Preparation, Characterization and Anti-Ulcer Efficacy of Sanguinarine Loaded Solid Lipid Nanoparticles.

AIM: This study was designed to develop sanguinarine-loaded solid lipid nanoparticles (SG-SLNs) and investigate its gastroprotective effect on ethanol-induced gastric mucosal lesions in mice. METHODS: SG-SLNs were prepared by high temperature melt-cool solidification method using glycerol monostearate as the lipid and a combination of lecithin with poloxamer 188 as the surfactants. Solid lipid nanoparticles (SLNs) were designed at varying lipid concentrations (5, 10, 15, and 20 mg/mL), surfactant mixture concentrations (6, 12, and 18 mg/mL), and drug contents (5, 10, 15, and 20 mg/mL) in "one factor at a time" fashion. Ethanol-induced gastric ulcer model was performed on Kunming mice to examine the anti-inflammatory activity of SG-SLNs and compare it with sanguinarine (SG). RESULTS: The high temperature melt-cool solidification method provided consistent production of SLNs with smaller size and high entrapment efficiency (EE%). The composition of optimal formulation was 10 mg/mL lipid concentration, 18 mg/mL surfactant mixture concentration, and 15 mg/mL drug amount. The mean particle size and EE% of optimized formulation were 238 ± 10.9 nm and 79 ± 2.8%, respectively. Additionally, SG-SLNs significantly decreased tumor necrosis factor-alpha and interleukin-6 levels in the serum and gastric tissue, and reduced the content of NO in serum, and strengthened the protection of mucosal membrane. Moreover, SG-SLNs treatment markedly inhibited ethanol-induced nuclear factor-kappa B (NF-κB) activation when compared with SG. CONCLUSIONS: SLNs could be potentially applied as a delivery system of SG. The protective effect of SG-SLNs is due to the suppression of NF-κB expression and subsequent pro-inflammatory cytokines release.

Supplementation with Quaternary Benzo(c)phenanthridine Alkaloids Decreased Salivary Cortisol and Salmonella Shedding in Pigs After Transportation to the Slaughterhouse.

The present study was aimed at evaluating the effect of herbal extracts supplementation, particularly quaternary-benzo(c)phenanthridine alkaloids (QBA), which have been previously demonstrated to have anti-inflammatory, antimicrobial, and immune-modulator effects. We investigated the role of QBA on stress response and Salmonella shedding in finishing pigs transported to the slaughterhouse. A total of 82 pigs were orally challenged with a Salmonella cocktail (day 0) containing Salmonella Meleagridis, Hartford, Bovismorbificans and Newport serovars and randomly assigned to three treatment groups after 2 wks (day [D] 14): T1, in-feed QBA; T2, in-feed and water-soluble QBA; CON, nonsupplemented). Pigs were transported to the slaughterhouse 2 weeks after intervention (D 28) and slaughtered after nearly 19 h (D 29). Saliva, fecal samples, and carcass swabs were collected from all pigs. Salivary cortisol, Salmonella shedding, and carcass contamination were measured. A high positive correlation (Spearman rank correlation coefficient range 0.82-0.93) between salivary cortisol and Salmonella shedding was found after transportation in all groups (p < 0.05). Only the CON group showed an increase in salivary cortisol after transportation (5.48 ng/mL; p < 0.0001) to concentrations that were higher than in T1 (2.73 ng/mL; p = 0.0002) and T2 (1.88 ng/mL; p < 0.0001). Salmonella prevalence and shedding decreased after transportation in pigs receiving the QBA intervention (p < 0.05), whereas the control group showed a significant increase in Salmonella shedding after transportation (p = 0.04). At D 28, pigs in T2 shed lower numbers of Salmonella as compared to T1 (1.3E + 02 CFU/mL versus 8E + 03 CFU/mL; p = 0.002). Additionally, carcass contamination by Salmonella was higher in the CON group than the treated groups (p = 0.01). The findings show QBA intervention was effective in reducing transportation stress of pigs, resulting in reduced Salmonella shedding and positively impacting animal welfare and pork safety.

Sanguinarine inhibits osteoclast formation and bone resorption via suppressing RANKL-induced activation of NF-κB and ERK signaling pathways.

