1.

1 INTRODUCTION

Nature provided different resources to get medicinal compounds to treat different ailments for
the benefit of human kind. Various chemical compounds were identified from plants, animal,
marine, mineral and microorganisms and later they became life saving drugs (Alamgir, 2017).
Plants were important reservoirs of new novel drug candidates as they are being played a key
role in drug discovery (Veeresham, 2012). Drugs of animal origin come from either whole
animal, parts (organs) and/or from glandular secretions. From the animal origin, heparin
obtained from Mexican medical leech Hirudo manillensis, and a potent analgesic compound
epibatidine obtained from skin extracts of the Ecuadorian poison frog Ameeregabilinguis
(Whittaker, 1969). Animal secretions include venoms and toxins from spider, snake,
scorpions and insects, were used in drug development process. Teprotide, a peptide obtained
from Brazilian viper Bothrops jararaca developed as antihypertensive agents, they are
captopril and cilazapril. Since ancient times, amphibians have been serving as potential
sources for new chemical compounds. These amphibians include, caecilians (order-
Gymnophiona), salamanders (order-Caudata), frogs and toads (order-Anura) (Harvey Pough,
2007) are from 6000 species. Amphibians are ectotherms and nocturnal in nature and also
their skin is permeable to ions, water, through the secretive action (Duellman and Trueb,
1994). Amphibians skin is pooled with plenty of mucus and poisonous glands and some of
the secretions helps in oxygen and carbon dioxide exchange for respiration (Alford and
Richards, 1999). Drugs developed from animal sources like batroxobin, a defibrinogenating
hemostatic agent from pit viper (Bothrops atrox and Bothrops moojeni) (Kini, 2006), captopril
a anti hypertensive agent from Brazilian pit viper venom (Waheed et al., 2017), and
eptifibatide, an anti coagulant drug from south-eastern pygmy rattle snake venom (Phillips
and Scarborough, 1997). Exenatide using in the treatment of Type-2 diabetes drug, derived
from saliva of the Gila monster lizard (Heloderma suspectum) (Furman, 2010). Ziconitide, an
N-type calcium channel blocker used for severe pain developed from cone snail (Safavi-
Hemami et al., 2019).
Drugs are substances that are used or intended to be used in the diagnosis, prevention,
treatment or cure of diseases. In early times, these substances were derived from natural
sources, of which plants took up the major share. With the introduction of technology, most
drugs today are manufactured synthetically in the laboratory. The major sources of drugs can
be grouped into the following

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 1

Drugs are obtained from six major sources:

1. Plant sources
2. Animal sources
3. Mineral/ Earth sources
4. Microbiological sources
5. Semi synthetic sources/ Synthetic sources
6. Recombinant DNA technology

1.2 Plant Sources:

Plant source is the oldest source of drugs. Most of the drugs in ancient times were
derived from plants. Almost all parts of the plants are used i.e. leaves, stem, bark, fruits and
roots.

Leaves:
a. The leaves of Digitalis Purpurea are the source of Digitoxin and Digoxin, which are cardiac
glycosides.

b. Leaves of Eucalyptus give oil of Eucalyptus, which is important component of cough syrup.

c. Tobacco leaves give nicotine.

d. Atropa belladonna gives atropine.

Flowers:
1. Poppy papaver somniferum gives morphine (opoid)
2. Vinca rosea gives vincristine and vinblastine
3. Rose gives rose water used as tonic.
Photo of Papaver somniferum by Evelyn Simak

Fruits:
1. Senna pod gives anthracine, which is a purgative (used in constipation)
2. Calabar beans give physostigmine, which is cholinomimetic agent

Seeds:
1. Seeds of Nux Vomica give strychnine, which is a CNS stimulant.
2. Castor oil seeds give castor oil.
3. Calabar beans give Physostigmine, which is a cholinomimetic drug.

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Roots:
1. Ipecacuanha root gives Emetine, used to induce vomiting as in accidental poisoning.
It also has amoebicidal properties.
2. Rauwolfia serpentina gives reserpine, a hypotensive agent.
3. Reserpine was used for hypertension treatment.

Bark:
1. Cinchona bark gives quinine and quinidine, which are antimalarial drugs. Quinidine
also has antiarrythmic properties.
2. Atropa belladonna gives atropine, which is anticholinergic.
3. Hyoscyamus Niger gives Hyosine, which is also anticholinergic.

Stem:
Chondrodendron tomentosum gives tuboqurarine, which is skeletal muscle relaxant used
in general anesthesia.

1.3Animal Sources:

1. Pancreas is a source of Insulin, used in treatment of Diabetes.

2. Urine of pregnant women gives human chorionic gonadotropin (hCG) used for the
treatment of infertility.
3. Sheep thyroid is a source of thyroxin, used in hypertension.
4. Cod liver is used as a source of vitamin A and D.
5. Anterior pituitary is a source of pituitary gonadotropins, used in treatment of
infertility.
6. Blood of animals is used in preparation of vaccines.
7. Stomach tissue contains pepsin and trypsin, which are digestive juices used in
treatment of peptic diseases in the past. Nowadays better drugs have replaced them.

