Biochemical and Structural Characterization of a Novel Bacterial Tannase From Lachnospiraceae bacterium in Ruminant Gastrointestinal Tract

Tannases are a family of esterases that catalyze the hydrolysis of ester and depside bonds present in hydrolyzable tannins to release gallic acid. Here, a novel tannase from Lachnospiraceae bacterium (TanALb) was characterized. The recombinant TanALb exhibited maximal activity at pH 7.0 and 50°C, and it maintained more than 70% relative activity from 30°C to 55°C. The activity of TanALb was enhanced by Mg2+ and Ca2+, and was dramatically reduced by Cu2+ and Mn2+. TanALb is capable of degrading esters of phenolic acids with long-chain alcohols, such as lauryl gallate as well as tannic acid. The Km value and catalytic efficiency (kcat /Km) of TanALb toward five substrates showed that tannic acid (TA) was the favorite substrate. Homology modeling and structural analysis indicated that TanALb contains an insertion loop (residues 341–450). Based on the moleculer docking and molecular dynamics (MD) simulation, this loop was observed as a flap-like lid to interact with bulk substrates such as tannic acid. TanALb is a novel bacterial tannase, and the characteristics of this enzyme make it potentially interesting for industrial use.

In Vitro Anti-HIV-1 Reverse Transcriptase and Integrase Properties of Punica granatum L. Leaves, Bark, and Peel Extracts and Their Main Compounds

In a search for natural compounds with anti-HIV-1 activity, we studied the effect of the ethanolic extract obtained from leaves, bark, and peels of Punica granatum L. for the inhibition of the HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) and integrase (IN) LEDGF-dependent activities. The chemical analyses led to the detection of compounds belonging mainly to the phenolic and flavonoid chemical classes. Ellagic acid, flavones, and triterpenoid molecules were identified in leaves. The bark and peels were characterized by the presence of hydrolyzable tannins, such as punicalins and punicalagins, together with ellagic acid. Among the isolated compounds, the hydrolyzable tannins and ellagic acid showed a very high inhibition (IC50 values ranging from 0.12 to 1.4 µM and 0.065 to 0.09 µM of the RNase H and IN activities, respectively). Of the flavonoids, luteolin and apigenin were found to be able to inhibit RNase H and IN functions (IC50 values in the 3.7–22 μM range), whereas luteolin 7-O-glucoside showed selective activity for HIV-1 IN. In contrast, betulinic acid, ursolic acid, and oleanolic acid were selective for the HIV-1 RNase H activity. Our results strongly support the potential of non-edible P. granatum organs as a valuable source of anti-HIV-1 compounds.

USE OF ULTRASOUND FOR EXTRACTION OF BIOLOGICALLY ACTIVE SUBSTANCES FROM TAMARIX HISPI-DA WILLD.

The extractions of biologically active substances from aerial part of Tamarix hispida Willd. over ultrasound have been studied. The results showed that the output of extractive substances using ultrasound was increased to 37.34% under the following conditions: 10% ethyl alcohol in a raw-extractant ratio (1 : 6) for 25 minutes at a 20–25 °С temperature and an ultrasound frequency of 35 kHz. The extraction was compared with maceration by yield and chemical composition. As a result of optimization, the output of components from the raw material is increased by 3–4 times at ultrasound frequency from 20 to 35 kHz and extraction time is significantly reduced from 48 hours to 30 minutes. It was found that the major substances were hydrolyzable tannins, the content of which are 10% (ultrasonic extraction) and 8% (maceration), respectively. Using UV-spectrometry, it was confirmed that the appearance of a characteristic maximum in the spectrum of the substance to be analyzed corresponds to the area of absorption of gallic acid and its derivatives, which indicates the content of these in the structure of hydrolyzable tannins. The substance shown significant antidiabetic activity (in vitro) at IC 50 = 3.94±0.14 μg/ml.

Effects of hydrolyzable tannin extract obtained from sweet chestnut wood (Castanea sativa Mill.) against bacteria causing subclinical mastitis in Thai Friesian dairy cows

Background and Aim: Hydrolyzable tannins are an important group of secondary plant metabolites, which are known for antimicrobial activity. This study aimed to assess the efficiency with which a hydrolyzable tannin extract from sweet chestnut wood (Castanea sativa Mill.) could inhibit mastitis-causing bacteria in vitro. Materials and Methods: The negative control used was sterile water, and the positive controls were penicillin and gentamicin. The treatments included five concentrations of hydrolyzable tannins (63, 190, 313, 630, and 940 mg/mL). In cows with subclinical mastitis, the bacteria causing the disease were isolated and identified. Then, the antibacterial activity of the hydrolyzable tannin extract was assessed by the disk diffusion method, by determining the minimum inhibitory concentration (MIC) and by determining the minimum bactericidal concentration (MBC). Results: Penicillin inhibited (p<0.01) the growth of Staphylococcus aureus, Streptococcus uberis, and Pseudomonas aeruginosa but could not inhibit (p>0.05) the growth of Streptococcus agalactiae, Escherichia coli, and Klebsiella pneumoniae. However, gentamicin and hydrolyzable tannins could inhibit (p<0.01) all isolated bacteria. Increasing the concentration of hydrolyzable tannin extract resulted in a quadratic increase in the inhibition zone diameter of S. aureus and S. agalactiae and a linear increase in the inhibition zone diameter of E. coli, K. pneumoniae, and P. aeruginosa. In addition, 630 and 940 mg/mL of hydrolyzable tannin extract showed the highest antibacterial activity against S. agalactiae and E. coli (p<0.01), while 940 mg/mL concentration had the highest antibacterial activity against K. pneumoniae (p<0.01). The MIC and MBC of the extract were 27.3-190 mg/mL and 58.8-235 mg/mL, respectively, with the MBC: MIC ratio being 2:1. Conclusion: The antimicrobial activity of the hydrolyzable tannin extract against subclinical mastitis bacteria was comparable to the antibiotics (positive controls) at concentrations over 630 mg/mL. Although these in vitro findings are promising, further research is needed to determine whether hydrolyzable tannins could be used to control or prevent subclinical mastitis in dairy cows.

