Enzyme inhibition and radical scavenging activities of aerial parts of Paeonia emodi Wall (Paeoniaceae)

The ethanolic extract derived from aerial parts of an indigenous medicinal plant Paeonia emodi was screened for enzyme inhibition activities against Urease (jack bean and Bacillus pasteurii) and alpha-Chymotrypsin. The extract was also investigated for its radical scavenging activity using DPPH assay. The crude extract was found to possess significant enzyme inhibition activities against jack bean (74%) and Bacillus pasteurii (80%) urease and a moderate activity (54%) against alpha-Chymotrypsin. The extract also displayed excellent (83%) radical scavenging activity. On the basis of these results, the crude extract was subsequently fractionated into n-hexane, chloroform, ethyl acetate, n-butanol and water fractions and tested independently for the aforesaid activities. Significant inhibitory activity against urease enzyme was observed for the ethyl acetate, n-butanol and water fractions while the n-hexane and chloroform fractions were devoid of any such activity. In the alpha-Chymotrypsin enzyme inhibition studies the activity was concentrated into the ethyl acetate fraction. All the fractions displayed potent radical scavenging activity. The crude extract and fractions thereof were also subjected to total phenolic content determination. A correlation between radical scavenging capacities of extracts and total phenolic content was observed in the majority of cases.     

滇重楼种子水浸液对三种植物种子萌发和幼苗生长的影响

利用滇重楼(Paris polyphylla Smith var.yunnanensis(Franch.)Hand.-Mazz.)种子外种皮和胚乳的水浸液对白菜(Brassica pekinensis(Lour.)Rupr.)、绿豆(Vigna radiata(Linn.)Wilczak)、小麦(Triticum aestivum L.)种子进行处理,研究滇重楼种子水浸液对3种植物种子萌发、幼苗生长和保护酶活性的影响,并利用GC-MS方法对滇重楼种子内源抑制物的成分进行分析。结果显示,不同浓度滇重楼外种皮、胚乳水浸液对上述3种受体植物的发芽率、苗高、根长及鲜重均产生影响,其作用强度和水浸液的浓度有关,总体上表现出低促高抑的双重浓度效应。滇重楼种子水浸液对白菜的影响作用最强,对绿豆的影响作用最弱,且胚乳水浸液的影响较外种皮强。不同浓度滇重楼种子外种皮和胚乳水浸液均能影响3种植物幼苗体内保护酶的活性,随着水浸液浓度的升高,叶片中超氧化物歧化酶(SOD)、过氧化物酶(POD)活性总体增加,与对照相比差异显著。白菜、小麦过氧化氢酶(CAT)活性减少,与对照相比差异显著;绿豆过氧化氢酶(CAT)活性增加,但与对照相比无显著差异。利用GC-MS方法从胚乳和外种皮水浸液中分别检出8种和2种物质。研究结果表明滇重楼种子中存在内源抑制物质,可能是导致种子休眠的原因;种子水浸液可能通过影响植物幼苗保护酶的活性进而影响其正常生长;有机酸类物质可能是滇重楼种子内源抑制物之一。     

Enzyme inhibition and radical scavenging activities of aerial parts of Paeonia emodi Wall. (Paeoniaceae)

The ethanolic extract derived from aerial parts of an indigenous medicinal plant Paeonia emodi was screened for enzyme inhibition activities against Urease (jack bean and Bacillus pasteurii) and α-Chymotrypsin. The extract was also investigated for its radical scavenging activity using DPPH assay. The crude extract was found to possess significant enzyme inhibition activities against jack bean (74%) and Bacillus pasteurii (80%) urease and a moderate activity (54%) against α-Chymotrypsin. The extract also displayed excellent (83%) radical scavenging activity. On the basis of these results, the crude extract was subsequently fractionated into n-hexane, chloroform, ethyl acetate, n-butanol and water fractions and tested independently for the aforesaid activities. Significant inhibitory activity against urease enzyme was observed for the ethyl acetate, n-butanol and water fractions while the n-hexane and chloroform fractions were devoid of any such activity. In the α-Chymotrypsin enzyme inhibition studies the activity was concentrated into the ethyl acetate fraction. All the fractions displayed potent radical scavenging activity. The crude extract and fractions thereof were also subjected to total phenolic content determination. A correlation between radical scavenging capacities of extracts and total phenolic content was observed in the majority of cases.     

