Seasonal and Geographic Dynamics in Bioproperties and Phytochemical Profile of Limonium algarvense Erben.

This study delved into the influence of ecological and seasonal dynamics on the synthesis of secondary metabolites in the medicinal halophyte Limonium algarvense Erben, commonly known as sea lavender, and examined their antioxidant and anti-inflammatory properties. Aerial parts of sea lavender were systematically collected across winter, spring, summer, and autumn seasons from distinct geographic locations in southern Portugal, specifically "Ria de Alvor" in Portimão and "Ria Formosa" in Tavira. The investigation involved determining the total polyphenolic profile through spectrophotometric methods, establishing the chemical profile via liquid chromatography electrospray ionization quadrupole time-of-flight mass spectrometry (LC-ESI-QTOF-MS/MS), and evaluating in vitro antioxidant properties using radical and metal-based methods, along with assessing anti-inflammatory capacity through a cell model. Results unveiled varying polyphenol levels and profiles across seasons, with spring and autumn samples exhibiting the highest content, accompanied by the most notable antioxidant and anti-inflammatory capacities. Geographic location emerged as an influential factor, particularly distinguishing plants from "Ria de Alvor". Seasonal fluctuations were associated with environmental factors, including temperature, which, when excessively high, can impair plant metabolism, but also with the presence of flowers and seeds in spring and autumn samples, which also seems to contribute to elevated polyphenol levels and enhanced bioproperties of these samples. Additionally, genetic factors may be related to differences observed between ecotypes (geographical location). This study underscores sea lavender's potential as a natural source of antioxidant and anti-inflammatory agents, emphasizing the significance of considering both geographic location and seasonal dynamics in the assessment of phenolic composition and bioactive properties in medicinal plant species.

The combination of pro-oxidative acting vanicosides and GLUT1 inhibitor (WZB117) exerts a synergistic cytotoxic effect against melanoma cells.

Vanicosides A and B isolated from Reynoutria sachalinensis rhizomes are disaccharide phenylpropanoid esters with proven antioxidant activity. Our earlier study showed the cytotoxic activity of vanicosides against melanoma cells, but the mechanism of cell death has not been elucidated. Based on the chemical structure of vanicosides, we proposed that they may induce cell death by generating reactive oxygen species (ROS) into melanoma cells. Moreover, the glucose molecule in their structure can affect the glucose transporters (GLUTs), upregulated in cancer cells. The A375 (melanotic) and C32 (amelanotic) melanoma cell lines were applied. Cell viability assay and ROS-Glo™ assay were performed before and after blocking of Glucose Transporter Type 1 (GLUT1) by WZB117. Fibroblasts and the SKOV-3 line were included in the study to test selectivity in the action of vanicosides and help to elucidate the mechanism of action. Upon incubation with vanicosides, high production of ROS occured, especially inside C32 cells, which was significantly reduced after GLUT-1 blocking. The A375 cells produced less ROS. Melanoma cells were simillary sensitive to the cytotoxic effects of vanicosides, which was clearly enhanced when vanicosides were used together with the WZB117 (GLUT1 inhibitor). The SKOV-3 line and the fibroblasts showed much less sensitivity to the cytotoxicity of vanicosides, also used together with WZB117. Moreover, no significant ROS formation was observed in these lines. The study proved that vanicosides generate ROS inside melanoma cells. These findings suggest that the combination of pro-oxidative acting vanicosides and GLUT1 inhibitors exerts a synergistic cytotoxic effect on melanoma cells.

Oral Wound Healing Potential of Polygoni Cuspidati Rhizoma et Radix Decoction-In Vitro Study.

