Indian Frog Research Articles

A New epoxy steroid isolated and characterized from Bufo melanostictus venom with potential IL-6 along with TNF-α inhibition and spectroscopic study for structure and vibrational properties using DFT

This paper deals with the study on the effects of a novel epoxy steroid (NES) extracted from the parotid gland of the Indian toad (Bufo melanostictus) on the liver, kidney, and colon. NES is a new chemical compound and it is first time reported with potential protective effects on organ toxicities from Indian toad venom. The Indian toad parotid secretion-derived chemical was isolated, identified as NES and tested for its potential of protective effect. NES was identified by using infrared, 1D, and 2D approaches. The in-silico investigations showed that it has a higher affinity for TNF-α and IL-6. By exposing mice to organ toxicity induced by Dextran Sodium Sulphate and Bisphenol A in the colon, kidneys, and liver, NES was able to target the production of IL-6 and TNF-α. NES significantly suppressed the IL-6 and TNF-α level in all selected organ toxicities in a dose a dependent manner and revealed prominent protection by inhibiting TNF-α. It was further confirmed by the histo-pathological recovery of the damaged cells and immunohistology suppression of CD 68 expression when compared to the induced groups. Further, the structural parameters of the new compound NES were determined from the optimized molecular geometry, approaching the quantum chemical computations employing density functional theory (DFT) using Becke's hybrid functional (B3LYP) along with 6–311++G(d,p) basis set. HOMO-LUMO energy gap was performed to assess the chemical reactivity and biological activity.

Incidence of Dermatophagy by Common Indian Toad Duttaphrynus Melanostictus (Schneider, 1799)

The skin of amphibians serves as a vital immune barrier that is essential for survival. In amphibians, the shedding of skin, or dermatophagy, is a multifaceted process influenced by factors such as species, age, and environmental conditions. This article explores an interesting incident of dermatophagy observed in Duttaphrynus melanostictus, the Common Indian Toad, within the Nature Garden of Amboli, Maharashtra. The toad exhibited the unique behaviour o consuming its own shed skin, a phenomenon not widely documented in amphibians. While dermatophagy has been studied in various reptiles, its purpose remains unclear. This observation provides insights into the ecological and behavioural aspects of amphibians, raising questions about the active or incidental nature of dermatophagy and its potential ecological correlates. Further research is warranted to unravel the precise reasons behind this intriguing behaviour in amphibians.

Regulatory Effect of Thyroxine on Calcium and Phosphorus Metabolism in Tissues of Common Indian Toad

Thyroxine (T4) plays a vital role in the metabolic processes of animals. It influences the storage and mobilisation of minerals and their utilisation according to the requirement of the body. It is also involved in the early development of vertebrates, especially in amphibian metamorphosis. It has shown anabolic and catabolic effects at a lower dose and higher dose, respectively. The present study aims to identify the role of thyroxine in regulating calcium and phosphorous metabolism in the blood, muscle and bone tissues of Bufo melanostictus. Toads of various sizes and different age groups were chosen, showing a positive correlation between body weight and snout to vent length, indicating overall continuous growth as observed for a large number of poikilothermic animals. As per an earlier report, thyroxine has been shown to have a direct catabolic effect on bone mineral homeostasis leading to an increase in bone mineral resorption and calcium loss through the kidney. Results of the present study indicate that thyroxine treatment in Bufo melanostictus caused depletion of calcium and phosphorous in muscle and bone, while their level increased in the serum at both doses (0.5 μg/gm and 2 μg/gm) during short term i.e. a one-day T4 treatment. Such results are very much consistent with many of the earlier obscurations that, T4 causes an increase in serum calcium and phosphorous levels, while their level in muscle and bone decreased. When the level of these two minerals falls in the serum due to any other condition, the bone resorption under the influence of thyroxine maintains the concentration in serum, consequently, there is a loss in bone density due to resorption and in muscle due to utilisation. However, if the exogenous supply is adequate, the loss caused as above is compensated for, in both bone and muscle.

