Evaluation of Antioxidant & Hepatoprotective Potential of Nicker Bean seed

extract

MINOR PROJECT

As the partial fulfillment of the requirement of minor project of

MASTER OF PHARMACY
IN
PHARMACOLOGY
Submitted By
AJAY DEWANGAN

Under guidance of
Dr.Hemant Sharma
(Principal)
 1. INTRODUCTION
1.1 Liver
Liver is the most important organ in the body. It plays a pivotal role in regulating various
physiological processes. It is also involved in several vital functions, such as metabolism,
secretion and storage. It has great capacity to detoxicate toxic substances and synthesize useful
principles. It helps in the maintenance, performance and regulating homeostasis of the body. It is
involved with almost all the biochemical pathways to growth, fight against disease, nutrient
supply, energy provision and reproduction. In addition, it aids metabolism of carbohydrate,
protein and fat, detoxification, secretion of bile and storage of vitamins. The role played by this
organ in the removal of substances from the portal circulation makes it susceptible to first and
persistent attack by offending foreign compounds, culminating in liver dysfunction.

Fig.1.1 Structure of Liver

1.1.1 Liver Anatomy

The liver is found in the upper right-hand side of the abdomen under the ribs below the
diaphragm. It is a dark reddish-brown organ that weighs about three pounds.

The liver has a right lobe and a left lobe. Blood enters the liver from the hepatic artery and the
hepatic portal vein and leaves the liver from the hepatic vein. Inside the two lobes is a network of
tubes, also called the biliary tree that carries bile from the liver to the intestine. Bile is a
substance that helps carry away wastes and is needed for the breakdown and absorption of
dietary fats. Each tube is called a duct. Smaller ducts connect to larger ducts. The larger ducts
join to form the hepatic duct. This duct network allows bile to drain out of the liver.

1.1.2 Liver functions

Liver identifies xenobiotics and metabolize them and make them suitable for elimination. This
involves chemical transformation (a) decreasing lipid solubility (b) change the biological
activity. Mainly smooth endoplasmic reticulum of liver principally participates in metabolism. It
is also called as metabolic clearing house of both exogenous and endogenous substances.
(Blumenthal D., et al., 2006) Drug metabolism takes place in 2 phases; Phase I and Phase II.
Phase I reactions involve oxidation, reduction, hydrolysis, hydration which makes them water
soluble and also generate metabolites which are more chemically active and potentially toxic.
Phase II reactions takes place in cytosol and involve conjugation with endogenous compounds
via transferase enzyme, a group of enzymes. Cytochrome P 450 is a enzymes are located in
endoplasmic reticulum, known as cytochrome P 450 terminal oxidase component of electron
transport chain. It is not a single enzyme but consists of a family closely related to 50 isoforms, 6
of them metabolize 90% of drugs.(Lynch T., et al., 2001)

1.1.3 Liver disease epidemiology

Hepatocellular injury is characterized by significant elevations in the aminotransferases in serum
which usually precede elevations in total bilirubin levels and alkaline phosphatase levels. Most
injuries occur within 1 year of initiating the offending agent. Hepatocellular injury can lead to
fulminant hepatitis with a corresponding 20% survival rate with supportive care. For those
patients who present with the combination of hepatocellular injury and jaundice, there is a 10%
mortality rate. Acarbose, allopurinol, fluoxetine, and losartan are capable of causing
hepatocellular injury. (Dipiro Joseph T., et al., 2008)
1.1.4 Liver disease

It is a broad term describing any single number of diseases affecting the liver. Many are
accompanied by jaundice caused by increased levels of bilirubin in the system. The bilirubin
results from the breakup of the hemoglobin of dead red blood cells; normally, the liver removes
bilirubin from the blood and excretes it through bile.

Diseases

 Hepatitis, inflammation of the liver, caused mainly by various viruses but also by some
poisons (e.g. alcohol), autoimmunity (autoimmune hepatitis) or hereditary conditions.
Diagnosis is done by checking levels of Alanine transaminase.
 Non-alcoholic fatty liver disease, a spectrum in disease, associated with obesity and
characterized as an abundance of fat in the liver; may lead to a hepatitis,i.e.
steatohepatitis and/or cirrhosis.

 Cirrhosis is the formation of fibrous tissue in the liver from replacing dead liver cells.
The death of the liver cells can be caused by viral hepatitis, alcoholism or contact with
other liver-toxic chemicals. Diagnosis is done by checking levels of Alanine transaminase
and Asparatine transaminase (SGOT).

 Haemochromatosis, a hereditary disease causing the accumulation of iron in the body,

eventually leading to liver damage.

 Cancer of the liver (primary hepatocellular carcinoma or cholangiocarcinoma and

metastatic cancers, usually from other parts of the gastrointestinal tract).

 Wilson’s disease, a hereditary disease which causes the body to retain copper.

 Primary sclerosing cholangitis, an inflammatory disease of the bile duct, likely

autoimmune in nature.

 Primary biliary cirrhosis, autoimmune disease of small bile ducts.

 Budd-Chiari syndrome, obstruction of the hepatic vein.

  Gilbert’s syndrome, a genetic disorder of bilirubin metabolism, found in about 5% of the
population.

 Glycogen storage disease type II, the build-up of glycogen causes progressive muscle
weakness (myopathy) throughout the body and affects various body tissues, particularly
in the heart, skeletal muscles, liver and nervous system.

There are also many pediatric liver diseases, including biliary atresia, alpha-1 antitrypsin
deficiency, alagille syndrome, and progressive familial intrahepatic cholestasis.

Liver diseases remain one of the major threats to public health and are a worldwide problem.
They are mainly caused by chemicals like acetaminophen (in large doses), excess consumption
of alcohol, infections and autoimmune disorders. Most of the hepatotoxic chemicals damage
liver cells mainly by inducing lipid peroxidation and other oxidative damages (Recknagel, 1983;
Wendel, et al., 1987; Dianzani, et al., 1991). Acetaminophen, a mild analgesic and antipyretic
drug, developed in the last century, causes serious liver necrosis in humans and in experimental
animals if taken in large doses (Lin, et al., 1995; Hinson, 1980). While alcohol is one of the main
causes of end stage liver disease worldwide, alcoholic liver disease is the second most common
reason for liver transplantation in the United States (Mandayam, et al., 2004). Due to increased
frequency of drinking and change of diet construction, such as the increase of fat content, the
incidence of liver diseases has increased in China, becoming another important risk factor for
morbidity and mortality in addition to viral hepatitis (Zhuang, et al., 2003). The spectrum of
alcoholic liver disease ranges from fatty liver to alcoholic hepatitis and ultimately fibrosis and
cirrhosis.

In spite of the tremendous advances in modern medicine, there is no effective drug available that
stimulates liver function, offer protection to the liver from damage or help to regenerate hepatic
cells (Chattopadhyay, 2003). It is therefore necessary to search for alternative drugs for the
treatment of liver diseases to replace currently used drugs of doubtful efficacy and safety.

