Almaty, 2009. Ecological design and standardization standardization.
Standardization of Formulations -...
Transcript of Standardization of Formulations -...
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 68
4
Standardization of Formulations
There are various factors which can affects the amount of active ingredients in herbs
and formulation which needs to be consider if we want better quality raw materials
from plant and hence it essential to standardize the raw material and formulations for
different parameters described in WHO guideline (Figure: 4.1)
Figure 4.1 Parameters of standardization for medicinal plant as
per WHO guidelines
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 69
Standardization parameters were established as per the WHO guidelines (WHO,
1998) of two Ayurveda formulations, one Unani formulation and one Siddha
formulation named Balacaturbhadrika churna (BC), Shringyadi churna (SC),
Safoof_E-Sana (SS) and Thirikadu choornam (TC) respectively for present study.
This chapter includes standard methodologies used for all the batches of formulations
and their raw materials which are summarized below with standard references
followed by result and discussions for the findings of the each selected formulations.
Material and Methods
4.1 Raw materials
All the raw material used for the preparation above mentioned formulations were
purchased from local market of Raipur (C.G.) 492010, INDIA and identified
morphologically and microscopically and compared with standard pharmacopoeia
monograph.
4.2 Organoleptic profile
The studies of organoleptic characters include color, consistency, odour and taste.
These parameters were established for the entire laboratory batch of BC, SC, SS, TC,
one marketed formulations of each and every raw ingredient of all the formulation
through the method described below (Wallis, 1967).
(i) Color
All the samples were taken in to watch glass and examined untreated under diffuse
daylight. They were observed for their color by naked eye.
(ii) Odour
All the samples were examined for their odour. The time interval among the two
smelling was kept two minutes to nullify the effect of previous smelling.
(iii) Taste
The samples were taken and examined separately for their taste on taste bud of the
tongue. The time interval among each sample was kept 15 minute, so as to make taste
buds available fresh every time.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 70
4.3 Physicochemical characteristics of crude drugs and formulations
The raw materials, lab formulation and marketed formulation of BC, SC, SS, TC
were subjected to evaluation of physical properties which are determined in form of
tap density, bulk density, angle of repose (Sinko, 2006), hausner ratio and carrs index
by using method (Gibson, 2001) as described below.
(i) Bulk density
Bulk density, ρb is defined as the mass of a powder divided by the bulk volume. A
sample of about 50 cm3 of powder that has previously been passed through a U. S.
Standard no. 20 sieve is carefully introduced into a 100 ml graduated cylinder. The
cylinder is dropped at 2-sec intervals on a hard wooden surface three times from a
height of 1 inch. The bulk density is then obtained by dividing the weight of the
sample in gm by the final volume in cm3 of the sample contained in the cylinder.
(ii) Tap density
Tap density, ρT is defined as the mass of a powder divided by the Tapped volume. A
sample of about 50 cm3 of powder that has previously been passed through a U. S.
Standard no. 20 sieve is carefully introduced into a 100 ml graduated cylinder. The
cylinder is dropped at 2-sec intervals on a hard wooden surface hundred times from a
height of 1 inch until no further decrease in the volume of powder takes place. The
tap density is then obtained by dividing the weight of the sample in gm by the final
volume in cm3 of the sample contained in the cylinder.
(iii) Angle of repose
A glass funnel is held in place with a clamp on ring support over a glass plate. The
glass plate is placed on a micro-lab jack. Approximately 100 g of powder is
transferred in to the funnel (that has previously been passed through a number 10
mesh size), keeping the orifice of funnel blocked by the thumb. As the thumb is
removed, the lab –jack is adjusted so as to lower the plate and maintain about 6.4 mm
gap between the bottom of funnel stem and top of the powder pile. When the powder
is emptied from the funnel, the angle of the heap to the horizontal plane is measured
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 71
with a protector. Measure the height of the pile (h) and the radius of the base(r) with
the ruler. The angle of repose is thus estimated by following formula.
(iv) Hausner Ratio
The Hausner ratio is calculated by the formula given below, where ρB is the freely
settled bulk density of the powder, and ρT is the tapped density of the powder.
(v) Carr index
The Carr index is an indication of the compressibility of a powder. It is calculated by
the following formula, where VB is the freely settled volume of a given mass of
powder, and VT is the tapped volume of the same mass of powder.
(vi) Determination of loss on drying
An excess of water in medicinal plant materials will encourage microbial growth, the
presence of fungi or insects, and deterioration following hydrolysis. Limits for water
content should therefore be set for every given plant material. This is especially
important for materials that absorb moisture easily or deteriorate quickly in the
presence of water. The presence of excess amount of moisture in crude drugs may
affect their quality. These parameters were established for the entire laboratory batch
of BC, SC, SS, TC, one marketed formulations of each and every raw ingredient of
all the formulation through the method described below (WHO, 1998).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 72
(vii) Gravimetric determination
Accurately weighed 5g of the air-dried material, is placed in a previously dried and
tare flat weighing bottle. The sample is dried in an oven at 100-105°C until two
consecutive weighing do not differ by more than 5mg.
(viii) Determination of foreign matter
Medicinal plant materials should be entirely free from visible signs of contamination
by moulds or insects, and other animal contamination, including animal excreta. No
abnormal odor, discoloration, slime or signs of deterioration should be detected. It is
seldom possible to obtain marketed plant materials that are entirely free from some
form of innocuous foreign matter. However, no poisonous, dangerous or otherwise
harmful foreign matter or residue should be allowed. All the formulation and its raw
material were examined for this parameter as per guideline (WHO, 1998).
Method
Accurately weighed (250g) sample of plant materials are spread in a thin layer and
foreign matter is sorted by using a magnifying lens (6x or 10x). The remainder of the
sample is sifted through a No. 250 sieve; dust is regarded as mineral admixture. The
portions of this sorted foreign matter are weighed and the content of each group is
calculated in grams per 100g of air-dried sample.
