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EVALUATION OF IN VITRO ACARICIDAL ACTIVITY OF Cyclea peltata (PADAL)
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-02-25) POONGHUZHALI R; Dr. Sujith. S.
The present study was conducted to identify the acaricidal activity of potent fraction/ extracts of Cyclea peltata (Padal) against Rhipicephalus (Boophilus) annulatus. The whole plant was collected locally, dried, powdered and extracted. The aqueous extract of C. peltata was prepared by using hot extraction process and the methanolic extract was done in Soxhlet extraction apparatus and concentrated under rotary vacuum evaporator. The methanolic extract fractionated using solvents n-hexane, dichloromethane, n-butanol and water in increasing order of polarity to acquire different fractions. Qualitative Phytochemical screening of all the extracts as well as GCMS and FTIR analysis of the most potent fraction was performed. The acaricidal activity was determined by evaluating various entomological parameters such as mean egg mass, index of egg laying, per cent adult mortality and per cent inhibition of fecundity. The most potent fraction (n-hexane) was screened for its safety in HepG2, hepatocellular carcinoma cell lines by MTT assay and the IC50 was calculated. The cells were exposed to AO/EB and JC-1 staining to assess the mechanism of cytotoxicity of the extract. The phytochemical analysis unveiled the presence of steroids, alkaloids, glycosides, flavonoids, terpenoids, saponins in aqueous extract and alkaloids, phenols, tannins, flavonoids, terpenoids, saponins in methanolic extract. The fractions of C. peltata showed the presence alkaloids, phenols, tannins, flavonoids, terpenoids, saponins. There was no significant difference in the weight of ticks exposed to different extracts/fractions of C. peltata compared to control. The maximum reduction in egg mass was observed with n-hexane fraction with values of 0.002 ± 0.00 g, followed by methanolic and aqueous extracts whereas, there was minimum reduction in egg mass when ticks were treated with dichloromethane, n-butanol and water fractions of C. peltata compared to control. The index of egg laying was minimum when ticks were treated with n-hexane fraction with a value 0.044 ± 0.15 g whereas it was maximum when ticks were treated with n-butanol fraction of C. peltata (0.359±0.15 g). The aqueous, methanolic and n-hexane fractions of C. peltata produced 100 per cent adult mortality whereas the other fractions produced nearly 80 per adult mortality. The n-hexane fraction of C. peltata showed almost 90 per cent inhibition of fecundity while minimum inhibition was seen with n-butanol treatment.From the results, it was evident that out of all the extracts and fractions considered, n-hexane fraction exhibited lower mean egg mass and induced very early adult tick mortality with 91.38 per cent inhibition of fecundity. Hence, n-hexane fraction of C. peltata was found to be most potent fraction and was selected for in vitro evaluation of safety in HepG2 cell lines. GC-MS analysis of the n-hexane potent fraction divulged the presence of alkaloids, flavonoids, phenols, terpenoids and saponins. FTIR analysisrevealed the presence of Decanol, octyl-beta-d-glucopyranoside, 9-octadecenoic acid, cis androsterone, 1,7-heptanediol, ethyl undecanoate etc,. A dose dependent cytotoxicity was shown by n-hexane fraction in HepG2 cells with decrease in cell viability. Based on the per cent inhibition, the IC50 of n-hexane fraction was calculated as 8 µg/mL. Acridine orange/Ethidium bromide staining (AO/EB) ascertained that most of the cells exposed to n-hexane fraction of C. peltata showed were in apoptotic stages and JC-1 staining elucidated that, the mitochondrial membrane potential declined in these cells. From the present study, it was evident that n-hexane fraction of C. peltataexhibited potent acaricidal activity against R. (B.) annulatus than other extracts/ fractions with effective cytotoxic property in HepG2 cells and can be used as a lead for the development of ectoparasiticide.
Open Access
EVALUATION OF PROTECTIVE EFFECT OF LEMONGRASS OIL AND CITRAL ON FATTY LIVER DISEASE IN RATS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-02-25) ADHEENA XAVIER; Dr. Suja Rani S.