1.2 Hepatotoxicity

1.2.1 Intrinsic Hepatotoxicity

One of two major categories of chemicals that can produce dose-dependent, hepatic injury, either
directly or indirectly via a metabolite, includes the intrinsic or direct (type A) hepatotoxicants.
These responses often can be anticipated from the known pharmacology of the drug, are
generally detectable in animal models, and occur more frequently or with greater severity when
exposure is increased, i.e., with increasing dose Levels or duration of dosing. Salicylates (Lewis,
1984) and acetaminophen are examples of analgesics that produce intrinsic liver toxicity. At
high blood levels, aspirin can produce hepatocellular injury, confirmed by liver biopsy studies
(focalnecrosis),with 10-to40-fold elevations for serum transaminases noted (Zimmerman,
1978).Chemotherapeutic agents often possess predictable, dose-dependent Hepatotoxicity (King,
et al., 1995), even though they do not intentionally target slowly dividing hepatocytes.

Tetracycline, methotrexate, mercaptopurine and possibly suldinac (Boel sterli et al., 1995) are
other examples of direct hepatotoxins. Intrinsic hepatotoxins produce injury in a large percentage
of exposed individuals after a short fixed latent period either by direct, non selective
physicochemical distortion of hepatocytes or by indirect presence with specific metabolic
processes leading to structural damage (Lewis, 1984).This type of toxicity can be alleviated by
dose reduction in patient populations.

1.2.2 Idiosyncratic Hepatotoxicity

Although a number of drugs, as for example some Antineoplastic agents (McDonald, et al.,
1984), Exhibit intrinsic toxicity in man or animals, most hepatotoxic drug reactions in humans
are considered to be idiosyncratic (King, et al., 1995), i.e., due to unusual susceptibility of an
individual. Occurring at therapeutic doses after a variable latent period, these responses are
characterized by an incidence of hepatic injury that is very low in frequency within a population,
dose independent, and not reproducible in experimental animals (Zimmerman, 1978). Due to
their low incidence, idiosyncratic responses are generally not detected in the drug development
process after a large number of patients have been treated. Although rare, serious adverse liver
responses may include fulminant hepatitis and cholestasis. These account for many drug induced
deaths worldwide. Drugs withdrawn from the market due to idiosyncratic drug reactions,
including or due solely to hepatotoxicity, include benoxaprofen, ibufenac, temafloxacin,
tenilicacid (Park, et al., 1992), nomifensine, and perhexilene. (Breckenridge, 1996).
Etiologically, these reactions are of two major types:

(1) Aberrant metabolism based responses leading to the accumulation of toxic metabolites
insusceptible individuals and

(2) Hypersensitivity or immune-based toxicity.

Other mechanisms may include abnormal receptor sensitivity, latent biochemical abnormalities,
or multi factorial causes (Park, et al., 1992). Almost all hepatotoxic reactions to antibacterials
(George and Crawford, 1996) or NSAIDs (Boelsterli, et al., 1995), especially of the indole,
pyrazole and propionic acid classes (Lewis, 1984) are idiosyncratic. Mechanisms may be
primarily metabolite dependent (isoniazid, diclofenac), hypersensitivity mediated (beta-lactams,
sulindac), or both (sulfonamides, erythromycin derivatives) (West phal, et al., 1994; Boelsterli,
et al., 1995). Some notable histamine receptor antagonists (H 2 and a number of anti depressants
are associated with hepatic idiosyncratic reactions, presumably mediated via chemically reactive
metabolites (Black, 1987; Pir mohammed, et al., 1992).

1.2.3 Immune system- based toxicity

Immune based injuries represent the other major mechanism for idiosyncratic responses in
humans. Auto immunity triggered by xenobiotics involves the modification of host tissues or
immune cells by the chemical so that self antigens are erroneously targeted; drug
hypersensitivity or allergy refers to a situation where the immune system responds in an
exaggerated or inappropriate manner (Burns, et al., 1996) by one of four mechanisms. Most
autoimmune diseases are associated with specific alleles of the major histocompatibility complex
(MHC) class II genes (Fronek, et al., 1991). The high level of polymorphism that is
characteristic of the MHC genes may lead to sub populations of individuals who are highly
immune to responses toward drugrelated antigens (Park, et al., 1992). Four major categories of
drugs known to cause autoimmune responses include hydrazines, derivatives of aromatic amines,
sulfhydryl containing compounds, and compounds with a phenol ring (Adams, et al., 1991).
Specific examples of drugs known or suspected of causing hepatitis via an autoimmune
mechanism include methyldopa, oxypehnisatin, isoniazid, nitrofurantoin, clometacin,
fenofibrate, papaverine and tienilic acid (Bigazzi, 1988). Although other non genetic factors may
be involved in the etiology of altered immune responses, the role of drug metabolism
polymorphism leading to bioactivation is an important one. Extra hepatic metabolism of some
drugs associated with a generalized type of idiosyncratic drug reaction mediated through the
immune system may also involve the liver. (Utrecht, 1988).

1.2.4 Mixed or Species-Relevant Toxicity

Some drugs appear to cause idiosyncratic hepatic injury in humans and yet can also produce
hepatotoxicity, perhaps by a different mechanism, in animal models at high doses. Thus, the
same drug can be an intrinsic hepatotoxicant in one or more animal models and yet cause an
idiosyncratic liver response in some humans. Some of the angiotensin converting enzyme
inhibitors used for the treatment of hypertension and congestive heart disease fall into this
category. Captopril, for example, has been associated with the rare incidence of hepatotoxicity in
humans (Rahmat, et al., 1985). In mice, acute high doses of captopril cause moderate increases
in ALT, decreases in hepatic GSH, and histological evidence of hepatic necrosis with 24h (Helli
well, et al., 1985). Enalapril maleate which has been reported to cause a rare but potentially
serious hepatotoxicity in humans was demonstrated by Jurim A Romet and Huang 1992 to
produce centrilobular necrosis and significant moderate increases in ALT and AST 24 h after
acute exposure in Fisher 344 rats.

1.3 Pattern of Drug Induced Liver Disease

1.3.1 Hepatocellular injury

Hepatocellular injury is characterized by significant elevations in the aminotransferases in serum

which usually precede elevations in total bilirubin levels and alkaline phosphatase levels.
(Navarro V., et al., 2006) Most injuries occur within 1 year of initiating the offending agent.
Hepatocellular injury can lead to fulminant hepatitis with a corresponding 20% survival rate with
supportive care. (Watkins P., et al., 2006)

For those patients who present with the combination of hepatocellular injury and jaundice, there
is a 10% mortality rate. (Bjornsson E., et al., 2006) Acarbose, allopurinol, fluoxetine, and
losartan are capable of causing hepatocellular injury. Hepatocellular injuries can be further
subdivided by specific histological patterns and clinical presentations. Centrolobular necrosis,
steatohepatitis (steatonecrosis), phospholipidosis, and generalized hepatocellular necrosis are
each identifiable by particular biopsy results and subtle differences in clinical presentation.

