12 - Chapter 4

"Formulation, standardization and evaluation of indigenous preparation
Chapter -4
MATERIALS AND METHODS
4.1. Gymnema sylvestre raw material
4.1.1. Gymnema sylvestre Authentication
A) Materials
Sr.No Name of materials Source
1 Gymnema sylvestre dried leaves Phyto-pharma Pvt. Ltd.
powder Kolhapur, India.
2 Gymnema sylvestre 75% Amasar Labs India Pvt. Ltd.
standardized extract Indor, India.
B) Method: Gymnema sylvestre consists of the dried leaflets of GS RBr. Family:
Asclepiadaceae
Synonyms: Meshashringi, Gurmar1
General appearance and Microscopic characteristic of the plant
were confirmed by the Phytopharma ltd Kolhapur. The extract of the Gymnema
sylvestre was procured from Amsar labs India Ltd. Indoor, along with the
certificate^of analysis. Thus the plants extract and plant powder raw material
was verified for originality.
Powder plant material was observed physically as well microscopically2
129 Govt. College ofPharamey, Karad

Formulation, standardization and evaluation ofindigenous preparation
4.1.2. Extraction of Gymnema sylvestre dried leaves
A) Materials
1 Hexane Loba chem. Pvt. Ltd. Mumbai
Q.
2 Ethanol
1
1
0
Q.
3 Purified water
1
1
0
-1
4 Cone. Hydrochloric acid -do-
5 Sodium hydroxide do--
6 Solxlet apparatus Borosil India ltd
7 Vacuum drier Lab India mumbai.
B) Method13
The crushed dried leaves of authenticated Gymnema sylvestre (GS) were
graded and disintegrated to required mesh size 20 - 40. Then this mass was
defatted with n-Hexane and subsequently washed with water. The whole mass
of the GS was extracted with the 1:1 or 55% alcoholic solution for 3 - 4 hours in
extraction apparatus. The micella was concentrated under, reduced pressure to
30 % of the original volume. The resultant extract was acidified to pH 3 by
hydrochloric acid. The residue thus obtained was considered as crude extract. It
was dried and purified to required strength.
130 Govt. College ofPharamcy, Karad

Formulation, standardization and evaluation of indigenous preparation
4.1.3. Enrichment of the extract to 25% strength and 75% strength3
A) Materials
1 Sodium hydroxide Loba chem. Pvt. Ltd. Mumbai
2 Purified water -do-
3 Cone. Hydrochloric acid -do-
4 Ethyl alcohol -do-
B) Method 3
About 30 gm of Gymnema sylvestre leaf , extract was taken and
dissolved in 500 ml distilled water, filter and discard the filtrate. The residue was
collected and dissolved in 0.1 N sodium Hydroxide and again acidify with 10 %
hydrochloric acid up to pH 1.5. The solution was stand at room temperature for
30 minutes and it was filtered through Whatman filter paper No. 1. The
precipitate was collected and dissolved in 20 ml 80 % ethanol. This filtrate was
combined and washed and evaporated to dry in oven under vacuum.

Formulation, standardization and evaluation of indigenous preparation"
Flow chart of the Extraction and Enrichment of the extract 75% strength
Size reduction of the dried leaves
1
Extraction with water ;
I
Extraction with 55% alcohol
}
Filtration
I
Vacuun^drying
Extract acidified to pH 3
I
Dissolved in 0.1 NaOH
i
Acidified with 0.1 N HCI
I
T
Drying
I
Standardization

4.2. Standardization 3.
4.2.1. Standardization of Gymnema sylvestre Extract.
A) Materials (section 4.2.1.a-g)
1 Ethyl alcohol Qualigens ltd mumbai.
2 1,4-dioxan Loba chem. Pvt. Ltd. Mumbai
, 3 Acetone --do--
4 Hydrochloric acid --do-
5 Potassium iodide -do--
6 Hypophosphorous -do-
7 Chloroform -do-
8 Acetone -do-
9 Vanillin Qualigens ltd mumbai.
10 Phosphoric acid -do-
11 Iodine Qualigens ltd mumbai.
12 Silica dish Borosil India ltd
13 Oven Lab India ltd.

B) Method3-4
In this section some physicochemical properties of Gymnema
sylvestre extract were tested for the verification. The methods employed in the
experiments were followed as described in the Indian pharmacopoeia. The
standard reagents were prepared as described in the Indian pharmacopoeia.
4.2.1 .a. General identity test for the extract of the Gs hydro alcoholic extract
The taste of the 1 % solution was determined.
1% solution was shaken with the water.
5 % solution was treated with the 2 ml of 0.1 N Hydrochloric acid.
4.2.1. b Loss on drying
Unit for expression: Loss on drying is the loss of weight expressed as % w/w.
Method:4
1 gm of the powder substance being examined was placed (Gymnema
sylvestre extract powder) in a weighing bottle previously dried in an oven at
105c. The substance was dried to constant weight or for one-hour time in an
oven within a specified temperature range of 100-110 c.
4.2.1. c. Solubility4
Unit for expression: solubility is expressed as % w/v.
In water: 1 gm of the substance was placed in 100 ml water in a conical flask. It
was stirred for 2-3 hours and filtered through previously weight filter paper.
Then the residue was dried along with the filter paper to obtain constant weight
and find out the quantity of the residue insoluble in water. The difference
between the total unfiltered qnitity gave the solibility of the substance in water.

