CYTOTOXICITY TESTING OF A NOVEL CROSSLINKED PRF (C-PRF) DEVELOPED FOR DENTAL PULP TISSUE ENGINEERING PURPOSE

Background- Whole pulp amputation followed by pulp space disinfection and ?lling with an arti?cial material causes loss of signi?cant amount of dentin leaving a non-vital and weakened tooth. Regenerative endodontics with its emerging ?eld of modern tissue engineering has demonstrated promising results using stem cells associated with scaffolds and responsive molecules. [1] Introduction- PRF was recognized as the “second generation” of this family of biomaterials. [6] PRF being tested in pulp tissue engineering by different research groups showed mixed results. (7,8) Research studies have shown that the interactions between the cells and their niche are closely related to physicochemical properties of the scaffolding materials [9, 10]. As PRF is a fragile gel its physical character needs to be improved by cross linking and thereby more longer period of liberation of its growth factors and delayed disintegration in physiological system. Aims and Objectives- Aim of our study was to prepare a very economical and autologous biomaterial for pulp tissue engineering by crosslinking of PRF with tannic acid. Our objective was to detect cytotoxic effect of tannic acid in PRF. Methods and Materials- We followed Choukroun et al. protocol to prepare PRF samples from whole venous blood collected from donors. PRF samples were then cross-linked in freshly prepared TA solution in dapendish for 10 minutes at room temperature. Concentrations of TA 1 wt% was used for preparing samples. After crosslinking, the gels were washed with normal saline for 5 min. to ensure that all excess TA was removed. The viability of cells cultured on the scaffolds was assessed through MTT assay (EZcountTM MTT cell Assay Kit, HiMedia, Mumbai, India). Observations- Both MTT Assay and Phalloidine staining showed favourable results of no clear cytotoxic effects of C-PRF. Conclusion- Based on the results of the cell viability analysis it can be concluded that none of the tannic acid crosslinked PRF created any clear cytotoxicity in the MC3T3 cells. So, C-PRF can safely be used as scaffold for dental pulp or similar tissue engineering purposes

Biopelícula cariogénica de Streptococcus mutans y efecto hemostático de ácido tánico en la hemorragia post-exodoncia / Streptococcus mutans cariogenic biofilm and hemostatic effect of tannic acid in post-extraction hemorrhage

La caries dental y la placa dental se encuentran entre las enfermedades más comunes en todo el mundo y son causadas por una mezcla de microorganismos y restos de alimentos. Tipos específicos de bacterias productoras de ácido, especialmente Streptococcus mutans, colonizan la superficie dental y causan daño a la estructura dental dura en presencia de carbohidratos fermentables, por ejemplo, sacarosa y fructosa. Por otro lado, el sangrado posterior a la extracción es una complicación reconocida y frecuente en la práctica dental, que se define como la pérdida de sangre que continúa más allá de las 8 a 12 horas después de la exodoncia. Existe una amplia gama de técnicas sugeridas y sustancias para el tratamiento del sangrado post-extracción, que incluyen intervenciones dirigidas tanto a causas locales como sistémicas. El ácido tánico es una de las sustancias astringente que precipitan proteínas, pero no penetran en las células, por lo que inciden solo en la capa superficial. Su objetivo se enfoca a robustecer la superficie para acrecentar su resistencia mecánica y reducir la exudación. El objetivo de este estudio fue determinar la presencia de S. mutans en las biopelículas dentales y al mismo tiempo evaluar la actividad del ácido tánico como inhibidor del sangrado profuso en las extracciones dentales. S. mutans se aisló en el 92,5% de los pacientes evaluados. Los tiempos de hemostasia post-exodoncia fue significativamente menor en el grupo de pacientes a los que se les aplicó el ácido tánico en comparación a los que no se les aplicó(AU)

Tooth decay and dental plaque are among the most common diseases worldwide and are caused by a mix of microorganisms and food debris. Specific types of acid-producing bacteria, especially Streptococcus mutans, colonize the tooth surface and cause damage to hard tooth structure in the presence of fermentable carbohydrates, for example, sucrose and fructose. On the other hand, post-extraction bleeding is a recognized and frequent complication in dental practice, defined as blood loss that continues beyond 8 to 12 hours after extraction. There is a wide range of suggested techniques and substances for the treatment of post-extraction bleeding, including interventions targeting both local and systemic causes. Tannic acid is one of the astringent substances that precipitate proteins, but does not penetrate the cells, so it affects only the superficial layer. Its objective is focused on strengthening the surface to increase its mechanical resistance and reduce exudation. The objective of this study was to determine the presence of S. mutans in dental biofilms and at the same time to evaluate the activity of tannic acid as an inhibitor of profuse bleeding in dental extractions. S. mutans was isolated in 92.5% of the patients evaluated. Post-extraction hemostasis times were significantly shorter in the group of patients who received tannic acid compared to those who did not(AU)