Novel Indole Derivative as the First P-glycoprotein Inhibitor from the Skin of Indian Toad (Bufo melanostictus)

To study the inhibitory effect of novel indole derivative (NID) from Indian toad skin (Bufo melanostictus) on permeability glycoprotein (P-gp). Dried Indian toad skin was used to isolate NID with column chromatography, and its structure was elucidated by infrared spectra, 13C nuclear magnetic resonance (NMR), 1H NMR spectra, and liquid chromatography-mass spectrometry. Female Wistar rats were used to determine LD50, in vitro permeability studies were done with the intestinal sac method, and in vivo pharmacokinetic studies were carried out to prove the P-gp inhibition using the rat model. The NID has shown increased clear permeability Papp (x10-6 cm/sec) significantly (p<0.001) from 1.04±0.11 to 2.90±0.08 in ileum 1.44±0.14 to 3.92±0.13 in jejunum this in vitro results confirmed that P-gp inhibited, this was further confirmed by in vivo studies in in vivo studies observed increased oral bioavailability of digoxin (DIG) significantly in NID treated groups from 3.26±0.25 to 7.47±0.18 ng/mL, the volume of distribution decreased from 232.56±64.59 to 86.57±7.04 L/kg. Area under the curve increased from 37.89±1.13 to 64.62±0.70 ng/mL/hr. This demonstrates NID increased the oral bioavailability of DIG significantly. Many compounds were isolated from the Indian toad skin. This NID was not reported earlier. Results demonstrate NID increased the oral bioavailability of DIG significantly. The isolated NID from Indian toad skin proved as a potent P-gp inhibitor in both in vitro and in vivo studies, and further studies are needed to develop as a possible new drug candidate.

Hematological Profile of Indian Bullfrog and Common Toad from Different Environmental Habitats

The wellbeing of human population depends on the ecosystem of earth. Amphibians from the ancient time represents a major part in the global diversity and play important role for the benefit of society worldwide. The aim of the present study was to investigate the comparative hematological study of Indian bullfrog (Hoplobatrachus tigerinus) from wetland and a common Indian toad (Duttaphrynus melanostictus) from terrestrial environment. In this study we have procured 10 sexually mature and disease-free Indian bullfrog (9.93±0.07 cm total length, 59.5±1.94 g weight) and 10 common Indian toad (8.26±0.26 cm total length, 78.6±1.36 g weight). The RBC and WBC count were found to be increased in frog than toad. Variation in RBC morphology which reveals the anemic condition in frog than that of toad. The study suggested that the environmental conditions have significant impact on status of frog and toad.

Mosquitoes (Uranotaenia sp.) biting an Indian Golden-backed Frog (Indosylvirana indica)

Mosquitoes (Uranotaenia sp.) biting an Indian Golden-backed Frog (Indosylvirana indica)

Influence of Toad Parotid Gland Secretion from Indian Toad (Bufo melanostictus) in Diabetic Rats: An Experimental Evidence of P-Glycoprotein Inhibition

