Formulation Development and Pharmacological Evaluation of ... · Formulation Development and Pharmacological Evaluation of Herbal Antimalarials Thesis Submitted in Partial Fulfillment

Formulation Development and Pharmacological

Evaluation of Herbal Antimalarials

Thesis Submitted in Partial Fulfillment

For the Award of Degree of

Doctor of Philosophy

In

Pharmacy

By

Sanjay Balasaheb Bhawar, M.Pharm.,

Registration Number: 0863600007

VINAYAKA MISSIONS UNIVERSITY

(Under section-3 of UGC Act 1956)

NH-47, ARIYANOOR, SALEM, TAMILNADU, INDIA

June- 2016

VINAYAKA MISSIONS UNIVERSITY, SALEM

CERTIFICATE BY THE GUIDE

I, Prof. (Dr.) B.S. Kuchekar, certify that the thesis entitled ―Formulation

Development and Pharmacological Evaluation of Herbal

Antimalarials‖ submitted for the degree of Doctor of Philosophy by Mr.

Sanjay Balasaheb Bhawar is the record of research work carried out by

him during the period from October 2008 to December 2015 under my

guidance and supervision and that this work has not formed the basis for

the award of any degree, diploma, associateship, fellowship or other titles

in this university or any other university or Institution of higher learning.

Date: Place: Dr. Bhanudas S. Kuchekar M.Pharm, PhD, FIC, LLB

Principal MAEER‘s Maharashtra Institute of Pharmacy,

Kothrud, Pune

VINAYAKA MISSIONS UNIVERSITY, SALEM

DECLARATION BY THE CANDIDATE

I, Sanjay Balasaheb Bhawar, declare that this dissertation/ thesis

entitled―Formulation Development and Pharmacological Evaluation of

Herbal Antimalarials‖ is a bonafide and genuine research work carried out

by me under the guidance of Dr. Bhanudas S. Kuchekar, Prinicipal,

MAEER‘s Maharashtra Institute of Pharmacy and this work has not formed

the basis for the award of any degree, diploma, associateship, fellowship

or other titles in this university or any other University or Institution of

higher learning.

Date:

Place: Sanjay Balasaheb

Bhawar

ACKNOWLEDGEMENT

I offer my sincerest gratitude to my supervisor, Dr. Bhanudas S.

Kuchekar, Professor and Principal, MAEER‘s Maharashtra Institute of

Pharmacy, Pune, who has supported me throughout my research work

with his patience, motivation, and immense knowledge. One simply could

not wish for such a genius and friendlier supervisor. My deepest gratitude

to Mrs. Kuchekar, whose motherly care never let me feel the stress of

completion of my thesis. .

I am very grateful to Dr Rajendran, Dean (Research), Vinayaka missions

University, Salem and Dr. B. Jaykar, Principal, Vinayaka Missions College

of Pharmacy, Salem for their kind co-operation, valuable guidance and

timely help throughout the study.

I am thankful to Padmabhushan Dr. Balasaheb Vikhe Patil, Chairman,

Pravara Rural Education Society, Loni for his blessings, encouragement

and motivation.

I express my deepest gratitude to Dr. Ashok Vikhe Patil, Executive

Chairman, Pravara Rural Education Society, for his kind support and

continuous encouragement.

I am thankful to Dr. S.A. Nirmal, Principal, Pravara Rural College of

Pharmacy, Loni for providing me necessary facilities, guidance and timely

help.

I am grateful to Oniosome laboratory, Chandigarh for in-vivo study. I am

also grateful to Dr. Salunke, Head, Dept. of Botany, PVP College Loni for

authentication of my plant.

―A friend in need is a friend indeed‖. I am thankful to Almighty God for

bestowing me with lovely friends Dr. Nachiket, Dr. Vijay, Dr. Amrutesh,

Abhijeet, and Santosh. They made this work look very easy and joyful.

Special thanks are due to Ravindrawithout whose brotherly help I could

never finish this Herculean task.

I thank my dear friend Vishal who stood behind me and supported me in

every ups and downs of my life and constantly encouraged me.

I am thankful to all my Colleagues, Friends and Students of Pravara

Rural College of Pharmacy and Pravara Rural Education Society for

helping me directly or indirectly in fulfillment of my work.

Thanks are due to Dr. R.P. Marathe, Dr. N.R. Jadhav, Dr. B.M. Patil, Dr.

Kiran Aher and Dr. Ashok Nighute for their constant motivation, support

and encouragement.

I feel proud to be son of my loving and caring Parents. They offered me

best of the best care and showered their blessings which gave me strength

for completion of this work. I am thankful to my beloved family members

Nilesh, Ashwini, Savita, Vanita and Girija for always being with me.

I thank my better half Mrs. Hemlata Bhawar for understanding me and

encouraging me to do the best . Her tender love, care and sometimes rage

forced me to work .

I can‘t forget my little angel Vardaanwhose ever smiling and charming

face recharged me with full energy and zeal whenever I was tired.

Last but not the least I thank Almighty God for His blessings and giving me

strength for completion of my thesis.

……Sanjay Balasaheb Bhawar

TABLE OF CONTENTS

Chapter Title Page No

1. INTRODUCTION 1

1.1Herbal Formulation 1

1.2Malaria 5

1.3 Floating Drug Delivery System 17

1.4 Plant profile 33

1.4.1 Chemical constituents 35

1.4.2 Uses 35

2. REVIEW OF LIETERATURE 38

3. NEED FOR THE STUDY 44

4. OBJECTIVES OF THE STUDY 46

5. PLAN OF WORK 47

6. POLYMER PROFILE 49

6.1 HPMC 49

7 EXCIPIENT PROFILE 54

7.1Psyllium husk 54

7.2 Sodium bicarbonate 58

7.3 Magnesium stearate 58

8. MATERIALS AND METHODS 60

8.1Plant material 60

8.2Pharmacognostic studies 60

8.3Extraction 64

8.4Preliminary phytochemical test 65

8.5Characterization of neem leaf extract 74

8.6Drug-excipient compatibility study 74

8.7Preparation of floating tablet 75

8.8Evaluation and characterization of

floating tablet

79

8.9Antimalarial screening 88

8.10Stability studies 90

8.11Statistical analysis 91

9. RESULTS AND DISCUSSION 92

9.1Pharmacognostic studies 92

9.2Phytochemical studies of extract 94

9.3TLC 95

9.4Preparation of Calibration curve using UV

spectrophotometer

100

9.5Preformulation studies 102

9.6Evaluation and characterization of

floating tablet

104

9.7Optimization of tablet formulation 110

9.8In-vivo gastro-retention study 112

9.9 Stability studies 113

9.10 Antimalarial activity 114

10. CONCLUSION 119

11. REFERENCES 121

LIST OF TABLES

Sr

No

Table No Captions Page No

1 Table 1.1 Different dosage forms 3

2 Table 1. 2 Marketed Products of FDDS 32

3 Table 6.1 Typical viscosity values for 2%

(w/v) aqueous solutions of

Methocelat 20°C

53

4 Table 7.1 Technical specification of Psyllium

husk powder

57

5 Table 8.1 List of Instruments/equipments 76

6 Table 8.2 List of chemicals and reagent 76

7 Table 8.3 Composition of floating tablet

formulation

78

8 Table 8.3 Stability conditions 91

9 Table 9.1 Morphological and organoleptic

characters

92

10 Table 9.2 Ash value 92

11 Table 9.3 Extractive values 93

12 Table 9.4 Foreign organic matter and

moisture content

93

13 Table 9.5 Summary of extraction 93

14 Table 9.6 Preliminary phytochemical tests 94

15 Table 9.7 TLC of Hydroalcoholic extract of A.

Indica for flavonoids

96


Indica for Alkaloids

97


Indicafor steroids

98

18 Table 9.10 TLC of Hydroalcoholic extract of

A.indicafor saponins

99

19 Table 9.11 Calibration curve data for Neem

leaf extract in 0.1 N HCI

100

20 Table 9.12 Evaluation of powder blend 105

21 Table 9.13 Post compression evaluation of

formulations

106

22 Table 9.14 Release kinetics for various

formulations

111

23 Table 9.15 Stability studies 113

24 Table 9.16 Survival over time of P. berghei 115

25 Table 9.17 Percent mean parasitemia in mice

receiving different treatments on 4th

day

117

LIST OF FIGURES

Sr

No.

Figure No. Captions Page No.

1 Fig 1.1 Malaria life cycle 10

2 Fig 1.2 Schematic localization of an

intragastric floating system and a high

density- system in the stomach.

22

3 Fig. 1.3 Hydrodynamically balanced system

(HBS).

25

4 Fig. 1.4 (A) Multiple-unit oral floating drug

delivery system. (B) Working principle

of effervescent floating drug delivery

system.

27

5 Fig. 1.5 Leaves of A. indica 33

6 Fig. 7.1 Psyllium seeds 54

7 Fig.8.1 Extraction of neem leaf 65

8 Fig. 8.2 Tablet compression machine 77

9 Fig. 9.1 TLC of hydroalcoholic extract of A.

Indica for flavonoids

96

10 Fig. 9.2 TLC of hydroalcoholic extract of

A.indicafor alkaloids

97


indica for Steroids

98


indica for Saponins

99

13 Fig. 9.5 Calibration of curve of neem extract 101

14 Fig. 9.6 FTIR spectra of hydroalcoholic extract of

A. indica

102

15 Fig. 9.7 FTIR spectra of HPMC K100M 103

16 Fig. 9.8 FTIR spectra of sodium bicarbonate 103

18 Fig. 9.9 FTIR spectra of physical mixture of

neem extract, HPMC and sodium

bicarbonate

104

19 Fig. 9.10 In vitro drug dissolution study for A1, A2

and A3

108

20 Fig. 9.11 In vitro drug dissolution study for A4 and

A5

109

21 Fig. 9.12 In vitro drug dissolution study for A6 and

A7

109

22 Fig. 9.13 X-ray photographs at different time

112

intervals of gastroretentive floating

tablets (a) X-ray at 0 hr (b) after 2 hr

(c) after 4 hr (d) after 6 hr (e) after 8 hr

(f) after 10 hr

23 Fig. 9.14 Percent mean protection in mice

receiving different treatments over

time (40 days post treatment)

116

LIST OF ABBREVIATIONS & SYMBOLS

ABBREVIATIONS

GRDDS Gastroretentive Drug Delivery System

ET Ethanol Extract

CR Controlled Release

CRDDS Controlled Release Drug Delivery System

CRDFS Controlled Released Dosage Formulation

GET Gastric Emptying Time

GIT Gastrointestinal Tract

GRDDS Gastroretentive Drug Delivery System

GRT Gastric retention Time

HBS HydrodynamicallyBalance System

HCl Hydrochloric Acid

KBr Potassium Bromide

P. berghei Plasmodium berghei

BBB Blood Brain Barrier

FDDS Floating Drug Delivery System

A. Indica Azadirachtaindica

DSC Differential Scanning Calorimetry

HPMC Hydroxy propyl methyl cellulose

BLT Buoyancy Lag Time

MST Mean Survival Time

FTIR Fourier Transform Infrared

RP HPLC Reverse Phase High Performance Liquid Chromatography

ICH International Conference on Harmonization

IR Infrared

RH Relative Humidity

TLC Thin Layer Chromatography

LBD Loose Bulk Density

TBD Tapped Bulk Density

ARM Artemether

Rf Retention Factor

RPM Rotation per minute

ppm Parts Per Million

S.D. Standard Deviation

SEM Scanning Electron Microscopy

IP Indian Pharmacopeia

USP United State Pharmacopeia

UV Ultraviolet

i. p. Intraperitoneal

ANOVA Analysis of Variance

SYMBOLS

% Percent

Ng nanogram

µg Microgram

Mg Milligram

G Grams

Kg Kilogram

Nm Nanometer

µm Micrometer

Mm Millimeter

Cm Centimeter

°C Degree Celsius

Sec Seconds

Min Minutes

Hr Hour

μL Microliter

mL Milliliter

L Liter

w/w Weight by weight

w/v Weight by volume

v/v Volume by volume

v/w Volume by weight

λmax Absorption maxima

R2 Regression coefficient

N Normality

ABSTRACT

Gastro retentive systems can remain in the gastric region for several hours

and hence prolongs the gastric residence time of drugs and improve the

bioavailability. The aim of project was to develop sustained release floating

matrix tablet for hydroalcoholic extract of neem using psyllium husk as

release controlling polymer along with synthetic polymer HPMC and

sodium bicarbonate as gas generating agent. The tablets were prepared

by direct compression method. Seven different formulations A1 to A7 were

prepared by varying the concentration of psyllium husk, HPMC, sodium

bicarbonate. Tablets were evaluated for pre and post compression

parameters like tablet thickness, hardness, weight variation, drug content,

friability, BLT and in vitro drug release. Results for angle repose, swelling

index, weight variation, drug content, thickness, hardness, % friability for

all the formulations were found in acceptable limit. In vitro drug release

was observed for 12 hours. The release pattern was best fitted to zero-

orde, Korsemeyer model for tablet formulations. It was optimized on the

basis of buoyancy time and in vitro drug release. The optimized

formulation was found to be A4 with 98.77% in vitro drug release in 12 h

and 212 sec buoyancy time. The BaSO4 tagged formulation, similar to A4

was tested in In-vivo gastric retention study in rabbits. It was observed

that formulation kept floating in stomach region till 10 hours. Formulations

containing combination of psyllium husk and HPMC K100M with sodium

bicarbonate as gas generating agent can be a promising way for

formulating gastroretentive drug delivery systems. Antimalaral screening

was done using mice survival study on swiss albino mice infected with

Plasmodium berghei. The animals were divided into eight groups each

consisting six animals based on treatment they received. Animals were

observed for 40 days post infection with P. berghei and number of mice

surviving in each group was recorded. Neem extract was given as single

drug and in combination with artemether to mice. Mice survival and %

parasitemia inhibition study showed that neem leaf extract has moderate

antimalarial activity compared to control group. Combination of neem–

artemether prolonged the survival time as compared to administration of

single drug at same dose. The suppressive action of combination was

superior with MST 36.4 days as compared to administration of single drug

(artemether) at the same dose with MST 31.8 days.

1 | P a g e

1. Introduction

1.1 Herbal Formulation

The increase in cases of multidrug resistance malaria has led to put

more efforts in research of new antimalarial agents. Plants traditionally

used to treat malaria can be diverse source to find new antimalarial drug

candidates. Combinations of novel antimalarial drugs are way forward to

improve therapeutic efficacy. Also they have ability to reduce the chances

of drug resistance. Globally, artemisinin based combination therapy is

mainstay to treat drug resistant malaria. But artemisinin derivatives have

shorter half life. They need partner antimalarial drug with longer half life.1,2

Azadirachta indica (A. indica),is one of the promising medicinal

plants, having a wide spectrum of well documented therapeutic activity. all

parts of the neem tree are known to possess a broad range of biological

properties. It has been extensively used in Ayurveda, Unani and

Homeopathic medicine. In Sanskrit it is called as ‗Arishtha‘ which means

‗reliever of sickness‘. Understanding the importance of the Neem tree the

US National Academy of Sciences has published a report in 1992 entitled

‗Neem-a tree for solving global problems‘. Neem has chemically diverse

biologically active constituents with enormous therapeutic potential.

Constituents like Nimbidin, azadirachtin and gedunin in neem tree are

reported to possess antimalarial activity. Irodin A obtained from Neem

2 | P a g e

leaves is found to be toxic to malaria parasite. Components of the alcoholic

extracts of leaves and seeds are effective against both chloroquine-

resistant and sensitive strains. The antimalarial potential of neem can be

explored in combination with artimisinin derivative provided that its plasma

concentration in the blood is maintained for a longer time and its release

from formulation is controlled. This can be achieved by designing a floating

drug delivery system for neem extract that will control the rate of release

for longer duration thus maintaining plasma concentration.

Natural products have become an integral part of human health care

system. There is growing interest worldwide in traditional medicines and

herbal products. Despite the achievement of synthetic chemistry and the

advances toward rational drug design, natural products are essential in

providing medicinal compounds as a starting point for development of

synthetic analogues.