1.4 Mineral Sources:

i. Metallic and Non-metallic sources:

1. Iron is used in treatment of iron deficiency anemia.

2. Mercurial salts are used in Syphilis.
3. Zinc is used as zinc supplement. Zinc oxide paste is used in wounds and in eczema.
4. Iodine is antiseptic. Iodine supplements are also used.
5. Gold salts are used in the treatment of rheumatoid arthritis.

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ii. Miscellaneous Sources:

1. Fluorine has antiseptic properties.

2. Borax has antiseptic properties as well.
3. Selenium as selenium sulphide is used in anti dandruff shampoos.
4. Petroleum is used in preparation of liquid paraffin.

1.5 Synthetic/ Semi synthetic Sources:

i. Synthetic Sources:

When the nucleus of the drug from natural source as well as its chemical structure is altered,
we call it synthetic.
Examples include Emetine Bismuth Iodide

ii. Semi Synthetic Source:

When the nucleus of drug obtained from natural source is retained but the chemical
structure is altered, we call it semi-synthetic.
Examples include Apomorphine, Diacetyl morphine, Ethinyl Estradiol, Homatropine,
Ampicillin and Methyl testosterone.

Most of the drugs used nowadays (such as antianxiety drugs, anti convulsants) are synthetic
forms.

1.6 Microbiological Sources:

1. Penicillium notatum is a fungus which gives penicillin.

2. Actinobacteria give Streptomycin.
3. Aminoglycosides such as gentamicin and tobramycin are obtained from streptomycis
and micromonosporas.

1.7 Recombinant DNA technology:

Recombinant DNA technology involves cleavage of DNA by enzyme restriction

endonucleases. The desired gene is coupled to rapidly replicating DNA (viral, bacterial or
plasmid). The new genetic combination is inserted into the bacterial cultures which allow
production of vast amount of genetic material.

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Advantages:

1. Huge amounts of drugs can be produced.

2. Drug can be obtained in pure form.
3. It is less antigenic.

Disadvantages:

1. Well-equipped lab is required.

2. Highly trained staff is required.
3. It is a complex and complicated technique.
Males can grow up to about 8 -10cm, while females are larger and can reach about 9 - 11 cm.
Life span is about approximately 10 years, undergoes hibernation from October to march.

1.8. Toads

Toads are distributed throughout the world except polar and arid regions but are present mainly
in areas on tropical and humid temperate climates. Each region is characterized by presence of
some species of these amphibians. The ability of toads to survive in such a broad diversity of
habitat types may be attributed to the evolution of many different morphological, physiological,
biochemical and behavioral adaptations. For animals with a permeable, naked skin which must
be kept at least slightly moist for cutaneous respiration, and able to maintain the body
temperature a few degrees above that of their surroundings. Toads adapted to an extraordinary
variety of habitats and climatic conditions, ranging from arid deserts to deep fresh water.
Although toads do not have venom inoculation system, they are considered venomous animals,
as the glands covering the whole surface of their skin secrete highly toxic venom (Russell,
1976). There are 34 genera and 410 species of toads throughout the continents (Yang et al.,
2015). In India about 311 species of existing orders, namely Anura, Caudata, and
Gymnophiona were reported (Vasudevan et al., 2001).

1.8.1. Indian toad

Duttaphrynus Melanostictus which is known by common Indian toad, locality throughout

India, also found in South-East Asia, grows up to overall 150 mm size. Habitat of Land includes
moist places, water tanks, garden, stream sides, leaf litter, and rocks. They are accessible below

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street lamps, bathrooms, water tanks and also in agricultural fields (Kv, 2012). They have
noticeable features like prominent cranial ridges, well-marked tympanum, cornified warts on
back, toe tips and fingers, webbing present in feet and elongated poison glands which is shown
in Fig 1.

Fig 1. Common Indian toad showing parotoid glands (Bufo melanostictus)

(Source: https://en.wikipedia.org/wiki/Duttaphrynus)

1.8.2 Scientific classification:

Kingdom: Animalia
Phylum : Chordata
Class : Amphibia
Order : Anura
Family : Bufonidae
Genus : Bufo
Species : Bufo melanostictus

1.8.3. Toad glands

Glands are of two types one is mucous glands and other is granular (poisonous) glands. Mucous
glands consists of transparent mucus (lubricant), glycoprotein’s (mucins, mucinigen)
carbohydrate-residues (Galactose, fucose and sialic acid). Granular glands consists of acrid
poisons or toxins, help in providing protection from predators like birds, mammals, snakes,
crocodiles (Noble and Noble, 1944; Toledo and Jared, 1995).