(ix) Determination of ash values
The total ash method is designed to measure the total amount of material remaining
after ignition. This includes both "physiological ash", which is derived from the plant
tissue itself, and "non-physiological" ash, which is the residue of the extraneous
matter (e.g. sand and soil) adhering to the plant surface. Acid-insoluble ash is the
residue obtained after boiling the total ash with dilute hydrochloric acid, and igniting
the remaining insoluble matter. This measures the amount of silica present, especially
as sand and siliceous earth. The percent total ash and Acid-insoluble ash was
determined for entire laboratory batch of BC, SC, SS, TC, one marketed formulations
of each and every raw ingredient of all the formulation. Detailed method for
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 73
determination of total ash and acid-insoluble ash is described below as per WHO
(WHO, 1998).
(x) Total ash
About 4g of the ground air-dried material is accurately weighed and Placed in a
previously ignited and tared silica crucible. The material is spread in an even layer
and ignited by gradually increasing the heat to a temperature of 500-600°C until it is
white, indicating the absence of carbon. The material is cooled in a desiccator and
weighed. The content of total ash is calculated in mg per g of air-dried material.
(xi) Acid-insoluble ash
To the crucible containing the total ash, 25 ml of hydrochloric acid is added, covered
with a watch-glass and boiled gently for 5 minutes. The watch-glass is rinsed with 5
ml of hot water and this liquid is added to the crucible. The insoluble matter is
collected on an ash less filter-paper and washed with hot water until the filtrate is
neutral. The filter-paper containing the insoluble matter is transferred to the original
crucible, dried on a hot-plate and ignited to constant weight. The residue is allowed to
cool in a suitable desiccator for 30 minutes, and then weighed without delay. the
content of acid-insoluble ash is calculate in mg per g of air-dried material.
(xii) Determination of Extractive values
The extractive values are recorded in alcohol and water with a view to study the
distribution of various constituents of BC, SC, SS, TC, one marketed formulations of
each and every raw ingredient of all the formulation. The method used for
determination of extractive value is described below.
Cold maceration
Accurately weighed 4.0g of coarsely powdered air-dried material is placed in a glass-
stopper conical flask and macerated with 100ml of the solvent for 6 hours, shaking
frequently, and then allowed to stand for 18 hours. The mixture is filtered rapidly
taking care not to lose any solvent. 25 ml of the filtrate is transferred to a tared flat-
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 74
bottomed dish and evaporated to dryness on a water-bath. The residue is dried at
105°C for 6 hours, cooled in a desiccator for 30 minutes and weighed without delay.
4.4 Phytochemical studies of crude drugs and formulations
To detect the presence of various phytoconstituents in formulations as well as in raw
materials phytochemical investigation is performed. The tests are performed on
alcohol and water extract. Qualitative phytochemical analyses were done for all
selected formulation and its raw materials (Kokate, 1994). Alkaloids, carbohydrates,
tannins and phenols, fixed oils and fats, saponins and gums and mucilage’s were
qualitatively analyzed.
(i) Alkaloids
The extracts were dissolved in diluted sulphuric acid and filtered. The filtrate was
treated with Mayer's, Dragendorfrs, Hager's and Wagner's reagents separately.
Appearance of cream, orange brown, yellow and reddish brown precipitates in
response to the above reagents respectively indicate the presence of alkaloids.
(ii) Carbohydrates
300 gm of aqueous and alcoholic extracts were dissolved in water and filtered. The
filtrate was treated with concentrated sulphuric acid and then with Molisch's reagent.
Appearance of pink to violet color indicates the presence of carbohydrates. The
filtrate was boiled with Fehling's and with Benedict's solutions. Formation of brick
red precipitate in Fehling's and Benedict's solutions is the positive result for reducing
sugars and non-reducing sugars respectively.
(iii) Tannins and phenols
Small quantity of alcoholic and aqueous extracts was dissolved in water and to that
ferric chloride solution (5%) or gelatin solution (1%) or lead acetate solution (10%)
was added. Appearance of blue color with ferric chloride or precipitation with other
reagents indicates the presence of tannins and phenols.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 75
(iv) Phytosterols
The extract is refluxed with solution of alcoholic potassium hydroxide till complete
saponification takes place. The saponification mixture is diluted with distilled water
and extracted with ether. The ethereal extract is evaporated and the residue is
subjected to Liebermann’s test and Liebermann burchard’s test.
(v) Gums and mucilage’s
About 10 ml of extract was slowly added to 25 ml of absolute alcohol with constant
stirring. Precipitation indicates the presence of gums and mucilage’s.
(vi) Fixed oils and fats
A drop of concentrated extract was pressed in-between two filter papers and kept
undisturbed. Oil stains on the paper indicate the presence of oils and fats.
(vii) Saponins
About one ml of the alcoholic and aqueous extracts were dissolved separately in 20
ml of water and shaken in a graduated cylinder for 15 minutes. Formation of one cm
layer of foam indicates the presence of saponins.
(viii) Protein and free amino acid
Small quantities of aqueous or alcoholic extract were dissolved in a few ml of water
and the solution is subjected to Millon’s, Biuret and Ninhydrin test.
(ix) Detection of Volatile oil
Accurately weighed (50g) powdered material is taken in a volatile oil estimation
apparatus and subjected to hydro distillation for the detection of volatile oil.
