Analysis of Bitter Almonds and Processed Products Based on HPLC-Fingerprints and Chemometry.

In the processing field, there is a saying that "seed drugs be stir-fried". Bitter almond (BA) is a kind of seed Chinese medicine. BA need be used after being fried. To distinguish raw bitter almonds (RBA) from processed products and prove the rationality of "seed drugs be stir-fried", we analyzed the RBA and five processed products (scalded bitter almonds, fried bitter almonds, honey fried bitter almonds, bran fried bitter almonds, bitter almonds cream) using RP-HPLC fingerprints and chemometric methods. The similarity between RBA and processed products was 0.733∼0.995. Hierarchically clustered heatmap was used to evaluate the changes in components. Principal component analysis (PCA) was used for classification, and all samples are distinguished according to RBA and five processing methods. Six chemical markers were obtained by partial least squares discriminant analysis (PLS-DA). The content and degradation rate of amygdalin and ß-glucosidase activity were determined. Compared with RBA, the content and degradation rate of amygdalin, and ß-glucosidase activity were increased in bitter almonds cream. The content and degradation rate were decreased, and ß-glucosidase was inactivated in other processed products. The above results showed that stir-frying had the best effect. The results showed that processing can ensure the stability of RBA quality, and the saying "seed drugs be stir-fried" is reasonable.

[Separation, characterization, and antiviral activity of colloidal phase state of Maxing Shigan Decoction].

This study focused on the separation, characterization, content determination, and antiviral efficacy research on colloidal particles with different sizes in Maxing Shigan Decoction(MXSG). The mixed colloidal phase of MXSG was initially separated into small colloidal particle segment(S), medium colloidal particle segment(M), and big colloidal particle segment(B) using ultrafiltration. Further fine separation was performed using size-exclusion chromatography. Dynamic light scattering(DLS) and transmission electron microscopy(TEM) were employed to characterize the size and morphology of the separated colloidal particles. UPLC-MS/MS was used to determine the content of ephedrine, amygdalin, glycyrrhizic acid, and the EDTA complexometric titration was used to measure the calcium(Ca~(2+)) content in different colloidal phases. Finally, a respiratory syncytial virus(RSV) infection mouse model was established using intranasal administration. The experimental groups included a blank group, a model group, a ribavirin group, an MXSG group, an S group, an M group, and a B group. Oral administration was given for treatment, and pathological changes in mouse lung tissue and organ indices were evaluated. The results of the study showed that the distribution of ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) content was not uniform among different colloidal segments. Among them, the B segment had the highest proportions of the three components, except for Ca~(2+), accounting for 46.35%, 53.72%, and 92.36%, respectively. Size-exclusion chromatography separated colloidal particles with uniform morphology in the size range of 100-500 nm. Compared to the S and M segments, the B segment showed an increased lung index inhibition rate(38.31%), spleen index, and thymus index in RSV-infected mice, and it improved the infiltration of inflammatory cells and lung injury in the lung tissue of mice. The complex components in MXSG form colloidal particles of various sizes and morphologies through heating, and small-molecule active components such as ephedrine, amygdalin, glycyrrhizic acid, and Ca~(2+) participate in the assembly to varying degrees. The main material basis for the antiviral effect of MXSG is the colloidal particles with certain particle sizes formed by the assembly of active components during the heating process.

Amygdalin: Toxicity, Anticancer Activity and Analytical Procedures for Its Determination in Plant Seeds.

Amygdalin (d-Mandelonitrile 6-O-ß-d-glucosido-ß-d-glucoside) is a natural cyanogenic glycoside occurring in the seeds of some edible plants, such as bitter almonds and peaches. It is a medically interesting but controversial compound as it has anticancer activity on one hand and can be toxic via enzymatic degradation and production of hydrogen cyanide on the other hand. Despite numerous contributions on cancer cell lines, the clinical evidence for the anticancer activity of amygdalin is not fully confirmed. Moreover, high dose exposures to amygdalin can produce cyanide toxicity. The aim of this review is to present the current state of knowledge on the sources, toxicity and anticancer properties of amygdalin, and analytical methods for its determination in plant seeds.

Inflammation, It's Regulation and Antiphlogistic Effect of the Cyanogenic Glycoside Amygdalin.