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1.8.4. Composition of glandular secretions

Toad poisons, excreted by both types of glands, are composed of proteins and peptides,
biogenic amines, steroids, and alkaloids (Gao et al., 2010; Garg et al., 2007a). Composition of
the toad poison depends on food, species and the environment in which they live (Batista et al.,
2011; Sciani et al., 2013). Biogenic amines include three catecholamines (dopamine, nor
epinephrine, and epinephrine) and tyrosine metabolites tyramine and octopamine (Farooqui
and Farooqui, 2010). Aromatic amines in toad skin found are indolealkylamines, bufogenines
and bufotoxins are the steroidal groups of toad poison components are cardiotonic glycosides
like cardenolides and bufadienolides based on their molecular arrangement. Bufadienolides in
toad poisons are named as cardiotonic glycosides eg. arenobufagin, bufalin, bufotalin,
cinobufagin, cinobufotalin, gamabufotalin, hellebrigenin, marinobufagin, resibufogenin and
telocinobufagin (Steyn and van Heerden, 1998). The Peptides in toad poisons are responsible
for having antimicrobial activity. Antimicrobial peptides(AMP) like buforin I from Bufo bufo
gargarizans (Cho et al., 2009), alyteserin 1 and alyteserin 2 from Alytes obstetricians (Conlon
et al., 2009a; König et al., 2012), maximin H5 from a skin cDNA of B. maxima (Lai et al.,
2002) are proved to have antimicrobial peptides in toad secretions (Baldo et al., 2015). Among
thealkaloidspumiliotoxinsallopumiliotoxins,homopumiliotoxins,samandarines,gephyrotoxins
,histrionicotoxins,batrachotoxins,epibatidine,decahydroquinolines, a diversity of izidines
(pyrrolizidines, indolizidines, quinolizidines, lehmizidines), pyrrolidines, piperidines,
varioustricyclics and spiropyrrolizidines were found in toad secretions (Daly et al., 2005).
Interestingly, till now there are no reports on Indian toad secretion, components as they are
from different ethnic background. So we investigated the possible pharmacological and
pharmacokinetic activities of paratoid glandular secretions.

1.8.5. Hibernation Period

Hibernation is a state of inactivity and metabolic depression in animals, characterized

by lower body temperature, slower breathing, and lower metabolic rate. Hibernation is not
inevitable to all amphibian species some of them are immune to hibernation. Bufo
melanostictus is a common Indian toad and like other anurans are markedly influenced by
changes in abiotic factors like temperature and water. Indian anurans undergo four different
physiological states (i) Hibernation or Wintering (Dec-Feb) and (ii) Aestivation or Summer
torpidity (Apr-June) are in active phases with low metabolic rates (Flanigan et al., 1991).

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During these two periods they are completely terrestrial. Breeding season is the period of high
sexual activities and is a completely aquatic phase which coincides with monsoon rain (van
Gelder et al., 1986). Post breeding, season is the period of hyperphagia which is a preparation
to undergo wintering and then summer torpidity. Amphibian skin is highly permeable to water
and it has been known that these animals absorb water (Fatmi et al., 2010; Jørgensen, 1994).
During terrestrial phase these anurans face high environmental temperature which leads to
evaporative cutaneous water loss and during aquatic phase they encounter the passive inflow
of water through skin and buccopharynx which may results in flooding of tissues. In bufo an
area of skin on the lower abdomen is termed as seat patch or pelvic patch. It constitutes about
10% of the skin area but is responsible for 70-85% of the total water uptake by dehydrated
toads. In Bufo melanostictus in order to adjust themselves to both aquatic and terrestrial life,
undergo a series of physiological adjustments to decrease osmotic gradients during the
breeding and post breeding periods, in order to reduce passive inflow of water through the skin.
In aestivation and hibernation they increase the osmotic gradient to actively absorb water from
the soil.
Hibernating animals conserve energy, especially during winter when food is short,
tapping energy reserves, and body fat at a slow rate. During pre-winter season they labor for
food, shelter where they could stay could stay during winter. Metabolic depression played a
crucial role during the period of hibernation.

Characteristics :
The top of the head has several bony ridges, along the edge of the snout (canthal ridge),
in front of the eye (preorbital), above the eye (supraorbital), behind the eye (postorbital), and a
short one between the eye and ear (orbitotympanic). The snout is short and blunt, and the space
between the eyes is broader than the upper eyelid width. The ear drum or tympanum is very
distinct and is at least as wide as two-thirds the diameter of the eye. The first finger is often
longer than the second and the toes are at least half webbed. A warty tubercle is found just
before the junction of the thigh and shank (subarticular tubercle) and two moderate ones are on
the shank (metatarsus). No skin fold occurs along the tarsus. The “knee” (tarsometatarsal
articulation) reaches the tympanum or the eye when the hind leg is held parallel along the side
of the body. The dorsal side is covered with spiny warts. The parotoids are prominent, kidney-
shaped, or elliptical and elongated, and secrete milky white Bufotoxin. The dorsal side is
yellowish or brownish and the spines and ridges are black. The underside is unmarked or
spotted. Males have a subgular vocal sac and black pads on the inner fingers that help in holding

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the female during copulation. Amphibians (Toads) obtain water by osmosis across their
ventral skin. A specialized region in the pelvic skin termed the seat patch, contains aquaporins
(AQPs) stimulates arginine vasotocin(AVT)(homologues to vasopressin) to facilitate
rehydration.(Shibata et al.,2011). Due to transport of water across their skin there may be a
chance of permiability across the biological membranes for various transporter subtrates

Table 1: Proven studies of Toad Toxins of different origins

Compound Molecular target

Bufalin Macrophages, eosinophils, lymphocytes, and neutrophils and

cytokines including IL-4, IL-5, and IL-13, NF-κB,COX-2, IL-1β,
IL-6, TNF-α.

