Development of the Potential for Cyanogenesis in Maturing Black Cherry (Prunus serotina Ehrh.) Fruits

Biochemical changes related to cyanogenesis (hydrogen cyanide production) were monitored during maturation of black cherry (Prunus serotina Ehrh.) fruits. At weekly intervals from flowering until maturity, fruits (or selected parts thereof) were analyzed for (a) fresh and dry weights, (b) prunasin and amygdalin levels, and (c) levels of the catabolic enzymes amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. During phase I (0-28 days after flowering [DAF]), immature fruits accumulated prunasin (mean: 3 micromoles/fruit) but were acyanogenic because they lacked the above enzymes. Concomitant with cotyledon development during mid-phase II, the seeds began accumulating both amygdalin (mean: 3 micromoles/seed) and the catabolic enzymes and were highly cyanogenic upon tissue disruption. Meanwhile, prunasin levels rapidly declined and were negligible by maturity. During phases II (29-65 DAF) and III (66-81 DAF), the pericarp also accumulated amygdalin, whereas its prunasin content declined toward maturity. Lacking the catabolic enzymes, the pericarp remained acyanogenic throughout all developmental stages.     

Tissue and Subcellular Localization of Enzymes Catabolizing (R)-Amygdalin in Mature Prunus serotina Seeds

In black cherry (Prunus serotina Ehrh.) homogenates, (R)-amygdalin is catabolized to HCN, benzaldehyde, and d-glucose by the sequential action of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase. The tissue and subcellular localizations of these enzymes were determined within intact black cherry seeds by direct enzyme analysis, immunoblotting, and colloidal gold immunocytochemical techniques. Taken together, these procedures showed that the two β-glucosidases are restricted to protein bodies of the procambium, which ramifies throughout the cotyledons. Although amygdalin hydrolase occurred within the majority of procambial cells, prunasin hydrolase was confined to the peripheral layers of this meristematic tissue. Highest levels of mandelonitrile lyase were observed in the protein bodies of the cotyledonary parenchyma cells, with lesser amounts in the procambial cell protein bodies. The residual endosperm tissue had insignificant levels of amygdalin hydrolase, prunasin hydrolase, and mandelonitrile lyase.     

Catalytic degradation of amygdalin by extracellular enzymes from Aspergillus niger

Amygdalin is a controversial anti-tumor natural product that has been used as an alternative cancer drug for many years. The anti-tumor mechanism and metabolism of amygdalin have been the focus of many studies. However, previous studies by our group demonstrated that amygdalin itself has no anti-tumor activity, but rather the active ingredients were determined to be amygdalin degradation products. To screen novel drugs with anti-tumor activity, the extracellular enzymes from Aspergillus niger were used to degrade amygdalin. Within 4 h of the catalytic reaction at 37°, amygdalin was rapidly degraded into four products. The products were then extracted and purified by column chromatography. By comparing the HPLC chromatograms, ¹H NMR, ¹³C NMR and MS data, the products were identified as mandelonitrile, prunasin, benzaldehyde and phenyl-(3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxy)-acetonitrile (PTMT), a novel hydroxyl derivative of prunasin. Furthermore, pharmacology studies of these compounds demonstrated that 10 mg/kg of PTMT significantly suppressed the growth of S-18 tumor cells within 11 days in a concentration-dependent manner.     

Pattern of the Cyanide-Potential in Developing Fruits : Implications for Plants Accumulating Cyanogenic Monoglucosides (Phaseolus lunatus) or Cyanogenic Diglucosides in Their Seeds (Linum usitatissimum, Prunus amygdalus)

The absolute cyanide content of developing fruits was determined in Costa Rican wild lima beans (Phaseolus lunatus), oil flax (Linum usitatissimum), and bitter almonds (Prunus amygdalus). The cyanide potential (HCN-p) of the lima bean and the almond fruit began to increase shortly after anthesis and then stopped before fruit maturity. In contrast, the flax inflorescence had a higher HCN-p in absolute terms than the mature flax fruit. At all times of its development the bean fruit contained the monoglucosides linamarin and lotaustralin. The almond and the flax fruits contained, at anthesis, the monoglucosides prunasin, and linamarin and lotaustralin, respectively, while, at maturity, only the corresponding diglucosides amygdalin, and linustatin and neolinustatin, respectively, were present.     

