Preparation and evaluation of unani medicine :Sherbath E Aynnunas(pineapple)

Abstract
Key words
Introduction
Materials and Methods
Analytical parameters
1.Prelimary test
3.Acid insoluble ash
4.Water soluble ash
5.Total solid content
6.Viscosity
7.Specific gravity
8.Total sugar content
9.Chemical test
10.Biochemical test to identify microorganism
11. Microbial load
12 .Self life

Guided by,
Dr.Ashok.B.S....M.Pharma,Ph.D
department of Pharmacognosy
Submitted By,
Veena.V
Thanushree.R
Suhasi.A
B.Pharma final year students
2020-21 batch
SKCVP

Unani System of Medicine refers to Graceo-Arabic medicine ,which is based on the
teachings of Greek physician Hippocrates and Roman physician Galen and developed
into an elaborate medical system in middle age era by Arabian and Persian physicians.
It is a descendent of Aesculapius and recognised as Father of Unani Medicine “.Unani
System is traditional healing through naturally obtained herbs and fruits ,vegetables and
drug product.
It have been the practised of many countries since ages ,as they were generally believed
to be non toxic natural products.
The WHO has estimated 80% of world population relies on traditional medicine on
primary health care.
India is one of the richest sources of medicine and aromatic plants.Because of rapid
progress of herbal drug industry in India for the last quater century ,an increases need is
felt to standardised the herbal products.
It is necessary to develop scientific protocols such as SOP,pharmacopeial
standards of the formulational Sherbath-E-Anynunnus

This formulation is used aliments of nausea,vomiting,thirst,weakness of
heart and liver.Studies relieved that microbial load and heavy metals such as
lead,cadmium,mercury,arsenic,were not detected in the drug formulation.
The data evoloved in the present work will aid in identifying the raw drug
used in finished product and will help to fix the scientific standards for
Sherbath –E-Anynuus.

The Unani System of Medicine has a long and impression record in Indian.It
was introduced in India by the Arabs and Persian around the 11th
century.Today India is one of the leading countries in so far as the practice of
Unani Medicine is concerned .It has the largest number of Unani educational
research and health care institutions.
Unani System originated in Greece.The foundations laid by Hippocrates.Unani
medicines got enriched by imbibing what was the best in the contemporary
middle east countries.
In India,Unani System of Medicine was introduced by Arabs and soon it took
firm roots in India.

The basic theory of Unani System is based upon the well known four humour
theory of Hippocrates.This presupposes the presence ,in the body,of four
humours viz ,blood,phlegm,yellow bile and balck bile.
Arkan(elements)
Mizaj(temperament)
Akhlat(humours)
Aaza(organs)
Arwah(spirits)
Quwa(faculties)
Afaal(functions

The word Sherbath is a soft drink or a liquor that incorpates white
sugar,misri,honey and gur dissolved in water.However,in Greeco-Arab
Pharmacopoeia,Sharbat implies ,a concentration liquor made from decotin or
fruit juices by adding sugar to yield quiam.
Sherbath is charactersized by sweet taste,good palatability.The reason bind
innovation of Sherbath dosage form lies in infact that most of the herbs ,is
taken raw are highly implatablilty.
So,to enhance palatability to prolong the storage period of medicines
,Sherbath dosage form was designed in Unani System of Medicines.Sherbath-
E-Aynnunas is in a concentration liquor or syrup form (Unani formulation)
which is widely used for nausea,thirst etc at dose of 15-30ml twice a day.
It is composed of Aabe Aynnunnas ,(Pineapple juice) ,Khande sufeed
(sugar).In the present study ,physicochemical evaluation of the Unani
formulation Sherbath- E- Anynnuas and its ingredients has been carried out
because these evaluations are surprisingly uncharted till date and
determination of these parameters are very essential to assure the quality
,safety,efficacy of this formulation.

Materials:
The fruit material such as Ananas cosmosus(pineapple) ia s tropical plant with
an edible fruit and the most economically significant plant in the family belong
to Bromeliacece is purchased from the local market of Chickaballapur,Karnataka
and also edible sugar is used.All the reagents and solvents used were of
analytical grade.
Preparation of formulation:
1.Clean and peel the outer layer of the pineapple fruit taken.
2.Wash and slice the fruit into small portion,so that it grind well.
3.The ratio of drug and water should be 1:1 ie 600g of drug:600ml of water.
4.Grind well and filter it proper to make the juicy liquefy.
5.Take twice the quantity of sugar ie 1:2 portion.
6.Add to the juicy fruit in a non stick container.
7.Boil the syrup in a low flame until the required consistency is obtained.
8.The pulp of the fruit should get disappear,boil till that.
9.The juice syrup and sugar should be melt completely at 80-95◦C