3-Dehydroshikimate dehydratase in mung bean cultured cells

Summary The activity of 3-dehydroshikimate dehydratase was detected in an extract prepared from cells of mung bean (Vigna mungo) that had been cultured in the presence of shikimate while such activity was not detectable in an extract prepared from cells cultured without shikimate. The enzyme was partially purified and characterized. The maximum activity of the enzyme was observed at pH 7.4. The activity was inhibited to a small extent by EDTA and sulfhydryl inhibitors. The partially purified enzyme was sensitive to thermal denaturation but was stabilized by Mg²⁺ ions. These results suggest that 3-dehydroshikimate dehydratase might be induced in mung bean cultured cells in the presence of shikimic acid.Abbreviations 2,4-D 2,4-Dichlorophenoxyacetic acid - DHS 3-dehydroshikimic acid - PCA protocatechuic acid - QA quinic acid - SA shikimic acid - SORase shikimate - NAEP oxidoreductase     

Changes in stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds

In crude extract of castor bean endosperm, isocitrate dehydrogenase (NADP⁺) (EC 1.1.1.42) was stable at 57°C at the beginning of seed germination as well as in maturing and dry seeds. The enzyme gradually became less thermostable as germination proceeded and became unstable after 4 days. Extract from 5-day-old endosperm reduced the thermostability of the thermostable enzyme. The destabilizing factor accumulated in the endosperm as germination progressed and was identified as ricinoleate. Ricinoleate destabilized the purified enzyme which was stabilized by isocitrate and Mg²⁺, but ricinoleate did not affect the activity of NADP⁺-isocitrate dehydrogenase itself. Stearate, oleate, palmitate and myristate were similar to ricinoleate in their effect on the thermostability of the enzyme. The thermolabile enzyme in the crude extract of 5-day-old endosperm was readily inactivated by trypsin and in low concentrations of buffer. The thermostable enzyme in the crude extract of 2-day-old endosperm was not affected by these treatments. The thermostable enzyme treated with ricinoleate showed the same instabilities as the thermolabile enzyme. The role of ricinoleate in ther germinating castor bean endosperm is discussed.     

Galactose inhibition of auxin-induced growth of mono- and dicotyledonous plants

Galactose inhibited auxin-induced cell elongation of oat coleoptiles but not that of azuki bean stems. Galactose decreased the level of UDP-glucose in oat coleoptiles but not in azuki bean hypocotyls. Glucose-1-phosphate uridyltransferase activity (EC 2.7.7.9), in a crude extract from oat coleoptiles, was competitively inhibited by galactose-1-phosphate, but that enzyme from azuki bean was not. A correlation was found between inhibition of growth by galactose and inhibition of glucose-1-phosphate uridyltransferase activity by galactose-1-phosphate using oat, wheat, maize, barley, azuki bean, pea, mung bean, and cucumber plants. Thus, it is concluded that galactose is converted into galactose-1-phosphate, which interferes with UDP-glucose formation as an analog of glucose-1-phosphate.     

Plantagoside, a novel alpha-mannosidase inhibitor isolated from the seeds of Plantago asiatica, suppresses immune response

A hot-water extract from the seed of Plantago asiatica showed a potent inhibitory activity against jack bean alpha-mannosidase, and a flavanone glucoside, plantagoside, was isolated as the inhibitor. Plantagoside was a specific inhibitor for jack bean alpha-mannosidase (IC50 at 5 microM) and appeared to be a non-competitive inhibitor of the enzyme. Whereas, negligible or weak inhibitory activities were observed for beta-mannosidase, beta-glucosidase, and sialidase tested. Plantagoside also inhibited alpha-mannosidase activities in mouse liver lysosomal and microsomal fractions, and the enzyme inhibitory activity in microsomal fraction was enhanced in the presence of glucosidase inhibitor, castanospermine. Plantagoside suppressed antibody response to sheep red blood cells and concanavalin A induced lymphocyte proliferation which was measured by [3H]thymidine incorporation.     

黑豆种皮花色苷酶法辅助提取工艺优化及其抗氧化活性分析

优化黑豆种皮花色苷复合酶法辅助提取工艺,并对其抗氧化活性进行评价。通过单因素试验和响应面法优化确定了黑豆种皮花色苷生物酶法辅助提取的最佳工艺为:复合酶(纤维素酶400 U/g,α-淀粉酶50 U/g),酶解温度50℃,液料比26∶1 mL/g,乙醇体积分数64%,酶解时间为59 min。在此条件下,提取花色苷的含量为2.019 mg/g。抗氧化试验研究表明,黑豆种皮花色苷的还原能力、对超氧阴离子自由基清除能力低于抗坏血酸,但对亚硝酸根离子和DPPH自由基、ABTS自由基的清除能力强于抗坏血酸。因此,生物酶法辅助提取是一种高效的黑豆种皮花色苷提取方法,且作为一种新型花色苷资源,黑豆种皮花色苷有着挖掘和应用价值。     