The traditional Indian medicinal system is based on phytochemicals

for the treatment of various diseases. A large number of phytochemicals

from medicinal plants are found to possess antiplasmodial activity. Use of

expensive allopathic drugs is associated with adverse effects. This has

provoked the need for the research into cheaper drugs with lesser side

effects, especially those empirically used in traditional systems of medicine

like Ayurveda, Homeopathy. Efforts are also being made to integrate

3 | P a g e

indigenous healthcare system with modern health facilities. Diverse

secondary metabolites from plants are sources of many commercially

important pharmaceutical compounds. Herbal products are generally in

unmodified form as concentrated extract. The biological activity is complex

to validate and can not be assigned to single entity. Mixture of constituents

play role in exerting therapeutic activity either additive or synergistic by

acting on multiple target site and pathways associated with

Pathophysiology of disease.3,4

1.1.1 Selection of Dosage form5

Herbal formulation shall mean a dosage form consisting of one or

more herbs or processed herb(s) in specified quantities to provide specific

nutritional, cosmetic benefits, and/or other benefits meant for use to

diagnose, treat, and mitigate diseases of human beings or animals and/or

to alter the structure or physiology of human beings or animals. Herbal

dosage form can be categorized into two types as follows

Table 1.1:Different Dosage forms

Ayurvedic dosage forms Modern dosage form

Churna

Bhasma

Powder

Tablet

4 | P a g e

Pill/gutica

Ark

Asava/Arishta

Avaleha

Kwatha

Gritha

Capsule

Liquid

Emulsion &

Suspension

injection

Ointment, cream &

Gel

Oral administration of drugs has been the most common and

preferred route for delivery of most therapeutic agents. It remains the

preferred route of administration investigated in the discovery and

development of new drug candidates and formulations. The popularity of

the oral route is attributed to patient acceptance, ease of administration,

accurate dosing, cost effective manufacturing methods, and generally

improved shelf-life of the product. For many drugs and therapeutic

indications, conventional multiple dosing of immediate release formulations

provides satisfactory clinical performance with an appropriate balance of

efficacy and safety. The rationale for development of an extended-release

formulation of a drug is to enhance its therapeutic benefits, minimizing its

side effects while improving the management of the diseased condition.

5 | P a g e

Besides its clinical advantages, an innovative extended-release

formulation provides an opportunity for a pharmaceutical company to

manage its product life-cycle. The lack of new chemical entities is forcing

many pharmaceutical companies to reformulate an existing conventional

formulation to an extended-release product as a strategy of life-cycle

management and retaining market share.

1.2 Malaria

Malaria is parasitic disease caused by the protozoan of the genus

plasmodium. It is a vector borne disease caused by bite of female

anopheles mosquito carrying parasite. Almost half of the world‘s population

mostly living in tropical and subtropical area is at risk of malaria. Malaria is

still one of the leading causes of death in the world. Infants below 5 years,

immune compromised patients, HIV infected patients, pregnant woman

and non immune travelers are at higher risk of infection.6

1.2.1 Types of malaria

There are four species of Plasmodium parasites, can cause malaria

infection in humans.

1) Plasmodium falciparum – causes malignant tertian malaria. It is a

severe form of the disease and if untreated it is rapidly fatal.

6 | P a g e

2) Plasmodium vivax- causes benign tertian malaria and these

parasites are mostly responsible for human infection and relapses

are common. The term tertian indicates that the attack of chills and

fever typically tend to recur every third day.

3) Plasmodium malariae – causes quartan malaria, an infection which

is not common. Relapses are rarer in P. malariae infection than in

vivax malaria. The term quartan indicates that the spikes of fever

come every fourth day.

4) Plasmodium ovale- causes a rare form of relapsing malaria. Its

periodicity is similar to vivax malaria i.e. it is tertian in nature but runs

a milder course and is more easily treated.7

1.2.2 Life cycle of Malaria parasites8

Life cycle of malaria parasite is divided into two phases asexual cycle in

humans and sexual cycle in mosquito. The human phase begins when an

infected female Anopheles mosquito bites man and injects sporozoits from

her salivary glands. The injected sporozoites rapidly leave the circulation

and localized in the liver parenchyma cells. Where it develops into primary

tissue schizonts and matures within 8-21 days to form merozoits. This is

called as preerythrocytic stage during which subject remains symptoms

free. On maturity the merozoits of all four plasmodium species are

released from liver cells and infect erythrocytes. At this point erythrocytic

7 | P a g e

stage begins. In all except falciparum malaria, a portion of merozoits

infects more liver tissue cells forming secondary tissue schizonts. This is

called as exoerythrocytic cycle which may continue for several years.

These secondary tissue schizonts are responsible for relapse of infection.

At the end of erythrocytic stage the infected red blood cells ruptures

releasing merozoits, pigments and other products into the blood. At this

point clinical symptoms of the disease appear in infected individual. Going

through several exoerythrocytic and erythrocytic cycles male and female

gamatocytes are formed from some erythrocytic parasites. These

gamatocytes if are ingested by female anopheles mosquito from blood

stream allow sexual cycle of parasite to proceed in the gut of mosquito.

Fertilization of female gamatocytes with male gamatocytes forms zygote

which develops in the gut wall as an oocyst. Sporozoits are eventually

developed from oocyst which makes their way to the mosquito‘s salivary

glands. Thus cycle is completed in both human being and mosquito.

1.2.3 Phases of life cycle

1.1.3.1 Exoerythrocytic phase

In this phase sporozoites multiply in liver cells to form merozoits without

any inflammatory reaction locally. These liver cells rupture releasing

thousands of merozoits into bloodstream. This phase takes 6 to 16 days

after first infection.

8 | P a g e

1.2.3.2 Dormant or hypnozoite phase

Infections because of P. falciparum and P. malarae have a single

exoerythrocytic form where invaded liver cells rupture and release

merozoits into bloodstream, while Infections with P. vivax and P. ovale

have two exoerythrocytic forms. Primary exoerythrocytic form causes liver

cells to rupture and release merozoits like P. falciparum and P. malariae

infection. But in other form known as secondary exoerythrocytic phase,

sporozoites in liver cells differentiate into hypnozoites. These hypnozoits

remain in dormant phase for several months to years. Hypnozoites have

ability to enter in exoerythrocytic schizogony, thus releasing merozoites

that invade the blood cells which lead to clinical relapse or delayed case.

1.2.3.3 Erythrocytic Phase

Released merozoites infect red blood cells and develop into trophozoites.

These trophozoites divide and develop forming 8-24 merozoites in each

cell. Upon completion of this phase, the host cells rupture and mature

merozoites are released into bloodstream. At this point clinical symptoms

associated with malaria infection like fever chills and other occur in host

body. The merozoites then invade fresh erythrocytes and goes through

another cycle. This process occurs repeatedly during the course of

infection. The length of this development cycle is 48 hours in vivax, ovale

and falciparum malaria, and 72 hours in P. malariae infections. Some

9 | P a g e

merozoites differentiate into sexual forms- male and female gametocytes

in invaded red blood cells and become ready for ingestion into female

anopheles mosquito for vector phase.

1.2.3.4 Vector Phase.

Anopheles mosquitoes ingest sexual forms of Plasmodium parasites from

infected host. In mosquito stomach, female macrogametocytes and male

microgametocytes mature and combine to form a zygote. These zygotes

undergo mitosis giving rise to ookinetes which ultimately develop into

oocysts. Thousands of motile sporozoits are formed from the oocysts.

Mature sporozoites move to salivary glands, and become ready to infect

human beings. The vector phase is completed in 8 to 35 days.

10 | P a g e

Fig 1.1: Life cycle of malaria parasite

Signs and symptoms

The clinical symptoms of malaria are seen after 8–25 days post infection.

Beginning of clinical manifestation of malaria shows typical flu like

symptoms and thus can be easily be confused with it.

11 | P a g e

The symptoms of malaria includes

a. Headache

b. Fever

c. Shivering

d. Joint Pain

e. Jaundice

f. Vomiting

g. Hemolytic anaemia

h. Hemoglobin in the urine

i. Retinal Damage

j. Convulsions

Paroxysm, a classical symptom of malaria, is repeatedly occurrence of

coldness followed by shivering, fever and sweating seen after every two

days in P. vivax and P. ovale and every three days in P. malariaeand

P. falciparum infection.

Severe malaria usually referred as falciparum malaria, is caused by

P. falciparum. Individuals with cerebral malaria exhibit neurological

symptoms, like abnormal postures, gaze palsy, seizures or coma.9,10

12 | P a g e

1.2.4 Complications11, 12

Malaria has several serious complications as enlisted below,

i. Respiratory distress which prevail in 25% of adults and 40% of

children in falciparum malaria.

ii. Renal failure because of black water fever, presence of hemoglobin

from lyzed RBC in urine

iii. Encephalopathy associated with cerebral malaria

iv. Spleenomegaly

v. Severe anaemia

vi. Hepatomegaly

vii. Hypoglycemia and hemoglobinuria

viii. Spontaneous bleeding, coagulopathy and shock

ix. Malaria in pregnancy may lead to stillbirths, abortion, infant mortality

and low birth weight.

x. Malaria infection with HIV increases mortality

1.2.5 Categories of antimalarial drugs

1. Causal prophylaxis

https://en.wikipedia.org/wiki/Complication_%28medicine%29

13 | P a g e

These agents prevent infection by its lethal effect on the plasmodia

during their preerythrocytic stages. Primaquine has causal

prophylactic properties

2. Suppressive treatment

A suppressive drug causes inhibition of erythrocytic stage of

development of parasite.

E.g. Chloroquine, Pyrimethamine and chloroguanide

3. Clinical cure

These agents interrupt erythrocytic schizogony of the plasmodia and

thereby terminate the clinical attack.

4. Radical cure

These agents eradicate both exoerythrocytic and erythrocytic

phases of the infection. Primaquine has high radical cure rate.

5. Suppressive cure

This refers to complete elimination of the malaria parasite from the

body by continued suppressive therapy.

6. Gametocytocidal therapy

These agents destroy the sexual forms of the malarial parasites in

human blood and thus eliminate reservoir of parasite from

mosquito.13,14

14 | P a g e

1.2.6 Classification of antimalarial agents

Antimalarial drugs are classified in two ways. First is based on chemical

structure while second on forms of the parasite against which they are

most effective.

1.2.6.1 Classification based on chemical structure

i. Cinchona alkaloid : Quinine

ii. 4-aminoquinolines: chlorquine, hydroxychloroquine, amodiaquine

iii. 8-aminoquinolines: Primaquine

iv. Acridine dyes : Quinacrine

v. Diaminopyrimidines : Pyrimethamine, Trimethoprim

vi. 4- Quinolone carbinolamines: Mefloquine

vii. Miscellaneous : Artemisinin derivative

1.2.6.2 Classification based on affected plasmodial stage

1. Primary tissue schizonticides: Drugs that destroy the primary tissue

schizonts in the liver soon after infection. E.g. Primaquine

2. Blood schizonticides: Drug that suppresses the symptoms of malaria

by destroying schizonts and merozoits in the erythrocytes. E.g.

Chloroquine, mefloquine, amodiaquine, quinine

3. Gametocides: Drugs that prevent infection of mosquitoes and spread

of infection. E.g. Primaquine

15 | P a g e

4. Sporocides: Drugs that eradicate malaria by preventing sporogony in

the mosquito. E.g. Chloroguanide

5. Secondary tissue schizont: they cure chronic relapsing fevers due to

infection by P. vivax, P. malariae and P. ovale.13, 14, 15

1.2.7 Artemisinin combination therapies

Artemisinin is herbal product extracted from Chinese plant Artemisia annua

L. traditionally used in China for the treatment of malaria. Artemisinin is a

sesquiterpene lactone having peroxide bridge which is believed to be

responsible for antimalarial action of the molecule. It is well tolerated in

human being and effective against asexual blood forms of the parasite. It is

active against both chloroquine sensitive and resistant strains of P.

falciparum. Artemisinin has poor water solubility and bioavailability which

severely limit its use. So many semisynthetic derivatives of artemisinin

have been developed including,

Artesunate- Water soluble derivative can be administered orally,

intramuscularly or intravenously

Artemether – Lipid soluble derivative can be administered orally,

intramuscularly or intravenously

Dihydroartemisinin- Lipid soluble derivatives16, 17

16 | P a g e

Artemisinin produces rapid clearance of systemic parasitemia and rapid

cure from the symptoms. But artemisinin and its derivatives are eliminated

rapidly.They are always given in combination with companion drug with

longer half life18. Use of artemisinin as monotherapy is discouraged by the

WHO guidelines as this can lead to development of drug resistance. Also

high rate of recrudescence is observed with the use of artemisinin as

monotherapy. Mechanism of action of artemisinin is yet not clear but it is

suggested that they are activated by reacting with haem and iron oxide

forming free radicals that damages susceptible protein in malarial

parasites19, 20. Artemisinin derivatives have wider therapeutic index with

mild adverse effects including nausea, vomiting, headache and abdominal

pain. WHO has included artemisinin combination therapy as first line of

drug treatment for treating both uncomplicated and complicated malaria21.

It is given in combination with drug like lumefantrine, mefloquine,

amodiaquine, chlorproguanil and sulfadoxine/ pyrimethamine. Artemether

17 | P a g e

and lumefantrine was the first fixed dose combination therapy

recommended by WHO22, 23. Other combinations of artemisinin are as

follows

Artemether plus lumefantrine,

Artesunate plus amodiaquine,

Artesunate plus mefloquine,

Artesunate plus sulfadoxine-pyrimethamine

Dihydroartemisinin plus piperaquine24, 25, 26

1.3 Floating drug delivery system

Oral administration is the most versatile, convenient and commonly

employed routeof drug delivery for systemic action. Indeed, for controlled

release system, oral route of administration has received more attention

and success because gastrointestinal physiology offers more flexibility in

dosage form design than other routes.27

Oral controlled release dosage forms have been developed for the

past three decades due to their considerable therapeutic advantages and

applications. The high level of patient compliance in taking oral dosage

forms is due to the ease of administration and handling of these forms.

18 | P a g e

Controlled Drug Delivery System provides drug release at a

predetermined, predictable and controlled rate to achieve high therapeutic

efficiency with minimal toxicity. Despite tremendous advancement in drug

delivery, oral route remains the preferred route for the administration of

therapeutic agents and oral drug delivery is by far the most preferable

route of drug delivery because of low cost of therapy. Ease of

administration leads to high levels of patient compliance and the

gastrointestinal physiology offers more flexibility in dosage form design

than most other routes. Consequently much effort has been put into

development of strategies that could improve patient compliance through

oral route.28

Gastroretentive systems

Variability in GI transit time is a concern for oral controlled drug

delivery systems. Drugs with a narrow absorption window in the GI tract

are particularly susceptible to variation in both bioavailability and time to

achieve peak plasma levels. If successful, gastroretentive controlled

release formulation could offer a potential solution to the problem by

offering a prolonged gastric residence lime. A drug that is released from

the dosage form in a controlled manner in the stomach will exit the

stomach together with gastric fluids and have the whole surface area of the

19 | P a g e

small intestine available for absorption. This type of drug deliver)' also

offers a potential for enhanced drug therapy for local conditions affecting

the stomach, for example antibiotic administration for Haemophilus pylori

eradication in the treatment of peptic ulcer. Attempts to achieve prolonged

gastric retention includes altering the density of the formulations and bio

adhesion to the stomach lining. Several strategies have been employed to

make the dosage forms float in the stomach.32Hydrodynamically balanced

system (IIBS) was the first formulation that used the floating property of a

device with density lower than water. HBS is a capsule containing drug,

gel-forming hydrophilic polymers (e.g. hydroxyl propylcelIulose) and some

hydrophobic fatty materials (e.g. stearates).Another approach includes ion

exchange resin beads loaded with bicarbonate, which on contact with

media containing hydrochloric acid; release carbon dioxide, causing the

resin to float Extension of the floating time is achieved by coating the

bicarbonate-coated beads with a semi-permeable membrane.26

1.3.1 Basic gastrointestinal tract physiology

Anatomically the stomach is divided into 3 regions: fundus, body,

and antrum (pylorus). The proximal part made of fundus and body acts as

a reservoir for undigested material, whereas the antrum is the main site for

mixing motions and act as a pump for gastric emptying by propelling

actions. Gastric emptying occurs during fasting as well as fed states. The

20 | P a g e

pattern of motility is however distinct in the 2 states. During the fasting

state an interdigestive series of electrical events take place, which cycle

both through stomach and intestine every 2 to 3 hours. This is called the

interdigestive myloelectric cycle or migrating myloelectric cycle (MMC),

which is further divided into following 4 phases as described by Wilson and

Washington.

1. Phase I (basal phase) lasts from 40 to 60 minutes with rare

contractions.

2. Phase II (preburst phase) lasts for 40 to CO minutes with intermittent

action potential and contractions. As the phase progresses the

intensity and frequency also increases gradually.

3. Phase III (burst phase) lasts for 4 to 6 minutes. It includes intense

and regular contractions for short period. It is due to this wave that

all the undigested material is swept out of the stomach down to the

small intestine. It is also known as the housekeeper wave. Phase IV

lasts for 0 to 5 minutes and occurs between phases II and I of 2

consecutive cycles. After the ingestion of a mixed meal, the pattern

of contractions changes from fasted to that of fed stale. This is also

known as digestive motility pattern and comprises continuous

contractions as in phase II of fasted state. These contractions result

in reducing the size of food particles (to less than 1 mm), which are

21 | P a g e

propelled toward the pylorus in a suspension form. During the fed

state onset of MMC is delayed resulting in slowdown of gastric

emptying rate. Scintigraphic Studies determining gastric emptying

rates revealed that orally administered controlled release dosage

forms arc subjected to basically 2 complications, that of short gastric

residence time and unpredictable gastric emptying rate.

1.3.2 Approaches for gastroretention

The various approaches that are used to prolong the gastric

residence time are as follows:-

High-density systems

Gastric contents have a density close to water (1.004 gcm3). When

the patient is upright small high-density pellets sink to the bottom of the

stomach (Fig 1.10) where they become entrapped in the folds of the

antrum and withstand the peristaltic waves of the stomach wall.