Sanguinarine is a natural plant extract that has been supplemented in a number of gingival health products to suppress the growth of dental plaque. However, whether sanguinarine has any effect on teeth and alveolar bone health is still unclear. In this study, we demonstrated for the first time that sanguinarine could suppress osteoclastic bone resorption and osteoclast formation in a dose-dependent manner. Sanguinarine diminished the expression of osteoclast marker genes, including TRAP, cathepsin K, calcitonin receptor, DC-STAMP, V-ATPase d2, NFATc1 and c-fos. Further investigation revealed that sanguinarine attenuated RANKL-mediated IκBα phosphorylation and degradation, leading to the impairment of NF-κB signaling pathway during osteoclast differentiation. In addition, sanguinarine also affected the ERK signaling pathway by inhibiting RANKL-induced ERK phosphorylation. Collectively, this study suggested that sanguinarine has protective effects on teeth and alveolar bone health.

Rise and fall of oral health products with Canadian bloodroot extract.

The rhizome of Sanguinaria canadensis (SC, bloodroot) contains an active principle with antimicrobial, antiinflammatory, antioxidative and immunomodulatory effects. For this reason SC extract has been added to toothpastes and mouthwashes in various concentrations. When tested separately, neither the toothpastes nor the mouthwashes with SC extract had any demonstrable clinical effectiveness against dental plaque and gingivitis. Although using them together twice a day seemed more effective than using placebo, more recent studies have shown conflicting results. Preclinical safety studies up to 2000, which did not include studies longer than 6 months, were thought not to indicate any appreciable potential for harm - to the oral mucosa in particular. In 2003, the FDA Subcommittee on Oral Health Care Drug Products for Over-the-Counter Human Use concluded from a review that using SC-containing products is safe. However, for reasons unknown, the review failed to consider publications between 1999 and 2001 that suggested a possible link between the use of SC-containing products and the pre-neoplastic lesion, leukoplakia. As it happened, bloodroot had already been removed (in 2001) from the formula of one of the most widely used products in question and the brand has since then disappeared altogether from the worldwide market.

Antiplatelet effect of sanguinarine is correlated to calcium mobilization, thromboxane and cAMP production.

Sanguinarine is a plant alkaloid present in the root of Sanguinaria canadensis and Poppy fumaria species. Sanguinarine has been used as an antiseptic mouth rinse and a toothpaste additive to reduce dental plaque and gingival inflammation. In this study, we investigated the antiplatelet effects of sanguinarine, aiming to extend its potential pharmacological applications. Sanguinarine inhibited platelet aggregation induced by arachidonic acid (AA), collagen, U46619 and sub-threshold concentration of thrombin (0.05 U/ml) with IC(50) concentrations of 8.3, 7.7, 8.6 and 4.4 microM, respectively. Sanguinarine (5-10 microM) inhibited 10-31% of platelet TXB(2) production, but not platelet aggregation induced by higher concentration of thrombin (0.1 U/ml). SQ29548, a thromboxane receptor antagonist, inhibited the AA-induced platelet aggregation but not TXB(2) production. Sanguinarine suppressed cyclooxygenase-1 (COX-1) activity (IC(50)=28 microM), whereas its effect on COX-2 activity was minimal. Sanguinarine (8, 10 microM) further inhibited the AA-induced Ca(2+) mobilization by 27-62%. In addition, SQ22536, an adenylate cyclase inhibitor, attenuated the inhibitory effect of sanguinarine toward AA-induced platelet Ca(2+) mobilization and aggregation. These results suggest that sanguinarine is a potent antiplatelet agent, which activates adenylate cyclase, inhibits platelet Ca(2+) mobilization, TXB(2) production as well as suppresses COX-1 enzyme activity. Sanguinarine may have therapeutic potential for treatment of cardiovascular diseases related to platelet aggregation.

The plant alkaloid Sanguinarine affects swine granulosa cell activity.

