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Aminu Ambi ADAMU


Download the complete PHARMACEUTICAL SCIENCES project topic and material (chapter 1-5) titled EVALUATION OF PHARMACOGNOSTICPARAMETERS AND HEPATOTOXIC EFFECTS OF EXTRACTSOF CASSYTHA FILIFORMIS LINN ON PARACETAMOL-INDUCED LIVER DISORDERS IN WISTER RATS here on PROJECTS.ng. See below for the abstract, table of contents, list of figures, list of tables, list of appendices, list of abbreviations and chapter one. Click the DOWNLOAD NOW button to get the complete project work instantly.



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Cassytha filiformis, a leafless and perennial vine with small scales as a replacement
of the leavesis currently being used in the treatment of various disease conditions
including jaundice without standardization.Microscopical evaluation,
chromatographic analysis (TLC, HPLC, LC-MS/MS), spectroscopic analyses
(NMR, FTIR, MS) and hepathoprotective studies were carried out with the view to
scientifically verify the potentials of this plant used in traditional medicine. The
results revealed the presence of some diagnostic microscopical features such as
paracytic stomata, unicellular covering trichomes with cystoliths, prismatic calcium
oxalate crystals and annular xylem vessels. Quantitative physical constants include
moisture contents (5.5 %), ash value (17 %), acid insoluble ash value (1 %), total
tannins (27.3 %), swelling index (165 %), water, alcohol and oil extractive indices
(20.6 %, 13.6 % and 1.6 % respectively). Trace metals detected in C. filiformis such
as Fe (165.4279 ppm), Mn (14.4093 ppm) and Ni (2.7933 ppm) which are essential
were higher than FAO/WHO (1984) permissible limit for edible plants. While
others:Pb (0.0568 ppm) Zn (0.1094 ppm), Cd (0.0103 ppm) and Cu (0.0535 ppm)
were found to be within the safety limit. Preliminary phytochemical screening of the
plant signifies the presence of alkaloids, tannins, flavonoids, saponins and steroids
Phytochemical constituents identified in ethyl acetate and methanol extracts of C.
filiformis include 3,3,O-di-O-methyl ellagic acid, catechin, chalcone compounds, p
hydroxybenzoic acid, isorhamnetin 3-O-rutinoside or isorhamnetin 3-O
neohesperidoside, kaemferol 3 rutinoside and 2-{cyclohex-2-en-1
yl(hydroxyl)methyl}-3-hydroxy-4-(2-hydroxyethyl)-3-methyl- oxoprolinate while
that of methanol fraction include 3,3,O-di-O-methyl etllagic acid, methy2
oxoprolinate, kaemferol 3 rutinoside, rutin and cathechinas revealed by the library
search on LC-MS/MS. Other compounds β-sitosterol and stigmasterol wereisolated
from petroleum ether extracts.The petroleum ether extract (500 mgkg-1) and
methanol (500 and 1000 mgkg-1) exhibited hepatoprotection properties on wistar
albino rats. These results could serve as bases for the use of the plant in traditional
medicine for the prevention of liver disorders.