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin. Received: 16 September 1997 / Received revision: 6 October 1997 / Accepted: 14 October 1997     

Decarboxylation of Oxalic Acid during Ripening of Banana Fruit (Musa sapientum L.)

ABSTRACT

Japanese apricot, Prunus mume Sieb. et Zucc., biosynthesizes the l-phenylalanine-derived cyanogenic glucosides prunasin and amygdalin. Prunasin has biological properties such as anti-inflammation, but plant extraction and chemical synthesis are impractical. In this study, we identified and characterized UGT85A47 from Japanese apricot. Further, UGT85A47 was utilized for prunasin microbial production. Full-length cDNA encoding UGT85A47 was isolated from Japanese apricot after 5?- and 3?-RACE. Recombinant UGT85A47 stoichiometrically catalyzed UDP-glucose consumption and synthesis of prunasin and UDP from mandelonitrile. Escherichia coli C41(DE3) cells expressing UGT85A47 produced prunasin (0.64 g/L) from racemic mandelonitrile and glucose. In addition, co-expression of genes encoding UDP-glucose biosynthetic enzymes (phosphoglucomutase and UTP-glucose 1-phosphate uridiltransferase) and polyphosphate kinase clearly improved prunasin production up to 2.3 g/L. These results showed that our whole-cell biocatalytic system is significantly more efficient than the existing prunasin production systems, such as chemical synthesis.     

Purification and characterization of the NADH:ferredoxinBPH oxidoreductase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400

Mucor circinelloides LU M40 and Penicillium aurantiogriseum P 35 produce extracellular β-glycosidases that are active on the cyanogenic glycoside amygdalin. From the culture broths of M. circinelloides, only one β-glycosidase could be identified, while two different enzymes – both having amygdalase activity – were found in culture broths of P. aurantiogriseum. The study of the mechanism of hydrolysis of the β-bis-glycoside amygdalin with purified enzymes from the two organisms indicated a possible sequential (two-step) reaction. In all cases, the first step of hydrolysis from amygdalin to prunasin was very rapid, while the second step from prunasin to cyanohydrin was much slower. No cyanohydrin lyase activity was found in the culture broths of either fungus. Received: 16 May 1997 / Accepted: 11 September 1997     

The enzymic hydrolysis of amygdalin

Chromatographic examination has shown that the enzymic hydrolysis of amygdalin by an almond beta-glucosidase preparation proceeds consecutively: amygdalin was hydrolysed to prunasin and glucose; prunasin to mandelonitrile and glucose; mandelonitrile to benzaldehyde and hydrocyanic acid. Gentiobiose was not formed during the enzymic hydrolysis. The kinetics of the production of mandelonitrile and hydrocyanic acid from amygdalin by the action of the beta-glucosidase preparation favour the probability that three different enzymes are involved, each specific for one hydrolytic stage, namely, amygdalin lyase, prunasin lyase and hydroxynitrile lyase. Cellulose acetate electrophoresis of the enzyme preparation showed that it contained a number of enzymically active components.     

Probing behavior of bird cherry-oat aphid Rhopalosiphum padi (L.) on native bird cherry Prunus padus L. and alien invasive black cherry Prunus serotina Erhr. in Europe and the role of cyanogenic glycosides

The probing behavior of bird cherry-oat aphid Rhopalosiphum padi was studied on its natural winter host in Europe, the bird cherry Prunus padus, and on the invasive black cherry Prunus serotina, on which spring generations of R. padi do not survive. The EPG-recorded behavior of R. padi on bird cherry and black cherry showed differences in crucial aspects of probing and feeding. The period of the pre-phloem penetration was twice as long and rarely interrupted in aphids on bird cherry as opposed to aphids on black cherry. On black cherry, there was a considerable delay between finding and accepting the phloem. Aphids that had sampled phloem sap either refused to ingest it or the ingestion periods were very short. Amygdalin and prunasin (cyanogenic glycosides present in leaves of Prunus) seriously impeded ingestion activities when applied in pure sucrose diet. The role of amygdalin and prunasin in winter host plant selection and host alternation in R. padi is discussed.     