Formulation, standardization and evaluation of indigenous preparation "
Solubility in alcohol
1 gm of the substance was placed in 100 ml alcohol in a conical flask. It was
stirred for 2-3 hr and filtered through the previously weighed filter paper. Then
the residue was dried along with the filter paper to obtain constant weight and
find out the quantity of the residue insoluble alcohol. The difference between
the total unfiltered qnitity gave the solibility of the substance in alcohol.
4.2.1. d. Heavy metal
Limit Tests for Heavy Metals 4

\
The specified quantity of the substance being examined was dissolved in an
organic solvent containing a minimum percentage of water, such as 1,4-dioxan
or acetone containing 15% v/v of water, and carry out Test A described in
Indian pharmacopoeia but prepare the standard by diluting lead standard
solution (100 ppm Pb) with the solvent used to prepare the test solution to
i i
contain 1 or 2 ppm of Pb, as specified. The standard solution exhibits a slightly
brown colour when compared to a solution prepared by treating in the same

\ '
manner a mixture of 10 ml of the solvent used to prepare ;the test solution and 2
ml of the solution being examined.
4.2.1. e. Total ash
Method I)
2 to 3 g of the ground drug was incinerated in tared silica dish at a temperature
not exceeding 450 until free from carbon, cool and weighed. Then the residue
was collected on an ash less filter paper, incinerated this residue and filter
paper, adds the filtrate and evaporated to dryness and ignited at a temperature

not exceeding 450. The percentage of ash was calculated with reference to the
air-dried drug.
4.2.1. f. Arsenic
Limit Tests for Arsenic 4
The prescribed quantity of the substance being examined was
added to a test tube containing 4 ml of concentrated hydrochloric acid and
about 5 mg of potassium iodide and added 3 mi of hypophosphorous reagent.
This mixture was heated on a water bath for 15 minutes by shaking
occasionally. Any colour produced is not more intense than that obtained in a
solution prepared in the same manner but using 0.5 ml of arsenic standard
solution (10 ppm As) in place of the substance being examined.
4.2.1. g. TLC of the Gymnema sylvestre extract4
Stationary phase: silica Gel G
Solvent system: Chloroform: acetone (95:5)
Developing agent: vanillin: phosphoric acid, Iodine vapours.
The TLCof Gymnema sylvestre extact was carried out on Silica Gel G as a
stationaray phase and chloroform: acetone mixture (95:5) as a mobile phase.
The procedure followed for the TLC was as per the Indian pharmacopiea.
136 Govt. College of Pharamcy, Karad

Formulation, standardization and evaluation ofindigenous preparation"
4.2.2.Estimation of crude Gymnemic acid from Gymnema sylvestre leaf extract.
Materials
1 Hydrochloric acid Loba chem. Pvt. Ltd. Mumbai
2 Potassium hydroxide do--
B) Method 3
3 gm of Gymnema sylvestre leaf extract was dissolved in 50 ml
distilled water. Then it was filtered and the filtrate acidified with 10 %
hydrochloric acid up to pH 1.5. It was stand at room temperature for 30 minutes
and filtered through Whatman filter paper No. 1. The precipitate was collected
and kept separately. The precipitate obtained from the filter paper was
dissolved in 20 ml 80 % ethanol. The filtrate was combined with washing of
alcohol and evaporated in preweighed beaker and dried in oven under vacuum
till a constant weight obtained. This was considered as the total Gymnemic
acid recovered from the Gymnema sylvestre extract. The percentage of total
Gymnemic acid was calculated.

4.2.3. Assay of the total Gymnemic acid by HPLC. 3,5
A) Materials

t
1 Potassium hydroxide AR Loba chem. Pvt. Ltd. Mumbai
2 Methyl alcohol Merck India ltd.
Acetoniltrile for HPLC Merck India ltd.
Acetic acid AR Merck India ltd.

3,5 '
B) Method
The method for high performance liquid chromatography, was
carried out using the following solutions (1) and (2).
For solution (1) an accurately weighed quantity of the preparation being
examined equivalent to 400 mg of GS extract was dissolved 50,ml of methanol,
pick up 5 ml of the stock solution and add 0.2 ml of 10% KOH and diluted to
100.00 ml with the mobile phase and filtered through 0.45 micron filter paper.
Solution (2) was prepared by dissolving 0.4 mg /ml of GS extract working
standard in the mobile phase.
The chromatographic procedure was earned out using (RP ODS
C18) stainless steel column (25 cm x 4.6 mm) Phenomene x 5 micron particle
i
size.), (b) 29 ml of water and 1 ml acetic acid, 70ml of acetoniltrile was mixed
and filtered the mixture through a membrane filter of pore size not more than
0.5 pm and degassing the same, with a flow rate of 0.5 ml per minute and (c)
detection at wavelength of about 240 nm.

