J.S.S. COLLEGE OF PHARMACY
Sri Shivarathreeshwara Nagara Mysore - 570015
DECLARATION BY THE CANDIDATE
I hereby declare that this dissertation/thesis entitled “PREPARATION AND
EVALUATION OF AN ANTACID AND ANTIULCER SUSPENSION
CONTAINING HERBAL DRUGS” is a bonafide and genuine research
work carried out by me under the guidance of Mr.R.S.Bhat, Assistant
Professor, Department of Pharmaceutics, J.S.S.College of Pharmacy, Mysore-15.
Date: Signature of the Candidate Place: Mr.Murthy Sudarshan Dakshina
J.S.S. COLLEGE OF PHARMACY
Sri Shivarathreeshwara Nagara Mysore - 570015
CERTIFICATE BY THE GUIDE This is to certify that the dissertation entitled “PREPARATION AND
EVALUATION OF AN ANTACID AND ANTIULCER SUSPENSION
CONTAINING HERBAL DRUGS” is a bonafide research work done by
Mr.Murthy Sudarshan Dakshina in partial fulfillment of the requirement
for the degree of Master of Pharmacy (Industrial Pharmacy).
Date: Signature of the guide Place: Mysore Mr.R.S.Bhat Assistant Professor Dept. of Pharmaceutics
J.S.S.College of Pharmacy Mysore-15.
CERTIFICATE
ENDORSEMENT BY THE HOD, PRINCIPAL/HEAD OF THE INSTITUTION This is to certify that the dissertation entitled “PREPARATION AND
EVALUATION OF AN ANTACID AND ANTIULCER SUSPENSION
CONTAINING HERBAL DRUGS” is a bonafide research work done by
Mr.Murthy Sudarshan Dakshina under the guidance of Mr.R.S.Bhat,
Assistant Professor, Dept. of Pharmaceutics, J.S.S.College of Pharmacy,
Mysore-15.
Seal & Signature of the HOD Seal & Signature of the Principal Dr. H.G. SHIVAKUMAR Dr. B.G.NAGAVI
Vice Principal & Head, Principal
Department of Pharmaceutics, J.S.S. College of Pharmacy
J.S.S. College of Pharmacy, Mysore.
Mysore. Date:
COPYRIGHT
DECLARATION BY THE CANDIDATE
I hereby declare that the Rajiv Gandhi University of Health Sciences,
Karnataka shall have the rights to preserve, use and disseminate this
dissertation / thesis in print or electronic format for academic / research
purpose.
Date: Signature of the Candidate Place: Mr. Murthy Sudarshan Dakshina
© Rajiv Gandhi University Of Health Sciences, Karnataka
ACKNOWLEDGEMENT
I am grateful to my esteemed guide Mr.R.S.Bhat, Asst.Professor, Dept.of
Pharmaceutics, for his generous support, valuable suggestions, inspiring encouragement,
and keen interest that greatly eased the task of completing my work.
My sincere thanks to Dr.B.G.Nagavi, Professor and Principal, J.S.S.College of
Pharmacy, Mysore and to Dr.H.G.Shivakumar, Professor and Head of Department of
Pharmaceutics, J.S.S.College of pharmacy, Mysore for providing the necessary facilities
required and moral support for carrying out this work.
I express my sincere gratitude to Mr.V.S.Datta Murthy, Head of the Department
of Pharmaceutics, PES College of Pharmacy, Bangalore and Dr.T.N.Nagraja, vice
principal J.S.S.Ayurveda Medical College, Mysore for providing invaluable guidance on
herbal drugs.
I sincerely thank M/S. Sami Labs Ltd, Mysore for providing me gift sample of
Glycyrrhetic acid.
I am thankful to Mr.P.K.Kulkarni, Asst.Professor, Dept.of Pharmaceutics,
J.S.S.College of pharmacy, Mysore for his valuable suggestions that helped to complete
my dissertation work without difficulty.
I am also very thankful to Mr.Mahadevan, Asst.Professor, Dept. of
Pharmacognosy, J.S.S.College of pharmacy, ooty for carrying out the HPTLC analysis of
the samples for the purpose of stability studies.
I am especially thankful to the teaching staff Mr.D.Vishakantegowda,
Mr.T.M.Pramodkumar, Mr.C.S.Satish, Mr. N.M.Mahesh, Mr.Madhusudhan Purohit and
I
Mr.D.H.P.Gowda and non-teaching staff Ms. Namitha shivanna, Mr.Somanna, Mr. Suresh
and Mr.Shankar for their cooperation towards my work.
I am extremely grateful to all the albino rats that have laid their life for my work
without which the work would have been half complete.
I remember all my friends Krishna, Sumit, Subhash, Shafiullah, Gunashekhar,
Satish, Shankar, Chetan, Rajesh, Harsha, Naveen Vishwnath, Manohar, Jayadev,
Kumarswamy, Venkatesh, Solanki, who by their honest opinions and diligence kept me
lively and all my juniors who by their honest opinions and diligence kept me lively.
I am always indebted to my family members for their overwhelming enthusiasm,
love and affection.
I wish to express my gratitude to umesh and manju for their invaluable help in
scientific illustrations and printing of manuscript.
I submit my humble pranamas to the lotus feet of Sri Shivarathreeshwara
Deshikendra Mahaswamiji.
I thank the Almighty for giving me courage and confidence when nobody else could
and for being there in every walk my of life.
Place:
Date: Mr. Murthy Sudarshan Dakshina
II
ABSTRACT
Gastric ulcer affects about 60% of the adults and about 80% of the child population
in the tropical countries. The herbs Glycyrrhiza glabra, Terminalia chebula, Terminalia
belerica, Emblica officinalis and mineral Turbinella rapa are reported in classical
Ayurvedic texts to possess anti-phlogistic activity, astringent activity and acid
neutralization activity that is desirable for the treatment of gastric ulcer. Hence they can be
combined in a suitable formulation to provide an effective treatment for gastric ulcer. The
individual herbs were identified by thin layer chromatographic analysis and evaluated for
the foreign organic matter, ash value, acid insoluble ash values, water-soluble and alcohol
soluble extractive values. The values obtained were within the limits prescribed in the
pharmacopoeia. The total tannin contents of the herbs Terminalia chebula, Terminalia
belerica and Emblica officinalis were estimated colorimetrically by Folin-Dennis method.
The mineral drug Shankha Bhasma was evaluated for its calcium carbonate content and
acid neutralization capacity as per the reported methods. The extracts of herbs Terminalia
chebula, Terminalia belerica and Emblica officinalis were obtained by cold maceration
process using 95% ethyl alcohol as the macerating solvent. These herbs and their extracts
were formulated into a suspension with Xanthan gum as the suspending agent to produce
cumulative antacid and anti-gastric ulcer activity. The concentration of the Xanthan gum
was varied as 0.2%, 0.25%, 0.3% and 0.35%.
The suspensions were then evaluated for the pH, Viscosity, sedimentation volume,
redispersibility, antacid and antiulcer activity. All the formulations showed a pH in the
basic range about 8.2 and ANC between 2-3mEq/ml. All the formulation showed high
sedimentation volume and good redispersibility .The formulation containing extracts of the
III
herbs showed significant decrease in the ulcer index as compared to one containing the
powders of these drugs The stability studies on the selected formulations were performed
at 30ºC/ 65%RH and 40ºC /75%RH for 90 days. The formulations were found to be stable
at the end of 90 days.
IV
LIST OF ABBREVIATIONS
λMax Absorption maxima
Conc. Concentration oC Degree centigrade
RH Relative Humidity
% Percentage
mEq Milliequivalents
ANC Acid neutralization
Capacity
hr Hour
R.T. Room temperature
μg Microgram
Rf Retention factor
mg Milligram
ml Milliliter
Min Minute
nm Nano meter
N Normality
% Percent / percentage
g grams
mcg microgram
SD Standard deviation
UV Ultra Violet
NLT Not Less Than
NMT Not more than
W/v Weight /volume
W/w Weight / weight
r.p.m Rotations per minute
V
CONTENTS
Sl.No Chapter Page No
1. Introduction 01
2. Literature Review 05
3. Materials And Methods 16
4. Results And Discussions 35
5. Conclusions 49
6.