The inflammatory reaction accompanies in part or in full any disease process in the vascularized metazoan. This complicated reaction is controlled by regulatory mechanisms, some of which produce unpleasant symptomatic manifestations of inflammation. Therefore, there has been an effort to develop selective drugs aimed at removing pain, fever, or swelling. Gradually, however, serious adverse side effects of such inhibitors became apparent. Scientific research has therefore continued to explore new possibilities, including naturally available substances. Amygdalin is a cyanogenic glycoside present, e.g., in bitter almonds. This glycoside has already sparked many discussions among scientists, especially about its anticancer potential and related toxic cyanides. However, toxicity at different doses made it generally unacceptable. Although amygdalin given at the correct oral dose may not lead to poisoning, it has not yet been accurately quantified, as its action is often affected by different intestinal microbial consortia. Its pharmacological activities have been studied, but its effects on the body's inflammatory response are lacking. This review discusses the chemical structure, toxicity, and current knowledge of the molecular mechanism of amygdalin activity on immune functions, including the anti-inflammatory effect, but also discusses inflammation as such, its mediators with diverse functions, which are usually targeted by drugs.

Bitter apricot ethanolic extract induces apoptosis through increasing expression of Bax/Bcl-2 ratio and caspase-3 in PANC-1 pancreatic cancer cells.

Pancreatic cancer is the fourth common cause of cancer death. Surgery and chemotherapy are the common treatment strategies for pancreatic cancer patients; however, the response rate is less than 20% at advanced stages. In recent years, growing interest has been dedicated to natural products. Bitter apricot seeds possess a number of pharmacological properties including antitumor activity and amygdalin from bitter apricot seeds can induce apoptosis. In this study, we investigated the cyto/genotoxic effects of bitter apricot ethanolic extract (BAEE) and amygdalin on human pancreatic cancer PANC-1 and normal epithelial 293/KDR cells. BAEE was assessed using high-performance liquid chromatography for the confirmation of the structure. The biological impacts of BAEE and amygdalin on PANC-1 and 293/KDR cells were evaluated by MTT assay, DAPI staining, AnnexinV/PI and Real-time qPCR analysis. BAEE and amygdalin inhibited cancer cell growth in a dose- and time-dependent manner. DAPI staining and flow cytometric analysis revealed fragmented nuclei and elevated numbers of early and late apoptotic cells, respectively. Also, increased Bax/Bcl-2 ratio and upregulation of caspase-3 further confirmed the occurrence of apoptosis in PANC-1 cells, but not in non-cancerous 293/KDR cells. These results indicate that BAEE could mediate apoptosis induction in cancer cells through a mitochondria dependent pathway. These findings suggest that BAEE functions as a potent pro-apoptotic factor for human pancreatic cancer cells without a significant effect on 293/KDR cells. Though, the potent anti-cancer components of BAEE should be further identified. Moreover, in vivo investigations are required to confirm bitter apricot ethanolic extract's clinical value as an anti-tumor drug.

Discovery of a Cryptic Depsipeptide from Streptomyces ghanaensis via MALDI-MS-Guided High-Throughput Elicitor Screening.

Microbial genomes harbor an abundance of biosynthetic gene clusters, but most are expressed at low levels and need to be activated for characterization of their cognate natural products. In this work, we report the combination of high-throughput elicitor screening (HiTES) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the rapid identification of cryptic peptide natural products. Application to Streptomyces ghanaensis identified amygdalin as an elicitor of a novel non-ribosomal peptide, which we term cinnapeptin. Complete structural elucidation revealed cinnapeptin as a cyclic depsipeptide with an unusual 2-methyl-cinnamoyl group. Insights into its biosynthesis were provided by whole genome sequencing and gene deletion studies, while bioactivity assays showed antimicrobial activity against Gram-positive bacteria and fission yeast. MALDI-HiTES is a broadly applicable tool for the discovery of cryptic peptides encoded in microbial genomes.

Elucidation of the Amygdalin Pathway Reveals the Metabolic Basis of Bitter and Sweet Almonds (Prunus dulcis).