In the chromatogram obtained with solution (1) the area of any peak (generally
first and most prominent peak is corresponds to Gymnemic acid) corresponding
GS extract in the chromatogram obtained with solution (2) was compared and
the percentage of the total gymnemic acid present was calculated.
Preparation of the working standard for the total gymnemic acid.3,5
3 gm of Gymnema sylvestre purified extract was taken and
dissolved in 0.25% of KOH solution. This solution was filtered and acidified with
10 % hydrochloric acid up to pH 1.5. This solution was stand at room
temperature for 30 minutes and filtered through Whatman filter paper No.1. The
precipitate was collected and dissolved in 50 ml 80 % ethanol and evaporated
80% ethanol up to volume of about 8-10 ml. Allow to stand for 2 hrs and filter
and dry in oven under vacuum till a constant weight obtained.
Calculation:
Quantity of total Gymnemic acid
_ A UCT xWtofS* potency of s tan dard

AUCSxWtofTx.{\.QQ% LOD of sample)
AUC T: peak area value of testing solution of (1) Considered as total gymnemic
acid
AUC S: Peak area value of standard solution' of (2) Considered as a total
gymnemic acid

4.2.4. Analytical method validation for Gymnemic acid by HPLC6.
A) Specificity of the method
The Specificity test includes the measurement of response on
high performance liquid chromatography, using the GS Working standard
solution at 240 nm.
The GS working standard solution contains 0.4 mg /ml of GS
extract working standard in the mobile phase.
The chromatographic procedure carried out using the method described in
section 4.2.3 pt detection-wavelength of about 240 nm.
B) Linearity of HPLC method:

>
Linearity was established by taking ten different samples of
different concentration
Procedure: The standard stock solution of GS extract of 'concentration
0.1 mg/ml) was taken and the chromatographic procedure followed as describe
in section 4.2.3.
C) Range: The range of an analytical procedure for GS extract was determined
by taking an interval between the upper and lower concentration of the GS
extract solution.
Limit for assay of an active sub.80 to 120% of the test concentration.
D) Accuracy:
Procedure: GS extract tablet powder was dissolved in 100 ml
saline buffer of pH 7.2 and it is diluted such a way to produce concentration
equivalent to 0.1 mg /ml.

/
The response was measured at 240 nm. This was considered as
a blank matrix reading. In this stock solution about 0.2ml, 0.4ml, 0.6ml, 0.8ml
and 1 ml of standard GS extract solution was added (spike). The response was
measured at 240 nm and recorded as a response and it is compared with the
response of blank.
The result of analysis was compared to the more amount added
for each spike and the average recovery of the GS extract is calculated.
E) Precisions
Repeatability:
Procedure: the analysis of the GS extract stock solution of
concentration 0.1 mg/ml is carried out by the HPLC method at 240 nm for
about six determinations and the average, standard deviation and relative
standard deviation was calculated.
4.2.5. Analytical method validation for Gymnemic acid from formulation by
using UV spectrophotometer 6'
A) Specificity: verification of the specificity of Gymnemic acid by UV
spectrophotometer and e max determination
Procedure:
1) 400 mg of the GS extract was dissolved in 100 ml of methanol.
2) Then this solution was further diluted with methanol to resulted final dilution
800 mcg/ml
3) Whether the absorbance was obtained or not it was checked at 203 nm to
321 nm by scanning through out the UV range.

4) The specificity was established by taking measurable absorbance.
5) The absorbance was recorded in a table and the maximum wavelength was
determined where the GS extract gave maximum absorbance.
c) Linearity of UV method:
Ci) Linearity of the GS extracts raw material
Procedure
Linearity was established by taking minimum five samples of
different concentrations. The standard stock solution of GS extract of
concentration 100mg /100 ml is prepared in a 100 ml volumetric flask with
saline buffer of pH 7.2.
It is filtered trough Whatman filter paper no 41.
1 ml of the stock solution was picked up and then further diluted to
10 ml with saline buffer of pH 7.2.
Similarly this stock solution was further diluted to achieve various concentration
Like to 0.02mg /ml, 0.04g /ml, 0.06mg/ml, 0.08 mg /ml, 0.1mg/ml.
The absorbance of this solution was taken at 240 nm and recorded.

j
The mean, standard deviation and correlation coefficient of the data was
determined
Cii) Linearity of the GS extracts tablet
Procedure: 10 tablets were crushed to form a powder. This powder equivalent
to about 100 mg of GS extract was Weighed and dissolved in 100 ml volumetric
flask with help of saline buffer pH 7.2.
It was filtered through Whatman filter paper no 41.

1 ml of the stock solution was then picked up about and then further diluteed to
10 ml with saline buffer of pH 7.2.
Similarly this stock solution was further diluted to achieve various
concentration such as 0.02mg /ml, 0.04g /ml, 0.06mg/ml, 0.08 mg /ml,
0.1 mg/ml.
The absorbance of this solution was taken at 240 nm and recorded.
The mean, standard deviation and correlation coefficient of the data was
determined.
D) Range: The range of an analytical procedure for GS extract was determined
by taking the interval between the upper and lower concentration of the GS
extract solution.
Thus a sample of concentration 0.05 mg/ml was prepared and the
absorbance was recorded and calculated for the content with reference to
calibration graph obtained in linearity.
If the result obtained equivalent or 1% then it was considered as
the range of the analysis. Limit for assay of an active sub.80 to 120% of the test
concentraction.
Similar procedure is carried out for the tablet formulation.
E) Accuracy:
Procedure: GS extract tablet powder was dissolved in 100 ml saline buffer of
pH 7.2 and it is diluted to produce concentration equivalent to 1 mg /ml. 0.2 ml
of this stock solution was taken and diluted with saline buffer of pH 7.2 to
produce 10 ml. The absorbance is measured at 240 nm. This, was considered

as a blank matrix reading. In this stock solution about 0.2 ml, 0.4ml, 0.6ml,
0.8ml amount of standard GS extract solution was added (spike). The
absorbance was measured at 240 nm. The results of analysis were compared
to the more amount added for each spike and the average recovery of the GS
extract was calculated.
F) Precisions
Sample repeatability:
Procedure:
The analysis of the GS extracts stock solution of concentration of
0.06 mg /ml was carried out by the UV spectrophotometer at 240 nm on about
six different determinations. The average, standard deviation and Relative
standard deviation was calculated.