4.5 Development of thin layer chromatography
(i) General
Thin layer chromatography (TLC) is the most commonly used methods for obtaining
chemical fingerprints and identification of the crude plant extracts. However, there
are several possibilities that may arise while using these techniques for standardizing
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 76
the crude extracts. It is possible that the plant materials collected from the same plant
in two different seasons can show different phytochemical fingerprint and therefore
different biological activity or two plants with identical taxonomy collected under
same environmental conditions can show different phytochemical fingerprint but
similar biological activity. In such situations comparisons of the phytochemical
profiles as an indicator of important constituents can act as a shortcut for identifying
biologically active constituents. Another possibility that may arise is when two
different plants showing similar phytochemical fingerprints show different biological
activity. In such situations bioassay guided fractionation or any other suitable method
is the only option in identifying the biologically active constituents. The TLC profile
was determined for BC, SC, SS, TC, one marketed formulations of each and every
raw ingredient of all the formulation. The method used for determination of TLC is
described below (Hoffman, 1966).
(ii) Experimental technique
Preparation and activation of plate
Aqueous slurry (1 part silica gel G and 3 part distilled water ) was triturated in a glass
pestle mortar and spread over the glass plates (10cm by 20cm) by pouring method
and allowed to air dried. The plates were activated for an hour at 110-120 degree and
placed in desiccators for cooling.
Preparation of sample solution
About 1% solution of methanolic extract was prepared and suspended impurities were
filtered off.
Saturation of chamber
The solvent system was prepared and poured into the TLC chamber. A filter paper
sheet was placed into it to provide rapid saturation and to prevent edging effect.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 77
Chamber was sealed by placing a glass plate at the mouth of chamber with paraffin
wax.
Application of spots
The spots off sample solutions were applied with the help of thin capillaries on the
plates, at a distance of about 1.5cm from the bottom and were allowed to dry in air. The
distance between two spots was kept at least 10mm.
Development of chromatograms
After saturation, the plates were placed in the chamber and the solvent was allowed to
run until a solvent height of approximately 15cm is attained from the point of spotting,
removed and it was marked. Then it was allowed to dry in air and sprayed with
detecting reagent and kept in oven for 5 minutes. Then Rf values were calculated.
Distance traveled solute front
Rf value =
Distance traveled solvent front
4.6 Swelling index determination
Many medicinal plant materials are of specific therapeutic or pharmaceutical utility
because of their swelling properties, especially gums and those containing an
appreciable amount of mucilage, pectin or hemicelluloses (WHO, 1998). The
swelling index was determined for BC, SC, SS, TC, one marketed formulations of
each and every raw ingredient of all the formulation.
(i) Method
Three determinations for 1g of coarsely powdered fruit were carried out, and shifted
into a 25ml glass stoppered measuring cylinder. The internal diameter of cylinder was
16mm; the length of the graduated portion was about 125mm and marked in 0.2 ml
divisions from 0 to 50 ml in an upward direction. 25 ml of water was added and the
mixture was shaken thoroughly every 10 minutes for 1hr. The mixture was allowed to
stand for 3hrs at room temperature. The volume in ml was measured which was
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 78
occupied by the reduced fruit powder. The mean value of the individual
determinations was calculated.
4.7 Foaming index determination
Many medicinal plant materials contain saponins that can cause persistent foam when
an aqueous decoction is shaken. The foaming ability of an aqueous decoction of plant
materials and their extracts is measured in terms of a foaming index. The Foaming
index was determined for BC, SC, SS, TC, one marketed formulations of each and
every raw ingredient of all the formulation. The method was used is as follow:
Method
1g of coarsely powdered fruit is accurately weighed and transferred to a 500ml
conical flask containing 100ml boiling water. Temperature was maintained for 30
minutes. Cooled and filtered decoction was transferred in to 100ml volumetric flask
and volume was made upto 100ml. the decoction was poured in to 10 stoppered test
tubes in successive portions in 1ml, 2ml, 3ml, 4ml, 5ml, 6ml, 7ml, 8ml, 9ml & 10ml
and shaken in a length wise motion for 15 seconds and allowed to stand for 15
minutes and d the height of foam is measured.
The foaming index was calculated by using the formula- 1000/A
A= the volume in ml of the decoction used for preparing the dilution in the tube
where foaming to a height of 1 cm was observed.
Results and Discussion
4.8 Balacaturbhadrika churna
Formulation was strictly prepared as prescribed in the official book of Ayurvedic
Formulary of India (2003). 50 grams of each ingredients which includes Ghana
(musta), Krsna (pippali), Aruna (ativisa) and Sringi (karkatasringi) were taken. All
the ingredients were weighed accurately and made fine powder by passing through
sieve no. 80. Fine powders were mixed geometrically in plastic tray and packed in
plastic container.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 79
Three sample batch of Balacaturbhadrika churna were prepared using above
mentioned methods and were named as BC-I, BC-II, BC-III. One Marketed
formulations named MF was purchased from local pharmacy store Raipur. These
samples were stored at optimized conditions of Temperature, light and moisture.
(i) Organoleptic profile and physical characteristics
Organoleptic profile of the formulations and each raw material were determined by
the method described earlier. Findings of the different organoleptic parameters is
summarized in (Table 4.1). Physical characteristics of all the three batches of
Balacaturbhadrika churna (BC-I, BC-II and BC-III), its marketed formulation (MF)
and all raw materials were established through the method described earlier and these
data are also recorded in (Table 4.1).