Chemical Composition, Antioxidant Activity, and Sensory Characterization of Commercial Pomegranate Juices

We undertook a qualitative and quantitative assessment of the bioactive compounds, volatile substances, sensory profile, and antioxidant activity of eight different commercial pomegranate juices (PJs) differing by cultivation area, processing (from concentrate (CPJ) or not (NCPJ)), and microbial stabilization. Punicalins were the main ellagitannins, whereas the predominant anthocyanin was cyanidin 3,5-diglucoside, followed by cyanidin 3-glucoside. Total phenols, tannins, hydrolyzable tannins, and anthocyanins in the investigated juices ranged from 1379.9 to 3748.8 mg gallic acid equivalent (GAE)/L, 394.8 to 895.2 mg GAE/L, 150.8 to 2374.2 mg ellagic acid/L, and 0 to 281 mg cyanidin 3-glucoside/L, respectively. Antioxidant activity, determined by DPPH˙, FRAP, and ABTS, was positively correlated with the total phenolic compounds and hydrolyzable tannins. Alcohols, acids, and furans were the volatile groups that best described the differences between juices. The most abundant volatile compound was 3-Furfural (8.6–879.9 µg/L) in those juices subjected to the concentration process and to high-temperature short-time (HTST) treatment, whereas it was not present in juice subjected to high-pressure processing (HPP). Processing also affected the juices’ sensory attributes: NCPJs were characterized by ‘red–purple’, ‘pomegranate fruit’, ‘fresh fruit’, and ‘overall intensity’ notes, while in CPJs these notes were not revealed or were masked by those related to heat treatment. Our results provide useful information on the importance of extraction methods and on the overall quality of PJ for the food industry.

Punicalagin in Cancer Prevention—Via Signaling Pathways Targeting

The extract of pomegranate (Punica granatum) has been applied in medicine since ancient times due to its broad-spectrum health-beneficial properties. It is a rich source of hydrolyzable tannins and anthocyanins, exhibiting strong antioxidative, anti-inflammatory, and antineoplastic properties. Anticancer activities of pomegranate with reference to modulated signaling pathways in various cancer diseases have been recently reviewed. However, less is known about punicalagin (Pug), a prevailing compound in pomegranate, seemingly responsible for its most beneficial properties. In this review, the newest data derived from recent scientific reports addressing Pug impact on neoplastic cells are summarized and discussed. Its attenuating effect on signaling circuits promoting cancer growth and invasion is depicted. The Pug-induced redirection of signal-transduction pathways from survival and proliferation into cell-cycle arrest, apoptosis, senescence, and autophagy (thus compromising neoplastic progression) is delineated. Considerations presented in this review are based mainly on data obtained from in vitro cell line models and concern the influence of Pug on human cervical, ovarian, breast, lung, thyroid, colorectal, central nervous system, bone, as well as other cancer types.

Ferroptosis-Inhibitory Difference between Chebulagic Acid and Chebulinic Acid Indicates Beneficial Role of HHDP

The search for a safe and effective inhibitor of ferroptosis, a recently described cell death pathway, has attracted increasing interest from scientists. Two hydrolyzable tannins, chebulagic acid and chebulinic acid, were selected for the study. Their optimized conformations were calculated using computational chemistry at the B3LYP-D3(BJ)/6-31G and B3LYP-D3(BJ)/6-311 + G(d,p) levels. The results suggested that (1) chebulagic acid presented a chair conformation, while chebulinic acid presented a skew-boat conformation; (2) the formation of chebulagic acid requires 762.1729 kcal/mol more molecular energy than chebulinic acid; and (3) the 3,6-HHDP (hexahydroxydiphenoyl) moiety was shown to be in an (R)- absolute stereoconfiguration. Subsequently, the ferroptosis inhibition of both tannins was determined using a erastin-treated bone marrow-derived mesenchymal stem cells (bmMSCs) model and compared to that of ferrostatin-1 (Fer-1). The relative inhibitory levels decreased in the following order: Fer-1 > chebulagic acid > chebulinic acid, as also revealed by the in vitro antioxidant assays. The UHPLC–ESI-Q-TOF-MS analysis suggested that, when treated with 16-(2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy free radicals, Fer-1 generated dimeric products, whereas the two acids did not. In conclusion, two hydrolyzable tannins, chebulagic acid and chebulinic acid, can act as natural ferroptosis inhibitors. Their ferroptosis inhibition is mediated by regular antioxidant pathways (ROS scavenging and iron chelation), rather than the redox-based catalytic recycling pathway exhibited by Fer-1. Through antioxidant pathways, the HHDP moiety in chebulagic acid enables ferroptosis-inhibitory action of hydrolyzable tannins.