4.3. Formulation development
4.3.1 Preformulation studies 7
The Preformulation studies consist of the physical as well as
chemical properties of the Gymnema sylvestre extract.
4.3.1.a-b. Physical and Chemical properties of the standardized Gymnema
sylvestre extract
A) Materials
1 Hydrochloric acid Qualigens ltd Mumbai.
3 Ethyl alcohol -do-
4 Sodium chloride Merck India ltd Mumbai

.
5 6.8 pH buffer Qualigens chemicals
B) Method: Some organoleptic properties were analysis by the sensory
observations like colour and appearance, odour, taste. The tests like Loss on
drying; pH of 1% solution, total ash, and solubility was performed as per the
standard protocol given in the official pharmacopoeia.

4.3.1.C. Bulk characterization 7
i) Fine particle characteristics
A) Materials
Gymnema sylvestre extract (75% standardizes)
Sieves of different mesh size (#36-#100)
B) Method 7
100 gm of the Gymnema sylvestre powder was taken on a sieve
shaker. The assembly was shake for 3-4 min and the weighed the material
retains on each sieve. By considering the Arithmetic mean of opening in micron
of sieve and % retain the mean average particle size was calculated by using
following formula
Average particle size =Mean of opening of sieve in micron X % retain
Wt of sample
ii) Hygroscopic Characters'
5 gm of the extract was placed in an open dish for about 0-24 hours exposure
at atmospheric temp and humidity and measure the % weight gain in the
sample. The % wt gain in each dish was found out.
iii) Bulk density
Apparent bulk density G/ml was determined by pouring presieved
material (#36) in to a graduated cylinder via a large funnel and measured the
%
volume and weighted as it was. The tap density was determined by placing a
same graduated cylinder on a mechanical taper apparatus, which was operated
to fix number of taps (i.e. 50).

iv) Powder flow properties 7
The measurement of the free flow property is a measurement of
compressibility. The %compressibility was given by following equation
% Compressibility = (Pt-Po)/Pt x 100
Where Pt is the tapped density and Po the initial bulk density.
The table gives the relation between compressibility and flow ability
Sr. no % Compressibility . Flow ability
1 5-15 Excellent
2 12-16 G.ood
3 18-21 Fair 1 ;
4 23-35 Poor
5 33-38 Very poor
6 I 40 onward Very very poor, v

I
v) Solubility analysis 7 i
The solubility values that are useful in early development are
solubility in distilled water, 0.9% NaCI, 0.01 M HCL, 0.1 M Hd and 0.1 M NaOH
all at room temp as well as at pH 7.4 buffer at 37c.
Procedure: 100 mg of Gymnema sylvestre drug powder was
placed in each four 100 ml volumetric flask and the volume was made up to 100
ml with 0.9% NaCI, 0.01 MHCL, 0.1 M NaOH I and 7.2 pH buffer solution
respectively. The flasks were sonicated for 5 minutes. These solutions were
filtered through the previously weighed filter paper and the residue was dried on
147 Govt. College ofPharamey, Karad

the filter paper until the constant weight obtained. The weight retained on filter
paper recorded.
vi) Solid state stability 7
The stability studies are usually the first quantitative assessment
of chemical stability of new a drug. These studies includes the experiments
under conditions similar to the handling, formulation storage and administration
of drug
The accelerated stability study of GS extract was conducted by
keeping the Gymnema sylvestre extract powder in the humidity oven at about
the 40C temp and relative humidity about 75% for about 3 months.


4.3.2.Formulations.
A) Materials and equipment7
1 Micro crystalline cellulose Alok cellulose, mumbai.
2 Sodium starch glycolate do-

!
3 Ethyl cellulose Merck India ltd mumbai.
4 Isopropyl alcohol -do--

i
5 Methyl acylic copolymer Corel Pharma chem. Ahemadabad
i
6 Ultra press D Ultra pharma diem. Ahemadabad.
\ 1
7 Starch 1 -n-do-
8 Di- calcium phosphate Enar research Laboratories. Mumbai
9 Cross Carmulose sodium Alok cellulose mumbai.

i i
10 Magnesium sterate -do-

-a.
1
11 Talc
0
12 Aerosil IP FMC ltd
13 Sodium laurel sulfate Merck India

ltd\ mumbai.
14 Tablet compression Kamavati. Eirig. Ltd.
machine

* Formulation, standardization and evaluation of indigenous preparation "
B) Methods7'8
4.3.2.a. Formulation of Gymnema sylvestre extended release tablet
Table 4.1 Formulation of Gymnema sylvestre extended release tablet
Sr.no Excipients/ Composition Quantities in mg / 600mg

tablet
T1 T2 T3 T4
1 Gymnema sylvestre (GS) extract

400 400 400 400
75%
2 Micro crystalline cellulose powder .110 100 95 90
3 Sodium starch glycolate 65 70 65 60
4 Ethyl cellulose 105 10 20 30
5 Isopropyl alcohol '300 300 300 300
6 Aerosil IP 05. 05. 05. 05.
7 Sodium lauryl sulphate 05. 05. 05. 05.
8 Magnesium stearate 05 05 05 05
9 Talc 05 05.. 05 05
10 Total 600 600 600 600
Abbrivations:
Gymnema sylvestre extended release tablet formulation No 1-4: (T1-T4)
Microcrystalline cellulose powder: MCCP
Sodium starch glycolate: SSG
Ethyl cellulose: EC
Isopropyl alcohol: ISP
150 Govt. College ofPharamcy, Ken-ad