7.
References
Appendices
51
54
VI
LIST OF TABLES
Sl.No Tables Page No
1. Formulation chart of the Suspension 30
2. Chart of ulcer score 34
3 Qualitative Analysis of Shankha Bhasma 35
4 Determination of foreign organic matter 36
5 Determination of Total ash value & acid insoluble ash value 37
6 Determination of alcohol &water soluble extractive value 38
7 TLC Identification of Individual Herbs 39
8 Standard curve of tannic acid in distilled water 40
9 Total tannin content in Triphala powder and extract 41
10 PH and viscosity of the suspension 42
11 Redispersibility and Sedimentation volume of suspension 43
12 Acid neutralization capacity of the suspension 44
13 Anti-ulcer activity of the suspension 45
14 Estimation of gallic acid by HPTLC &TLC ofsuspension at 0th Day and 90th Day
46
15
16
Viscosity, pH and ANC of the suspension at 0th, 30th, 60th and 90th day Organoleptic properties of the suspensions at 0th, 30th, 60th and 90th day
47
48
VII
LIST OF FIGURES
Sl.No Figures/Graphs Page No
1. UV Spectra of tannic acid in distilled water. 25
2. TLC identification of the herbs 39(a)
3. Calibration curve of tannic acid in distilled water at 703 nm
40
4. Particle size distribution data of suspension 41
5. Histopathological studies of anti-ulcer activity 45(a)
6. HPTLC graph of gallic acid (standard) 46(a)
7. HPTLC graph of gallic acid (FI-3) at 0th Day 46(b)
8. HPTLC graph of gallic acid (FII-2) at 0th Day 46(c)
9. HPTLC graph of gallic acid (FI-3) at 90th Day at 30°C/65%RH 46(d)
10. HPTLC graph of gallic acid (FI-3) at 90th Day at 30°C/65%RH 46(e)
11. HPTLC graph of gallic acid (FII-2) at 90th Day at 40°C/75%RH 46(f)
12. HPTLC graph of gallic acid (FII-2) at 90th Day at 40°C/75%RH 46(g)
13. TLC of the formulations at 0th Day and 90th Day 46(h)
VIII
Ayurveda the traditional system of medicine continues to be matchless so far its perfection
in health care is concerned.It's time tested theurapeutics based on the use of natural
resources has broken all the barriers and is gradually making inroads in the domain of the
allopathy and rightly gaining more popularity not only at home but abroad too.
The word "Ayurveda" is a combination of the two words "Ayuh" and "Veda"
which literally means knowledge about the life .The origin of the Ayurveda is based on the
vedas which are said to be the oldest classics of the world written between 10,000-4,000
B.C.1
Gastric (mainly peptic) ulcer is one of the major factors affecting about 60% of the
human adults and about 80% of the child population in the tropical countries individuals. It
is interesting to note that nearly 50% of the Indian population harbors the disease.
Peptic ulcer as a disease is characterized by a group of disorders described
lesions of the upper gastrointestinal tract (with stomach and duodenum being the most
affected areas
Peptic ulcer is defined as the pathological lesions and ulcers of any portion of the
gastrointestinal tract exposed to the acid activated pepsin. Even though the acid and pepsin
are the contributing factors, current reasoning incorporates additional factors into the
etiology of the disease.
Gastric ulcers also occur due to the intense irritation of the various reflexogenous
zones and portions of the nervous system as well as due to the unfavorable emotions also.
The dependence of the gastric ulcers on the seasons has also been noted, mostly prevalent
1
in the spring and autumn seasons. Further several studies also indicate that gastric ulcers
result from the malnutrition in the intestinal mucosa.
The appearance of the gastric ulcers has also been attributed to the antral
infection of the gastric mucosa by the organism, Helicobacter pylori.
The various factors that are responsible for causing peptic ulcers can be categorized into
the following: -
General factors
Vagal effects, hormonal effects (histamine), insufficient circulation, shock, general
ischemia etc
Constitutional and environmental factors
Sex, age, temperament, family history, social class, geographical difference, occupation
etc.
Local factors in the stomach
Aggressive factors
Hydrochloric acid, Pepsin, refluxed bile, on-steroidal Anti-inflammatory Drugs,
Alcohol, Proteolytic enzymes, ingested irritants, bacterial toxins, Physicochemical trauma,
etc
Defensive factors
Mucus, bicarbonates, blood flow, restitution of epithelium, prostaglandins,etc.
Microbial factors
Helicobacter pylori infection.
It is generally acknowledged that an ulcer results from an imbalance between the
Aggressive gastric factors and the defensive (resistance) factors.2, 3
2
It is a firm belief that the drugs of the plant origin have very little or no side effects and in
view of this property herbal drugs are preferred over the synthetic drugs. The practitioners
of the Ayurveda since ages have been reaping the advantages of the medicinal plants in
one form or the other, offering comparatively cheaper and safer health remedies to the
masses. The use of the plant derived crude drugs for the treatment and cure drugs for the
treatment and cure of the chronic and refractive diseases is a healthy sign of our great
tradition.
Hence the present study involved preparing an antacid and anti-ulcer
suspension containing these herbal drugs.
3
OBJECTIVES
. To carry identification tests for the herbs and minerals.
. To carry out the monographic analysis of the ingredients.
. To extract the active principles from the herbs.
. To prepare the suspension and carry out the invitro evaluation.
. To evaluate the anti-ulcer activity of the Prepared Suspension.
4
Gastric and duodenal ulcers, gastrooesophageal reflux disease are the gastrointestinal
disorders sharing a common abnormality: too much acid and pepsin activity for the local
tissue resistance. The therapy for these disorders is directed at the correction of an apparent
imbalance between the acid and pepsin activity and the mucosal resistance. The success of
the therapy is measured in terms of ulcer healing, symptom control, relapse rate etc.
The strategy adopted for the treatment of the gastric ulcers may involve one or more of the
following mechanism of action: -
Inhibition or neutralization of the gastric acid.
Inhibition of the pepsin activity.
Resistance to pepsin activity.
Inhibition of the bile reflux or absorption of the bile.
Gastro protective actions like
a) Increased bicarbonate secretion at the mucus gastric fluid interface.
b) Increased mucus secretion.
c) Increased surfactant formation.
d) Increased cell migration (cell restitution).
e) Increased cell division.
f) Normalization of the cell metabolism.
g) Anti-helicobacter pylori compounds.
h) Preventing the degradation of gastro- protective Prostaglandins.3
5
Herbal compounds including have been used for a variety of the disorders of the
gastrointestinal tract. The herbal drugs are considered to be safe for use, easily available at
cheaper cost and produce minimal side effects.
Goel R.K, Pathak N.K.R have studied the anti-ulcer properties of trunk,
bark and wood chips of Tectona grandis on aspirin induced gastric ulcers and histamine
induced duodenal ulcers in rats. They have reported that the plant parts reduced the
increased peptic activity induced by aspirin and also increase in sialicacid and mucin
secretion. The active constituent was reported to be lapachol.4
Banerjee RS et al have studied the antiulcer activity of shilajit a
herbomineral compound on pylorus ligation induced and aspirin induced gastric ulcer and
cystamine induced and histamine induced duodenal ulcers in rats.They have reported that it
exhibited its anti-ulcer activity by reducing acid pepsin secretion and increase in mucin
secretion.5
Sairam K et al have reported the anti-ulcer activity of the centella Asiatica
whole plant on cold restraint stress and aspirin induced gastric ulcers in rats. They have put
forth that the fresh juice of the plant exhibited anti-ulcer activity by increasing the mucin
secretion and increasing the lifespan of the mucosal cells.6
Glycyrrhiza glabra, which is known as the yashtimadhu in Ayurveda, is
known to possess anti-phlogistic activity (increasing the mucus secretion and its viscosity).
