FUNCTIONAL ANALYSIS OF MEDICINAL PLANTS USING SYSTEMS BIOLOGY APPROACHES

Plant derived medicine is an important source of life saving drugs, but the genome information of most important medicinal plants is still unavailable. The need of the hour is to identify more functional genes and enzymes that control secondary metabolite production in medical plants, develop new methods for systematics, engineer resistance to number of biotic and abiotic stresses, and develop new conservation strategies, more genomics, proteomics and metabolomics information needs to be produced. In this review, a brief overview of various omic technologies and its applications to medicinal and aromatic plants are discussed.Â

ANTICOAGULANT AND ANTIPLATELET ACTIVITIES OF JACKFRUIT (ARTOCARPUS HETEROPHYLLUS) SEED EXTRACT

  Objective: The current study focuses on the anticoagulant and antiplatelet activities of aqueous seed extract of Jackfruit (AqSEJ).Methods: Anticoagulant effect of AqSEJ was tested using plasma recalcification time, mouse tail bleeding time, Activated Partial Thromboplastin Time (APTT) and Prothrombin Time (PT). Antiplatelet activity was examined by platelet aggregation studies using agonists such as ADP, Collagen and Epinephrine.Results: The AqSEJ enhanced the clotting time of citrated human plasma from control 200±10 s to 740±14 s. The anticoagulant activity of AqSEJ was further strengthened by in-vivo mouse tail bleeding assay. The i. v. injection of AqSEJ significantly prolonged the bleeding time in a dose dependent manner. The recorded bleeding time was>10 min (P<0.01) at the concentration of 30 μg against the PBS treated control of 1.48±0.10 min with the IC50 values 37.5 μg/ml and 47.5 μg/ml respectively. Interestingly, AqSEJ specifically prolonged the clot formation process of only APTT but not PT, revealing the anticoagulation triggered by the extract could be due to its interference in an intrinsic pathway of the blood coagulation cascade. Furthermore, AqSEJ inhibited the agonists such as ADP, epinephrine and collagen induced platelet aggregation of about 66.7%, 39.2% and 37.0% respectively at the concentration of 200 μg.Conclusion: AqSEJ showed anticoagulant and antiplatelet activities. Hence, it may serve as a better alternative for thrombotic disorders.Â

Purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract that exhibits anticoagulant and antiplatelet activity

The current study deals with the purification and characterization of non-enzymatic glycoprotein (NEGp) from flax seed buffer extract. Sephadex G-100 and DEAE-A25 column chromatography techniques were employed to isolate NEGp. NEGp showed single sharp band at 29 kDa region on 10% SDS-PAGE, and under reduced and non-reduced conditions revealed its monomeric nature. Besides, NEGp taken up the PAS stain at 29 kDa region reveals the presence of carbohydrate moiety. Purity of NEGp was adjudged by RP-HPLC, as it revealed a single sharp peak at the retention time of 3.4 min. The exact molecular mass of NEGp was found to be 26 kDa which was confirmed by MALDI-TOF. Circular di-chromism spectra of NEGp showed 12.0% alpha-helix, 24.3% alpha-helix turn and 63.7% random coils without beta pleated sheets. NEGp was found to exhibit anticoagulant activity by extending clotting time of both platelet rich plasma and platelet poor plasma from control 240 s to 1800 s and 280 s to 2100 s respectively at the concentration of 8 mu g. NEGp inhibited the agonists such as ADP, epinephrine and arachidonic acid induced platelet aggregation in washed platelets. The percentage of inhibition was found to be 70%, 80% and 60% respectively. While, it did not interfere in thrombin, PAF and collagen induced platelet aggregation. NEGp did not hydrolyse RBC membrane, devoid of haemorrhagic and edema inducing properties in experimental mice. (C) 2020 Elsevier B.V. All rights reserved

EMILIN2 Regulates Platelet Activation, Thrombus Formation, and Clot Retraction

<div><p>Thrombosis, like other cardiovascular diseases, has a strong genetic component, with largely unknown determinants. EMILIN2, Elastin Microfibril Interface Located Protein2, was identified as a candidate gene for thrombosis in mouse and human quantitative trait loci studies. EMILIN2 is expressed during cardiovascular development, on cardiac stem cells, and in heart tissue in animal models of heart disease. In humans, the EMILIN2 gene is located on the short arm of Chromosome 18, and patients with partial and complete deletion of this chromosome region have cardiac malformations. To understand the basis for the thrombotic risk associated with EMILIN2, EMILIN2 deficient mice were generated. The findings of this study indicate that EMILIN2 influences platelet aggregation induced by adenosine diphosphate, collagen, and thrombin with both EMILIN2-deficient platelets and EMILIN2-deficient plasma contributing to the impaired aggregation response. Purified EMILIN2 added to platelets accelerated platelet aggregation and reduced clotting time when added to EMILIN2-deficient mouse and human plasma. Carotid occlusion time was 2-fold longer in mice with platelet-specific EMILIN2 deficiency, but stability of the clot was reduced in mice with both global EMILIN2 deficiency and with platelet-specific EMILIN2 deficiency. <i>In vitro</i> clot retraction was markedly decreased in EMILIN2 deficient mice, indicating that platelet outside-in signaling was dependent on EMILIN2. EMILIN1 deficient mice and EMILIN2:EMILIN1 double deficient mice had suppressed platelet aggregation and delayed clot retraction similar to EMILIN2 mice, but EMILIN2 and EMILIN1 had opposing affects on clot retraction, suggesting that EMILIN1 may attenuate the effects of EMILIN2 on platelet aggregation and thrombosis. In conclusion, these studies identify multiple influences of EMILIN2 in pathophysiology and suggest that its role as a prothrombotic risk factor may arise from its effects on platelet aggregation and platelet mediated clot retraction.</p></div