Fatty liver disease is on the rise across the world, necessitating an urgent quest for effective remedies, particularly those with minimal adverse effects. Medicinal plants and its secondary metabolites especially the essential oils have recently been considered as prospective pharmacological agents for the prevention and management of various hepatic and metabolic impairments. The present study was therefore undertaken to evaluate the protective effect of lemon grass oil (LGO) and citral on experimentally induced hepatic lipidosis in rats. Male Sprague-Dawley rats were divided into six groups of six animals each. Group I received 0.1 per cent tween 80 (vehicle/normal control) and group II received both CCl4 treatment and tween 80 (CCl4 control), while group III to VI received LGO and citral at 45 and 90 mg/kg body weight dose levels respectively, along with CCl4 treatment. The vehicle/test substances were administered orally for 14 days on daily basis and all the animals except from normal control group were given CCl4 subcutaneously on 9th, 10th, 11th and 12th day of experiment for the induction of fatty liver. The CCl4 induced abnormal levels of serum biomarker enzymes (ALT, AST and ALP), hepatic lipid levels and hepatic antioxidant parameters (LPO and GSH) could be significantly restored after the treatment with LGO/citral at both the dose levels. The histological examination of liver revealed near normal architecture in LGO at 90 mg/kg and citral at both 45 and 90 mg/kg treated groups, indicating their remarkable lipotropic potential. Moreover, LGO and citral were found to be safe at the orally tested limit dose level of 2000 mg/kg as per OECD guidelines. The phytochemical profile of LGO as analysed by GC-MS revealed the presence of alpha and beta citral as the major constituents. In silico docking of citral against PPARα further disclosed high binding affinity, suggesting its role as a plausible target of action for LGO and citral. Besides, the treatment with LGO/citral for 14 days significantly attenuated the CCl4 induced rise in PPARα gene expression in liver and even restored to the level of normal control. Thus, the present findings of the study established the protective effect of LGO and citral on fatty liver disease, which could be attributed to its lipotropic, antioxidant and PPARα modulatory effects.
Open Access
IN VIVO PHARMACOKINETICS OF MEROPENEM ALONE AND IN COMBINATION WITH CATIONIC ANTIMICROBIAL PEPTIDE, CECROPIN A-(1-7) MELITTIN IN BROILER CHICKEN
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-06) VEENA; Dr. Sanis Juliet
Meropenem is a carbapenem class of antibiotics with a broad spectrum of activity against gram-positive, gram-negative, and anaerobic bacteria. Cationic antimicrobial peptides (AMPs) are a potential class of chemicals that can be used in conjunction with conventional antibiotics to treat a variety of infections. Imprudent use of antibiotics without knowledge of its pharmacokinetics and pharmacodynamics is one of the reasons for development of resistance. And in poultry, studies on the pharmacokinetics of meropenem alone or its combination with other antimicrobial agents are meagre when compared to the other species. Hence, the present study was conducted to investigate the pharmacokinetics of meropenem alone and in presence of a synthetic cecropin A-(1-7) melittin (CAMA) following single intravenous administration of 24 mg/kg in broiler chicken. Blood and intestinal contents were collected at predetermined time intervals after administration alone and in combination with CAMA at 0.65 mg. The concentration of meropenem in plasma and intestinal contents were analysed by HPLC. The pharmacokinetics of meropenem, as well as meropenem in combination with CAMA was best described by a two-compartmental model for plasma samples whereas for the intestine it followed a non-compartmental model. Meropenem was detected in the plasma of birds till 4 h when administered alone whereas in combination with CAMA up to 48 h. Kinetic parameters of both the groups indicated a rapid rate of distribution reinforced by the high tissue-to-blood ratio, whereas, in combination with CAMA significantly higher values were noted for Cp(max), AUC, T1/2β and MRT. The concentration in the intestinal content was lower than the plasma for initial time points and was maintained at almost a steady level till 48 h with approximately half of the initial plasma concentration indicating that meropenem was actively secreted into the intestine following intravenous administration. The study thus revealed that the presence of CAMA significantly altered the pharmacokinetic behaviour of meropenem in healthy broiler birds showing a rapid, but limited distribution with slower elimination.