Almond (Prunus dulcis) is the principal Prunus species in which the consumed and thus commercially important part of the fruit is the kernel. As a result of continued selection, the vast majority of almonds have a nonbitter kernel. However, in the field, there are trees carrying bitter kernels, which are toxic to humans and, consequently, need to be removed. The toxicity of bitter almonds is caused by the accumulation of the cyanogenic diglucoside amygdalin, which releases toxic hydrogen cyanide upon hydrolysis. In this study, we identified and characterized the enzymes involved in the amygdalin biosynthetic pathway: PdCYP79D16 and PdCYP71AN24 as the cytochrome P450 (CYP) enzymes catalyzing phenylalanine-to-mandelonitrile conversion, PdUGT94AF3 as an additional monoglucosyl transferase (UGT) catalyzing prunasin formation, and PdUGT94AF1 and PdUGT94AF2 as the two enzymes catalyzing amygdalin formation from prunasin. This was accomplished by constructing a sequence database containing UGTs known, or predicted, to catalyze a ß(1→6)-O-glycosylation reaction and a Basic Local Alignment Search Tool search of the draft version of the almond genome versus these sequences. Functional characterization of candidate genes was achieved by transient expression in Nicotiana benthamiana Reverse transcription quantitative polymerase chain reaction demonstrated that the expression of PdCYP79D16 and PdCYP71AN24 was not detectable or only reached minute levels in the sweet almond genotype during fruit development, while it was high and consistent in the bitter genotype. Therefore, the basis for the sweet kernel phenotype is a lack of expression of the genes encoding the two CYPs catalyzing the first steps in amygdalin biosynthesis.

Reconfigured Cyanogenic Glucoside Biosynthesis in Eucalyptus cladocalyx Involves a Cytochrome P450 CYP706C55.

Cyanogenic glucosides are a class of specialized metabolites widespread in the plant kingdom. Cyanogenic glucosides are α-hydroxynitriles, and their hydrolysis releases toxic hydrogen cyanide, providing an effective chemical defense against herbivores. Eucalyptus cladocalyx is a cyanogenic tree, allocating up to 20% of leaf nitrogen to the biosynthesis of the cyanogenic monoglucoside, prunasin. Here, mass spectrometry analyses of E. cladocalyx tissues revealed spatial and ontogenetic variations in prunasin content, as well as the presence of the cyanogenic diglucoside amygdalin in flower buds and flowers. The identification and biochemical characterization of the prunasin biosynthetic enzymes revealed a unique enzyme configuration for prunasin production in E. cladocalyx This result indicates that a multifunctional cytochrome P450 (CYP), CYP79A125, catalyzes the initial conversion of l-phenylalanine into its corresponding aldoxime, phenylacetaldoxime; a function consistent with other members of the CYP79 family. In contrast to the single multifunctional CYP known from other plant species, the conversion of phenylacetaldoxime to the α-hydroxynitrile, mandelonitrile, is catalyzed by two distinct CYPs. CYP706C55 catalyzes the dehydration of phenylacetaldoxime, an unusual CYP reaction. The resulting phenylacetonitrile is subsequently hydroxylatedby CYP71B103 to form mandelonitrile. The final glucosylation step to yield prunasin is catalyzed by a UDP-glucosyltransferase, UGT85A59. Members of the CYP706 family have not been reported previously to participate in the biosynthesis of cyanogenic glucosides, and the pathway structure in E. cladocalyx represents an example of convergent evolution in the biosynthesis of cyanogenic glucosides in plants.

In vitro and in silico anticancer activity of amygdalin on the SK-BR-3 human breast cancer cell line.

In spite of several studies that have shown the cytotoxic effects of amygdalin on the different cancer cell lines, however, the chemopreventive potential of amygdalin on the breast cancer cell line is not completely understood. We investigated the effect of amygdalin on the cell death and the level of pro-apoptotic Bax protein and anti-apoptotic Bcl-2 protein in SK-BR-3 human breast cancer cell line. The cell viability of SK-BR-3 cells was evaluated by MTT assay in different concentration of amygdalin. The level of Bax and Bcl-2 in SK-BR-3 cells were measured by western blot analysis. For statistical analysis, One-way ANOVA was used for the comparison of Bax and Bcl-2 protein level and percent of cell viability between groups. The molecular docking studies of amygdalin within the Bcl-2 (PDB ID: 4LVT) and HER2 (PDB ID: 3RCD) active site, were performed using AutoDock 4.2.5. Amygdalin induced a significant reduction of cell viability in SK-BR-3 after 24-h treatment in a dose-dependent manner. Also, amygdalin causes an increase in pro-apoptotic Bax protein and a decrease in anti-apoptotic Bcl-2 protein expression in the SK-BR-3 cells. Molecular docking studies showed that amygdalin interacts with the active site amino acids of Bcl-2 and HER2 through hydrogen bonding and some hydrophobic interactions. Amygdalin can induce apoptotic death in SK-BR-3 cells by increasing pro-apoptotic Bax protein and decreasing anti-apoptotic Bcl-2 protein expression. The results suggest that amygdalin may be a valuable candidate for the treatment of breast cancer, especially in HER2 positive cells.