The study was conducted to improve the oral bioavailability of glyburide (GLY) with Indian Toad Parotid Gland Secretions (TPGS). P-glycoprotein is an efflux transporter cellular protein and effluxes xenobiotics and drugs to the outside of cells lead to decreased concentration of drugs at the target site. P-gp inhibitors essentially increase the levels and there is a need for new P-gp inhibitors to develop for the improvement of the oral bioavailability of P-gp substrate drugs because the existing inhibitors have serious side effects. This study was aimed to describe the P-gp inhibitory action from TPGS, Bufo melanostictus, in diabetic rats by using glyburide as p-gp substrate. Acute toxicity studies showed 300 mg/kg as toxic dose and 50 mg/kg was selected as study dose according to OECD 423. LC-HRMS study conducted to identify the new compounds. Apparent permeability (Papp) was estimated by non-everted sac method (In Vitro) with rat jejunum and ileum to confirm the P-gp inhibitory activity of TPGS by using fexofenadine (FEX) as P-gp substrate. In in-vivo protocol rats grouped into 4 groups (n=6), the first one is normal, second diabetic, third GLY 30 mg/kg, and fourth group GLY+ TPGS, 50 mg/kg for the single and multiple-dose treatment study. The spectrometric analysis revealed the new compounds, and TPGS Papp (X10-6 cm/s) in rat jejunum and ileum was significantly increased from 2.0±0.1 to 6.4±0.3 and 1.2±0.3 to 3.0±0.3 respectively. Blood glucose concentration in rats more than 250 mg/dl were considered as diabetic and in single, multiple-dose interaction studies (SDI, MDI) the concentrations decreased from 140.0±2.0 and 122.0±2.2 µg/dl respectively. The pharmacokinetic parameters like Cmax, Cl and in SDI, MDI and significant increase of C max and AUC t and decrease of Cl was observed. The above results conclude that TPGS had the potential P-gp inhibitory activity and improved the oral bioavailability of GLY significantly. Subsequent experimentation is warranted to chemically characterize the compounds from TPGS as potential new P-gp inhibitors.

Cellular lactate and pyruvate are key intermediates for intra-cellular energy metabolism regulated by Lactate dehydrogenase (LDH) in age-related phenomenon

Ageing in animals is accompanied by changes in cellular function. Although the cause of aging appears to be multifactorial, the alterations in cellular functions are the consequence of changes in biochemical composition of cells since a number of enzymes and metabolites regulate the overall activities of the cell. There is a great deal to be known about the interrelationship between growth, sexual maturity and aging in animals. The poikilothermic vertebrates would serve as suitable animal models in which these parameters could be easily manipulated through variation. In the present study we have used the non-mammalian vertebrate common Indian toad to find the glycolytic enzyme activity (Lactate dehydrogenase-LDH) during ageing. We have investigated the LDH activity, pyruvate and lactate content in liver and muscle and compared if there is any biochemical distinctiveness in the ageing pattern.

Response of Ultimobranchial and Parathyroid Glands of the Indian Skipper Frog, Euphlyctis cyanophlyctis to Cadmium Toxicity

Background: Cadmium toxicity has been shown in aquatic animals but the effect on frog’s endocrine glands is not known. We investigated the effects of cadmium on ultimobranchial and parathyroid glands of Indian skipper frog, Euphlyctis cyanophlyctis. Methods: Frogs were exposed to cadmium chloride for short and long terms and sacrificed after 24, 48, 72 or 96 h (short-term) and after 5, 10, 15 and 30 days (long-term). Results: After 48h of cadmium exposure, serum calcium levels decreased and persisted for 96h. After 96h of exposure, weak staining of the ultimobranchial cells was noted, and the nuclear volumes decreased. The histological structures of parathyroid cells remained unchanged. Frogs exposed to cadmium exhibited decreased serum calcium levels from day 10 to day 30. After 15 days of cadmium exposure, ultimobranchial cells exhibited a decline in staining response and the nuclear volume also decreased. The follicular epithelia of the ultimobranchial glands appeared to be reduced at certain areas. After 30 days of cadmium exposure, the nuclear volume was further decreased and the follicular epithelia shrunk and appeared as a single layer. Cellular degeneration and vacuolization were also noticed in the gland. After day 15 of cadmium exposure, the nuclear volume of the parathyroid cells increased with elongated and hyperchromatic appearance. These changes were exaggerated on day 30 of cadmium exposure and few cell lyses appeared among parathyroid cells. Conclusions: Cadmium adversely affected the calcium regulating glands, ultimobranchial and parathyroid glands of the Indian frogs, Euphlyctis cyanophlyctis.