Table 4.1 Organoleptic and physical characteristics of Balacaturbhadrika
churna and its raw materials
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI (Pistacia
integerrima), BC (Balacaturbhadrika churna), MF (Marketed formulation)
S
N Name Colour Odour Taste
Tap
density
Bulk
density
Angle
of
repose
Hausn
er
ratio
Carr’s
index
1 CR Dark
brown Pleasant Pungent 0.62 0.50 28.62 1.24 20
2 PL Dark
Brown
Charact
eristic Pungent 0.66 0.52 31.28 1.26 21
3 AH Ash
color
Charact
eristic Bitter 0.60 0.50 29.86 1.20 17
4 PI Light
brown Pungent Bitter 0.52 0.41 24.34 1.26 21
5 BC-I Light
brown
Charact
eristic Pungent 0.50 0.40 27.36 1.25 20
6 BC-II Light
brown
Charact
eristic Pungent 0.52 0.41 26.24 1.26 21
7 BC-III Light
brown
Charact
eristic Pungent 0.62 0.50 28.62 1.24 20
8 MF Light
brown
Charact
eristic Pungent 0.62 0.52 27.45 1.19 16
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 80
Discussion
Evaluation of organoleptic characters of raw materials Ghana (musta), Krsna
(pippali), Aruna (ativisa) and Sringi (karkatasringi) was performed and characters are
recorded. Laboratory batches of Balacaturbhadrika churna (BC-I, BC-II and BC-III)
and one marketed preparations were also evaluated for organoleptic characters. The
results for the marketed formulations (MF and Laboratory formulations are found
comparable. The above mentioned organoleptic data are very useful for the
preliminary identification of raw material and formulations.
The value of angle of repose for raw materials Cyperus rotundus, Piper longum,
Aconitum heterophy, Pistacia integerrima, lab formulation and marketed formulation
were 28.62, 31.28, 29.86, 24.34, 27.36, and 27.45 respectively which shows good
flow properties of prepared lab formulation. The flow properties of lab (BC-I) and
marketed formulations were also confirmed by Hausner’s ratio and Carr’s index; it
was found 1.25, 20, and 1.19, 16 respectively and indicates good flow characteristics.
(ii) Determination of foreign matter and loss on drying
Raw materials, Laboratory batches of Balacaturbhadrika churna (BC-I, BC-II and
BC-III) and one marketed preparation were also evaluated for foreign matter and loss
on drying through the methods described earlier in this chapter. The results of both
parameters are tabulated in (Table 4.2).
Table 4.2 Loss on drying and foreign matter content in Balacaturbhadrika
churna and its raw materials
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI (Pistacia
integerrima), BC (Balacaturbhadrika churna), MF (Marketed formulation)
SN Name %LOD
±S.D. (n=6)
%Foreign Matter
±S.D. (n=6)
1 CR 3.32±0.268 1.41±0.212
2 PL 3.78±0.642 1.32±0.064
3 AH 3.29±0.382 1.08±0.121
4 PI 3.02±0.196 1.86±0.226
5 BC-I 3.24±0.146 NIL
6 BC-II 3.58±0.206 NIL
7 BC-III 3.84±0.224 NIL
8 MF 3.88±0.292 NIL
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 81
Discussion
The amount of moisture in the crude drugs should be minimized in order to prevent
decomposition of either due to chemical changes or due to microbial contamination.
The percent moisture content for BC-I, BC-II and BC-III are 3.24±0.146, 3.58±0.206
and 3.84±0.224, while it is 3.88±0.292 for MF. The moisture content of formulation
was within acceptable range (<8%) thus implying that the formulation can be stored
for a long period and would not easily be attacked by microbes. The percent of
foreign matter was found to be 1.41±0.212 for Cyperus rotundus, 1.32±0.064 for
Piper longum, 1.08±0.121 for Aconitum heterophy and 1.86± 0.0226 for Pistacia
integerrima. Laboratory formulations of Balacaturbhadrika churna were prepared
after removal of foreign matter. In examination no poisonous, dangerous and harmful
foreign matter
or residue was found.
(iii) Ash values determination
The percent total ash and acid-insoluble ash was determined for each batch of
Balacaturbhadrika churna, its one marketed formulations and separately its raw
materials as per above mentioned method. The results are recorded in (Table 4.3).
Total ash value of Cyperus rotundus, Piper longum, Aconitum heterophy, Pistacia
integerrima, lab formulation and marketed formulation were 7.346±0.346,
5.032±0.624, 2.981±0.243, 4.621±0.334, 8.148±0.337 and 19.633±0.552
respectively. The value of total ash in marketed formulation is comparatively high in
comparison to lab formulation may be because of the higher amounts of inorganic
components present in marketed formulation. Acid-insoluble ash value of prepared
lab formulations were 3.281 ± 0.286 and 5.041 ± 0.368 for lab and marketed
formulation respectively shows that a small amount of the inorganic component is
insoluble in acid it indicates adulteration of raw ingredients by substance like silica,
rice husk is very less in both formulation. Low acid-insoluble ash value may also
affect amount of the component absorbed in the gastrointestinal canal when taken
orally.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 82
Table 4.3 Percentage ash value of Balacaturbhadrika churna and its raw
materials
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima), BC (Balacaturbhadrika churna), MF (Marketed
formulation)
(iv) Determination of extractive values
The percent extractive values were determined in various solvents ranging from non
polar semi polar to polar behavior. The extractive values are recorded in alcohol and
water with a view to study the distribution of various constituents of
Balacaturbhadrika churna (BC-I, BC-II and BC-III), Marketed formulation (MF) and
its raw materials. The method for determination of extractive value in different
solvent are described earlier in this chapter. The findings were recorded in terms of
percentage extractive values in (Table 4.4).