Efecto radioprotector del ácido tánico y vinos de la var vitis vinífera L. cv tannat en Saccharomyces cerevisiae / Radioprotective effect of tannic acid and wines of the vitis vinifera L. cv tannat on Saccharomyces cerevisiae / Proteção contra radiação por vinho tinto var tannat e ácido tanico em Saccharomyces cerevisiae

El vino tinto variedad Vitis vinifera L. cv Tannat en los últimos años ha tomado relevancia por su alta concentración de polifenoles, esto le podría significar un rol protector sobre el genoma disminuyendo la formación de lesiones oxidativas. Los efectos a nivel celular de las radiaciones ionizantes en blancos como el ADN, componentes de cascadas de transducción de señales, resultan en lesiones letales, mutagénicas y recombinogénicas y en retardos en el ciclo celular. Se utilizó como modelo eucariota poblaciones de Saccharomyces cerevisiae en fase exponencial expuestas a radiación gamma (200 Gy) en presencia, o ausencia, de vino Tannat (10 % v/v) o de ácido tánico (60 µg/mL). Se estimaron las probabilidades de sobrevida y frecuencia mutagénica en distintas condiciones. Las muestras celulares expuestas a radiación ionizante presentaron una fracción de sobrevida de 0.21 ± 0.02 mientras que en las muestras irradiadas en presencia de vino Tannat o de ácido tánico la fracción de sobrevida fue de 0.33 ± 0.03 y 0.30 ± 0.03 respectivamente. Se observó en las poblaciones irradiadas un aumento significativo de la probabilidad de mutagénesis. En el caso de los tratamientos combinados se observó que la frecuencia mutagénica fue significativamente menor (gamma Tannat: 33%, gamma ácido tánico: 45% ). Estos resultados preliminares podrían indicar radioprotección moderada por parte de los compuestos estudiados, efecto que podría explicarse por las interacciones redox del ácido tánico y polifenoles contenidos en el vino con los radicales libres formados por las radiaciones ionizantes, además de la activación de vías de reparación genómica.

The red wine variety Vitis vinifera L. cv Tannat in recent years has gained relevance due to its high concentration of polyphenols, this could mean a protective role on the genome, reducing the formation of oxidative lesions. The effects at the cellular level of ionizing radiation on targets such as DNA, components of signal transduction cascades, result in lethal, mutagenic and recombinogenic lesions and delays in the cell cycle. Exponential phase populations of Saccharomyces cerevisiae exposed to gamma radiation (200 Gy) in the presence or absence of Tannat wine (10% v / v) or tannic acid (60 µg / ml) were used as a eukaryotic model. The probabilities of survival and mutagenic frequency in different conditions were estimated. Cellular samples exposed to ionizing radiation presented a survival fraction of 0.21 ± 0.02, while in samples irradiated in the presence of Tannat wine or tannic acid, the survival fraction was 0.33 ± 0.03 and 0.30 ± 0.03 respectively. A significant increase in the probability of mutagenesis was observed in irradiated populations. In the case of the combined treatments, it was observed that the mutagenic frequency was significantly lower (Tannat gamma: 33%, Tannic acid gamma: 45%). These preliminary results could indicate moderate radioprotection by the compounds studied, an effect that could be explained by the redox interactions of tannic acid and polyphenols contained in wine with the free radicals formed by ionizing radiation, in addition to the activation of genomic repair pathways.

A variedade de vinho tinto Vitis vinifera L. cv Tannat nos últimos anos tem ganhado relevância devido à sua alta concentração de polifenóis, o que pode significar um papel protetor do genoma, reduzindo a formação de lesões oxidativas. Os efeitos no nível celular da radiação ionizante em alvos como o DNA, componentes de cascatas de transdução de sinal, resultam em lesões letais, mutagênicas e recombinogênicas e atrasos no ciclo celular. Populações de fase exponencial de Saccharomyces cerevisiae expostas à radiação gama (200 Gy) na presença ou ausência de vinho Tannat (10% v / v) ou ácido tânico (60 µg / ml) foram utilizadas como modelo eucariótico. Foram estimadas as probabilidades de sobrevivência e frequência mutagênica em diferentes condições. As amostras celulares expostas à radiação ionizante apresentaram uma fração de sobrevivência de 0,21 ± 0,02, enquanto nas amostras irradiadas na presença de vinho Tannat ou ácido tânico, a fração de sobrevivência foi de 0,33 ± 0,03 e 0,30 ± 0,03, respectivamente. Um aumento significativo na probabilidade de mutagênese foi observado nas populações irradiadas. No caso dos tratamentos combinados, observou-se que a frequência mutagênica foi significativamente menor (Tannat gama: 33%, ácido tânico gama: 45%). Esses resultados preliminares podem indicar radioproteção moderada pelos compostos estudados, efeito que pode ser explicado pelas interações redox do ácido tânico e polifenóis contidos no vinho com os radicais livres formados pela radiação ionizante, além da ativação de vias de reparo genômico.