Cyanogenic glycosides in leaves of Perilla frutescens var. acuta

Besides 7-(2-O-β-D-glucuronyl-β-D-glucuronyloxy)-5,3′,4′-trihydroxyflavone, scutellarin, rosmarinic acid and caffeic acid, two cyanogenic glycosides have been isolated from the dried leaves of Perilla frutescens var. acuta. One of them is prunasin and the other is (R)-2-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyloxy)-phenylacetonitrile, a new isomer of amygdalin.     

Bitterness in almonds

Bitterness in almond (Prunus dulcis) is determined by the content of the cyanogenic diglucoside amygdalin. The ability to synthesize and degrade prunasin and amygdalin in the almond kernel was studied throughout the growth season using four different genotypes for bitterness. Liquid chromatography-mass spectrometry analyses showed a specific developmentally dependent accumulation of prunasin in the tegument of the bitter genotype. The prunasin level decreased concomitant with the initiation of amygdalin accumulation in the cotyledons of the bitter genotype. By administration of radiolabeled phenylalanine, the tegument was identified as a specific site of synthesis of prunasin in all four genotypes. A major difference between sweet and bitter genotypes was observed upon staining of thin sections of teguments and cotyledons for beta-glucosidase activity using Fast Blue BB salt. In the sweet genotype, the inner epidermis in the tegument facing the nucellus was rich in cytoplasmic and vacuolar localized beta-glucosidase activity, whereas in the bitter cultivar, the beta-glucosidase activity in this cell layer was low. These combined data show that in the bitter genotype, prunasin synthesized in the tegument is transported into the cotyledon via the transfer cells and converted into amygdalin in the developing almond seed, whereas in the sweet genotype, amygdalin formation is prevented because the prunasin is degraded upon passage of the beta-glucosidase-rich cell layer in the inner epidermis of the tegument. The prunasin turnover may offer a buffer supply of ammonia, aspartic acid, and asparagine enabling the plants to balance the supply of nitrogen to the developing cotyledons.     

Utilization of Amygdalin during Seedling Development of Prunus serotina

Cotyledons of mature black cherry (Prunus serotina Ehrh.) seeds contain the cyanogenic diglucoside (R)-amygdalin. The levels of amygdalin, its corresponding monoglucoside (R)-prunasin, and the enzymes that metabolize these cyanoglycosides were measured during the course of seedling development. During the first 3 weeks following imbibition, cotyledonary amygdalin levels declined by more than 80%, but free hydrogen cyanide was not released to the atmosphere. Concomitantly, prunasin, which was not present in mature, ungerminated seeds, accumulated in the seedling epicotyls, hypocotyls, and cotyledons to levels approaching 4 [mu]mol per seedling. Whether this prunasin resulted from amygdalin hydrolysis remains unclear, however, because these organs also possess UDPG:mandelonitrile glucosyltransferase, which catalyzes de novo prunasin biosynthesis. The reduction in amygdalin levels was paralleled by declines in the levels of amygdalin hydrolase (AH), prunasin hydrolase (PH), mandelonitrile lyase (MDL), and [beta]-cyanoalanine synthase. At all stages of seedling development, AH and PH were localized by immunocytochemistry within the vascular tissues. In contrast, MDL occurred mostly in the cotyledonary parenchyma cells but was also present in the vascular tissues. Soon after imbibition, AH, PH, and MDL were found within protein bodies but were later detected in vacuoles derived from these organelles.     

On the metabolism of amygdalin. 2. The distribution of beta-glucosidase activity and orally administered amygdalin in rats

The organs of 15-day-old rats had the highest capability to hydrolyze amygdalin and prunasin, and most of this activity is concentrated in the tissues of the small and large intestines. The activity decreased with age. In adult rats, the ability of the organs to hydrolyze prunasin is higher than that of amygdalin and is concentrated in the spleen, large intestine, and kidney (35.0, 15.0, and 8.9 micrograms prunasin hydrolyzed . h-1 . g tissue-1). Minced tissues of the liver, spleen, kidney, and stomach contain more hydrolytic capability than the homogenate of these organs, while the reverse is the case with the small and large intestines. When 30 mg amygdalin was orally administered to adult rats, its distribution after the 1st h was as follows: stomach (0.89 mg), small intestine (0.78 mg), spleen (0.36 mg), large intestine (0.30 mg), kidney (0.19 mg), liver (0.10 mg), and serum (5.6 micrograms/mL). At the end of the 2nd h, the highest amygdalin content was found in the large intestine (0.79 mg).     

