Boron trifluoride etherate on silica-A modified lewis acid reagent (VII). Antitumor activity of cannabigerol against human oral epitheloid carcinoma cells

Geraniol (1), olivetol (2), cannabinoids (3 and4) and 5-fluorouracil (5) were tested for their growth inhibitory effects against human oral epitheloid carcinoma cell lines (KB) and NIH 3T3 fibroblasts using two different 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay and sulforhodamine B protein (SRB) assay. Cannabigerol (3) exhibited the highest growth-inhibitory activity against the cancer cell lines.     

Screening of promising chemotherapeutic candidates from plants against human adult T-cell leukemia/lymphoma (III)

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature peripheral T lymphocytes caused by human T-cell lymphotropic virus type I (HTLV-I). In our previous paper, 214 extracts from 162 plants were screened to elucidate the anti-proliferative principles against HTLV-I-infected T-cell lines. In this study, 245 extracts from 182 plants belonging to 61 families were further tested against two HTLV-I-infected T-cell lines (MT-1 and MT-2). Potent anti-proliferative effects were exhibited against MT-1 and MT-2 cells by 52 and 60 of the 245 extracts tested, respectively. Of these, two extracts showed strong inhibitory activity (EC₅₀ values 0.1–1 μg/mL; +++) against both cells, 7 extracts showed moderate inhibitory activity (EC₅₀ values 1–10 μg/mL; ++), and 43 extracts showed weak inhibitory activity (EC₅₀ values 10–100 μg/mL; +), whereas the remaining extracts did not show any activity (EC₅₀ values >100 μg/mL; –) against MT-1 cells. On the other hand, 10 extracts showed moderate inhibitory activit and, 48 extracts showed weak inhibitory activity, whereas the remaining extracts did not show any activity against MT-2 cells. Extracts from the aerial parts of Annona reticulata and A. squamosa showed the most potent inhibitory activity and three aporphine alkaloids were isolated from their extracts as the active principles by activity-guided fractionation.     

Action of Δ9-tetrahydrocannabinol on membrane-bound monoamine oxidase activity

In vivo administration (ip) of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) to adult rats under acute conditions (10 and 50 mg/kg) produced a dose-dependent stimulation of monoamine oxidase (MAO; monoamine: O₂ oxidoreductase (deaminating), EC 1.4.3.4) activity in blood platelets and in mitochondrial preparations of hypothalamus and heart. Chronic treatment (10 mg/kg/day for 15 consecutive days) produced a smaller stimulating effect on MAO activity of these tissues. In vitro treatment with Δ⁹-THC produced (1) a stimulatory effect in hypothalamic MAO up to a concentration of 4 μg/mg of protein, (2) an inhibitory effect on heart MAO, and (3) a slight though not insignificant inhibitory effect on platelet MAO. Kinetic studies of in vivo and in vitroΔ⁹-THC-treated enzyme (MAO) are discussed on the basis of K_m and V_max values obtained from Linewwaver-Burk plots. When KSCN, KNO₃, and urea were used, they produced a significant inhibitory effect on MAO activity in all these tissues, even in the presence of Δ⁹-THC, indicating that a characteristic interaction between membrane-bound MAO and Δ⁹-THC can be disrupted by these chaotropic agents.     

In vitro antimicrobial and acetylcholinesterase inhibitory activities of coumarins from <Emphasis Type="Italic">Ferulago</Emphasis><Emphasis Type="Italic">carduchorum</Emphasis>

Ferulago carduchorum (Apiaceae) is an endemic plant of Iran. From the hexane and ethyl acetate extracts of F. carduchorum seven coumarins, one flavonoid and one steroid were isolated using column chromatography with silica gel and Sephadex LH₂₀ as the stationary phases. Antimicrobial activity of the isolated compounds was examined by a broth microdilution method. Acetylcholinesterase (AChE) inhibitory activity of isolated coumarins was also investigated. The isolated compounds were identified as suberosin, suberenol, bergapten, xanthotoxin, isopimpinellin, prantschimgin, β-sitosterol and hesperetin by comparison of their NMR and MS spectral data with those reported in the literature. Evaluation of the minimum inhibitory concentration (MIC) for each compound showed that hesperetin (flavonoid) was the most potent antimicrobial agent against a gram-positive bacterium (Staphylococcus aureus) and among the coumarins, bergapten had the best activity against S. aureus and Candida albicans. All coumarins inhibited AchE enzyme, in which xanthotoxin showed the most inhibitory among them (IC₅₀ = 39.64 µM). Our results indicate that isolated coumarins are effective against the tested bacterial strains and have AchE inhibitory activity suggesting their potential for commercial applications.     

