyl ITCs were detected in the volatile extracts. However, their corresponding GSLs were not detected using UHPLC-DAD-MS/MS. Thus, they are suggested to be formed during GC-MS analysis via thermolysis of 5-(methylsulfinyl)pentyl- and 6-(methylsulfinyl)hexyl ITCs, respectively. Volatile isolates were tested for their cytotoxic activity using MTT assay. EXT and MHG showed the best cytotoxic activity against human lung cancer cell line A549 during an incubation time of 72 h (IC50 18.8, and 33.5 µg/mL, respectively), and against breast cancer cell line MDA-MB-231 after 48 h (IC50 6.0 and 11.8 µg/mL, respectively). These activities can be attributed to the ITCs originating from 3 and 4.

Endothelial Nitric Oxide Mediates the Anti-Atherosclerotic Action of Torenia concolor Lindley var. Formosama Yamazaki.

Torenia concolor Lindley var. formosama Yamazaki ethanolic extract (TCEE) is reported to have anti-inflammatory and anti-obesity properties. However, the effects of TCEE and its underlying mechanisms in the activation of endothelial nitric oxide synthase (eNOS) have not yet been investigated. Increasing the endothelium-derived nitric oxide (NO) production has been known to be beneficial against the development of cardiovascular diseases. In this study, we investigated the effect of TCEE on eNOS activation and NO-related endothelial function and inflammation by using an in vitro system. In endothelial cells (ECs), TCEE increased NO production in a concentration-dependent manner without affecting the expression of eNOS. In addition, TCEE increased the phosphorylation of eNOS at serine 635 residue (Ser635) and Ser1179, Akt at Ser473, calmodulin kinase II (CaMKII) at threonine residue 286 (Thr286), and AMP-activated protein kinase (AMPK) at Thr172. Moreover, TCEE-induced NO production, and EC proliferation, migration, and tube formation were diminished by pretreatment with LY294002 (an Akt inhibitor), KN62 (a CaMKII inhibitor), and compound C (an AMPK inhibitor). Additionally, TCEE attenuated the tumor necrosis factor-α-induced inflammatory response as evidenced by the expression of adhesion molecules in ECs and monocyte adhesion onto ECs. These inflammatory effects of TCEE were abolished by L-NG-nitroarginine methyl ester (an NOS inhibitor). Moreover, chronic treatment with TCEE attenuated hyperlipidemia, systemic and aortic inflammatory response, and the atherosclerotic lesions in apolipoprotein E-deficient mice. Collectively, our findings suggest that TCEE may confer protection from atherosclerosis by preventing endothelial dysfunction.

The ancient Thracian endemic plant Haberlea rhodopensis Friv. and related species: A review.

ETHNOPHARMACOLOGICAL RELEVANCE: Haberlea rhodopensis (HR) use dates back to the Thracian and Roman periods. Bulgarians call it Orpheus flower and exploit its leaves for making tea and extracts with detoxifying, tonic, restorative and rejuvenating effects. HR was traditionally applied in wound healing and treatment of cattle diseases. AIM OF THE STUDY: The general aim of the review was to analyze the progress of phytochemical and pharmacological studies on HR, focusing on its radioprotective and immunomodulating effects. MATERIALS AND METHODS: The main source material for the review was collected using several global search engines with the phrase: Haberlea rhodopensis, as well as Bulgarian books and dissertations. RESULTS: HR metabolite profile includes large amounts of free sugars, polyols, polysaccharides (PS), flavonoids, phenolic acids and carotenoids. The radioprotective effect of 70% ethanolic leaf extract (70HREE) is explained by preservation of lymphocytes, other blood cells and testicular tissue from aberration under γ-radiation via stimulation of antioxidant enzymes and neutralization of free radicals. The extract immunomodulating activity results from raised antibody response, stem and neutrophil cell count, complement system activation, anti-tumour and anti-inflammatory effects. The detoxifying, restorative, rejuvenating and wound healing plant properties known to ethnomedicine were supported by radioprotective and immunomodulating studies. CONCLUSIONS: Metabolites of phenolic origin involved in HR resurrection are supposed to contribute to its radioprotective, immunomodulatory, anti-mutagenic and anti-aging effects. However, there is no chemical characterization of 70HREE in the investigations with humans and animals. Structure-activity relationship studies on HR immunomodulating and radioprotective compounds, and on their mode of action are required. They should include not only phenols but PS and other unexplored molecules. The metabolic activity of phagocytes, platelets and lymphocytes triggered by HR extracts has to be examined to elucidate their immunostimulatory potential. HR formulations can be tested in cosmetic, food and medical products as adjuvants to treat infectious, chronic inflammatory and tumour diseases, and especially in patients undergoing radiotherapy.