A "turn off-on" fluorescent nanoprobe consisting of CuInS2 quantum dots for determination of the activity of ß-glucosidase and for inhibitor screening.

A fluorescent "turn off-on" nanoprobe is described for highly sensitive and selective determination of the activity of the enzyme ß-glucosidase (ß-Glu). Firstly, cysteine modified CuInS2 quantum dots (Cys-CuInS2 QDs) were prepared from indium(III) and copper(II) salts and the presence of thiourea. The red fluorescence of the Cys-CuInS2 QDs, with excitation/emission maxima at 590/656 nm, is quenched by Cu(II). However, in the presence of ß-Glu and the cyanogenic glycoside, enzymatic hydrolysis leads to the formation of cyanide. The latter competitively binds to Cu(II) owing to its high affinity for cyanide. This restores the fluorescence of the Cys-CuInS2 QDs. Under the optimum conditions, fluorescence increases linearly in the 0.5-700 U·L-1 ß-Glu activity range. The detection limit is 0.2 U·L-1. The nanoprobe was applied to analyze spiked soil samples, and satisfactory results were obtained. The average recoveries of ß-Glu were in the range of 96-103%, and the RSD was lower than 4.0%. The fluorescent probe can also be used to screen for ß-Glu inhibitors as demonstrated for castanospermine as an example. Graphical abstractSchematic representation of the fluorescent nanoprobe for ß-glucosidase activity detection and inhibitor screening by taking advantage of the fluorescence (FL) "turn-off" and "turn-on" feature of cysteine capped CuInS2 quantum dots (Cys-CuInS2 QDs).

Potential toxicity of cyanogenic glycoside amygdalin and bitter apricot seed in rabbits-Health status evaluation.

Amygdalin is one of the most studied secondary metabolites of Prunus genus. It is a cyanogenic glycoside which was initially obtained from the bitter almonds seeds and is a major component of the seeds of plants, such as apricots, almonds, peaches, apples and other rosaceous plants. The views of scientists on the use of amygdalin have been contradictory for many years, partly because toxicokinetics and metabolism of amygdalin still have not been adequately explored. The present in vivo study was designed to reveal whether pure amygdalin intramuscularly injected or apricot seeds oral consumption induce changes in overall health status of rabbit as a biological model. A total of 60 adult rabbits were randomly divided into five groups. The control group received no amygdalin while the two experimental groups E1 and E2 received a daily intramuscular injection of amygdalin at doses 0.6 and 3.0 mg/kg bw. The experimental groups E3 and E4 were fed crushed bitter apricot seeds (Prunus armeniaca L.), at doses 60 and 300 mg/kg bw, mixed with commercial feed for rabbits. Blood collection was carried out after 14 days. Biochemical, haematological and antioxidant enzymes activity analysis were performed and statistically evaluated. A short-term amygdalin administration had negligible impact on biochemical parameters-mainly level of urea, bilirubin, cholesterol. Haematological profile of rabbits was influenced very slightly-non-significant platelet count and platelet percentage increase, erythrocytes count and haemoglobin decrease. SOD activity of rabbits decreased significantly (p > 0.05) after apricot seeds consumption (102.3 U/ml) in comparison to control (117.4 U/ml). Differences might be connected to diverse metabolism by different administration routes and at the same time by the presence of other substances in apricot seeds (phytosterols, polyphenols, fatty acids). However, a short-term consumption had only slight effect on health status of rabbits and at recommended doses did not represent risk for their health.

Identification and Analysis of Amygdalin, Neoamygdalin and Amygdalin Amide in Different Processed Bitter Almonds by HPLC-ESI-MS/MS and HPLC-DAD.