1.3.2 Centrolobular necrosis

Centrolobular necrosis is often a dose-related, predictable reaction secondary to drugs such as

acetaminophen; however, it also can be associated with idiosyncratic reactions, such as those
caused by the anesthetic halothane. Also called direct or metabolite-related hepatotoxicity,
centrolobular necrosis is usually the result of the production of a toxic metabolite. The damage
spreads outward from the middle of a lobe of the liver. Patients suffering from centrolobular
necrosis tend to present in one of two ways, depending on the extent of necrosis. Mild drug
reactions, involving only small amounts of parenchymal liver tissue, may be detected as
asymptomatic elevations in the serum aminotransferases. If the reaction is diagnosed at this
stage, most of these patients will recover with minimal cirrhosis and thus minimal chronic liver
impairment. More severe forms of centrolobular necrosis are accompanied by nausea, vomiting,
upper abdominal pain, and jaundice. (Fontana R.J., et al., 1999)
These reactions are predictable, often dose-related effects in the liver caused by specific agents.
When taken in overdose, acetaminophen becomes bioactivated to a toxic intermediate known as
Nacetyl- p-benzoquinone imine (NAPQI). NAPQI is very reactive, with a high affinity for
sulfhydryl groups. The amino acid glutathione provides a ready source of available sulfhydryl
groups within the hepatocyte. When the liver’s glutathione stores are depleted and there are no
longer sulfhydryl groups available to detoxify this metabolite, it begins to react directly with the
hepatocyte. Replenishingthe liver’s sulfhydryl capacity through the administration of
Nacetylcysteine early after ingestion of the overdose halts this process. (Buckley N.A., et al.,
1999)
During the first hours after ingestion, some patients report mild symptoms of nausea and
vomiting, but no elevations of the commonly measured liver enzymes are seen. Not for 40 to 50
hours after ingestion do serum elevations in the liver enzymes begin. (Black M., et al., 1980)

1.3.3 Steatohepatitis
Steatohepatitis (also known as steatonecrosis) is a specialized type of acute necrosis resulting
from the accumulation of fatty acids in the hepatocyte. drugs or their metabolites that cause
steatonecrosis do so by affecting fatty-acid oxidation within the mitochondria of the hepatocyte.
Hepatic vesicles become engorged with fatty acids, eventually disrupting the homeostasis of the
hepatocyte. The liver biopsy is marked by a massive infiltration by poly morphonuclear
leukocytes, degeneration of the hepatocytes, and the presence of Mallory bodies. Alcohol is the
drug that most commonly produces steatonecrotic changes in the liver. When alcohol is
converted into acetaldehyde, the synthesis of fatty acids is increased. (Agarwal D.P., et al., 1989)
When the hepatocyte has become completely engorged with microvesicular fat, it often breaks
open, spilling into the blood. If enough hepatocytes break open, an inflammatory response
begins. If the offending agent is withdrawn before significant numbers of hepatocytes become
necrotic, the process is completely reversible without long-term sequelae. Patients experiencing
steatohepatitis may present with abdominal fullness or pain as their only complaint. Patients with
more severe steatonecrosis will present with all the symptoms characteristic of alcoholic hepatitis
such as nausea, vomiting, steatorrhea,abdominal pain, pruritus, and fatigue.

1.3.4 Phospholipidosis

Phospholipidosis is the accumulation of phospholipids instead of fatty acids. The phospholipids

usually engorge the lysosomal bodies of the hepatocyte. (Lullman H., et al., 1975) Amiodarone
is associated with this reaction. Patients treated with amiodarone who develop overt hepatic
disease tend to have received higher doses of the drug. These patients also have higher
amiodarone–to–N-desethyl-amiodarone ratios, indicating a greater accumulation of the parent
compound. Amiodarone and its major metabolite N-desethyl-amiodarone remain in the liver of
all patients for several months after therapy is stopped. Usually the phospholipidosis develops in
patients treated for more than 1 year. The patient can present with either elevated
aminotransferases or hepatomegaly; jaundice is rare. (Chang C.C., et al., 1999)
Signs & Symptoms of Hepatotoxicity & Liver Failure

Hepatotoxicity is a term used to describe damage caused to the liver by certain chemicals.
Common culprits behind this condition are prescription drugs, recreational drugs and even
certain herbal supplements and vitamins. It is critical to understand the signs and symptoms
associated with hepatotoxicity, as left untreated it can result in liver failure.

Jaundice
Jaundice is a condition that causes yellowing in the whites of the eyes (sclera) as well as the skin.
It is the result of an excessive buildup of bilirubin in the blood. Bilirubin is a yellowish brown
chemical in bile, a substance produced by the liver. As the liver loses the ability to effectively
break down these chemicals, they store up in the body, which results in jaundice.

Discoloration of urine and feces

Presence of bilirubin in stool is what gives it its dark color. When it is not properly processed in
the liver, it can cause stools to be pale in color. Moreover, this excess buildup of bilirubin can
become present in other body fluids such as urine. Urine that is dark in color can be an indication
of excess bilirubin, a sign of liver damage.

Chronic itching of the skin

When the human liver is compromised, a common symptom is extreme itching and irritation of
the skin. Less is known about why this phenomenon occurs. Some speculate it is the result of the
liver's inability to filter the common toxins and irritants that enter the human body. With
nowhere to go, these toxins can sometimes accumulate in the skin, causing the itch.

Swollen legs and/or abdomen

In severe cases of hepatotoxicity and liver failure, excess fluids can accumulate in the body.
Typically, these fluids collect in the legs and cause swelling, a condition known as edema. This
fluid can also accumulate in the abdomen, causing swelling and pain. When fluid accumulates in
the abdomen, it is known as ascites.

Easy bruising and bleeding

Of the many jobs the liver is responsible for, one of the most critical is the production of proteins
that aid in the clotting of blood. As less of this protein is made available, it becomes easier for
the body to become bruised. Frequent nosebleeds are often another symptom of this lack of
clotting proteins.

1.5 Biochemical Tests Used for the Detection of Hepatic Injury

Three categories of non invasive laboratory tests are used to identify the type and extent of drug
hepatotoxicity based on the presence or absence of specific markers in the blood of exposed
individuals. The first category of clinical assays is that used to assess hepatocellular damage
leading to liver cell necrosis. Evidence of this type of injury is based on the detection the hepatic
transaminases, alanine aminotransferase (ALT), and aspartate aminotransferase (AST). These are
not normal components of blood and serve no known function outside the organ of origin. A
third specific marker of hepatocellular damage, serum F protein, has been recently described
(Foster, et al., 1989), although it is not yet as widely used as the transaminases. Based on liver
biopsy specimens, chronic ALT elevations in symptomatic patients have also been associated
with fatty liver (Hulteranz, et al., 1986). Infact, nonalcoholic Steatosis has been cited as a very
common cause of chronic ALT elevations in the general population. ( Craxi, et al., 1996).
Thus, transaminase elevations are indicative of hepatocellular death and fatty degeneration in
particular, but can also be used in conjunction with other serum enzymes for distinguishing on
hepatocellular injury as described below.These group of tests includes markers for hepatobiliary
(chlolestatic) effects.This type of injury includes biliary obstruction or hepatic infiltrative
processes resulting in the retention of bile acids in liver and leading to drug induced jaundice if
severe. Serum markers include AlkP from the cell canalicular membrane, 5nucleotidase (5NT),
and GGT. However, marked serum AlkP elevations, especially when accompanied by 5NT or
GGT elevations, suggest mechanical bile duct obstruction, primary sclerosingcholangitis,
primary biliary cirrhosis, or drug induced hepatitis (Herlong, 1994). Thus, serum enzyme profiles
as opposed to individual enzyme changes are used for diagnostic purposes, especially for
monitoring hepatobiliary effects.
The final category of diagnostic procedures is based upon altered liver function. These are
methods tha tmonitor serum albumin, cholesterol, prothrombintime, or serum bilirubin as general
indicators of the synthetic and general metabolic capacity of the liver (Fregia, et al., 1994) as
opposed to marking some specific toxic injury. The serum bilirubin assay will indicate liver
injury; however, elevated serum enzyme assays as described above usually reflect hepatotoxicity
earlier. (Herlong, 1994)
Although drug associated hepatic dysfunction is uncommon in general severely altered liver
function, especially those processes leading to coagulation disorders or bilirubin encephalopathy,
are indicative of severe hepatic injury.