Polygoni Cuspidati Rhizoma et Radix (syn. rhizomes of Reynoutria japonica Houtt.) is a pharmacopoeial raw material in Europe and China. In traditional medicine, one of the applications for Reynoutria japonica rhizomes is wound healing. In a recent in vitro study, we demonstrated that ethanol and acetone extracts from this herbal drug have the potential to heal oral gum wounds. However, considering that a majority of herbal medicines have been traditionally administered as water decoctions, in the present study, a decoction of Reynoutria japonica rhizomes was prepared and detailed tests to determine its in vitro gingival wound healing activity were conducted. We used the primary human gingival fibroblasts (HGF) incubated with a decoction to determine cell viability (MTT assay), cell proliferation (the confocal laser scanning microscope-CLSM), and cell migration (wound healing assay). Moreover, the collagen type III expression was examined using immunocytochemical staining. The studied decoction was qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. The Folin-Ciocalteu assay was used to determine the total phenols and tannins content. Additionally, HPLC-RI analysis of decoction and the previously obtained ethanol and acetone extracts was used to determine the composition of saccharides. Low concentration (from 50 to 1000 µg/mL) of decoction after 24 h incubation caused a significant increase in HGF cell viability. No cytotoxic effect was observed at any tested concentration (up to 2000 µg/mL). The lowest active concentration of decoction (50 µg/mL) was selected for further experiments. It significantly stimulated human gingival fibroblasts to proliferate, migrate, and increase the synthesis of collagen III. Phytochemical analysis showed significantly fewer polyphenols in the decoction than in the ethanol and acetone extracts tested earlier. In contrast, high levels of polysaccharides were observed. In our opinion, they may have a significant effect on the oral wound healing parameters analyzed in vitro. The results obtained encourage the use of this raw material in its traditional, safe form-decoction.

Betulin and Its Derivatives Reduce Inflammation and COX-2 Activity in Macrophages.

Betulin is a heavily studied natural compound for its use as an anticancer or pro-regenerative agent. The structural similarity between betulin to steroids gives rise to the idea that the substance may as well act as an anti-inflammatory drug. This study is the first to describe the anti-inflammatory properties of betulinic acid, betulin, and its derivatives with amino acids 1,4-diaminebutane (Dab), 1,3-diaminepropane (Dap), Ornithine (Orn), and lysine (Lys) on murine macrophages from lymphoma site. The compounds were compared to dexamethasone. To establish the response of the macrophages to the natural compounds, we tested the viability as well as sensitivity to the inflammatory signaling (IFNγR). IL-6 secretory properties and HSP-70 content in the cells were examined. Furthermore, we characterized the effects of compounds on the inhibition of cyclooxygenase-2 (COX-2) activity both in the enzymatic assays and molecular docking studies. Then, the changes in COX-2 expression after betulin treatment were assessed. Betulin and betulinic acid are the low-cytotoxicity compounds with the highest potential to decrease inflammation via reduced IL-6 secretion. To some extent, they induce the reorganization of IFNγR with nearly no effect on COX-2 activity. Conversely, Bet-Orn and Bet-Lys are highly cytotoxic and induce the aggregation of IFNγR. Besides, Bet-Lys reduces the activity of COX-2 to a higher degree than dexamethasone. Bet-Orn is the only one to increase the HSP-70 content in the macrophages. In case of IL-6 reduction, all compounds were more potent than dexamethasone.

Botox Therapy for Hypertrophy of the Masseter Muscle Causes a Compensatory Increase of Stiffness of Other Muscles of Masticatory Apparatus.

Little is known about the nature of masseter muscle hypertrophy. We investigated the masseter muscle stiffness change after a single intra-masseteric session of Botox injections in people with benign bilateral masseter hypertrophy and the effect of the treatment on the stiffness of the temporalis muscle. Stiffness of the muscles was measured with shear-wave elastography at baseline and 3 weeks after Botox injections in 22 otherwise healthy people. Before the treatment, the stiffness of the masseter was lower than of the temporalis muscle (10.18 ± 1.67 kPa vs. 11.59 ± 1.54 kPa; p = 0.002). After the treatment, this difference increased (6.38 ± 1.34 vs. 13.10 ± 1.92; p < 0.0001). The drop in the stiffness of the masseter muscle was symmetrical (left side by 3.78 kPa; right side by 3.83 kPa). No differences between the left and right sides of the face in terms of muscle stiffness were observed. The study shows that Botox injections reduce stiffness of the masticatory muscles which, in turn, increases the stiffness of the temporalis muscles. Due to the knock-on effect of the change in the masseter function on the other masticatory muscles, simultaneous evaluation and treatment of the temporalis muscle may be required to ensure the desired functional and cosmetic effect.