Sanguinarine (SA) has been used in toothpastes and oral rinse products and has been recently applied to livestock nutrition. This study was undertaken to examine the reproductive-related consequences of this feeding supplementation, evaluating SA effects (10, 100 and 300 nM) on swine granulosa cell steroidogenesis, oxidative enzyme activities (peroxidase, catalase and superoxide dismutase), and proliferation. Since angiogenesis is fundamental for follicle development, we also tested the impact of SA exposure on vascular endothelial growth factor (VEGF) production. SA had no effect on proliferation and did not alter progesterone production, although it significantly reduced estradiol synthesis at the two highest concentrations tested (100 and 300 nM). SA addition to granulosa cell culture inhibited VEGF production and increased peroxidase and catalase activities at all tested concentrations while superoxide dismutase activity was increased only at 300 nM. Our data suggest that SA can negatively affect some key biochemical parameters of ovarian granulosa cells.

Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation.

Nitidine chloride (NC), a bioactive alkaloid isolated from Zanthoxylum nitidum, has been used as a herbal ingredient in toothpaste that prevents cavities for decades. It also displays potential antitumor and anti-inflammation properties. However, its anticatabolic effect on bone is not known. We investigated the effect of NC on osteoclastogenesis, bone resorption and RANKL-induced NF-κB and NFATc1 signalling. In mouse-derived bone marrow monocytes (BMMs), NC suppressed RANKL-induced multinucleated tartrate-resistant acid phosphatase (TRAP)-positive osteoclast formation and bone resorption in a dose dependent manner. NC attenuated the expression of osteoclast marker genes including cathepsin K, D2, calcitonin receptor, NFATc1, and TRAP. Further, NC inhibited RANKL-activated NF-κB and NFATc1 signalling pathways. In vivo study revealed that NC abrogated oestrogen deficiency-induced bone loss in ovariectomized mice. Histological analysis showed that the number of osteoclasts was significantly lower in NC-treated groups. Collectively, our data demonstrate that NC suppressed osteoclastogenesis and prevented OVX-induced bone loss by inhibiting RANKL-induced NF-κB and NFATc1 signalling pathways. NC may be a natural and novel treatment for osteoclast-related bone lytic diseases.

Improved sanguinarine production via biotic and abiotic elicitations and precursor feeding in cell suspensions of latex-less variety of Papaver somniferum with their gene expression studies and upscaling in bioreactor.

Elicitors play an important role in challenging the plant defense system through plant-environment interaction and thus altering the secondary metabolite production. Culture filtrates of four endophytic fungi, namely, Chaetomium globosum, Aspergillus niveoglaucus, Paecilomyces lilacinus, and Trichoderma harzianum were tested on embryogenic cell suspensions of latex-less Papaver somniferum in dose-dependent kinetics. Besides this, abiotic elicitors salicylic acid, hydrogen peroxide, and carbon dioxide were also applied for improved sanguinarine production. Maximum biomass accumulation (growth index (GI) = 293.50 ± 14.82) and sanguinarine production (0.090 ± 0.008 % dry wt.) were registered by addition of 3.3 % v/v T. harzanium culture filtrate. Interestingly, it was further enhanced (GI = 323.40 ± 25.30; 0.105 ± 0.008 % dry wt.) when T. harzanium culture filtrate was employed along with 50 µM shikimate. This was also supported by real-time (RT) (qPCR), where 8-9-fold increase in cheilanthifoline synthase (CFS), stylopine synthase (STS), tetrahydroprotoberberine cis-N-methyltransferase (TNMT), and protopine 6-hydroxylase (P6H) transcripts was observed. Among abiotic elicitors, while hydrogen peroxide and carbon dioxide registered low level of sanguinarine accumulation, maximum sanguinarine content was detected by 250 µM salicylic acid (0.058 ± 0.003 % dry wt.; GI = 172.75 ± 13.40). RT (qPCR) also confirms the downregulation of sanguinarine pathway on CO2 supplementation. Various parameters ranging from agitation speed (70 rpm), impeller type (marine), media volume (2 l), inoculum weight (100 g), and culture duration (9 days) were optimized during upscaling in 5-l stirred tank bioreactor to obtain maximum sanguinarine production (GI = 434.00; 0.119 ± 0.070 % dry wt.). Addition of 3.3 % v/v T. harzanium culture filtrate and 50-µM shikimate was done on the 6th day of bioreactor run.