Title page i
Declaration ii
Certification iii
Dedication iv
Acknowledgement v
Abstract vii
Table of Contents ix
List of Tables xiv
List of Figures xv
List of Plates xvii
List of Appendices xix
List of Abbreviations xx
1.1 Introduction 1
1.2 Statement of Research Problems 5
1.3 Justification of the Study 7
1.4 Aims and Objectives of the Study 7
2.1 Description of the family lauraceae 8
2.1.1 Description of the genus Cassytha 8
2.1.2 Morphological description of the specie C. filiformis 8
2.1.3 Ethomedical uses of C. filiformis 11
2.1.4 Phytochemical constituents of C. filiformis 12
2.1.5 Biological activity of C. filiformis 15
2.2 Hepatotoxicity and its Mechanism 16
2.2.1 Pharmacological evaluation of hepatoprotective plants 18
2.3 Conventional Management for Liver Disorders 21
2.4 Contributions of Metals to Potential Treatments for Hepatic Disorders 22
2.5 Medicinal Plants and Natural Products Used in the Management of Liver disorder. 25
2.6 Research Techniques inPharmacognosy 32
3.1 Collection, Identification and Processing of Cassytha filiformis 33
3.1.1 Collection of C. filiformis 33
3.1.2 Identification of C. filiformis 33
3.1.3 Processing of C. filiformis 33
3.2 Equipment, Solvents and Reagents 33
3.2.1 List of Equipment and other laboratory apparatus 33
3.2.2 Solvents 35
3.2.3 Preparation of Reagents / Solutions 35
3.3 Pharmacognostic Studies of Cassytha filiformis 37
3.3.1 Macroscopical studies of C. filiformis 37
3.3.2 Microscopical studies of C. filiformis 38
3.4 Phytochemical Studies of C. filiformis 40
3.4.1 Test for alkaloids 40
3.4.2 Test for cyanogenic glycosides 41
3.4.3 Test for cardiac glycosides 41
3.4.4 Test for Tannins 42
3.4.5 Test for antraquinones (Borntrager’s reaction) 42
3.4.6 Test for flavonoids 42
3.4.7 Test for saponins 43
3.4.8 Test for terpenoids 44
3.5 Determination of Numerical Standards of C. filiformis 44
3.5.1 Determination of Water-soluble Extractive Values 44
3.5.2 Determination of Alcohol-soluble Extractive Values 45
3.5.3 Determination of moisture content (loss on drying) 45
3.5.4 Determination of total ash 45
3.5.5 Determination of acid-insoluble ash 46
3.5.6 Determination of bitterness value 46
3.5.7 Determination of total tannins 48
3.5.8 Determination of swelling index 49
3.5.9 Determination of crude lipid 50
3.5.10 Determination of crude fibres 50
3.6 Extraction of powdered C. filiformis 51
3.7 Analysis of metals of the powdered C. filiformis using atomic absorption spectrophotometry 52
3.8 Chromatographic and Spectroscopic Studies 52
3.8.1 Thin layer chromatography of the extracts 52
3.8.2 Column Chromatographic separation of petroleum ether extract 52
3.8.3 Melting Point analysis 53
3.8.4 Spectroscopy of isolated compound 5B 53
3.8.5 High performance liquid chromatography (HPLC) 54
3.8.6 Liquid chromatography mass spectrometry/mass spectrometry (LCMS/MS)
3.9 Hepatoprotective Studies of Cassytha filiformis 55
3.9.1 Animals 55
3.9.2 Drugs 55
3.9.3 Test of acute toxicity of C. filiformis 55
3.9.4 Experimental Design 56
3.9.5 Histopathological studies of C. filiformis 57
Chapter 4.0 RESULTS 58
4.1 Collection, Identification and Processing of Cassytha filiformis 58
4.1.1 Collection ofC. filiformis 58
4.1.2 Identification ofC. filiformis 58
4.2 Equipment, Solvents and Reagents 58
4.2.1 Equipment 58
4.2.2 Solvents/Reagents 58
4.3 Pharmacognostic Studies of Cassytha filiformis 59
4.3.1 Macroscopic and Organoleptic Properties of C. filiformis 59
4.3.2 Microscopic Examination of C. filiformis 59
4.3.3 Chemomicroscopical examination of powdered C. filiformis 63
4.4 Preliminary Phytochemical Screening of C. filiformis 65
4.5 Numerical Standard of C. filiformis 67
4.6: Extraction of Powdered C. filiformis 69
4.7 Analysis of metals detected in powdered C. filiformis 69
4.8 Chromatography and Spectroscopy 71
4.8.1: Thin layer chromatography Profile of extracts 71
4.8.2: Column chromatography of petroleum ether extract 75
4.8.3 Melting point of 5B 79
4.8.4 Spectroscopic analysis 81
4.8.5: HPLC analysis for ethyl acetate and methanol extracts from C. filiformis 87
4.8.6: LCMS/MS analysis for ethyl acetate and methanol fractions from
C. filiformis 92
4.9 Hepatoprotective Studies of Cassytha filiformis 108
4.9.1 Acute toxicity studies on extracts of C. filiformis extracts 108
4.9.2 Histopathological studies 110


1.1 Introduction

The role of plants in the treatment of disease is exemplified by their employment in
all the major systems of medicine irrespective of the underlying philosophical
premise. As example, we have the western medicine with origin in Mesopotamia and
Egypt, the Unani (Islamic) and the Ayurvedic (Hindu) system and in Western Asia
and the Indian subcontinent and those of the Orient (China, Japan, Tibetetc.). There
is a great wealth of knowledge concerning the medicinal,narcotic and other properties
of plants that is still transmitted orally from generation to generation by tribal
societies, particularly those of the tropical Africa, North and South America and the
Pacific countries (Evans, 2009). These are areas containing the world’s greatest
number of plant species, not found elsewhere, and with the westernization of so