Six New Coumarin Glycosides from the Aerial Parts of Gendarussa vulgaris.

Six new coumarin glycosides, genglycoside A-F (1-6), were isolated from the aerial parts of Gendarussa vulgaris, along with ten known analogues (7-16). Their structures were unambiguously established on the basis of extensive spectroscopic data and HPLC analysis. The cytotoxic activities of all isolated compounds were evaluated by MTT assay. Compound 12 showed the most potent cytotoxicity in Eca-109, MCF-7, and HepG2 cell lines. By the preliminary structure-activity relationships, it was firstly discovered that the glycosylation or esterification at 7,8-dihydroxy or 7-hydroxy drastically reduced the cytotoxic activity of the parent coumarin.

In vitro propagation of lemon verbena: a plant native of South America

In vitropropagation increases the supply and commercialisation of products of interest. For this, optimising the growing conditions and the composition of the culture medium is crucial to benefit the full development of the plants. Thus, the objective was to evaluate the in vitropropagation of Aloysia triphyllaon different culture media, with varying agar and sucrose concentrations. The experiment was conducted as a completely randomised design, 3×3×3 factorial scheme, with three culture media (MS, JADS and WPM), three sucrose concentrations (8, 10 and 12 g L-1) and three agar concentrations (15, 30 and 45 g L-1), with three replicates each and experimental units composed of one plant per replicate. After 25 days of cultivation, the fresh and dry mass of the plants, numbers of leaves, numbers of nodes, plant lengths, numbers of oxidised leaves, hyperhydricity and acclimatization percentages were evaluated. The WPM medium resulted in a reduced fresh mass, reflecting in the low hyperhydricity observed in the explants, and favoured the acclimatization of the plants. Thus, the WPM medium with sucrose (15 g L-1) and agar (12 g L-1) is recommended as the medium most suitable for the in vitroregeneration of Aloysia triphylla.

CJK-7, a Novel Flavonoid from Paulownia tomentosa Triggers Cell Death Cascades in HCT-116 Human Colon Carcinoma Cells via Redox Signaling.

BACKGROUND: Colon cancer is the second most common cancer to cause death worldwide. About half of colon cancers patients require adjuvant therapy to control relapse following surgical resection. Therefore, abolition of tumor cell progression using an effective chemotherapeutic agent holds a feasible approach to treat patients suffering from colon cancer. In the present study, we evaluated the effects of geranylated flavonoid CJK-7, isolated from Paulownia tomentosa on HCT-116 human colon carcinoma cells. MATERIALS AND METHODS: The effects of CJK-7 as an active component on HCT-116 cells programmed cell death and its underlying molecular mechanism were examined by using MTT assay, morphological assessment, H2DCFDA staining, Fura-2AM staining, Hoechst-33342 staining, comet assay, Acridine orange staining, mitochondrial membrane potential (ΔΨm) assay and Western blot analyses. RESULTS AND CONCLUSION: The results revealed that, CJK-7 was capable of inducing caspase-dependent cell death events in cancer cells. Moreover, it was involved in up-regulation of autophagy signaling as evidenced by enhanced expression of LC3I/II. We also noticed stimulated expression of endoplasmic reticulum stress markers and phosphorylation of c-Jun NH2-terminal kinase (JNK), which was associated with up-regulated expression of p53, PUMA, Atg5 and Beclin-1, and down-regulation of Bcl-2, stressing the interaction of ROS on the aforementioned signaling. Furthermore, exposure to ROS scavengers (N-acetyl-l-cysteine (NAC), and JNK-specific inhibitor SP600125) significantly reversed the effects of CJK-7 by down-regulating apoptosis and autophagy signatures in HCT-116 cancer cells. Collectively our findings clarify the ROS-dependent regulatory effect of CJK-7 on programmed cell death signaling events in HCT-116 cancer cells while depicting its virile pro-oxidant capacity.