Age-specific digestion of Tenebrio molitor (Coleoptera: Tenebrionidae) and inhibition of proteolytic and amylolytic activity by plant proteinaceous seed extracts

Tenebrio molitor L. (Coleoptera: Tenebrionidae), is an international and serious pest of stored products. So far nothing is known about the activity for each growth stage digestive enzyme regarding this insect species. Thus, the aim of the current study was to get in depth analysis of the stage specific digestion and to investigate the effect of cereal (wheat cultivars including MV17, Aflak, Sivand, Saymon, and Zare) and legume (bean) seed extracts on the two main digestive enzymes i.e. α-amylases and proteases. Therefore, gut enzymes were extracted using distilled water and wheat cultivars and bean seed proteinaceous compounds were extracted using 0.1 M NaCl. Results showed that a steady state increase in the number and amount of digestive enzyme activities from first to fourth instar larvae was seen in both enzyme and in gel assays. In the first instar larvae (L1) only one band of α-amylase activity was seen (A1), whereas in the second (L2), third (L3), fourth (L4) and fifth (L5) instar larvae as well as in the adult (A) more than one amylase band (up to 4 isoenzymes) was seen. The same pattern was observed for α and β glucosidases and proteases. Probit analysis showed that bean and MV17 inhibited the amylase activity with an I₅₀ of 9.73 and 7.4 μg, respectively. The same cultivar seed extract inhibited protease activity with I₅₀s of 11.54 and 6.5 μg proteins. It is concluded that proteinaceous extract of cereals and bean seeds have a strong potential to be used in this pest management.     

Diacylglycerol acyltransferase in maturing oil seeds of maize and other species

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was detected in the microsomal fractions of maturing maize scutellum, soybean cotyledon, peanut cotyledon, and castor bean endosperm. The activity detected was high enough to account for the in vivo rate of triacylglycerol synthesis. The activity of the maize enzyme was characterized using diolein micelles prepared by sonication in Tween 20 as the substrate. The activity was highest at pH values of 6 to 7. The activity was proportional to the amount of enzyme added, and the reaction rate was linear for about 2 minutes. The enzyme was not inactivated by Tween 20, Zwitterion 3-08, Triton-X 100, and cholate, but was inactivated completely by sodium dodecyl sulfate. The enzyme was active on linoleoyl coenzyme A (CoA), palmitoyl CoA, and oleoyl CoA, although the activity was highest on linoleoyl CoA. Endogenous diacylglycerol was present in the microsomes, and the enzyme activity was only partially dependent on the addition of external diolein. Subcellular fractionation of the total scutellum extract in sucrose density gradients was performed. By comparing the migration of the enzyme between rate and equilibrium centrifugation, and between equilibrium centrifugation in the presence and absence of magnesium ions in the preparative media, the enzyme was shown to be associated with the rough endoplasmic reticulum. Some of the above findings on the maize enzyme were extended to the enzymes from castor bean, soybean, and peanuts.     

Fixation of carbon dioxide in the dark by the malic enzyme of bean and oat stem rust uredospores

Malic enzyme was found in both bean rust and cat stem rust uredospores. In bean rust uredospores it was shown to catalyze the formation of pyruvic acid from l-malic acid and to synthesize malic acid from pyruvic acid and CO₂. The malic enzyme from bean rust uredospores was specific for NADP and dependent on manganous ions for activity. The specific activity of the bean rust malic enzyme in crude extracts of ungerminated uredospores was approximately 6 times greater than that found in crude extracts obtained from germinated uredospores. The malic enzyme was also found in extracts obtained from healthy and rust-infected bean leaves. The specific activity of the enzyme was approximately 2 to 5 times greater in partially purified extracts obtained from the infected bean tissue at 6 days after inoculation. The specific activity of the malic enzyme in crude extracts obtained from oat stem rust uredospores was 2 times greater than the specific activity of this enzyme in crude extracts obtained from bean rust uredospores. Phosphoenolpyruvate carboxylase activity could not be demonstrated in crude extracts obtained from the ungerminated uredospores of the bean rust fungus.     