Formulation of a Topical Tannic Acid and Chitosan Gel Haemostatic Drug Delivery System for Treatment of Wounds and Abrasions

Haemostatic agents accelerate blood clotting and help in wound healing processes for wounds or abrasions. The purpose of this study was to prepare and evaluate a haemostatic gauze impregnated with chitosan andtannic acid as two effective haemostatic agents. The haemostatic gauze was easy to prepare and produce by means of a simple solvent casting method followed by lyophylisation. Nine different concentration combinations of tannic acid and chitosan were impregnated onto a 50mm x 50mm 8 ply gauze strip and were prepared and evaluated for dissolution into simulated body fluid (SBF) and finally tested for in vitroblood clotting ability versus Quikclot©(a commercially imported haemostatic gauze).There was an initial quick release of tannic acid from the haemostatic gauze strips within 60 secs and thereafter a constant release of tannic acid. The invitroblood clotting ability was found to be better in all the formulated haemostatic gauze strips than that of Quikclot©. Best clotting extent was achieved from 0.5% w/vchitosan and 1% w/vtannic acid gauze strips at 180 secs. In conclusion, the combination of tannic acid and chitosan haemostatic gauze strips developed in this study presents an inexpensive and effective alternative to importing haemostatic gauzes and bandages

Tannic acid-induced apoptosis in FaDu hypopharyngeal squamous cell carcinoma

Tannic acid (TA) is a water-soluble polyphenol compound found in various herbal plants. We investigated the chemopreventive effects of TA on FaDu hypopharyngeal squamous carcinoma cells. In an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, TA showed dose-dependent cytotoxicity with a half maximal inhibitory concentration (IC50) of 50 µM. Cell cycle analysis and immunofluorescence imaging demonstrated that under low-dose (25 µM) treatment, FaDu cells were arrested in G2/M phase, and as the dose of TA was increased, apoptosis was induced with the increase of cell population at sub-G1 phase. The expressions of various cyclins, including cyclin D1 and cyclin-dependent kinases (CDK-1 and CDK-2), were down-regulated at low doses of TA, whereas apoptotic effectors such as cleaved caspase 3, cleaved caspase 7, and poly (ADP-ribose) polymerase (PARP) were expressed in a dose-dependent manner in Western blotting. In addition, TA-induced apoptosis of FaDu cells might be mediated by the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase pathway, with the upregulation of p-AKT/p-PKB (phosphorylated protein kinase B) and p-ERK. Overall, our data support the hypothesis that TA is a potential candidate agent for the treatment of hypopharyngeal cancer.

Effect of Tannic Acid on Transport of Diaco-Aconitine in Caco-2 Cell Model / 中国药学杂志

OBJECTIVE: To study the effects of three aconitine transporters in Caco-2 cells and tannin (tannic acid) in Terminalia on the transport of three di-ester aconitine (aconitine, meoaconitine and hypaconitine) in Aconitum chinensis. METHODS: The components were detected by UPLC/Q exactive MS in terms of the cumulative transshipment volume of three aconitine and the apparent permeability coefficient Papp as indicators to investigate the two-way transport behaviors of three aconitine in the Caco-2 cell model, as well as the proportion of tannic acid, and the changes of the transport behaviors of three aconitine. RESULTS: There was a positive correlation between the cumulative transshipment volume of aconitine and the incubation time. There was no statistical difference in Papp values of the three aconitine, and the efflux effect was significantly stronger than the absorption, with an external ratio close to 1.5.When the compatibility ratio of three aconitine with tannic acid was 1∶1 and 1∶0.5, the absorption of aconitine was significantly inhibited (P was 0.05), but the transport behavior with effluent was not significantly affected. CONCLUSION: Aconitine, meoaconitine, and hypaconitine in P. aeruginosa are mainly passive transporters, and may involve in efferent proteins, which are a kind of drug with good absorption.When mixed with tannic acid, the absorption and transshipment of three kinds of alkali were significantly reduced, which proves that Terminalia chebula could detoxify aconitum by inhibiting its absorption.

Assessment of flatulence causing agents in Chickpea (Cicer arietinum L.) and their possible removal

Abstract Flatulence and fullness of stomach is one of the most common problem associated with chickpea primary due to presence of some oligosaccharides and phenols. In this investigation Desi and Kabuli varieties were compared for these oligosaccharides and phenolic compounds. Furthermore, the effect of different processing and cooking methods such as soaking, cooking and germination in the reduction of these antiphysiological factors were are also studies. Maximum tannic acid (0.90 ± 0.20%) was observed in Parbat and C-44 while minimum (0.60 ± 0.04%) in Karak-2. Stachyose contents ranged between 1.10 ± 0.05 (Karak-3) to 1.42 ± 0.02% (Parbat) while raffinose was 0.63 ± 0.05(Karak-3) to 0.81 ± 0.02% (Dasht). The highest tannic acid content was reduced up to 50% in C-44 by cooking of 72 hours germinated seeds. Stachyose and raffinose contents were completely removed after 72 hours germination. Present studies revealed that cooking after germination is the most effective method to reduce the anti-nutritional factors of chickpea. Individually, soaking and cooking also contributed to the loss of the same factors but to a lesser extent.