Bleeding and Thrombus Formation in E2^-/- and E2p^-/- Mice.

<p>A. D. Tail Bleeding/Rebleeding Assay. Bleeding time (1^st) is between the start of the bleeding and cessation of the bleeding. Clot stability (rebleeding) time (CS) is measured as the time between the cessation of the bleeding and the start of the second bleeding time (2^nd). Bars are mean±SEM, n = 9–11. Statistical Analysis, One-way ANOVA. B, E. Carotid Occlusion Time. Bars are mean±SEM, n = 9–10. Statistical analysis, t-test. C,F. Patency (percent mice with open carotid 4hr after treatment), WTc mice (4/10), E2^-/- mice (6/9). WTp mice (3/5), E2p^-/- (9/10).</p

Platelet and Plasma Mixing Experiments.

<p>A,B. WW–WTc platelets and plasma, WD–WTc platelets and E2^-/- plasma, DD–E2^-/- platelets and plasma, DW–E2^-/- platelets and WTc plasma. Number of platelets per assay, 1x10⁸ per mL, pooled from 3 mice, 1μM ADP. Bars are mean±SEM, n = 3 performed in triplicate. Statistical analysis, one-way ANOVA, Newman-Keuls post-test, **P = 0.01</p

Aggregation of platelets from E2^-/- and E2p^-/- mice.

<p>A,C. E2^-/- mice. B,D. E2p^-/- mice. A,B. Representative (3–5 experiments repeated 2–3 times with each platelet preparation) aggregometer tracing in PRP induced by 5μM ADP with platelets adjusted to 1.2x10⁸ per ml. C,D. Maximum amplitude from the platelet aggregometry tracings. The aggregation of PRP was induced with 1 μM or 5μM ADP added to PRP with platelets at adjusted to 0.8–1.2x10⁸ per mL pooled from 3 mice. Bars are mean±SEM, performed in triplicate. Statistical analysis, one-way ANOVA, Newman-Keuls post-test **P = 0.01, ***P = 0.001. E2^-/- (EMILIN2 deficient), E2p^-/- (EMILIN2 platelet deficient)</p

Role of E2 in Clotting Time and Clot Retraction.

<p>A. Clotting time in WTc and E2^-/- mouse PPP and PRP was induced by CaCl₂. B. Clotting time of PRP upon addition of varying concentrations of recombinant EMILIN2 (rE2). rE2 protein was preincubated with PRP or PPP prior to addition of CaCl₂. C. Clot retraction. Plasma clotted with 1 U/mL thrombin and 5mM CaCl₂, maintained at 37°C, and photographed from 0–120 min after the thrombin addition.</p

Generation of EMILIN2 Deficient Mice.

<p>A. Schematic of Wildtype Allele, Targeted Allele, Targeted Allele (after Neo deletion), Mutant Allele (after crossing with a Cre deletor). Bands: E2-/-––530bp, LoxP––350bp, WT––250bp. B. PCR EMILIN2 genotyping. CMV-cre = 100 bp (not shown) C. qRT-PCR EMILIN2 in mice. D. Western blot of 2 μL plasma from WT and E2-/-mice immunostained with primary mouse antiserum #407 (1:5000) to E2 and secondary (goat anti mouse 1:5000). Std, Standard purified E2 protein = 50ng. E. Genotyping PCR of platelet EMILIN2 conditional knockout mice. Pf4-cre = 450 bp (not shown). E2-/- (global EMILIN2 deficient), E2p-/- (EMILIN2 platelet deficient)</p

Platelet aggregation is impaired in E2^-/- mice in response to ADP, collagen or thrombin and E2 increases aggregation of both WTc and E2^-/- platelets.

<p>A,B,C. Platelet aggregation measured in platelet rich plasma (1 x 10⁸ platelets/assay). Maximum amplitude (% Light Transmission). A. 2.5 μM ADP, B. 1 μg collagen, C. 0.5 U/mL thrombin. Bars are the mean±SEM of 3–5 separate experiments performed in triplicate of platelets pooled from 3 mice. Statistical analysis, one-way ANOVA. **P < 0.001 ***P < 0.001. WTc (black bar), E2^-/- (white bar).</p