Sodium lauryl sulphate: SSL
Preparation Method: 7,8
Dry Mixing:
Gymnema sylvestre extract 400gm, MCCP. 100 gm, SSG 60 gm
was taken and passed through #30-mesh sieve in the sifter. The sieved
materials was loaded in the mixer and mixed for 20 minutes.The material from
mixture was removed and 300ml of 10% Ethyl cellulose solution in IPA was
added in the blended powder and it was mixed for 20 min in a mixer.The dump
mass was passed through the sieve no 10 and then dried at 45c for 2 hours.
| 1
The dried mass was pass through oscillatory granulaitor with 10-mesh sieve.
The sized granules were collected in a plastic bag and weighed. The quantity of
the dried granules was noted. The granules thus obtainbed were mixed for 2
minutes in mass mixer. The weighed quantity of SLS, Mg Sterate, talc and
I
Colloidal Silicon dioxide (Aerosil) were taken and passed through 36-mesh
i
sieve. Then this material was blended with dry granules in mass mixer and
i
lubricated thoroughly for10 minutes. The quantities o1j the lubricated granules
were noted.
Compression (Tablet Formation):
After confirming tableting machine, hopper and dies punches were
clean, the round shaped die punches of diameter 11.6 + 0.30 mm and thickness
5.0 0.30 mm wt adjusted. The weight of the tablet was adjested approximately
600 mg in standard concave shape. Then compression machine was opreted
continuously to form a desired tablet. After compression of 1000 tablets the
/
/
"
above parameters are checked and recorded. The compressed tablets were
then packed in an airtight polythene bag. The analysis of the tablet was carred
out as per the IP1996 for the specified parameter.
4.3.2.b. Formulation development: formulation of Gymnema sylvestre extended
release granules.7,8
Table 4.2 : Formulation development: formulation of Gymnema sylvestre
extended release granules
Sr.no Excipients/ Composition Quantities .in mg / 600mg
granules
. ! / ' :!* :
G1 ! iG 2:: G 3
ji * i i
G4
* I
1 Gymnema, sylvestre (GS) extract \ I
! '1
400 400'1 ;400 400
75% |
: I
2 Micro crystalline cellulose powder 110 100 , 95 90
i
3 Sodium starch glycolate 65 70 , 65 60

>i
i
i

4 Ethyl cellulose 05 10 20 30
i
! 1
i
< i
5 Isopropyl' alcohol 300 300.; 1300 300

I'
6 Aerosil IP 05. 05. j 05. 05.
7 Sodium lauryl sulphate 05. : 05. 05. 05.

i
8 Methacrylic acid copolymer Type
10 10 i *
1.0 10
A
, i
Total 600 600 600 600
Gymnema sylvestre extended release granules formulation No 1-4: (G1-G 4)
-*Carad
152 Govt. College ofPharo-
/ /
"
Procedure:
The same procedure of the extended release tablet was carried
out up to drying stage of the granules. 2 gm of Methacrylic acid copolymer Type

i
A (L-100) was weighted and dissolved in 200 ml of Isopropyl alcohol with high
speed stirring. 0.2 gm of Polyethelene glycol was added in it and mixes it
uniformly. This coating solution was spraied on the dried granules in a coating
pan and then dried this coated granules in oven at 60C. The coating of the
granules is carried out for the masking of bitter and, astringent taste of the
* i
Gymnema sylvestre granules.

4.3.2.C. Formulation development: formulation of Gymnema sylvestre
immediate release tablet7,8
Table 4.3 Formulation development: formulation of Gymnema sylvestre
immediate release tablet
Sr. Excipients/Composition Quantities in mg / 600mg

no tablet
Ti1 Ti2 Ti3 Ti4
1 Gymnema sylvestre (GS) extract 75% 400 400 400 400
2 Micro crystalline cellulose powder 100 , 60 0 0
3 Sodium search glycolate 0 15 50 0

' Ii
4 Ethyl cellulose 0 f' 0 0 0
5 Isopropyl alcohol 0 0 ; 0 0
i
6 Aerosil IP 05. 05. 05. 10
7
Sodium laury! sulphate 05 05 05 10
A
Methacryiic acid copolymer Type A. Qi 0 n o
oj
9 Ultra press D 0 0 170
10 Starch 55 80 30 0
11 Dicalcium phosphate 2$, 25 30 0
0i'.
12 Cross Carmellose sodium 0 70 0
13 Magnesium stearate 05 05 1 05 05
j
14 Talc o5: 05.! 05 05.
Total 600 600 600 600
Gymnema sylvestre immediate release tablet formulation No 1-4: (Ti1-Ti4)

"Formulation, standardization and evaluation ofindigenous preparation
Preparation Method7,8:
Dry Mixing: 400 gm of Gymnema sylvestre extract was taken and
. other materials from the table (no. 2 to no 6) weremixed and sieved through 30-
mesh sieve with respect to the formula A-D.
The weighed quantity of SLS, Mg Sterate, talc and Colloidal
Silicon dioxide (Aerosil) were taken arid passed through 36-mesh sieve. Then
this material was blended with dry granules in mass mixer and lubricated
thoroughly for10 minutes. The quantities of the lubricated granules were noted.
Compression (Tablet Formation):

After confirming tableting machine, hopper and dies punches were

I
clean, the round shaped die punches of diameter 1116 it 0.30 mm and thickness

5.0+ 0.30 mm wt adjusted. The weight of the tablet was adjested approximately
600 mg in standard concave shape. Then compression machine was opreted
continuously to form a desired tablet. After compression of 1000 tablets the

' i
above parameters are checked and recorded. The* compressed tablets were
' IiJ
then packed in an airtight polythene bag. The analysis; of the tablet was earned
i i
out as per the IP1996 for the specified parameter.