(Na+ + K+)ATPase inhibition by Palythoa extracts--chemical nature of the inhibitor and kinetics of inhibition.

Crude toxic extracts obtained by ethanol extraction from the coelenterate Palythoa caribaeorum were shown to possess strong (Na⁺ + K⁺)ATPase inhibitory activity on enzyme preparations from the electroplax of Etectrophorus electricus. The toxic and inhibitory effects were foun to be separable. Chromatographie, spectrophotofluorimetric, electrophoretic and biological data demonstrate that the inhibitor is serotonin. It is a non-competitive inhibitor for Na⁺ and ATP but is a competitive inhibitor for K⁺. In enzyme preparations of a specific activity of 1.5 μM P_i/min, I₅₀ is of the order of 1 mM.     

Hair-Loss Preventing Effect of Grateloupia elliptica

This study was conducted to evaluate the effect of Grateloupia elliptica, a seaweed native to Jeju Island, Korea, on the prevention of hair loss. When immortalized rat vibrissa dermal papilla cells were treated with extract of G. elliptica, the proliferation of dermal papilla cells significantly increased. In addition, the G. elliptica extract significantly inhibited the activity of 5α-reductase, which converts testosterone to dihydrotestosterone (DHT), a main cause of androgenetic alopecia. On the other hand, the G. elliptica extract promoted PGE₂ production in HaCaT cells in a dose-dependent manner. The G. elliptica extract exhibited particularly high inhibitory effect on LPS-stimulated IL-12, IL-6, and TNF-α production in lipopolysaccharide (LPS)-stimulated bone marrow-derived dendritic cells. The G. elliptica extract also showed inhibitory activity against Pityrosporum ovale, a main cause of dandruff. These results suggest that G. elliptica extract has the potential to treat alopecia via the proliferation of dermal papilla, 5α-reductase inhibition, increase of PGE₂ production, decrease of LPS-stimulated pro-inflammatory cytokines and inhibitory activity against Pityrosporum ovale.     

5-substituted 2′-deoxyuridines: Correlation between inhibition of tumor cell growth and inhibition of thymidine kinase and thymidylate synthetase

A large variety of 5-substituted 2′-deoxyuridines (dUrds) and 2′-deoxyuridylates (dUMPs) have been evaluated for their inhibitory effects on the thymidine (dThd) kinase or thymidylate (dTMP) synthetase isolated from mouse leukemia L1210 cells. The most potent inhibitors of dThd kinase were 5-chloro-, 5-bromo- and 5-iodo-dUrd. Their K_i/K_m values ranged from 0.57 to 0.82. All dUrd analogs tested showed competitive kinetics with respect to dThd. However, there was little, if any, correlation between the inhibitory effects of the compounds on L1210 cell growth and their inhibitory activities against dThd kinase (r = 0.16). The most potent inhibitors of dTMP synthetase were (in order of decreasing activity): 5-nitro-dUMP > 5-formyl-dUMP > 5-fluro-dUMP > 5-oxime of 5-formyl-dUMP > 5-azidomethyl-dUMP > (E)-5-(2-bromovinyl)-dUMP. The k_i/K_m values for these compounds ranged from 0.001 to 0.665. All dUMP analogs tested showed competitive kinetics with respect to dUMP (if not preincubated with the enzyme at 37°). There was a strong correlation (r = 0.833) between the inhibitory effects of these compounds on L1210 cell growth and their inhibitory activities against dTMP synthetase. Thus, the suppressive action of 5-substituted dUrd derivatives on tumor cell growth would involve prior conversion of the nucleoside analogs to the corresponding 5′-monophosphates followed by an inhibition of dTMP synthetase.     