It is also reported in various literatures that liquorice inhibits the enzymes that degrade the
gastrocyto-protective Prostaglandins E and Fα thus raising their concentration.7 Joshi
V.K.et al have reported the anti-ulcer activity of liquorice on pylorus ligation and cold
6
restraint stress induced gastric ulcers in rats. They have reported that water decoction of
the root enhances the mucosal defensive factors and thus exhibits anti-ulcer activity.8
Terminalia Chebula, Terminalia belerica and Emblica officinalis are astringent
compounds. In Ayurveda these are combined in equal proportions to give an Ayurvedic
preparation “TRIPHALA CHURNA”, which is being used for a variety of the ailments.
These have a property of forming a complex with the proteins. This complex has been
proved to be resistant to the proteolytic enzymes.9 Rege N.N et al have reported the anti-
ulcer activity of fresh juice of the fruits Emblica officinalis on aspirin induced, pylorus
ligation induced and ethanol induced gastric ulcers in rats.They have reported that
antiulcer activity is exhibited by enhancement of mucosal defensive factors10.
Shankha Bhasma, which is the ash of conch shell, contains mainly calcium carbonate has
the property to neutralize the acid.11
The properties that these drugs possess and the strategies that have been put forth for the
treatment of gastric ulcers prompted us to prepare a polyherbal formulation containing
these herbs and minerals and assess the extent of antacid and antiulcer activity.
The anti-ulcer activities of the suspension claiming anti-ulcer activity were
studied. It was found that suspension containing varatika, Dugdhapashana, Mouktika
shukti powders and extract of liquorice and which was processed in amalaki, guduchi and
punarnava showed significant decrease in ulcer index 12.
7
Suspension as a dosage form offers the following advantages13
1) As the disintegration step of the tablets and capsules are absent, it permits quicker
onset of action,
2) As the particle size of the suspension is small, it permits quicker dissolution of the
drug in the gastrointestinal fluids and hence quicker onset of the action.
3) Suspension has the advantage of the flexibility of the dosage that is particularly
advantageous for an antacid and antiulcer suspension since symptoms of acidity
depend upon a variety of conditions and varies from patient to patient.
4) Suspension has the advantage of ease of administration than tablets and capsules.
Requirements of a good suspension14
1) Suspended particles should not settle down to form a hard cake.
2) Suspended materials settled down should be easily redispersible.
3) Suspension should have a very good viscosity so that it can be easily poured out of
the bottle and should have elegant appearance, smell and taste.
4) The particles should remain dispersed for sufficient time for dose to be
administered.
8
Individual monographs of the drugs are briefly described below 15, 16
TERMINALIA CHEBULA (Haritaki)
Biological Source
It consists of the dried, ripe and fully matured fruits of Terminalia chebula Retz.
Belonging to the family Combretaceae.
Other Names
Hindi :- Harara, Bal-har.
Malayalam :- Kadukka.
Telgu :- Karaka, Karakkaya.
Kannada : - Alalekayi.
English :- Chebulic myrobalan , Indian gallnut.
Phytochemistry
Myrobalan fruits are an important source of the tannins. The tannins of the myrobalan
fruits are of Pyrogallol type that on hydrolysis yields chebulic acid and d-galloyl glucose.
Chebulagic acic, chebulinic acid ellagic acid and gallic acid are the other constituents of
the myrobalan.
Marker constituent Gallic Acid.
9
TERMINALIA BELERICA (Vibhitaki)
Biological Source
It consists of the dried ripe fruits of the Plant, Terminalia belerica Roxb.belonging to the
family Combretaceae.
Other Names
Hindi :- Bhaira, Bahera.
Malayalam :- Tannikai, Tanni.
Telgu :- Tani.
Kannada : - Tarekayi.
English :- Beleric myrobalan .
Phytochemistry
The fruit contains about 20-30% of the tannins. It contains gallic acid, ellagic acid,
Phyllemblin, ethyl gallate and galloyl glucose. It also contains most of the sugars as
reported in the myrobalan.
Marker Constituent Gallic Acid
10
EMBLICA OFFICINALIS (Amalaki)
Biological Source
It consists of the fresh or dried fruits of Emblica officinalis Gaerth belonging to the family
Euphorbiaceae.
Other Names
Hindi :- Amla, Aonla.
Malayalam :- Nelli.
Tamil :- Nelli.
Kannada : - Nelli,Amlalaka.
English :- Indian Gooseberry,Emblic myrobalan.
Phytochemistry
It contains the hydrolysable tannins as the major bioactive constituents along with gallic
acid and ellagic acid. It also contains alkaloids Phyllantidine and Phyllantine,Pectin and
minerals. It also contains Vitamin C.
Marker Constituent Gallic Acid.
11
GLYCYRRHIZA GLABRA (Yashtimadhu)
Biological Source
It consists of dried, peeled or unpeeled roots and stolons of Glycyrrhiza glabra Linn.
belonging to the family Leguminosae.
Other Names
Hindi :- Mulethi.
Sanskrit :- Jyesthamadhu.
Tamil :- Atimadhuram.
Kannada : - Atimadhura.
English :- Liquorice.
Phytochemistry
The chief constituent of liquorice is the triterpenoid saponin glycyrrhizin which is the
potassium and calcium salt of the glycyrrhizinic acid. Other constituents are flavonoids,
Liqueritin and Liquertigenin, Isoliqueritin, glycyramarin resins, asparagin, sucrose.fats and
glucose.
Marker constituent Glycyrrhetic acid.
12
Conch shell ash (Shankha Bhasma)
Biological Source: -
It is the ash of the conch shell of the shell fish Turbinella rapa belonging to the family
mollusca. It is prepared by soaking the shell in the limejuice and calcinating covered
crucible ten to twelve times and finally reducing it to the powder (ash).
Other Names: -
Sanskrit :- Shankha Bhasmam.
Telgu :- Sehkham.
Kannada : - Shankha Bhasma.
English :- Conch Shell Ash.
Constituents: -
It is mainly composed of calcium carbonate (about 94%). In minor amounts it also
contains magnesium, manganese and traces amounts of other minerals.
Marker Constituent Calcium Carbonate
13
Xanthan Gum NF 17
Description
It is a natural anionic biopolysaccharide made up of different monosaccharides, mannose,
and glucose and glucuronic acid.
It is soluble in water but insoluble in organic solvents. This gum can tolerate upto 50% of
water miscible organic solvents.
Xanthan gum exhibits pronounced pseudoplastic rheology with a definite yield point.
Enzymes that degrade other natural polymers such as cellulose do not affect Xanthan gum
that is due to the uniform polymer structure and shielding of the polymer backbone side
chains.
This polymer is resistant to shear depolymerisation.
Xanthan gum has a good stability from pH 1 to 12.
Xanthan gum has good temperature stability. Its viscosity is not affected by temperature
changes.
Other features of the Xanthan gum are that it exhibits high viscosity at low concentration
and compatible with the divalent and polyvalent ions such as calcium salts, with tannins
etc.
14
Sorbitol Solution 70% (Non-Crystallizing) 18,19
Sorbitol solution 70% (Non-Crystallizing) is an aqueous solution of hydrogenated, partly
hydrolyzed starch.
Description
It is clear, colorless or faintly yellow, syrupy liquid and odorless and is miscible with
water.
Uses
It is widely used as a vehicle in sugar free formulations. It is used as a stabilizer in drug
and vitamin formulations. It is widely used in antacid suspensions as a stabilizer,
sweetening vehicle and as an anti-caking agent.
Concentrations to be used
Sorbitol solution (70%) can be used upto a concentration of 70%.
15
INSTRUMENTS
Digital Balance Shimadzu Corporation, Japan.
Electric Furnace Tempo Instruments and Equipments
Pvt. Ltd., Mumbai.
Microscope Labfit.
Digital pH Meter Systronics.
Silica Crucibles
Stage and Eyepiece micrometer Erma, Japan.
TLC developing Chamber and Plates
UV-VIS Spectrophotometer UV-1601 Shimadzu Corporation,
Japan
Brookefields Viscometer model DV-III+ Modrobs Grant,Germany
HPTLC Apparatus Cadrach
Homogeniser Shreeji Chemicals,Mumbai
Hot Air Oven Tempo Instruments and Equipments
Pvt. Ltd.,Mumbai
16
MATERIALS
Plant Materials
Glycyrrhiza glabra Linn. NKCA Pharmacy, Mysore
Terminalia chebula NKCA Pharmacy, Mysore
Terminalia belerica NKCA Pharmacy, Mysore
Emblica officinalis NKCA Pharmacy,Mysore
Turbinella rapa Ashwini Oushadalaya,Mysore
Herbal Extracts Himalaya Drug Company, Bangalore
CHEMICALS
Acetone Thomas Baker Chemicals Ltd., Mumbai.