Tannic acid-Aluminum (Ⅲ) coordination-enabled microcapsules for pH-responsive protein vaccine delivery / 国际生物医学工程杂志

Objective To establish a simple and gentle antigen loading method to prepare pH-responsive and biodegradable microcapsules for efficient antigen delivery.Methods Co-precipitation method was used to embed chicken egg albumin (OVA) in CaCO3 particles.Then,TA and Al (Ⅲ) were coated on the surface of CaCO3 particles template by metal-organic coordination bonds.The CaCO3 template was removed from disodium edetate to obtain TA-Al(Ⅲ) microcapsules carrying OVA,i.e.the OVA@TA-Al(Ⅲ) microcapsules.The microcapsules were characterized by field emission scanning electron microscopy,transmission electron microscopy,X-ray energy spectrometry and atomic force microscopy.The distribution of OVA in the microcapsules was observed by laser scanning confocal microscopy.The cumulative release rate of OVA in the microcapsules at different pH phosphate buffers was also investigated.The cytotoxicity of the microcapsules on immortalized mouse dendritic cells DC2.4 was observed by thiazolyl blue assay.The phagocytosis of the microcapsules by DC2.4 cells was observed by laser scanning confocal microscopy.Results The results of field emission scanning electron microscope and transmission electron microscopy showed that the OVA@TA-Al(Ⅲ) microcapsules have a intact structure and a hollow and collapsed appearance with a diameter of about 4 μm.X-ray energy spectrum showed that there are five kinds of elements,i.e.C,O,Al,Si and Na,in the microcapsules,among which C,Al and some O elements belong to the composition of the microcapsules.Atomic force microscopy showed that the microcapsules have an ultra-thin wall,and the walls of the microcapsules are uniform in thickness (about 16 nm).Laser scanning confocal microscopy showed that OVAs were evenly distributed in the CaCO3 particles.Moreover,the pH sensitivity of the coordination bond makes the OVA@TA-Al(Ⅲ) microcapsules have pH responsiveness.In addition,the microcapsules also have good biocompatibility,and the DC2.4 cells also have good phagocytic ability to the microcapsules.Conclusion A simple and gentle antigen-encapsuling method was developed to achieve effective antigen payload and pH responsive delivery.The prepared microcapsules are expected to be used as a novel antigen delivery vector for clinical research.

Tannic acid enhances cisplatin effect against hepatocellular carcinoma HepG2 by endoplasmic reticulum stress pathway / 重庆医学

Objective To investigate the synergistic effects of tannic acid(TA) and cis-dichlorodiamine platinum(CDDP) on hepatocellular carcinoma HepG2 cells and its activation situation of endoplasmic reticulum stress(ERS) pathway.Methods Human hepatocellular carcinoma HepG2 cells were divided into the control group,TA group,CDDP group and TA+CDDP group,and were cultured in vitro for 24 h.The growth inhibitory effect of medication on HepG2 cells was detected by MTT assay.The pharmacodynamics synergistic effect between the two drugs was analyzed by the drug interaction index,drug dose reduction index and equivalent graphical method.The nucleus changes were observed by DAPI staining.Real time fluorescent quantitative PCR(q-RT-PCR) and Western blot were used to detect the levels of ERS markers glucose regulated protein (GRP)78 and GRP94.Results TA and CDDP had dose-dependent growth inhibition effect on HepG2 cells,their median effective concentrations(IC50) were 360.00 μmol/L and 1.80 μg/mL respectively.The combination treatment of 180.00 μmol/L TA and 0.90 μg/mL CDDP on HepG2 cells could enhance the inhibitory effect on cell growth.Ta and CDDP had synergistic effect for inhibiting hepatocellular carcinoma cells growth.Compared with the TA group and CDDP group,cell shrinkage and rounding were accelerated in the combined group,apoptotic cells were increased,nuclear had pyknosis,irregular edge and dense staining,nuclear fragmentations were increased and the expressions of GRP78 and GRP94 were up-regulated.Conclusion TA can enhance the effect of CDDP on anti-hepatic carcinoma HepG2 cells,and the synergy mechanism may be related to the activation of ERS pathway.