Antiglycoxidative Properties of Extracts and Fractions from Reynoutria Rhizomes.

Hyperglycemia, when sustained over a long time in diabetes mellitus (DM), leads to biochemical and cellular abnormalities, primarily through the formation of advanced glycation end-products (AGEs). In the treatment of diabetes, beside blood-sugar-lowering medications, a consumption of herbal products that can inhibit the AGEs' formation is recommended. This study investigated the in vitro antiglycoxidative potential of extracts and fractions from the rhizomes of Japanese, Giant, and Bohemian knotweeds (Reynoutria japonica (Houtt.), R. sachalinensis (F. Schmidt) Nakai, and R.× bohemica Chrtek et Chrtkova). Their effects on glycooxidation of bovine and human serum albumin were evaluated by incubation of the proteins with a mixture of glucose and fructose (0.5 M) and 150 µg/mL of extract for 28 days at 37 °C, followed by measuring early and late glycation products, albumin oxidation (carbonyl and free thiol groups), and amyloid-ß aggregation (thioflavin T and Congo red assays). The highest antiglycoxidative activity, comparable or stronger than the reference drug (aminoguanidine), was observed for ethyl acetate and diethyl ether fractions, enriched in polyphenols (stilbenes, phenylpropanoid disaccharide esters, and free and oligomeric flavan-3-ols). In conclusion, the antiglycoxidative compounds from these three species should be further studied for potential use in the prevention and complementary treatment of DM.

In Vitro Gingival Wound Healing Activity of Extracts from Reynoutria japonica Houtt Rhizomes.

Rhizomes of Reynoutria japonica Houtt. are a traditional Chinese medicinal herb (Polygoni cuspidati rhizoma, hu zhang) used for treatment of numerous diseases including wound healing support. The aim of this study was to provide evidence for the value of this herbal drug's traditional use as a gingival healing treatment as well as to obtain the most active extract. In vitro studies were performed using primary human gingival fibroblasts (HGFs) with determination of viability (MTT assay), cell proliferation (the confocal laser scanning microscope (CLSM) was used to visualize histone 3 expression), cell migration (wound healing assay), and evaluation of the expression of collagen type III (immunocytochemical staining) after incubation with extracts from R. japonica rhizomes (25% or 40% ethanol or 60% acetone). In addition to these extracts, commercial dental rinse (containing chlorhexidine digluconate 0.2%) was tested as the gold standard of choice for gum healing in dental practice. The studied extracts were qualitatively and quantitatively characterized using the validated HPLC/DAD/ESI-HR-QTOF-MS method. Total phenols and tannins content were determined using the Folin-Ciocalteu assay. Low concentration of all extracts after 24 h incubation caused significant increase in HGF viability. This effect was most pronounced at a concentration of 50 µg/mL, which was selected for further experiments. All extracts (at 50 µg/mL) stimulated HGF to proliferate, migrate, and increase collagen III synthesis, but with different strength. The highest stimulated proliferation and migration activity was observed after incubation with 25% EtOH, which according to phytochemical analysis may be related to the highest content of resveratrol and an appropriate composition of procyanidins. The 25% EtOH extract from R. japonica rhizomes appears to be a promising gingival wound healing agent worthy of animal and clinical trials.

Reynoutria Rhizomes as a Natural Source of SARS-CoV-2 Mpro Inhibitors-Molecular Docking and In Vitro Study.