Aminotransferases
Aminotransferases are a group of enzymes that catalyze the reversible transfer of the amino
group from amino acid to anoxo acid. ALT and AST shunt and their amino acid and oxo acid
substrates into several intermediate pathways. Cytosolic ALT is associated with the utilization of
pyruvate in glycolysis, mitochondrial ALT is involved in the conversion of alanine to Pyruvate
for gluconeogenesis, and AST plays an important role in the transport of reducing equivalents
across the mitochondrial membrane (Sakagishi, 1995; Rej, 1989).
Hepatocellular damage with the subsequent disruption of the plasma membrane allows leakage
of intra cellular enzymes such as ALT or AST into the bloodstream. With some hepatotoxicants,
increased hepatics ynthesis of aminotransferases has also been suggested as a source ofi ncreased
serum Enzyme levels in hepato cellular injury. (Pappas, 1986)
The range of normal is determined by either cut off values of 2 SD or 97.5 percentile cutoffs in a
population without known disease. Due to half-lives of approximately 17h for AST and 47 h for
ALT, the presence of these enzymes in serum is considered an indicator of recent hepatocyte
injury. (Scheig, 1996)
Some drugs actually decrease the activities of serum ALT and AST. Examples include oxodipine
which causes hepatic damage and yet decreases ALT activities in dogs and rats (Waner, et al.,
1991) cefazolin which significantly depresses ALT and AST activities in rat liver (Dhrami, et al.,
1979), and isoniazid which significantly inhibits hepatic and serum AST inrodents (Yamada, et
al., 1984) Inaddition to inhibitory effects by anti vitamin B6 compounds, transaminase levels are
effected by other nutritional events. For example,healthy human volunteers in gestinga choline
deficient diet for 4weeks showed significantly elevated levels of ALT compared their
cohorts(Zeisel, et al., 1991) A discussion of the characteristics of each of these important
enzymes follows.
ALT
ALT(L-alanine:2-oxoglutalate amino transferase) is a pyridoxal enzyme which L-alanine
Catalyzes the reversible interconversion of keto glutarate to pyruvate L glutamate with peridoxal
phosphateas a coenzyme.The presence of low Levels of ALT in the peripheral circulation
represents normal cell turnover or release from non vascular sources. Drug related increases in
aminotransferase activity are typically transitory with values returning to within normal
reference limits within a few weeks (Rej, 1989). ALT is widely distributed. Human isozymesare
found in the cytosol and mitochondria of liver, kidney,and skeletal and cardiac muscles
(Sakagishi,1995).
Mitochondrial ALT comprises only a small portion of the tissue activity and has not been
demonstrated in normal human serum. The largest pool of ALT is in the cytosol of hepatic
parenchymal cells. (Sherman, 1991).Considerable differences in both the organ distribution and
intracellular compartmentalization of ALT have been found among species (Hoffmann, et al.,
1989). Some non human primates, for example, show little or no ALT organ specificity
(Clampitt, et al., 1978). However, overall, serum ALT is one of the most universal markers for
hepatic injury acrosss pecies. In the clinical laboratory, the measurement of ALT is a routine part
of serum chemistry panels used to assess hepatic injury. ALT values in healthy blood donors,
however, can be influenced by age, sex, dietary change, geographic allocation, ethnicity, obesity,
long term acetaminophen use, alcohol use, and marital status (Sherman,1991), a complicating
factor when monitoring human populations for transaminase elevations following drug exposure.
Even the difference in mean ALT values between males and females in a donor population can
be significant (Mijovic, et al., 1987). ALT Activities are elevated for a few days following major
abdominal orthoracic surgery (Stricker, et al., 1992) And can also be elevated by the disease
being treated.The incidence of hepatic damage due to cotrimaoxazole, for example, is around
20% higher in AIDS patients (Westphal, et al., 1994) compared to other diseases. Cognizant of
these modulating factors in clinical populations, ALT is the single most important indicator of
hepatocellular injury for preclinical animal studies, clinical trials, and postmarketing monitoring.

AST
AST is found in both the cytosol and mitochondria of hepatocytes (Herlong, 1994), but high
tissue levels are also found in heart, skeletal muscle, kidney, brain, and pancreas (Rej, 1989).
Accordingly, muscle trauma and surgery by itself can lead to AST serum elevations (Clermont,
et al., 1967). An estimated 60–70% of AST activity in human hepatocytes is localized with in
mitochondria (Schmidt, et al., 1990). Hence, when found in blood, AST is considered to be a
sensitive indicator of mitochondrial damage, especially in the hepatic centrilobular regions which
are particularly sensitive to toxic and hypoxic liver injury (Schmidt, et al., 1990). It should be
noted however that depending upon the assay used a number of drugs can reportedly produces
purious elevations in AST (Davis, 1989). Serum AST is affected to a greater degree by alcohol
consumption than ALT (Lewis, 1984).Within these limitations AST in conjunction with ALT is
a very important marker of hepatic injury.
1.6 Mechanism of Hepatotoxicity caused by Different Agents

Damage to the liver is not due to the drug itself but to a toxic metabolite (N-acetyl-p-
benzoquinone imine NAPQI or NABQI) which is produced by cytochrome P 450 enzymes in the
liver. (Wallace J.L., et al., 2004), in overdoses large amount of NAPQI is generated which
overwhelm the detoxification process and lead to damage to liver cells. Nitric acid also plays role
in inducing toxicity. (James L.P., et al., 2003) the mechanisms of hepatotoxicity caused by
NSAIDs were documented to be both idiosyncratic and dose dependant. Aspirin and
phenylbutazone are associated with intrinsic hepatotoxicity; idiosyncratic reaction has been
associated with ibuprofen, sulindac, phenylbutazone, piroxicam, diclofenac and indomethacin.
Enlarged liver is a rare side effect of long term steroid use in children. (Iancu T.C., et al., 1986)

Carbon tetrachloride

Liver injury due to carbontetrachloride in rats was first reported in1936 (Cameron GR et al.,
1936) and has been widely and successfully used by many investigators. (Shirwaiker A., et al.,
2006) Carbontetrachloride is metabolized by cytochrome P450 in endoplasmic reticulum and
mitochondria with the formation of CCl 3O, a reactive oxidative free radical, which initiates lipid
peroxidation. (Zimmerman M.D., et al., 1976)

Administration of a single dose of CCl4 to a rat produces, within 24 hrs, a centrilobular necrosis
and fatty changes. The poison reaches its maximum concentration in the liver within 3 hrs of
administration. Thereafter, the level falls and by 24 hrs there is no CCl 4 left in the liver.
(Dawkins, et al., 1963) The development of necrosis is associated with leakage of hepatic
enzymes into serum. Dose of CCl4: 0.1 to 3 ml/kg I.P.