Cinobufagin Caspase-3, hypoxia-inducing factor-1 alpha,Cortactin Notch

pathway

Arenobufagin P-53 pathway ADP-ribose polymerase, light chain 3-II

Gamabufotalin COX-2

Telocinobufagin CD4, CD8, IL-2, IL-12, IFN-γ, TNF-α, IL-4

(Garg et al., 2008)

1.9 Oxidative Stress

Today animals and human beings were exposed with environmental and biological factors like
changes in food habits, alteration in feed, climatic differences, transportation, many stressful
conditions, therapeutic and prophylactic activities (Rahal et al., 2014). Now a day’s human
kind is facing chronic diseases like cancer, hypertension, asthma, diabetes, cardiovascular
diseases and other ailments due to oxidative stress. One survey, suggests that diet plays a

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crucial role for the control of these chronic diseases (Kumar et al., 2011b; Kumar et al., 2010a).
Fruits and vegetables are having a protective effect against many ailments due to presence of
natural antioxidant system (Upadhayay et al., 2012). Generation of harmful reactive oxygen
species (ROS) and reactive nitrogen species (RNS) free radicals, causing potential damage to
the biological systems inside our body is called as oxidative stress and nitrosative stress
(Kovacic and Jacintho, 2001; Ridnour et al., 2005). ROS comprises unstable molecular oxygen
species (O2) like hydroxyl radical (HO∙) and superoxide radical (O2∙) and non radical molecules
like hydrogen peroxide (H2O2). Mitochondrion is the key center for ROS synthesis which
generates ATP through a series of oxidative phosphorylation practices, during this reduction
of one or two electrons takes place, leads to the formation of O2∙ or H2O2. Other sources for
the formation of ROS are involvement of xanthine oxidase (Dorsam et al., 2000), NAD (P)H
oxidases (Cheng et al., 2001), paroxysmal oxidases (Dvorakova et al., 2000) and cytochrome
P-450 enzymes (Geiszt et al., 1997).

1.9.1 Oxidative Stress and Diseases

Oxygen free radicals are potent initiators of enhanced levels of lipoperoxidation which leads
to some metabolic disease states like diabetes and some CNS disorders (Cherubini et al., 2005).
Oxidative stress modifies proteins which changes the antigenic profile of the self proteins,
enhances the antigenicity leads to several autoimmune diseases like systemic lupus
erythematosus (Scofield et al., 2005), diabetes mellitus (Trigwell et al., 2001) and diffuse
scleroderma (Casciola-Rosen et al., 1997). Due to impaired antioxidant defense system, all
pathogens in the host tissues makes more susceptible to bacterial and viral infections
(Halliwell, 1989).

1.9.2 Antioxidants

The antioxidant system consists of enzymes like superoxide dismutase (SOD) and catalase
(CAT) and antioxidants such as ascorbic acid, vitamin E, reduced glutathione (GSH) and other
non-protein thiols (Winston and Di Giulio, 1991). Amphibians skin especially Toad skin is
exposed to various endogenous and exogenous oxidative stress sources (Kohen, 1999)
susceptible to biological or non-biological injuries, like microorganism infections, predation,
radiation and physical wounds (Clarke, 1997). Skin peptides had shown most represented
antioxidant activity as they are related to amino acidic composition, secondary structures and
arrangements. Presence of reductive cysteine in toads might be responsible for antioxidant
activity (Xu et al., 2017).

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1.10 Inflammation

Inflammation is a tissue response to injury, cell death, ischemia, cancer and degeneration
(Lucas et al., 2006). Innate immune response and adaptive immune response are responsible
for inflammation (Rock et al., 2011; Waisman et al., 2015). The innate immune system means
involving the activity of mast cells, dendrite cells and macrophages, whereas adaptive
immune systems means involvement of B and T cells activity. All inflammatory mediators,
especially nuclear factor k B induces acute and chronic inflamation (Montgomery and
Bowers, 2012). In the 1st century AD it was described that 4 cardinal signs of inflammation
as swelling or edema (Tumor), redness (Rubor), pain (Dolor) and heat (Calor) (Punchard et
al., 2004). In acute and chronic inflammation stages the response is in three different phases.
In first phase, an increase in vascular permeability resulting in oozing out of fluids into the
interstitial space from the blood, in second phase, leukocytes infiltrations occurs from the
blood into the tissue and in third phase, formation of granuloma and repairing of tissue
happens (Eddouks et al., 2012). Some of the following well known animal models for
assessing the anti-inflammatory activity in rats fall under acute, sub acute and chronic types

1.5.1. Acute inflammation models

Commonly used acute models for anti-inflammatory activity in laboratory are Carrageenan-
induced paw edema in rats (Winter et al., 1962), histamine-induced paw edema in rats (Dray,
1995), acetic-acid (Whittle, 1964) and xylene induction models (Kou et al., 2005), ear edema
models (Evans et al., 1971; Griswold et al., 1998; Romay et al., 1998), MPO-assay (Bradley et
al., 1982).

1.5.2. Sub-acute inflammation models

Commonly used subacute models are carrageenan-induced granuloma pouch model (Selye,
1953) and formalin-induced paw edema (Turner, 2013).