SN Name
Total ash
(% w/w ± S.D.,
n=6)
Acid insoluble ash
(% w/w ± S.D.,
n=6)
Water Ssluble ash
(% w/w ± S.D.,
n=6)
1 CR 7.346± 0.346 0.756 ± 0.031 13.431± 0.387
2 PL 5.032 ± 0.624 1.302 ± 0.346 23.163± 0.736
3 AH 2.981 ±0.243 1.324 ± 0.078 38.263±0.642
4 PI 4.621 ±0.334 2.418 ± 0.249 18.725±0.354
5 BC-I 8.148 ± 0.337 3.281 ± 0.286 45.602 ± 0.414
6 BC-II 8.682 ± 0.221 3.641 ± 0.129 45.649 ± 0.882
7 BC-III 8.248 ± 0.662 3.368 ± 0.148 44.892 ± 0.648
8 MF 19.633 ± 0.552 5.041 ± 0.368 51.403 ± 0.223
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 83
Table 4.4 Extractive values of Balacaturbhadrika churna and its raw
materials
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima), BC (Balacaturbhadrika churna), MF (Marketed
formulation)
Alcohol-soluble and water soluble extractive values of ingredients and formulation
are depicted in (Table 4.4), which shows 39.294±2.226 and 30.662±0.472 alcohol-
soluble extractive value for lab and marketed formulation respectively which is
higher than water soluble extractive value of both formulations. Higher ethanol-
soluble extractive value implies that ethanol is a better solvent of extraction for the
formulation than water.
(v) Qualitative phytochemical studies
Results of the phytochemical screening of the raw materials, lab formulation and
marketed formulation of Balacaturbhadrika churna are concluded in (Table 4.5). One
notable difference as a result of the methods of extraction is the possibility that the
alkaloids in Piper longum and Pistacia integerrima are more soluble in ethanol, the
reason why the presence of that group was not detectable in the aqueous extract.
SN Name Alcohol-soluble extractive Water-soluble extractive
1 CR 19.482±0.468 24.268±0.442
2 PL 28.282 ± 0.368 12.246±2.638
3 AH 37.442±2.664 20.842±2.425
4 PI 48.726±1.263 12.856±1.424
5 BC-I 39.294±2.226 20.224±0.682
6 BC-II 38.964±1.662 20.124±0.228
7 BC-III 39.229±1.926 20.103±0.771
8 MF 30.662±0.472 19.331±1.552
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 84
Furthermore, where more than one test was conducted for the detection of a chemical
group such as the alkaloids, no differences in the results were observed for the
different tests.
Out of the nine phytochemical groups investigated, seven namely carbohydrate,
glycosides, tannins, flavonoids, fixed oil and proteins were detected in the ethanolic
extract of lab and marketed formulations however the aqueous extracts of both
formulations shows the presence of saponins with previously stated seven
phytochemical groups. Steroids were absent in all the ingredients and formulations
for both methods of extraction.
(vi) Development of thin layer chromatography
The TLC was performed for each batch of Balacaturbhadrika churna (BC-I, BC-II
and BC-III), marketed formulation (MF) and separately for raw materials. The
experimental technique used for preparation and development of TLC plate is given
earlier (Table 4.6).
Table 4.5 Development of solvent system and TLC plate
Particulars Description
Solvent System (Best) Toluene: ethyl acetate (70:30v/v)
Adsorbent Silica gel
Detecting method Ultra Violet lamp at 366 nm
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal Formulations 85
Table 4.6 Phytochemical characterization of Balacaturbhadrika churna and its raw materials
Test
Ethanolic extract Aqueous extract
CR PL AH PI BC-I BC-
II
BC-
III MF CR PL AH PI
BC-
I
BC
-II
BC-
III MF
Alkaloids + + + + + + + + + - + - + + + +
Carbohydrates + + + + + + + + + + + + + + + +
Glycosides - + - + + + + + + + + + + + + +
Tannins + + + + + + + + + + + + + + + +
Flavonoids + - + + + + + + - - + + - - - +
Fixed oil + - + - + + + + - - - + + + + +
Saponins - - - - - - - - + - + - + + + +
Proteins and
Amino acids + - + + + + + + + - + + + + + +
Steroids - - - - - - - - - - - - - -
- : Absent, + : Present, CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI (Pistacia integerrima), BC
(Balacaturbhadrika churna), MF (Marketed formulation)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 86
Table 4.7 TLC profile of Balacaturbhadrika churna and its raw material
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima), BC (Balacaturbhadrika churna), MF (Marketed
formulation)
(vii) Determination of swelling index
The swelling index was determined for each batch of each batch of Balacaturbhadrika
churna (BC-I, BC-II and BC-III), marketed formulation (MF) and separately for raw
materials. The finding are reported in experimental technique used for preparation
and development of TLC plate is given earlier (Table 4.8).
SN Name No. of Spots Rf values
1 CR 05 0.27,0.42,0.53,0.59,0.64
2 PL 07 0.08,0.21,0.27,0.42,0.50,
0.53,0.57,
3 AH 05 0.17,0.26,0.38, 0.50,0.80
4 PI 03 Overlapping
5 BC-I 10 0.08, 0.17, 0.21, 0.26,
0.27,0.42,0.50, 0.53,0.57,
6 BC-II 10 0.08, 0.17, 0.21, 0.26,
0.27,0.42,0.50, 0.53,0.57,
7 BC-III 10 0.08, 0.17, 0.21, 0.26,
0.27,0.42,0.50, 0.53,0.57,
8 MF 08 0.08, 0.17, 0.21,
0.27,0.42,0.50,0.57,
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 87
Table 4.8 Swelling index of Balacaturbhadrika churna and its raw materials
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima), BC (Balacaturbhadrika churna), MF (Marketed
formulation)
(viii) Determination of foaming index
The Foaming index was determined for all the formulations and its ingredients
includes CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima). The method used is depicted earlier in this chapter.
(ix) Determination of microorganisms
The BC-I and BC-II and BC-III, Marketed formulation (MF) and its raw materials CR
(Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI (Pistacia
integerrima) were tested for presence of Escherichia coli, Salmonella spp.,
Staphylococcus aureus as per the standard method described in WHO (1998). The
results shows that all above mentioned microorganism were absent in all formulation
and ingredients (Table 4.9).