Anisaldehyde Loba Chemie Pvt.Ltd., Mumbai
Acetic Acid Glacial Ranbaxy Fine Chemicals Ltd.,New Delhi.
Ammonium Oxalate Reachem Lab. Chemicals Pvt. Ltd., Chennai
Calcium Carbonate Thomas Baker Chemicals Ltd.,Mumbai
Chloroform Thomas Baker Chemicals Ltd.,Mumbai
Carmoisine Creative Aromatics Pvt. Ltd., Chennai
17
Dimethylpolysiloxane Sigma Chemicals Ltd., Bangalore
Ethyl Acetate Thomas Baker Chemicals Ltd.,Mumbai
Ethyl Alcohol Reachem Lab.Chemicals Pvt. Ltd., Chennai
Formic Acid Thomas Baker Chemicals Ltd.,Mumbai
Folin Dennis Reagent S.D.Fine chemicals Pvt.Ltd.
Gallic Acid Thomas Baker Chemicals Ltd.,Mumbai
Glycyrrhetic Acid Sami Labs. Ltd., Mysore
Iodine Thomas Baker Chemicals Ltd.,Mumbai
Menthol Crystals Loba Chemie Pvt. Ltd., Mumbai.
Methanol Reachem Lab. Chemicals Pvt. Ltd., Chennai.
Methyl Paraben Loba Chemie Pvt.Ltd., Mumbai
Potassium Permagnate Loba Chemie Pvt.Ltd., Mumbai
Silica Gel GF254 Thomas Baker Pvt.Ltd., Mumbai
Sodium Carbonate Reachem Lab.Chemicals Pvt.Ltd.,Chennai
Sodium hydroxide Reachem Lab.Chemicals Pvt.Ltd.,Chennai
Sorbitol 70% liquid Loba chemie Pvt.Ltd., Mumbai.
Sulphuric Acid Ranbaxy Fine Chemicals, New Delhi.
Tannic Acid Thomas Baker Pvt.Ltd., Mumbai
Toulene Ranbaxy Fine Chemicals, New Delhi.
Xanthan Gum Loba Chemie Pvt. Ltd.,Mumbai
18
Reagents
Chloroform Water
Shaken 2.5 ml of Chloroform water with about 700 ml of distilled water until dissolved
and made the volume to 1000 ml with distilled water.
Anisaldehyde Sulphuric Acid
0.5 ml of Anisaldehyde is mixed with 10 ml of glacial acetic acid, followed by 85 ml of
methanol and 5 ml of concentrated Sulphuric acid. The TLC Plate with about 10 ml heated
at 100 ºC for about 5-10 min and then evaluated in the normal light.
19
METHODS
Qualitative and Quantitative chemical tests for Shankha Bhasma.20, 21
Qualitative tests
Test for the presence of calcium
a) Solution of Bhasma in dilute hydrochloric was treated with ammonium oxalate.
b) Solution of Bhasma in dilute hydrochloric was treated with potassium ferrocyanide.
Test for the presence of carbonate
a) Bhasma was treated with dilute hydrochloric acid and the liberated gas was passed
through limewater.
b) To about 5ml of the Bhasma solution in dilute hydrochloric acid, added 2ml of
10%w/v solution of magnesium sulphate was added.
Test for the presence of magnesium
The precipitate obtained from ammonium oxalate was filtered and the filtrate was
concentrated highly. To this concentrate solution sodium hydroxide pellets were added
and dissolve and then heated to remove ammonia.
Test for the presence of Manganese
The filtrate obtained after separation of calcium oxalate was treated with ammonium
sulphide, well stirred and left overnight.
20
Determination of calcium carbonate content in Shankha Bhasma
This was determined by redox titration method. The oxalate ion from calcium oxalate was
titrated against standard potassium permanganate. Potassium permanganate is a self –
indicator. The reactions are mentioned below: -
5CaC2O4 + 2KMn O4 + 8H2 SO4 2MnSO4 + K2 SO4 + 5CaSO4 + 8H2 O+ 10CO2
Procedure
Accurately weighed about 0.25 g of Bhasma and dissolved in 5ml of Hydrochloric acid
(1:1) and then added 50ml of 0.5N of ammonium oxalate and two drops of methyl orange
indicator. Then 10% of aqueous ammonia was added to it drop wise till pink color of the
solution turned to yellow, it was then heated to about 70-80 ºC & left for 1 hr. Then filtered
through whattman filter paper no.41 and washed thoroughly with excess of dilute (0.1N)
Ammonium Oxalate solution. The precipitate obtained from Bhasma was dissolved in 1:1
Hydrochloric acid again and then reprecipitated and washed as above. The precipitate
obtained from the Bhasma was washed thoroughly with distilled water till filtrate showed
negative test for oxalate (no discoloration of acidic KMnO4). The precipitate was then
dissolved quantitatively in 70ml of 10%Sulphuric acid and volume was made upto 100ml
with the 10% Sulphuric acid. The solution was then titrated against standard potassium
permanganate till a drop gave slight pink color persistent for 1min.
21
Determination of the antacid activity22
This was determined by Acid neutralization capacity test as mentioned in the USP. As
per the USP, an antacid substance has to raise the gastric pH above 3.5 within 15 min.
In this test, the number of milliequivalents of acid neutralized by 1 gm of Bhasma was
determined. Based upon the Acid neutralizing capacity, the dose of the Shankha Bhasma
was calculated and incorporated .
Procedure
Accurately weighed about 500mg of the shankha Bhasma and to it 30 ml of the
standardized 1N hydrochloric acid was added. Then it was stirred for 15 min. accurately
timed after the addition of the acid. Then it was titrated immediately (in a period not to
exceed an additional 5min.) with 0.5N sodium hydroxide to attain a stable pH of 3.5.
Calculated the number of milliequivalents of acid consumed and the result was expressed
in terms of milliequivalents of acid consumed per gm of Bhasma tested. The ANC in mEq
was calculated as shown below:
Each ml of IN Hcl consumed = 1mEq. Of acid consumed.
The same was converted into ANC (mEq) / gm of the Bhasma.
22
Monographic Studies on the herbal drugs23
Foreign Organic Matter.
Weighed 100 g of the sample and spread out as a thin layer and separated the foreign
organic matter. It was again weighed and percentage was calculated and values were
compared with the Pharmacopeial limits.
Total Ash
Incinerated 3 g of the drug accurately weighed in a silica crucible at a temperature not
exceeding 450 ºC until free of carbon. It was then cooled and weighed. Then calculated the
percentage ash with reference to the air-dried drug.
Acid Insoluble ash
Boiled the ash as obtained above for 5 min. with about 25ml of dilute hydrochloric acid.
The insoluble matter was collected on an ashless filter paper, washed with hot water and
then ignited to a constant weight. Calculated the percentage of the acid insoluble ash with
reference to the air-dried drug.
Alcohol Soluble extractive value
Macerated 5 g of coarsely powdered air-dried drug with about 100ml of ethanol in a closed
flask for 24 hrs, frequently shaken for 6 hrs and allowed to stand for 18 hrs. Then it was
filtered and 25 ml of the filtrate was evaporated to dryness in a tared flat-bottomed shallow
dish. Calculated the percentage of the alcohol soluble extractive with reference to the air-
dried drug.
Water Soluble extractive value
The same procedure was carried out as described under alcohol soluble extractive value
But here the extracting solvent used was chloroform water. The percentage of water-
soluble extractive value was calculated with reference to the air-dried drug.
23
Preparation of the Triphala powder24
Accurately weighed about 100 gms of the herbs Terminalia Chebula,Terminalia belerica
and Emblica officinalis. These were then passed individually through 120# mesh
separately. Then equal quantities of these herbs were taken in the mortar and then mixed
with the help of the pestle to give triphala powder. This powder was then used for
subsequent procedures.