1.5.3. Chronic inflammation models

Cotton pellet-induced granuloma in rats (Crunkhorn and Meacock, 1971) and glass rod
granuloma (Al-Harbi et al., 1994) are used to assess the chronic anti-inflammatory activities.

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1.5.4. λ- Carrageenan-Induced Paw Edema in Rats

Edema formation is due to carrageenan which shows biphasic phenomenon. Histamine and
serotonin release takes place in the initial phase. Prostaglandins, protease and lysosome
release happens in the late phase (Posadas et al., 2004; Vinegar et al., 1969). Inflammation is
induced by S.C injection of 1 ml of 0.1% carrageenan into the sub planter region of rat hind
paw, which is due to plasma extravasation, increased tissue water and plasma protein
exudation, along with neutrophil extravasation, and which is due to the arachidonic acid
metabolism (Chatpalliwar et al., 2002). This method is adopted to test our TPGS anti-
inflammatory activity using Lambda (λ) Carrageenan.

1.5.5. Toad secretions as anti-inflammatory

• Bufalin inhibits the cyclooxygenase-2 (COX-2), nitric oxide synthase (iNOS),

interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor α(TNF-α) (Wen
et al., 2014).
• Cane toad skin aqueous extracts (CTSAE) exhibited the in vitro antiiflammatory activty
by inhibiting the expression and release of IL-6 and TNF-α (Zulfiker et al., 2016).

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2. REVIEW OF LITERATURE

Sousa-filho et al.,2016 Identified 104 protiens from Rhinella schneideri parotoid gland
(RsPP) by Tandem mass spectrometric analysis. Proteolytic Activity, Clotting Activity,
Antibacterial Activity, Anti-Inflammatory activity was studied.

Wen et al., 2014 Reported the anti-inflammatory and analgesic effects of bufalin a major
component of “Chan-su.” in which bufalin down regulated the expression levels of nitric oxide
synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1𝛽 (IL-1𝛽), interleukin-6 (IL-6), and
tumor necrosis factor-𝛼 (TNF-𝛼).

Kim et al.,(2008) Studied the inhibitory effects of Chan-Su on NO production in

lipopolysaccharide (LPS)-stimulated BV2 microglial cells. Also on the production of
prostaglandin E2 (PGE2) and COX-2. Proinflammatory cytokine (TNF-α, IL-1β and IL12) in
dose dependent manner

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3. AIMS AND OBJECTIVE

Aim
To determine the in-vitro anti-inflammatory activity of TPGS.

Objectives
To collect secretions from Toad parotoid glands.
To Explore the biological activities of TPGS

Work Done:
• Collected the Toad bufo melanostictus.

• Collected the Toad parotoid gland secretions (TPGS)

• Identified the standard methods to be followed for invite evaluation.

PLAN OF WORK
a) Collection of Toad Parotoid Glandular Secretions (TPGS)
b) Evaluating the Anti-inflammatory activity

COLLECTION
Toads: Toads collected were in the vicinity of Vaageswari college campus
buildings Karimnagar, Telangana, and India.

Secretion: The secretion is obtained by mechanical compression of both

glands from living individuals. These yellowish and doughy
secretions were collected on the surface of the watch glass and
after, they were released. Secretions are air dried and grounded in
to powder

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Fig. 2: Collection, drying, grinding process of Parotoid gland secretion s of Indian toad
(Bufo melanostictus)

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Fig. 3: CERTIFICATE OF AUTHENTICATION

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METHODOLOGY: INTRODUCTION
• From the amphibians Indian toads plays an important role for the new chemical
compounds from the animal source.
• Toads belongs to Anura order lives in diverse range of moist environment, during the
demanding situations like self defending with their predators they expel noxious,
venomous and poisonous chemical substances (Daly et al.,1987).

• Different types of chemical substances are found in skin glands of toad make them
unique to find out new chemical entities from their secretions (Clark et al ., 1997)

• Antioxidant activity of proteins from animal-derived products used in treating the

diseases which might be associated with oxygen free radical mediated tissue damages.
• Free radicals are generated in oxidative stress leads to diseases like cancer,
hypertension, neuronal problems, Alzheimer’s disease, slight cognitive impairment,
Parkinson’s disease, alcohol prompted liver disease, ulcer, ageing and
atherosclerosis(Safavi-Hemami et al .,2019; Valko et al.,2007).

• The inflammation is a tissue response to a damage/trauma characterized by the signs of

heat, pain, redness, swelling, and lack of functioning (Kumar et al.,2004).

• Over production of free radicals leads to activation of a complex enzymes, and releases
inflammatory and pro-inflammatory mediators and they are involved in the
inflammatory process.