Floating systems

These have a bulk density lower than the gastric content. They

remain buoyant in the stomach for a prolonged period of time, with the

potential for continuous release of drug. Eventually, the residual system is

emptied from the stomach. Gastric emptying is much more rapid in the

fasting state and floating systems rely heavily on the presence of food to

retard emptying and provide sufficient liquid for effective buoyancy.28

22 | P a g e

Fig 1.2: Schematic localization of an intragastric floating system and

a high density- system in the stomach.

Floating drug delivery system

Floating drug delivery systems (FDDS) or hydrodynamically

controlled systems are low-density systems that have sufficient buoyancy

to float over the gastric contents and remain buoyant in the stomach

without affecting the gastric emptying rate for a prolonged period of time.

While the system is floating on the gastric contents, the drug is released

slowly at the desired rate from the system after release of drug; the

residual system is emptied from the stomach. This results in an increased

Gastric retention time and a belter control of the fluctuations in plasma

drug concentration. However, besides a minimal gastric content needed to

allow the proper achievement of the buoyancy retention principle, a

23 | P a g e

minimal level of floating force (F) is also required to keep the dosage form

reliably buoyant on the surface of the meal . Many buoyant systems have

been developed based on granules, powders, capsules, tablets, laminated

films and hollow microspheres.29, 30

1.3.3 Drug candidates suitable for FDDS

Drugs that have narrow absorption window in GIT (e.g.L-DOPA,

paminobenzoic acid, furosemide, riboflavin).

Drugs those are locally active in the stomach (e.g. misoprostol,

antacids).

Drugs those are unstable in the intestinal or colonic environment

(e.g. captopril, ranitidine HCI, metronidazole).

Drugs that disturb normal colonic microbes (e.g. antibiotics used for

the eradication of Helicobacter pylori, such as tetracycline,

clarithromycin, amoxicillin).

Drugs that exhibit low solubility at high pH values (e.g. diazepam,

chlordiazepoxide)

The two approaches used in designing intragastric floating systems

are as follows

24 | P a g e

Hydrodynamically balanced systems

These are single-unit dosage forms, containing one or more gel-

forming hydrophilic polymers. Hydroxypropylmethylcellulose (HPMC) is the

most common used excipient, although hydroxyethylcellulose (HEC),

hydroxypropylcellulose (HPC), sodium carboxymethylcellulose (NaCMC),

agar, carrageenans or alginic acid are also used. The polymer is mixed

with drug and usually administered in a gelatin capsule. The capsule

rapidly dissolves in the gastric fluid, and hydration and swelling of the

surface polymers produces a floating mass. Drug release is controlled by

the formation of a hydrated boundary at the surface. Continuous erosion of

the surface allows water penetration to the inner layers, maintaining

surface hydration and buoyancy (Fig. 1.3). Incorporation of fatty excipient

gives low-density formulations and reduced penetration of water, reducing

the erosion.

25 | P a g e

Fig. 1.3:Hydrodynamically balanced system (HBS).

The main drawback is the passivity of the operation. It depends on

the air sealed in the dry mass centre following hydration of the gelatinous

surface layer and hence the characteristics and amount of polymer.

Effective drug delivery depends on die balance of drug loading and the

effect of polymer on its release profile. A variety of strategies has been

employed to improve efficacies of the floating HBS. Some investigators

developed bilayer formulations in which one layer conferred the buoyancy

and the other controlled the drug release.

Gas-generating systems

Floatability can also be achieved by generation of gas bubbles. CO:

can be generated in situ by incorporation of carbonates or bicarbonates,

which react with acid—either the natural gastric acid or co-formulated as

citric or tartaric acid. The optimal stoichiometric ratio of citric acid and

sodium bicarbonate for gas generation is reported to be 0.76:1. An

26 | P a g e

alternative is to incorporate a matrix with entrapped of liquid, which forms a

gas at body temperature. The approach has been used for single and

multiple unit systems. In single unit systems, such as capsules or tablets,

effervescent substances arc incorporated in the hydrophilic polymer and

CO; bubble are trapped in the swollen matrix .In vitro, the lag time before

the unit floats is <1 min and the buoyancy is prolonged for 8 to 10 h. Drug

and excipients can be formulated independently and the gas generating

unit can be incorporated into any of the layers Further refinements involve

coating the matrix with a polymer which is permeable to water, but not to

CO2.The main difficulty of such formulation is to find a good compromise

between elasticity, plasticity and permeability of the polymer. Multiple unit

systems avoid the "'all or nothing" emptying process. However, it is

essential that the units remain dispersed and suspended individually in the

gastric fluid and not agglomerate into a mass floating at the top of the

stomach.31,32

1.3.4 Classification of floating drug delivery systems (FDDS)

Floating drug delivery systems are classified depending on the use

of 2 formulation variables:

i. Effervescent and

ii. No effervescent systems.

I) Effervescent floating dosage forms

27 | P a g e

These arc matrix types of systems prepared with the help of

swellable polymers such as methylcellulose and chitosan and various

effervescent compounds, e.g. sodium bicarbonate, tartaric acid, and citric

acid. They arc formulated in such a way that when in contact with the

acidic gastric contents, CO2 is liberated and gets entrapped in swollen

hydrocolloids, which provides buoyancy to the dosage forms.

Fig.1.4:(A) Multiple-unit oral floating drug delivery system. (B)

Working principle of effervescent floating drug delivery system.

II) Non-Effervescent Floating Dosage Forms

Non-effervescent floating dosage forms use a gel forming or

swellable cellulose type of hydrocolloids, polysaccharides, and matrix-

forming polymers like polycarbonate, polyacrylate, polymethacrylate, and

polystyrene. The formulation method includes a simple approach of

thoroughly mixing the drug and the gel-forming hydrocolloid. After oral

administration this dosage form swells in contact with gastric fluids and

28 | P a g e

attains a bulk density of <1. The air entrapped within the swollen matrix

imparts buoyancy to the dosage form. The so formed swollen gel-like

structure acts as a reservoir and allows sustained release of drug through

the gelatinous mass. 33, 34, 35, 36

Applications of floating drug delivery systems

Floating drug delivery offers several applications for drugs having

poor bioavailability because of the narrow absorption window in the upper

part of the gastrointestinal tract. It retains the dosage form at the site of

absorption and thus enhances the bioavailability37. These are summarized

as follows.

1) Sustained drug delivery

HBS systems can remain in the stomach for long periods and hence

can release the drug over a prolonged period of time. The problem of short

gastric residence time encountered with an oral CR formulation hence can

be overcome with these systems. These systems have a bulk density of GI

as a result of which they can float on the gastric contents. These systems

are relatively large in size and passing from the pyloric opening is

prohibited. Recently sustained release floating capsules of nicardipine

hydrochloride were developed and were evaluated in vivo.38,39

2) Site-Specific Drug Delivery

29 | P a g e

These systems are particularly advantageous for drugs that are

specifically absorbed from stomach or the proximal part of the small

intestine, e.g, riboflavin and furosemide. Furosemide is primarily absorbed

from the stomach followed by the duodenum. It has been reported that a

monolithic floating dosage form with prolonged gastric residence time was

developed and the bioavailability was increased. AUG obtained with the

floating tablets was approximately 1.8 times those of conventional

furosemide tablets. A bilayer-floating capsule was developed for local

delivery of misoprostol, which is a synthetic analogue of prostaglandin El

used as a protectant of gastric ulcers caused by administration of NSAIDs.

By targeting slow delivery of misoprostol to the stomach, desired

therapeutic levels could be achieved and drug waste could be reduced.40

3) Absorption enhancement

Drugs that have poor bioavailability because of site specific

absorption from the upper part of the gastrointestinal tract are potential

candidates to be formulated as floating delivery systems, thereby

maximizing their absorption.41

4) Enhanced bioavailability:

The bioavailability of riboflavin CR-GRDF is significantly enhanced in

comparison tothe administration of non GRDF CR polymeric formulations.

There are severaldifferent processes, related to absorption and transit of

30 | P a g e

the drug in the gastrointestinaltract, that act concomitantly to influence the

magnitude of drug absorption.42, 43

5) Minimized adverse activity at the colon:

Retention of the drug in the HBS systems at the stomach minimizes

the amount ofdrug that reached the colon. Thus, undesirable activities of

the drug in colon may be prevented. This pharmacodynamic aspect

provides the rationale for GRDFformulation for betalactam antibiotics that

are absorbed only from the small intestine,and whose presence in the

colon leads to the development of microorganism‘sresistance.44,45

6) Reduced fluctuations of drug concentration:

Continuous input of the drug following CRGRDF administration

produces blood drugconcentrations within a narrower range compared to

the immediate release dosageforms. Thus, fluctuations in drug effects are

minimized and concentration dependentadverse effects that are

associated with peak concentrations can be prevented. Thisfeature is of

special importance for drugs with a narrow therapeutic index.46, 47

1.3.6 Advantages of Floating Drug Delivery System

Floating dosage systems are important technological drug delivery

systems with gastric retentive behavior and offer several advantages in

drug delivery. These advantages Include,

31 | P a g e

1. Improved drug absorption, because of increased GRT and more

time spent by the dosage form at its absorption site.

2. Controlled delivery of drugs.

3. Delivery of drugs for local action in the stomach.

4. Minimizing the mucosal irritation due to drugs, by drug releasing

slowly at controlled rate.

5. Acidic substances like aspirin cause irritation on the stomach wall

when come in contact with it. Hence HBS formulation may be useful

for the administration of aspirin and other similar drugs.

6. Administration of prolongs release floating dosage forms, tablet or

capsules, will result in dissolution of the drug in the gastric fluid.

They dissolve in the gastric fluid would be available for absorption in

the small intestine after emptying of the stomach contents. It is

therefore expected that a drug will be fully absorbed from floating

dosage forms if it remains in the solution form even at the alkaline

pH of the intestine.

7. When there is a vigorous intestinal movement and a short transit

time as might occur in certain type of diarrhea, poor absorption is

expected. Under such circumstances it may be advantageous to

keep the drug in floating condition in stomach to get a relatively

better response.

32 | P a g e

8. Treatment of gastrointestinal disorders such as gastro esophageal

reflux.

9. Simple and conventional equipment for manufacture.

10. Ease of administration and better patient compliance.

11. Site-specific drug delivery. 48, 49, 50

Table1.2: Marketed Products of FDDS51

Sr.

No.

Dosage Form Drugs Brand Name Company,

Country

1) Floating

Controlled

Release Capsule

Levodopa,

Benserazide

MODAPARR Roche

Products,

USA

2) Effervescent

Floating Liquid

alginate

Preparation

Aluminium

hydroxide.

Magnesium

carbonate

LIQUID

GAVISONRR

Glaxo Smith

Kline, INDIA

3) Floating Liquid

alginate

Preparation

Floating

Liquid

alginate

Preparation

TOPALKANR Pierre Fabre

Drug,

FRANCE

4) Colloidal gel

forming FDDS

Ferrous sulphate CONVIRONR Ranbaxy,

INDIA

5) Gas-generating

floating Tablets

Ciprofloxacin FRAN ODR Ranbaxy,

INDIA

Plant Profile

33 | P a g e

1.4 PLANT PROFILE

Azadirachta indica A.Juss (Meliaceae)

Fig.1.5: Leaves of A. indica

Taxonomical Hierarchy:

Kingdom

Division

Order

Family

Genus

Species

:

:

:

:

:

:

Plantae

Magnoliophyta

Sapindalcs

Meliaceae

Azadirachta

A. indica

Plant Profile

34 | P a g e

Part used : Leaves

Common Names

Synonym

Sanskrit

Hindi

Marathi

:

:

:

:

:

:

MeliaAzadirachta

Arishta

Nim

Limba / Balantnimba

Geographical distribution:

It is distributed all over in India and also cultivated all over the world.

Botanical description:

Leaves: Leaves are alternate, imparipinnate; leaflets are subopposite

serrate, very unequal at base.

Flowers: Hermaphrodite, in axillary panicles. Calyx is 5- lobed. Petals 5

in number, free, imbricate. Staminal tube is little shorter than the petals,

cylindric, widening above 9-10 lobed at the apex, the lobes truncate,

again slightly toothed; anthers within the tube opposite to and shorter

than the lobes. Ovary is 3-celled; style is elongate, slender, stigma is

shortly cylindric, 3-lobed; ovules are 2 in each cell, collateral.52

Fruits: 1-seeded drupe, endocarp is woody.

Plant Profile

35 | P a g e

Seed: Ellipsoid; cordate atthebase; radical is superior.

Ayurvedic Description: 52

Rasa - tikta, kasaya

Guna - laghu

Veerya - sheeta

Vipak - katu

1.4.1Chemical constituents

Number of chemicals isolated from leaves like limoniods and cyclic

triand tetrasulphides. It also contains azadirachtin, meliantriol and salanin.

The leaves contain nimbinene, 6-desacetyhiimbinene, nimbandiol,

nimbolide, quercetinand β- sitosterol.

Active principles from leaves of A. indica

Nimbidin, Azadirachtin and geduin arereported to possess

antimalarial activity.53

1.4.2 Medicinal Uses

Young leaves are astringent; used in leprosy, skin

diseases, rheumatismleucoderma, piles and reduces inflammation.

Plant Profile

36 | P a g e

Young branches are anthelmintic, good for cough, asthma, piles, tumors

and urinary discharge. Unripe fruit used in tumors, piles and toothache.

Other uses

i. Poultice of leaves for swollen glands, bruises and sprains.

ii. Fresh leaf-tea used for malaria

iii. Tree and root barks have been used for malaria, jaundice, and for

intestinal parasitism.

iv. Edible pulp of the fruit used for hemorrhoids.

Ayurvedic uses

i. Leaf- leprosy, intestinal parasites, eye problems, skin and gastric

ulcers

ii. Bark - pain and fever.

iii. Flower-bile suppression, intestinal worms and phlegm

iv. Fruit - piles, intestinal worms, urinary disorder, nose

bleeding,phlegm, eye problem, diabetes, wounds and leprosy.

v. Twig- Cough, Asthma, plies, intestinal worms, spermatorrhoea, urinary

disorders, diabetes.

vi. Gum - ringworms, scabies, wounds and ulcers.

vii. Seed pulp and oil- leprosy and intestinal worms.54

Plant Profile

37 | P a g e

Folk uses

1. Young tender branches are chewed for tooth brushing use.

2. Leafs oil .is used as „a local antiseptic and insecticide.

3. Neem oil may be useful for gingivitis.

4. In the rural areas, burning of leaves and seed used as mosquito

repellant.

5. Neem oil has been shown to possess some spermicidal and

contraceptive properties when used intravaginally.

6. Use of neem oil animals showed lowering of glucose.

Commercial uses

i. Neem extracts used in the manufactures of toothpaste for its

antibacterial properties.

ii. Fresh seed oil has a strong garlic odor and is an ingredient for insect

sprays.55

Literature Review

38 | P a g e

2. REVIEW OF LITERATURE

1. Deshpande et al (2014) have studied neem leaves with an objective

to evaluate antimalarial activity of nimbidin, azadirachtin and

gedunin present in neem leaves. Antimalarial activity was tested in

mice infected with Plasmodium berghie using mice survival study. It

was demonstrated that neem leaf constituents possess antimalarial

activity.56

2. Akin Osanaiye et al (2013) have studied neem leaves with an

objective to isolate an active ingredient irodin A from Neem leaves

and to evaluate it for antimalarial activity. The antimalarial activity of

irodine A was tested by Peters 4 days test. The toxicity against

causative strains of malaria was found significant. The components

effective against both chloroquine-resistant and sensitive strains of

malarial parasite found in alcoholic extracts of leaves and seeds.57

3. Siddique et al (2004) have studied combination of neem and

artemisinin to explore the antimalarial potential of neem in

combination with artimisinin derivative. The mean percent

parasitemia and percent suppression of parasitemia was tested in

infected mice. It was demonstrated that combination of neem and

Literature Review

39 | P a g e

artemisinin is more effective. But the study also demonstrated that

half life of neem is short. 58

4. Dhiman et al (2012) have studied psyllium husk with an objective to

evaluate its potential as carrier to control the drug release. Various in

vitro parameters like buoyancy lag time, % drug release and swelling

index were studied. It was observed that psyllium husk possess

good swelling and gelling properties.Psyllium husk has high affinity

for water (swelling index is about 20 times in volume).59

5. Raval JA et al(2007) investigated the effects of formulation and

processing parameters on the release of ranitidine HCl from the

prepared floating matrix tablets. The release rate was modified by

varying the type of matrix tablet polymers, the tablet geometry

(radius) and addition of water soluble or water insoluble diluents.