(x) Determination of arsenic and heavy metals
Limit test for arsenic
The limit test for arsenic was performed (The Pharmacopoeia of India, 1966) for
each batch of Balacaturbhadrika churna (BC-I, BC-II and BC-III), marketed
SN Name Swelling index ±S.D.(n=6)
1 CR 4.223±0.281
2 PL 3.921±0.701
3 AH 3.481±0.229
4 PI 3.121±0.662
5 BC-I 3.878±0.268
6 BC-II 3.842±0.364
7 BC-III 3.889±0.825
8 MF 3.009±0.227
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 88
formulation (MF) and ingredients as per the methods described above. The findings
are recorded in (Table 4.9).
Limit test for heavy metals
The amount of heavy metals for each batch of Balacaturbhadrika churna (BC-I, BC-II
and BC-III), marketed formulation (MF) and raw materials were estimated for lead by
matching the intensity of color with that of the standard stain using I.P. method. The
inferences are recorded in (Table 4.9).
Table 4.9 Limits of arsenic and heavy metals in Balacaturbhadrika churna and
its raw material
SN Name Standard
stain
Color intensity as
compared to
standard strain E. coli
Salmonel
la sp
S.
Aureus
Inference
as per
WHO Arsenic
Heavy
metal
(Lead)
1. CR
1 ppm Less Less absent absent absent Permissible
2. PL
1 ppm Less Less absent absent absent Permissible
3. AH
1 ppm Less Less absent absent absent Permissible
4. PI
1 ppm Less Less absent absent absent Permissible
5. BC-I
1 ppm Less Less absent absent absent Permissible
6. BC-II
1 ppm Less Less absent absent absent Permissible
7.
BC-
III 1 ppm Less Less absent absent absent Permissible
8. MF
1 ppm Less Less absent absent absent Permissible
CR (Cyperus rotundus), PL (Piper longum), AH (Aconitum heterophy), PI
(Pistacia integerrima), BC (Balacaturbhadrika churna), MF (Marketed
formulation)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 89
Arsenic and heavy metals are below the specified limit in all ingredients, lab and
marketed formulations. Tests were also performed for specific pathogen, E. coli,
Salmonella species and S. aureus which are found absent. This ensures the level of
safety of formulation.
(xi) Development of fingerprinting method
The Fingerprinting method was developed for each batch of Balacaturbhadrika
churna, its one marketed formulation and for its raw materials by using sophisticated
instrument UV, HPLC and HPTLC.
Development of UV spectroscopy fingerprinting method
The UV spectroscopy fingerprinting method was developed via estimation of piperine
which is an important content in Balacaturbhadrika churna and its raw materials. All
the chemicals and solvents were used of A.R. Grade. Balacaturbhadrika churna and
its ingredients were estimated for their piperine contents against standard piperine
solution on UV-Visible Spectrophotometer (Shimadzu, UV-1700, Pharmaspec).
Preparation of piperine extract of Balacaturbhadrika churna
The powdered Balacaturbhadrika Churna (1gm) was refluxed with 60 ml ethanol for
1 hour. The extract was filtered and the marc left was re reflux with 40 ml of ethanol
for another 1hours. Filtered and combine the filtrate. The ethanol extract was
concentrated under vacuum till the semisolid mass is obtained. The residue was
dissolved in 75 ml ethanol and filter through sintered glass funnel (G-2) by vacuum
filtration assembly. The filtrate was centrifuged at 2000 rpm for 20 minutes, the
supernatant was collected in 100 ml volumetric flask and volume was made with
ethanol.
The same procedure was performed for each batch of Balacaturbhadrika Churna and
separately powdered Piper longum (Pippali). As other ingredients does not contain
piperine is not included in present study.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 90
Preparation of standard solution of piperine
The absorbance characteristics show that piperine follow Beer Lambert’s law within
the concentration range 2-10 µg/ml at the λ-max of 342.5 nm (Table 4.10) (Figure
4.2). The determination of formulations was carried out through UV
spectrophotometer at 342.5 nm for piperine.
Preparation of standard curve of piperine
A series of calibrated 10 ml volumetric flask were taken and appropriate aliquots of
the working standard solution of piperine were withdrawn and diluted up to 10 ml
with ethanol. The absorbance was measured at absorption maxima 342.5 nm (Figure
4.2) against the reagent blank prepared in similar manner without the piperine. The
absorption maxima and Beer’s law limit were recorded and data that prove the
linearity and obey Beer’s law limit were noted.
Table 4.10 Standard curve parameter of piperine
The estimation of piperine content of the Balacaturbhadrika churna and powder of
Piper longum (Pippali) was carried out separately. The concentration of piperine
content in raw material was found to be 1.852 ± 0.241 w/w in Piper longum. The
content of piperine in different batches of Balacaturbhadrika churna was found to be
0.435 ± 0.030 %, 0.424 ± 0.001 %, 0.430 ± 0.004 % and 0.346 ± 0.002 % w/w
respectively for lab formulation (BC-I, BC-II, BC-III) and marketed formulation
(MF) (Table 4.11). The developed method was found to be reliable, accurate, precise
and sensitive.
S.No. Parameter Value
1
2
3
4
5
Absorption Maxima
Beer’s Law limit
Regression equation (y= bx+a)
Correlation coefficients (r2)
Accuracy (%)
342.5 nm
2-10 g/ml
y= 0.174x -0.318
r2 = 0.962
99.14
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 91
Figure 4.2 UV scan of piperine in ethanol
Figure 4.3 Standard curve of piperine in ethanol at 342.5 nm
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 92
Table 4.11 Spectrophotometric determination of piperine content
Name Piperine Content (% w/w ± SD)
Piper longum 1.852 ± 0.241
BC-I 0.435 ± 0.030
BC-II 0.424 ± 0.001
BC-III 0.430 ± 0.004
MF 0.346 ± 0.002
P<0.001 (highly significant)
Precision and accuracy
The method was validated for precision and accuracy, by performing the recovery
studies at two levels by adding known amount of piperine extract of
Balacaturbhadrika churna, of which the piperine content have been estimated
previously. The data were obtained and recovery was calculated (Table 4.12).