• Carrageenan causes edema with a biphasic phenomenon. Before 1 hr (initial phase) is

a launch of bradykinin, histamine, serotonin and substance P. After 1 hr (late section)
is a contribution of neutrophil infiltration, prostaglandin generation and launch of the
neutrophil-derived free radicals, nitric oxide (NO) and pro-inflammatory cytokines
including tumor necrosis factor(TNF-α), and interleukin-1 β (IL-1 β).(Arteel et al
.,2003)

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4.1. MATERIALS AND METHODS
4.1.1. Materials:
4.1.1.1. Chemicals:
➢ Bovine albumin
➢ HCL (1N)
➢ Tris HCL Buffer (pH 7.4)
➢ Casein (0.8% w/v)
➢ Perchloric acid (2ml of 70%)
➢ 10% Normal Saline
➢ 10% RBC Suspension
➢ Phosphate Buffer
➢ Diclofenac sodium
➢ Linolic acid
➢ Borate Buffer (pH 9.0)
4.1.1.2. Animals:
Indian toads of weighing 25gm were collected

4.1.2. METHODS
4.1.2.1.Inhibition of albumin denaturation
• The anti-inflammatory activity of Paratoid glandular (TPGS) was studied by
using inhibition of albumin denaturation technique which was studied according
to Mizushima et al and Sakat et al followed with minor modifications.
• The reaction mixture was consists of TPGS and 1% aqueous solution of bovine
albumin fraction, pH of the reaction mixture was adjusted using small amount of
1N HCl.
• The sample extracts were incubated at 37 ºC for 20 min and then heated to 51 º
C for 20 min, after cooling the samples the turbidity was measured at 660nm.(
UV Visible Spectrophotometer Model 371, Elico India Ltd)
• The experiment was performed in triplicate.
• The Percentage inhibition of protein denaturation was calculated as follows:
Percentage inhibition = (Abs Control –Abs Sample) X 100/ Abs control.

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4.1.2.2. Antiproteinase action:
• The test was performed according to the modified method of Oyedepo et al and
Sakat et al.
• The reaction mixture (2 ml) was containing 0.06 mg trypsin, 1 ml 20 mM Tris
HCl buffer (pH 7.4) and 1 ml test sample of different concentrations (100 - 500
µg/ml).
• The mixture was incubated at 37oC for 5 min and then 1 ml of 0.8% (w/v)
casein was added.
• The mixture was incubated for an additional 20 min. 2 ml of 70% perchloric
acid was added to arrest the reaction.
• Cloudy suspension was centrifuged and the absorbance of the supernatant was
read at 210 nm against buffer as blank.
• The experiment was performed in triplicate. The percentage inhibition of
proteinase inhibitory activity was calculated.
• Percentage inhibition = (Abs control –Abs sample) X 100/ Abs control.

4.1.2.3. Membrane stabilization:

Preparation of Red Blood cells (RBCs) suspension
• The Blood was collected from healthy human volunteer who has not taken any
NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) for 2 weeks prior to the
experiment and transferred to the centrifuge tubes.
• The tubes were centrifuged at 3000 rpm for 10min and were washed three
times with equal volume of normal saline.
• The volume of blood was measured and re constituted as 10% v/v suspension
with normal saline

4.1.2.4. Heat induced haemolysis:

• The reaction mixture (2ml) consisted of 1 ml test sample of different
concentrations (100 - 400 µg/ml) and 1 ml of 10% RBCs suspension, instead of
test sample only saline was added to the control test tube.
• Aspirin was used as a standard drug. All the centrifuge tubes containing
reaction mixture were incubated in water bath at 56 ºC for 30min.

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 19

 • At the end of the incubation the tubes were cooled under running tap water. The
reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of
the supernatants was taken at 560 nm.
• The experiment was performed in triplicates for all the test samples. The
• Percentage inhibition of Haemolysis was calculated as follows:
Percentage inhibition = (Abs control –Abs sample) X 100/ Abs control

4.1.2.5. Hypotonicity-induced
• Different concentration of TPGS (100-400µg/ml), reference sample, and control
were separately mixed with 1ml of phosphate buffer, 2ml of hyposaline and
0.5ml of HRBC suspension.
• Diclofenac sodium (100µg/ml) was used as a standard drug. All the assay
mixtures were incubated at 370c for 30minutes and centrifuged at 3000rpm.
• The supernatant liquid was decanted and the haemoglobin content was estimated
by a spectrophotometer at 560nm.
• The percentage hemolysis was estimated by assuming the haemolysis produced
in the control as 100%.
Percentage protection = 100- (OD sample/OD control) x 100

4.1.2.6. Anti-lipoxygenase activity

• Anti-Lipoxygenase activity was studied using linoleic acid as substrate and
lipoxidase as enzyme.
• Test samples were dissolved in 0.25ml of 2M borate buffer pH 9.0 and added
0.25ml of lipoxidase enzyme solution (20,000U/ml) and incubated for 5 min at
250C. After which, 1.0ml of lenoleic acid solution (0.6mM) was added, mixed
well and absorbance was measured at 234nm.
• Indomethacin was used as reference standard.
• The percent inhibition was calculated from the following equation, % inhibition=
[{Abs control- Abs sample}/Abs control] x 100 A dose response curve was
plotted to determine the IC50 values.
• IC50 is defined as the concentration sufficient to obtain 50% of a maximum
scavenging capacity. All tests and analyses were run in triplicate and averaged.

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 20

4.1.3. STATISTICAL ANALYSIS

Results are expressed as Mean ± SD. The difference between experimental groups was
compared by One-Way Analysis Of Variance (ANOVA) followed by Dunnet Multiple
comparison test (control Vs test) using the software Graph Pad Prism.