The highly porous co-polymer [poly (styrene-divinyl benzene)]

provided a low density and, thus, excellent in vitro floating behavior

of the tablets at a concentration of 15%w/w. It was established that

floating behavior of low density drug delivery system could be

successfully combined with accurate control and prolongation of the

drug release pattern.60

Literature Review

40 | P a g e

6. Rao M et al (2007) performed the evaluation of effervescent floating

matrix tablet formulation of salbutamol sulfate using full factorial

design. An increase in the concentration and viscosity grade of the

polymer resulted in a decrease in the release rate, but it was found

that at a higher concentration of HPMC, the viscosity grade did not

significantly affect the drug release. Optimized effervescent floating

tablets of salbutamol sulfate were successfully prepared and a good

correlation was observed between predicted and actual values of the

dependent variables chosen for the study. Viscosity grade of HPMC

did not significantly impact the floatability of the dosage form. It was

concluded that a combination of HPMC, stearic acid and sodium

bicarbonate can be used to increase the gastric residence time of the

dosage form up to 12 hrs.61

7. Jadhav KR et al (2007)carried out the development and in-vitro

evaluation of an oral floating matrix tablet formulation of diltiazem

hydrochloride. The results of factorial design indicated that a high level

of both Methocel K100M CR (X1) and Compritol 888 ATO (X2) favors

the preparation of floating controlled release of DTZ tablets. The

linear regression analysis and model fitting showed that all these

formulations followed Korsmeyer and Peppas model, which had a

higher value of correlation coefficient (r).While tablet hardness had little

Literature Review

41 | P a g e

or no effect on the release kinetics and was found to be a

determining factor with regards to the buoyancy of the tablets.62

8. Patel DM et al (2007)developed a floating tablet of carbamazepine

using melt granulation technique. Bees wax was used as a

hydrophobic meltable material and HPMC, sodium bicarbonate, and

ethyl cellulose were used as matrix agent, gas generating agent and

floating enhancers respectively. Based on the results it was

concluded that the addition of matrixing polymer, HPMC K4 M,

and gas generating agent, sodium bicarbonate was essential to

achieve in vitro buoyancy. A systematic study using a simplex

lattice design revealed that the amount of HPMC K4 M, sodium

bicarbonate and EC had a significant effect on Flag, t50 and t80.

Thus, by selecting a proper optimization technique, proper

balance of formulation variables can be achieved rapidly with

minimum efforts to produce required in vitrobuoyancy and drug

dissolution profile.63

9. Chattopadhyay et al., (2004) reported antiulcer activity of neem leaf

extract. The extract of neem dose-dependently inhibits gastric

lesions induced by restraint–cold stress, ethanol and indomethacin.

In stress ulcer model, neem extract is more effective than ranitidine

Literature Review

42 | P a g e

but less effective than omeprazole. Mechanism of antiulcer effect of

neem (Azadirachtaindica) leaf extract is due to its action on H+-K+-

ATPase. 64

10. Khosla et al(2000) reported antinociceptive activity of

Azadirachtaindica (neem) in rats Tail flick reaction time was

significantly increased in rats both with leaf extract and seed oil.

Naloxone pretreatment partially reversed the antinociceptive action

of both leaf extract and seed oil. GAA induced writhing was reduced

with both neemextact and seed oil. Neemextract was more potent

than seed oil. 65

11. Badam et al. (1999) evaluated in vitro antiviral activity of neem

(Azadirachtaindica. A. Juss) leaf extract against group B

coxsackieviruses. Antiviral activity of methanolic extract fraction of

leaves of neem (Azadirachtaindica A. Juss) (NCL-11) was studied

for its antiviral activity and mechanism of action against Coxsackie B

group of viruses.66

The literature survey revealed that neem leaf extract has antimalarial

activity and combination of neem extract and artemether is more effective.

But neem has major disadvantage of low biological half life. There is no

Literature Review

43 | P a g e

work done so far on controlled floating drug delivery system of neem

extract to improve its half life.

Need for the Study

44 | P a g e

3. NEED FOR THE STUDY

Resistance to first line drugs to treat malaria is the prime problem in

controlling it.

Artemisinin(ARM)based combinations have several distinct

advantages in that they produce rapid clinical and parasitological

cure, there is as yet no documented parasite resistance.

Artemisinin derivatives are eliminated rapidly and have short half life.

A general principle in ACT is to have a partner drug with a longer

half-life than artemisinin, so that the residual parasites not cleared by

ARM are eliminated by the partner drug. However concentrations of

the partner drug below threshold levels are issues to be dealt with in

the long-term use of these ACTs.

In this context, studies have shown that A. indica has potential

antimalarial activity anda new combination therapy with ARM and A.

Indica can be promising.

Both the drugs have short half-lives, faster elimination.

In present study an attempt is being made to prepare floating

delivery of A. indicawhich will maintain the concentrations of drug in

blood throughout treatment period and will reduce the dosing

Need for the Study

45 | P a g e

frequency. Thus it could reduce the chances of developing drug

resistance problems.

Objectives of Study

46 | P a g e

4. OBJECTIVES OF STUDY

The objective of study is to formulate floating drug delivery system

so as,

1. To study drug-excipient interaction and preformulation parameters

2. To prepare and optimize floating tablet of neem extract.

3. To study release pattern of drug form the formulation by fitting to

various dissolution models.

4. To study in vivo gastro retention behavior of optimized formulation.

5. To study stability of optimum formulation

6. To evaluate antimalarial activity of neem extract in combination with

artemether.

Plan of Work

47 | P a g e

5. PLAN OF WORK

5.1 Collection, procurement and authentication of plant:

5.2 Pharmacognostic and phytochemical studies:

5.2.1 Ash Value

5.2.2 Extractive value

5.2.3 Foreign organic matter and moisture content

5.2.4 Preliminary phytochemical tests

5.3 Preformulation studies:

5.3.1 UV spectrophotometric determination

5.3.2 FTIR analysis

5.3.3 Evaluation of powder blend

5.4 Pre-compression parameters:

5.4.1 Bulk density and tapped density

5.4.2 Angle of repose

5.4.3 Compressibility

5.4.4 Hausners ratio

Plan of Work

48 | P a g e

5.5 Preparation of floating tablet containing neem extract

5.6 Evaluation of formulation:

5.6.1 Hardness and Friability

5.6.2 % drug content

5.6.3 Swelling index

5.6.4 In-vitro dissolution studies

5.7 Optimization of tablet and mathematical modelingof

dissolution data

5.8 In Vivo gastro retention study –X ray imaging

5.9 In vivo antimalarial activity of extract in combination with

artemether

5.9.1 Mean Survival Time

5.9.2 % mean parasitemia

Polymer and Excipient Profile

49 | P a g e

6. POLYMERPROFILE

6.1 Hydroxy propyl methyl cellulose

Nonproprietary Names

BP: Hypromellose

JP: Hydroxypropylmethylcellulose

PhEur: Hypromellosum,

USP: Hypromellose

Synonyms

Hydroxypropyl methyl ether

Hydroxyl propyl methylcellulose

HPMC

Methocel,

Methyl hydroxyl propylcellous

Chemical Name

Cellulose, 2-hydroxypropyl methyl ether

Molecular Weight

Approximately 10,000-1,500.000.

Description

It is tasteless, odorless and white granular

powder.


50 | P a g e

Structural formula

Functional category

It is used as

Film forming agent

Release controlling polymer

Protective colloid

Viscosity modifying agent

Binder in tablet dosage form.

Applications in pharmaceutical formulation or technology

Hypromellose is widely used in almost all dosage forms. At low

concentration, It is used as a binder in both wet and dry-granulation

processes. While at high concentration, high-viscosity grade HPMC is


51 | P a g e

used to retard the release of drugs from a matrix. At moderate

concentration HPMC is used for film forming solutions in film coating of

tablets. In the preparation of aqueous filmcoating solutions, lower-viscosity

grades are used. While in organic solutions higher-viscosity grades are

used. It is also used as a suspending and thickening. It forms clear solution

with few undispersed fibres and so is preferred in formulating ophthalmic

preparations. It is also used as thickening agent in artificial tear solutions.

It is widely used as suspending agent in formulating liquid dosage

forms. It is used as stabilizing agent in gels and ointments. Being

hydrophilic colloid it prevent agglomeration or co-acervation of particles

and inhibits sedimentation of particles in to hard cake.

It is also used as adhesive in bandages in manufacturing of

capsules. It acts as wetting agent for hard contact lenses. It has many

applications in cosmetics and food products too.

General properties

pH1% w/w aqueous solution : 5.5-8.0

Density (bulk) : 0.341 g/ml

Tap Density : 0.557 g/ml

True Density : 1.326 g/ml

Melting point : Chars at 225-2300C.


52 | P a g e

Glass transition temperature: 170-1800C.

Solubility :

Soluble in water, forming a viscous colloidal solution.

Insoluble in chloroform, ether and ethanol (95%)

Soluble in,

Mixtures of ethanol and dichloromethane,

Mixtures of methanol and dichloromethane

Mixtures of water and alcohol

Certain grades are soluble in aqueous acetone solutions, mixtures of

dichloromethane and propan-2-ol, and other organic solvents.

Specific gravity : 1.27

Viscosity :

HPMC is available in wide variety of viscosity grades. Solutions prepared

in organic solvents are more viscous as compared to aqueous solutions.

Viscosity of solutions increases with concentration. In aqueous solution, it

is first dispersed and allowed to hydrate in about 30% of required amount

of water and remaining water is added with vigorous shaking at 80- 90°C

and finally cold water should be added to produce required volume.67, 68, 69,

70


53 | P a g e

Table 6.1: Typical viscosity values for 2% (w/v) aqueous solutions of

Methocel. Viscosities measured at 20°C.

Methocel grade Nominal Viscosity (mPas)

K100LVP 100 80-120

K4M 4 000 3 000-5 600

KI5MP 15 000 12 000-21 000

K100MP 100 000 80 000-120 000

E4MP 4 000 3 500-5 600

E10MPCR 10 000 8 000-13 000

E3 PREMLV — 2.4-3.6

E5 PREMLV — 4-6

B6 PREM.LV — 5-7

E15PREM.LV — 12-18


54 | P a g e

7.1 Psyllium husk71, 72, 73

Common Names

Isabgol, Ispaghula,Sand Plantain, Psyllium Seed, Indian plantago, spogel,

psyllium

Fig 7.1 Psyllium seeds

Source

It is white fibrous material obtained from coating of Psyllium seed. It

is a mucilage coating around the seeds. It is used as dietary fiber. From

the several species, Psyllium seed husk of plantago ovate is considered as

of best quality with highest content of fiber. Psyllium husk is made up of

epidermis and adjacent layers removed from the dried seeds.

It has many important Pharmaceuticals, Nutraceuticals and

Medicinal application. It is generally used as a laxative.


55 | P a g e

Properties of Psyllium husk

It is white, translucent, thin, boat shaped seed. It is odorless with bland,

mucilaginoustaste.

It has high affinity to water

It has no habitforming tendency

It produces gel by absorbing water and lubricates bowel

It is chemically inert

It is not digested or absorbed by the body in any case

Psyllium husk powder

The Psyllium Husk and powder is a product consisting of the

epidermis and adjacent layers of the dried seeds.

Psyllium powder is a pulverized form of the Husk.

Appearance:

Color: Light Brown to dark brown

Composition

Psyllium fiber can be fractionated into three components


56 | P a g e

A highly (greater than 80%) fermentable component totaling 15–20%

of Psyllium weight

An unfermentable (less than 20%) component comprising 10–15%

of Psyllium weight

A poorly (30%) fermentable bulk-forming component constituting 55–

60% of Psyllium weight

Psyllium is composed of Xylose (59%) and arabinose (22.3%), uronic

acid content (6.1%), glucose (3.5%), rhamnose (3.0%), galactose (3.7%),

mannose (1.6%) and also scanty ribose content (0.01%). The gel forming

ability of Psyllium is because of unfermentable components of Psyllium. It

has ability to absorb 2-3 gm of water per gram of powder.

Psyllium husk powder – Technical specification

Psyllium Husk is available in various grades on the basis of purity

and mesh size. Psyllium Husks and Psyllium Seeds are graded according

to the purity of the material.

\


57 | P a g e

Table 7.1: Technical specification of Psyllium husk powder

Quality 99% Pure 98% Pure 95% Pure 85% Pure

Mucilloid content 99% Min. 98% Min. 95% Min. 85% Min.

Light extraneous

matter

0.5% 1.5% 4.0% 14.0%

Heavy extraneous

matter

0.5% 0.5% 1.0% 1.0%

Swell volume 60ml/g 60ml/g 45ml/g 40ml/g

Moisture vontent Not more than 10.0%

Total ash Not more than 4.0%

Acid Insoluble ash Not more than 0.75%

Sieve 30 mesh to 100 mesh as per specific

requirement


58 | P a g e

7.2 Sodium bicarbonate

Empirical formula and

NaHCO3

Molecular weight: 84.0 gm/mole

Action and Use

Antacid, buffer solution, used in treatment of electrolyte deficiency.

Appearance

White and crystalline powder.

Solubility

It is soluble in water, practically insoluble in ethanol. When heated in

the dry state or in solution, it gradually changes into sodium carbonate74, 75

7.3 Magnesium stearate

Action and use

Excipient, lubricant.

Description

Magnesium stearate is a mixture of magnesium salts of different

fatty acids consisting mainly of stearic (octadecanoic) acid [(C17H35COO) 2

Mg] and palmitic (hexadecanoic)[(C15H31COO) 2 Mg] acid with minor


59 | P a g e

proportions of other fatly acids. It contains not less than 4.0% and not more

than 5.0 % of Mg, calculated with reference to the dried substance. Fatty

acid fraction contains not less than 40.0 % of stearic acid and the sum of

stearic acid and palmitic acid is not less than 90.0%.

Characters

A white or almost white, very fine, light powder, greasy to the touch,

practicallyinsoluble in water and in ethanol75, 76


60 | P a g e

8. MATERIALS AND METHODS

8.1 Plant material

8.1.1 Collection and procurement

The Leaves of A.indica was collected as and when required from

Ahmednagar district (M.S). The leaves of plant were dried under shade

away from direct sunlight. The dried parts were cleaned and coarsely

powdered in grinder and powder material was passed through 120 mesh to

remove fine powders and coarse powder was used for extraction.

8.1.2 Authentication

The plant was authenticated by Dr. K.J. Salunke Head and

Associate professor Botany Department of Padmashri Vikhe Patil College,

Pravaranagar( M.S.) through comparing morphological features.

8.2 Pharmacognostic studies

8.2.1 Macroscopy

Organoleptic characters, extra feature and macroscopical details of

all parts of plant were carried out.

8.2.2 Evaluation of physical constants77

8.2.2.1 Determination of foreign organic matter


61 | P a g e

Five grams of air dried coarsely powdered drug was spread in a thin

layer. The sample was inspected with the unaided eye or with the use of

6X lens. The foreign organic matter was separated manually as completely

as possible. Sample was weighed and percentage of foreign organic

matter was determined from the weight of the drug taken.

8.2.2.2 Determination of moisture content:

Accurately weighed glass Stoppered shallow weighing bottle, and

was dried. 2 gm of sample was transferred to the bottle and covered, the

weight was taken and sample was distributed evenly and poured to a

depth not exceeding 10 mm. Then loaded bottle was kept in oven and

stopper was removed. The sample was dried to constant weight. After

drying it was collected to room temperature in a desiccator. Weighed and

calculated loss on drying in terms of percent w/w.

8.2.2.3 Ash value

Ash value is used to determine quality and purity of crude drug. Ash

value contains inorganic radicals like phosphates, carbonates and silicates

of sodium, potassium, magnesium and calcium etc. sometimes inorganic

variables like calcium oxalate, silica, carbonate content of the crude drug


62 | P a g e

affects 'total ash value'. Such variables are then removed by treating with

acid and then acid insoluble ash value is determined.

Determination of total ash

Accurately weighed 2 gm of the air-dried crude drug was taken in a

tarred silica dish and incinerated at a temperature not exceeding 450°C

until free from carbon, cooled in a desiccator and weight was taken. The

process was repeated till constant weight was obtained. The percentage of

ash was calculated with reference to air-dried drug.

Water soluble ash

The ash, obtained as per the method described above boiled for 5

minutes with 25 ml of water, filtered, and collected the insoluble matter in a

Gooch crucible, washed with hot water and ignited for 15 minutes at a

temperature not exceeding 450°C and weight was taken. The percentage

of water-soluble ash was calculated with reference to air-dried drug.

Acid insoluble ash

The ash obtained as per method described above and boiled with 25

ml of 2M hydrochloric acid for 5 minutes, filtered, and collected the


63 | P a g e

insoluble matter in a Gooch crucible or on an ash less filter paper, washed

with hot water, ignited, and cooled in a desiccator and weighed. The

percentage of acid-insoluble ash was calculated with reference to the air-

dried drug.

8.2.2.4 Extractive values

Different extractive values like alcohol soluble extractive, water

soluble extractive values were performed by standard method.

Determination of water-soluble extractive value

Five gm of air dried coarsely powdered drug was macerated with

100 ml of chloroform water in a closed flask for 24 hours and it was shaken

frequently during first 6 hours and allowed to stand for 18 hours. Then it

was filtered, 25 ml of the filtrate was evaporated in a fiat shallow dish and

dried at 105°C and weighed. Percentage of water-soluble extractive value

was calculated with reference to air-dried drugs.