Paracetamol

Paracetamol, a widely used analgesic and antipyretic drug, produces acute liver damage in high
doses. Paracetamol administration causes necrosis of the centrilobular hepatocytes characterized
by nuclear pyknosis and eosinophilic cytoplasm followed by large excessive hepatic lesion. The
covalent binding of N-acetyl-P-benzoquinoneimine, an oxidative product of paracetamol to
sulphydryl groups of protein, result in lipid peroxidative degradation of glutathione level and
thereby, produces cell necrosis in the liver. Dose of Paracetamol: 1 gm/kg P.O. (Kanpur V., et
al., 1994).

1.7 Oxidative Stress and role of Antioxidants

Oxidation refers to transfer of electrons from a substance to an oxidizing agent. Oxidation

reactions results in production of free radicals which immediately start chain reactions that result
in damage to the living cells. Metabolism in majority of complex living organisms requires
oxygen for its survival .But, oxygen being, a highly reactive molecule damages living organisms
by producing reactive oxygen species. These reactive oxygen species produced in the living cells
include hydrogen peroxide (H2O2 ) , hypochlorous acid (HOCl) and free radicals such as the
hydroxyl radical (·OH) and the superoxide anion (O2) . The hydroxyl radical is unstable and will
react rapidly and non-specifically with most of the biological molecules.This oxidant damage the
cells by starting chemical chain reactions such as lipid peroxidation, or by oxidizing DNA or
proteins. Damage to DNA result in serious problems possibly cancer, if not reversed by DNA
repair mechanisms. Damage to proteins result in enzyme inhibition, denaturation and protein
degradation. During metabolism, the use of oxygen generates highly reactive species such as the
superoxide anion which is produced as a by-product of several steps in the electron transport
chain. The reduction of coenzyme Q in complex III, results in the formation of highly reactive
free radical as an intermediate (Q·−). This intermediate, being unstable results in electron
"leakage", where the electrons jump directly to oxygen and form the superoxide anion, instead of
moving through the normal series of well-controlled reactions of the electron transport chain.
An antioxidant is a molecule that slows or prevents the oxidation of the molecules. Antioxidants
terminate these chain reactions by removing free radical intermediates and inhibit other oxidation
reactions by being oxidized themselves. As a result, antioxidants are often considered as
reducing agents such as thiols, ascorbic acid, polyphenols .
Antioxidants are classified into two broad divisions, depending on whether they are soluble in
water (hydrophilic) or in lipids (hydrophobic). In general, water-soluble antioxidants react with
oxidants in the cell cytosol and the blood plasma, while lipid-soluble antioxidants protect cell
membranes from lipid peroxidation. Although oxidation reactions are essential for survivals,
they can also be damaging. Hence, plants and animals maintain complex systems of multiple
types of antioxidants, such as glutathione, vitamin C, and vitamin E as well as enzymes such as
catalase, superoxide dismutase and various peroxidases. Low levels of antioxidants or inhibition
of the antioxidant enzymes, cause oxidative stress and may damage or kill the living cells. As
oxidative stress is an important part of many human diseases, the use of antioxidants in
pharmacology is intensively studied, particularly in the treatment of stroke and
neurodegenerative diseases. Antioxidants are widely used as ingredients in the dietary
supplements in order to maintain health and to prevent diseases such as cancer and coronary
heart disease.
These compounds may be synthesized in the body or obtained from the diet. The different
antioxidants are present at a wide range of concentrations in body fluids and tissues, some such
as glutathione or ubiquinone mostly present within the cells, while others such as uric acid are
more evenly distributed. The action of an antioxidant thus depends on the proper function of
other members of the antioxidant system. The extent of protection provided by any one
antioxidant also depends on its concentration, its reactivity towards the particular reactive
oxygen species and the status of the antioxidants with which it interacts.

Antioxidant protection system in biological system

Endogenous Antioxidants
• Bilirubin
• Thiols, e.g., glutathione, lipoic acid, N-acetyl cysteine
• NADPH and NADH
• Ubiquinone (coenzyme Q10)
• Uric acid
• Enzymes:
– Copper/zinc and manganese-dependent superoxide dismutase (SOD)
– Iron-dependent catalase
– Selenium-dependent glutathione peroxidise
Dietary Antioxidants
• Vitamin C
• Vitamin E
• Beta carotene and other carotenoids and oxycarotenoids, e.g., lycopene and lutein
• Polyphenols, e.g., flavonoids, flavones, flavonols, and Proanthocyanidins
Metal Binding Proteins
• Albumin (copper)
• Ceruloplasmin (copper)
• Metallothionein (copper)
• Ferritin (iron)
• Myoglobin (iron)
• Transferrin (iron). (Duy Thai, et al., 1997)
 2. RATIONALE OF THE STUDY

A detailed study of the ethnomedicinal background, Review of available literatures and several
research articles on Nicker bean (Caesalpinia bonducella); it was known that that the plant
Caesalpinia bonducella contains Phytoconstituents such as alkaloids,glycosides, flavonoids,
terpenoids etc. which were responsible for several pharmacological activities like Anti-
inflammatory Anti-pyretic, Analgesic, Anxiolytic, Anti-hyperglycemic, Immunomodulatory,
Antibacterial and Anti-oxidant .

Based on Anti-oxidant activity, Flavonoids and Total phenolic content it can be assumed that the
active constituents of Caesalpinia bonducella can also have hepatoprotective activity. Hence
seed of the plant was taken for its hepatoprotective action.

The aim of present study is:

 To study the physicochemical and phytochemical property of plant Ceasalpinia

bonducella.
 To evaluate the in Vivo Hepatoprotective activity of Caesalpinia bonducella seed
extracts by
 Paracetamol induced hepatotoxicity in rats.
 CCl4 induced hepatotoxicity in rats.
 3(A). Work Done

4.1 Preliminary work

 Literature review.

 Plant selection.

 Plant authentification.

 Preparation of crude extract.

4.2 Pharmacognostical screening.

 Screening of Seeds.

 Screening of powder.

 Screening of crude extract.

 3(B).Work to be done

Evaluation of antioxidant activity

 DPPH method

 Reducing Power method

Screening of Antioxidant and Hepatoprotective activity in rats by :

 Antioxidant activity determination by DPPH and reducing Power method

 Paracetamol induced hepatotoxicity in rats.

 CCL4 induced hepatotoxicity in rats.

 Histopathological study.
 4. PLANT PROFILE

Nicker Bean

Picture No. 4.1 Caesalpinia bonducella whole plant

Kingdom: Plantae

Order: Fabales

Family: Caesalpineaceae

Genus: Caesalpinea

Species: C. bonducella

4.1.1 Synonyms:

Sanskrit: Kakachika, Karanja and Latakaranja

Hindi: Kathkaranj

Bengali: Nata

English: Fever nut

Urdu: Akitmakit

French: Bois canic

4.1.2 Geographical Distribution:

An armed liana, up to 15 m in height, found up to an altitude of 1,000 m in Himalaya and wild

throughout the plains of India and; it is also found in deltaic region of western, eastern and
southern India 1. Found particularly along the seacoast throughout the hotter parts of India,
Burma and Sri Lanka. (Kapoor L.D., et al., 2002)

4.1.3 Ayurvedic Description:

Properties: Rasa-katu, tikta; Guna-laghu, rooksha, teekshna; Veerya-ushna; Vipak-katu. Action

and Uses: Kapha, vat samak, sotha har, badana sthapan, dipan, anuloman, krimighan, rakt
sodhak, swashar, mutral, jwaraghan.