Cytotoxicity and apoptotic signalling cascade induced by chelidonine-loaded PLGA nanoparticles in HepG2 cells in vitro and bioavailability of nano-chelidonine in mice in vivo.

Poor oral bioavailability of chelidonine, a bio-active ingredient of Chelidonium majus, showing anti-cancer potentials against cancer cells with multidrug resistance, makes its optimal use rather limited. To address this problem, we encapsulated chelidonine in biodegradable poly(lactide-co-glycolide) (PLGA) polymers and evaluated nano-chelidonine's (NCs) anti-cancer efficacy vis-à-vis free chelidonine (FC) against HepG2 cells and also evaluated its bioavailability in mice. Physicochemical characteristics indicated that stable spherical NC were formed in nanometer size range (123±1.15 nm) with good yield (86.34±1.91%), better encapsulation efficiency (82.6±0.574%), negative surface charge (-19.6±2.48 mV) and ability of prolonged and sustained release of chelidonine. Fourier transform infrared analysis revealed that NC resembled similar peaks as that of FC suggesting effective encapsulation in PLGA. NC exhibited rapid cellular uptake and stronger apoptotic effect (∼46.6% reduced IC50 value) than FC, blocking HepG2 cells at G2/M phase. p53, cyclin-D1, Bax, Bcl-2, cytochrome c, Apaf-1, caspase-9 and caspase-3 expressions also corroborated well to suggest greater anticancer potentials of NC. Our in vivo studies demonstrated NC to be more bio-available than FC and showed a better tissue distribution profile without inducing any toxicity (100 mg/kg bw) in mice. Unlike FC, NC could permeate into brain tissue, indicating thereby NC's better potentials for use in therapeutic oncology.

[Experimental study of the inhibitory effects of Chelidonium majus L. extractive on Streptococcus mutans in vitro].

PURPOSE: To study the inhibitory effects of Chelidonium majus L. extractive on the growth of Streptococcus mutans in vitro, and to explore its mechanism in caries prevention. METHODS: Streptococcus mutans 25175 was chosen as the experimental bacterium. The Chelidonium majus L. extractives chelidonine and chelerythrine were double diluted to different concentrations by two-fold dilution. The inhibitory effect of Streptococcus mutans was measured by slip diffusion method. The minimal inhibitory concentration(MIC) was also determined. 0.16% liquor hibitane was used as positive control. Spearman correlation was used for statistical analysis. RESULTS: Inhibition zone of Streptococcus mutans appeared in some concentration of chelerythrine, but no inhibition zone in each concentration of chelidonine. The MIC of chelerythrine was 0.78 mg/ml which determined by liquid culture medium. The concentration of chelerythrine was highly related to the inhibitory zone of Streptococcus mutans (r=0.99, P<0.01). CONCLUSION: The antibacterial activity of Chelidonium majus L. extractive chelerythrine on Streptococcus mutans was significant,and the antibacterial activity of the concentration 100 mg/ml was higher than that of 0.16% liquor hibitane (19.4 mm), indicating that chelerythrine can be used as an agent for prevention of dental caries.

The effect of chelerythrine on cell growth, apoptosis, and cell cycle in human normal and cancer cells in comparison with sanguinarine.

We compared the effects of chelerythrine (CHE) and sanguinarine (SA) on human prostate cancer cell lines (LNCaP and DU-145) and primary culture of human gingival fibroblasts. CHE and SA treatment of cell lines for 24 h resulted in (1) inhibition of cell viability in a dose-dependent manner in all tested cells (as evaluated by MTT test and bromodeoxyuridine incorporation assay); (2) dose-dependent increase in DNA damage in all tested cells (as evaluated by DNA comet assay); (3) changes in apoptosis (assessed by western blot analysis and TUNEL assay); and (4) significant induction of cyclin kinase inhibitors p21(Waf1/Cip1) and p27(Kip1) in prostate cancer cells (identified by western blot analysis). Our study demonstrates that CHE had significant cytotoxic effect, independent of p53 and androgen status, on human prostate cancer cell lines. Normal gingival fibroblasts and DU-145 cells were more sensitive to the treatment with both alkaloids than were LNCaP cells. CHE and SA may be prospective natural molecules for use in the treatment of prostate cancer owing to their involvement in apoptosis and cell cycle regulation.