many of the people of these zones there is pressing need to record local knowledge
before it is lost forever. In addition, with the extermination of plant species
progressing at an alarming rate in certain regions, even before plant have been
botanically recorded, much less studied chemically and pharmacologically, the need
arises for increased efforts directed towards the conservation of gene pools (Evans,
Since plant taxa are defined by their morphology (more specifically on the
morphology of the flowering parts), identification techniques have relied almost
entirely on physical examination of the specimen. However, drying and powdering
alters or destroys diagnostic features and the plant parts traded in commerce may not
include the parts necessary for establishing botanical identity. Classical description in
compendia reflect this approach with physical descriptions that include appearance,
color, order, and fracture. Advances in microscopy, chemical spot test and modern
separation techniques, coupled with highly sensitive detectors and powerful software
packages have extended the capability of plant identifications. When used in
combination, these techniques may enable the investigator to confidently establish
the botanical nature of even the most highly processed herbal products (Arias and
Murray, 2009).One of the major driving forces behind the continued demand for
novel bioactive molecular entities is the need to develop new therapies for disorders
that are associated with our modern lifestyle such as, cardiovascular diseases,
ischemic heart diseases malignant neoplasms and liver disorders; this necessitate the
replacementof older drugs that have been used against bacteria, viral and parasitic
infections. Over the last few decades, there has been a marked decline in the efficacy
of wide range of medications owing partly to the development of resistant strains of
parasites and microorganisms. Unfortunately, the timeline for acquiring such
resistance has diminished dramatically since the first reported case in 1950, and one
of the major causes has been the widespread misuse and overuse of antibiotics (Arias
and Murray, 2009).
The search for natural bioactive molecules as potential drugs can be conducted
through at least three major disciplines or activities:
i) Ethno-pharmacology: this is the study of indigenous medical systems that
connect the ethnography of health and healing with the physiologic
relevance of its medical practices.
ii) Ethnobotany and the associated traditional knowledge: this is one of the
most useful approaches to get information on plant species since it
facilitates targeted searches.
iii) Chemotaxonomy or Phylogenetic approach: this method can be employed
to target a specific taxonomic group containing classes of compounds that
are similar to those present in the species, genera, families that have
previously exhibited high-hitrates for a particular type of bioactivity.
iv) High throughput-based bioprospecting programs: this is an approach
using robotic technology to screen thousands of samples per day.
The current approach to drug discovery, based on the search for molecular diversity
from natural sources, involves a number of complementary activities that necessitates
multi- and interdisciplinary approaches and a multitude of players and different
professionals. The combination of such activities can be broadly categorized with
respect to the associated disciplines of natural product chemistry and Pharmacognosy
(Arias and Murray, 2009).
Plant products as important sources of drugs have been used in diseases of many
organs including that of the liver despite its complexity. The liver is an essential and
the largest internal body organ which performs over different 500 functions (EASL,
2010). It plays a significant role in the maintenance, performance and regulation of
homeostasis of the body. It is involved in almost all the biochemical pathways to
growth, fight against diseases, nutrient supply, energy provision and reproduction.
The major functions of the liver are carbohydrate, protein and fat metabolism,
detoxification, secretion of bile and storage of vitamin. Thus, to maintain a healthy
liver is a crucial factor for overall health and well-being.
The complexity of the liver structure and frequency of its exposure to drugs and
foods that might cause harm therefore, make it susceptible to many kinds of diseases,
including hepatitis, cirrhosis, fatty liver, liver cancers and genetic diseases. The liver
has a unique ability to regenerate itself. Liver diseases are among the most serious
ailments (Samir, 2001) and have become some of the major causes of morbidity and
mortality in man and animals all over the globe and hepatotoxicity due to drugs
appears to be the most common contributing factor (Nadeemet al., 1997). In 1998,
Liver diseases ranked tenth among the diseases causing death in USA of which
chronic liver diseases and cirrhosis was the prevalent. In UK, there is increase in
liver disease with reports of rising morbidity and mortality, particularly in younger
age groups (Kaner et al., 2007).
