Antimutagenic activity against trp-P-1 of the edible Thai plant, Oroxylum indicum vent.

A methanolic extract of Oroxylum indicum strongly inhibited the mutagenicity of Trp-P-1 in an Ames test. The major antimutagenic constituent was identified as baicalein with an IC50 value of 2.78+/-0.15 microM. The potent antimutagenicity of the extract was correlated with the high content (3.95+/-0.43%, dry weight) of baicalein. Baicalein acted as a desmutagen since it inhibited the N-hydroxylation of Trp-P-2.     

Structural Elucidation of Rotihibin B by Tandem Mass Spectrometry

The mutagenicity and desmutagenicity of extracts of soybeans heated at 225 ± 5°C were investigated by the Ames test. The soybeans were refluxed in water, methanol, or diethylether for 2h. The aqueous and methanol extracts (2–4 mg/plate) of the heated soybeans exhibited strong desmutagenic activity of 43–92% against heterocyclic amines (Trp-P-1, Glu-P-2, IQ, MeIQx, PhIP), while no mutagenicity was observed. The desmutagenicity of the heated soybean extracts remained even after denaturation by 0.1 N HCI in vitro and absorption by the rat small intestine. The desmutagenic mechanism for heated soybeans was evaluated, and it was verified that the soybean extract exhibited its desmutagenicity by blocking the mutagenicity of activated Trp-P-1, and not by inhibiting the S9 enzyme system.     

Preparation of baicalein from baicalin using a baicalin-β-D-glucuronidase from Aspergillus niger b.48 strain

Baicalin-β-d-glucuronidase was produced from a culture of Aspergillus niger b.48 strain using Scutellaria root extract as an enzyme inducer, purified and characterized. The enzyme’s molecular weight was approximately 45 kDa; its optimal operating temperature and pH were 50 °C and 5.0, respectively. The enzyme specifically hydrolysed 7-O-β-d-glucuronide of baicalin into baicalein, weakly hydrolysed β-d-glucuronide of p-nitrophenyl-β-d-glucuronide and p-phenolphthalein-β-d-glucuronide, but did not hydrolyse β-d-glucuronide of glycyrrhizin. The Michaelis constant (Km) was 21.74 mM; Vmax was 11.63 mM/h. Common metallic ions almost did not effect enzyme activity; greater than 10 mM/L Cu²⁺ and greater 50 mM/L Fe³⁺ ion strongly inhibited enzyme activity. The use of pure enzyme in baicalin conversion to baicalein was costly, the crude baicalin-β-d-glucuronidase from A. niger b.48 strain was used in the preparation of baicalein from baicalin to keep costs low. The optimum conditions for baicalein production from crude enzyme reaction were 1% baicalin reacting for 20 h–24 h at pH 5.0 and 50 °C. Here, 10.7 g baicalein was obtained from 20 g baicalin using the crude enzyme, and the molar yield was 88.4 %. Therefore, active baicalein was successfully produced at low cost from baicalin using a non-transgenic crude enzyme from A. niger b.48.     

Tyrosinase and α-glucosidase inhibitory potential of compounds isolated from Quercus coccifera bark: In vitro and in silico perspectives

Bark of Quercus coccifera is widely used in folk medicine. We tested tyrosinase and α-glucosidase inhibitory effects of Q. coccifera bark extract and isolated compounds from it. The extract inhibited tyrosinase with an IC₅₀ value of 75.13 ± 0.44 µg/mL. Among the isolated compounds, polydatin (6) showed potent tyrosinase inhibition compared to the positive control, kojic acid, with an IC₅₀ value of 4.05 ± 0.30 µg/mL. The Q. coccifera extract also inhibited α-glucosidase significantly with an IC₅₀ value of 3.26 ± 0.08 µg/mL. (-)-8-Chlorocatechin (5) was the most potent isolate, also more potent than the positive control, acarbose, with an IC₅₀ value of 43.60 ± 0.67 µg/mL. According to the kinetic analysis, 6 was a noncompetitive and 5 was a competitive inhibitor of tyrosinase, and 5 was a noncompetitive α-glucosidase inhibitor. In the light of these findings, we performed in silico molecular docking studies for 5 and 6 with QM/MM optimizations to predict their tyrosinase inhibition mechanisms at molecular level and search for correlations with the in vitro results. We found that the ionized form of 5 (5i) showed higher affinity and more stable binding to tyrosinase catalytic site than its neutral form, while 6 bound to the predicted allosteric sites of the enzyme better than the catalytic site.     