More than a year has passed since the world began to fight the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the Coronavirus disease 2019 (COVID-19) pandemic, and still it spreads around the world, mutating at the same time. One of the sources of compounds with potential antiviral activity is Traditional Chinese Medicinal (TCM) plants used in China in the supportive treatment of COVID-19. Reynoutria japonica is important part of the Shu Feng Jie Du Granule/Capsule-TCM herbal formula, recommended by China Food and Drug Administration (CFDA) for treatment of patients with H1N1- and H5N9-induced acute lung injury and is also used in China to treat COVID-19, mainly combined with other remedies. In our study, 25 compounds from rhizomes of R. japonica and Reynoutria sachalinensis (related species), were docked into the binding site of SARS-CoV-2 main protease. Next, 11 of them (vanicoside A, vanicoside B, resveratrol, piceid, emodin, epicatechin, epicatechin gallate, epigallocatechin gallate, procyanidin B2, procyanidin C1, procyanidin B2 3,3'-di-O-gallate) as well as extracts and fractions from rhizomes of R. japonica and R. sachalinensis were tested in vitro using a fluorescent peptide substrate. Among the tested phytochemicals the best results were achieved for vanicoside A and vanicoside B with moderate inhibition of SARS-CoV-2 Mpro, IC50 = 23.10 µM and 43.59 µM, respectively. The butanol fractions of plants showed the strongest inhibition of SARS-CoV-2 Mpro (IC50 = 4.031 µg/mL for R. sachalinensis and IC50 = 7.877 µg/mL for R. japonica). As the main constituents of butanol fractions, besides the phenylpropanoid disaccharide esters (e.g., vanicosides), are highly polymerized procyanidins, we suppose that they could be responsible for their strong inhibitory properties. As inhibition of SARS-CoV-2 main protease could prevent the replication of the virus our research provides data that may explain the beneficial effects of R. japonica on COVID-19 and identify the most active compounds worthy of more extensive research.

Short (6 mm) and Regular Dental Implants in the Posterior Maxilla-7-Years Follow-up Study.

Short 6 mm dental implants are considered as an alternative to the maxillary sinus elevation and bone augmentation procedure where there is a reduced alveolar ridge height. The aim of this study was to compare the implant survival rate between short dental implants (6 mm) and regular length implants (11-13 mm) when placed in combination with bone grafting and loaded with a single non splinted crown, seven years after placing the implant. It was conducted as a controlled clinical study of 30 patients with partial edentulism in the posterior maxilla. The protocol included radiological and clinical evaluation of the C/I ratio (length of the superstructure divided by the length of the implant crestal part), marginal bone level (MBL), ultrasonography measurement of soft tissue surrounding implant (STT), patient-reported outcomes, and biological and technical complications. A total number of 28 implants (93%) remained integrated during follow-up period. MBL of 0.50 and 0.52 mm was observed for short implants and regular implants, respectively. MBL was checked for correlation with STT, and a negative correlation was found between MBL: STT. Our study has demonstrated a significantly lower implant survival rate for short implants compared to regular implants (87% compared to 100%). Despite the loss of several implants, good clinical results were achieved in the remaining implants in both groups. It is, therefore, worth considering short implants as an alternative to regular implants with a sinus lift surgery.

Phenolic Compounds of Reynoutria sp. as Modulators of Oral Cavity Lactoperoxidase System.

Lactoperoxidase (LPO) together with its (pseudo)halogenation cycle substrates, H2O2 and thiocyanate ions oxidized to hypothiocyanite ions, form one of the main systems involved in antimicrobial defense within the oral cavity. In bacterial diseases such as dental caries, lactoperoxidase is oxidized to a form known as Compound II, which is characterized by its inability to oxidize SCN-, resulting in a decreased generation of antimicrobial products. Reynoutria sp. rizome extracts, due to their high polyphenol content, have been tested as a source of compounds able to regenerate the antimicrobial activity of lactoperoxidase through converting the Compound II to the native LPO state. In the presented study, acetone extracts of R. japonica, R. sachalinensis, and R. x bohemica, together with their five fractions and four selected polyphenols dominating in the studied in extracts, were tested toward lactoperoxidase reactivating potential. For this purpose, IC50, EC50, and activation percentage were determined by Ellman's method. Furthermore, the rate constants for the conversion of Compound I-Compound II and Compound II-native-LPO in the presence of extracts, extracts fractions, and selected polyphenols were determined. Finally, the ability to enhance the antimicrobial properties of the lactoperoxidase system was tested against Streptococcus mutans. We proved that Reynoutria sp. rhizome is the source of lactoperoxidase peroxidation cycle substrates, which can act as activators and inhibitors of the antimicrobial properties of that system. The presented study shows that the reactivation of lactoperoxidase could become a potential therapeutic target in prevention and treatment support in some infectious oral diseases.