Potential therapeutic activity of Phlogacanthus thyrsiformis Hardow (Mabb) flower extract and its biofabricated silver nanoparticles against chemically induced urolithiasis in male Wistar rats.

Urolithiasis is a painful disorder in which stones are formed in the kidney, bladder or urethra. There are no proper therapeutic treatments available for kidney stones and people suffering from larger stones have to undergo surgery which has many side effects. A natural remedy with therapeutic effects that can dissipate and remove even the larger stones would eliminate the need of a surgery and the risks associated with it. The flowers of Phlogacanthus thyrsiformis used in culinary recipes in the north eastern India are also widely used as a folklore medicine for the treatment of kidney stones and liver disorders. The aim of this study was to evaluate the prophylactic and therapeutic activity of the aqueous extract of P. thyrsiformis flowers and its biofabricated silver nanoparticles against struvite urinary stones and calcium oxalate kidney stones. A kidney stone inhibition study was carried out on struvite stones grown in gel medium and calcium oxalate stones in rat models using an aqueous extract of P. thyrsiformis flowers and its biofabricated silver nanoparticles. The aqueous extract of P. thyrsiformis flowers and their biofabricated silver nanoparticles, obtained by a green synthetic method, were used to treat struvite urinary stones in vitro and calcium oxalate kidney stones in vivo. Struvite stones were grown in tubes by gel diffusion technique and were treated with varying concentrations of the extract and its nanoparticles. The size of the struvite stones was monitored for 96h using a travelling microscope. Calcium oxalate stones were induced in male Wistar rats by feeding ethylene glycol-ammonium chloride mixture for 14days. Both, prophylactic and therapeutic activities were evaluated by analyzing the urine, serum and histopathological parameters of the rats. The qualitative screening of water extract unveiled the presence of flavonoids as a major constituent. Both, the extract and the nanoparticles effectively reduced the size of struvite stones in vitro and eliminated calcium oxalate stones in Wistar rats in vivo. The potent therapeutic activity of both extract and silver nanoparticles was observed as compared to preventive activity. Anti-urolithiatic potency can be attributed to the presence of flavonoids.

In Vitro Reversible and Time-Dependent CYP450 Inhibition Profiles of Medicinal Herbal Plant Extracts Newbouldia laevis and Cassia abbreviata: Implications for Herb-Drug Interactions.

This study evaluated the effects of Newbouldia laevis and Cassia abbreviata extracts on CYP450 enzyme activity. Recombinant CYP450 enzyme and fluorogenic substrates were used for evaluating inhibition, allowing the assessment of herb-drug interactions (HDI). Phytochemical fingerprinting was performed using UPLC-MS. The herbal extracts were risk ranked for HDI based on the IC50 values determined for each CYP enzyme. Newbouldia laevis inhibited CYP1A2, CYP2C9, and CYP2C19 enzyme activities with Ki of 2.84 µg/mL, 1.55 µg/mL, and 1.23 µg/mL, respectively. N. laevis exhibited a TDI (4.17) effect on CYP1A2 but not CYP2C9 and CYP2C19 enzyme activities. Cassia abbreviata inhibited CYP1A2, CYP2C9, and CYP2C19 enzyme activities showing a Ki of 4.86 µg/mL, 5.98 µg/mL, and 1.58 µg/mL, respectively. TDI potency assessment for Cassia abbreviata showed it as a potential TDI candidate (1.64) for CYP1A2 and CYP2C19 (1.72). UPLC-MS analysis showed that Newbouldia laevis and Cassia abbreviata possess polyphenols that likely give them their therapeutic properties; some of them are likely to be responsible for the observed inhibition. The observations made in this study suggest the potential for these herbal compounds to interact, especially when co-administered with other medications metabolized by these CYP450 enzymes.