Open Access
METABOLISM AND DISPOSITION OF PHYTOL FOLLOWING ORAL ADMINISTRATION IN RATS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2023-02-06) MERY S JOHN; Dr. Sanis Juliet
Phytol is a diterpene member of the long-chain unsaturated acyclic alcohols with many significant bioactivities. Phytol has recently gained interest for its potential role in cancer prevention and its efficacy in certain central nervous system disorders. Because of its potential as a future drug candidate, the present study was designed to investigate the in vitro metabolism and disposition of phytol followingrepeated oral administration in rats. Phytol, suspended in one per cent carboxy methyl cellulose was administered orally for seven consecutive days once daily at a dose rate of 100 mg/kg to the test group. Blood samples were collected at 0.25, 6,12, 24, 48, 72, 96, 120, 144, and 168 h post-drug administration, processed and subsequently analysed for parent compound and its metabolites using Gas chromatography-Mass spectrophotometer (GC-MS). The GC-MS conditions, sample extraction, and analytical methods for the quantification of phytol and its metabolites from blood and tissues were optimized and validated. After seven days of consecutive administration, six rats each from the experimental and control group were humanely sacrificed as per ethical guidelines. Tissue samples were collected for residue analysis and histopathology. Livers from control rats were used for in vitro metabolism studies. Repeated administration of phytol did not significantly (P>0.05) affect the feed, water intake, body weight gain, and relative organ weight when compared with that of control rats. Phytol was readily absorbed from the gastrointestinal tract and was detected at all sampling times between day 1 andday 7. The plasma concentration after repeated administration of phytol for seven consecutive days follows a wavy pattern. The maximum concentration of 0.53±0.22 and 0.49±0.156 µg/mL was detected on the first and fifth day respectively post drug administration. The concentration of the metabolites in plasma remained higher than that of the parent compound. Phytol was detected in all the organs except the brain, whereas phytanic acid was also detected in the brain. Histopathological evaluation revealed changes in their tissue architecture in the lungs, kidney, heart, and liver except for the brain. There was no detectable concentration of the two metabolites in the incubated S9 fraction till 120 hr of incubation. However, the concentration of the parent decreased withthe increase in time. On repeated administration, phytol is retained in vital organs and cause renal and hepatic toxicities.
Open Access
PHARMACOKINETIC EVALUATION OF PHYTOL IN RATS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCS, POOKODE, WAYANAD, KERALA VETERINARY AND ANIMAL SCIENCES UNIVERSITY, 2022-10-27) SARANYA C; Dr. Sanis Juliet
Phytol (3,7,11,15- tetramethylhexadec-2-en-1-ol) is a diterpene and a branched-chain unsaturated acyclic fatty alcohol, abundantly present in nature as a part of the chlorophyll molecule. Phytol is metabolised in the liver into phytanic acid and pristanic acid. Phytol possesses a wide range of bioactivities including antioxidant, cytotoxic, autophagy and apoptosis-inducing, anti-nociceptive, anti-inflammatory, immune-modulating, antimicrobial, antianxiety, and anticonvulsant effects. So far no data is available on the pharmacokinetics of phytol in any animal species. The present study was conducted to investigate the pharmacokinetics of phytol after the oral and intravenous administration and to assess the residual retention of phytol and its metabolites in different organs. A quantifiable GC-MS method was developed for the determination of phytol and its metabolites in rat plasma and different tissues. Single dose of phytol was given orally and intravenously to separate groups of rats at a dose rate of 250 mg/kg and 25 mg/kg respectively. Blood was collected at predetermined time intervals up to 24 h and 168 h post intravenous and oral administration respectively. The blood concentrations were determined and pharmacokinetic analysis were done using PK Solver, version. 