Liver diseases may be classified as acute or chronic hepatitis (inflammatory liver
diseases), hepatosis (non-inflammatory diseases) and cirrhosis (degenerative disorder
resulting in fibrosis of the liver). Causative factors of liver disorders include; virus
infection, exposure to, or consumption of, certain chemicals, e.g. the excessive
inhalation of chlorinated hydrocarbons or over indulgence in alcohol; medication
with, chemotherapeutic agents and possibly plant material such as those containing
pyrrolizidine alkaloids; contaminated food containing toxins such as aflatoxins or
peroxides in oxidized edible oils; ingestion of industrial pollutants, including
radioactive material. Drug abuse in Western society and poor sanitary conditions in
Third World countries are contributing factors to the above. The predominant type of
liver disease varies according to country and may be influenced by local factors
(Evans, 2009).
Except for the use of appropriate vaccine for the treatment of hepatitis caused by
viral infections, there are few effective cures for liver disease (Evans, 2009). It is
therefore necessary to search for other drugs for the treatment of liver diseases
(Adewusi and Afolayan, 2010).In recent years many researchers have examined the
effects of plants used traditionally to support liver function and treat diseases of the
liver. In most cases, research has confirmed traditional experience by discovering the
mechanisms and modes of action of these plants as well as reaffirming the
therapeutic effectiveness of certain plants or plant extracts in clinicalstudies, a typical
example is the use of sylimarin from Silybum marianum in treatment of liver
diseases. Hundred plants have been examined for use in a wide variety of liver
1.2 Statement of Research Problems
The liver, the largest and most complex organ in human body has a wide range of
function making it vulnerable to a variety of disorders. Liver diseases such as
jaundice, hepatitis and fatty liver diseases are very common and large public health
problem in the world and account for a high morbidity and mortality (Kumar et al.,
2011). The principal causative factors for the liver diseases in developed countries
are excessive alcohol consumption, and viral-induced chronic liver diseases while in
the developing countries the most frequent causes are environmental toxins, parasitic
disease, hepatitis B and C viruses, and hepatotoxic drugs (certain antibiotics,
chemotherapeuticagents, high doses of paracetamol, carbon tetrachloride,
thioacetamide etc.) (Saleem et al., 2010). Liver damage is associated with cellular
necrosis, increase in tissue lipid peroxidation and depletion in the tissue GSH levels
(Mascolo et al., 1998).
There is no rational therapy for the treatment of liver disorders and management of
liver diseases is still a challenge to modern medicine (Chandrasekar et al., 2004).
Conventional medicine is now pursuing the use of natural products such as herbs to
provide support that the ailing liver needs on a daily basis (Gayatri et al., 2011). The
traditional system of medicine like Ayurveda and Sindda system of medicine, Unani
system, Chinese system and African traditional system have played a significant role
in the management of liver disorders in the past and continue to play a major role
(Mulla et al., 2009).
Medicinal plants constitute the corner stone of traditional medicine and the
formulations of these medicinal plants are used in various traditional medical
practices for the management of liver disorders (Bagban et al., 2012). However, there
is paucity of data on the validation of the folkloric claims of these medicinal plants.
Similarly, there is absence of pharmacognostic and chemotaxonomic biomarkers for
the identification of potential hepatoprotective medicinal plants. C. filiformis is one
of the medicinal plants which has enjoyed wide patronage among the traditional
practitioner of Northern Nigeria in the management of many diseases, including
jaundice. However, there is little or no data on the validation of this claim.
1.3 Justification of the Study
Cassytha filiformis is reputed to be beneficial in the management of jaundice and
other liver diseases in traditional medicine. Validation of its hepatoprotective
property will provide scientific basis for the use of the plant in traditional medicine.
The results of the study could also provide useful information for the inclusion of the
plant in Pharmacopoeia.
The pharmacognostic and spectroscopic study of the plant could provide useful
information in formulating C. filiformis as drug. The possible correlation of the
activity of the extracts with the constituents will lead to development of safer
hepatoprotective drug of plant origin.
1.4 Aims and Objectives of the Study
To set pharmacognostic standardforC. filiformis
To isolate the phytochemical constituents of C. filiformis.
To evaluate the effects of C. filiformis extracts on the liver of Wister albino rats.


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