Spectrum-effect relationship of anti-inflammatory effect of different part from Polygonum capitatum / 中草药

Objective To study the spectrum effect relationship between the water and ethanol extracts of Polygonum capitatum and its anti-inflammatory activity. Methods The inflammatory model of mice RAW264.7 cells induced by lipopolysaccharide (LPS) was established, and the release of inflammatory factors was detected by NO and TNF-α kit. The relationship of chemical constituents of P. capitatum was established by using the partial least square method to associate the fingerprint data established by UPLC-MS method and the pharmacodynamics index of different extracts of P. capitatum. Results Different extracts of P. capitatum had no cytotoxicity in the range of concentration less than 250 mg/L, and all of them had anti-inflammatory effect, which had a dose-effect relationship. By using partial least square method, the correlation degree between TNF-α and chromatographic peak was compared. The results showed that the correlation among the peaks 24, 17, 22, 23, 20 and the anti-inflammatory effect was positive, and the anti-inflammatory effect was negatively correlated with the peaks 11, 1, 7, 15, 5, 3. From the analysis of the five peaks, the results showed that all the four peaks were flavonoids except for the tannic acid at peak 17. Conclusion Different extracts of P. capitatum inhibited the inflammatory response of RAW264.7 cells. The flavonoids in P. capitatum had significant anti-inflammatory activity. The results showed that quercetin, tannic acid, and hyperoside had great contribution to the anti-inflammatory effect of P. capitatum.

Tannic acid enhances cisplatin-induced apoptosis of hepatocellular carcinoma HepG2 cells by activating ATF6-CHOP pathway / 实用医学杂志

Objective To detect the activation levels of endoplasmic reticulum stress ATF6-CHOP pathway,and to investigate the molecular mechanism of tannic acid (TA) combined with cisplatin (CDDP) against hepatocellular carcinoma.Methods HepG2 cells were cultured with 180 μM TA or/and 0.9 μg/ml CDDP for 24 h or 48 h.The levels of ATF6 (ATF6α),ATF6B (ATF6β) and CHOP were analyzed by real-time fluorescence quantitative PCR (q-RT-PCR) and western blot.Results We found that after 24 h or 48 h,compared with the control group,ATF6 mRNA and protein levels,ATF6B protein level,CHOP mRNA and protein levels were significantly increased in the TA group,CDDP group or combination group (P ＜ 0.01 or P ＜ 0.05).Conclusion TA can combine with CDDP to increase the levels of endoplasmic reticulum stress ATF6-CHOP pathway,and the ATF6-CHOP pathway may be one of the molecular mechanisms synergistic anti-hepatocellular carcinoma effect of TA and CDDP on HepG2 cells.

Altered indirect hemagglutination method for easy serotyping of Haemophilus parasuis / Método de Hemaglutinação Indireta alterado para facilitar a sorotipificação de Haemophilus parasuis

Glässer's disease is an emergent bacterial disease that affects swine husbandries worldwide causing important economic losses. The aetiological agent, Haemophilus parasuis, is currently divided in fifteen serovars but an increasing number of non-typeable serovars have been reported. Indirect hemagglutination (IHA) is indicated as a serotyping method for H. parasuis. In the present study, we describe an additional step that aims to work around a possible obstacle in the original protocol that may compromise the outcome of this assay. We observed that the choice of anticoagulant for blood collection influences and/or impairs spontaneous adsorption of H. parasuis antigens on sheep red blood cells (SRBCs). However, regardless of the anticoagulant used, chemical treatment of SRBCs with tannic acid induces a stable antigen adsorption (sensitization step). The addition of 1% BSA to SRBCs washing buffer and to antisera dilution augments IHA specificity. Tannic acid treated SRBCs combined with thermo-resistant H. parasuis antigens increases the assay resolution. Thus, our results demonstrate an improvement in the technique of H. parasuis serotyping that will prove valuable to understand Glässer's disease epidemiology and to better characterize serovars involved in outbreaks.(AU)

A Doença de Glässer é uma doença bacteriana emergente que afeta a produção de suínos em todo o mundo e causa importantes perdas econômicas. O agente etiológico, Haemophilus parasuis, é atualmente dividido em quinze sorovares; no entanto, um número crescente de cepas não tipificáveis tem sido relatado. O teste de hemaglutinação indireta (IHA) tem sido utilizado para a sorotipificação de H. parasuis. Neste estudo, descrevemos uma alteração no protocolo original de IHA e que supera uma limitação específica que pode comprometer o uso geral deste ensaio. Descobrimos que o tipo de anticoagulante utilizado para coletar os eritrócitos ovinos (SRBCs) pode comprometer a adsorção espontânea dos antígenos do H. parasuis. Por outro lado, o tratamento químico dos SRBCs com ácido tânico promove uma adsorção antigênica estável (passo de sensibilização) e independente do anticoagulante utilizado. O uso de 1% de BSA durante as lavagens dos SRBCs e na diluição dos antissoros incrementa a especificidade da IHA e, a combinação dos SRBCs tratados quimicamente com antígenos de H. parasuis termo-resistentes aumentam a resolução da IHA. Nossos resultados destacam uma melhoria na principal técnica de sorotipificação de H. parasuis, que auxiliará diretamente no entendimento da epidemiologia da Doença de Glässer e na caracterização dos sorovares envolvidos em surtos da doença.(AU)

Quantification of astringency for traditional Chinese medicine based on animal preference index and electronic tongue / 中国中药杂志