2.0. The animals were sacrificed on 7, 14, 21 and 28 days following oral and seven days post intravenous administration and organs were collected for residue analysis of phytol and its metabolites. Phytol and its metabolites were present in plasma in detectable concentrations at all sampling times after oral and intravenous administration. The plasma concentration of pristanic acid was higher followed by phytanic acid and phytol. Remarkable double peaks for phytol were observed in plasma concentration-time curve following oral administration whereas after intravenous administration the plasma concentration of phytol decreased rapidly in zig-zag manner during the first hour and more slowly thereafter. Pharmacokinetics of orally administered phytol in rats fitted best into a non-compartment model. The absorption of phytol into blood was very rapid with the time to reach maximum concentration in plasma ranges from 2.19 to 4.23 h with 95% confidence interval. The terminal half-life (t1/2), maximum plasma concentration (Cmax) and mean resident time (MRT) were 84.53 h, 1.38 μg/mL, and 117.74 h respectively. The apparent volume of distribution (Vd) and clearance were found to be 1015.03 ± 136.54 mL/g and 9.34 ± 1.25 mL/g/h respectively. Additionally, from six hour onwards it is best fitted into a two compartmental model as evident by the concentration against time curve. Phytol tends to accumulate in the brain, seminal vesicle and small intestine of rats after 28th day post administration. In addition, phytol was also detected in the liver, lung, adrenal gland, stomach, kidney, heart, reproductive organs, caecum and spleen after seventh day of post administration. Phytanic acid was detected in almost all the organs with highest concentration in stomach, small intestine and caecum. After intravenous administration of phytol, the plasma concentrations of phytol and its metabolites was detected till 24 h. Pharmacokinetics of intravenously administered phytol in rats also fitted best into a non-compartment model. The Cmax, and Tmax were 0.08 ± 0.02 µg/mL and 0.73 ± 0.37 h respectively. The terminal elimination half-life (T1/2) and MRT were 68.32 ± 22.14 h and 98.09 ± 32.05 h respectively. The steady-state volume of distribution (Vdss) and apparent volume of distribution during terminal phase (Vdλ) and clearance were 516.21 ±78.28 mL/g 520.37 ± 76.73 mL/g and 7.28 ± 1.57 mL/g/h respectively. Phytol was detected in the brain, liver, kidney, adrenal gland, seminal vesicle, fat, small intestine and caecum seven days post intravenous administration. Phytanic acid was detected in almost all the tissues. In the fat tissue, the peak area of phytol could not be separated as it merged with areas of other fatty acid components. In conclusion, it was found that phytol was rapidly absorbed from gastro intestinal tract after oral administration with wider distribution and slow elimination. Phytol possesses all favourable pharmacokinetic parameters for a possible therapeutic agent.
Open Access
EVALUATION OF WOUND HEALING ACTIVITY OF ANDROGRAPHOLIDE AND HORDENINE
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, 2022-04-02) VIDYAVARSHA S. J.; Bibu John Kariyil
Wound healing is a multidimensional process that involves four overlapping phases, including hemostasis, inflammation, proliferation, and remodelling, to restore damaged or wounded tissue. Wound infections impede wound healing and lead to chronicity. Staphylococcus aureus is the most common bacteria causing wound infections. Due to the haphazard use of antibiotics in humans and animals, methicillin-resistant S. aureus (MRSA) has developed resistance to practically all commercially available antibacterial drugs. Hence, the present study is envisaged to assess the wound healing activity of andrographolide and hordenine on infected wounds. The study was designed to evaluate the antibacterial activity and wound healing activity of andrographolide and hordenine. Kirby-Bauer agar disc diffusion method and modified resazurin microtiter plate assay were used to determine the antibacterial activity. MTT and scratch assays were used to evaluate the cell viability