4.1.4 Pharmacognostic studies:

Macroscopic Characteristics

Leaves: Leaves are with large, leafy, branched, basal appendages; 30-60 cm. long; petioles
prickly; stipules a pair of reduced pinnae at the base of the leaf each furnished with a long
mucronate point; pinnae 6-8 pairs, 5-7.5 cm. long, with a pair of hook stipulary spines at the
base. Main leaf axis armed with stout, sharp, recurved spines, divided into 4-8 pairs of secondary
branches.

Leaflet: Leaflets 6-9 pairs, 2-3.8 by 1.3-2.2 cm, membranous, elliptic-oblong, obtuse, strongly
mucronate, glabrous above more or less puberulous beneath; petioloules very short; stipels of
short hooked spines.

Flowers: Flowers in dense (usually) long-peduncled terminal and supraaxillary racemes dense at
the top, lax downward, 15-25 cm. long; pedicels very short in bud, elongating to 5 mm. in flower
and 8 mm. in fruits, brown-downy; bracts squarrose, linear, acute, reaching 1 cm. long, fulvous
hair. Calyx 6-8 mm. long,fulvous hairy; lobes obovate- oblong, obtuse.Petals oblanceolate,
yellow. (Handa S.S., et al., 1996)
Seeds: Seed coat is hard, glossy, and greenish to ash grey in colour. And is traversed by circular
and vertical faint markings of the cracks, forming uniform rectangular to squarish rectulations all
over the surface Seeds 1-2, oblong, lead-colored, 1.3 cm. long.. A raised hilum with remains of
the stalk lies in the centre of the dark spot, at the narrow edge of the seed. Adjacent to the hilum,
lays a faint coloured circular to oval elevated micropyle. In dry seed, kernel gets detached from
the testa. Testa is about 1-1.25 mm in thickness and is composed of three distinct layers, the
outermost - thin and brittle, the middle one - broad, fibrous and dark – brown and the innermost
– white and papery. The seed is exalbuminous. The kernel surface is furrowed and ridged, hard,
pale yellowish – white, circular to oval, flattened and about 1.23- 1.75 cm. in diameter.. A scar of
the micropyle lies at one end of the kernel, from where arises a prominent ridge demarking the
two cotyledons of the embryo. Plumule – radical axis is thick, cylindrical and straight. Taste is
very bitter and odour is nauseating and unpleasant. (Sharma B.M., et al., 1972)

Picture no.4.2 Seeds of Caesalpinia bonducella

Microscopic Characteristics

Seeds: Seeds show a palisade layers which are composed of vertical, columnar, and laterally
closed appressed cells. Thickenings are present on the walls of palisade cells which in tangential
section appear as 6-10 denticulate projections into the lumen of cells. Then after that there is the
layer of bearer cells and a thick zone of parenchymatous cells. The majority of bearer cells are T-
shaped, thick walled and nonlignified.

Identification Test
Powder does not show any fluorescence when exposed to ultraviolet light. However, the extract
in 1% NaOH solution ethyl alcohol and solvent ether emitted a green fluorescence under
ultraviolet light.

4.1.5 Lethal dose

LD50 of the extract is higher than 2000 mg/kg and no changes were observed in any behavioral
parameters in Rats. (Sagar K., et al., 2010)

4.1.6 Phytochemistry

Phytochemical screening of methanolic extracts revealed the presence of alkaloids, saponins,

flavonoids, tannins and steroids (Gupta, et al., 2004). The methanol extract of C. bonducella
leaves containing flavonoids and triterpenoids, the antioxidant defense system has been
evaluated (Gupta, et al., 2005). The phenolic compound has several functions such as singlet and
triplet oxygen quenchers, free radical scavengers, peroxide decomposers, enzyme inhibitors and
synergists (Zhang, et al., 2004). The qualitative analysis has shown the presence of phenol in all
the extracts performed other than the hexane for C. bonducella.

Isolated constituents

Alkaloids: There is controversial reports exist regarding the presence of alkaloids in C. crista.
Earlier workers detected an alkaloid “Natin” in the plant but could not confirm the presence. The
presence of alkaloid in the seed(Iyenger M., et al., 1965) and twigs. (Puri H.S., et al., 1980) and
its absence in stem and leaf was indicated in later reports.

Glycosides: First non- alkaloidal bitter principle isolated from the seed of C. crista was Bonducin
(Bonducellin) (Dymock, et al., 1890). It was detected as a glycoside and was said to be sulphur
containing compound. But later on, the compound (C20H28O8- m. p.119.200C) was found to be
devoid of sulphur. The structural formula of Bonducin (a homoisoflavone) has been well
established recently. (Purshotaman K.K., et al., 1982)
Saponin: was reported in seed, but later on was found to be devoid of this (Kapoor V.P., et al.,
1971). Number of enzymes like protease, urease, amylase, peroxidase, catalase and oxidase has
been reported in the seed. (Vinayak, et al., 1929)

Terpenoids (1, 5, 6, 7, 14-Voucapanepentol derivative) : Caesalpin (C24H32O8) (1-ketone 6, 7-

diacetylcassane) M. W. -448.512, ß-caesalpin (C20H28O6) (1-ketone 5, 6, 7, 14-tetrahydroxy
voucapanone) M. W. -364.438 and a-caesalpin (C34H56O7) (o-tetradecanoyl voucapane
diterpenoid) M. W.-576.812 were the first three bitter cassane/voucapane diterpenoids isolated
from the seeds of C. crista .( Khuda I.Q.M., et al.,1963) Determination of the functional group,
other chemical aspects , structure elucidation (Canonica L., et al.,1966) etc. were exhaustively
studied by number of workers. d– caesalpin (C20H30O6) (1a, 5a, 6a, 7a, 7ß, 14ß-cassane) M.W.
-366.453 is hydrolysed product of caesalpin and a reduced product of a-caesalpin and ß-
caesapin. a-caesalpin on hydrolysis yields acetic acid, myristic acid and a crystalline bitter
compound (C20H30O6). The structural relationship of a, ß and? caesalpins with vinaticole,
vouncapenic and cassaic acids have been established (Francesca P., et al., 1968). Three more
Caesalpins E caesalpin, F caesalpin and Y caesalpin have also been isolated from kernels of C.
crista. Y caesalpin, a minor constituent, is closely related to d–caesalpin (Purshotaman K.K., et
al., 1982) and F caesalpin is closely related to E caesalpin. (Balman A., et al., 1980)