Determination of alcohol-soluble extractive value

Five gm of air-dried coarsely powdered drug was macerated with

100 ml of ethanol of specified strength in a closed flask for 24 hours and it


64 | P a g e

was shaken frequently during first 6 hours and allowed to stand for 18

hours. Then it was filtered, during filtration precaution was taken against

loss of ethanol, 25 ml of the filtrate was evaporated in a flat shallow dish

and dried at 105°C and weighed. Percentage of ethanol soluble extractive

value was calculated with reference to air-dried drugs.77.

8.3 Extraction

The leaves of A. indica were collected and shade dried and then

pulverized in grinder. About 100 gm powdered leaves utilized for extraction

was passed through 120-mesh sieve to remove fine powder and coarse

powder was used for extraction.

Solvent used - Petroleum ether and ethanol.

Technique - Continuous hot extraction method.

The powdered Neem leaves were extracted with Petroleum ether for

removal of coloring matter by defattation process using continuous soxhlet

extraction method. The completion of extraction was indicated by taking

sample out of siphon tube on TLC plate and placing it in iodine chamber.

Absence of colored spot on plate indicates complete extraction. The

defatted neem leaves were refluxed with water & alcohol (1:1) for 3 hrs to

get hydroalcoholic extract. The extraction temperature was maintained at

503C with constant shaking. The extract was filtered and concentrated to


65 | P a g e

get a thick paste & after it freeze dried to get powder. The extract was

stored in airtight container.78

Fig. 8.1: Extraction of neem leaf

8.4Preliminary phytochemical evaluation of extracts

8.4.1 Preliminary phytochemical test80

Test for Carbohydrates

i) Molish's test

2-3 ml of extract, add few drops of α- naphthol solution in alcohol.

Shake and add concentrated sulphuric acid from sides of the test tube.


66 | P a g e

Violet ring is formed at the junction of two liquids which shows presence of

carbohydrates.

Test for reducing sugar

Fehling's test

Five ml of extract solution was mixed with 5 ml Fehling's solution

(equal mixture of Fehling's solution A and B) and boiled. Development of

brick red precipitate indicates the presence of reducing sugars.

Benedict's test

Mix equal volume of Benedict's regent and extract solution in test

tube. Heat in a boiling water bath for 5 min. Solution appears green, yellow

or red depending on amount of reducing sugar present.

Test for monosaccharides

Barfoed's test

Mix equal volume of Barfoed's reagent and extract solution. Heat for

1-2 minute in boiling water bath and cool. Development of red precipitate

indicates presence of monosaccharides.

Test for Proteins

Biuret test

The extract was treated with 1 ml of 10 percent sodium hydroxide

solution and heated. A drop of 0.8 percent copper sulphate solution was


67 | P a g e

added to the above mixture. The formation of purple violet color indicates

the presence of proteins,

Millon's test:

The extract was treated with 2 ml of Millon's reagent. Formation of

white precipitate indicates the presence of proteins and amino acids.

Test for Amino acids

Ninhydrin test

The extract was treated with Ninhydrin reagent at pH range of 4-8

and boiled. Formation of purple color indicates the presence of amino

acids.

Test for Steroids

Liebermann-Burchard test

10 mg extract was dissolved in 1 ml of chloroform and 1 ml of acetic

anhydride was added following the addition of 2 ml of concentrated

sulphuric acid from the side of the test tube. Formation of reddish violet

color at the junction indicates the presence of steroids.

Liebermann's test:

To 2 ml of the extract a few ml of acetic anhydride was added and

gentle heated. The content of the test tube were cooled and 2 ml of


68 | P a g e

concentrated sulphuric acid was added from the side of the test tube.

Development of blue color gave the evidence for presence of steroids.

Salkowski test:

One ml of concentrated sulphuric acid was added to 10 mg of extract

dissolved in 1 ml of chloroform. A reddish brown color exhibited by

chloroform layer and green fluorescence by the acid layer suggests the

presence of steroids.

Test for Glycosides

Anthraquinone glycosides:

Borntrager'stest:

To 3 ml extract add dilute sulphuric acid, boil and filter. To the cold

filtrate, add equal volume benzene or chloroform shake well. Separate

organic solvent. Add ammonia, the ammonical layer turns pink or red

indicates the presence of anthroquinone glycoside.

Cardiac glycoside:

Keller-killani test:

To 2 ml of extract, glacial acetic acid, one drop 5 % Ferric chloride

and concentrated sulphuric acid was added. Presence of cardiac

glycosides is indicated by formation of reddish brown color at junction of

the two liquid layers and upper layer appeared bluish green.


69 | P a g e

Test for saponins

Foam formation test

One ml solution of the extract was diluted with 20 ml distilled water

and shaken in a graduated cylinder for 15 minutes. The development of

stable foam indicates the presence of saponins.

Test for flavonoids

Shinoda test

To the extract 5 ml (95%) ethanol and few drops of con. HCI and 0.5

g of magnesium turnings was added gives pink color indicates presence of

flavonoids.

Lead acetate test

Few drops of 10 percent lead acetate are added to the extract.

Development of yellow colored precipitate confirms the presence of

flavonoids.

Test for Alkaloids

Dragendroff's test

0.1 ml dilute hydrochloric acid and 0.1 ml Dragendroff's reagent was

added in 2 ml of extracts in test tube. Formation of orange brown

precipitate indicates the presence of alkaloids.

Mayer's test


70 | P a g e

Two ml of extract was taken in a test tube. 0.2 ml of dilute

hydrochloric acid and 0.1 ml of Mayer's reagent were added. Formation of

yellowish buff precipitate indicates the presence of alkaloids.

Hager’s test

Two ml of extract was allowed to react with 0.2 ml dilute hydrochloric

acid and 0.1 ml of Hager‘s reagent. Formation of yellowish precipitate

indicates the presence of alkaloids.

Wagner's test

Two ml of extract was treated with 0.2 ml dilute hydrochloric acid

and 0.1 ml of Wagner's reagent. Formation of reddish brown precipitate

indicates the presence of alkaloids.

Test for tannins and phenolic compounds

5% Ferric chloride test

Five ml of extract solution was allowed to react with 1 ml of 5 percent

ferric chloride solution. Greenish black coloration indicates the presence of

tannins.

Potassium dichromate test

2-3 ml of extract solution, mix with 2 ml of Potassium dichromate.

The formation of red precipitate indicates presence of tannins.

Bromine water test


71 | P a g e

Two ml of extract solution mix with 2 ml of bromine water.

Discoloration of bromine water indicates presence of tannins

Dilute nitric acid test

Two ml ofextract solution was allowed to react with few drops of

dilute nitric acid solution. Formation of reddish to yellow color indicates the

presence of tannins.

Test for vitamin-C

1 ml of extract solution with 2 ml of water and add 1 drop of freshly

prepared 5 % w/v solution of sodium nitroprusside and 1 ml of dilute

sodium hydroxide solution. Add 0.6 ml of HCl, stir it. The yellow color turns

to blue indicate presence of vitamin- C.

8.4.2 Thin layer chromatography

Steps involved in performing TLC of extracts

Preparation of TLC plate

The slurry of adsorbent media (silica gel-G) in distilled water was

prepared and the slurry was poured on the TLC glass plates to obtain a

thin layer.

Activation of TLC plate: Plate was heated in oven for 30 min. at

105°C for activating TLC plate.


72 | P a g e

Sample application: The capillary was dipped into the solution to

be examined and was applied the by touching the sample capillary

to the thin layer plate at a point about 2 cm from the bottom. Air-

dried the spot.

Chamber saturation: The glass chamber for TLC should be

saturated with mobile phase. Mobile phase was poured into the

chamber and capped with lid. Allowed saturating about 30 min.

Chromatogram development: After the saturation of chamber and

spotting of samples on plate, it was kept in chamber. The solvent

level in the bottom of the chamber must not be above the spot that

was applied to the plate, as the spotted

Material will dissolve in the pool of solvent instead of undergoing

chromatography. The solvent was allowed to run around 10-15 cm

on the silica plate

Visualization: Plates were removed and were examined visually,

under UV or suitable visualizing agent (Vanillin-sulphuric acid,

Methanolic ferric chloride solution) after that Rf was calculated by

following formula.


73 | P a g e

Detection of Steroids

Solvent system used: Toluene: Ethyl acetate (9:1)

Spray reagent: Vanillin-Sulphuric acid reagent

Detection of Alkaloids

Solvent system used: Toluene: Ethyl acetate: Diethylamine (80:20:10)

Spray reagent: Sulphuric acid reagent (1% solution of concentrated

sulphuric acid inethanol.)

Detection of Flavonoids

Solvent system used:Ethyl acetate: Formic acid: Acetic acid: Water

(100:11:11:26)

Spray reagent:Anisaldehyde-Sulphuric acid reagent

Detection of Saponins

Solvent system used: Ethyl acetate: Formic acid: Acetic acid: Water

(100:11:11:26) Spray reagent: Anisaldehyde-Sulphuric acid reagent

Detection of Tannins

Solvent system used: Ethyl acetate: Formic acid: Acetic acid: Water

(100:11:11:26) Spray reagent: Ferric Chloride reagent.


74 | P a g e

8.5 Characterization of neem leaf extract

Calibration curve

25.0mg of the drug was dissolved in 25 ml 0.1 N HCI. 2.5ml of this

solution was taken and diluted up to 25 ml with 0.1 N HCI. This solution

(l00ug/ml) was used as the stock solution for the preparation of further

dilutions. Dilutions were made using 0.1 N HCI. The calibration curve was

obtained by recording the absorbance on a UV spectrophotometer at

λmax280 nm.

8.6. Drug excipient compatibility study

Preformulation studies

Preformulation is defined as phase of research and development

process where physical, chemical and mechanical properties of a new

drug substance are characterized alone and when combined with

excipients, in order to develop stable, safe and effective dosage form A

thorough understanding of physicochemical properties may ultimately

provide a rationale for formulation design, or support the need for

molecular modification or merely confirm that there are no significant

barriers to the compounds development. Hence, preformulation studies on

the obtained sample of drug for identification and compatibility studies

were performed.


75 | P a g e

Fourier Transform Infrared (FTIR) Spectroscopy studies and HPTLC

were used for the evaluation of physicochemical compatibility and

interactions, which helps in the prediction of interaction of the drug with

polymers, diluents and lubricants used in case tablet formulations. Positive

interactions sometimes have a beneficial effect as far as desired release

parameters are concerned.

8.6.1 Fourier Transform Infrared Spectroscopy (FTIR) analysis

The IR spectra of Neem leaf extract and all the tablet formulations

were recorded using Fourier Transform Infra-Red spectrophotometer

(FTIR) (Jasco FT/IR-4100) with diffuse reflectance principle. Sample

preparation involved mixing the samples with potassium bromide (KBr),

triturating in glass mortar and finally placing in the sample holder. The

spectrum was scanned over a frequency range 450-400 cm-1.81

8.7 Preparation of floating tablet formulation

8.7.1 Instruments and materials used

Instruments


76 | P a g e

Table 8.1: List of Instruments/equipments

Instrument' Equipment Make Model no.

UV Spectrophotometer UB VarianCary 100 scan EL 08053091

Dissolution test apparatus Electro Lab TDT-08L

Tablet compression

Machine

Rimek (8 station Rotary) Minipress -II

Tablet Hardness tester Monsanto -

FTIR Spectrophotometer Varian 640 IR

Roche friability tester Lab Hosp, Kumar

Vernier caliper Jashbin

Enterprises,Mumbai

Materials

Table 8.2: List of chemicals and reagent

Ingredients Supplier

Hydroxy propyl methyl cellulose

K100M

Colorcon laboratories, Mumbai

Psyllium husk local market, Maharashtra

Talc MERCK Pharmaceuticals, Mumbai

Sodium bicarbonate S.D.FINE Laboratories Pvt. Ltd.,

Mumbai

Magnesium stearate S.D.FINE Laboratories Pvt. Ltd


77 | P a g e

8.7.2 Preparation of floating tablet containing neem extract

In the present study, all the tablets were formulated by direct

compression technique using HPMC K100M and other ingredients like

psyllium husk, magnesium stearate, talc and sodium bicarbonate. All

ingredients were passed through sieve no # 80 and weighed accurately.

The extract, HPMC K100M, sodium bicarbonate and psyllium husk were

mixed properly in a mortar and pestle to get a uniform tablet blend. Finally

talc and magnesium stearate were mixed with the blend. The tablet blend

was then weighed individually according to the formula and compressed

into tablets using 10 station tabletting machine. The different formulations

were labeled A1-A8 as per composition given in Table82

Fig. 8.2: Tablet Compression Machine


78 | P a g e

Table 8.3: Composition of floating tablet formulation

Ingredients mg A1 A2 A3 A4 A5 A6 A8

Neem extract 250 250 250 250 250 250 250

Psyllium husk 85 100 125 100 100 100 100

HPMC K100M 50 50 50 40 60 50 50

Sodium bicarbonate 100 100 100 100 100 90 110

Talc 20 20 20 20 20 20 20

Magnesium stearate 5 5 5 5 5 5 5


79 | P a g e

8.8 Evaluation and characterization of tablets

In recent years, there has been great demand for plant derived

products in developed countries. Due to lack of infrastructures, skilled

manpower, reliable methods and stringent regulatory laws most of these

manufacturers produce their product on very tentative basis.

In order to have a good coordination between the quality of raw

materials, in process materials and the final products, it has become

essential to develop reliable, specific and sensitive quality control methods

using a combination of classical and modern instrumental method of

analysis. Standardization is an essential measurement for ensuring the

quality control of the herbal drugs.. It also means adjusting the herbal drug

preparation to a defined content of a constituent or a group of substances

with known therapeutic activity respectively by adding excipients or by

mixing herbal drugs or herbal drug preparations. "Evaluation" of a drug

means confirmation of its identity and determination of its quality and purity

and detection of its nature of adulteration.

The herbal formulation in general can be evaluated schematically as

to formulate the medicament using raw materials collected from different

localities and a comparative chemical efficacy of different batches of

formulation are to be observed. The preparations with better clinical

efficacy are to be selected. After all the routine physical, chemical and


80 | P a g e

pharmacological parameters are to be checked for all batches to select the

final finished product and to validate the whole manufacturing process.

8.8.1 Evaluation of formulation 83,84,85

8.8.1.1 Evaluation of powder blend

Angle of repose

10gm of powder was passed through funnel and the pile was

formed. The height and weight of the pile were measured and the angle of

repose was calculated by using the formula:

𝐴𝑛𝑔𝑙𝑒 𝑜𝑓 𝑟𝑒𝑝𝑜𝑠𝑒 𝜃 = tan−1𝑕𝑒𝑖𝑔𝑕𝑡

𝑟𝑎𝑑𝑖𝑢𝑠

Bulk density

Both loose bulk density and tapped bulk density were determined for

powder blend. A quantity of 2gm powder of each formula, previously

shaken to break agglomerates was taken into a 10 ml measuring cylinder.

Initial volume was observed and cylinder was tapped for a fixed time till

100 tapings. LBD (loose bulk density) and TBD (tapped bulk density) were

calculated using following formula:

𝐿𝐵𝐷 = 𝑤𝑒𝑖𝑔𝑕𝑡𝑜𝑓𝑝𝑜𝑤𝑑𝑒𝑟

𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑝𝑎𝑐𝑘𝑖𝑛𝑔


81 | P a g e

𝑇𝐵𝐷 =𝑤𝑒𝑖𝑔𝑕𝑡 𝑜𝑓 𝑝𝑜𝑤𝑑𝑒𝑟

𝑡𝑎𝑝𝑝𝑒𝑑 𝑣𝑜𝑢𝑚𝑒 𝑜𝑓 𝑝𝑎𝑐𝑘𝑖𝑛𝑔

Carr's compressibility index

The Carr's compressibility index was calculated by calculating the

tapped and bulk density using the 100 ml measuring cylinder.

Compressibility is calculated by the formula.

𝐶𝑎𝑟𝑟𝑠𝑐𝑜𝑚𝑝𝑟𝑒𝑠𝑠𝑖𝑏𝑖𝑙𝑖𝑡𝑦𝑖𝑛𝑑𝑒𝑥 =𝑇𝐵𝐷 − 𝐿𝐵𝐷

𝑇𝐵𝐷 × 100

8.8.1.2 Evaluation of floating tablets

Tablet hardness

The hardness of tablets from all the batches was determined using

the Monsanto hardness tester.

Friability

For each formulation, the friability of 20 tablets was determined

using the Roche friabilator. In this test tablets were subject to the

combined effect of shock and abrasion by utilizing a plastic chamber which

revolves at a speed of 25 rpm, dropping the tablets to a distance of 6

inches in each revolution. A sample of pre weighted 20 tablets was placed

in Roche friabilator which was then operated for 100 revolutions i.e. 4 min.


