Development of an Enzyme-Linked Immunosorbent Assay for Specific Detection of Mulberroside A in Mulberry (Morus alba L.) Using Anti-mulberroside A Polyclonal Antibody

An enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibody (PAb) against mulberroside A (MuA), a major active component found in the root bark of mulberry (Morus alba L.). MuA was conjugated with the carrier protein bovine serum albumin for immunization to rabbits. The results showed that the antibodies were specific only for MuA and have very low specificity for its aglycone, oxyresveratrol. The ELISA assay was suitable for quantitating MuA in the range of 0.17–15.62 μg/mL with a relative standard deviation (RSD) of less than 5 % for both intra- and inter-assay precision levels. The recovery rates of MuA in the samples were in the range of 97.6–101.4 % with a RSD of less than 5 %. The developed ELISA exhibited a good correlation value (R ²?=?0.994) with the standard high-performance liquid chromatography method in the crude extracts of plant samples. These results suggest that the developed ELISA method using PAb against MuA can be applied to determine MuA content with high specificity, rapidity, and simplicity in mulberry samples. The developed ELISA method described could prove to be useful as an analytical tool for quality control of mulberry and their products.     

Identification of hemocyanin as a novel non-cross-reactive allergen from the giant freshwater shrimp Macrobrachium rosenbergii

Scope : Sensitization to giant freshwater shrimp Macrobrachium rosenbergii (Mr) was recently reported. However, the allergens have yet to be identified. This study aimed to identify and characterize a novel allergen of Mr shrimp. Methods and results: Extracted proteins were separated and purified by anion and in some experiments, size‐exclusion chromatography. Serum IgE from shrimp allergic donors identified a candidate protein, which was characterized by LC‐MS/MS. The specificity of IgE binding was tested using immunoblotting and inhibition ELISA. The IgE‐binding profiles from 12 of 13 Mr allergic subjects that were pre‐incubated with an extract of Penaeus monodon showed residual binding to ～60–80 kDa proteins. The 60–80 kDa IgE‐bound proteins were fractionated in the flow‐through of anion chromatography showing a high IgE reactivity. Peptides identified by LC‐MS/MS showed the proteins closely match subunits of hemocyanin (Hcs). Purified Hcs from hemolymph markedly inhibited binding of IgE from sera of Mr allergic subjects to solid‐phased Mr proteins in inhibition ELISA. Conclusion: Hcs were identified as heat‐stable, non‐cross‐reactive, high‐molecular‐weight (MW) allergens from Mr shrimp. Since circulatory organs are not always removed during food preparation, high concentrations of Hcs may be present along with shrimp meat, which contains the known cross‐reactive tropomyosin protein.     

Determination of Iriflophenone 3‐C‐β‐d‐Glucoside From Aquilaria spp. by an Indirect Competitive Enzyme‐linked Immunosorbent Assay Using a Specific Polyclonal Antibody

Polyclonal antibody against iriflophenone 3‐C‐β‐d ‐glucoside (IP3G), a major compound from the leaves of Aquilaria spp., was produced for the development of an enzyme‐linked immunosorbent assay (ELISA). The results showed that the antibodies were specific for IP3G. The produced antibody has low cross reactivity with iriflophenone 3,5‐C‐β‐d ‐diglucopyranoside (13%), genkwanin 5‐O‐β‐primeveroside (3.55%) and no cross reactivity found in other compounds. The range of ELISA assay extends from 100 to 1560 ng/mL with coefficient of variation (CV) 1.19% to 2.07% for intra‐assay and 3.76% to 7.15% for inter‐assay precision levels. The recovery rates of IP3G in the leaves of Aquilaria spp. were in the range of 96.0% to 99.0% with CV 4.50% to 5.32%. A correlation between ELISA and high‐performance liquid chromatography methods was obtained when analysis of IP3G in the plant samples (R² = 0.9321). These results suggest that the developed ELISA method can be applied to determine IP3G content with high specificity, rapidity, and simplicity. The developed immunosorbent assay in this study provides a useful tool for the analysis of IP3G in plant samples and products.     

Inhibitory Mechanisms of Lusianthridin on Human Platelet Aggregation

Lusianthridin is a phenanthrene derivative isolated from Dendrobium venustum. Some phenanthrene compounds have antiplatelet aggregation activities via undefined pathways. This study aims to determine the inhibitory effects and potential mechanisms of lusianthridin on platelet aggregation. The results indicated that lusianthridin inhibited arachidonic acid, collagen, and adenosine diphosphate (ADP)-stimulated platelet aggregation (IC₅₀ of 0.02 ± 0.001 mM, 0.14 ± 0.018 mM, and 0.22 ± 0.046 mM, respectively). Lusianthridin also increased the delaying time of arachidonic acid-stimulated and the lag time of collagen-stimulated and showed a more selective effect on the secondary wave of ADP-stimulated aggregations. Molecular docking studies revealed that lusianthridin bound to the entrance site of the cyclooxygenase-1 (COX-1) enzyme and probably the active region of the cyclooxygenase-2 (COX-2) enzyme. In addition, lusianthridin showed inhibitory effects on both COX-1 and COX-2 enzymatic activities (IC₅₀ value of 10.81 ± 1.12 µM and 0.17 ± 1.62 µM, respectively). Furthermore, lusianthridin significantly inhibited ADP-induced suppression of cAMP formation in platelets at 0.4 mM concentration (p < 0.05). These findings suggested that possible mechanisms of lusianthridin on the antiplatelet effects might act via arachidonic acid-thromboxane and adenylate cyclase pathways.     

Development of Anti-Asiaticoside Polyclonal Antibody-Based Immunoassay and Applications for Centella asiatica Products and Human Serum

A sensitive indirect competitive enzyme-linked immunosorbent assay (ELISA) was developed using polyclonal antibody (PAb) against asiaticoside (AS), one of the triterpenoid saponins found in Centella asiatica (Linn). AS-bovine serum albumin conjugate was immunized to rabbits for producing PAb. The results showed that the antibodies were specific only for AS and very low specific for madecassoside whose basic skeleton is almost the same as AS. The range of the assay extends from 0.05?C25 ??g/ml of AS. A good correlation between ELISA and high-performance liquid chromatography methods was obtained when analysis of AS in the crude extracts of plant samples. In addition, the products containing C. asiatica in various preparations were determined AS content by competitive ELISA. The results showed that the product from tea bag preparation gave the highest yield of AS content (35.59?mg/g dry wt) comparing to other preparations. In order to evaluate the matrix effect of the serum for AS immunoassay, the standard curves of AS in different media were observed. Standard curve of the serum was similar to the water media and both curves showed the measurement range of 0.20?C6.25???g/ml. The developed ELISA method can be used for quality assessment of C. asiatica and their products including AS detection in serum samples.