Processing is a traditional pharmacy technology based on traditional Chinese medicine theory. The traditional Chinese medicine (TCM) ingredients should be processed before being used as a medicine. Processed bitter almonds are widely used in the clinic in TCM for the treatment of cough and asthma. In this work the amygdalin profile of three producing areas in China was determined, with respect to three differently processed bitter almond products: raw, stir-fried and scalded. Identification of the compounds was done by using high performance liquid chromatography coupled to electrospray ionization mass spectrometry (HPLC-ESI-MS/MS). Results indicated that amygdalin, neoamygdalin and amygdalin amide were identified in the different processed bitter almonds. Meanwhile, amygdalin was used as a standard to calculate the quantification of amygdalin and the concentration ratio of neoamygdalin and total amygdalin by HPLC-DAD. The data suggested that composition of amygdalin isomers in bitter almonds was influenced by the processing method. It also gives a new understanding of the processing principle of bitter almonds. Moreover, the classification of different processed bitter almonds can be achieved on the basis of amygdalin isomers levels.

Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats via inhibiting MMP2/9, TIMP1/2 and the TGF-ß/Smad signaling pathways.

AIM: Chinese medicine CGA formula consists of polysaccharide from Cordyceps sinensis mycelia (CS-PS), gypenosides and amygdalin, which is derived from Fuzheng Huayu (FZHY) capsule for treating liver fibrosis. In this study we attempted to confirm the therapeutic effects of CGA formula in dimethylnitrosamine (DMN)-induced liver fibrosis in rats, and to identify the mechanisms of anti-fibrotic actions. METHODS: Rats were injected with DMN (10 mg·kg(-1)·d(-1), ip) for 3 consecutive days per week over a 4-week period. The rats then were orally administered with CGA formula (CS-PS 60 mg·kg(-1)·d(-1), gypenosides 50 mg·kg(-1)·d(-1) and amygdalin 80 mg·kg(-1)·d(-1)) daily in the next 2 weeks. CS-PS, gypenosides or amygdalin alone were administered as individual component controls, whereas colchicine and FZHY were used as positive controls. Serum biomarkers were measured. Hepatic injury, collagen deposition and stellate cell activation were examined. The MMP activities, expression of TIMP protein and proteins involved in the TGF-ß1/Smad signaling pathways in liver tissues were assayed. RESULTS: In DMN-treated rats, administration of CGA formula significantly decreased serum ALT, AST and total bilirubin and hepatic hydroxyproline levels, increased serum albumin level, and attenuated liver fibrosis as shown by histological examination. Furthermore, these effects were comparable to those caused by administration of FZHY, and superior to those caused by administration of colchicine or the individual components of CGA formula. Moreover, administration of CGA formula significantly decreased the protein levels of α-SMA, TGF-ß1, TGF-ß1 receptor (TßR-I), p-TßR-I, p-TßR-II, p-Smad2, p-Smad3, TIMP1 and TIMP2, as well as MMP2 and MMP9 activities in liver tissues of DMN-treated rats. CONCLUSION: Chinese medicine CGA formula ameliorates DMN-induced liver fibrosis in rats, and this effect was likely associated with the down-regulation of MMP2/9 activities, TIMP1/2 protein expression and the TGF-ß1/Smad signaling pathways in the liver.

[Amygdalin - structure and clinical significance]. / Amigdalina - budowa i znaczenie kliniczne.

In this publication we described amygdalin. It was isolated for the first time in the 19th century. Amygdalin is called interchangeably vitamin B17 or laetrile. Since more than a hundred years, there has been reports about its unique anticancer properties. We tried to introduce the present knowledge about therapeutic efficacy of laetrile. Most of these studies has been made in the in vitro environment. The lack of appropriate studies forced scientists to examine the positive influence of amygdalin on many diseases like: bladder cancer, prostate cancer, cervical cancer, colon cancer, promyelocytic leukemia, chronic kidney disease, psoriasis and other.

Quantitative analysis of amygdalin and prunasin in Prunus serotina Ehrh. using (1) H-NMR spectroscopy.

INTRODUCTION: Prunus serotina is native to North America but has been invasively introduced in Europe since the seventeenth century. This plant contains cyanogenic glycosides that are believed to be related to its success as an invasive plant. For these compounds, chromatographic- or spectrometric-based (targeting on HCN hydrolysis) methods of analysis have been employed so far. However, the conventional methods require tedious preparation steps and a long measuring time. OBJECTIVE: To develop a fast and simple method to quantify the cyanogenic glycosides, amygdalin and prunasin in dried Prunus serotina leaves without any pre-purification steps using (1) H-NMR spectroscopy. METHODS: Extracts of Prunus serotina leaves using CH3 OH-d4 and KH2 PO4 buffer in D2 O (1:1) were quantitatively analysed for amygdalin and prunasin using (1) H-NMR spectroscopy. Different internal standards were evaluated for accuracy and stability. The purity of quantitated (1) H-NMR signals was evaluated using several two-dimensional NMR experiments. RESULTS: Trimethylsilylpropionic acid sodium salt-d4 proved most suitable as the internal standard for quantitative (1) H-NMR analysis. Two-dimensional J-resolved NMR was shown to be a useful tool to confirm the structures and to check for possible signal overlapping with the target signals for the quantitation. Twenty-two samples of P. serotina were subsequently quantitatively analysed for the cyanogenic glycosides prunasin and amygdalin. CONCLUSION: The NMR method offers a fast, high-throughput analysis of cyanogenic glycosides in dried leaves permitting simultaneous quantification and identification of prunasin and amygdalin in Prunus serotina.