Phenylalanine derived cyanogenic diglucosides from Eucalyptus camphora and their abundances in relation to ontogeny and tissue type

The cyanogenic glucoside profile of Eucalyptus camphora was investigated in the course of plant ontogeny. In addition to amygdalin, three phenylalanine-derived cyanogenic diglucosides characterized by unique linkage positions between the two glucose moieties were identified in E. camphora tissues. This is the first time that multiple cyanogenic diglucosides have been shown to co-occur in any plant species. Two of these cyanogenic glucosides have not previously been reported and are named eucalyptosin B and eucalyptosin C. Quantitative and qualitative differences in total cyanogenic glucoside content were observed across different stages of whole plant and tissue ontogeny, as well as within different tissue types. Seedlings of E. camphora produce only the cyanogenic monoglucoside prunasin, and genetically based variation was observed in the age at which seedlings initiate prunasin biosynthesis. Once initiated, total cyanogenic glucoside concentration increased throughout plant ontogeny with cyanogenic diglucoside production initiated in saplings and reaching a maximum in flower buds of adult trees. The role of multiple cyanogenic glucosides in E. camphora is unknown, but may include enhanced plant defense and/or a primary role in nitrogen storage and transport.     

Cyanogenic polymorphism in Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill (Myrtaceae)

Plant cyanogenesis, the release of cyanide from endogenous cyanide-containing compounds, is an effective herbivore deterrent. This paper characterises cyanogenesis in the Australian tree Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill for the first time. The cyanogenic glucoside prunasin ((R)-mandelonitrile β-D-glucoside) was determined to be the only cyanogenic compound in E. polyanthemos foliage. Two natural populations of E. polyanthemos showed quantitative variation in foliar prunasin concentration, varying from zero (i.e. acyanogenic) to 2.07 mg CN g^-1 dry weight in one population and from 0.17 to 1.98 mg CN g^-1 dry weight in the other. No significant difference was detected between the populations with respect to the mean prunasin concentration or the degree of variation in foliar prunasin, despite significant differences in foliar nitrogen. Variation between individuals was also observed with respect to the capacity of foliage to catabolise prunasin to form cyanide. Moreover, variation in this capacity generally correlated with the amount of prunasin in the tissue, suggesting genetic linkage between prunasin and β-glucosidase.     

Exolytic hydrolysis of toxic plant glucosides by guinea pig liver cytosolic beta-glucosidase.

We demonstrate that although the guinea pig liver cytosolic beta-glucosidase does not catalyze the hydrolysis of gentiobiose, it does hydrolyze, disaccharide-containing glycosides such as p-nitrophenyl-beta-D-gentiobioside (Glc beta 1----6Glc beta-pNP) and mandelonitrile-beta-D-gentiobioside (amygdalin). Furthermore, we establish that the enzyme attacks disaccharide glycosides exolytically; specifically, we document the exolytic deglucosylation of amygdalin and the generation of the intermediate monosaccharide glycoside mandelonitrile-beta-D-glucoside prior to the formation of the aglycone (mandelonitrile). We also show that the cytosolic beta-glucosidase catalyzes the hydrolysis of various phenolic (e.g. arbutin and salicin) and cyanogenic plant glucosides (e.g. prunasin). Using the everted gut-sack technique, we demonstrate that the plant glucosides, amygdalin, prunasin, and vicine, are transported across the small intestine of the guinea pig efficiently and without being hydrolyzed. Based on these data we speculate that the cytosolic beta-glucosidase may participate in biotransformation of toxic plant glucosides.     