A potent platelet aggregation inhibitor purified from Agkistrodon halys (mamushi) snake venom

By means of gel filtration on Sephadex G-75, DEAE-Sephadex A-50 column chromatography and three gel filtrations on Sephadex G-75, a potent platelet aggregation inhibitor was purified from Agkistrodon halys snake venom and shown to be a single peptide chain, as judged by SDS-polyacrylamide gel electrophoresis. The purified platelet aggregation inhibitor was an acidic protein with a molecular weight of 14,000 and possessed phospholipase A₂ activity. Its inhibitory activity on platelet aggregation was heat stable (at 96°C, 30 min) in an acidic medium (pH 5.5), while its phospholipase A enzymatic activity was heat labile under the same conditions. Its inhibitory activity on platelet aggregation induced by thrombin, sodium arachidonate, collagen or ionophore A-23187 was non-competitive and dose-dependent with a similar id₅₀ (～ 11 μg/ml). It exerted its inhibitory action without pre-incubation with platelet suspension, however, its inhibitory effect could be moderately increased after longer incubation (30 min).     

Cyclophosphamide, 2,2-dimethylaziridines and other alkylating agents as inhibitors of serum cholinesterase.

The effects of cyclophosphamide (CTX) and of alkylating agents containing aziridine or 2,2-dimethylaziridine moieties on the procaine esterase activity of horse serum cholinesterase were investigated. The results indicated that CTX is a competitive, reversible inhibitor of the enzyme, while all the other agents studied caused irreversible inhibition. However, there was no over-all correlation between the cholinesterase inhibitory activities of these agents and their alkylating reactivities toward the model nucleophile 4-(p-nitrobenzyl)pyridine (NBP). The kinetics of inhibition were consistent with the formation of a reversible enzyme alkylating agent complex prior to the irreversible inactivation of the enzyme. In the case of the ring-C-unsubstituted aziridines (TEM. TEPA and AB-100), the inactivation process could be described by the Main equation from which a dissociation constant (K_d) and a reaction rate constant (k₂)wcre calculated. The 2,2-dimethylaziridines(AB-132. AB-163 and TEPA-132) readily hydrolyzed. with rapid loss of alkylating activity (vs NBP). Simultaneously, the cholinesterase inhibitory activities of AB-132 and AB-163 significantly increased, reached a maximum and then gradually decreased on further hydrolysis; in contrast, TEPA-132 showed progressive loss of inhibitory activity. These results indicate that both AB-132 and AB-163 (but not TEPA-132) hydrolyze with the formation of an unstable intermediate(s) having little or no alkylating activity but acting as a potent, irreversible cholinesterase inhibitor(s).     

In vitro inhibition of CYP2B1 monooxygenase by β-myrcene and other monoterpenoid compounds

β-myrcene (MYR) is an acyclic monoterpene found in the essential oils of several useful plants such as lemongrass (Cymbopogon citratus), hop, bay, verbena and others. Recently it has been reported that MYR as well as lemongrass oil blocked the metabolic activation of some promutagens (e.g., cyclophosphamide and aflatoxin B1) in in vitro genotoxicity assays. The present study was performed to evaluate the inhibitory effects of MYR and some other monoterpenoid compounds on microsomal enzymes involved in the activation of genotoxic substances. The effects of MYR and other monoterpenes on the activity of pentoxyresorufin-O-depenthylase (PROD), a selective marker for CYP2B1, was determined in a pool of liver microsomes prepared from phenobarbital-treated rats. The effect of MYR on the activity of ethoxyresorufin-O-deethylase (EROD), a marker for CYP4501A1, was investigated in liver microsomes of untreated rats. Results revealed that MYR had almost no effect on EROD (IC₅₀>50 μM), but produced a concentration-dependent inhibition of PROD activity (IC₅₀=0.14 μM). The analysis of alterations produced by MYR on PROD kinetic parameters (Lineweaver-Burk plot) suggested that inhibition is competitive (K_i=0.14 μM). The inhibitory effects of seven other monoterpenes on PROD activity (pentoxyresorufin 5 μM) were also studied and the IC₅₀ were as follows: (−)-α-pinene, 0.087 μM; (+)-α-pinene, 0.089 μM; d-limonene, 0.19 μM; α-terpinene, 0.76 μM; citral, 1.19 μM; citronellal, 1.56 μM, and (±) camphor, 7.89 μM. The potent inhibitory effects on CYP4502B1 suggest that MYR, and other monoterpenes, interfere with the metabolism of xenobiotics which are substrates for this isoenzyme.     