4.4.4. Evaluation of the Gymnema sylvestre formulation:
A) Materials 47'8
4.4.4. a-c. List of materials.
3 Acetoniltrile for HPLC Merck India ltd Mumbai
4 Potassium dihydrogen Merck India ltd [Mumbai
phosphate
4.4.4.b.list of apparatus.
Sr.No Name of apparatus Source

i
I
1 Vernier caliper Vigo scientific mumbai
2 Friability apparatus Eelectrolab ltd
3 Dissolution apparatus Eelectrolab ltd

l'
4 Disintegration apparatus Eelectrolab

I ltd
5 Hardness tester Vigo scientific mumbai
B) Methods4'78
Weight variation test of tablet:
Procedure: Weigh accurately 10 uncoated tablets individually and calculated
the average weight and weight variation.
Thickness: The thickness of the tablet is measured on vernier caliper.


Diameter: The diameter of the tablet is measured on vernier caliper.
Friability: procedure
A sample of twenty tablets was taken and placed the tablets on a
sieve no. 100. The loose dust was removed by air pressure. The tablet sample
was accurately weighed and places the tablets in the drum. The drum Rotated
for 100 times and tablets were removed. Weighed the tablets to the nearest
milligram and found out the mass loss.
A maximum loss of 1% of the mass of the tablets tested is
considered to be acceptable for most products.
Expression of the results: The friability is expressed as the loss of mass and it is
calculated as a percentage of the initial mass.
Dissolution:
Apparatus II (Paddle apparatus)
In apparatus two the whole assembly was same as apparatus one
except that in the stirring element the basket is replaced by a paddle. The blade
passes through the diameter of the shaft so that the bottom of the blade is flush
with the bottom of the shaft. The shaft was positioned so that its axis is within 2
mm of the axis of the vessel and the lower edge of the blade was 23 to 27 mm
from the inside bottom of the vessel. The apparatus operates such a way that
the paddle rotates smoothly and without significant wobble.
Method:4'78
900 ml of the saline phosphate buffer of pH 7.4 as a dissolution
medium, free from dissolved air, was introduced into the vessel of the

"
apparatus. The dissolution medium was heated to between 36.5 and 37.5.
One tablet of Gymnema sylvestre extract was added in each vessel. Care was
taken to ensure that air bubbles are excluded from the surface of the tablet. The
apparatus was operated immediately at the speed of rotation 50 rpm. The
dissolution mediumwas kept in between 36.5 and 37.5. The samples were
drawn at every one-hour interval. Each ml of this sample under test was diluted
with 9ml of dissolution Medium. The absorbance of each sample was token at
the wavelength 240 nm in comparison with a Standard solution having a known

* l
concentration of GS extract working standard the same Medium.
Acid Stage
Medium: 0.1 N hydrochloric acid; 900 ml.
Acid Stage 1000 ml of 0.1 N hydrochloric acid was kept in the vessel, and
assemble the apparatus. Allow the medium to equilibrate to a temperature of 37
0.5. One tablet of Gymnema sylvestre extract was added in each vessel.
Care was token to ensure that air bubbles are excluded from toe surface of the
tablet. Apparatus was covered, and operated toe apparatus; for 1 hour at the
rate of 50 revolutions per min. After 2 hours of operation ini 0.1 N hydrochloric
acid, withdraw an aliquot of the fluid, and proceed immediately as directed
under Buffer Stage. Perform an analysis of toe aliquot using toe Procedure
specified in the test for total gymnemic acid test by HPLC method. From 0.1 N
hydrochloric acid remaining in vessel, added 20.0 ml of 5 N! sodium hydroxide,
and stir for 5 minutes. The amount of GS extract was determined using the
procedure specified in the test for total gymnemic acid test by HPLC method

Formulation, standardization and evaluation ofindigenous preparation"
Buffer stage:
Medium: 7.4 pH Saline phosphate buffer; 900 ml.
Apparatus 2 (paddles constructed of or coated with polytef being used): 50 rpm.
Procedure At the end of 2 hours, each Tablet was removed from the
individual vessels, and subjected to the test in the Buffer stage in Apparatus 2
with the speed: of 50 rpm. At the end of 60 minutes, the amount of total GS
extract was determined using the procedure specified in the test for total
gymnemic acid test by HPLC method.

i
Tolerances
' , C
Tolerances (for products 400-mg tablet): The percentage of the claimed amount
of GS extract dissolved should not more than 45% (Q) in 120 minutes, 60% (Q
0) in 180 minutes, and not less than 70% (Q 0 0) in 480 minutes of the claimed
amount of GS extract dissolved in it.
Evaluation of GS dispersible tablet
Dispersible tablet are coated or uncoated tablet intended to be disperse in water

i
before administration giving homogeneous dispersion.
Test: disintegration: dispersible tablet disintegrate within 3 minutes when
examine by the test under disintegration but using water at 15-25 c
Fineness of dispersion: the two tablets were placed in 100 ml of water and
stired until completely disperse to produce a smooth dispersion, which passes
the screen of mesh size 710 micron.