Behavior,Parasitism, Morphology,and Biochemistry of Criconemella xenoplax and C. ornata on Peach

Host-parasite relationships of Criconemella xenoplax and C. ornata on Nemaguard peach and common bermudagrass were determined in the greenhouse. Criconemella xenoplax reproduced on peach and reduced root volume, height, and dry stem weight after 6 months, compared with the noninfested check. Numbers of C. ornata did not increase on peach or influence peach growth, but they did reduce dry top weight and root volume of common bermudagrass, compared with C. xenoplax. Criconemella xenoplax and C. ornata produced the enzyme β-glucosidase and were capable of metabolizing prunasin, but only C. xenoplax produced β-cyanoalanine synthase to detoxify the cyanide released from prunasin. The apparent inability of C. ornata to detoxify cyanide is one explanation why numbers of this species did not increase on peach. Criconemella xenoplax and C. ornata can be distinguished by using stylet length, vaginal configuration, and shape of the anterior head region.     

Cyanogenic glycosides from the rare Australian endemic rainforest tree Clerodendrum grayi (Lamiaceae)

The cyanogenic diglycoside lucumin ((R)-mandelonitrile-β-d-primeveroside) and monoglucoside prunasin ((R)-mandelonitrile-β-d-glucoside) were isolated from the foliage of the rare Australian rainforest tree species Clerodendrum grayi (Lamiaceae). This is the first reported isolation of the diglycoside lucumin from vegetative tissue (foliage), and the first reported co-occurrence of lucumin and prunasin. Furthermore, unusually, the diglycoside lucumin was the most abundant cyanogen accounting for approximately 60% of total cyanide in a leaf tissue.     

In silico authentication of amygdalin as a potent anticancer compound in the bitter kernels of family Rosaceae

Amygdalin a naturally occurring compound, predominantly in the bitter kernels of apricot, almond, apple and other members of Rosaceae family. Though, amygdalin is used as an alternative therapy to treat various types of cancer but its role in cancer pathways has rarely been explored yet. Therefore, present study was intended with the aim to investigate the alleged anti-cancerous effects of amygdalin specifically on PI3K–AKT–mTOR and Ras pathways of cancer in human body. Computational modelling and simulation techniques were used to assess the effect of amygdalin on PI3K-AKT-mTOR and Ras pathways using different level of dosage. It was observed that amygdalin had direct and substantial contribution to regulate PI3K-mTOR activities on threshold levels while the other caner pathways were effected indirectly. Consequently, amygdalin is a down-regulator of a cancer within a specified amount and contribute considerably to reduce various types of cancer in human. Furthermore, in-vitro and in-vivo analyses of amygdalin could be of helpful to authenticate its pharmacological effects.     

Prunasin hydrolases during fruit development in sweet and bitter almonds

Amygdalin is a cyanogenic diglucoside and constitutes the bitter component in bitter almond (Prunus dulcis). Amygdalin concentration increases in the course of fruit formation. The monoglucoside prunasin is the precursor of amygdalin. Prunasin may be degraded to hydrogen cyanide, glucose, and benzaldehyde by the action of the β-glucosidase prunasin hydrolase (PH) and mandelonitirile lyase or be glucosylated to form amygdalin. The tissue and cellular localization of PHs was determined during fruit development in two sweet and two bitter almond cultivars using a specific antibody toward PHs. Confocal studies on sections of tegument, nucellus, endosperm, and embryo showed that the localization of the PH proteins is dependent on the stage of fruit development, shifting between apoplast and symplast in opposite patterns in sweet and bitter cultivars. Two different PH genes, Ph691 and Ph692, have been identified in a sweet and a bitter almond cultivar. Both cDNAs are 86% identical on the nucleotide level, and their encoded proteins are 79% identical to each other. In addition, Ph691 and Ph692 display 92% and 86% nucleotide identity to Ph1 from black cherry (Prunus serotina). Both proteins were predicted to contain an amino-terminal signal peptide, with the size of 26 amino acid residues for PH691 and 22 residues for PH692. The PH activity and the localization of the respective proteins in vivo differ between cultivars. This implies that there might be different concentrations of prunasin available in the seed for amygdalin synthesis and that these differences may determine whether the mature almond develops into bitter or sweet.