Calcium binding properties of rat heart plasma membrane and inhibition by structural analogues of dl-propranolol

The isolation and characterization of a plasma membrane preparation from rat heart is described. Enzymatic, chemical, and electron microscopic analysis revealed a relative lack of contamination with nuclear, mitochondrial, ribosomal, and sarcoplasmic reticulum membrane. One calcium binding site (K_d) = 265 μM, B_max = 65 $\text{nmoles}\text{mg}$ protein) was detected by equilibrium dialysis. Monovalent metal ions exhibited 10–100-fold less inhibition potency than divalent metal ions when analyzed by competitive inhibition of calcium binding. The range of K_i values found for divalent metal ions was similar to the K_d value for calcium. La⁺³ produced a potent non-competitive inhibition. A large variety of structural analogues of d,l-propranolol, many of which have been shown to lack β-adrenergic blocking activity, were competitive inhibitors of the calcium binding activity, with K_i values ranging from 40–900 μM. Electrophilic, hydrophobic, and diamino substituents greatly increased the inhibitory activity. There was no significant difference between related tertiary and quaternary amines. The experimental antiarrhythmic agent UM 272 had the least ability to inhibit calcium binding to the cardiac plasma membrane preparation (K_i = 795 μM). However, UM 424, another experimental antiarrhythmic agent, had an inhibitory activity similar to dl-propranolol (k_i = 115 μM and 108 μM, respectively).     

In vitro study on α-amylase inhibitory activity of an Indian medicinal plant, Phyllanthus amarus

Objective:

The objective of this study was to evaluate the α-amylase inhibitory activity of different extracts of Phyllanthus amarus against porcine pancreatic amylase in vitro.

Materials and Methods:

The plant extracts were prepared sequentially with ethanol, chloroform, and hexane. Each extract was evaporated using rotary evaporator, under reduced pressure. Different concentrations (10, 20, 40, 60, 80, and 100 μg/mL) of each extract were made by using dimethyl sulfoxide (DMSO) and subjected to α-amylase inhibitory assay using starch azure as a substrate. The absorbance was read at 595 nm using spectrophotometer. Using this method, the percentage of α-amylase inhibitory activity and IC₅₀values of each extract was calculated.

Results:

The chloroform extract failed to inhibit α-amylase activity. However, the ethanol and hexane extracts of P. amarus exhibited appreciable α-amylase inhibitory activity with an IC50 values 36.05 ± 4.01 μg/mL and 48.92 ± 3.43 μg/mL, respectively, when compared with acarbose (IC₅₀value 83.33 ± 0.34 μg/mL).

Conclusion:

This study supports the ayurvedic concept that ethanol and hexane extracts of P. amarus exhibit considerable α-amylase inhibitory activities. Further, this study supports its usage in ethnomedicines for management of diabetes.     

Antioxidant activity of selected plant species; potential new sources of natural antioxidants

The aim of this study was to examine six plants from Serbia for their potential antioxidant activity. Therefore, six antioxidant activity assays were carried out, including: total antioxidant capacity, DPPH free-radical scavenging, the inhibitory activity toward lipid peroxidation, Fe³⁺- reducing power, Fe²⁺- chelating ability and hydroxyl radical scavenging activity. Total phenolic and flavonoid contents were also determined for each alcoholic extract. Cotinus coggygria extract contained the highest amount of total phenols (413 mg GAE /g dry extract), while the highest proportion of flavonoids was found in the Echium vulgare methanol extract (105 mg RU/g). Cotinus coggygria and Halacsya sendtneri alcoholic extracts showed the highest total antioxidant capacity (313 and 231 mg AA/g dry extract), as well as DPPH free-radical scavenging (IC₅₀ = 9 and 99 μg/ml), inhibitory activity toward lipid peroxidation (IC₅₀ = 3 and 17 μg/ml) and reducing power. Whereas, the greatest hydroxyl radical scavenging activity, as well as ferrous ion chelating ability showed Echium vulgare, Echium rubrum and Halacsya sendtneri.     