Current evaluation method for astringency is mainly focused on human sensory evaluation. However, it is subjective, vague, and short of assessment indicators for objective quantification. In this paper, the quantification method for astringent intensity of traditional Chinese medicine was established based on the animal preference index and electronic tongue in vitro and in vivo. Firstly, the standard substance of astringency, tannic acid, was used for the methodology optimization and validation of two-bottle preference test. It was determined that the standard experimental animals were female rats of 140-180 g. The functional relationship between concentration of tannic acid and preference index was obtained Y= ln(1.682 6－0.441 66X), r=0.997 3. Then the typical astringent Chinese herbs Chebulae Fructus, Ardisiae Japonicae Herba, Canarii Fructus, Catechu, and Arecae Pericarpium were evaluated by the optimized method. Their corresponding concentration of tannic acid was converted by the concentration-preference index relationship through preference index. Their astringency was equivalent to 0.56, 0.29, 0.24, 0.34, 0.25 g•L⁻¹ tannic acid. Finally, the results were verified by electronic tongue. The correction analysis between Euclidean distance in PCA and preference index and concentration of tannic acid converted by samples showed a high correlation through pearson correlation analysis. The above results indicated that the method was objective, true and reliable. The method provided a reliable tool for the quantification of astringency and evaluation of taste masking effect for Chinese medicines, and also offered a new idea and model for the quantification of taste in the pharmaceutical and food fields.

Screening of ligninase-producing bacteria from south east Pahang peat swamp forest soil

Aims: Research on lignin degradation capability is previously restricted exclusively to fungal enzymes. However, recent studies had successfully revealed several soil bacterial strains that were able to produce ligninolytic enzymes. These bacterial ligninolytic enzymes were claimed to be more specific in catalysing cleavage of certain linkages between phenolic units of lignin polymers as compared to fungal enzymes. The present study focuses on screening for ligninaseproducing bacteria isolated from South East Pahang Peat Swamp Forest (SEPPSF) soil using agar-based assay. Methodology and results: Thirteen isolates used in this study, which were selected based on distinctive colony morphology from our previous isolation work, showed decolourisation zone on Azure B plates screening. The ratio of decolourisation zones were measured to the ratio of the colony size and the biggest ratio was 2.22 by isolate AR1. Only 4 out of the 13 isolates were able to grow on lignin plates. Subsequently, the 4 isolates, AR3, AR8, AR10 and AR13 were tested on M1 agar supplemented with 3 ligninolytic enzyme indicator compounds which were tannic acid (TA), guaiacol and 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) respectively. All four isolates showed growth on TA plates while only AR10 showed a clear brown coloration. An Intense reddish-brown colour formation was observed around the colony of isolates AR3 and AR10 on guaiacol plates while none exhibited green coloration around the colonies when tested on ABTS plates. Conclusions, significance and impacts of study: Isolate AR10 that was identified as Serratia sp. was perceived to be a potential ligninase-producer, though in-depth analysis has to be conducted in the future to determine the specific ligninolytic enzyme activities and characteristics. The application of different substrates is essential to investigate the ligninolytic potential and reaction of those bacterial enzymes towards different indicator compounds. This study is a preliminary endeavour concerning potential ligninolytic enzymes from bacteria as biocatalysts in various industrial processes. This is the first report on preliminary study for ligninolytic activities of soil bacteria from SEPPSF soil.

Efeito do ácido tânico sobre a toxicidade induzida pela 6-OHDA em células PC12, um modelo in vitro de doença de Parkinson

A doença de Parkinson (DP) é a segunda forma mais comum de doença neurodegenerativa nos idosos. É caracterizada pela perda progressiva de neurônios dopaminérgicos na substância negra pars compacta, levando a uma severa redução do conteúdo de dopamina no estriado. O modelo utilizando células PC12, uma linhagem celular de feocromocitoma de rato, exposta a diferentes toxinas que recapitulam mecanismos de morte celular na DP, vem sendo utilizado como screeningpara testar agentes neuroprotetores. Dentre os principais mecanismos que levam à morte celular nas doenças neurodegenerativas, incluindo a DP, a inflamação, a apoptose e o estresse oxidativo assumem um papel importante.O Ácido Tânico (AT) é um polifenol com efeito antioxidante do tipo quelante e sequestrador de radicais livres. O objetivo do presente estudo foi avaliar o possível efeito citoproterordo AT sobre a citotoxicidade induzida por 6-OHDA em células PC12...

Parkinson's disease (PD) is the second most common neurodegenerative disease in the elderly. It is characterized by progressive loss of dopaminergic neurons in the substantia nigra pars compacta, leading to a severe reduction in striataldopamine content. The PC12 cells are a cell line of rat pheochromocytoma and represents an experimental model of PD when it is exposed to toxins like the 6-hydroxydopamine (6-OHDA). This recapitulate differents mechanisms of cell death in PD and has been used as a screening test for neuroprotective agents. Inflammation, apoptosis and oxidative stress play an important role among the major mechanisms that lead to cell death in neurodegenerative diseases including PD. The tannic acid (TA) is a polyphenol that has a antioxidant effect as a chelating and sequestering free radicals. The aim of this study was to evaluate the possible citoproteror effect of TA on the cytotoxicity induced by 6-OHDA in PC12 cells...