Formation of amygdalin/ß-cyclodextrin derivatives inclusion complexes for anticancer activity assessment in human cervical carcinoma HeLa cell line.

Nanoencapsulation has gained considerable attention because of its unique features and advantages in anticancer drug delivery. Amygdalin (AMY) is an anticancer compound, showing limitations in its applications by low stability. Herein, the inclusion complexes (ICs) of AMY with ß-cyclodextrin (ßCD), and its derivatives such as 2-hydroxypropyl-ßCD (HPßCD) and methyl-ßCD (MßCD) were fabricated. The fabricated AMY/CD-ICs were thoroughly evaluated using Fourier-transform infrared spectroscopy, powder X-ray diffraction, thermogravimetric/differential thermal analysis, proton nuclear magnetic resonance, ultraviolet-visible diffuse reflectance spectroscopy, and photoluminescence techniques. Double reciprocal profile study of the absorption and fluorescence spectra revealed that the AMY formed the ICs with ßCD derivatives at a guest/host stoichiometric ratio of 1/1. The thermal stability of AMY was enhanced as the IC formation aid observed by the shift of thermal degradation temperature of AMY from the range of âˆ¼ 220-250 °C to > 295 °C. Theoretical analyses of the energetic, electronic, and global reactivity parameters of the AMY/CD-ICs were evaluated using the PM3 method. Further assessment of the dissolution diagrams of AMY/CD-ICs revealed a burst release profile. In addition, cell toxicity was evaluated using the MTT assay, and the results showed that AMY/CD-ICs had significantly more efficacious in inhibiting HeLa cancer cells than AMY. These results proved that the IC formations with CDs significantly enhanced the anticancer activity of AMY.

Anti-tumoral Immunity and Chemo-preventive Effectiveness of Herbal Extracts of Curcumin, Ginger, Clove and Amygdaline in Ehrlich Ascites Carcinoma-Challenging Mice.

BACKGROUND: Due to its systemic toxicity, traditional chemotherapy of tumors is being taken into consideration. Herbal therapy, containing phytochemical polyphenol derivatives such as Curcumin (Cur), Ginger (Gin), Cloves (Clov) and Amygdaline (Amyg), is one of the numerous complementary and alternative approaches as an anti-cancer therapy and holds great promise for cancer chemo-prevention with fewer side effects. AIM: The current study was designated to assess anti-tumoral immunity and anti-cancer and chemo-preventive effectiveness of herbal extracts of Cur, Ginger, Clov and Amyg in Ehrlich Ascites Carcinoma (EAC)-challenging mice. METHODS: Chemo-preventive efficacy of herbal extracts of Cur, Gin, Clov and Amyg were analyzed in vivo by examination of the apoptosis rate of EAC tumor cells by flow cytometry. The total numbers of EAC cells, splenocytes counts and leucocytes count with their differentials relative % in peripheral blood (PB) of EACchallenging mice were investigated. RESULTS: EAC-challenging mice treated with herbal extracts of Cur, Gin, Clov and Amyg showed a marked decline in EAC tumor cell count and a noticeable increase in apoptosis rate of EAC tumor cells, a remarkable decrease in serum level of cancer antigen 125 (CA-125) with an obvious increase in the number of splenocytes comparing to that in EAC-challenging mice treated with PBS alone. Moreover, the data indicated an insignificant change in the total leucocytes count and their differentials relative % of eosinophil, neutrophils, monocytes and lymphocytes in EAC-challenging mice treated with Cur and Amyg, but these parameters were markedly increased in EAC-challenging mice injected with Gin and Clov compared to that in EAC-challenging mice treated with PBS alone. CONCLUSION: To conclude, the herbal extracts of Cur, Gin, Clov and Amyg may have anti-tumoral immunity and anti-cancer potency and potential to reduce the resistance to cancer conventional chemotherapy and exert cancer chemo-protective approaches with low adverse effects. Further research is necessary to determine the regimen's toxicity on various tissues and organs and to connect the diagnostic and therapeutic approaches used in the regimen's biomedical use.