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 21

5. Results and discussion
5.1. Inhibition of albumin denaturation
Protein Denaturation is a process in which proteins lose their tertiary structure and secondary
structure by application of external stress or compound, such as strong acid or base, a
concentrated inorganic salt, an organic solvent or heat. Most biological proteins lose their
biological function when denatured. Denaturation of proteins is a well documented cause of
inflammation. As part of the investigation on the mechanism of the anti-inflammation activity,
ability of TPGS to inhibit protein denaturation was studied. It was effective in inhibiting heat
induced albumin denaturation. Maximum inhibition of 62% was observed at 400 µg/ml.
Aspirin, a standard anti-inflammation drug showed the maximum inhibition 65% at the
concentration of 100 µg/ml compared with control (Table 2).

Table 2: Effect of TPGS on albumin denaturation

Treatment Concentration Absorbance at % Inhibition of
(µg/ml) 660nm albumin
denaturation
Control - 0.981 ± 0.010 -
TPGS 100 0.786 ± 0.009 ns 19
TPGS 200 0.593 ± 0.010** 39
TPGS 400 0.372 ± 0.011** 62
Aspirin 100 0.342 ± 0.010** 65
Each value represents the mean ± SD. N=3, Experimental group were compared with control **p<0.01,
considered extremely significant. TPGS: Toad Parotoid Glandular Secretion

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 22

 EFFECT OF TPGS ON ALBUMIN DENATURATION

1.5

ABSORBANCE
1.0

0.5

0.0
Control TPGS TPGS TPGS Aspirin
GROUPS

Fig 4: Effect of TPGS on albumin denaturation

% INHIBITION OF
ALBUMIN DENATURATION
150

100
% Inhibition

50

0
l 0 0 0 IN
ntro 10 20 40 PR
o GS GS GS S
C A
TP TP TP

GROUPS

Fig. 5: % Inhibition of TPGS on Albumin denaturation

5.2. Proteinase Inhibitory Action

Neutrophils are known to be a rich source of serine proteinase and are localized at lysosomes.
It was previously reported that leukocytes proteinase play an important role in the development

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 23

of tissue damage during inflammatory reactions and significant level of protection was
provided by proteinase inhibitors. TPGS exhibited significant anti proteinase activity at
different concentrations as shown in Table 2. It showed maximum inhibition of 60% at
400µg/ml. Aspirin showed the maximum inhibition 52% at 100µg/ml.

Table 3: Effect of TPGS on Proteinase inhibitory action

Treatment Concentration Absorbance at % Inhibition of
(µg/ml) 660nm proteinase action
Control - 0.982 ± 0.011 -
TPGS 100 0.812 ± 0.011 ns 17
TPGS 200 0.708 ± 0.010** 27
TPGS 400 0.385 ± 0.009** 60
Aspirin 100 0.466 ± 0.011** 52
Each value represents the mean ± SD. N=3, Experimental group were compared with control
**p<0.05, ns-non significant. TPGS: Toad Parotoid Glandular Secretion

EFFECT OF TPGS ON PROTEINASE

INHIBITORY ACTION

1.5
ABSORBANCE

1.0

0.5

0.0
Control TPGS TPGS TPGS Aspirin
GROUPS

Fig 6: Effect of TPGS on proteinase inhibitory action

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 24

 % INHIBITION OF
PROTEINASE INHIBITORY ACTION
150

% Inhibition 100

50

0
ol 0 0 0
iri
n
tr 10 20 40 p
on G
S
G
S
G
S
A
s
C P P P
T T T

GROUPS

Fig. 7: Effect of TPGS on proteinase inhibitory action

5.3. Heat Induced Haemolysis

The extract was effective in inhibiting the heat induced haemolysis at different concentrations.
The results showed that TPGS at concentration 400µg/ml protect significantly

Table 4: Effect of TPGS on Heat Induced Haemolysis of Erythrocyte

Treatment Concentration Absorbance at % Inhibition of
(µg/ml) 660nm haemolysis

Control - 0.962 ± 0.011 -

TPGS 100 0.801 ± 0.011 ns 16

TPGS 200 0.716 ± 0.006** 25

TPGS 400 0.373 ± 0.003** 61

Aspirin 100 0.463 ± 0.011** 51

Each value represents the mean ± SD. N=3, Experimental group were compared with control
**p0.05, non significant. TPGS: Toad Parotoid Glandular Secretion

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 25

 EFFECT OF TPGS ON
HEAT INDUCED HAEMOLYSIS OF ERYTHROCYTE

ABSORBANCE 1.5

1.0

0.5

0.0
ControlTPGS TPGS TPGS Aspirin
GROUPS

Fig 8: Effect of TPGS on Heat induced haemolysis of erythrocyte

% INHIBITION OF
HEAT INDUCED HAEMOLYSIS OF ERYTHROCYTE
150
% Inhibition

100

50

0
l 0 0 0 n
ntro 10 20 40 p iri
S S S
C o
PG PG PG As
T T T
GROUPS

Fig. 9: Effect of TPGS on Heat induced haemolysis of erythrocyte

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 26

5.4 Hypotonicity Induced Haemolysis
The results showed that TPGS at concentration range of 200-400µg/ml protect significantly
(p<0.01) the erythrocyte membrane against lysis induced by hypotonic solution (Table 4).
Diclofenac sodium (100µg/ml) offered a significant (p<0.01) protection against the damaging
effect of hypotonic solution. At the concentration of 400µg/ml, TPGS showed maximum of
61% protection, whereas, Diclofenac sodium (100µg/ml) showed 63% inhibition of RBC
haemolysis when compared with control.