The prepared sample was subjected for analytical parameters such as
physicochemical studies such as :
1.Prelimary test
2.Total ash
3.Acid insoluble ash
4.Water soluble ash
5.Total solid content
6.Viscosity
7.Specific gravity
8.Total sugar content
9.Chemical test
10.Biochemical test to identify microorganism
11. Microbial load
12.Self life

Colour : Yellow
Taste : Sweeter and tar
Odour : Aromatic odour
PH : PH paper is used to determine the PH of the drug and PH of the drug is
acidic in nature (2).

Determination of Ash value :
Procedure:
Incinerate about 2 g to 3 g weighed of the grounded drug in a tarred platinum or
silica dish at a temperature exceeding 450 ˚C until free from carbon , cool and
weigh .
If the carbon free ash cann’t be obtained in this way , exhaust the charred mass
with hot water ,collect the residue on an Ashless filter paper , incinerate the
residue and filter paper , add the filterate evaporate to dryness , and ignite at a
temperature not exceeding 450 ˚C .
Calculate the %of ash with reference to the air dried drug .
Calculation :
Weight of the drug - 2 g
Weight of the crucible and drug – 29.5 g
Weight of the crucible and Ash - 27g
Total Ash – 0.5 g
Percentage of total ash = × 100
%of Total Ash= 100
%of Total Ash= 100 %

Determination of acid insoluble ash :
Boil the ash obtained for 5 minutes with 25 ml of dilute Hcl , collect the
insoluble matter in a gooch crucible , or on an ashless filter paper , wash
with hot water and ignite to constant weight .
Calculate the %of acid insoluble ash with reference to the air dried drug
Calculation :
Percentage of acid insoluble ash = × 100
%of acid insoluble ash = = 60%

Determination of water soluble ash
Boil the ash for 5 minutes with 25 ml of water .
Collect insoluble matter in a gooch crucible or on an ashless filter paper ,
wash with hot water , and ignite for 15 minutes at a temperature not
exceeding 450˚
Substract the weight of the insoluble matter from the weight of the ash .
The difference in weight represents the water soluble ash .
Calculate the % of water soluble ash with reference to the air dried drug.
Calculation:
Percentage of water soluble ash =
Percentage of water soluble ash = = 35 %

Total solids are measured by weighing the amount of solids present in a
known volume of sample. This is done by weighing a china dish , filling it
with a known volume , evaporating the water in an oven and completely
drying and the residue and then weighing the china dish with the residue .
Calculation :
Weight of the china dish = 49.5 g
Weight of the china dish with sample =59.6 g
Weight of the sample after drying = 58.5 g
Percentage of total solids =
%of total solids = = 98.15%

Viscosity is a measure of a fluids resistance to flow.
It describes the internal friction of a moving fluids .
Procedure :
Clean the viscometer by the water and ethanol and dry it .
pipette until the small bulge is full.
Put viscometer vertically in water bath at the desired temperature .
Let the liquid to flow through the capillary tube with run time when the liquid
reaches the mark shown on the viscomoter and then stop the time when
liquid reaches the bottom mark .
Repeat the experiment and record the results
Repeat the experiments to other liquids .
Change the temperature and calculate the viscosity .

Temperature(˚
C)
Time (sec)
Average time Viscosity ( poise)
Relativ
e
viscosi
ty
1 2 3
Water (25˚) 27.8 sec 26.6 sec 26 sec 26.84 sec 0.891 poise
1.2 poise
Liquid(30◦) 20.6 sec 22 sec 21.8 sec 21.73 sec 1.07 poise

=
Where, =viscosity of liquid
viscosity of water =0.891 poise
time of liquid
= time of water
density of liquid
density of water = 0.997 g /
Relative viscosity by the relationship
= =
= 1.200 Poise

The Specific gravity of a liquid is the relative weight of that liquid compared
to an equal volume of water .
Specific gravity bottles are used to determine the liquid densities by
measuring the difference between an empty and filled bottle and dividing by
an equal volume of water to find the specific gravity of the substance .
) = 28 g
)= 82 g
) =102 .8 g
Calculation :
Weight of empty specific gravity bottle (
Weight of specific gravity bottle and water (
Weight of specific gravity bottle and sample (
Mass = -
Mass = 102. 8 - 28 = 74. 8 g
Specific gravity =
Density of object = = = 1.49 g / ml
Density of water = 1 g / ml
Specific gravity = = 1.49 g / ml