Reserved Food Materials of the Kernals

The kernals contains fatty oil (20-24%); starch, sucrose, two phytosterols, one of them identified
as sitosterol, and a hydrocarbon (mp 58-590C) identified as heptocosane (Tummin Katti M.C., et
al., 1930) Ghatak’s investigated the presence of noncrystalline bitter glycoside bonducin, a
neutral saponin, starch, sucrose, an enzyme, and yellow oil from seeds kernels. A white
amorphous bitter substance (0.035%) has been reported. (Ghatak N.G., et al., 1934) The oil,
which is thick and pale yellow with a disagreeable smell, has the following characteristics:
saponification value 197.9; sp gr. 0.926; acetyl value 35.6; iodine value 111.0; acid value 8.5;
and unsaponified matter 1.1%. The constituents of fatty acid are stearic, palmitic, oleic, linoceric,
linolenic, and a mixture of unsaturated acid of low molecular weights 15, 18, 45. Seed kernels of
C. crista content protein which varies from 7.4 to 18.4 to 25.3 percentage. Amino acids
composition was also studied by number of workers, are as follows: aspartic acid-9.5%, lysine-
7.9%, glycine-6.9%, leucine-6.3%, histidine-5.1%, isoleucine-5.1%, serine-3.8%, r-amino-
butyric acid-3.7%, tyrosine-3.7%, citrulline-3.6%, glutamic acid-3.6%, threonine-3.6%, arginine-
3.4%, proline-3.3%, L-alanine-2.5%, methionine-2.1%, phenyl alanine-1.4%, cystine-1.2%,
valine-1.2% and tryptophan-0.8%. The amino acid substrate specificity of glutamyl-t-RNA
synthetase prepared from the seed was also studied 48. Number of workers studied seed protein
of Caesalpinaceae by chemotaxonomic view point. (Handbuch H., et al., 1972) The non-protein
amino acids detected in the seed were r-ethylidene glutamic acid, r-methylene glutamic acid, r-
ethyl glutamic acid and traces of r-OH-r-methyl gltamic acid and B-OH r-methyl glutamic acid,
accumulation of r-methyl glutamic acid being extremely large. Some of the common
carbohydrates reported in the seed are pentoan (16.8%), starch (6.1%) and water soluble
mucilage (4.4%). (Kapoor V.P., et al., 1971)
 5. REVIEW OF LITERATURE

Pharmacological studies

Many Pharmacological activities of Caesalpinia bonducella have been reported treatment

of tumors, inflammation antiviral, antiasthmatic, antiamebic, and antiestrogenic (Negi NC,
1958, Adesina and liver disorders (Kirtikar KR, 1975), antipyretic, antidiuretic, anthelmintic,
antibacterial, anticonvulsant, antiviral, antiasthmatic, antiamebic, and antiestrogenic (Negi
N.C., 1958 ; Adesina SK, 1982; Dhar M.L., et al., 1968; Gayaraja S., et al., 1978;
Raghunathan K., et al., 1982), and other activities. The present study was therefore
undertaken to investigate some of the folkloric claims especially the use of the plant as
a treatment of inflammation (Kirtikar K.R., 1975).

Previous work on Caesalpinia bonducella

1. Anti-diarrheal activity of the nuts of Caesalpinia bonducella Flem. (Iyenger M., et al.,
1965)
2. Antibacterial activityof Caesalpinia Bonducellaseeds. ( Saeed M.A., et al., 2001)

3. Oral Antidiabetic Activities of Different Extracts of Caesalpinia bonducella Seed

Kernels. (Parameshwar S., et al., 2002)

4. Advanced studies on the hypoglycemic effect of Caesalpinia bonducella F. in type 1 and

2 diabetes in Long Evans rats. (Chakrabarty S., et al., 2003)

5. Antitumor Activity and Antioxidant Status of Caesalpinia bonducella Against Ehrlich

Ascites Carcinoma in Swiss Albino Mice. (Gupta M., et al., 2004)
 6. Antifilarial activity of Caesalpinia bonducella against experimental filarial infection.
(P.K. Murthy., et al., 2008)

7. Anxiolytic Activity of Seed Extract Of Caesalpinia Bonducella (Roxb) In Laboratory.

( Rao V. N., et al., 2008)

8. Studies on anti-inflammatory, antipyretic and analgesic properties of Caesalpinia

bonducella F. seed oil in experimental animal models. (Shukla S., et al., 2009)

9. Antioxidant activity and total phenolic content of ethanolic extract of Caesalpinia

bonducella seeds. (Shukla S., et al., 2009)

10. Immunomodulatory activities of the ethanolic extract of Caesalpinia bonducella seeds.

( Shukla S,. et al., 2009)

Other valuable literatures

1. Hepatoprotective activity of an ethanolic extract of stems of anisochilus carnosus against

carbon tetrachloride induced hepatotoxicity in rats. (Venkatesh P., et al., 2011)
2. Hepatoprotective herbal drug, silymarin from experimental pharmacology to clinical
medicine. (Pradhan S.C., et al., 2006)
3. Protective effect of livactine against ccl4 and paracetamol induced hepatotoxicity in adult
wistar rats. (Mayuren C., et al., 2010)
 6.MATERIALS AND METHODS

6.1 PRELIMINARY WORK

Gathering sufficient information from vivid articles and journals it was concluded that there is
scope to explore some more pharmacological activities in the plant Caesalpinia bonducella.
Hence it was selected for further studies.

6.1.1 Authentication of Plant

Plant was identified and authentified by Dr. Zia Ul Hasan, Head of Department, Safia College of
Science,Bhopal (M.P.) Voucher specimen no. 282/bot/safia/11.

6.1.2 Collection of Plant Material

Herb was collected from Moolchand and phoolchand shop, jummerati, bhopal (M.P). The plant
was collected in the month of July- Aug 2019. Seeds were separated and were made completely
clean and dust free.

6.1.3 Drying and Size Reduction of Plant Material

The plant material was dried under shade. It was pulverized to coarse powder with the help
of mixer grinder. The coarse powder was passed through sieve No.20 to maintain uniformity
and packed into airtight container and stored in cool and dry place. This material was used for
the further study.

6.1.4 Preparation of Crude Extract

500 gram of powdered plant material was extracted using a Soxhlet apparatus with 500 ml 90%
ethanol and 500 gram with distilled water at 60–70°C for 35 complete cycles. The extracts were
dried at 30–40°C. (Tiwari U., et al., 2004)

Picture No.6.1 Extraction process

6.2 PHARMACOGNOSTICAL SCREENING

5.2.1 Screening of seeds

Colour The seed colour was dark Green.

Odor Characteristic

Taste Acrid

Size 2-2.5cm length, 0.5-1cm width

Texture Soft

Fracture Tough.

5.2.2 Screening of seed powder (Physiochemical analysis)

Loss on Drying
10 gm of the powdered drug was weighed in a tarred petridish. It was dried at 105°C for 1 hour
in hot air oven and then reweighed. Loss on drying was determined by calculating the initial and
final weight.

Total Ash value

About 5 gm accurately weighed powdered drug was incinerated in a silica dish at a temperature
not exceeding 450°C until free from carbon in muffle furnace. It was then cooled and weighed.
The % w/w of ash with reference to the air-dried drug was calculated.

Acid insoluble ash value

1 gram ash was boiled for 5 minute with 25ml hydrochloric acid by covering the crucible with a
watch-glass on water bath then cooled. The watch-glass was rinsed with 5 ml of hydrochloric
acid and this liquid was added in to the crucible. Then the content was filtered on a previously
weighed filter paper and filtrate was dried and weighed. Acid insoluble ash value was
determined by calculating the % content remaining after deducting the weight of filter paper.

