Augmented rescue of macroglobulins by supplementation of anti-snake venom with methanolic extract of Andrographis paniculata in Naja naja envenomation.

Proteins of the macroglobulin family are prime targets of venom enzymes in snake bite. A massive reduction in the active concentration of these multifunctional proteins in snake bite, makes the living system vulnerable to dysregulation. This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in rescuing human alpha 2-macroglobulin (A2MG) and its homologues in rat plasma, from inactivation by Naja naja (N.N) venom enzymes. In-vitro experiments were conducted with heparinized human plasma and in-vivo experiments with female Wistar rats. Along with appropriate controls, there were 3 test groups in in-vitro and 8 test groups in in-vivo experiments. The in-vitro test groups were exposed to N.N venom for zero, 30 or 90 min prior to incubation with ASV or MAP or reduced ASV supplemented with MAP and incubated for 16 h at 37 °C. Chymotrypsin-bound esterase (CTBE) activity of A2MG was estimated. Rats were administered the venom intramuscularly and treated with ASV/MAP/ASV + MAP. CTBE activity of macroglobulin homologues was measured on day 1, 7 and 14. Survival of animals was noted. In human plasma, addition of ASV or MAP or ASV + MAP prevented loss of A2MG activity maximally to the extent of 88-100% (p = 0.001). In rats, reduced concentration of ASV supplemented with MAP showed complete rescue of macroglobulin homologues and 90% survival. The compulsive evidence from this study, underscores the merits of using this multipronged strategy in rescuing the macroglobulins and improving survival in envenomation due to N.N.

Evaluation of the merit of the methanolic extract of Andrographis paniculata to supplement anti-snake venom in reversing secondary hemostatic abnormalities induced by Naja naja venom.

Increasing evidence suggests a sizable involvement of hemotoxins in the morbidity associated with envenomation by the Indian spectacled cobra, Naja naja (N.N). This study investigates the ability of Indian polyvalent anti-snake venom (ASV), methanolic extract of Andrographis paniculata (MAP) and their combination in reversing the hemostatic abnormalities, viz. activated partial thromboplastin time(aPTT), prothrombin time(PT) and thrombin time(TT) in citrated plasma. These parameters were assessed in 2 groups of experiments. Group 1: Without the prior incubation of plasma with venom and Group 2: With prior incubation of plasma with venom for 90 min at 37°C. Venom caused significant (p < 0.001) prolongation in aPTT (175%), PT (49%) and TT (34%) in Group 1 and ASV could completely bring them back to normal. MAP showed a concentration-dependent reversal in aPTT, normalization of PT and prolongation of TT. When low concentration of ASV was supplemented with MAP, their combined effect in normalizing aPTT and PT improved by 37% and 26% respectively when compared to ASV alone. In Group 2, venom caused significant (p < 0.001) prolongation in aPTT (231%), PT (312%) and TT (245%). ASV had limited effect in reversing aPTT (52%), TT (31%) but completely normalized PT. MAP was marginally effective in reversing the prolonged aPTT and PT but caused further prolongation of TT. Combination of ASV and MAP was more effective than ASV alone in reversing venom-induced increase in aPTT (52%) and PT (29%). The study proved that, a drastic reduction of ASV by 70%, could be effectively supplemented by MAP in combating hemostatic abnormalities induced by NN venom.

Anti-snake venom and methanolic extract of Andrographis paniculata: a multipronged strategy to neutralize Naja naja venom acetylcholinesterase and hyaluronidase.