Procedure :
Weigh accurately 2 g from the sample interested .
Transfer it into 200 ml beaker and add nearly 100 ml distilled water .
Boil the sample for 10 – 15 mins while stirring in a water bath .
Add 3ml of concentrated H 2SO 4 into sample and again boil for 3 mins .
Filter the sample into 250 ml volumetric flask using fluted filter paper.
Sample dilution :
Take 2.5 ml from the sample into 100ml volumetric flask and volume up to
the mark .
Again take 5 ml from above sample into 50 ml conical flask and add 15 ml
concentrated H 2SO 4 , 5 ml of 5 %phenol as previously done .
Measure the absorbance with respect to the colour development at 492 nm
using UV visible spectrophotometer .

Sl no.
Test Observation Inference
1 Molish test :
Take the test sample and add α naphthol
dissolved in ethanol and add con. Sulphuric
acid
Violet colour
is appeared
Indicates the
presence of
carbohydrate
s.
2
Fehling’s test :
Take sample and add fehling’s reagent A
and B and boil .
Yellow or
brownish colour
is formed
Indicates the
presence of
carbohydrate
s.
3 Benedict’s test:
Take 2ml of benedict’s reagent , boil for 5
mins then add 5 – 6 drops of sample
Yellow colour
is obtained .
Indicates the
prensence of
carbohydrate
s.

4
Blue colour is
obtained
Indicate the
presence of
polysaccharide
5
Iodine test:
4-5 drops of sample and add
1ml of iodine
Barfoed’s test:
2ml of sample+2ml of barfoed
reagent and boil
Deep blue colour
is obtained
Indicates the
presence of
reducing sugar.
6 Test for protein
Sample+biuret reagent
purple
Colour changes to Indicates the
presence of
proteins.
7
boiling water bath for 30 min.
Cool the solution and observe
the crystals under microscope
Osazone test:
2ml of test solution+3ml of Formation of
phenyl hydrazine Hcl +keep in a yellow crystals of
osazone needle
shaped
Indiactes the
presence of gulco-
osazone.

Formation of yellow crystals of Osazone needle
shape

8
Test for amino
acids
1ml of sample+40% of NaOH
+α- napthol+bromine water 5-
10 drops
Red colour
complex
Absence of
amino acids.
9 Test for
tannins:
Lead acetate
Test:
1ml of sample + 3 drops of
lead aceate
Cream
colour
precipitate
Absence of
tannins
1
0
Ferric
test
1ml of sample + distilled water
+ 2 drops of Fecl 3
Green
precipitate
Absence of
tannins
11 Test
alkaloids
Sample + dragendroff’s
reagent
Orange –
red
precipitate
Indicates the
presence of
alkaloids

1
2
Mayer’s
reagent
Sample + mayer’s
reagent
Cream
colour
precipitate
Indicates the
prrsence of
alkaloids
1
3
Wagner’s
test
Sample + wagner ‘s
reagent
Buff
colour
precipitat
e
Indicates the
presence of
alkaloids
1
4
Glycoside’s
test
Borntranger’s
test:
Sample + 5-10 ml of dilute HCL + boil and No pink
to red filter then add benzene + ammonia
colour is
seen solution equally , filter and shake well.
Absence of
antraquinon
e
glycosides.

1
5
Legal test
Drug sample + pyridine add No pink to
red sodium nitroprusside solution colour
is seen to make alkaline
Absence
of
gylcosides
1
6
Test for
steroids
Sample + acetic anhydride
+ boil,then cooled , add 1
ml of H 2 SO 4 along the
sides of test tube
No green
colour
Absence of
steriods

a) Carbohydrates fermentation test :
composition of sugar broth medium:
• Peptone – 1g
• Meat extract – 0.3g
• Nacl – 0.5 g
• Distilled – 100ml
• Indicator – 0.008 g
• Sugar – 0.5 g ( glucose / lactose /sucrose)
Procedure:
• Take 3 different durham’s tube with different sugar broth .
• Sterile the durham tubes
•After sterilization , the tubes are cooled down to room temperature and inoculated with
cell suspension in aspetic condition
• Tubes are inoculated at 37 ˚c for 24 hours
After incubation , the tubes are examined for acid and gas production .

catalase enzyme is present in living plants and animals ( aerobic and anaerobic
microorganisms utilise oxygen and production of
H 2 O 2 is toxic to the cells .
And its affects the cells enzymes system so to avoid this toxic effect .It produces
catalyse enzyme .
Role of catalyse enzyme is to convert,
H 2 O 2 - - - - - - - - - - > H 2 O + O 2 .
This reaction is an irreversible .
Purpose :
To detect the bacteria production of catalyse enzyme by bacteria
Used to determine whether the microorganisms of aerobic or anerobic.
Methods :
Requirments : Bacterial culture , sterile test tube dropper ,wire loop.
Procedure :
A sterile tube is taken and with the help of a dropper 5 to 6 drops of H 2 O are added .
A colony of a test culture is picked up and inserted in H 2 O 2 containing testtube
The test tube is observed for the powder of effervescence that is bubble formation this
reaction occurs immediately after aadition of culture .
Report:
No effervence test culture shows negative catalase.