Preparation of the Triphala extract 25
100 g of each of the herbs Terminalia chebula, Terminalia belerica and Emblica officinalis
(obtained after passing through 120#) were separately macreated with 95% of ethyl alcohol
for 4 days. Then it was filtered and the filtrate was evaporated to dryness (at a temperature
not to exceed 50ºC) when gummy mass was obtained. It was further dried at a temperature
not to exceed 50ºC in a vacuum drier .The powder obtained was passed through 120#.
Then the equal quantities of the extracts obtained from these herbs were mixed to get the
triphala extract. This was used for subsequent procedures.
Spectrophotometric estimation of tannins in the triphala powder and triphala extract
by Folin-Dennis Method 26,27
Principle
The method is based upon the oxidation of the molecules containing a phenolic hydroxyl
group. The tannins reduce phosphotungustomolybdic acid in alkaline medium to produce a
highly colored blue solution, the intensity of which is proportional to the concentration and
hence to the amount of the tannins and can be estimated against the standard tannic acid
solution at a wavelength of 700nm.
24
Fig1. Determination of the λmax. Of the tannic acid in distilled water.
Preparation of the standard solution of tannic acid in distilled water
Accurately weighed 100mg of tannic acid and dissolved in 100ml of distilled water in a
100 ml of volumetric flask. Then 10ml of the resulting solution was taken in another 100
ml volumetric flask and diluted upto the mark with the distilled water to give a
concentration of 100μg/ml solution. From this solution was pipetted out
0.2,0.4,0.6,0.8,1.0,1.2 and 1.4 ml into a series of 10 ml volumetric flask. To these solutions
were added 1ml of (1:10) folin’s reagent and 0.8 ml of 7.5% sodium carbonate solution.
Allowed to stand for the development of the color and then volume was made upto 10 ml
with distilled water. Then recorded the absorbance of the solution and plotted the
calibration graph.
25
Preparation of the sample solution
a) For Triphala powder
100 mg of the triphala powder was taken in 100ml volumetric flask and to this was added
95% of ethanol upto the mark, shaken well and then filtered. Then taken 1ml of the filtrate
in a 10ml volumetric flask, added 1ml of (1:10) Folin’s reagent and 0.8ml of 7.5% sodium
carbonate solution. Allowed to stand for the development of color and recorded the
absorbance at 703nm.The concentration corresponding to the absorbance was recorded
from the calibration curve of tannic acid and expressed as tannic acid equivalents.
b) For Triphala extract
Accurately weighed quantity of 100mg of the extract was taken in 100ml volumetric flask
and volume made upto the mark with 95% ethanol. Then 1ml of the filtrate was taken in a
10ml volumetric flask, added 1ml of (1:10) Folin’s reagent and 0.8ml of 7.5% of sodium
carbonate solution. Allowed to stand for development of color (about 30min.).Then
recorded the absorbance at 703nm.The concentration corresponding to the absorbance was
read from the calibration curve of tannic acid and expressed as tannic acid equivalents.
26
Thin layer chromatography 28,29,30,31
TLC provided for the entire drug in the monographs includes the identification of the drug
based on its major chemical constituents as markers. Silica gel GF254 plates of uniform
thickness were prepared and activated at 105ºC for 30min. and the plates were spotted with
the drug extract using capillary tube. The spotted plates were dried at R.T. The solvents
used were of analytical grade. Solvent system was prepared and the chromatographic
chamber was saturated for 30 min. The plates were placed in the chamber and left for the
development. Using different reagents did visualization of the spots and Rf values were
calculated.
Rf = Distance traveled by the spot
Distance travelled by the solvent front
Terminalia Chebula - gallic acid
Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid: Formic acid (20:45:20:5)
Sample Preparation :- Powdered sample was extracted with methanol.The methanol extract
was concentrated and residue dissolved in methanol.
Detection :- Anisaldehyde Sulphuric Acid.
Terminalia belerica - gallic acid
Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid: Formic acid (20:45:20:5)
Sample Preparation :- Powdered sample was extracted with methanol.The methanol extract
was concentrated and residue dissolved in methanol.
Detection :- Anisaldehyde Sulphuric Acid.
27
Emblica officinalis - gallic acid
Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid: Formic acid (20:45:20:5)
Sample Preparation :- Powdered sample was extracted with methanol.The methanol extract
was concentrated and residue dissolved in methanol.
Detection :- Anisaldehyde Sulphuric Acid.
Glycyrrhiza glabra - glycyrrhetic acid
Mobile Phase : -Toulene: Ethylacetate: Glacial acetic acid (9:1:0.5)
Sample Preparation :- Shaken 1 gm of the drug with about 20ml of chloroform for 15
min..Filtered and discarded the filtrate. Then refluxed the marc for
1hr with 30m 0.5M Sulphuric acid. Cooled and shaken the unfiltered
mixture with chloroform(2x20ml) and concentrated the combined
chloroform extract.Dissolved the residue in 1.0ml of chloroform :
methanol(1:1)mixture.
Standard Preparation :-Refluxed 5mg of glycyrrhizin with 20ml of 0.5 M Sulphuric
acid.Cooled and extracted with chloroform(2x10 ml) . Evaporated
the combined chloroform extract and dissolved the residue in 1.0
ml of Chloroform: methanol (1:1) mixture.
Detection: - Anisaldehyde Sulphuric Acid.
28
PREPARATION OF THE SUSPENSION 32,33
1) In a 100 ml beaker about 40 ml of the distilled water was taken and to
this was added 35.0 gms of Sorbitol solution 70% and mixed well.
2) To the above mixture was added, Xanthan gum (in varying
concentration) and started stirring using a homogeniser.
3) In another beaker, methyl paraben was dissolved in a very little
quantity of alcohol and added to the above mixture.
4) Drugs and /or their extracts were passed through 120# sieve and then
was added in small quantity to the above mixture and stirred for 30
min.
5) Then dissolved the color in water and added to the above mixture.
Lastly flavor was added and the volume was made upto the mark.
29
FORMULATION CHART OF THE SUSPENSION
Ingredients
Quantity in g/ 100 ml
Formulation code
F I-1 FI-2 FI-3 FI-4 FII-1 FII-2 FII-3 FII-4
Powders
Glycyrrhiza glabra extract 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0
Triphala Churna 2.0 2.0 2.0 2.0 - - - -
Turbinella rapa 16 16 16 16 16 16 16 16
Triphala Churna Extract - - - - 2.0 2.0 2.0 2.0
Xanthan Gum 0.20 0.25 0.30 0.35 0.20 0.25 0.30 0.35
Sorbitol 70% liquid 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0
Distilled water to make 100ml 100ml 100ml 100ml 100ml 100ml 100ml 100ml
Preservative (methyl paraben) 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
Color q.s q,s q.s q.s q.s q.s q.s q.s
Flavor (Menthol) 0.003 0.003 0.003 0.003 0.003 0.003 0.003 0.003
30
Evaluation of the suspension34
1) Particle size distribution
This was accomplished by using an optical microscope, a calibrated eyepiece that was
calibrated by using a stage micrometer.
Calibration of the eyepiece micrometer
The smallest division of the stage micrometer represents 0.01 mm in length.
Kept the eyepiece and the stage micrometer.
Focused under high power
Seen the superimposing
The stage micrometer was replaced with the slide of the object, which was suspended in
the paraffin oil. Sample was uniformly spread on a clean glass slide. Then placed under
microscope and the size was measured. The size obtained was multiplied by calibration
factor to get the actual dimension. The average value was taken. The details are mentioned
in page no.
Physico-chemical characterization of the suspension
pH measurement
pH of the prepared formulation was tested by using the calibrated systronic pH meter. The
prepared suspension formulations were taken separately in different glass beakers. The
electrodes were dipped in the formulations and pH was noted.
31
Viscosity
The viscosity of the prepared formulations were tested by using Brookfield’s viscometer,
attached with spindle no.21.The prepared formulations were placed in separate 100ml
beakers. The spindle of the viscometer was introduced into the formulation and the reading
was set to zero. The viscosity measured at 200 r.p.m was noted.
Ease of redispersibility
The conventional formulation was allowed to settle completely in a measuring cylinder.