Table 4.12 Recovery study
S.no. Amount of piperine ( g/ml)
Recovery% Sample Added Estimated SD
(n=6)
1
2
100
100
50
100
148.71 0.34
198.5 0.26
99.04 0.29
99.25 0.44
Mean 99.14
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 93
(xii) HPLC fingerprinting method for formulations
The present study is an attempt to develop the chromatographic fingerprint method
for Balacaturbhadrika churna by High-performance liquid chromatographic method
using Piperine as a standard, which is as an important and major content in
formulation. RP- HPLC methods for determination of piperine from the fruits of
Piper longum, Balacaturbhadrika churna (BC-I, BC-II and BC-III) and one marketed
formulation (MF) have been developed by using following methods. A C18 LUNA
(5 micron 25 cm×4.6 mm) column from Phenomenex a binary gradient high-
pressure liquid chromatograph (Shimadzu HPLC class VP series) with two LC–10
AT VP pumps, variable wavelength programmable UV/Visible SPD 10 AVP were
used. The mobile phase consisted methanol. The flow rate was 1.0 mL/min. The
wavelength of detection was 343 nm and the injection volume was 10 μl.
Extraction of piperine from Balacaturbhadrika churna
The powdered Balacaturbhadrika churna (1gm) was refluxed with 60 ml methanol for
1 hour. The extract was filtered and marc was re refluxed with 40 ml of methanol for
another 1hours. Filtered and combine the filtrate. The methanol extract was
concentrated under vacuum till the semisolid mass is obtained. The residue was
dissolved in 75 ml methanol and filter through sintered glass funnel (G-2) by vacuum
filtration assembly. The filtrate was centrifuged at 2000 rpm for 20 minutes, the
supernatant was collected in 100 ml volumetric flask and volume was made with
ethanol. The same procedure was performed for each batch of Balacaturbhadrika
churna, it’s marketed formulation MF and powdered Piper longum solution.
Calibration curve of standard piperine
The stock solution of piperine was prepared by dissolving 10.0 mg of piperine in
100.0 mL methanol, creating a 100 μg/mL solution of piperine. This solution was
diluted with the solvent as needed to prepare different standard solutions (2, 4, 6, 8,
10, 12, 14, 16, 18 and 20μg/mL).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 94
Linearity
Standard solutions (2, 4, 6, 8, 10,--------18, 20μg/mL), each in three replicates, were
injected into the system. The method of linear regression was used for data
evaluation. Peak area ratios of standard compounds were plotted against theoretical
concentrations of standards. Linearity was expressed as a correlation coefficient.
Figure 4.4 Range of linearity
Precision
The precision of the method was tested by injecting a standard solution of piperine
(20μg/mL and 2μg/mL) three times. Peak areas were determined and compared.
Precision was expressed as percentage relative standard deviation (Table 4.13).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 95
Table 4.13 Validation parameters of piperine
Figure 4.5 HPLC chromatogram of piperine
S.No. Parameter Value
1 Absorption Maxima 343 nm
1 Retention time 3.808min (Figure 4.5)
2 Beer’s Law limit 2-20 g/ml
3 Regression equation (y= bx+a) y= 62.34x -23.47
4 Intercept (a) 23.47
5 Slope (b) 62.34
6 Correlation coefficients (r2) r
2 = 0.999
7 Precision (n=3 % RSD) 0.354
8 Accuracy (%) 99.65
9 Limit of quantification(LOQ) 0.449 g/ml
10 Limit of detection(LOD) 0.103 g/ml
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 96
Reproducibility
Inter and intra-day variation was performed by injecting the standard solutions (2, 4,
6, 8, 10, --------18, 20μg/mL), each in three replicates, twice on the same day, and
once on the next day and Peak areas are determined and compared (Table 4.14).
Table 4.14 System repeatability
Concentration (μg/mL) Day 1 peak area Day 1 peak
area
Day 2 peak
area
2 94.57± 0.56 94.02± 0.85 94.47± 0.25
4 217.84± 0.98 217.12± 0.78 217.24± 0.35
6 346.72± 0.57 346.21± 0.56 346.24± 0.35
8 482.53± 0.21 482.14± 0.24 482.87± 0.11
10 617.14± 0.11 617.18± 0.10 617.3± 0.10
12 733.45± 0.68 733.16± 0.99 733.24± 0.24
14 852.33± 0.36 852.54± 0.15 852.74± 0.28
16 968.41± 0.63 968.14± 0.24 968.85± 0.65
18 1088.12± 0.12 1088.89± 0.15 1088.14± 0.01
20 1222.11± 0.02 1222.14± 0.15 1222.78± 0.03
Mean ± S.D. (n = 3).
Determination of limit of quantitation and limit of detection
The limit of detection (LOD) is the lowest amount of analyte in a sample which can
be detected but not necessarily quantitated as an exact value. The limit of quantitation
(LOQ) is the lowest amount of analyte which can be quantitatively determined with
suitable precision. The LOD and LOQ of the developed method were determined by
injecting progressively low concentration of the standard solution and the lowest
concentrations assayed (Table 4.13).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 97
Estimation of Piperine in Balacaturbhadrika churna
The appropriate aliquots from piperine extract of each batch of Balacaturbhadrika
churna, its marketed formulation and powdered Piper longum were withdrawn in 10
ml volumetric flask separately. The corresponding concentrations of piperine against
respective peak areas value were determined using the piperine calibration curve
(Table 4.15).