Disintegration for dispersible Tablets
Disintegration test4,7,8
APPARATUS
The apparatus was consists of a basket-rack assembly, a 1000-
mL, low-form beaker, 138 to 155 mm in height and having an inside diameter of
97 to 110 mm for the immersion fluid, a thermostatic arrangement for heating
the fluid between 35 and 39, and a device for raising and lowering the basket
in the immersion fluid at a constant frequency rate between 29 and 32 cycles
per minute through a distance of not less than 5.3 cm and not more than 5.7
cm. The volume of the fluid in the vessel was such that at the highest point of
the upward stroke the wire mesh remains at least 2.5 cm beiow the surface of
the fluid and descends to not less than 2.5 cm from the bottom of the vessel on
the downward stroke. The time required for the upward stroke is equal to the
time required for the downward stroke, and the change in stroke direction is a
smooth transition, rather than an abrupt reversal of motion. The basket-rack
assembly moves vertically along its axis. There was no appreciable horizontal
motion or movement of the axis from the vertical. Basket-rack Assembly The
basket-rack assembly consists of six open-ended transparent tubes, each 7.75
0.25 cm long and having an inside diameter of 20.7 to 23 mm and a wall 1.0
to 2.8 mm thick; the tubes were held in a vertical position by two plastic plates,
each 8.8 to 9.2 cm in diameter and 5 to 7 mm in thickness, with six holes, each
22 to 26 mm in diameter, equidistant from the center of the plate and equally
spaced from one another. Attached to the under surface of the lower plate is a

woven stainless steel wire cloth, which has a plain square weave with 1.8- to
2.2- mm mesh apertures and with a wire diameter of 0.63 0.03 mm. The parts
of the apparatus are assembled and rigidly held by means of three bolts
passing through the two plastic plates. A suitable means is provided to suspend
the basket-rack assembly from the raising and lowering device using a point on
its axis.
Procedure4,7,8
Uncoated Tablets 1 tablet was place in each of the six tubes of
the basket and the apparatuswas operated, using water maintained at 37 2
as the immersion fluid. At the end of the time limit of specified in of three
minutes, the basket was lifted from the fluid, and the tablets were observed for
all of the tablets have been getting disintegrated completely or not.
Dissolution study: Gymnema sylvestre immediate release/dispersibie
tablet.
Medium: 7.4 pH saline Phosphate buffer.
Apparatus 2: 50 rpm.
Time: 60 minutes.
Procedure: same as per the Gymnema sylvestre extended
release tablet was adopted except the acid stage. The amount of Gymnema
sylvestre extract dissolved in dissolution medium was determined, in
comparison with a standard solution having a known concentration of
Gymnema sylvestre Extract in the same medium at the time of use. Using UV
spectrophotometer at 240 nm wavelengths the analysis was carried out.

Tolerances not less than 80% (Q) of the claimed amount of Gymnema
sylvestre extract is dissolved in 60 minutes.
4.5. Design of the in vivo dissolution study
4.5.1. Clinical subject:9
Gs extended release granules (3.00 gm) were administered to 6
fasting healthy male volunteer (six male) of 24-30 year of age. All volunteers
were free from the symptoms of the renal damage and any of the diabetic
mellitus symptoms. The volunteer were attending the Dr. Desai clinic and were
continuously monitored. The information on the health aspect and the
background of any therapy in the past was' obtained through the questionnaire
and by the personal interview. During the study duration the volunteer were
given adequate instructions on the food. During the study they also learnt to
/
recognize hypoglycemic episode and were advised to consume one
teaspoonfui of sugar or to drink glucose containing beverages when they
experience any symptom of hypoglycemia (a tendency for fainting, dry feeling in
the mouth, weakness in the volunteer were the limb.) The volunteer included in
the trial only after informed consent.
The every volunteer under study were investigated for the fasting
blood glucose level serum total gymnemic acid concentration at the beginning
of the study and also after 13 hr. the blood samples were collected after
regular interval at 3 hours (8.00,11.00,14.00,17.00,20.00 hours).

4.5.2. Clinical testing. 9'10
Venous blood samples were drawn under fasting conditions into
the tubes containing the disodium salt of ethylene diamine tetracetic acid
(1 mg/ml) as an anticoagulant another samples was collected as sodium
fluoride as a anticoagulant for blood glucose estimation by the o-toludine colour
reaction as a modified method by sasaki and matsui(1972). The serum
separation was carried out by centrifugation and subjected for the estimation of
total gymnemic acid by the method developed and explained in section 4.2.12.
The serum was separated from the cell within 2 hr of blood collection, labeled
and store in refrigerator until the assay of total gymnemic add were made using
\
HPLC method of analysis at research center ICPA health product ltd.
Ankaleshwar.

4.5.3. Estimation of total gymnemic acid extract from the plasma sample5
Estimation of gymnemic acid from plasma by HPLC method:
Test solution and standard solutions of equal concentrations are
subjected to HPLC separately. The Column used is L1 (5C 4.6mm x 250 mm)
using mobile phase Acetonitrile-water-acetic acid (70:29:1) in Isocratic system
for 0-20 min. Flow rate: ,1.5 ml/min at Wavelength: 220 nm
Preparation of the Test solution:
Take all the vials containing the about 1.0 ml of the plasma
sample (free from the blood cells) These vials were then stirred for about 20-25
minutes on the sonicator and filter through o.45 micron pall nylon filter paper to
remove the colloidal particle present in the blood serum that may interfere in the
HPLC analysis. Then filtrate is subjected to the HPLC analysis by employing
method describes above.
Calculation: the calculation of the concentration of the total
gymnemic acid from the plasma carried out by the comparison against the
calibration graph of Gymnema sylvestre extract.
Reference . .
1) The Wealth oif' India, a directory of Indian raw material and industrial
products. CSIR1956, vol-4, pp. 276-77.