Table 5: Effect of TPGS on Hypotonicity induced haemolysis of erythrocyte

Treatment Concentration Absorbance at % Inhibition of
(µg/ml) 660nm haemolysis
Control - 0.975 ± 0.010 -
TPGS 100 0.817 ± 0.011 ns 16
TPGS 200 0.698 ± 0.011** 28
TPGS 400 0.374 ± 0.004** 61
Diclofenac sodium 100 0.360 ± 0.034** 63
Each value represents the mean ± SD. N=3, Experimental group were compared with control
**p<0.05, non significant. TPGS: Toad Parotoid Gland Secretion

EFFECT OF TPGS ON
HYPOTOXICITY INDUCED HAEMOLYSIS OF ERYTHROCYTES
1.5
ABSORBANCE

1.0

0.5

0.0
Control TPGS TPGS TPGS Aspirin
GROUPS

Fig 10: Effect of TPGS on Hypotonicity induced haemolysis of erythrocyte

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 27

 % INHIBITION OF HYPOTOXICITY
INDUCED HAEMOLYSIS OF ERYTHROCYTE
150

% Inhibition

100

50

0
ol 0 0 0 m
tr 10 20 40 iu
on S S S d
C PG PG PG so
T T T
n ac
fe
lo
ic
D
GROUPS

Fig. 11 Effect of TPGS on Hypotonicity induced haemolysis of erythrocyte

5.5 Anti-lipoxygenase activity

The establishment of new invitro test systems has stimulated the screening of TPGS aiming to
find leads for the development of new drugs. The animal lipoxygenase pathway is in many
respects equivalent to the ‘arachidonic acid cascades’ in animals. For this reason, the in vitro
inhibition of lipoxygenase constitutes a good model for the screening of TPGS with anti-
inflammatory potential. LOXs are sensitive to antioxidants and the most of their action may
consist in inhibition of lipid hydroperoxide formation due to scavenging of lipidoxy or lipid
peroxy- radical formed in course of enzyme peroxidation. This can limit the availability of lipid
hydroperoxide substrate necessary for the catalytic cycle of LOX. TPGS has been checked at
100,200,400µg/ml, it showed 09, 16, 39, 62 anti-lipoxygenase inhibition respectively. From
these result, the strongest inhibition was obtained at concentration 400µg/ml. The standard
Indomethacin showed a 53% inhibition at a concentration of 100µg/ml. At the concentration
of 100 and 200µg/ml, TPGS not showed significant difference (p <0.05) when compared with
control (Table 5). The results obtained from our studies on TPGS have shown a potential anti-

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 28

inflammatory activity. The TPGS extracts inhibited the lipoxygenase enzyme activity. This
indicates that animal TPGS is more useful in studies of inflammation and in various related
physiological studies and diseases such as cancer, neurological disorder etc.

Table 6: Effect of TPGS on Lipoxygenase inhibitory action

Treatment Concentration Absorbance at % Inhibition of
(µg/ml) 660nm lipoxygenase action

Control - 0.887 ± 0.011 -

TPGS 100 0.802 ± 0.010 ns 9

TPGS 200 0.739 ± 0.049 ns 16

TPGS 400 0.336 ± 0.054** 62

Indomethacin 100 0.416 ± 0.011** 53

Each value represents the mean ± SD. N=3, Experimental group were compared with control
**p<0.05, non significant. TPGS: Toad Parotoid Glandular Secretion

EFFECT OF TPGS ON LIPOXYGENASE

INHIBITION ACTION
1.0

0.8
ABSORBANCE

0.6

0.4

0.2

0.0
Control TPGS TPGS TPGS Aspirin
GROUPS

Fig 12: Effect of TPGS on Lipoxygenase inhibitory action

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 29

 % INHIBITION OF
LIPOXYGENASE INHIBITORY ACTION
150
% Inhibition

100

50

0
ol

in
10

20

40
tr

c
ha
on

S
G

et
C

TP

TP

TP

m
do
In

GROUPS

Fig. 13: Effect of TPGS on Lipoxygenase inhibitory action

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 30

CONCLUSION

In the present work TPGS is proved to be having in-vitro anti-inflammatory activity at 500
ug/ml. Toad Paratoid glandular secretions having antioxidant, anti-inflammatory Therefore
further studies are required to isolate the new chemical compounds in pure form from Toad
parotoid gland secretion of Bufo melanostictus as they may become new drug candidates.

DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 31

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ERRATA

S.No Page No Printed as To be printed as

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DEPARTMENT OF PHARMACOLOGY, VAAGESWARI COLLEGE OF PHARMACY 37