In vitro and in silico characterization of angiogenic inhibitors from <Emphasis Type="Italic">Sophora interrupta</Emphasis>

Sophora interrupta Bedd, (Fabaceae) is used in Indian folk medicine to treat cancer. Angiogenesis is one of the crucial characteristics of cancer metastasis and is regulated by vascular endothelial growth factor (VEGF). In this study, we examined the antiangiogenic properties of the root ethyl acetate extract of Sophora interrupta by various methods. In vitro antioxidant activity (100–600 μg/ml) of S. interrupta ethyl acetate (SEA) extract was evaluated by DPPH and ABTS, anti-inflammatory activity (50, 100 and 150 μg/ml) by estimating nitric oxide (NO) levels, anti-angiogenic activity (200 and 500 μg/ml) was validated by chorio allantoic membrane (CAM) assay and in silico molecular dynamic (MD) simulations analyses (25 ns) were performed to identify the anti-angiogenic compounds extracted from root extract. The antioxidative activity of SEA extract at IC₅₀ (200?±?0.6 μg/mL) is equal to that of ascorbic acid at IC₅₀ (50?±?0.6 μg/mL), and the anti-inflammatory activity of SEA extract at IC₅₀ (150?±?0.2 μg/mL) was inhibited significantly by nitric oxide (NO) production. The SEA extract significantly reduced the sprouting of new blood vessels at ID₅₀ 500?±?0.13 μg/mL in the CAM assay. Gas chromatography–mass spectrometry analysis of the SEA extract detected 34 secondary metabolites, of which 6a,12a-dihydro-6H-(1,3)dioxolo(5,6)benzofuro(3,2-c)chromen-3-ol (maackiain) and funiculosin formed strong hydrogen bond interactions with Lys 920, Thr 916 and Cys 919 (2H), as well as Glu 917 of VEGFR2, and these interactions were similar to those of the anti-angiogenic compound axitinib. Significant findings in all the assays performed indicate that SEA extract has potential anti-angiogenic compounds that may interfere with VEGF-induced cancer malignancy.     

Iron-chlorophyllin-mediated conversion of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)) into its nitroso derivative

Early work from our laboratory has shown that the mutagenicity of heterocyclic amines in Salmonella can be inhibited by hemin and chlorophyllins. We have speculated that the inhibition is a result of complex formation between heterocyclic amines and the pigments, and the speculation has been given a line of experimental evidence. We have now found that ferric-chlorophyllin (Fe-chlorophyllin) can modify the mutagenicity of 3-hydroxyamino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NHOH)), a metabolically activated form of 3-amino-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2). The mutagenicity of Trp-P-2(NHOH)) in Salmonella typhimurium TA 98 (without S9) was strongly inhibited by an addition of an equimolar Fe-chlorophyllin in the pre-incubation mixture. Fe-chlorophyllin also inhibited the mutagenicity of 2-hydroxyamino-6-methyldipyrido[1,2-a:3′,2′-d] imidazole (Glu-P-1(NHOH)). A rapid change in the UV spectrum of a mixture of Trp-P-2(NHOH) and Fe-chlorophyllin was observed. Analysis by high performance liquid chromatography showed that Trp-P-2(NHOH) was converted into 3-nitroso-1-methyl-5H-pyrido[4,3-b]indole (Trp-P-2(NO)), the mutagenic potency of which is a quarter of that of Trp-P-2(NHOH). Furthermore, the mutagenicity of Trp-P-2(NO), in turn, was inhibited by Fe-chlorophyllin. We conclude that the suppression of the mutagenicity of Trp-P-2(NHOH) is ascribable to the oxidative function of Fe-chlorophyllin, coupled with its ability to form complex formation with the planar surface of the heterocyclic amine molecules.     