The cylinder was inverted through 180 degrees and the number of inversions necessary to
restore a homogenous suspension was determined.
Sedimentation Volume (VS)
Sedimentation volume considers the ratio of the ultimate height (HU) of the sediment to the
initial height (HS) of the total suspension as the suspension settles in a cylinder under
standard conditions. The larger the fraction the better is the suspendibility. It is calculated
as follows: VS = HU/HS
Acid Neutralization Capacity
This was performed as per USP method by taking 5 ml of the suspension. The procedure is
same as described for Shankha Bhasma.
32
Stability studies 35,36,37
Stability studies were carried out on the selected formulations taking gallic acid as the
marker compound for 90 days at two temperatures i.e. 30 ºC /65% RH and 40 ºC / 75%
RH. The samples were withdrawn at regular intervals for 30 days for a total period of 90
days. HPTLC studies were performed to ascertain the stability data. The HPTLC graphs
were taken and discussed under results and discussion.
Anti-ulcer activity studies38
Antiulcer activity studies on the formulations were carried out on albino rats of either sex
weighing 200-250 g. The Histopathological studies were performed to ascertain the ulcer
prevention. The procedure is briefly described below: -
The rats were randomly divided into four groups, each group containing six animals.
Group 1: - control group i.e. no drug formulation was given.
Group 2: - ulcer was induced.
Group 3: - Animals pretreated with formulation containing herbal powders
@0.2ml/animal.
Group 4: - Animals pretreated with formulation containing herbal extracts @0.2ml/animal
33
The overnight fasted rats belonging to all the groups were administered 1ml of absolute
alcohol. After 1 hr of administration of the ulcerogen all the rats were anaesthetized and
sacrificed by cervical dislocation. The stomachs were removed, and opened along the
greater curvature, washed with normal saline and observed under 100X for the presence of
the ulcers. The ulcers were scored as below:
0 Normal colored stomach. 0.5 Red coloration.
1 Spot ulcer 1.5 Hemorrhagic streaks
2 Ulcers equal to 3mm but less than
5mm
3 Ulcers greater than 5
Mean ulcer score for each group was determined and ulcer index (UI) was calculated as
under:
UI = (n. lesion 1) + (n. lesion 2) + (n. lesion 3) + …….
n. Animals
34
Qualitative and Quantitative Analysis of Shankha Bhasma:
Table No. 1 Qualitative tests
Test
Observations
Inference
Tests for calcium
White precipitate
Calcium Present
Tests for Carbonate
White Precipitate
Carbonate Present
Tests for Magnesium
White Precipitate
Magnesium Present
Tests for Manganese
Green precipitate in soluble in dil. HCL.
Manganese Present
Table No.2 Quantitative tests
Observations
Parameter
Trial 1
Trial 2
Trial 3
Mean ± S.D
1) Calcium carbonate Content (%)
94.1
93.6
95.3
94.33±0.873
2) Acid Neutralization Capacity (mEq/gm)
16.6
17.2
17.0
16.93±0.305
35
Monographic analysis of the herbs.
The monographic analysis of all the herbal ingredients was performed with
reference to the Herbal Pharmacopoeia of India and the Ayurvedic Pharmacopoeia of
India. Both these Pharmacopoeias suggest tests like foreign organic matter, total ash, acid
insoluble ash, water-soluble extractive and alcohol soluble extractive for the purity and
identification of the individual drugs. The values of the monographic analysis are reported
along with the standard values for comparison purposes. The results are within the range of
the pharmacopoeias.
Table No. 3: Foreign organic matter of herbal drug. Drug Obtained value
(In percentage) Pharmacopeial Standards
I II III Mean ±S.D
Terminalia Chebula 0.85 0.74 0.45 0.68±0.206 NMT 1%
Terminalia belerica 1.17 1.26 1.24 1.22±0.047 NMT 2%
Emblica officinalis 2.2 2.36 2.81 2.45±0.316 NMT 3%
Glycyrrhiza glabra 1.53 2.11 2.63 2.09±0.5503 *NS
*NS = not specified *S.D = Standard deviation, n=3
36
Table No. 4: Determination of total ash value.
Obtained value (In percentage)
Drug
I II III Mean ±S.D*
Pharmacopeial limits
Terminalia Chebula
3.41 3.241 3.042 3.23±0.184 NMT 5%
Terminalia belerica
5.824 5.833 5.714 5.79±0.066 NMT 7%
Emblica officinalis 4.933 4.582 4.621 4.712±0.192 NMT 5%
Glycyrrhiza glabra 6.51 7.23 7.81 7.183± 0.6513
NMT 10%
*S.D. = Standard deviation, n=3 Table No.5: Acid insoluble ash value.
Obtained value (In percentage)
Drug
I II III Mean ±S.D*
Pharmacopeial limit
Terminalia Chebula
1.8 1.782 1.923 1.835± 0.0767
NMT 5%
Terminalia belerica
0.866 0.814 0.823 0.834± 0.0277
NMT 1%
Emblica officinalis 0.933 0.925 0.907 0.9223± 0.010
NMT 2%
Glycyrrhiza glabra 1.87 2.32 1.95 2.0467± 0.2401
NMT 2.5%
S.D*. = Standard deviation, n=3
37
Table No.6: Determination of extractive values: Alcohol soluble extractive values.
Obtained value (In percentage)
Drug
I II III Mean ±S.D*.
Pharmacopeial limit
Terminalia Chebula
44.26 46.22 40.38 43.62±2.972 NLT 40%
Terminalia belerica
25.84 27.20 27.54 26.86±1.311 NLT 8%
Emblica officinalis 40.6 43.18 49.36 44.38±4.5016 NLT 40%
Glycyrrhiza glabra 12.96 13.92 15.12 14.0±1.082 NLT 10%
S.D*. = Standard deviation, n=3 Table No.7: Extractive values: Water-Soluble extractive values.
Obtained value (In percentage)
Drug
I II III Mean ±S.D*.
Pharmacopeial limit
Terminalia Chebula
62.02 65.92 62.34 63.42±2.165 NLT 35%
Terminalia belerica
39.2 37.76 41.08 39.34±1.664 NLT 60%
Emblica officinalis 52.4 54.8 63.2 56.8±5.570 NLT 50%
Glycyrrhiza glabra 24.2 23.84 25.08 24.373± 0.6379
NLT 20%
S.D* = Standard deviation, n=3
38
Table No.8: TLC Identification of the Individual Herbs
HERBAL DRUGS RF Value
Obtained Literature
1) Glycyrrhiza glabra 0.45 0.46
2) Terminalia Chebula 0.66 0.68
3) Terminalia belerica 0.70 0.68
4) Emblica officinalis 0.69 0.68
The Thin Layer Chromatographs of all the herbal ingredients were taken and
are shown on next page.
39
TLC Photographs of the herbal drugs
STD SAMPLE STD SAMPLE Terminalia belerica Terminalia chebula
STD SAMPLE STD SAMPLE Emblica officinalis Glycyrrhiza glabra
39(a)
Estimation of tannin content by Folin-Dennis Method.
Table No.9: Standard graph of the tannic acid by Spectrophotometric method.
Sl.No. Concentration. (μg/ml)
Absorbance Mean±S.D*.
1 2 0.1278±0.012 2 4 0.2235±0.025 3 6 0.3307±0.023 4 8 0.4673±0.041 5 10 0.5769±0.044 6 12 0.6665±0.043 7 14 0.7823±0.051 8 Triphala Powder 0.1755±0.034 9 Triphala extract 0.3297±0.046
S.D*= Standard deviation, n=3 Fig. No.2: Calibration curve of tannic acid in distilled water at 703 nm.
Stan d ad r d g r ap h o f T an n ic Acidy = 0 .0 5 6 4 xR 2 = 0 .9 9 8 3
0
0 .2
0 .4
0 .6
0 .8
1
0 2 4 6 8 1 0 1 2 1 4
Conce ntra tion M cg/m l
Abs
orba
nce
Quantity of tannins present = Conc. from graph x Vol. taken x Dilution Factor (%W/w) 1000
40
Table No.10. Total tannin Content HERBAL DRUGS Tannin content (%w/w)
Triphala churna powder 29.1
Triphala churna extract 59.5
Standard deviation, n=3
Physicochemical Characterization of the suspension.