82 | P a g e

The tablets were then dusted and reweighed. Percent friability (%F) was

calculated as follows,

% 𝐹 =𝑙𝑜𝑠𝑠 𝑖𝑛 𝑤𝑒𝑖𝑔𝑕𝑡

𝐼𝑛𝑖𝑡𝑖𝑎𝑙 𝑤𝑒𝑖𝑔𝑕𝑡× 100

Thickness

Thickness of all tablets was measured using the Vernier caliper.

Tablet weight variation

Twenty tablets were randomly selected and accurately weighed.

Results are expressed as mean values ± SD.

Drug content uniformity

Ten tablets were individually weighed and crushed. A quantity of

powder equivalent to the mass of 10 mg was dissolved in 10 ml of 0.1N

HCI. The solution was filtered through a membrane filter (0.45 um). The

drug content was determined by UV spectroscopy at a wavelength of 280

nm after a suitable dilution with 0.1 N HCI. Each sample was analyzed in

triplicate. The standard curve of neem leaf extract was taken using

different concentrations and the slope and intercept was calculated from

the standard curve. Then concentration of the sample solution was

calculated by using the formula


83 | P a g e

𝑋 = 𝑌 − 𝐶𝑀

Where, X= concentration in µg/ml.

Y=absorbance of solution at 280 nm.

C= intercept of standard curve.

M= slope of standard curve.

Further, the % drug content was calculated from the concentration

using the equation as follows% drug content = Concentration of sample

solution X 100 Equivalent concentration of drug taken. The equivalent

concentration of drug taken in this case was 10 mg.

Swelling index

For calculating the swelling index, the previously weighed tablets

were placed in the 100 ml beaker containing 0.1 N HCI. The tablets were

removed at the time interval of 1 hr for 8 hours, the excess quantity of

solution was removed and tablets were weighed. The swelling index (SI)

was calculated using the formula:

𝑆𝑤𝑒𝑙𝑙𝑖𝑛𝑔 𝑖𝑛𝑑𝑒𝑥 =𝑊𝑡 − 𝑊0

𝑊0 × 100

Where, Wt= Final weight of tablets at time t.

Wo= Initial Weight of tablets.


84 | P a g e

Buoyancy lag time (BLT) and total buoyancy time

BLT is the time required for the formulation to float in the medium

and the total buoyancy period is the time for which the formulation remains

afloat in the medium. The BLT and total buoyancy time of all the

formulations was calculated by placing the tablets in 100 ml of 0.1 N HCI

for 18 hours.

In vitro drug release studies

Drug release studies of the prepared floating tablets were

performed, in triplicate, in a USP Dissolution Tester Apparatus, type-II

(Paddle method) (Dissolution tester, Electrolab) at 38 ± 0.5°C. The paddles

rotated at a speed of 50 rpm. The tablets were placed into 900 ml of 0.1 N

HCl solution (pH 1.2). Aliquots of 5 ml were withdrawn from the dissolution

apparatus at different time intervals and filtered through a cellulose acetate

membrane (0.45 µm). The drug content was determined

spectrophotometrically at a wavelength of 280 nm. At each time of

withdrawal, 5 ml of fresh medium was replaced into the dissolution flask.

Cumulative percent drug release for formulations from A1-A8 was

calculated.

8.8.2 Kinetic Model for release of drug from formulation86, 87


85 | P a g e

i) Zero order kinetic model

Zero order describes the system where the release rate of drug is

independent of itsconcentration. The equation is

At = A0 + K0t

Where, At is the amount of drug dissolved intime t, A0 is the initial amount

of drug and K0 is the zero order release constant.This relationship describe

the dissolution of drug from modified release Pharmaceutical dosage form

like some transdermal system and matrix tablet with low soluble drugs in

coated forms.

ii) First Order Kinetic Model

The dissolution phenomenon of a solid particle in a liquid media is because

of surface action and dependent on concentration of drug in reservoir.

𝐿𝑜𝑔𝑄𝑡 = 𝐿𝑜𝑔𝑄0 +𝐾1

2.303

Where, Qt is the amount of drug dissolved intime t, Q0 is the initial amount

of drug in the solutionand K1 is the first order release constant. The plot of

log cumulative drug release vs. time yields a straight line with slope of-

K/2.303. This relationship describes thedrug dissolution in pharmaceutical

dosage forms suchas those containing water soluble drugs in

porousmatrices.


86 | P a g e

iii) Higuchi Matrix Model

Higuchi describes drug release as a diffusionprocess based in the Fick‘s

law, proportional to square root of time. This model is based on following

equation

𝑄 = 𝐾𝐻 √𝑡

Where, KH is the Higuchi dissolutionconstant.

iv) Hixson Crowell Model

For this model to be valid drug powder should have uniformed size

particles. This model is based on equation whichexpresses rate of

dissolution based on cube root ofweight of particles. This is expressed by

the equation,

M01/3 - Mt

1/3 = k t

Where, M0 is the initial mass of drug in thepharmaceutical dosage form, Mt

mass of powder dissolved in time‗t‘ and k cube root dissolution rate

constant. It evaluates the dissolution with changes in surface area.

v) Korsmeyer-Peppas Model

Korsmeyer Peppas derived a simple relationship which describes drug

release from a polymeric system.


87 | P a g e

𝑄𝑡

𝑄∞

= 𝑘𝑡𝑛

Where Qt/Qαis fraction of drug dissolved at time t

K is constant includes structural and geometrical characteristics of

formulation

n= diffusion exponent which represent drug release mechanism

Where, n=1 signifies that release follows zero order kinetics

n=0.5 signifies that release is by fickian diffusion

0.5< n<1 signifies that release is through anomalous diffusion

In vivo gastro retention study88

The digital X-ray obtained for radio-opaque placebo tablet in rabbits

provides the evidence of floating nature of formulation in rabbit's stomach.

The BaSO4 tagged formulation, similar to formulation A4 was observed in

stomach region till 10 hours. The protocol for in vivo study was approved

by the Institutional Animal Ethics Committee of Pravara Rural College of

Pharmacy, Pravaranagar. In vivo study of the final formulation (A4) was

performed using the Albino rabbit by an X-ray imaging method. Three

albino rabbits were selected for the study. The animals were fasted


88 | P a g e

overnight with free access to water and a radiograph was made just before

the administration of the floating tablet to ensure the absence of any radio-

opaque material in the stomach. The formulation was administered by the

natural swallowing by the rabbit followed by 50 ml of water. The

radiographic imaging was taken in a supine position and the distance

between the sources of X-rays and the animal was kept constant for all

imaging; thus, the observation of the floating tablet movement could be

easily noticed. Gastric radiography was carried out at the 2 h time intervals

for a period of 10 h using an X-ray machine (WiproGEDX-300 with the

horizontal X-ray system, model SI-0146-3128).

8.9 Antimalarial screening 89, 90, 91

8.9.1 Mice survival study

Male Swiss albino mice were used for the study. Tap water and

mouse feed were provided ad libitum. Plasmodium berghei (ANKA strain)

erythrocytic stages were maintained by serial passaging of infected blood

in male Swiss albino mice.

Method

Animals were divided into eight groups based on the treatment. Mice

were injected intraperitoneally with 107P. bergheiinfected mouse

erythrocytes. Control group (group no 1) was only given 5% Tween 60.


89 | P a g e

Mice in group no 2 received neem extract orally as a suspension in corn oil

at a dose equivalent to 5mg of neem extract on day 1, 2 and 3. Animals in

group no 3, 4 and 5 received combination of neem extract (5mg on day 1,

2 and 3) and i.p injection of artemether at single dose of 0.5, 1 or 1.5 mg

respectively on day 1. While artemether was injected intraperitonially as a

suspension in 5% tween 60 to group no 6, 7 and 8 as single dose of 0.5, 1

or 1.5 mg respectively.

The survival time (over 40 days post treatment) of mice infected with

the erythrocytic stages of P. berghei was compared in different groups.

Determination of mean survival time

Mortality was monitored daily and the number of days from the time

of inoculation of the parasite up to death was recorded for each mouse in

the treatment and control groups throughout the follow up period. The

mean survival time (MST) for each group was calculated as:

𝑀𝑆𝑇 =𝑆𝑢𝑚 𝑜𝑓 𝑠𝑢𝑟𝑣𝑖𝑣𝑎𝑙 𝑡𝑖𝑚𝑒 𝑖𝑛 𝑎𝑙𝑙 𝑚𝑖𝑐𝑒 𝑖𝑛 𝑔𝑟𝑜𝑢𝑝 (𝑑𝑎𝑦𝑠)

𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑚𝑖𝑐𝑒 𝑖𝑛 𝑡𝑕𝑎𝑡 𝑔𝑟𝑜𝑢𝑝

8.9.2 Percent mean parasitemia in mice 92

Parasitemia was monitored by light microscopy (oil immersion,

1000× magnification) by examining thin smears of blood from the tail veins


90 | P a g e

of the mice. Blood films are made by applying 4.5 microlitres of blood to

microscope slides as soon as the specimen is received. Thin blood films

were fixed in methanol and stained with Giemsa stain immediately after

slide production. The parasitemia level was determined by counting, in

random fields of the microscope, the number of parasitized RBCs. The

percent of infected RBCs were determined by enumerating the number of

infected RBCs in relation to the number of uninfected RBCs. A minimum of

500 RBCs was counted per sample.

% 𝑃𝑎𝑟𝑎𝑠𝑖𝑡𝑒𝑚𝑖𝑎 =𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑖𝑛𝑓𝑒𝑐𝑡𝑒𝑑 𝑅𝐵𝐶

𝑇𝑜𝑡𝑎𝑙 𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑× 100

Inoculation of P. berghei to mice was done on day 0, whilepercent

mean parasitemia was measured on day 1, 2, 3 and 4. The blood samples

were collected after 4 hours of receiving treatment as per specified above

and at the same time on next days.

8.10 Stability studies

Stability studies were conducted on the optimized tablet batches to

assess their stability after storage. They were packed in Aluminium

pouches and stored under the following conditions for a period as

prescribed.


91 | P a g e

Table 8.3: Stability conditions

Sr. No Study Storage condition

1 Long term 25°C±2'C/60%RH±5% RH

2 Intermediate (30 °C±2°C/65%±5%RH)

3 Accelerated 40'C±2°C/85%RH±5%RH

The tablets were withdrawn after a period of 1, 2 and 3 months and

analyzed for percent drug content, floating time, swelling index and

dissolution profile.

8.11 Statistical analysis

All data were expressed as mean ± SD. The statistical analysis of all

the observations was carried out using one-way ANOVA followed by a

multiple comparison test of Tukey, where necessary. P<0.05 was

considered as significant compared with the control group and all data

were analyzed at a 95% confidence interval.

RESULTS AND DISCUSSION

92 | P a g e

9. RESULTS AND DISCUSSION

9.1. Pharmacognostic studies

As per WHO guideline Pharmacognostic study of all plant parts like

Macroscopy, physical parameters, and extractive values were studied.

9.1.1 Macroscopy

Table 9.1: Morphological and organoleptic characters

Sr. No. Parameter A. indica

1. Color Green

2. Odor Characteristic

3. Taste Bitter

4. Size leaflets 9-l5in.long

5. Shape Alternate, imparipinnate |

9.1.2Ash Value

Table 9.2: Ash Value

1. Total ash 7.75%

2. Water- soluble ash 1.75%

3. Acid insoluble ash 0.7%


93 | P a g e

9.1.3 Extractive Values

Table 9.3 : Extractive Values

Sr. No. Parameter Extractive

value

1. Alcohol soluble extractive value 10.2%

2. Water soluble extractive value 23.8%

9.1.4 Foreign organic matter and moisture content

Table 9.4 : Foreign Organic Matter and Moisture Content

Sr. No. Parameter A.indica

1. Foreign organic matter 0.9%

2. Moisture content 0.5%

9.1.5Preliminary screening of extracts

Table 9.5: Summary of extraction

Extracts Color Nature Percentage Yield (%

W/W)

A.indicaextract Dark Green Sticky

powder 15.08%


94 | P a g e

9.2Phytochemical evaluation

Preliminary phytochemical tests

Table 9.6: Preliminary phytochemical tests

Sr. No. Test Perform A. indica

1. Test for carbohydrate

Molish's test -

Fehling test -

Benedicts test +

Barfoed's test -

2. Test for Proteins

Biuret Test -

Millions Test -

3. Test for amino acids

Ninhydrinetest -

4. Test for Steroids

Salkowski test +

Libermann test -

Libermann

Burchard

Reaction

+

5. Test for Glycosides

Cardiac -

Anthraquinone

6. Test for Saponin

Foam test

+


95 | P a g e

7. Test for Flavonoids

Shinoda test +

Lead acetate test +

8. Test for Alkaloids

Dragondroffs test +

Mayer's test +

Hager's test +

Wagner's test +

9. Test for Tannins and phenolic compounds

5% Ferric chloride test +

Potassium dichromate test -

Bromine water test +

Oil. Nitric acid test -

10. Test for Vitamin

Vitamin C -

+ Indicates presence of Constituents

- Indicates absence of Constituents.

9.3 Thin layer chromatography

Thin layer chromatography technique carried out for separation,

isolation, and identification of constituents present in the hydroalcoholic

extracts.

TLC of flavonoids


96 | P a g e

Table 9.7:TLC of Hydroalcoholic extract of A. Indicafor flavonoids

Sr.

No.

Chemical

constituent

Mobile Phase Spraying

reagent

Color of

spot

Rf

1. Flavonoids Ethyl acetate

Formic acid: acetic

acid:H20

(100:11:11:26)

Anisaldehyde -

Sulfuric acid.

Yellowish

green

0.6

Fig. 9.1: TLC of Hydroalcoholic extract of A. Indica for flavonoids


97 | P a g e

TLC of alkaloids-

Table 9.8: TLC of Hydroalcoholic extract of A. Indica for Alkaloids

Fig. 9.2 : TLC of Hydroalcoholic extract of A.indicafor alkaloids

Sr.

No.

Chemical

constituent


reagent

Color of

spot

Rf

I. Alkaloids Toluene: ethyl

acetate;

diethylamine

(70:20:10)

10%H2SO4 in

ethanol

Violet -

blue

0.35


98 | P a g e

TLC of Steroids

Table 9.9: TLC of Hydroalcoholic extract of A. Indica for Steroids

Fig. 9.3: TLC of Hydroalcoholic extract of A. indica for Steroids.

Sr.

No.

Chemical

constituent


reagent

Color of

spot

Rf

1. Steroids Toluene:

Ethylacetate (9:1)

Vanillin-

Sulfuric acid

Pink 0.59


99 | P a g e

TLC of Saponins

Table 9.10: TLC of Hydroalcoholic extract of A.indicafor Saponins

Fig.9.4: TLC of Hydroalcoholic extract of A. indica for Saponins.

Sr.

No.

Chemical

constituent


reagent

Color

of

spot

Rf

I. Saponin Ethylacetate: formic

acid: acetic acid:H20

(100:11:11:26)

Anisaldehyde -

Sulfuric acid.

green 0.9


100 | P a g e

9.4Preparation of calibration curve using UV spectrophotometer

Various concentrations of neem extract in 0.1 N HCL were scanned

on UV spectrophotometer and absorbance values for various

concentrations were noted. A standard curve was developed. It followed

the linear relationship between concentration and absorbance as shown in

figure

Table 9.11- Calibration curve data for neem leaf extract in 0.1 N HCI

Sr. No Concentration (µg/ml) Absorbance

1 0 0.00

2 5 0.051

3 10 0.0956

4 15 0.1390

5 20 0.1823

6 25 0.2213

7 30 0.2657


101 | P a g e

Fig.9.5 : Calibration curve of neem extract

Analytical Parameter

Parameters Value

Wavelength of detection 280 nm

Beer‘s law limit 1-50 mcg/ ml

Regression equation Conc.

= 114.43 Abs. + -0.579

Correlation coefficient 0.9994

0

0.05

0.1

0.15

0.2

0.25

0.3

0 5 10 15 20 25 30 35

Ab

so

rba

nc

e

Concentration


102 | P a g e

9.5 Preformulation study

Compatibility Studies

9.5.1 FTIR analysis

Neem extract, excipients and physical mixtureintopowderedformwas

scanned between 4000cm-1to 450 cm-1.The resultant spectrum obtained

shownin figure 2 to 5. Presence of peaks for aldehydic C-H stretching

(around 2940 cm-1), C=C group (around 1625 cm-1) and Germinal methyl

group (around 1350 cm-1) were indicative of terpenoid group of compounds

present in the aqueous neem extract. Above peaks were seen in FTIR

spectra of neem extract and physical mixture of neem extract and excipient

which suggested physical compatibility between them.

Fig. 9.6: FTIR spectra of hydroalcoholic extract of A. indica


103 | P a g e

Fig. 9.7: FTIR spectra of HPMC K100M

Fig. 9.8: FTIR spectra of sodium bicarbonate


104 | P a g e

Fig. 9.9: FTIR spectra of physical mixture of neem extract, HPMC and

sodium bicarbonate

9.6Evaluation and characterization of tablets

9.6.1 Evaluation of powder blend

Powder blends of seven formulations (A1 to A7) were evaluated

for angle of repose, bulk density and Carr's compressibility index (Table

3). The results showed that the pre compressed blend has good flow

property. The angle of repose for all formulation was found between 24-30

except A1 and A3, which represents excellent flow property.