Characterization of a multi-tolerant tannin acyl hydrolase II from Aspergillus carbonarius produced under solid-state fermentation

Background Tannases are enzymes with biotechnological potential produced mainly by microorganisms as filamentous fungi. In this context, the production and characterization of a multi-tolerant tannase from Aspergillus carbonarius is described. Results The filamentous fungus A. carbonarius produced high levels of tannase when cultivated under solid-state fermentation using green tea leaves as substrate/carbon source and tap water at a 1:1 ratio as the moisture agent for 72 h at 30°C. Two tannase activity peaks were obtained during the purification step using DEAE-Cellulose. The second peak (peak II) was purified 11-fold with 14% recovery from a Sepharose CL-6B chromatographic column. The tannase from peak II (tannase II) was characterized as a heterodimeric glycoprotein of 134.89 kDa, estimated through gel filtration, with subunits of 65 kDa and 100 kDa, estimated through SDS-PAGE, and 48% carbohydrate content. The optimal temperature and pH for tannase II activity was 60°C and 5.0, respectively. The enzyme was fully stable at temperatures ranging from 20-60°C for 120 min, and the half-life (T1/2) at 75°C was 62 min. The activation energy was 28.93 kJ/mol. After incubation at pH 5.0 for 60 min, 75% of the enzyme activity was maintained. However, enzyme activity was increased in the presence of AgNO3 and it was tolerant to solvents and detergents. Tannase II exhibited a better affinity for methyl gallate (Km = 1.42 mM) rather than for tannic acid (Km = 2.2 mM). Conclusion A. carbonarius tannase presented interesting properties as, for example, multi-tolerance, which highlight its potential for future application.

Estimation of Nuclear DNA Content in Tannin-rich Medicinal Plant Cornus officinalis by Flow Cytometry / 中草药·英文版

Objective: The amount of nuclear DNA (C-value) is a key biodiversity character that provides strong unifying elements in revealing the phylogenetic regularity and relationship between genome size and functional traits for plant resource. The estimation of C-values could primarily extend our knowledge on the genetic background and genome diversity for medicinal plants, and thereby the variation of pharmacological constituents and phylogenetic mechanism of medicinal plant taxa will be revealed. However, a large number of medicinal plants (e.g. Cornus officinalis) typically contain a series of secondary metabolites, especially tannic acid, which would significantly affect the estimation of DNA content by flow cytometry (FCM). Methodological discussions and improvement need to be made to solve this problem. Methods: Two isolation buffers LB01 and Otto 1 were selected to prepare nuclear suspension with additional treatments of pre-soaking and centrifugation combination of gradient centrifugal force and duration. The best isolation and estimation methods were determined by FCM measurement in C. officinalis. Results: The dry leaves were pre-soaked in Otto I buffer for 15 min and the Otto I nuclear suspension was centrifugated at 1.0103 g for 2 min. The results showed that debris and nuclei were better separated and the scatterplots of good quality were obtained with low coefficient of variation (CV). Contrarily, the nuclear DNA content of C. officinalis could not be accurately estimated for nuclei extracted by LB01 buffer. Finally, 2C-value and genome size of C. officinalis were first estimated as 5.92 pg and 2893 Mbp, respectively. Conclusion: The new methods proposed here are able to accurately estimate DNA content of C. officinalis, which provides valuable references for the estimation of genome size in other tannin-rich medicinal plants. © 2014 Tianjin Press of Chinese Herbal Medicines.

Tannic acid inhibits the proliferation of SW620 cell lines by downregulating TMEM16A expression / 重庆医学