The Antifungal Activity of Skin Secretion and its Extract of Indian Toad Bufo melanostictus

The Antifungal Activity of Skin Secretion and its Extract of Indian Toad Bufo melanostictus

Occurrance of Aplectana macintoshii (Stewart, 1914) Travassos, 1931 (Nematoda: Cosmocercidae) in Indian Skipper Frog, Euphlyctis cyanophlyctis (Anura: Dicroglossidae) at Raniganj Town, Paschim Bardhaman, West Bengal

The Indian skipper frog, Euphlyctis cyanophlyctis (Schneider, 1799), is a common frog found in West Bengal. A total of six frogs (E. cyanophlyctis) were collected and examined for nematode infection from Raniganj town of Asansol coal-field area, West Bengal. Six male and 11 female nematodes were recovered from the rectum of two out of six, E. cyanophlyctis. On examination the recovered nematodes were found to be, Aplectana macintoshii (Stewart in Rec Indian Mus 10:165–193, 1914) Travassos, 1931. Aplectana macintoshii is reported for the first time from Asansol coal-field area. This species can be identified by the presence of lanceolate shaped gubernaculum, one pair of similar and equal spicules and seven pairs of postanal caudal papillae, all are ventral in position and a single papilla on the tail tip. This communication intends to record Asansol industrial area as a new geographical area for the parasite A. macintoshii.

Indian toad (Bufo melanostictus, Schneider) skin extract induces apoptosis and shows cytotoxic effect on Ehrlich ascites carcinoma (EAC) cells

Indian toad (Bufo melanostictus) skin extract (TSE) was evaluated for cytotoxic and apoptogenic activities on Ehrlich ascites carcinoma (EAC) cells of EAC bearing Swiss albino male mice. LD50 of TSE was found to be 400 mg kg-1, (i.v.) and 750 mg kg-1, (i.p.) in mice. EAC cells (1-2 ´ 105 / mouse) were inoculated (i.p.) into mice (20.6 ± 0.14 gm) and the status of EAC cell proliferation and viability were studied in control and TSE treated groups. TSE (10, 20, 30 mg kg-1 d-1, i.p. for 10 d) significantly inhibit EAC cell growth in dose dependent manner. TSE (50, 100 mg kg-1 d-1, i.p. for 1 d) significantly reduced the viability of EAC cells and decreased the MTT values compared to control cells. TSE induced DNA breakage was reflected in DNA ladder and single cell gel electrophoresis (comet assay). TSE significantly (P&lt;0.001) increased the number tailed cells and length-width ratio of DNA mass in EAC cells as compared to control. Fluorescent microscopy of TSE treated cells showed significant increase in number of early and late apoptotic cells compared to the control EAC cells. Apoptotic index of TSE treated cells was significantly (P&lt;0.001) higher than that of the control cells. Changes in serum LDH, β2-microglobulin in TSE treated EAC mice and our earlier studies indicated the involvement of immunomodulation which may indirectly be associated with the anticancer activity of TSE.&#x0D; Keywords: Toad skin extract; Bufo melanostictus; Ehrlich ascites carcinoma; Cytotoxic agent;

A new locality record for Leith’s Leaping Frog, Indirana leithii (Boulenger 1888), from Dhulda Village, Dangs District, Gujarat, India

A new locality record for Leith’s Leaping Frog, Indirana leithii (Boulenger 1888), from Dhulda Village, Dangs District, Gujarat, India

Vitamin A induced homeotic hindlimb formation on dorsal and ventral sides of regenerating tissue of amputated tails of Japanese brown frog tadpoles.

When anuran tadpoles are treated with vitamin A after tail amputation, hindlimb-like structures can be generated instead of the lost tail part at the amputation site. This homeotic transformation was initially expected to be a key to understanding the body plan of vertebrates. Unfortunately, homeotic limb formation has been reproduced in only some Indian frog species and a European species, but not in experimental anurans such as Xenopus laevis or Rana catesbeiana. Consequently, this fascinating phenomenon has not been well analyzed, especially at the molecular level. In addition, the initial processes of ectopic limb development are also unclear because morphological changes in the early phases have not been analyzed in detail. In this study, we report the induction of homeotic transformation using Japanese brown frogs and present a detailed morphological analysis. Unexpectedly, the ectopic limbs developed not only at the ventral sites, but also at the dorsal sites of the tail regenerates of vitamin A-treated tadpoles. The relationship between position and axial orientation of ectopic limbs suggested the double duplication of positional value order along the rostral-caudal axis and the dorsal-ventral axis of the tail regenerates.