Proanthocyanidins and Flavan-3-Ols in the Prevention and Treatment of Periodontitis-Antibacterial Effects.

Flavan-3-ols and their oligomeric forms called proanthocyanidins are polyphenolic compounds occurring in several foodstuffs and in many medicinal herbs. Their consumption is associated with numerous health benefits. They exhibit antioxidant, anti-inflammatory, cytoprotective, as well as antimicrobial activity. The latter property is important in the prevention and treatment of periodontal diseases. Periodontitis is a multifactorial polymicrobial infection characterized by a destructive inflammatory process affecting the periodontium. Using non-toxic and efficient natural products such as flavanol derivatives can significantly contribute to alleviating periodontitis symptoms and preventing the disease's progress. Therefore, a comprehensive systematic review of proanthocyanidins and flavan-3-ols in the prevention and treatment of periodontitis was performed. The present paper reviews the direct antibacterial effects of these compounds against periodontic pathogens. The immunomodulatory effects, including animal and clinical studies, are included in a separate, parallel article. There is significant evidence supporting the importance of the antibacterial action exerted by proanthocyanidins from edible fruits, tea, and medicinal herbs in the inhibition of periodontitis-causing pathogens.

Proanthocyanidins and Flavan-3-ols in the Prevention and Treatment of Periodontitis-Immunomodulatory Effects, Animal and Clinical Studies.

This paper continues the systematic review on proanthocyanidins and flavan-3-ols in the prevention and treatment of periodontal disease and covers the immunomodulatory effects, and animal- and clinical studies, while the other part discussed the direct antibacterial properties. Inflammation as a major response of the periodontal tissues attacked by pathogenic microbes can significantly exacerbate the condition. However, the bidirectional activity of phytochemicals that simultaneously inhibit bacterial proliferation and proinflammatory signaling can provide a substantial alleviation of both cause and symptoms. The modulatory effects on various aspects of inflammatory and overall immune response are covered, including confirmed and postulated mechanisms of action, structure activity relationships and molecular targets. Further, the clinical relevance of flavan-3-ols and available outcomes from clinical studies is analyzed and discussed. Among the numerous natural sources of flavan-3-ols and proanthocyanidins the most promising are, similarly to antibacterial properties, constituents of various foods, such as fruits of Vaccinium species, tea leaves, grape seeds, and tannin-rich medicinal herbs. Despite a vast amount of in vitro and cell-based evidence of immunomodulatory there are still only a few animal and clinical studies. Most of the reports, regardless of the used model, indicated the efficiency of these phytochemicals from cranberries and other Vaccinium species and tea extracts (green or black). Other sources such as grape seeds and traditional medicinal plants, were seldom. In conclusion, the potential of flavan-3-ols and their derivatives in prevention and alleviation of periodontal disease is remarkable but clinical evidence is urgently needed for issuing credible dietary recommendation and complementary treatments.

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis Against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

Antimicrobial Efficacy of Different Decontamination Methods as Tested on Dental Implants with Various Types of Surfaces.