Table No. 10: Particle Size Distribution data
1 division of eyepiece micrometer = 13.1 μm
0
5
10
15
20
25
30
#19.
5
#32.
5
#45.
5
#58.
5
#71.
5
#84.
5
#97.
5
Mean particle size in microns
% fr
eque
ncy
From the particle size distribution data it can be seen that most of the particles lies between
50-75 micrometers.
41
TABLE NO.11: pH and Viscosity
PARAMETER pH
Viscosity (in cps)
Formulation code
Trial1 Trial 2 Trial 3 Mean±S.D Trial1 Trial 2 Trial 3 Mean±S.D
FI-1
7.88
8.02
8.16
8.02±0.14
30.0
30.3
29.7
29.967± 0.3512
FI-2
8.22
8.17
8.29
8.227± 0.06028
40.2
41.3
40.8
40.767± 0.5508
FI-3
8.09
8.11
8.32
8.173± 0.1274
50.7
50.3
51.2
50.73± 0.4509
FI-4
7.96
8.21
8.05
8.07± 0.1266
61.6
60.3
61.4
61.1± 0.70
FII-1
7.90
8.11
8.12
8.0467± 0.1321
38.5
39.7
40.3
39.5± 0.9165
FII-2
8.0
8.21
7.98
8.063± 0.1263
51.4
52.8
50.6
51.6± 1.114
FII-3
8.22
8.37
8.16
8.25± 0.1082
63.3
62.7
62.1
62.7±0.60
FII-4
8.15
7.95
8.26
8.12± 0.1572
76.6
77.9
80.3
78.267± 1.877
1) The results show that the pH of all the formulations lies around 8.0, which is desirable
for an antacid suspension.
2) The viscosity data show that the viscosity of the suspension containing herbal extracts is
more than those containing herbal powders. This may be attributed to the fact that the
herbal extracts being sticky in nature contribute some value to the viscosity of the
suspension. 42
Table no. 12: sedimentation volume and redispersibility PARAMETER
Formulation code
Sedimentation volume
Redispersibility
FI-1
0.88
2
FI-2
0.91
3
FI-3
0.93
3
FI-4
0.96
4
FII-1
0.90
3
FII-2
0.92
3
FII-3
0.95
4
FII-4
0.98
5
Standard deviation, n=3
1) Hence from the above data, it can be concluded that as all the formulations are
characterized by high sedimentation volume, the suspension may be flocculated
suspension.
2) From the redispersibility data it can be seen that formulations FI-2, FI-3 FII-1 and FII-2
have good redispersibility with good consistency.
43
Table no. 12: Acid Neutralization Capacity PARAMETER Acid Neutralization Capacity (mEq/ml)
Formulation code
Trial1 Trial 2 Trial 3 Mean±S.D
FI-1
2.65
2.85
2.81
2.77±0.1058
FI-2
3.0
2.91
2.52
2.81±0.2551
FI-3
2.55
2.98
2.31
2.613± 0.3395
FI-4
2.86
3.15
2.99
3.0± 0.1453
FII-1
2.58
2.38
2.40
2.453±0.1102
FII-2
1.93
2.31
2.90
2.38±0.4888
FII-3
2.45
3.0
2.20
2.55±0.4093
FII-4
2.20
2.19
2.54
2.31± 0.1769
1) From the above data it is evident that the acid neutralization capacity of all the
formulations lies between 2-3 mEq/ml. The minimum acid that has to be neutralized by
an antacid should be 12 mEq-40 mEq and hence 5ml of the above formulations
satisfies the requirement.
44
ANTI-ULCER ACTIVITY
TABLE NO.13: Ulcer Index
GROUP ULCER INDEX
Control 1.01 ± 0.68
Ulcerated 8 .68 ± 1.51
Treated (FI-2) 3.57± 2.14*
Treated (FII-2) 2.11±1.07*
Standard deviation, n=6
P < 0.05 as compared to the ulcerated group , * indicates statistically significant
From the above data is very obvious that the Suspension containing the herbal extracts
shows a decrease in the ulcer index as compared to the one containing the fine powders of
these drugs.
Histolopathological Studies
The tissue samples were fixed in 10% buffered formalin and processed with paraffin wax.
For this study 5mm sections were stained with haematoxylin and eosin. The extent and
depth of the ulceration and hemorrhage were evaluated.
Statistical Analysis
Statistical Analysis was carried out by using one-way ANOVA followed by Duncan’s
multiple comparison tests. All the results obtained in the study were compared with each
group. P values than 0.05 were considered stastically significant.
The Photos of Histopathological studies were taken and discussed on next page
45
Histopathological studies:
The fig.no.1 indicates the normal mucosa of the stomach with mucus and presence of
chief cells.
The fig. No. 2 indicates the ulcerated mucosa with prominent loss of mucus, chief
cells.
The fig no.3 indicates the pretreated mucosa with suspension containing herbal
powders that shows less loss of mucus and chief cells.
The fig no. 4 indicates the mucosa pretreated with suspension containing herbal
extracts with near normal appearance to that of normal mucosa.
ANTI-ULCER ACTIVITY STUDIES
NORMAL MUCOSA ULCERATED MUCOSA
TREATED MUCOSA FII-2 TREATED MUCOSA FI-3
45(a)
HPTLC method for estimation of gallic acid
Table no.14: Percentage of gallic acid by HPTLC Method
Formulation Temperature Days % Gallic acid
0th Day 11.54 30ºC/65%RH
90th day 11.52
0th Day 11.54
FI-3
40ºC/75%RH
90th day 11.49
0th Day 19.63 30ºC/65%RH
90th day 19.59
0th Day 19.63
FII-2 40ºC/75%RH
90th day 19.57
The HPTLC data shows that there is no significant change in the concentration of gallic
acid at the end of 90 days at two temperatures. The graphs are shown on next page.
Table No.15: TLC of the suspension at 0th and 90th Day
Rf value Formulation Temperature Days
Obt. Std.
0th Day 0.67 0.68 30ºC/65%RH
90th day 0.66 0.68
0th Day 0.67 0.68
FI-3
40ºC/75%RH
90th day 0.66 0.68
0th Day 0.67 0.68 30ºC/65%RH
90th day 0.69 0.68
0th Day 0.67 0.68
FII-2 40ºC/75%RH
90th day 0.69 0.68
The TLC Photographs are shown on next Page. TLC Photographs shows no secondary
spots, which shows that there is no degradation of the product during storage.
46
TLC of the Formulations at 0th Day TLC of the formulations at 90th Day
G 1 2 3 4 5 6 G 1 2 3 4 5 6
std samples std samples
G : gallic acid G : gallic acid
46(h)
Table no.15: Viscosity, pH and acid neutralization capacity.
Formulation
Temperature/
Humidity
Days Viscosity pH Acid Neutralization
capacity
0th Day 50.73±0.4509 8.17±0.1274 2.61± 0.3395
30th day 50.64±0.4487 8.12±0.1258 2.60± 0.3346
60th day 50.56±0.4410 8.11±0.1192 2.59±0.3277
30ºC/65%RH
90th day 50.43±0.4361 8.10±0.1132 2.58±0.3201
0th Day 50.73±0.4509 8.17±0.1274 2.61± 0.3395
30th day 50.57±0.4438 8.16±0.1265 2.60± 0.3313
60th day 50.45±0.4397 8.16±0.1259 2.59± 0.3298
FI-3
40ºC/75%RH
90th day 50.23±0.4337 8.15±0.1247 2.58±0.3288
0th Day 51.63±1.114 8.06±0.1263 2.38±0.4888
30th day 51.32±1.111 8.05±0.1257 2.32±0.4873
60th day 50.81±1.109 8.04±0.1252 2.27±0.4864
30ºC/65%RH
90th day 49.45±1.085 8.0±0.1179 2.19±0.4856
0th Day 51.66±1.114 8.06±0.1263 2.38±0.4888
30th day 51.54±1.108 8.04±0.1253 2.29±0.4865
60th day 51.43±1.104 8.03±0.1247 2.23±0.4857
FII-2
40ºC/75%RH
90th day 51.39 ±1.080 7.9±0.1243 2.15±0.4790
47
Table no.16: Organoleptic properties.