and cell migration of L929 mouse skin fibroblast cells. Angiogenesis activity was determined by chorioallantoic membrane (CAM) assay. In vivo MRSA infected excision wound healing study on male Wistar rats was conducted to evaluate the wound healing potential of andrographolide and hordenine. Both andrographolide and hordenine did not show any zone of inhibition against MRSA and S. aureus. While hordenine showed MIC of 1000 µg/mL against MRSA and S. aureus. An increase in the per cent mean cell viability in a concentration dependent manner from 10 to 100 µg/mL was observed. Among the treatment groups, andrographolide treated cells showed highly significant (P<0.001) per cent mean cell viability. Andrographolide and hordenine treated L929 cells reduced the cellular gap significantly (P<0.001) at day 3. Among the two treatments, the reduction in cellular gap was significant (P=0.002) for hordenine treated L929 cells. Andrographolide treatment increased the primary and secondary blood vessels significantly (P<0.001) than the hordenine treatment. Andrographolide and hordenine treatments significantly (P≤0.001) increased the number of tertiary and quaternary blood vessels. Andrographolide and hordenine showed significant (P=0.017, P=0.003 respectively) reduction in the per cent reduction in wound from day 6 onwards. The per cent reduction in wound was comparable for both the treatments on day 15. Significant (P<0.001) increase in hydroxyproline content in hordenine treated group was observed than the andrographolide treated group. The hexosamine content in both the andrographolide and hordenine treated groups were comparable. On histopathological examination, fibroblast proliferation, angiogenesis, collagen formation were confirmed. Andrographolide and hordenine accelerated wound healing by proliferation and migration of fibroblasts, and angiogenesis. Hence andrographolide and hordenine could be incorporated into the treatment of MRSA infected wound. The antibacterial activity of hordenine could be utilised against S. aureus and MRSA. Furthermore, the study could be extended to exploit the mechanism of antibacterial and wound healing properties of andrographolide and hordenine.
Open Access
RECEPTOR PHARMACOLOGY OF PHYTOL ON RAT INTESTINE
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES POOKODE, 2022-02-15) MEGHA MADHUSUDHANAN; Sanis Juliet
Receptors act as mediators between the drug molecule and the physiological responses elicited. The activity of any compound depends on its interaction with the intracellular and trans-membrane receptors. Receptor binding and functional assays help in characterisation of novel receptors and receptor subtypes. Phytol is a diterpenoid has gained importance in therapeutics recently because of its numerous pharmacological activities. The studies on interaction of phytol with known classical receptors are meagre in the literature. Therefore, the present study was undertaken to assess the binding of phytol with the muscarinic, GABAnergic and histaminergic receptors in rat ileum using a Digital Dales mono bath with isometric transducer. The EC50 and IC50 of phytol in presence of agonist and antagonist of muscarinic cholinergic, GABAergic and histaminergic receptors were derived. Pre-treatment with submaximal doses of phytol attenuated the contractile response to acetylcholine (ACh). The activity of phytol as a muscarinic blocker was comparable with that of solifenacin while the relaxation potential for subsidence of pre-contracted intestine with ACh was incomplete and less effective. The response of phytol in presence of GABA and histamine receptor agonists and antagonists were not well appreciated. In conclusion, phytol produced weak spasmolytic effect in the rat ileum mediated through muscarinic receptors. Further, downstream pathway studies are required to understand the mechanism by which phytol induced relaxation of the rat ileum.
Open Access
COMPARATIVE STUDY OF BERBERINE AND CAPSAICIN AS EFFLUX PUMP INHIBITORS IN QUINOLONE RESISTANT STAPHYLOCOCCUS AUREUS ISOLATES FROM BOVINE MASTITIS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2021-08-30) ARYA MOHAN; Nisha A.R.