BACKGROUND Peri-implantitis is an inflammatory reaction affecting both hard and soft tissues surrounding dental implants. This pathological condition is caused by a polymicrobial aggressive biofilm that colonizes the implant and abutment surface at the peri-implant crevice level. The present in vitro study evaluated different methods of implant surface decontamination and assessed whether the type of the implant surface influences the results. MATERIAL AND METHODS The study was conducted in an in vitro model of peri-implantitis using 30 implants. The implants were divided into 3 equal groups based on the surface characteristics: machined-surface, sand-blasted and acid-etched, and HA-coated. Implants were coated with E. coli biofilm. After an incubation period, they were decontaminated with 4 different methods: sonic scaler application, sonic scaler application with the chemical agent Perisolv® combination, Er: YAG laser treatment, and PDT therapy with methylene blue as a photosensitizer. RESULTS The highest level of decontamination was achieved for machined-surface implants and for the combined chemical-mechanical and Er: YAG laser treatment. CONCLUSIONS The results of our study suggest that the method of implant decontamination should be customized to the type of implant surface.

Evaluation of the three methods of bacterial decontamination on implants with three different surfaces.

BACKGROUND: The main goal of the treatment of the peri-implantitis is to decontaminate the surface of the implant, thereby enabling further treatment involving, e.g., guided bone regeneration. Since new implants of the rougher surface were introduced to the common dental practice, decontamination is even more difficult. OBJECTIVES: The aim of the study was to evaluate 3 different methods of decontaminating implants with 3 different surfaces. MATERIAL AND METHODS: A total of 30 dental implants with 3 different surface types (machined, sandblasted, and acid-etched (SLA) and hydroxyapatite (HA)-coated) were used in the study. Each group of implants was coated with Escherichia coli biofilm and cultivated. Afterwards, the implants were transferred to the jaw model and treated with a different method: sonic scaler mechanical debridement with a Woodpecker PT5 sonic scaler (1st group), and mechanical debridement with sonic scaler and with the combination with chemical agent Perisolv® (2nd group), and with Er:YAG laser treatment (3rd group). Each implant was treated with the specific method and sent for further microbiological evaluation. RESULTS: The highest level of decontamination was achieved for machined-surface implants and the lowest for HA-coated implants. The method with the highest biofilm reduction was the scaler and Perisolv® group. The highest level of decontamination of HA-coated implants were achieved for Er:YAG laser irradiation method. CONCLUSIONS: In the following paper, the superiority of combined chemical-mechanical method of decontaminating the surface of the implant on SLA and machined-surface implants was proved. On the contrary, Er:YAG laser irradiation was reported as the best option for decontamination of the HA-coated implants. In our opinion, it is a significant finding, revealing that the method of peri-implantitis management should be considered in accordance to the type of the surface of the implant (customized to the surface of the implant).

Chemical Composition of East Asian Invasive Knotweeds, their Cytotoxicity and Antimicrobial Efficacy Against Cariogenic Pathogens: An In-Vitro Study.

BACKGROUND Giant knotweeds originating from East Asia, such as Reynoutria japonica, and Reynoutria sachalinensis, and their hybrid such as Reynoutria x bohemica, are invasive plants in Europe and North America. However, R. japonica is also a traditional East Asian drug (Polygoni cuspidati rhizoma) used in Korean folk medicine to improve oral hygiene. The aim of this study was to evaluate the antibacterial activity of acetone extracts of Reynoutria species against dominant caries pathogen such as Streptococcus mutans and alternative pathogens, as well as characterize the phytochemical composition of extracts and examine their cytotoxicity. MATERIAL AND METHODS Ultrasonic extraction was used to obtain polyphenol-rich extracts. The extracts were characterized by HPLC-DAD-ESI-MS. To test bacterial viability, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) against S. mutans, S. salivarius, S. sanguinis, and S. pyogenes were determined. The cytotoxicity of the extracts to human fibroblasts derived from gingiva was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RESULTS The R. japonica extract had the highest bacteriostatic and bactericidal activity against pathogens causing caries, mainly dominant caries pathogen S. mutans (mean MIC 1000 µg/mL and MBC 2000 µg/mL), which was most likely associated with a higher content of stilbene aglycons and anthraquinone aglycons in the extract. Moreover, the R. japonica extract demonstrated the lowest cytotoxic effect on human fibroblasts and exhibited cytotoxic activity only at the concentration causing the death of all S. mutans. CONCLUSIONS The results indicate that the R. japonica acetone extract can be considered as a natural, antimicrobial agent for caries control.