Possible involvement of β₁ receptors in various emetogen-induced increases in salivary amylase activity in rats

We investigated the inhibitory effects of β₁- or β₂-adrenoceptor (AR) antagonists on salivary amylase secretion produced by various emetic agents, such as cisplatin, apomorphine, and lithium chloride (LiCl), or the non-emetic agent β_1/2-AR agonist isoprenaline in rats. We also determined the inhibitory effect of metoclopramide, a dopamine D₂–receptor antagonist, on increases in the salivary amylase activity induced by apomorphine or granisetron, a 5-HT₃–receptor antagonist, on LiCl-induced increased salivary amylase activity. Isoprenaline (0.01 mg/kg, s.c.) produced an increase in salivary amylase and the increase was inhibited by the β_1/2-AR antagonist propranolol (5 mg/kg, s.c.) and β₁-AR antagonist atenolol (2 mg/kg, s.c.) but not by the β₂-AR antagonist butoxamine (8 mg/kg, s.c.). The increased amylase activity induced by cisplatin (15 mg/kg, i.v.), apomorphine (3 mg/kg, s.c.), or LiCl (120 mg/kg, i.p.) was inhibited significantly by atenolol (2 mg/kg, s.c.) but not by butoxamine (8 mg/kg, s.c.). In addition, increases in amylase activities induced by apomorphine and LiCl were inhibited significantly by metoclopramide (10 mg/kg, i.v.) and granisetron (3 mg/kg, i.v.), respectively. These results suggest that salivary amylase secretion induced by various emetogens is involved in β₁-adrenoceptor activity and that salivary amylase activity is useful to detect emetogens with no direct β₁-AR activation in rats, a species that does not exhibit vomiting.     

Effects of ginsenoside on G protein-coupled inwardly rectifying K+ channel activity expressed in Xenopus oocytes

Recently, we provided evidence that ginsenoside, the active component of Panax ginseng, uses the pertussis toxin-insensitive Gα_q/11-phospholipase C-β3 signal transduction pathway to increase Ca²⁺-activated Cl⁻ currents in the Xenopus oocyte. Other investigators have shown that stimulation of receptors linked to the Gα_q-phospholipase C pathway inhibits the activity of G protein-coupled inwardly rectifying K⁺ (GIRK) channels. In the present study, we sought to determine whether ginsenoside influenced the activity of GIRK 1 and GIRK 4 (GIRK 1/4) channels expressed in the Xenopus oocyte, and if so, the underlying signal transduction mechanism. In oocytes injected with GIRK 1/4 channel cRNA, bath-applied ginsenoside inhibited the high K⁺ solution-elicited GIRK current (EC₅₀: 4.9±4.3 μg/ml). Pretreatment of the oocyte with pertussis toxin reduced the high K⁺ solution-elicited GIRK current by 49%, but it did not alter the inhibitory effect of ginsenoside on the GIRK current. Prior intraoocyte injection of cRNA(s) coding Gα_q, Gα₁₁ or Gα_q/Gα₁₁, but not Gα_i2 or Gα_oA, attenuated the inhibitory ginsenoside effect. Injection of cRNAs coding Gβ₁γ₂ also attenuated the ginsenoside effect. Preincubation of GIRK channel-expressing oocytes with phospholipase C inhibitor, {1-[6-((17b-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione} (U73122), or protein kinase C inhibitor, staurosporine or chelerythrine, blocked the inhibitory ginsenoside effect on the GIRK current. Intraoocyte injection of bis (o-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid (BAPTA), a free Ca²⁺ chelator, had no significant effect on the action of ginsenoside. Taken together, these results suggest that ginsenoside inhibits the activity of the GIRK 1/4 channel expressed in the Xenopus oocyte through a pertussis toxin-insensitive and Gα_q/11-, phospholipase C- and protein kinase C-mediated signal transduction pathway.     

Synthesis of 6-deoxymollugins and their inhibitory activities on tyrosinase

A series of 6-deoxymollugins were prepared five steps from benzaldehyde and its derivatives via phenylboronic acid-catalyzed chromenylation as a key step. Their inhibitory activities against tyrosinase from mushroom were evaluated to show that the parent, methyl 2,2-dimethyl-2H-benzo[h]chromene-5-carboxylate (9a) showed best and promising inhibitory activity at IC₅₀ = 18.3 μM.     