Objective To investigate the effects of Tannic acid on the proliferation of human colon cancer SW 620 cell line and the mRNA and protein levels of TMEM16A .Methods Human colon cancer cell line SW620 were divided into the low dose(50 μmol/L) ,high dose(100 μmol/L) ,they were cultured for 48 h or 72 h separately .Control groups were cultured in the medium with DMSO .The proliferation of SW620 cell line was detected by the MTT assay at different time points (48 h or 72 h) .The cell cycle and apoptosis in the Tannic acid-treated groups were detected by flow cytometry .RT-PCR and Western blotting were used to de-termine the mRNA and protein levels of TMEM16A separately .All data were analyzed using the one-way analysis of variance (ANOVA) ,SNK test by the SPSS software .Results Compared with the control group ,the proliferation of SW620 cell line was significantly inhibited after the treatment by Tannic acid at the concentration of 50 μmol/L and 100 μmol/L for 48 h or 72 h(t=15 .35 ,P0 .05) ,and increased apoptotic rate when compared with control group (F=545 .3 ,P<0 .01) .The value of 3H-TdR and 3H-Leucine incorporation of SW620 cells treated with Tannic acid(100 μmol/L) 48 h and 72 h separately ,were obviously decreased as compared with that of control group (P<0 .05) .In the low dose treated groups (50 μmol/L) ,the mRNA levels in 48 h group and 72 h group were(0 .633 ± 0 .009) and(0 .621 ± 0 .011) ,and in the high dose treated groups (100 μmol/L) ,the mRNA levels in 48 h group(0 .64 ± 0 .15) and 72 h group(0 .63 ± 0 .11) ,were lower than the control group(F=7 .645 ,P< 0 .05) .After treating SW620 with Tannic acid for 48 h and 72 h ,in the low dose groups ,the protein expression of TMEM16A were(0 .68 ± 0 .14) and(0 .65 ± 0 .12) ,and in the high dose groups ,the protein expression of TMEM16A were(0 .64 ± 0 .15) and(0 .63 ± 0 .11) were decreased when compared with the control group (1 .28 ± 0 .06)(F=4 .508 ,P<0 .05) .Conclusion Tannic acid arrested SW620 at G1-S phase and decrease the mRNA and protein expression of TMEM16A .

Ameliorating effect of tannic acid on high glucose or AGEs induced oxidative stress and micro-inflammatory state in glomerular mesangial cells / 吉林大学学报(医学版)

Objective To investigate the effect of tannic acid on glomerular mesangial cells (GMC),and to clarify the mechanism of tannic acid in improving the pathological changes of diabetic nephropathy (DN)from the aspect of oxidative stress and micro-inflammation. Methods The glomerular mesangial cells were treated with glucose (30 mmol·L-1 )or advanced glycosylation end-products (AGEs)bovine serum albumin(BSA)(250 mg·L-1 )and then different concentrations of tannic acid (10,20,40 and 80μmol·L-1 )were added into the GMC.The cells cultured by normal glucose or treated with BSA were used as control groups and then the level of malonic dialdehyde (MDA), glutathione peroxidase (GSH-Px ), superoxide Dismutase (SOD ), CAT (Catalase ) activities and 8-hydroxy-2′-deoxyguanosine(8-OHdG)levels in the culture supernatant 48 h after culture were determined by colorimetry and ELISA method. The expressions of intercellular cell adhesion molecule-1 (ICAM-1 ) protein, monocyte chemotactic protein 1 (MCP-1 ) and ICAM-1 mRNA in GMC were detected by immunohistochemical staining and RT-PCR method.Results Compared with high glucose and AGEs groups,the MDA levels in tannic acid groups were reduced significantly(P<0.05);the activities of GSH-Px,SOD and CAT were increased significantly(P<0.05 or P<0.01);the 8-OHdG levels in annic acid groups were significantly reduced (P<0.05). Compared with high glucose and AGEs groups,the expressions levels of ICAM-1 protein in 40 and 80μmol· L-1 tannic acid groups were decreased (P<0.05 ). The mRNA expressions levels of MCP-1 and ICAM-1 were significantly lower than those in high glucose group (P<0.01 ).Conclusion Tannic acid could protect GMC against the damage of oxidative and inflammatory mediators,thereby delaying and improving the glomerular lesions of DN.

Antioxidative Properties of an Extract of Hygrocybe conica, a Wild Edible Mushroom

Hygrocybe conica (HC), a wild mushroom commonly consumed by the indigenous people (Orang Asti) in Peninsular Malaysia, was assessed for its antioxidant content. Methods: The HC mushroom was extracted using distilled water and the crude extract partitioned using different solvents and open column chromatography to evaluate its potential antioxidant properties. The mushroom extract was partitioned using liquid-liquid extraction into the hexane (F1), chloroform (F2), butanol (F3) and formic acid (F4) fractions. Based on solvent polarity, the water extract of the mushroom was fractionated into non-polar (FI), semi-polar (FII), and polar fractions (FIII) using open column chromato�graphy. Antioxidant capacities were determined using DPPH, ABTS, and ferric reducing antioxidant power (FRAP) assays while Folin-Ciocalteu reagent assay was used to determine total phenolic content (TPC). Results: The HC extract had the highest TPC and DPPH scavenging capacity compared to its extract fractions. TE values (ABTS assay) of F2 and F4 were not significantly higher than the HC extract. Among the extract fractions of different polarities, FIII had the highest antioxidant capacities (DPPH and FRAP) compared to FI and FII while FRAP values of these fractions were not significantly lower than the FRAP value of HC extract. The HC extract had significantly lower antioxidant capacity than antioxidant standards (ascorbic acid and BHA). Tannic acid as the main bioactive component in HC mushroom was detected using HPLC method. The presence of phenolics in HC extract was also confirmed using TLC. Conclusion: Due to the presence of potent phenolic components, the mycelia of HC could be consumed for potential antioxidative benefits.