Phytochemical Diversity in Rhizomes of Three Reynoutria Species and their Antioxidant Activity Correlations Elucidated by LC-ESI-MS/MS Analysis.

The rhizome of Reynoutria japonica is a well-known traditional herb (Hu zhang) used in East Asia to treat various inflammatory diseases, infections, skin diseases, scald, and hyperlipidemia. It is also one of the richest natural sources of resveratrol. Although, it has been recently included in the European Pharmacopoeia, in Europe it is still an untapped resource. Some of the therapeutic effects are likely to be influenced by its antioxidant properties and this in turn is frequently associated with a high stilbene content. However, compounds other than stilbenes may add to the total antioxidant capacity. Hence, the aim of this research was to examine rhizomes of R. japonica and the less studied but morphologically similar species, R. sachalinensis and R. x bohemica for their phytochemical composition and antioxidant activity and to clarify the relationship between the antioxidant activity and the components by statistical methods. HPLC/UV/ESI-MS studies of three Reynoutria species revealed 171 compounds, comprising stilbenes, carbohydrates, procyanidins, flavan-3-ols, anthraquinones, phenylpropanoids, lignin oligomers, hydroxycinnamic acids, naphthalenes and their derivatives. Our studies confirmed the presence of procyanidins with high degree of polymerization, up to decamers, in the rhizomes of R. japonica and provides new data on the presence of these compounds in other Reynoutria species. A procyanidin trimer digallate was described for the first time in, the studied plants. Moreover, we tentatively identified dianthrone glycosides new for these species and previously unrecorded phenylpropanoid disaccharide esters and hydroxycinnamic acid derivatives. Furthermore, compounds tentatively annotated as lignin oligomers were observed for the first time in the studied species. The rhizomes of all Reynoutria species exhibited strong antioxidant activity. Statistical analysis demonstrated that proanthocyanidins should be considered as important contributors to the total antioxidant capacity.

Fractal dimension analysis a supplementary mathematical method for bone defect regeneration measurement.

AIM OR PURPOSE: The geometry of a bone defect is very complex. Its shape is too complicated to measure or compare with other bone defects using only traditional measuring methods Traditional measuring techniques based on the histomorphometric analysis of a bone specimen require supplementary measuring. For the fractal dimension analysis (FDA) mathematic formulas are used to describe complicated and chaotic shapes. The FDA offers a possibility of a comparison between complicated and complex shapes such as a histological image of a bone defect. The aim of this study was to evaluate the FDA of bone defects as a supplementary method for a defect regeneration assessment. MATERIALS AND METHODS: For the purpose of this study, microscopic photographs of bone specimens stained with hematoxylin and eosin obtained during a block biopsy were used. The bone blocks used in this study were obtained during a rat animal model study. Specimens were collected from 36 Wistar rats where a cranial defect was created and augmented with five different novel biomaterials and compared to the unfilled defect in the control group. New bone formation in every specimen was histomorphometrically measured by two independent operators and compared to FDA measurements. RESULTS: Both traditional and FDA techniques have shown statistically significant differences between bone formation in test groups compared to the control one; on the other hand, no statistically significant difference was found between other groups. The Pearson's r-test was conducted to measure the linear dependence (correlation) between standard measurements and the FDA, and a positive linear correlation was found -r=0.94. CONCLUSIONS: The FDA can be used as a supplementary method for bone regeneration measurements.

Isolation and Determination of Phenolic Glycosides and Anthraquinones from Rhizomes of Various Reynoutria Species.