Compressibility index value was minimum for A5 (13.18) and maximum for

A1 as 17.30.


105 | P a g e

Table 9.12: Evaluation of powderblend

Formulation

No.

Angle of

Repose (°)

LBD

(g/cm2)

TBD

(g/cm2)

Compressibility

Index

A1 23.54±1.2 0.4137±0.05 0.5064±0.09 17.30±1.3

A2 24.57±0.5 0.5678±0.04 0.6542±0.06 13.20±0.9

A3 22.24±0.8 0.5490±0.1 0.6341±0.07 13.42±0.5

A4 26.07±0.4 0.4675±0.06 0.5462±0.04 14.40±0.6

A5 25.54±2.1 0.4642±0.05 0.5231±0.05 13.18±1.2

A6 24.46±1.3 0.5548±0.03 0.6742±0.05 17.20±2.1

A7 27.36±1.4 0.5450±0.02 0.6473±0.06 15.81±0.9

*Data is expressed as mean± S.D

9.6.2 Evaluation of floating tablets

Various formulations of floating tablet were evaluated for physical

parameters such as hardness, thickness, weight variation, % friability, drug

content and swelling index, the results are shown in Table 4. The total

weight of each formulation was maintained constant; the weight variations

of the tablets were within the permissible limits. Tablet thickness was also

used to assess the quality of tablets. The thickness of floating tablets


106 | P a g e

ranged from 8.36 to 8.68 mm. Friability test of all the formulations was

found satisfactory showing enough resistance to the mechanical shock and

abrasion with % friability being less than 1%. Drug content in all

formulations was calculated and the presence of active ingredient ranged

between acceptable limit of 95 to 105%. There was marked variation in

swelling index for various formulations.

Table 9.13: Post compression evaluation of formulations

Formul

ation

no.

Tablet

weight

(mg)

Tablet

thi-

ckness

(mm)

Friability

%

% drug

content

Hardne

ss

kg/cm2

Swelling

index

Buoyanc

y lag

time

(sec.)

A1 505.1

±0.56

8.54±0.

07

0.551±0.

06

97.45±0.

56

3.1±0.1

3

178.45±2.

34

298±2.2

8

A2 526.4

±0.14

8.64±0.

04

0.641

±0.10

98.27±0.

75

3.3±0.2

5

234.66±3.

75 271±1.5

A3 552

±0.35

8.58±0.

03

0.66±0.1

5

98.17±0.

34

3.2±0.0

6

245.56±2.

56

239 ±

1.76

A4 513.5

±0.95

8.50±0.

05

0.531

±0.09

97.96±0.

78

3.1±0.1

5

201.45±1.

34

212 ±

1.8

A5 535.6

±0.23

8.43±0.

08

0.634±0.

13

98.59±1.

56

3.4±0.0

8

256.55±3.

13

302 ±

2.57

A6 515.3

±0.87

8.36±0.

02

0.426±0.

16

96.39±0.

50

3.3±0.1

2

208.44±1.

57

252 ±

1.2

A7 534.2

±0.98

8.68±0.

10

0.723±0.

15

97.19±1.

34

3.5±0.3

4

205.45±2.

18

294 ±

2.73

*Data is expressed as mean± S.D


107 | P a g e

The in vitro buoyancy studies in 0.1 N HCl (pH 1.2), revealed

buoyancy variations for all the formulations (Table 5). Sodium bicarbonate

was used as the effervescent base which generates carbon-di-oxide gas in

the presence of hydrochloric acid present in dissolution medium. The gas

generated was trapped and protected within the gel (formed by hydration

of HPMC K100M and psyllium husk), thus decreasing the density of the

tablet. As the density of the tablet falls below 1 (density of water), the tablet

becomes buoyant. The tablet mass decreased progressively due to

liberation of CO2 and release of drug from the matrix. On the other hand,

as solvent penetrated the polymer layer, the swelling of HPMC K100 M

caused an increase in volume of the tablet. The combined effect was a net

reduction in density of the tablets, which prolongs the duration of floatation

beyond 12 h. The buoyancy lag time of all formulations was in the range 3

to 5 min.

Effect of different concentrations of psyllium husk on in vitro release

was as shown in figure 6. As the concentration of psyllium husk increased

from 75 (A1) to 125 mg (A3) per tablet, the percent cumulative drug

release in 12 h decreased with 96.33 ±1.9 %, 95.99 ± 1.4 and 91.66 ±2.6

for A1, A2 and A3 respectively. The slow release of the drug was attributed

to the gelling properties of psyllium husk.


108 | P a g e

Effect of different concentrations of HPMC K100M on in vitro release

was as shown in figure 7. As the concentration of HPMC K100M was

increased from 40 (A4) to 60 mg (A5), drug release decreased from 98.77

± 2.3 % to 94.53 ± 1.89 %. With the increase in polymer concentration

there was an increase in the diffusion path length of the drug, retarding the

drug release.

The effect of sodium bicarbonate on in vitro drug was shown in

figure 8. In such systems, sodium bicarbonate acts as a gas-generating

agent. As the concentration was increased from 90 (A6) to 110 mg (A7)

per tablet, the drug release was decreased from 96.03 ± 1.7 % to 90.55 ±

2.4 %.

Fig. 9.10: In vitro drug dissolution study for A1, A2 and A3


109 | P a g e

Fig. 9.11: In vitro drug dissolution study for A4 and A 5

Fig. 9.12: In vitro drug dissolution study for A6 and A7


110 | P a g e

9.7 Optimization of tablet formulation

Based upon the buoyancy time and % cumulative drug release, the

formulations were optimized. The buoyancy time of all formulations was

found in the range 3-5 min. The % cumulative drug release was in the

range 90.55 -98.77%. The optimized formulation was found to be A4 with

the buoyancy time 212 seconds and % cumulative drug release 98.77%.

Mathematical modeling and release kinetics

The release pattern of all the formulations was calculated using PCP

Disso v2.0.8.5 software. All the formulations were fitted for zero order

release, first order release, Higuchi matrix model, Hixson and Crowell

powder dissolution model and Korsmeyer- peppas model. The data

obtained are represented in Table 5. None of the formulations followed

first-order kinetics, which was confirmed by the poor correlation coefficient

values. All formulations best fitted both zero-order (R2 =0.9534–0.9871)

and Korsemeyer and Peppas equation (R2 =0.9734-0.9926). The value for

diffusional exponent n was found between 0.5 (suggesting Fickian diffusion

controlled drug release) and 1.0 (swelling-controlled drug release). For all

formulations, the value of n was in the range 0.5523-0.8178 indicating non-

fickian anomalous transport wherein the drug release mechanism was

controlled by both diffusion and polymer swelling.


111 | P a g e

Table 9.14: Release Kinetics for various formulations

FOR

mul

a

tion

cod

e

Zero

order

Correlati

on

coefficie

nt

(R2)

First

order

Correlati

on

coefficie

nt (R2)

Matrix

Correlati

on

coefficie

nt

(R2)

Hixson

Crowell

Correlati

on

coefficie

nt

(R2)

Korsmeyer-

Peppas

Best fit

model

Correlation

coefficient

(R2)

Diffusion

al

exponent

(n)

A1 0.9652 0.9089 0.9455 0.9890 0.9843 0.7962 Zero

order

A2 0.9766 0.9118 0.9723 0.9095 0.9915 0.5523

Korsmey

er

peppas

A3 0.9534 0.8657 0.9534 0.9863 0.9874 0.6213 Zero

order

A4 0.9540 0.9260 0.9422 0.9715 0.9926 0.8154

Korsmey

er

peppas

A5 0.9603 0.9244 0.9219 0.9348 0.9734 0.4815

Korsmey

er

peppas

A6 0.9547 0.9160 0.9633 0.9941 0.9886 0.8178 Hixson

Crowell

A7 0.9871 0.9201 0.9657 0.9502 0.9814 0.5214

Korsmey

er

peppas


112 | P a g e

9.8 In vivo gastro retention study

The digital X-ray obtained for radio-opaque placebo tablet in rabbit

provides the evidence of floating nature of formulation in the rabbit's

stomach. The BaSO4 tagged formulations, similar to formulation A4 were

observed in the stomach region as shown in Fig.9.13. It was observed that

formulation kept floating in the rabbit stomach till 10 hours.

Fig. 9.13: X-ray photographs at different time intervals of

gastroretentive floating tablets X-ray (a) at 0 h. (b) after 2 h. (c) after 4

h. (d) after 6 h. (e) after 8 h. (f) after 10 h


113 | P a g e

9.9Stability studies

Stability studies were conducted at accelerated condition 40±2°C

/75±5 % RH for 1 month. Data for stability study was shown in the table

no.25

Table 9.15:Stability studies

Formulation Day‘s condition % drug

content

% Drug release Swelling

index 1 hr 2 hr 6 hr 10 hr

A4

Initial result 98.77 % 27.57 36.87 78.64 92.35 201.45 ±

1.34

1, months

Accelerated 98.73 % 27.30 36.15 77.98 91.94

201.43 ±

1.34

No visible changes in the appearance of the tablets were observed

at the end of the storage period. Form the data (Table no 25) it was

concluded that after 1 month accelerated conditions, no significant change

in release profile was observed. There was significant increase in the

floating lag time in case of formulation, this may due to reaction of sodium

bicarbonate with water. Due to this density of formulation was increased

and this lead to increase in floating time. There is no significant change in


114 | P a g e

Hardness, friability and swelling index. Thus it can be concluded that all

formulations were physically stable.

9.10 Antimalarial activity

9.10.1 Mice survival study

The survival time (over 40 days post treatment) of mice infected with the

erythrocytic stages of P. berghei was compared in different groups as

showed in table 10. All mice in control group died in 8 days post exposure

to infection. Animals treated with only neem extract failed to survive

beyond 10 days. While animals treated with 0.5 mg of artemether have

survived little longer than treated with neem extract with 40% survival on

day no 16. Animals in the group 7, showed 40% survival on day 20 and

100% mortality by day 25. Survival rate for animals in group 6 was 40% on

day 25 but 100 % mortality by day 40. Combination of neem–artemether

prolonged the survival time as compared to monotherapy at same dose

while survival rate in group 3 and 4 were found 60% and 80% respectively

by the day 40.


115 | P a g e

Table 9.16:Survival over time of P. berghei-infected mice

Treatm

ent

Rou

te

Dose

Mg

mi

ce

No. of mice surviving on the following

day after treatment

MST

(day)

0 2 4 6 8 1

0

1

2

1

6

2

0

2

5

3

0

4

0

Neem

extract

Oral 3 Days

5mg/ d

5 5 5 5 4 2 2 1 0 8.6

neem

extract

+

artemet

her

combin

ation

Oral

i.p.

3 Days

5mg/d

+1.5mg

5 5 5 5 5 5 5 5 5 5 4 4 4 36.4

Oral

i.p.

3 Days

5mg/d

+1 mg

5 5 5 5 5 5 5 5 5 4 4 3 3 32.8

Oral

i.p

3 Days

5mg/d

+0.5mg

5 5 5 5 5 5 5 4 2 1 0 16.2

Artemth

er

i.p. 1.5 mg 5 5 5 5 5 5 5 5 4 4 3 3 3 31.8

i.p. 1.0 mg 5 5 5 5 5 5 5 3 2 2 2 0 18.2

i.p. 0.5 mg 5 5 5 5 4 4 2 2 0 10.6

Control 5 5 5 5 4 2 0 6.8


116 | P a g e

Fig. 9.14 : Percent mean protection in mice receiving different

treatments over time (40 days post treatment), * (n=5)

9.10.2 Percent mean parasitemia

There was marked variation in percent parasitemia in mice receiving

control, artemether, neem extract and combination of neem- arthemether

for the first 3 days as showed in table no 11. The mean percent

parasitemia is measured after 2 hrs of i.p. injection of p. berghei on day 0

0

20

40

60

80

100

120

0 2 4 6 8 10 12 16 20 25 30 40

Control

Gr -1

Gr -2

Gr -3

Gr -4

Gr -5

Gr -6

Gr - 7

% M

ean

pro

tect

ion

d


117 | P a g e

while after 4 hours after treatment on day 1, 2 and 3. There was drastic

increase in percent mean parasitemia in control mice within 3 days from

3.16% to 83.56%. Administration of neem-artemether combination reduced

the parasitemia in mice by 77% compared to that in control mice. Such

suppressive action was superior to that of artemether alone at the same

dose.

Table 9.17: Percent Mean parasitemia in mice receiving different

treatments on 4th day

Treatment Route Dose No. of mice

tested

% mean Parasitemia

on 4th day

Control 5 56.51 ± 5.2

Neem

extract

oral 3 Days

5mg/ d

5 46.12 ±4.9*

neem

extract +

artemether

combination

Oral

i.p.

3 Days

5mg/ d

+1.5 mg

5 7.16 ± 0.9

Oral

i.p.

3 Days

5mg/ d

+1.0 mg

5 10.58 ± 1.8

Oral 3 Days 5 19.91 ± 1.9


118 | P a g e

i.p 5mg/ d

+ 0.5 mg

Artemther i.p. 1.5 mg 5 9.05 ± 1.5**

i.p. 1.0 mg 5 13.12 ± 2.2**

i.p. 0.5 5 25.26 ±2.2

Data are expressed as mean ±SD (n = 5); *P<0.05, **P<0.001 compared

to control

Conclusion

119 | P a g e

10. CONCLUSION

1. The drug excipient interaction study was conducted using FTIR

spectrometry. It was observed that there is no incompatibility

between drug and excipients as characteristic peak at 2940 was

observed in all spectra.

2. Floating tablets of hydroalcoholic neem leaf extract using psyllium

husk, HPMC K100M, talc, sodium bicarbonate and magnesium

stearate were prepared and optimized. Formulated tablets were

within acceptable official limits for various physicochemical

parameters.

3. The drug release pattern from all seven formulations were studied by

fitting to various dissolution models. Formulation A4 showed good

floating behavior with BLT 212 sec and better controlled drug

release of 98.77% in comparison to other formulations. Floating

tablets best fitted to Korsmeyer-Peppas model and zero-order

kinetics with R2 value 0.9715 and 0.9540 respectively.

Conclusion

120 | P a g e

4. In vivo gastro retention study of optimized formulation A4 was

conducted in rabbits. The study confirmed floating behavior of

BaSO4 tagged formulation till 10 hour.

5. Antimalarial activity of neem leaf extract and combination of neem

extract-artemether was conducted in mice infected with Plasmodium

berghie. Mice survival and % parasitemia inhibition study showed

that neem leaf extract has moderate antimalarial activity compared

to control group with % mean parasitemia on 4th day 46.12 and

56.51 % respectively. The suppressive action of combination was

superior with MST 36.4 days as compared to administration of single

drug (artemether) at the same dose with MST 31.8 days.

References

121 | P a g e

REFERENCES

[1] Warrell DA. Clinical Features of Malaria. In: Gilles HM, Warrell DA, Editors.

Bruce-Chwatt‘s Essential Malariology. 3rded. London: Arnold; 1993. p. 37-

49.

[2] Valecha N, Tripathi KD. Artemisinin: Current status in malaria. Indian J

Pharmacol 1997; 29(2): 71-75.

[3] Sonalkar MY, Nitave SA, Kagalkar A. A review on neem plant. World J

Pharm Pharmaceutical Sci 2014; 3 (4), 590-598

[4] Biswas K, Chattopadhyay I, Banerjee RK, Bandyopadhyay U. Biological

activities and medicinal properties of neem (Azadirachta indica). Current

Sci 2002; 82(11): 1336-1345.

[5] Chhibber S, Sharma N. Medicinal and therapeutic potential of neem

(Azadirachta indica): A review. Inter J of Sci Res Pub 2014; 4(5): 1-5.

[6] Bledsoe GH. Malaria primer for clinicians in the United States. Southern

Med J2005; 98(12): 1197-204.

[7] Sturm A, Amino R, Regen T, Retzlaff S, Rennenberg A, Krueger A, et. al.

Manipulation of host hepatocytes by the malaria parasite for delivery into

liver sinusoids. Science 2006;313: 1287-1490.

[8] www.chsrd.aua.am/malaria. Malaria life cycle & pathogenesis. Malaria in

Armenia. Accessed October 12, 2011.

http://www.chsrd.aua.am/malaria

References

122 | P a g e

[9] Idro R, Otieno G, White S, Kahindi A, Fegan G, Ogutu B et al. Decorticate,

decerebrate and opisthotonic posturing and seizures in Kenyan children

with cerebral malaria. Malaria J 2005;4 (57):1-10

[10] Boivin MJ, Effects of early cerebral malaria on cognitive ability in

Senegalese children.J of Develop and Behavioral Pediat 2002; 23 (5):

353–64.

[11] Holding PA, Snow RW. Impact of Plasmodium falciparum ,malaria on

performance and learning: review of the evidence.Am J of Trop Med and

Hyg 2001; 64 (1): 68–75.