Haberlea rhodopensis: pharmaceutical and medical potential as a food additive.

This review discusses the potential of Haberlea rhodopensis as a food additive. The following are described: plant distribution, reproduction, cultivation, propagation and resurrection properties; extraction, isolation and screening of biologically active compounds; metabolite changes during dehydration; phytotherapy-related properties such as antioxidant potential and free radical-scavenging activities, antioxidant skin effect, antibacterial activity, cytotoxic activity and cancer-modulating effect, radioprotective effect, chemoprotective effect, immunologic effect; present use in homoeopathy and cosmetics, pharmacological and economical importance; perspectives based on the ethnobotanical data for medicinal, cosmetic or ritual attributes. H. rhodopensis showed unique medical and pharmaceutical potential, related to antioxidant, antimicrobial, antimutagenic, anticancer, radioprotective, chemoprotective and immunological properties. H. rhodopensis extracts lack any cytotoxic activity and could be used in phytotherapy. The metabolic profiling of H. rhodopensis extracts revealed the presence of biologically active compounds, possessing antiradical and other physiological activities, useful for design of in vitro synthesised analogues and drugs.

Composition, Cytotoxic and Antimicrobial Activities of Satureja intermedia C.A.Mey Essential Oil.

In this study, the essential oil (EO) constituents from the aerial parts of Satureja intermedia C.A.Mey were detected by GC and GC/MS. The antimicrobial activity of EO on oral pathogens and its cytotoxicity to human cancer cells were determined by the microbroth dilution method and the crystal violet staining method, respectively. Thirty-nine compounds were identified and the main EO constituents were γ-terpinene (37.1%), thymol (30.2%), p-cymene (16.2%), limonene (3.9%), α-terpinene (3.3%), myrcene (2.5%), germacrene B (1.4%), elemicine (1.1%) and carvacrol (0.5%). The S. intermedia EO showed a concentration-dependent decrease in viability of Hep-G2 (hepatocellular carcinoma) and MCF-7 (breast adenocarcinoma) human cancer cell lines (p < 0.05). Antimicrobial screening of S. intermedia EO demonstrated slight antibacterial and antifungal activities against Streptococcus mutants, S. salivarius, Enterococcus faecalis, Staphylococcus aureus, Candida albicans and C. glabrata. Further preclinical studies are needed to assess the efficacy and safety of S. intermedia EO as a new promising anticancer agent.

Antinociceptive activity of the ethanolic extract, fractions, and aggregatin D isolated from Sinningia aggregata tubers.