Endo-[beta]-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence

Lettuce (Lactuca sativa L.) endosperm cell walls isolated prior to radicle emergence underwent autohydrolysis, the rate of which was correlated with whether radicle emergence would subsequently occur. Extraction of endosperm cell walls with 6 M LiCl suppressed autohydrolysis, and the desalted extract possessed activity that was capable of hydrolyzing purified locust bean galactomannan but not arabinogalactan, carboxymethylcellulose, glucomannan, polygalacturonic acid, tomato galactomannan, or native lettuce endosperm cell walls. Some hydrolytic activity was detected on endosperm cell walls if they were modified by partial trifluoroacetic acid hydrolysis or pretreatment with guanidinium thiocyanate. In extended incubations the cell wall enzyme extract released only large molecular mass fragments from locust bean galactomannan, indicating primarily endo-activity. Galactomannan-hydrolyzing activity in the cell wall extract increased as a function of imbibition time and was greatest just prior to radicle emergence. Thermoinhibition (imbibition at 32[deg]C) or treatment with abscisic acid at a temperature optimal for germination (25[deg]C) suppressed both germination and endosperm cell wall mannanase activity, whereas alleviation of thermoinhibition with gibberellic acid was accompanied by significant enhancement of mannanase activity. We conclude that a cell wall-bound endo-[beta]-mannanase is expressed in lettuce endosperm prior to radicle emergence and is regulated by the same conditions that govern germination.     

A Vacuolar Processing Enzyme in Maturing and Germinating Seeds: Its Distribution and Associated Changes during Development

Proprotein precursors of vacuolar components are transportedfrom endoplasmic reticulum to the dense vesicles, and then targetedto the vacuoles, where they are processed proteolytically totheir mature forms by a vacuolar processing enzyme. Immunoelectronmicroscopy of the maturing endosperm of castor bean (Ricinnscommunis) revealed that the vacuolar processing enzyme is selectivelylocalized in the dense vesicles as well as in the vacuolar matrix.This indicates that the vacuolar processing enzyme is transportedto vacuoles via dense vesicles as does IIS globulin, a majorseed protein. During seed maturation of castor bean, an increasein the activity of the vacuolar processing enzyme in the endospermpreceded increases in amounts of total protein. The enzymaticactivity reached a maximum at the late stage of seed maturationand then decreased during seed germination concomitantly withthe degradation of seed storage proteins. We examined the distributionof the enzyme in different tissues of various plants. The processingenzyme was found in cotyledons of castor bean, pumpkin and soybean,as well as in endosperm, and low-level processing activity wasalso detected in roots, hypocotyls and leaves of castor bean,pumpkin, soybean, mung bean and spinach. These results suggestthat the proprotein-processing machinery is widely distributedin vacuoles of various plant tissues. (Received July 11, 1993; Accepted August 17, 1993)     

A procedure for purifying jack bean urease for clinical use

Urease with a purity meeting the requirements of analytical use was purified from jack bean meal through steps consisting of 20% acetone extraction, heat treatment, acid precipitation, and lyophilization. For extraction of urease, one part of bean meal was mixed with 5 parts of 20% acetone containing 1 mM EDTA and 1 mM 2-mercaptoethanol, and stirred at 20 degrees C for 5 min. Milky substances in the extract were removed by heat treatment. Urease in the clear yellow supernatant was precipitated by adjusting the pH of the solution to 5.4 with citric acid. The acid precipitated urease was neutralized by dissolving in 0.015 M phosphate buffer, pH 8.5 (final pH 6.8 to 7.0) and then lyophilized. By this procedure, the purity of the enzyme was increase 14.7 fold, the recovery of activity was 63%, and the yield was 6.75 g from 1 kg of bean seeds. The specific activity of the preparation was 411 units/mg protein (240 units/mg solid), and the free ammonia content was less than 0.01 microgram per unit. Some other proteins were present in the urease preparation as examined by gel filtration and gradient polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 480,000. However, two urease activity bands with molecular weight of 230,000 and 480,000 were observed in the polyacrylamide gel electrophoregram. From the result of determination of blood urea nitrogen (BUN), this simple purification procedure could be used for practical preparation of urease from jack bean meal for clinical analysis.     