Giant knotweeds of the genus Reynoutria (syn. Fallopia)-Reynoutria japonica, Reynoutria sachalinensis, and a hybrid of them, Reynoutria x bohemica-are noxious invasive plants in Europe and North America. R. japonica is a traditional East Asian (Japan and China) drug (Polygoni cuspidati rhizoma). Recently, it has been included in European Pharmacopoeia as one of the traditional Chinese medicinal herbs. In this study, a reversed-phase high performance liquid chromatography method with diode array detector and time-of-flight mass spectrometry was developed and validated for the profiling of rhizomes from European invasive populations and Polygoni cuspidati rhizoma purchased in China. Twenty-five compounds were identified, mainly stilbenes, anthraquinones, flavan-3-ols, and phenylpropanoid esters. Tatariside B, hydropiperoside, vanicoside C, a new compound (3,6-O-di-p-coumaroyl)-ß-fructofuranosyl-(2 → 1)-(2'-O-acetyl-6'-O-feruloyl)-ß-glucopyranoside) were reported for the first time in these raw materials. Six compounds from three phytochemical classes-stilbenes: piceid and resveratrol; anthraquinones: emodin and physcion; hydroxycinnamic sucrose esters: vanicosides A and B-were quantified using the validated method. R. japonica from China contained twice as many stilbenoids than samples from Poland (piceid 14.83 mg/g dm vs. 7.45 mg/g and resveratrol 1.29 mg/g vs. 0.65 mg/g). R. sachalinensis rhizomes contained lower quantities of anthraquinones and no detectable stilbenes, which together with higher amounts of hydroxycinnamic glycosides makes it easily distinguishable from the other two. The phytochemical profile of R. x bohemica was intermediate between the two parent species.

Inhibition of Glycation-induced Cytotoxicity, Protein Glycation, and Activity of Proteolytic Enzymes by Extract from Perovskia atriplicifolia Roots.

BACKGROUND: Protein glycation and glycotoxicity belong to the main oxidative-stress related complications in diabetes. Perovskia species are used in Asian folk medicine as antidiabetic herbs. OBJECTIVE: The aim of this study was to verify the ability of the methanolic extract from Perovskia atriplicifolia Benth. roots to diminish glycation of albumin and to prevent cell damage in vitro. Furthermore, we tested the extract for in vitro antioxidant activity and inhibition of elastase and collagenase. MATERIAL AND METHODS: The aqueous methanol extract was analyzed by UHPLC-MS for the content of polyphenols and terpenoids. The prevention of glycated albumin-induced cell damage was tested in four mammalian cell lines (peripheral blood mononuclear cells, human embryonic kidney cells - HEK293, normal human fibroblasts, and Chinese hamster ovary cells) with the 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazoly)-3-(4-sulfophenyl) tetrazolium assay. RESULTS: Glycated albumin is significantly more toxic than native human serum albumin (LC50 from 35.00 to 48.34 µg/mL vs. 5.47-9.10 µg/mL, respectively). The extract, rich in rosmarinic acid (344.27 mg/g dry mass), mitigated the glycated albumin toxicity, and increased glycated albumin-treated cell survival by more than 50%. The inhibition of advanced glycation endproduct formation was confirmed by monitoring conformational changes. The free radical scavenging activity was higher than Trolox and metal reducing power was one-third to half that of ascorbic acid. The activity of elastase and collagenase was inhibited by 54.75% ± 6.87% and 60.03% ± 7.22%, respectively. CONCLUSIONS: The results confirm antiglycative and antiglycotoxic potential of Perovskia root and its traditional antidiabetic use. The high activity can be attributed to rosmarinic acid abundance. SUMMARY: Perovskia is a small genus of aromatic shrubby plants growing in arid regions of Central and South Asia. Different parts are used in folk medicine as antiparasitic, anti-infectious and antidiabetic remedy. Here, we have studied the extract from roots for inhibition of: glycation-induced cytotoxicity, human serum albumin glycation, inflammation-related enzymes, as well as for antioxidant activity. Result: the extract from P. atriplicifolia roots inhibited protein glycation and AGE-induced toxicity in cell cultures. The mechanism is likely to rely on the antioxidant activity of high content of rosmarinic acid. Abbreviations used: AGE: advanced glycation end-products; DPPH: 2,2-diphenyl-1-picrylhydrazyl; HSA: human serum albumin.