Antimicrobial resistance which causes failure of antibiotic therapy has become a global issue nowadays. Bacteria develop resistance towards an antibacterial agent via many mechanisms, the most important one is the drug efflux mediated through efflux pumps. Bacterial resistance causes reduction in the usage of several chemotherapeutic agents in the treatment of bacterial diseases. In this context, research on natural products from plants which can be used as plausible candidates to combat antimicrobial resistance will be of utmost importance. Hence, a study was undertaken to assess the antibacterial activity and resistance modulation through down regulation of norA, norB, and norC by two plant alkaloids berberine and capsaicin against quinolone-resistant S. aureus from bovine mastitis. Disc diffusion assay and broth microdilution assay were performed to assess the antibacterial activity of berberine and capsaicin alone (at various concentrations viz 8 g/L to 0.25 g/L) and in combination of antibiotics with enrofloxacin, norfloxacin and nalidixic acid. Resistance modulating potential through efflux pump inhibition by berberine and capsaicin in combination with quinolones at sub inhibitory concentration in down regulating norA, norB, and norC was executed by reverse transcription and quantitative real-time PCR. Antibiofilm assay was conducted for various combinations of quinolones with berberine and capsaicin using congo red agar plate method. In silico studies were carried out using autodock software to analyse the binding of berberine and capsaicin to NorA protein. The findings of disc diffusion assay revealed that berberine and capsaicin in combination with enrofloxacin, norfloxacin and nalidixic acid increased the zone of inhibition significantly (p-value < 0.05) in a dose-dependent manner. Checkerboard assay showed that berberine lowered the MIC of antibiotics tested up to eightfold and capsaicin up to fourfold. The combination of enrofloxacin with berberine exhibited synergy and with capsaicin exhibited partial synergy. The combination of norfloxacin with test compounds exhibited partial synergy and the combination of berberine and capsaicin with nalidixic acid exhibited additivity. Gene expression studies indicated downregulation in the expression of norA and norC genes, while norB was not expressed in any of the isolates. Berberine and capsaicin in combination with quinolones could inhibit the biofilm formation by S. aureus isolates in a dose dependent manner. In silico studies revealed that capsaicin bind to NorA protein. To consolidate, berberine and capsaicin can be promising drug molecules in modulating the drug resistance mediated by the Nor efflux pumps in S. aureus from bovine mastitis. Thus, berberine and capsaicin can be used as an adjunct to antibiotics for the treatment of bacterial diseases.
Open Access
EFFECT OF LEMONGRASS OIL AND CITRAL IN COMBATING ANTIBIOTIC RESISTANCE IN STAPHYLOCOCCUS AUREUS ISOLATES FROM BOVINE MASTITIS
(COLLEGE OF VETERINARY AND ANIMAL SCIENCES MANNUTHY, THRISSUR, 2020-03-24) JISHA S.; Suja Rani S.
Antibiotic resistance imposes severe therapeutic and economic burden in the treatment of mastitis and may threaten the success of antimicrobial chemotherapy. Essential oils and their constituents are gaining great importance in the recent times as promising antimicrobial and resistance modifying agents. Hence, the present study was conducted to evaluate antibacterial, resistance modifying and antibiofilm activities of lemongrass oil (LGO) and its major active principle citral, individually as well as in combination with methicillin/oxacillin against methicillin resistant Staphylococcus aureus (MRSA) isolated from bovine mastitic milk. Among the 80 mastitic milk samples collected, 58 S. aureus organisms were isolated using culture and biochemical characteristics, in which 16 isolates were identified as MRSA by antibiogram. Nevertheless, only six isolates among the MRSA revealed presence of mecA and hence selected for further evaluation study. The test substance LGO was subjected to GC-MS analysis prior to evaluation and level of the citral content was found to be 72 per cent approximately. The findings of the study indicated significant antimicrobial activity of LGO and citral as evident from their minimum inhibitory concentration (MIC) and diameter of zone of inhibition. Lemongrass oil and citral produced remarkable inhibition of biofilm formation and preformed biofilm, manifested as significantly reduced minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC). Furthermore, the combination therapy of LGO and citral with methicllin/oxacillin, as appraised by checkerboard method revealed synergistic interaction in both antimicrobial and antibiofilm assays, establishing the implication of combination strategy in mitigating antibiotic resistance. Moreover, in silico molecular docking study revealed that citral possesses binding affinity for mecA via hydrogen bond and hydrophobic interactions. Thus, present findings are indicative of the vital role of LGO and citral in alleviation of methicllin/oxacillin resistance via its synergistic effect with the resistant antibiotics, down regulation of mecA and antibiofilm activities.

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