Casbene synthetase: regulation of phytoalexin biosynthesis in Ricinus communis L. seedlings. Purification of casbene synthetase and regulation of its biosynthesis during elicitation

Castor bean (Ricinus communis L.) seedlings responded to stress by producing the antifungal diterpene, casbene. Casbene synthetase, the enzyme catalyzing the production of casbene from geranylgeranyl pyrophosphate, was purified 4700-fold to a final specific activity of 4.2 nkat/mg protein by a combination of ion-exchange and dye-ligand chromatographic procedures. Approximately 500 micrograms of purified enzyme was recovered from 1600 seedlings that had been infected with the fungus, Rhizopus stolonifer. The purified enzyme showed a single protein band, by Ag staining, of Mr 59,000 +/- 1000 after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoretic analysis of the immunoprecipitate obtained from a crude enzyme extract and polyclonal rabbit antibodies raised against the purified enzyme revealed no contaminants or cross-reacting components. In vitro translation of polysomal RNA pools obtained from healthy castor bean seedlings and seedlings at various times after exposure to pectic fragment elicitors coupled with immunoprecipitation showed that healthy seedlings have nondetectable levels of casbene synthetase mRNA and that seedlings exposed to elicitor show a rapid increase in casbene synthetase mRNA which reaches a maximum after 6 h. Casbene synthetase activity increases to a maximum 10 h after elicitation under comparable conditions. These results show that increases in the activity of mRNA for casbene synthetase after elicitation by pectic fragments precede the appearance of casbene synthetase activity as would be expected if the enzyme were being synthesized de novo.     

Observations on the enzymatic synthesis of a beta-1,4-glucan by a soluble preparation form mung bean(Phaseolus aureus)

An enzyme extract of mung bean roots and hypocotyls (Phaseolus aureus) that catalyzes the synthesis of a β-1,4-glucan from guanosine 5′-diphosphate-d-glucose was prepared by a modification of the method of T.-Y. Liu and W. Z. Hassid (1970, J. Biol. Chem.245, 1922–1925). Its activity was not increased by any of those factors that have contributed to the marked improvement in the performance of various cell-free polysaccharide-synthesizing systems from other organisms. Evidence is presented to suggest that in the mung bean system a stable precursor of the cell wall polysaccharide or intermediate in its synthesis is formed by incubation of the enzyme with guanosine 5′-diphosphate-d-mannose.     

In Vitro Synthesis of Ureidohomoserine by an Enzyme from Jack Bean (Canavalia ensiformis) Leaves

An enzyme was extensively purified from jack bean leaves (Canavalia ensiformis L.) which produced o-ureidohomoserine from l-canaline and carbamyl phosphate. The most highly purified preparations catalyzed both this reaction and citrulline synthesis from ornithine and carbamyl phosphate, and the ratio of the two activities remained nearly constant during purification. When hydrated jack bean seeds were the enzyme source, ornithine carbamyltransferase (EC 2.1.3.3) activity was high but synthesis of ureidohomoserine was barely detectable. Both ornithine carbamyltransferase and the ureidohomoserine synthesizing enzyme had similar Km values for carbamyl phosphate. The purification data suggest that one enzyme may catalyze both reactions in jack bean leaves.     

Characterization of the Bacillus subtilis WL-3 mannanase from a recombinant Escherichia coli

A mannanase was purified from a cell-free extract of the recombinant Escherichia coli carrying a Bacillus subtilis WL-3 mannanase gene. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. Optimal conditions for the purified enzyme occurred at pH 6.0 and 60 degrees C. The specific activity of the purified mannanase was 5,900 U/mg on locust bean gum (LBG) galactomannan at pH 6.0 and 50 degrees C. The activity of the enzyme was slightly inhibited by Mg(2+), Ca(2+), EDTA and SDS, and noticeably enhanced by Fe(2+). When the enzyme was incubated at 4 degrees C for one day in the presence of 3 mM Fe(2+), no residual activity of the mannanase was observed. The enzyme showed higher activity on LBG and konjac glucomannan than on guar gum galactomannan. Furthermore, it could hydrolyze xylans such as arabinoxylan, birchwood xylan and oat spelt xylan, while it did not exhibit any activities towards carboxymethylcellulose and para-nitrophenyl-beta-mannopyranoside. The predominant products resulting from the mannanase hydrolysis were mannose, mannobiose and mannotriose for LBG or mannooligosaccharides including mannotriose, mannotetraose, mannopentaose and mannohexaose. The enzyme could hydrolyze mannooligosaccharides larger than mannobiose.     

Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

Occurrence of glucuronokinase in various plant tissues and comparison of enzyme activity of seedlings and green plants

Glucuronokinase (EC 2.7.1.43) activity was detected in etiolated seedlings of corn, mung bean and soybean. Biosynthesis of glucuronokinase is not limited to seedlings, because expanding green leaves of corn produced almost as much glucuronokinase activity as etiolated seedlings when data were expressed on the basis of soluble protein. The enzyme was also present in extracts of tobacco callus and Lilium longiflorum pollen, with more enzyme activity obtained from pollen than any other source. Detection of glucuronokinase in green leaves of of mung bean was precluded by the presence of an enzyme inhibitor.