Liquid chromatography/tandem mass spectrometry study of anti-inflammatory activity of Plantain (Plantago L.) species

To evaluate anti-inflammatory activity of selected Plantago species (P. lanceolata L. and P. major L.) an optimized in vitro test for determination of cyclooxygenase-1 (COX-1) and 12-lipoxygenase (12-LOX) inhibition potency was undertaken. By using intact cell system (platelets) as a source of COX-1 and 12-LOX enzymes and highly sensitive and specific LC–MS/MS technique for detection of main arachidonic acid metabolites formed by COX-1 and 12-LOX, this test provides efficient method for evaluation of anti-inflammatory potential of plant extracts and isolated compounds. Our results validated the well-known COX-1 inhibitory activity of P. lanceolata and P. major methanol extracts (concentration required for 50% inhibition (IC₅₀) was 2.00 and 0.65 mg/ml, respectively). Furthermore, 12-LOX inhibitory activity of examined extracts was reported for the first time (IC₅₀ = 0.75 and 1.73 mg/ml for P. lanceolata and P. major, respectively). Although renowned inhibitors, such as acetylsalicylic acid and quercetin showed higher activity, this study verifies P. lanceolata and P. major as considerable anti-inflammatory agents.     

Chemical constituents and their acetyl cholinesterase inhibitory and antioxidant activities from leaves of Acanthopanax henryi: potential complementary source against Alzheimer’s disease

The aim of this study was to investigate chemical constituents of the leaves of Acanthopanax henryi, and their antioxidant, acetyl cholinesterase inhibitory activities. Caffeoyl quinic acid derivates and flavonoids were obtained from A. henry, through column chromatography technologies, and the content of major constituents was determined by the HPLC–UV method. Anti-oxidant activity of the isolated metabolites was evaluated by free radical scavenging (DPPH, ABTS radicals) and superoxide anion scavenging. The results showed that di-caffeoyl quinic acid derivates had stronger antioxidant activity than positive controls (ascorbic acid, trolox and allopurinol). Acetyl cholinesterase inhibitory activity was estimated on the constituents, among which, quercetin, 4-caffeoyl-quinic acid and 4,5-caffeoyl quinic acid were found to have strong acetyl cholinesterase inhibitory activity with IC₅₀ values ranging from 62.6 to 121.9 μM. The present study showed that some of the tested constituents from the leaves of A. henryi exhibit strong antioxidant and acetyl cholinesterase inhibitory effects. This suggest that the leaves of A. henryi can be used as a new natural complementary source of acetyl cholinesterase inhibitors and anti-oxidant agents, thus being a promising potential complementary source against Alzheimer’s disease.     

Vanillin derivatives as the selective small molecule inhibitors of FtsZ

A series of vanillin derivatives have been designed and synthesized and their biological activities were also evaluated as potential inhibitors of FtsZ. These compounds were assayed for antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. Compounds with potent antibacterial activities were tested for their FtsZ inhibitory activity. Compound 4u showed the most potent antibacterial activity with MIC of 0.28 μg/mL against E. coli strains and exhibited the most potent FtsZ inhibitory activity with polymerization IC₅₀ of 2.1 μM. Docking simulation was performed to position compound 4u into the FtsZ active site to determine the probable binding conformation.     

Isolation and characterization of a trypsin inhibitor from the skin secretions of Kaloula pulchra hainana

Amphibian skin secretions contain many bioactive compounds. A trypsin inhibitor termed KPHTI was purified from the skin secretions of frog Kaloula pulchra hainana by successive ion-exchange and gel-filtration chromatography. KPHTI is a single chain glycoprotein, with an apparent molecular weight of 23 kDa in SDS-PAGE. It is a competitive inhibitor and effectively inhibits trypsin catalytic activity on peptide substrate with the inhibitor constant (K_i) value of 27 nM. KPHTI shows no inhibitory effect on chymotrypsin, thrombin, elastase, and subtilisin. The N-terminal sequence of KPHTI is DHEVTS, which shows no similarity with other known trypsin inhibitors. DTT apparently affected the inhibitory activity of KPHTI. But it was not sensitive to temperature and pH range, which suggested that it possessed stable trypsin inhibitory activity in natural environment, and maybe play an important role in against predators.