Formulation
Temperature/
Humidity
Days Color Odour Taste
0th Day No Change No change No Change
30th day color change No Change No Change
60th day color change No Change No Change
30ºC/65%RH
90th day color change No Change No Change
0th Day No Change No Change No Change
30th day color change No Change No Change
60th day color change No Change No Change
FI-3
40ºC/75%RH
90th day color change No Change No Change
0th Day No Change No Change No Change
30th day color change No Change No Change
60th day color change No Change No Change
30ºC/65%RH
90th day color change No Change No Change
0th Day No Change No Change No Change
30th day color change No Change No Change
60th day color change No Change No Change
FII-2 40ºC/75%RH
90th day color change No Change No Change
48
From the Present study we can conclude the following: -
1) From the particle size distribution data it can be concluded that the suspension is a
coarse dispersion with particle size predominantly lying between 50-75 micrometers.
2) The suspensions containing the herbal extracts show greater viscosity than those
containing the herbal powders for the same concentration of the suspending agent. This
difference may be due to the fact that the extracts obtained are sticky in nature that may
contribute to the viscosity of the formulation.
3) The suspension containing herbal powders and 0.3% concentration of Xanthan gum
shows the optimum quality of the suspension with regards to the consistency and
redispersibility, while in case of suspension containing herbal extracts, 0.25%
concentration of the Xanthan gum shows the optimum quality of the suspension with
regards to the consistency and redispersibility. Hence these formulations were selected
for in vivo studies and stability studies.
4) All the formulations show an acid neutralization capacity between 2-3 mEq /ml .
5) All the formulations show the pH of around 8.0 that is the desirable pH for an antacid
suspension.
6) All the formulations show high sedimentation volume and good ease of redispersibility
except FII-4 which has poor ease of redispersibility.
7) The HPTLC graphs indicate that there are no interactions between the ingredients
during the stability studies.
8)The TLC Photographs taken during stability studies show no secondary spots
that indicates that there is no degradation-taking place during stability studies.
49
9) From the data on the viscosity, pH and acid neutralization capacity of the
suspension, which was evaluated during the stability studies, indicates that there is no
change in these parameters.
10) The TLC Photo graphs that were taken during stability studies show no secondary
spots that indicate that there is no degradation-taking place during stability studies.
11) The extracts obtained from the herbs show significantly greater Antiulcer Activity
than those of the powdered drugs. This may be due to the fact that the extracts may
contain more concentration of the active ingredients responsible for antiulcer activity.
12) Thus efforts to formulate an herbal antacid suspension were found to be
satisfactory.
Future Work that can be carried out: -
1) The Anti-ulcer activity can be evaluated by varying the concentrations of the herbal
Powders / extracts.
2) The extracts of other antiulcer herbs may also be evaluated to find out extent of
anti- ulcer activity.
50
1. Prof. Vaidya Banwari lal gaur, Prof.Vaidya Satyanarayana Sharma,” Researches
in Ayurvedic: Past and Present”: 55-60,129-137,85-89
2. S.M.Jain and D.D.Santani,”Peptic ulcer disease and status of current drug
therapy”, Indian Drugs, Vol.31 (9): 395-399.
3. V.Sihorkar et al,”Helicobacter Pylori: Pathogenesis and current therapeutic
strategies”, Indian Drugs, Vol.36 (7), July 99: 420-429.
4. Pandey B.L et al,” Effect of Tectonia grandis linn. On experimental ulcers and
gastric secretion”, Indian Journal of medicinal Research 1982,Vol.76 : 224-228.
5. Goel R.K.et al,” Antiulcerogenic and anti-inflammatory studieswith Shilajit “,
Journal of Ethnopharmacology,Vol.29 :95-103
6. Sairam K et al ,” Effect of centella Asiatica on Physical and chemical factors
inducedgastric ulceration and secretion “, Indian Journal of Experimental Biology
1997, Vol.39 : 65-68
7. WHO monographs on selected medicinal plants, Vol.1, WHO Geneva1999: 183-
194.
8. De B Maiti et al,” Effect of some sitavirya drugs on gastric secretion and ulceration
“,Indian Journal of Experimental Biology
9. Brady , Tylor ,” Textbook of Pharmacognosy” fifth edition : 77-79
10. Rege NN et al ,” Adaptogenic properties of six rasayana herbs used in Ayurvedic
medicine “, Phytotherapy Research 1999,Vol. 13 : 275-291
11. Ayurvedic Formulary of India Part1, second edition, Govt. of India: 245
51
12. “ A comparative study of marketed herbal liquid antacid formulations : in vitro
antacidactivity,Anti-ulcer activity and microbial standardisation”,Indian Drugs Vol.
40 (3) march 2003 :182-185 ,
13. M.E.Aulton,”Pharmaceutics: The science of Dosage form Design”, second
edition: 334-342,351-353.
14. C.K.Kokate et al, “Textbook of Pharmacognosy”, eleventh edition, feb.99: 216-
218,224-225.
15. Siddhinandan Mishra,”Ayurvedic Rasashastra”: 62-63.
16. Ainley wade et al ,” Hand Book of Pharmaceutical excipients”, Part –2, second
edition : 562-563.
17. Ainley wade et al ,” Hand Book of Pharmaceutical excipients”, Part –1 second
edition : 304-305,310-313.
18. Indian Pharmacopoeia, Vol.2,1996 :709
19. Lalla J.K. et al, Preparation, characterization and analysis of Shankha Bhasma,
Indian Drugs 39(3), March 2002,152-157
20. Dr.S.L.Khalale and Dr.R.J.Pandey,”Physical-chemical study of Kapardica
Bhasma”, Reasearches in Ayurvedic: Past and Present: 235-242
21. United States Pharmacopoeia, Convention.Inc.Rockville, 1996,ed. XXIII, 1684-
1686
22. Ayurvedic Pharmacopoeia of India Part-1, Vol.-1,first edition, ministry of health
and family welfare, New Delhi: 4-6,26,47-48,127-128
23. Lalla J.K. etal, Triphala Churna-From Raw Materials to Finished Products,
Indian Drugs, Vol.38 (2), Feb.2001: 87-93.
52
24. Munira momin et al,”Development and evaluation of Triphala Formulation”,
Indian Journal of Pharmaceutical Sciences, Vol.66, No.4.Jul-Aug 2004: 427-432
25. Determination of Total Phenolics, Journal of Agriculture and food chemistry,
Vol.50, no.1, 2002: 81-86.
26. Jain U.K. and Dixit V.K.,”Spectrophotometric estimation of tannins from
Chyavanprash”, Indian Drugs, Vol, 41(8) August 2004: 469-472
27. Indian Herbal Pharmacopoeia, Vol.1, 1998: 89-98
28. Indian Herbal Pharmacopoeia, Vol.2, 1999: 50-57
29. Dr.Pulok.Mukherjee,”Quality Control for Herbal Drugs”, first edition
2002,Business horizons Pharmaceutical Publishers, 323-325,729-731,738-740
30. Ukani M.D, Nanavati D.D,” Chromatography of Herbal Compounds: TLC in
Ayurveda “, Indian Drugs, Vol.35 (3), March 98: 153-162.
31. Herbert Liebermann et al,” Pharmaceutical dosage forms: Disperse systems”,
Vol.1, second edition: 17-43,287-309,377-412.
32. Leon Lachmann et al,” Theory and Practice of Industrial Pharmacy”, Third
edition, Varghese Publishing House, Bombay: 479-501.
33. Gilbert Banker et al,” Modern Pharmaceutics”, Third edition: 310-318.
34. Lalla J.K. et al,” An Application of HPTLC to Alternate medicine- Qualitative
and Quantitative evaluation of Ayurvedic formulation Triphala Churna”,
Journal of Planar Chromatography-Modern TLC.
35. ICH Guidelines for stability testing of Pharmaceutical Products.
36. S.K.Kulkarni et al,” Hand Book of Experimental Pharmacology”: 84-87
53
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