Take a clean grease free slide and as it take a drop of sterile water and
with the help of a sterile wire loop pick a colony of a test culture
Further with the help of a dropper add 3 to 4 drops of H 2 O 2 .
All this procedure should be carried out in sterile conditions.
After the addition of H 2 O 2 the slide is observed for formation of
effervescence that is bubbles .
Report :
No effervescence is obtained within 20 sec , then catalase test is
observed as negative .

Streak the surface of a urea agar slant with a portion of a well isolated colony
or inoculated slant .
Leave the cap loosely and incubate the tube at 35˚C to 37˚C
In ambient air for 48 hours to 7 days .
Examine for the development of a pink colour for as long as 7 days .
Report :
Formation of pink colour , uraese test shows positive .
D) Indole test:
Take a sterilized test tubes containing 4 ml of tryptophan broth
Innoculate the tube aspectically by taking the growth 18 to 24 hrs
Incubate the tube at 37 ˚ C for 24 -48 hours .
Add 0.5 ml of kovac’s reagent to the broth culture .
Observe for the presence or absence of ring formation.
Report :
Absence of ring formation .

Hydrogen sulphide is produced if the sulphur compound is reduced by the
bacterial strain .
The test thus determines whether the microbes reduces sulphur containg
compounds to sulphides during the process of metabolism .
The black colour acts as an indicator for the presence of hydrogen sulphide .
Media :
Triple sugar iron media :
Inoculate the test organisms into TSIA media and incubate it at 24 hours .
Observe for blackening of media for 7 days.
Report :
No black colour produced hydrogen sulphide test shows negative .

Sl no. chemicals Grams /liter
1. Protease peptone 10.000 g
2. Beef extract 1.000 g
3. Mannitol 5.000 g
4. Sodium chloride 5.000 g
5. Phenol red 0.018 g
6. Final Ph (at 25˚C) 7.4 0.2 ml

Suspend 21.02 g in 1000 ml water and mix well .
Heat if neccessry to dissolve the medium completely .
Distribute in fermentation tubes ( tubes containing inverted durham Tubes ) .
sterilize by autoclaving at 15 Ibs pressure (121˚C) for 15 mins.
Report :
No gas production in mannitol broth.

Microbial load ( viable count , spore former , mold and yeast count) was
determined using serial dilution and spread plate method . bacterial
colonies in tap water ranges from the maximum value of 1.05×104 to the
minimum value of 2.55×108 CFU / ml , former colonies in tap water , range
from 1.30×102 to 2.75 ×105 CFU / ml.
CFU / mm = 187

Make the dilution series from a sample .
Pipette 0.1 ml from the appropriate desired dilution series onto the center
of the surface of the agar plate .
Dip the L- shaped glass spreader into alcohol .
Flame the glass spreader ( hockey stick ) over a Bunsen burner .
Spread the sample evenly over the surface of agar using the sterile glass
spreader , carefully rotating the petridish underneath at the same time .
Incubate the plate at 37 ˚C for 24 hours .
Calculate the CFU value of the sample . Once you count the colonies,
multiply by the appropriate dilution factor to determine the number of CFU /
ml in the original sample .

The shelf of a product begins from the time the food prepared or
manufactured .
Its length is dependent on many factors including the types of ingredients
, manufacturing process , type of packaging and how the food is stored .
It is indicated by labelling the product with date mark .
Procedure :
Take four different containers and sterile them .
Then close the lid of container tightly , place the air tight container in
different climate conditions and different temperature zone. such as ,
In Humid condition
In refrigerator – 2 to 4 ˚C
In normal condition ( room temperature )
Under light
Keep the air tight containers for 6 to 7 months as mentioned .
Report :
The containers which were kept in different climatic conditions were good
and stable for 6 months.

From B.Pharmacy final year students of
2020-21 from SKCVP

Preparation and evaluation of unani medicine :Sherbath E Aynnunas(pineapple)

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