The study investigates the ability of methanolic extract of Andrographis paniculata (MAP) to supplement polyvalent anti-snake venom (ASV) in inhibiting neurotoxic enzyme acetylcholinesterase (AChE) and 'spreading factor' hyaluronidase from Naja naja (N.N) venom. AChE and hyaluronidase activity were measured in 100 or 200 µg of crude venom, respectively, and designated as 'control'. In Test Group I, enzyme assays were performed immediately after the addition of ASV/MAP/ASV + MAP to the venom. Inhibition of AChE by ASV (100-367 µg) was 12-17%, and of hyaluronidase (22-660 µg) was 33-41%. Under the same conditions, MAP (100-400 µg) inhibited AChE and hyaluronidase to the extent of 17-33% and 17-52%, respectively. When ASV (220 µg) and MAP (100-200 µg) were added together, AChE and hyaluronidase were inhibited to a greater extent from 39-63 to 36-44%, than when either of them was used alone. In Test Group 2, the venom was incubated with ASV/MAP/ASV + MAP for 10-30 min at 37 °C prior to the assay which enhanced AChE inhibition by 6%, 82% and 18% respectively, when compared to Test Group I. Though there was no change in inhibition of hyaluronidase in the presence of ASV, MAP could further increase the extent of inhibition by 27% and ASV + MAP upto 4%. In Test Group III, venom and substrate were incubated for 90 min and hyaluronidase activity was measured after the addition of inhibitors. Here, ASV + MAP caused increased inhibition by 69% compared to ASV alone. The study confirms the ability of phytochemicals in MAP to contribute to a multipronged strategy by supplementing, thereby augmenting the efficacy of ASV.

Can the methanolic extract of Andrographis paniculata be used as a supplement to anti-snake venom to normalize hemostatic parameters: A thromboelastographic study.

ETHNOPHARMACOLOGICAL RELEVANCE OF ANDROGRAPHIS PANICULATA: The whole plant (including leaves and roots) is used in traditional Ayurveda and Siddha medicine to treat various clinical conditions such as fever, respiratory tract infections, colic pain, liver disorders, diabetes, hypertension, and inflammation. It is also used as an antidote for snake-bite, poisonous bites of insects and recommended as a dietary supplement to boost immunity. AIM OF THE STUDY: In-vitro thromboelastographic evaluation of the efficacy of methanolic extract of Andrographis paniculata (MAP) and polyvalent anti-snake venom (ASV) in neutralizing the Naja naja (N.N) venom-induced changes in hemostatic parameters. MATERIALS AND METHODS: Thromboelastographic evaluation of hemostatic parameters was initiated by adding 3µg N.N venom to citrated whole blood from healthy volunteers. The effect of different concentrations of ASV and MAP in neutralizing the toxicity of N.N venom were studied in two groups. In group 1 experiments, citrated whole blood (340µl) was mixed with N.N venom (3µg), immediately followed by successive addition of ASV (5µl, 8µl and 15µl) or MAP (15µg, 30µg, 60µg and 120 µg) or combination of ASV and MAP (3µl ASV+30µg MAP and 3µl ASV+60µg MAP). In group 2 experiments, N.N venom was incubated with whole blood for 90 minutes at 37°C, followed by successive addition of ASV (5µl, 10µl, and 15µl) or MAP (30µg and 60µg) or combination of ASV and MAP (5µl ASV+30µg MAP and 5µl ASV+60µg MAP). RESULT: In Group 1 experiments, N.N venom caused significant (p<0.05) increase in R-time, K-time, LY30% and a decrease in angle and MA. Optimum effect on hemostatic parameters was observed at a concentration of 8µl ASV, where all the deleterious effects of the venom were completely reversed. Similarly, the addition of MAP to the assay system could reproduce results as ASV, in reversing the deleterious effects of the venom. This occurred in a concentration-dependent manner, from 15µg-60µg, with the optimum results at 60µg. When ASV concentration was reduced to 3µl and supplemented with MAP (30µg or 60µg), the positive supplementary effect of MAP was demonstrated. In Group 2 experiments, N.N venom caused significant (p<0.05) changes in all TEG parameters, with most deleterious changes observed in MA and LY30% compared to Group 1 experiments. ASV when added in increasing concentrations (5-15µl), had beneficial effects only on K-time, angle, and MA. When added together with ASV, MAP (30µg or 60µg) could significantly (p<0.05) supplement the effect of ASV (5µl) in improving R-time, K-time, and angle. CONCLUSION: This in-vitro study demonstrates the effectiveness of MAP as a supplement to ASV in combating the deleterious effects of N.N venom on hemostasis. However, further in-vivo experiments in animal models are required to substantiate these effects.

PBL Trigger Design by Medical Students: An Effective Active Learning Strategy Outside the Classroom.