Antimutagenicity of sweetpotato (Ipomoea batatas) roots

Antimutagenicity of the water extracts prepared from the storage roots of four varieties of sweetpotato with different flesh colors was investigated using Salmonella typhimurium TA 98. The extract from the whole roots of the purple-colored Ayamurasaki variety effectively decreased the reverse mutation induced not only by Trp-P-1, Trp-P-2, IQ, B[a]P, and 4-NQO but also by dimethyl sulfoxide extracts of grilled beef. Comparison of the inhibitory activity of the extracts from the normal Ayamurasaki and its anthocyanin-deficient mutant one suggested that the anthocyanin pigment in the flesh decreases the mutagenic activity of the mutagens as heterocyclic amines. Two anthocyanin pigments purified from purple-colored sweet-potato, 3-(6,6'-caffeylferulylsophoroside)-5-glucoside of cyanidin (YGM-3) and peonidin (YGM-6) effectively inhibited the reverse mutation induced by heterocyclic amines, Trp-P-1, Trp-P-2, and IQ in the presence of rat liver microsomal activation systems.     

Macerating Activity of endo-Polygalacturonase Produced by Saccharomyces fragilis

The effect was examined of aqueous dialyzates from 16 kinds of vegetables and fruits on the mutagenicity of some mutagens toward Salmonella typhimurium TA 100. Each dialyzate inhibited the mutagenicity of Trp-P-2, and the antimutagenicity was retained even after heating at 100°C for 20 min. Dialyzates of burdock, eggplant, spinach and apple also inhibited the mutagenicity of Trp-P-l, benzo[a]pyrene, sterigmatocystin, aflatoxin Bl, 2-(2-furyl)-3-(5-nitro-2-furyl)-acrylamide and N-methyl-N′-nitroso-N-nitrosoguanidine. The dialyzates of apple reacted with S9 mix and Trp-P-2. A Sepharose CL 6B gel filtration study of the dialyzates of apple indicated that the antimutagenic activity of these dialyzates on Trp-P-2 and AF-2 was mainly detectable in the polyphenol-rich fractions.     

Neuroprotective Effects of Hydroalcoholic Extract of Ocimum sanctum Against H2O2 Induced Neuronal Cell Damage in SH-SY5Y Cells via Its Antioxidative Defence Mechanism

Oxidative stress mediates the cell damage in several ailments including neurodegenerative conditions. Ocimum sanctum is widely used in Indian ayurvedic medications to cure various ailments. The present study was carried out to investigate the antioxidant activity and neuroprotective effects of hydroalcoholic extract of O. sanctum (OSE) on hydrogen peroxide (H₂O₂)-induced oxidative challenge in SH-SY5Y human neuronal cells. The extract exhibited strong antioxidant activity against DPPH, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical and hydroxyl radicals with IC₅₀ values of 395 ± 16.2, 241 ± 11.5 and 188.6 ± 12.2 μg/ml respectively, which could be due to high amount of polyphenols and flavonoids. The observed data demonstrates 41.5 % cell survival with 100 μM H₂O₂ challenge for 24 h, which was restored to 73 % by pre-treatment with OSE for 2 h. It also decreased the lactate dehydrogenase leakage and preserved the cellular morphology. Similarly OSE inhibited lipid peroxidation, DNA damage, reactive oxygen species generation and depolarization of mitochondrial membrane. The extract restored superoxide dismutase and catalase enzyme/protein levels and further downregulated HSP-70 over-expression. These findings suggest that OSE ameliorates H₂O₂ induced neuronal damage via its antioxidant defence mechanism and might be used to treat oxidative stress mediated neuronal disorders.     