Water soluble ash value

1 gram ash was boiled for 5 minute with 25ml distilled water by covering the crucible with a
watch-glass on water bath then cooled. The watch-glass was rinsed with 5 ml of distilled water
and this liquid was added in to the crucible. The % of remaining content was deducted from
initial % of ash taken (i.e. 100%) to determine the water soluble ash value.
Foaming Index
1 gm coarse powder was weighted and transferred to a 500 ml conical flask containing 100 ml of
water. It was maintained at moderate boiling for 30 minute on water bath. It was cool and filtered
in to a 100 ml volumetric flask. Volume was diluted by adding sufficient amount of water. The
decoction was poured in test tube, and then shaken in a lengthwise motion for 15 seconds. They
were allowed stand for 15 minutes and the height of foam was measured to determine the
foaming index. (Khandelwal K.R., 2002; Pradhan P., et al., 2010)

6.2.3 Screening of crude extracts (Qualitative Phyotochemical Analysis)

The crude extracts obtained by solvent extraction were subjected to various qualitative tests to
detect the presence of common chemical constituents as:

• Alkaloids
• Glycosides
• Carbohydrates
• Phytosterols
• Saponins
• Tannins
• Flavonoids
• Proteins

Tests for Alkaloids

Dragendorff’s test
To the 1 ml of extract, add 1 ml of Dragendorff’s reagent (potassium bismuth iodide solution).
An orange-red precipitate indicates the presence of alkaloids.

Mayer’s test
To the 1 ml of extract, add 1 ml of Mayer’s reagent (Potassium mercuric iodide solution).
Whitish yellow or cream coloured precipitate indicates the presence of alkaloids

Tests for Glycosides

Legal’s test
Dissolve the extract in pyridine and add sodium nitroprusside solution to make it alkaline. No
formation of pink to red colour shows absence of glycosides.
Baljet’s test
To 1ml of the test extract, add 1ml of sodium picrate solution and the yellow to orange color
reveals the presence of glycosides.

Tests for carbohydrate

Benedict’s test
To 5ml of Benedict’s reagent, add 1ml of extract solution and boil for 2 minutes and cool.
Formation of red precipitate shows the presence of sugars.

Molisch's test
A small fraction from the respective extracts was taken in ethanol separately and a few drops of
20% w/v solution of α-napthol in ethanol (90%) were added to it. After shaking well, about 1 ml
of concentrated sulphuric acid was allowed to flow carefully by the side of the test tube. A
reddish violet ring at the junction of the two layers indicated the presence of carbohydrates.

Tests for steroids

Salkowski test
The extract was dissolved in chloroform and equal volume of conc. H₂SO4 was added.
Formation of bluish red to cherry color in chloroform layer and green fluorescence in the acid
layer represents the steroidal components in the tested extract.

Liebermann-Burchard test
A small portion from each extract was taken with about 1 ml of acetic anhydride and dissolved
by warming. The contents were cooled and a few drops of concentrated sulphuric acid were
added in each case by the sides of the test tube. Appearance of blue colour indicated the presence
of sterols.

Test for Proteins

Biuret test
Add 1ml of 40% sodium hydroxide solution and 2 drops of 1% CuSO4 solution till a blue color
is produced, and then add to the 1ml of the extract. Formation of pinkish or purple violet color
indicates the presence of proteins.

Tests for Saponins

A little fraction from the various extracts were boiled with about 1 ml of distilled water and
shaken. Appearance of a characteristic foam formation indicated the presence of Saponins.
Aqueous and alcoholic extracts were tested directly.
A little fraction from various extracts was taken with about 2 ml of distilled water. A small
quantity of sodium carbonate was added to each and shaken. The characteristic foam formation
indicated the presence of Saponins. Aqueous and alcoholic extract were tested directly.

Tests for Tannins

A small fraction of the residue from each extract was dissolved in about 2 ml of distilled water
separately and filtered. The filtrate was tested with the Ferric chloride solution. Appearance of a
blue to bluish green or bluish-black colour indicated the presence of tannins

Tests for Flavonoids

Shinoda test
To the test solution add few magnesium turnings and concentrated hydrochloric acid dropwise,
pink scarlet, crimson red or occasionally green to blue colour appears after few minutes.

Alkaline reagent test

To the test solution add few drops of sodium hydroxide solution, intense yellow color is formed
which turns to colorless on addition of few drops of dilute acid indicate presence of Flavonoids. .
(Khandelwal K.R. 2002, Pankaj Pradhan et al.2010)
 RESULTS AND DISCUSSION

6.1 PHARMACOGNOSTICAL SCREENING

Table 6.1- Seed characteristic

S.No. Characters Observation

1 Color Greyish

2 Odour Characteristic

3 Taste Acridic

4 Size 2-2.5 cm length,1.5-2 cm width

5 Texture Smooth

6 Fracture Tough

6.2 PHYSIOCHEMICAL ANALYSIS

 Table 6.2 Physiochemical analysis

S.No. Parameters Observations

1 Loss on drying 0.1%

2 Foaming index Less than 20 %

3 Total Ash value 2.95%

4 Acid insoluble ash value 1.27%

5 Water soluble ash value 1.19%

Table 6.3 Extractive values of Ceasalpinia Bonducella in following solvents.

Plant Ethanol Water

Ceasalpinia 0.9 % 0.7%

Bonducella

Table 6.4 Percentage yield of Successive Solvent extracts of Ceasalpinia Bonducella in

following solvents.

S. NO. Extracts Percentage yield

1 Ethanol 1.4%
2 Aqueous 1.8%

6.3 PHYTOCHEMICALS SCREENING

Table 6.5 Phytochemicals Screening in Succesive Solvent extract of Ceasalpinia Bonducella
seeds..

S.No. Phytochemicals Test Ethenolic extract Aqueous extract

1 Alkaloids Mayer’s test, - -

Dragendorff’s test
2 Tannins Ferric chloride test - +

3 Flavonoids Shinoda’s Test + +

4 Glycosides Legal’s test ,Baljet’s + +

test
5 Carbohydrates Benedict’s test - +

6 Amino acid Biuret test - -

7 Fixed oil & Fats Spot test - +

8 Steroid Salkowski test - -

9 Saponins By shaking the extract + +

in test tube

(+ Present, - Absent)
 SUMMARY AND CONCLUSION

The study have been designed to evaluate the Hepatoprotective activity of Ceasalpinea
bonducella seed extract against albino rats in acute experimental liver damage induced by carbon
tetrachloride and paracetamol. The preliminary Pharmacognostical studied of seed was studied
and result was tabulated in tabble6.1.The physiochemical parameter was studied and result was
tabulated in tabble6.2.The extractive value in ethanol and aqueous were found to be 1.4% &
1.8% respectively. Qualitative phytochemical analysis of the plant extract showed the presence
of majority of compounds like carbohydrates, flavonoids, terpenoids, glycosides, amino acids,
tannins and saponins.
 Significance of work

Phytochemicals are biologically active, naturally occurring chemical compounds found in plants,
which provide health benefits for human health. Literature survey revealed that Nicker bean seed
showed significant amount of carbohydrates, flavonoids, terpenoids, glycosides, amino acids,
tannins and saponins. Amongst these Phytoceuticals flavonoids terpenoids showed potent
antioxidants potential. Looking all this aspect the study is design for investigation of
hepatoprotective potential of the whole seed extract (ethanolic and aqueous) of Caesalpinia
bonducella plant.
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