INTRODUCTION: Problem Based Learning (PBL) is known world over as an effective, active learning strategy with many benefits for the student. Usually, in medical schools, PBL triggers are designed by a well-trained group of faculty from basic and clinical sciences. The challenge was whether this task could be given to students in the first year of their curriculum and be executed by them effectively. AIM: To enhance active learning, comprehension and critical thinking with a view to promote horizontal and vertical integration between subjects. MATERIALS AND METHODS: Student volunteers of the first year MBBS course (n=10), who had been exposed to the curriculum for approximately 38 weeks and were familiar with the PBL process were recruited for the study. In addition to a handout on the topic 'gout', they were given the freedom to access any resource in the university library to construct the PBL triggers. The PBL triggers were vetted by two faculties. In addition to a focus group discussion with students, students' and faculty's responses were collected on a Likert scale. RESULTS: Students opined that the exercise helped improve their comprehension (100%), critical thinking abilities (90%) and clinical orientation to the topic (100%). They felt that designing a PBL trigger was a relevant active learning strategy (100%) and would help them answer questions on this topic better in the future (90%). The clinicians who examined the PBL triggers, felt that they were of good quality and that the process was a good tool for vertical integration between basic and clinical sciences. DISCUSSION: The results prove that students when given a challenge will rise to the occasion. Unfamiliarity with the nuances of a disease did not prevent them from going the extra mile to achieve their target. By taking part in this exercise, students benefitted in many ways and got a holistic understanding of the topic. CONCLUSION: PBL trigger design can be introduced as an active learning strategy for students in medical schools where PBL is part of the curriculum. It promotes integration across subjects and is very effective in augmenting student motivation.

Plasma proteinase inhibitory activity in ectopic and molar pregnancy.

Antitryptic, antichymotryptic and alpha 2- macroglobulin activities were measured in sera of normal nonpregnant and normal pregnant women and women with tubal ectopic pregnancy and molar pregnancies in the first 5 to 7 weeks of pregnancy calculated from the last menstrual period. While alpha 2-macroglobulin decreased in early normal pregnancy compared to nonpregnant state (p<0.001), in ectopic and molar pregnancies there was an increase in alpha 2- macroglobulin activity (p < 0.001), as compared to nonpregnant and normal pregnant women. Antitryptic activity did not increase in normal and ectopic pregnancy, however was increased in molar pregnancy (p < 0.01). Antichymotryptic activities did not show a change either in normal pregnancy or in cases of ectopic and molar pregnancy. Drop in alpha 2- macroglobulin activity to near normal levels in ectopic, 6 weeks post surgery, correlated well with the decrease in ß-hCG. However, in molar pregnancy, alpha 2- macroglobulin remained elevated even when the ß-hCG levels in serum returned to zero 10 weeks after surgery. The studies suggest a major role for circulating proteinase inhibitors especially alpha 2-macroglobulin in regulating proteinase activity in normal, ectopic and molar pregnancy.

Serum alpha-2-macroglobulin, antitrypsin and antichymotrypsin activities in patients receiving treatment with cyclosporine.

Cyclosporine has been reported to function as an inhibitor of the chymotrypsin like activity of proteasome. We hypothesized that the administration of an exogenous proteinase inhibitor may affect the activities of the naturally occurring serum anti proteinases. The aim of this study was to observe the pattern of alteration of serum alpha 2 macroglobulin (AMG), alpha 1- antitrypsin (AT) and alpha 1-antichymotrypsin (ACT) activities in renal transplant patients receiving the immunosuppressive drug, cyclosporine. Patients (97) who had received a single renal allograft were inducted into the study. Subjects were on a twice-daily dosage of cyclosporine capsules. Trough (Co) and two-hour post dose (C 2) cyclosporine levels were regularly estimated and all patients had stable creatinine levels. In 5 newly transplanted patients, antiproteinase activities were estimated weekly over a 4-week period as their cyclosporine doses were gradually tapered. Average serum activities of ACT and AMG in the transplant group were significantly less than in the control group (p<0.002 and p<0.003 respectively). AT and ACT activities fell gradually over 4 weeks. AMG activities showed a biphasic pattern, initially falling by almost 50% in the second week, increasing marginally in the third week and decreasing to less than 50% of the activities observed in the first week. Serum antiproteinase activities of serum alpha 2 macroglobulin (AMG), alpha 1-antitrypsin (AT) and alpha 1-antichymotrypsin (ACT) were found to be altered in renal transplant patients receiving cyclosporine.