Differential effects of Oroxylum indicum bark extracts: antioxidant, antimicrobial, cytotoxic and apoptotic study

Stem bark of Oroxylum indicum (L) (SBOI) is used by ethnic communities of North East India as health tonic and in treating diseases of humans and animals. The objective of this research was to carry out a detailed investigation including total phenolic and flavonoid content, antioxidant, antimicrobial, cytotoxic and apoptotic activities of different solvent extracts of SBOI and to establish correlation between some parameters. Among petroleum ether (PE), dichloromethane and methanol (MeOH) extract of SBOI, MeOH extract contained the highest amount of total phenolic (320.7 ± 34.6 mg Gallic acid equivalent/g extract) and flavonoid (346.6 ± 15.2 mg Quercetin equivalent/g extract) content. In vitro antioxidant activity (IC₅₀ 22.7 μg/ml) was highest in MeOH extract (p > 0.05) and also a significant inverse correlation was observed between phenolic (r = 0.886)/flavonoid (r = 0.764) content and corresponding DPPH IC₅₀. Only MeOH extract inhibited both bacteria and fungi. Although, individual extract showed cytotoxicity on HeLa cells with characteristic features of apoptosis, PE extract caused maximum cytotoxicity (IC₅₀ of 112.3 μg/ml, p < 0.05) and apoptotic activity (33.2 % sub-G0/G1 population) on HeLa cells. But, there was a significant non-inverse correlation of the MTT IC₅₀ with total phenolic (r = 0.812, p < 0.05)/flavonoid (r = 0.998, p < 0.05) content in the three solvent extracts. TLC analysis showed three unique compounds in PE extract which may have a role in apoptosis mediated cytotoxicity. These results called for futher chemical characterisation of MeOH and PE extract of SBOI for specific bioactivity.     

The Piperaceae Amides I: Structure of Pipercide,A New Insecticidal Amide from Piper nigrum L.

It was evidenced that mutagenic principles in tryptophan pyrolysate, 3-amino-1,4-dimethyl-5H pyrido(4,3-b) indole and 3-amino-1-methyl-5H pyrido(4,3-b) indole (abbreviated as Trp-P-1 and Trp-P-2, respectively) bind to DNA without activation by rat liver microsomes. The bindings of Trp-P-1 and Trp-P-2 were not random and did not introduce strand scissions into DNA. Trp-P-1 bound more easily than Trp-P-2. The bindings of these mutagenic principles to DNA were concluded by using negatively superhelical simian virus 40 (SV40) DNA from following experimental data. (1) The intensity of ethidium bromide (EtBr)-DNA fluorescence by illumination with UV light and the electrophoretic mobility of superhelical DNA in agarose gel decreased as a function of the amounts of Trp-P-1 and Trp-P-2. (2) In vitro RNA synthesis catalyzed by Escherichia coli DNA-dependent RNA polymerase and nick-translation catalyzed by Escherichia coli DNA polymerase I (Kornberg enzyme) were inhibited significantly on DNA treated with Trp-P-1 and Trp-P-2. (3) The negative superhelicity of SV40 DNA introduces unpaired regions into DNA. These regions can be cleaved by single-strand-specific S1 endonuclease to generate unit length linear duplex molecules. It was found that this S1-sensitivity of DNA decreased by treatment with Trp-P-1. (4) The cleavage patterns of Trp-P-1 treated DNA with five restriction endonucleases were investigated. The protection of the cleavage site by the drug was observed against HincII, HindIII and EcoRII, whereas not against HaeIII and HinfI. These results show that the binding of Trp-P-1 to DNA is not random. Identical results were also obtained in Trp-P-2.

However, the bindings of Trp-P-1 and Trp-P-2 were not so tight, and phenol extraction of the complex dissociated these drugs from DNA.     

Scavenging Effect of Various Tea Extracts on Superoxide Derived from the Metabolism of Mutagens

We determine the superoxide formed in the self-degradation of mutagens activated by cytochrome enzymes and evaluated the scavenging effect of various tea extracts. Benzo[a]pyrene (B[a]P) and 2-amino-6-methyldipyrido(1,2-a:3′,2′-d)imidazole (Glu-P-1) each produced a large amount of superoxide after activation by cytochrome enzymes. However, 2-amino-3-methyl-imidazo(4,5-f)quinoline (IQ), 3-amino-1,4-dimethyl-5H-pyridol(4,3-b)indole (Trp-P-1) and alfatoxin B₁ (AFB₁) failed to generate a significant amount of superoxide. The addition of a tea extract to the reaction system marked inhibited the derivation of superoxide from Glu-P-1. However, the tea extracts showed weaker inhibition of the B[a]P-mediated formation of superoxide. Among the four teas tested, the oolong tea extract tended to exhibit the strongest inhibitory effect. Our results suggest that the chemopreventive efficacy of a tea extract is partly associated with its antioxidative activity.     