1
Ir
2) Zhen H, Xu S, Pan X. The pharmacognostical identification of peel of
Gymnema sylvestre Zhong Yao Cai. 2001 Feb;24(2):95-7
3) V. Rajpal. Testing and extraction methods of medicinal herbs Eastern

/
publication, new Delhi, Vol. 1 page no: 140-150
164 Govt. College^ofPharamcy, Karad

4) Indian pharmacopoeia 1996
5) Suzuki K, Ishihara S, Uchida M, Komoda Y. Yakugaku Zasshi.
Quantitative analysis of deacylgymnemic acid by high-performance liquid
chromatography1993 Apr; 113(4): 316-20.
6) ICH guidline for analylitica! method validation cited bywww.ich.org
7) Lachman liberman. Theory practice of industrial pharmacy 3rd edition ,
Varghese publication Mumbai.
8) US pharmacopoeia 26,2003
9) Bsskaran K, et al. J Ethnopharmacol. 1990 Oct; 30(3): 295-300
10) ICH guideline for clinical efficacy cited bywww.ich.org

Formulation, standardization and evaluation ofindigenous preparation "
4.6 In vivo in vitro correlation 1
A level C correlation was used to study correlation between in
vitro and in vivo data. Level C was chosen for an easy method to find rough
in vitro/in vivo correlation. Level C correlation was determined using the in
vitro parameters time at which 50% of drug had dissolved (t 50%) and
amount dissolved in 3 hours (D 3h) and the in vivo parameters C max, t max.
Each in vivo parameter was plotted against an in vitro parameter and
correlation coefficient was calculated.
Refrence:
1) United state Pharmacopiea (26) 2003

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"Formulation, standardization and evaluation of indigenous preparation

MATERIALS AND METHODS

4.1. Gymnema sylvestre raw material

4.1.1. Gymnema sylvestre Authentication

Sr.No Name of materials Source

1 Gymnema sylvestre dried leaves Phyto-pharma Pvt. Ltd.

powder Kolhapur, India.

2 Gymnema sylvestre 75% Amasar Labs India Pvt. Ltd.

standardized extract Indor, India.

B) Method: Gymnema sylvestre consists of the dried leaflets of GS RBr. Family:

Synonyms: Meshashringi, Gurmar1

General appearance and Microscopic characteristic of the plant

was verified for originality.

Powder plant material was observed physically as well microscopically2

129 Govt. College ofPharamey, Karad

4.1.2. Extraction of Gymnema sylvestre dried leaves

Sr.No Name of materials Source

1 Hexane Loba chem. Pvt. Ltd. Mumbai

5 Sodium hydroxide do--

6 Solxlet apparatus Borosil India ltd

7 Vacuum drier Lab India mumbai.

The crushed dried leaves of authenticated Gymnema sylvestre (GS) were

extraction apparatus. The micella was concentrated under, reduced pressure to

30 % of the original volume. The resultant extract was acidified to pH 3 by

was dried and purified to required strength.

130 Govt. College ofPharamcy, Karad

4.1.3. Enrichment of the extract to 25% strength and 75% strength3

Sr.No Name of materials Source

1 Sodium hydroxide Loba chem. Pvt. Ltd. Mumbai

2 Purified water -do-

3 Cone. Hydrochloric acid -do-

4 Ethyl alcohol -do-

About 30 gm of Gymnema sylvestre leaf , extract was taken and

precipitate was collected and dissolved in 20 ml 80 % ethanol. This filtrate was

combined and washed and evaporated to dry in oven under vacuum.

131 Govt. College ofPharamcy, Karad

Size reduction of the dried leaves

132 Govt. College ofPharamcy, Karad

4.2.1. Standardization of Gymnema sylvestre Extract.

A) Materials (section 4.2.1.a-g)

Sr.No Name of materials Source

1 Ethyl alcohol Qualigens ltd mumbai.

2 1,4-dioxan Loba chem. Pvt. Ltd. Mumbai

4 Hydrochloric acid --do-

5 Potassium iodide -do--

9 Vanillin Qualigens ltd mumbai.

10 Phosphoric acid -do-

11 Iodine Qualigens ltd mumbai.

12 Silica dish Borosil India ltd

13 Oven Lab India ltd.

133 Govt. College ofPharamcy, Karad

In this section some physicochemical properties of Gymnema

experiments were followed as described in the Indian pharmacopoeia. The

standard reagents were prepared as described in the Indian pharmacopoeia.

The taste of the 1 % solution was determined.

1% solution was shaken with the water.

5 % solution was treated with the 2 ml of 0.1 N Hydrochloric acid.

4.2.1. b Loss on drying

1 gm of the powder substance being examined was placed (Gymnema

sylvestre extract powder) in a weighing bottle previously dried in an oven at

oven within a specified temperature range of 100-110 c.

Unit for expression: solubility is expressed as % w/v.

In water: 1 gm of the substance was placed in 100 ml water in a conical flask. It