[12] Trampuz A, Jereb M, Muzlovic I, Prabhu R. Clinical review: Severe

malaria. Crit Care 2003;7 (4): 315-23.

[13] Kain K, Harrington M, Tennyson S, Keystone J. Imported malaria:

prospective analysis of problems in diagnosis and management. Clin Infect

Dis 1998;27 (1): 142-49.

[14] Mockenhaupt F, Ehrhardt S, Burkhardt J, Bosomtwe S, Laryea S,

Anemana S, et.al. Manifestation and outcome of severe malaria in children

in northern Ghana. Am J Trop Med Hyg 2004; 71 (2): 167-72.

[15] Carter JA, Ross AJ, Neville BG, Obiero E, Katana K, Mung'ala-Odera V,

et.al. Developmental impairments following severe falciparum malaria in

children. Trop Med Int Health 2005;10: 3-10.

References

123 | P a g e

[16] Meis J, Verhave J, Jap P, Sinden R, Meuwissen J. Malaria parasites--

discovery of the early liver form. Nature 1983; 302 (5907): 424-6.

[17] Trager W, Jensen JB. Human malaria parasites in continuous culture".

Science 1976; 193(4254): 673-85.

[18] Farcas GA, Soeller R, Zhong K, Zahirieh A, Kain KC. Real-time

polymerase chain reaction assay for the rapid detection and

characterization of chloroquine-resistant Plasmodium falciparum malaria in

returned travelers. Clin Infect Dis 2006;42: 622–27.

[19] Djimde A, Doumbo OK, Cortese JF. A molecular marker for chloroquine-

resistant falciparum malaria. N Engl J Med 2001; 344 (4): 257–263.

[20] Escalante A, Freeland D, Collins W, Lal A. The evolution of primate

malaria parasites based on the gene encoding cytochrome b from the

linear mitochondrial genome. Proc Natl Acad Sci 1998;95 (14): 8124-29.

[21] Kaufman T, Ruveda E. The quest for quinine: those who won the battles

and those who won the war. Angew Chem Int Ed Engl 2005;44 (6): 854-

85.

References

124 | P a g e

[22] Joy D, Feng X, Mu J, Furuya T, Chotivanich K, Krettli A, Ho M, et.al. Early

origin and recent expansion of Plasmodium falciparum. Science 2005;300

(5617): 318-21.

[23] Hay S, Guerra C, Tatem A, Noor A, Snow R. The global distribution and

population at risk of malaria: past, present, and future. Lancet Infect Dis

2004; 4 (6): 327-36.

[24] Korenromp E, Williams B, de Vlas S, Gouws E, Gilks C, Ghys P, Nahlen B.

Malaria attributable to the HIV-1 epidemic, sub-Saharan Africa. Emerg

Infect Dis 2005; 11 (9): 1410-9.

[25] Abu-Raddad L, Patnaik P, Kublin J. Dual infection with HIV and malaria

fuels the spread of both diseases in sub-Saharan Africa. Science 2006;314

(5805): 1603-16.

[26] Geyer M, editor. Antimalarial drug combination therapy-Report of WHO

technical consultation. Geneva: WHO; 2001.

[27] Robinson JR, Lee VHL. Controlled drug delivery: fundamentals and

applications, 2nd ed. NY: Marcel Dekker, Inc.; 1987.

[28] Chien YW. Novel drug delivery systems, 2nd ed. NY: Marcel Dekker, Inc.;

1992.

[29] Lachman L, Lieberman HA, Lachman kaling JL, editors. The theory and

practice of industrial pharmacy, 3rd ed. Bombay: Varghese Publishing

House; 2007.

References

125 | P a g e

[30] Aunachalam A. Floating drug delivery systems: A review. Int J Res

PharmSci 2011; 2(1) 76-83.

[31] Fell JT, Whitehead L, Collet H. Prolonged gastric retention using floating

dosage forms, Pharm Technol 2000;24(3): 82-90.

[32] Menon A, Ritschel WA, Sakar A. Development and evaluation of a

monolithic floating dosage form for Furosemide. J Pharm Sci 1994; 239-

245.

[33] Moursy NM, Afifi NN, Ghorab DM. Formulation and evaluation of sustained

release capsules of Nicardipine Hydrochloride, Pharmazie 2003;58(1): 38-

43.

[34] Hilton AK, Deasy PB. In vitro and in vivo evaluation of an oral sustained

release floating dosage form of Amoxicillin Trihydrate, Int. J. Pharm 1992;

86:79-88.

[35] Vedha BN. The recent developments on gastric floating drug delivery

systems: an overview, Int J Pharmtech Res 2010; 2(1): 524-534.

[36] Sanjay S, Joshi V, Barpete PK. Gastro retentive drug delivery system:

Current approaches. J Pharm Res 2009; 2(5): 881-886.

[37] Shaha SH. Gastro-retentive floating drug delivery system, Asian J of

Pharm Sci 2009; 4 (1): 65-80

References

126 | P a g e

[38] Vidyadhara S, Rao PR, Prasad JA. Development and In-Vitro Kinetic of

Propanolol Hydrochloride Controlled Release Matrix Tablets. The Indian

Pharmacist 2006; 2: 66-70.

[39] Gopalakrishnan CS. Floating Drug Delivery Systems: A Review. J of

Pharm Sci and Tech 2011; 3 (2): 548- 554.

[40] Talukdar R, Fassihi R. Gastroretentive delivery systems: hollow beads.

Drug DevInd Pharm 2004; 30: 405-12.

[41] Hilton AK, Deasy PB. In vitro and in vivo evaluation of an oral sustained

Release floating dosage form of amoxicillin trihydrate. Int J Pharm1992;

86:79‐88.

[42] Basak SC, Nagswara RK, Manavalan R. Development and in-vitro

evaluation of an oral floating Matrix tablet formulation of ciprofloxacin. Ind J

of Pharm.Sci 2004; 66(3):313-316.

[43] Choi BY, ParkHJ, Hwang SJ, Park JB. Preparation of alginate beads for

floating drug delivery system: Effect of CO2 generating agents. Int J Pharm

2002; 239: 81-91.

[44] Arora S, AliJ, AhujaA, Khar RK, BabootaS. Floating Drug Delivery system:

A Review. AAPS PharmaSciTech 2002; 6(03): 221-28.

[45] Reddy LHV, MurthyRSR. Floating Dosages Systems in Drug Delivery. Crit

Rev in Therap Drug Carrier Sys 2002; 19(6):553-585.

References

127 | P a g e

[46] RahmanZ, AliM, KharRK. Design and evaluation of bilayer floating tablet of

captopril. Acta Pharm 2006; 56: 49-57.

[47] Murata Y, Saraki N, Miyamoto E, Kawashima S. Use of floating alginate

gel beads for stomach specific drug delivery. Eur J Pharm Biopharm 2002;

50: 221-226.

[48] El-Kamel AH, Sokar MS, Al Gamal SS, Naggar VF. Preparation and

evaluation of ketoprofen floating oral delivery system. Int J Pharm 2001;

220: 13-21.

[49] Singh BN, Kim KH. Floating Drug Delivery system, an approach to oral

controlled drug delivery via gastric retention. J Cont Rel 2000; 63: 253-259.

[50] ShivakumarHS, Shakante VG, Pramodkumar TM. Floating controlled drug

delivery system for prolonged Gastric Residence: A Review.Ind JPharm

Educ 2004; 38(4): 172-178.

[51] IchikawaSW,Miyake Y. A new multiple unit oral floating dosage system in

preparation and in vitro evaluation of floating and sustained release

characteristics, J of Pharm Sci 2006; 80(11): 115-21.

[52] Bulgarelli E, Forni E, Beranabei MT. Effect of matrix composition and

process conditions on casein gelatin beads floating properties.Int J Pharm

2000; 198: 157-165.

References

128 | P a g e

[53] Ahmedullah M, Nayar MP. Red data book of Indian plants, (Peninsular

India). Vol-4. Calcutta: Botanical Survey of India;1999.

[54] Aditi G, Bhandari BS, Rai N. Antimicrobial activity of medicinal plants

Azadirachtaindica A. Juss, Allium cepa L. and Aloe vera L. J Phama Tech

Res 2011; 3: 1059-1065.

[55] Bhat SS, Girish K.Neem – A Green Treasure. Electronic J Bio2008; 4:102-

111.

[56] Deshpande PK.Phytochemical analysis and evaluation of antimalarial

activity of Azadirachtaindica,The Pharma Innovation J 2014; 3 (9): 12-16.

[57] Akin-Osanaiye.Antimalarial effect of neem leaf and neem stem bark

extracts on Plasmodium berghei infected in the pathology and treatment of

malaria.Internat J of Res in Biochem and Biophy 2013; 3 (1): 7-14.

[58] Siddiqui BS.Tetracyclic triterpenoids from the leaves of

Azadirachtaindica,Phytochem 2004; 65 (2): 2363–2367.

[59] Dhiman B, Prashar B. Psyllium: A potential carrier to control the drug

delivery. Internat Res J of Pharm 2012; 3 (7): 39- 44.

[60] ,Raval JA, Patela JK, Michael F, ALib N, Patela MM. Ranitidine

hydrochloride floating atrix tablets based on low density powder: effects of

References

129 | P a g e

formulation and processing parameters on drug release. Asian journal of

pharmaceutical sciences 2007; 2 (4): 130-142.

[61] Rao MRP, Sonar GS, Mandsaawale RR, Vanshiv SD. Evaluation of

effervescent floating matrix tablet formulations of salbutamol sulfate using

full factorial design.Asian journal of pharmaceutics 2007; 1:43-49.

[62] Jadhav KR, Gambhire MN, Ambade KW, Kurmi SD, Kadam VJ.

Development and In Vitro evaluation of an oral floating matrix tablet

formulation of diltiazem hydrochloride. AAPS PharmSciTech 2007; 8

(3):73.

[63] Patel DM, Patel NM, Pandya NN,Jogani PD. Formulation and optimization

of carbamazepine floating tablets. Indian J Pharm Sci 2007; 69 (6): 763-67.

[64] Chattopadhyay I, Nandi B, Chatterjee R, Biswas K, Bandyopadhyay U,

Banerjee RK. Mechanism of antiulcer effect of Neem (Azadirachta indica)

leaf extract: effect on H+-K+-ATPase, oxidative damage and apoptosis.

Inflammopharmacol 2004; 12:153–17

[65] Khosla P, Sangeeta B, Jain K, Srivastva RK. Antinociceptive activity of

azadirachtaindica(neem) in rats.Ind J phamacol 2000; 32: 372-374.

References

130 | P a g e

[66] BadamL, Joshi SP, Bedekar SS.In vitroantiviral activity of neem

(Azadirachta indica)A extract against group B Coxsackie viruses. J

Commun Dis 1999; 31: 79-90.

[67] Asthana RK, Srivastava A, Singh AP, Deepali, Singh SP, Nath G,

Srivastava R, Srivastava BS. Identification of an antimicrobial entity from

the cyanobacterium Fischerella sp. isolated from bark of Azadirachta

indica(Neem) tree. J Appl Phycol 2006; 18: 33–39.

[68] Shravan KD, Ramakrishna R, Santosh KM, Kannappan N.In

vivoAntidiabetic evaluation of Neem leaf extract in alloxan induced rats. J

Appl Pharm Sci 2011; 1: 100-105.

[69] Lin LY. Hydroxy propyl methyl cellulose.In: Hand Book of Pharmaceutical

Excipients.Great Britain: American Pharmaceutical Association and the

Pharmaceutical Society of Great Britain; 1986.p.187-92.

[70] Indian Pharmacopoeia. Hydroxy propyl methyl cellulose. Vol II.New Delhi:

Ministry of Health and Welfare, Government of India; 1996.

[71] Verma A, Mogra R. Psyllium (Plantago ovata)Husk: A wonder food forgood

health. International Journal of Science and Research 2015; 4 (9): 1581-

85.

[72] Al-Assaf S, Phillips GO, Williams PA, Takigami S, Dettmar P, HavlerMA.

Molecular weight, tertiary structure, water binding and colon behaviour of

ispaghula husk fibre. Proceedings of the Nutrition Society 2011; 62:211-21.

References

131 | P a g e

[73] Tewari D,Anjum N, Tripathi YC. Phytochemistry and pharmacology of

Plantago ovata: a natural source of laxative medicine. WJ of Pharma Res

2014; 3(9): 361-372.

[74] Raymond CR, Paul JS, Marian E. Quinn Handbook of Pharmaceutical

Excipients. 6th ed.NY: Pharmaceutical Press and American Pharmacists

Association; 2009:404

[75] Great Britain. British Pharmacopoeia. Vol II 2001. International ed.London:

Department of Health; 2009 : 2381

[76] The Merck Index- An Encyclopedia of drugs, chemicals and biologicals.

13thed.New Jersey: Merck and CO.Inc.; 1996:385

[77] Khandelwal KR. Practical Pharmacognosy Techniques And Experiments.

Pune: Nirali Prakashan; 2012 :157

[78] Das BK, Mukherjee SC, Sahu BB, Murjani G. Neem (Azadirachta indica)

extract as an antibacterial agent against fish pathogenic bacteria. Indian J

Exp Biol 1999; 37:1097–1100.

[79] Dholi SK, Raparla R, Mankala SK, Nagappan K.Invivo Antidiabetic

evaluation of Neem leaf extract in alloxan induces rats. J of Appl Pharma

Sci 2011; 01:100-105.

[80] Deshpande PK, Gothalwal R, Pathak AK.Phytochemical analysis and

evaluation of antimalarial activity of Azadirachta indica. The Pharma

Innovation Journal 2014; 3 (9): 12-16.

References

132 | P a g e

[81] Aswatharam HN, Lachake P, Kaushik U, Shreedhara CS. Formulation and

evaluation of floating tablets of liquorice extract. Pharmacognosy Res

2010; 2 (5): 304–308.

[82] Nanjwade BK, Adichwal SA, Nanjwade VK, Gaikwad KR, Thakare SA,

Manvi FV. Development and evaluation of gastroretentive floating tablets

of Glipizide based on effervescent technology. J Drug Metab Toxicol 2012;

3 (3): 1-5.

[83] Swarnakamala Chinthala CH, Reddy Kota KS, Hadassah M, Metilda EH,

Sridevi S. Formulation and evaluation of gastroretentive floating tablets of

gabapentin using effervescent technology. Int J Pharm Biomed Res 2012;

3(4): 202-208.

[84] Pawar HA, Dhavale R. Development and evaluation of gastroretentive

floating tablets of an antidepressant drug by thermoplastic granulation

technique. Beni-suef Univ Journal of basic And Applied Sciences 2014; 3

(1): 122-132.

[85] Yasir M, Asif M, Bhattacharya A, Bajpai M. Development and evaluation of

gastroretentive drug delivery system for theopylline

using psyllium husk. Internat J of ChemTech Res 2010; 2(2): 792-799.

[86] Costa P, Manuel J, Lobo S. Modeling and comparison of dissolution

profiles. Eur J of Pharm Sci 2001; 13: 123–133.

http://www.ncbi.nlm.nih.gov/pubmed/?term=Ram%20HN%5Bauth%5D

References

133 | P a g e

[87] Ramteke KH, Dighe PA, Kharat AR, Patil SV. Mathematical Models of

Drug Dissolution: A Review. Sch Acad J Pharm 2014; 3(5): 388-396.

[88] Patil SH, Talele GS.Formulation development and in vitro and in vivo

evaluation of gastroretentive floating drug delivery system of Lafutidine.

Asian J of Pharmaceutics 2013; 7 (2): 68-74.

[89] Akin-Osanaiye BC, Nok AJ, Ibrahim S, Inuwa HM, Onyike E, Amlabu E,

Haruna E. Antimalarial effect of neem leaf and neem stem bark extracts on

Plasmodium berghei infected in the pathology and treatment of malaria.

International J of Res in Biochem and Biophy 2013; 3 (1): 7-14.

[90] Vathsala PG, Dende C, Nagaraj VA, Bhattacharya D, Das G, Rangarajan

PN, Padmanaban G. Curcumin-arteether combination therapy of

Plasmodium berghei-infected mice prevents recrudescence through

immunomodulation. PLoS One 2012; 7(1): 1-10.

[91] Nandakumar DN, Nagaraj VA, Vathsala PG, Rangarajan PG,

Padmanaban G. Curcumin-artemisinin combination therapy for malaria.

Antimicrob Agents Chemother 2006; 50(5): 1859-60.

[92] http://www.cdc.gov/dpdx/resources/pdf/benchAids/malaria/Parasitemia_a

nd_LifeCycle.pdf: 1-2

http://www.asiapharmaceutics.info/searchresult.asp?search=&author=Satish+H+Patil&journal=Y&but_search=Search&entries=10&pg=1&s=0

http://www.asiapharmaceutics.info/searchresult.asp?search=&author=Gokul+S+Talele&journal=Y&but_search=Search&entries=10&pg=1&s=0

Documents

Formulation Development and Pharmacological Evaluation of ... · Formulation Development and Pharmacological Evaluation of Herbal Antimalarials Thesis Submitted in Partial Fulfillment