In Vitro α-Glucosidase Inhibition and Antioxidative Potential of an Endophyte Species (Streptomyces sp. Loyola UGC) Isolated from Datura stramonium L

Endophytic actinomycetes isolated from Datura stramonium L. was evaluated for its effects against in vitro α-glucosidase inhibition, antioxidant, and free radical scavenging activities. Based on microbial cultural characteristic and 16S rRNA sequencing, it was identified as Streptomyces sp. loyola UGC. The methanolic extract of endophytic actinomycetes (MeEA) shows remarkable inhibition of α-glucosidase (IC₅₀ 730.21 ± 1.33 μg/ml), scavenging activity on 2,2-diphenyl-picrylhydrazyl (DPPH) (IC₅₀ 435.31 ± 1.79 μg/ml), hydroxyl radical (IC₅₀ 350.21 ± 1.02 μg/ml), nitric oxide scavenging (IC₅₀ 800.12 ± 1.05 μg/ml), superoxide anion radical (IC₅₀ 220.31 ± 1.47 μg/ml), as well as a high and dose-dependent reducing power. The MeEA also showed a strong suppressive effect on rat liver lipid peroxidation. Antioxidants of β-carotene linoleate model system revels significantly lower than BHA. The total phenolic content of the extract was 176 mg of catechol equivalents/gram extract. Perusal of this study indicates MeEA can be used as natural resource of α-glucosidase inhibitor and antioxidants.     

Total phenolic and flavonoid contents and antioxidant activity of ginger (Zingiber officinale Rosc.) rhizome,callus and callus treated with some elicitors

The present study was aimed at determining total phenolic and flavonoid contents and studying the antioxidant activity of ginger (Zingiber officinale Rosc.) rhizome and callus, 6-gingerol and 6-shogaol and callus treated with elicitors. Petroleum ether (PE) and chloroform: methanol (1:1, v/v) (CM) extracts were prepared by maceration. Highest total phenolic content was obtained from the CM extract (60.34?±?0.43?mg gallic acid/g) of rhizome while callus showed lower content detected in the CM extract (33.6?±?0.07?mg gallic acid/g). Flavonoids were only detected in rhizome (CM extract 40.25?±?0.21?mg quercetin/g). Both rhizome extracts exhibited good antioxidant activity with higher activity recorded in PE extract (IC₅₀ value 8.29?±?1.73?μg/mL). Callus extracts revealed lower antioxidant activity (IC₅₀ value 1265.49?±?59.9?μg/mL obtained from CM extract). 6-gingerol and 6-shogaol displayed high antioxidant activity in both assays with IC₅₀ 4.85?+?0.58_DPPH and 5.35?±?0.33_ABTS μg/mL for the former and IC₅₀ 7.61?±?0.81_DPPH and IC₅₀ 7.05?±?0.23_ABTS μg/mL for the latter. Treatment of callus with elicitors showed significant (p?<?0.05) effects in enhancing phenolic content and related antioxidant activity. The highest significant increase in phenolic content (37% and 34%) and antioxidant activity in DPPH assay (34% and 30%) was observed in callus treated with 100?mg/L yeast extract and 50?mg/L salicylic acid respectively. Therefore, studying the effect of the elicitation of ginger cultured tissues in phenolic accumulation would be of immense importance for pharmacological, cosmetic and agronomic industries.     