Frog mucus peptide kills flu virus

The slimy mucus that coats the skin of an Indian frog contains peptides that destroy the H1 influenza virus, according to a team of researchers led by Joshy Jacob of Emory University. One of these flu-killing peptides appears to be harmless to human cells, making it a promising antiviral candidate to fight the flu (Immunity 2017, DOI: 10.1016/j.immuni.2017.03.018). The slimy surface of Hydrophylax bahuvistara and other amphibians could become a siren call for researchers searching for antimicrobial compounds. The mucus contains many molecules that protect the animals against bacterial and viral pathogens, and the slime is relatively easy to isolate: Researchers need to just give the frogs a small electric shock, collect the released mucus, and let the amphibians hop away. Jacob’s team focused on one 23-amino-acid peptide, which the researchers named urumin after a deadly whip sword found in the same region as the frog’s native habitat. Most antiviral

An Amphibian Host Defense Peptide Is Virucidal for Human H1 Hemagglutinin-Bearing Influenza Viruses

Although vaccines confer protection against influenza A viruses, antiviral treatment becomes the first line of defense during pandemics because there is insufficient time to produce vaccines. Current antiviral drugs are susceptible to drug resistance, and developing new antivirals is essential. We studied host defense peptides from the skin of the South Indian frog and demonstrated that one of these, which we named "urumin," is virucidal for H1 hemagglutinin-bearing human influenza A viruses. This peptide specifically targeted the conserved stalk region of H1 hemagglutinin and was effective against drug-resistant H1 influenza viruses. Using electron microscopy, we showed that this peptide physically destroyed influenza virions. It also protected naive mice from lethal influenza infection. Urumin represents a unique class of anti-influenza virucide that specifically targets the hemagglutinin stalk region, similar to targeting of antibodies induced by universal influenza vaccines. Urumin therefore has the potential to contribute to first-line anti-viral treatments during influenza outbreaks.

Seven new species of Night Frogs (Anura, Nyctibatrachidae) from the Western Ghats Biodiversity Hotspot of India, with remarkably high diversity of diminutive forms.

The Night Frog genus Nyctibatrachus (Family Nyctibatrachidae) represents an endemic anuran lineage of the Western Ghats Biodiversity Hotspot, India. Until now, it included 28 recognised species, of which more than half were described recently over the last five years. Our amphibian explorations have further revealed the presence of undescribed species of Nights Frogs in the southern Western Ghats. Based on integrated molecular, morphological and bioacoustic evidence, seven new species are formally described here as Nyctibatrachus athirappillyensis sp. nov., Nyctibatrachus manalari sp. nov., Nyctibatrachus pulivijayani sp. nov., Nyctibatrachus radcliffei sp. nov., Nyctibatrachus robinmoorei sp. nov., Nyctibatrachus sabarimalai sp. nov. and Nyctibatrachus webilla sp. nov., thereby bringing the total number of valid Nyctibatrachus species to 35 and increasing the former diversity estimates by a quarter. Detailed morphological descriptions, comparisons with other members of the genus, natural history notes, and genetic relationships inferred from phylogenetic analyses of a mitochondrial dataset are presented for all the new species. Additionally, characteristics of male advertisement calls are described for four new and three previously known species. Among the new species, six are currently known to be geographically restricted to low and mid elevation regions south of Palghat gap in the states of Kerala and Tamil Nadu, and one is probably endemic to high-elevation mountain streams slightly northward of the gap in Tamil Nadu. Interestingly, four new species are also among the smallest known Indian frogs. Hence, our discovery of several new species, particularly of easily overlooked miniaturized forms, reiterates that the known amphibian diversity of the Western Ghats of India still remains underestimated.