The present study investigated the effects of the ethanolic extract (ESa), fractions, and compounds isolated from Sinningia aggregata in male Swiss mice on carrageenan-induced paw edema, neutrophil migration, mechanical hyperalgesia, formalin-induced nociception, and lipopolysaccharide-induced fever. The ESa did not alter edema, neutrophil migration, or fever at any of the doses tested. However, the ESa reduced phase II of formalin-induced nociception and carrageenan-induced mechanical hyperalgesia. The petroleum ether (PE) and ethyl acetate (EA) fractions and aggregatin D (AgD; isolated from the EA fraction) reduced formalin-induced nociception. Anthraquinones from the PE fraction were ineffective. AgD also inhibited carrageenan-induced mechanical hyperalgesia. Neither the ESa nor AgD altered thermal nociception or motor performance. Local administration of AgD also reduced hyperalgesia induced by carrageenan, bradykinin, tumor necrosis factor-α, interleukin-1ß, cytokine-induced neutrophil chemoattractant, prostaglandin E2, and dopamine but not hyperalgesia induced by forskolin or dibutyryl cyclic adenosine monophosphate. The positive control dipyrone reduced the response induced by all of the stimuli. Additionally, glibenclamide abolished the analgesic effect of dipyrone but not the one induced by AgD. AgD did not change lipopolysaccharide-induced nitric oxide production by macrophages or the nociception induced by capsaicin, cinnamaldehyde, acidified saline, or menthol. These results suggest that the ESa has important antinociceptive activity, and this activity results at least partially from the presence of AgD. AgD reduced mechanical hyperalgesia induced by several inflammatory mediators through mechanisms that are different from classic analgesic drugs.

Lignans from the stems and leaves of Brandisia hancei and their effects on VEGF-induced vascular permeability and migration of HRECs and DLAV formation in zebrafish.

In our continuing search for novel antiangiogenic agents, a new lignan glycoside, (7R,8R)-1-(4-O-ß-d-glucopyranosyl-3-methoxyphenyl)-2-{2-methoxy-4-[1-(E)-propene-3-ol]-phenoxyl}-propane-1,3-diol (1), along with three known lignans (2-4), were isolated from the 80% EtOH extract of Brandisia hancei stems and leaves. These isolates (1-4) were subjected to an in vitro bioassay to evaluate their effects on vascular endothelial growth factor (VEGF)-induced vascular permeability and migration of human retinal endothelial cells (HRECs). Of the compounds tested, compound 1 resulted in the greatest reduction in VEGF-induced vascular permeability by about 31.5% at 10 µM compared to the VEGF-treated control. In the migration assay, compounds 1 and 2 significantly decreased VEGF-induced HREC migration. Furthermore, zebrafish embryos treated with compounds 1 and 2 showed mild reductions of dorsal longitudinal anastomotic vessel (DLAV) formation.

Mimulone-induced autophagy through p53-mediated AMPK/mTOR pathway increases caspase-mediated apoptotic cell death in A549 human lung cancer cells.

Anticancer properties and mechanisms of mimulone (MML), C-geranylflavonoid isolated from the Paulownia tomentosa fruits, were firstly elucidated in this study. MML prevented cell proliferation in a dose- and time-dependent way and triggered apoptosis through the extrinsic pathway in A549 human lung adenocarcinoma cells. Furthermore, MML-treated cells displayed autophagic features, such as the formation of autophagic vacuoles, a primary morphological feature of autophagy, and the accumulation of microtubule-associated protein 1 light chain 3 (LC3) puncta, another typical maker of autophagy, as determined by FITC-conjugated immunostaining and monodansylcadaverine (MDC) staining, respectively. The expression levels of LC3-I and LC3-II, specific markers of autophagy, were also augmented by MML treatment. Autophagy inhibition by 3-methyladenine (3-MA), pharmacological autophagy inhibitor, and shRNA knockdown of Beclin-1 reduced apoptotic cell death induced by MML. Autophagic flux was not significantly affected by MML treatment and lysosomal inhibitor, chloroquine (CQ) suppressed MML-induced autophagy and apoptosis. MML-induced autophagy was promoted by decreases in p53 and p-mTOR levels and increase of p-AMPK. Moreover, inhibition of p53 transactivation by pifithrin-α (PFT-α) and knockdown of p53 enhanced induction of autophagy and finally promoted apoptotic cell death. Overall, the results demonstrate that autophagy contributes to the cytotoxicity of MML in cancer cells harboring wild-type p53. This study strongly suggests that MML is a potential candidate for an anticancer agent targeting both autophagy and apoptotic cell death in human lung cancer. Moreover, co-treatment of MML and p53 inhibitor would be more effective in human lung cancer therapy.