A potential bio‐control agent from baical skullcap root against listeriosis via the inhibition of sortase A and listeriolysin O

Listeria monocytogenes (LM) is a classical model intracellular pathogen and the leading cause of listeriosis, which has long been a global public health issue. The successful infection of LM is related to a series of virulence factors, such as the transpeptidase enzyme sortase A (SrtA) and listeriolysin O (LLO), which are crucial for bacterial internalization and escape from phagosomes respectively. It is speculated that targeting multiple virulence factors may be due to a synergistic effect on listeriosis therapy. In this study, an active flavonoids component of Scutellaria baicalensis Georgi, baicalein, was found to potently block both listerial SrtA catalyzed activity and LLO hemolytic activity within 16 μg/mL. After pretreatment with baicalein, 86.30 (±11.35) % of LM failed to associate with Caco‐2 cells compared to the LM without preincubation (regarded as 100% internalization). Furthermore, baicalein addition may aid in bacterial degradation and clearance in macrophagocytes. During a 5 h observation, LM in cells incubated with baicalein showed significantly decreased vacuole escapes and sluggish endocellular growth. In addition, baicalein directly prevented LM‐induced cells injury and mice fatality (survival rate from 10.00% to 54.55% in 4 days post‐intraperitoneal injection). Taken together, as an antagonist against SrtA and LLO, baicalein can be further developed into a biotherapeutic agent for listeriosis.     

LC-ESI-MS/MS Chemical Characterization,Antioxidant and Antidiabetic Properties of Propolis Extracted with Organic Solvents from Eastern Anatolia Region

Geographic conditions (altitude, climate, and local flora) lead to significant differences in the chemical composition of propolis. Therefore, more research is needed for propolis in different geographical regions. So, the aim of this study was to evaluate the phenolic profile, total phenolic content, antioxidant, and antidiabetic properties of Pülümür propolis from Turkey. Methanol (MeOH), chloroform (CHCl₃), and hexane extracts of propolis were analyzed. LC-ESI-MS/MS analysis of the extracts showed that the most abundant phenolic compound is caffeic acid in the MeOH extract (2943.12±11.12 μg phenolics/g extract), while on the other hand, CHCl₃ extract had the highest total phenolic content (125.75±1.02 mg GAE/g extract). Antioxidant activity was measured using ABTS and DPPH assays, whereas CHCl₃ extract (IC₅₀=6.35±0.11 and 28.84±0.10 μg/mL, respectively) and MeOH extracts (IC₅₀=5.04±0.07 and 28.80±0.09 μg/mL, respectively) showed relatively high antioxidant activity. The MeOH extract showed better antidiabetic activity than the standard compound, acarbose (IC₅₀=0.544 and 0.805 mg/mL, respectively).     

Cholinesterase inhibitory activity of tinosporide and 8-hydroxytinosporide isolated from Tinospora cordifolia: In vitro and in silico studies targeting management of Alzheimer’s disease

Tinosporide and 8-hydroxytinosporide isolated from Tinospora cordifolia were evaluated for acetylcholinesterase (AChE) and butylcholinesterase (BuChE) inhibitory activities. The structure of the compound was confirmed by spectroscopic analysis, whereas cholinesterase inhibition was investigated by Ellman method using donepezil as standard drug and the data were presented as IC₅₀ (μg/ml ± SEM). Furthermore, donepezil, tinosporide and 8-hydroxytinosporide were executed for docking analysis. The results from the isolated compounds TC-16R confirmed as tinosporide promisingly inhibited AChE with IC₅₀ value of 13.45 ± 0.144, whereas TC-19R confirmed as 8-hydroxytinosporide moderately inhibited AChE with IC₅₀ value of 46.71 ± 0.511. In case of BuChE inhibition, the IC₅₀ values were found to be 408.50 ± 17.197 and 317.26 ± 6.918 for tinosporide and 8-hydroxytinosporide, respectively. The in silico studies revealed that the ligand tinosporide fit with the binding sites and inhibited AChE. Overall, the study findings suggested that tinosporide would be a complementary noble molecule of donepezil which is correlated with its pharmacological activity through in vitro studies, while 8-hydroxytinosporide modestly inhibited BuChE and the results are very close to the standard donepezil.     

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average V_max of 0.86±0.01 μmol min^–1mL^–1 and K_M of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (K_i=64.0±0.53 nM), competitively inhibited by bacitracin (K_i=0.16±0.01 mM) and bestatin (K_i=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC₅₀ value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn²⁺ and Ca²⁺, for which a mixed inhibition mechanism was observed (K_i values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca²⁺ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn²⁺ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.