Unearthing the Fossorial Tadpoles of the Indian Dancing Frog Family Micrixalidae.

Tadpoles of the monotypic Indian dancing frog family Micrixalidae have remained obscure for over 125 years. Here we report the discovery of the elusive tadpoles of Micrixalus herrei from the sand beds of a forested stream in southern Western Ghats, and confirm their identity through DNA barcoding. These actively burrowing tadpoles lead an entirely fossorial life from eggs to late metamorphic stages. We describe their internal and external morphological characters while highlighting the following features: eel-like appearance, extensively muscularized body and tail, reduced tail fins, skin-covered eyes, delayed development of eye pigmentation in early pre-metamorphic stages (Gosner stages 25–29), prominent tubular sinistral spiracle, large transverse processes on vertebrae II and III, ankylosed ribs on transverse processes of vertebra II, notochord terminating before the atlantal cotyle-occipital condyle junction, absence of keratodonts, serrated well-formed jaw sheaths, and extensive calcified endolymphatic sacs reaching sacrum posteriorly. The tadpole gut contains mostly fine sediments and sand. We discuss the eel-like morphology and feeding habits of M. herrei in the context of convergence with other well-known fossorial tadpoles. This discovery builds the knowledge base for further comparative analyses and conservation of Micrixalus, an ancient and endemic lineage of Indian frogs.

First report of Lividin and Spinulosain peptides from the skin secretion of an Indian frog.

Here, we report two novel peptides identified from the skin secretion, having homologies to Lividin and Spinulosain, of an endemic frog, Hydrophylax bahuvistara, of Western Ghats. This is the first report of these peptides from Indian frogs and first identification of Lividin from the Hydrophylax genus. Both peptides exhibited weak antimicrobial activity but very low haemolytic activity. The problems of naming amphibian host defense peptides (HDPs) are also discussed.

Detection of antidiabetic activity by crude paratoid gland secretions from common Indian toad (bufomelano stictus).

Background:Amphibians have provided a remarkable array of biological active compounds, which are secreted from socalled granular skin glands which serve to protect the amphibians from predators due to its noxious effects on buccal tissue and at least in the case of some peptides, to protect from bacterial (or) protozoan infections. Given the respiratory and antimicrobial functions of amphibian skin, it is likely that some of the novel molecules found in amphibian granular gland secretions might be of use in the treatment of skin and respiratory infections. Secretions from common Indian toad (Bufo melanostictus) a member of Bufonidae family has the history of medicinal use however the anti-diabetic activity is not reported. The present study is aimed to determine whether paratoid gland extract have any influence on the diabetes and the pharmacokinetics and pharmacodynamics of glimepiride (GLM) in normal and diabetic rats.Materials and Methods:An aqueous and methanolic extracts of paratoid glandular secretions were prepared, air dried and used to determine the antidiabetic activity in rats. The blood sampling was done at preset time intervals between 0, 0.5, 1, 2, 4, 6, 8 and 12 h, using heparinized capillaries. The blood glucose levels are estimated by glucose oxidase-peroxidase method, and reversed-phase high-performance liquid chromatography is used to determine the pharmacokinetic parameters of GLM using glibenclamide as an internal standard.Results:Both the aqueous and methanolic extracts produced better glycemic control in diabetic rats, and methanolic extract is better than the aqueous extract. Serum concentrations of GLM increased at 2nd h, and the percentage glucose reduction is maximal at the 4th h with both aqueous and methanolic extracts of paratoid secretions of common Indian toad.Conclusions:Paratoid gland secretions of the common Indian toad is antidiabetic, in addition it has beneficial effects in combination with GLM. Further, it requires the systematic structure elucidation of the compounds and pharmacokinetic studies to explore the beneficial effects.