Allosteric indicator displacement enzyme assay for a cyanogenic glycoside.

Indicator displacement assays (IDAs) represent an elegant approach in supramolecular analytical chemistry. Herein, we report a chemical biosensor for the selective detection of the cyanogenic glycoside amygdalin in aqueous solution. The hybrid sensor consists of the enzyme ß-glucosidase and a boronic acid appended viologen together with a fluorescent reporter dye. ß-Glucosidase degrades the cyanogenic glycoside amygdalin into hydrogen cyanide, glucose, and benzaldehyde. Only the released cyanide binds at the allosteric site of the receptor (boronic acid) thereby inducing changes in the affinity of a formerly bound fluorescent indicator dye at the other side of the receptor. Thus, the sensing probe performs as allosteric indicator displacement assay (AIDA) for cyanide in water. Interference studies with inorganic anions and glucose revealed that cyanide is solely responsible for the change in the fluorescent signal. DFT calculations on a model compound revealed a 1:1 binding ratio of the boronic acid and cyanide ion. The fluorescent enzyme assay for ß-glucosidase uses amygdalin as natural substrate and allows measuring Michaelis-Menten kinetics in microtiter plates. The allosteric indicator displacement assay (AIDA) probe can also be used to detect cyanide traces in commercial amygdalin samples.

Functional characterization, homology modeling and docking studies of ß-glucosidase responsible for bioactivation of cyanogenic hydroxynitrile glucosides from Leucaena leucocephala (subabul).

Glycosyl hydrolase family 1 ß-glucosidases are important enzymes that serve many diverse functions in plants including defense, whereby hydrolyzing the defensive compounds such as hydroxynitrile glucosides. A hydroxynitrile glucoside cleaving ß-glucosidase gene (Llbglu1) was isolated from Leucaena leucocephala, cloned into pET-28a (+) and expressed in E. coli BL21 (DE3) cells. The recombinant enzyme was purified by Ni-NTA affinity chromatography. The optimal temperature and pH for this ß-glucosidase were found to be 45 °C and 4.8, respectively. The purified Llbglu1 enzyme hydrolyzed the synthetic glycosides, pNPGlucoside (pNPGlc) and pNPGalactoside (pNPGal). Also, the enzyme hydrolyzed amygdalin, a hydroxynitrile glycoside and a few of the tested flavonoid and isoflavonoid glucosides. The kinetic parameters K (m) and V (max) were found to be 38.59 µM and 0.8237 µM/mg/min for pNPGlc, whereas for pNPGal the values were observed as 1845 µM and 0.1037 µM/mg/min. In the present study, a three dimensional (3D) model of the Llbglu1 was built by MODELLER software to find out the substrate binding sites and the quality of the model was examined using the program PROCHEK. Docking studies indicated that conserved active site residues are Glu 199, Glu 413, His 153, Asn 198, Val 270, Asn 340, and Trp 462. Docking of rhodiocyanoside A with the modeled Llbglu1 resulted in a binding with free energy change (ΔG) of -5.52 kcal/mol on which basis rhodiocyanoside A could be considered as a potential substrate.

[Chemical constituents from the linseed meal].

Ten compounds were isolated from the 70% ethanol extract of linseed meal (Linum usitatissimum L) through a combination of various chromatographic techniques, including silica gel, macroporous adsorbent resin, Sephadex LH-20, and preparative HPLC. On the basis of spectroscopic data analysis, they were elucidated as 1-methylethyl-2-O-beta-D-glucopyranosyl-(1" --> 6')-beta-D-glucopyanoside (1), linustatin (2), neolinustatin (3), lotaustralin (4), linamarin (5), deoxyguanosine (6), deoxyadenosine (7), (+)-pinoresinol-4'-O-beta-D-glucopyranoside (8), 4-O-beta-D-glucopyranosylvanillyl alcohol (9) and tachioside (10), separately. Among them, compound 1 is a new compound, and compounds 6, 8 and 10 were isolated from the linseed meal for the first time.