Table 4.15 Estimation of Piperine Content in Balacaturbhadrika churna (HPLC)
S.no. Name Piperine content
%w/w
1 Piper longum 1.852 ± 0.124
2
Balacaturbhadrika churna
BC-I 0.435 ± 0.025
BC-II 0.424 ± 0.241
BC-III 0.430 ± 0.262
MF 0.346 ± 0.762
Mean SD (n=3),
Recovery studies
The recovery studies performed at three levels by adding known amount of piperine
to extract of Balacaturbhadrika churna, of which the piperine content has been
estimated previously. The data were obtained and recovery calculated (Table 4.16).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 98
Table 4.16 Recovery study
S.No
Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.27 0.24 0.160 0.14 99.51
2 100 100 199.41 0.68 0.341 0.39 99.70
3 100 150 249.42 0.34 0.136 0.20 99.76
Mean 0.212 0.242 99.65
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
(xiii) Development of HPTLC fingerprinting method
The HPTLC fingerprinting method was developed for each batch of
Balacaturbhadrika churna, its marketed formulation and powdered Piper longum via
estimation of piperine, which is an important content in Balacaturbhadrika churna.
All the chemicals and solvents used were of A.R. Grade.
Preparation of piperine extract of Balacaturbhadrika churna
The piperine extract of Balacaturbhadrika churna were prepared by refluxing the
powdered Balacaturbhadrika churna (1gm) with 60 ml methanol for 1 hour. The
extract was filtered and marc was re refluxed with 40 ml of methanol for another
1hours. Filter and combine the filtrate. Concentrate the methanol extract under
vacuum till the semisolid mass is obtained. Dissolve the residue in 75 ml methanol
and filter through sintered glass funnel (G-2) by vacuum filtration assembly. The
same procedure was performed for each batch of Balacaturbhadrika churna, marketed
formulation MF and separately powdered Piper longum solution (100 ml) for their
piperine extract were prepared.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 99
Equipment
The instrument used for the estimation, was Camag Linomat V semi automatic
sample applicator, Camag TLC scanner 3, CATS V.4.06 software for interpretation of
the data, Hamilton syringe and Camag twin trough chamber. The pre coated silica gel
G 60 F 254 was used as stationary phase, obtained from E. Merck. The piperine were
well resolved on the precoated silica gel G 60 F 254 on aluminum sheets, the mobile
phase was toluene: ethyl acetate (70:30v/v), chamber saturation time 20 min,
migration distance 70 mm, wavelength scanning at 335 nm, band width 8 mm, slit
dimension 5 * 0.45 mm, scanning speed 20 nm/sec, and the source of radiation was a
deuterium lamp. All the solvents used were of AR grade, obtained form S. D. Fine
Chemicals Ltd., Mumbai. Marketed formulation of Balacaturbhadrika churna were
purchased from a local pharmacy store.
Preparation of standard solution
Standard piperine (98%) was procured from Sigma Aldrich Pvt. Ltd. A standard
solution of piperine was prepared with accurately weighed 1mg into a 10 ml
volumetric flask. The content was dissolved in methanol, and volume was made up to
10 ml. The method was validated for linearity, accuracy, limit of detection, limit of
quantification, inter-day and intra - day assay precision, repeatability of measurement,
and repeatability of sample application.
Estimation of piperine
The appropriate aliquots from piperine extract of each batch of Balacaturbhadrika
churna, marketed formulation MF and Piper longum withdrawn in 10 ml volumetric
flask separately. The corresponding concentrations of piperine against respective peak
area were determined using calibration curve of piperine. The results are cited in
(Table 4.18).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 100
Table 4.17 Validation parameters
S. No. Parameter Value
1 Rf 0.24±0.06
2 Linearity (ng/spot) 10-30 ng
3 Correlation coeificients r2 0.9989
4 LOD(ng /spot) 5 ng
5 LOQ(μg /spot) 0.3 µg
Rf : Retention factor, , LOD : Limit of detection, LOQ: Limit of quantification
Table 4.18 Estimation of piperine content in Balacaturbhadrika churna (HPTLC)
S.no. Name Piperine content %w/w
1 Piper longum 1.92 ± 0.08
2
Balacaturbhadrika churna
BC-I 0.442 ± 0.005
BC-II 0.431 ± 0.023
BC -III 0.430 ± 0.081
MF 0.362 ± 0.242
Mean SD of six determinations
Recovery studies
The recovery studies performed at three levels were done by adding known amount of
piperine to extract of Balacaturbhadrika churna of which the piperine content have
been already estimated. The observations recorded and recovery was calculated
(Table 4.19).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 101
Table 4.19 Recovery study
S.No
Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.37 0.24 0.161 0.14 99.58
2 100 100 199.50 0.70 0.351 0.40 99.75
3 100 150 249.52 0.34 0.136 0.20 99.81
Mean 0.216 0.246 99.713
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
Figure 4.6 HPTLC chromatogram of piperine ( Rf = 0. 24)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 102
(xiv) Development of Method for Stability Testing
All the three batches BC-I, BC-II, BC-III of Balacaturbhadrika churna were subjected
to stability studies at accelerated condition at 45 0C at 75% Relative humidity for six
month. The sample were withdrawn at intervals of time (1,3 and 6 months) and
evaluated previously developed parameter colour, odour, taste, moisture content,
volatile oil content and piperine content. The changes in value of above parameters
with respect to developed parameters are recorded in (Table 4.20).
Table 4.20 Accelerated stability testing at 45oC Temperature of
Balacaturbhadrika churna
S.
No
.
Param
eter
Formu
lations
Time duration (after months) Inferenc
e
1 3 6
1 Colour
BC-I
BC-II
BC-III
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
No
change
2 Odour
BC-I
BC-II
BC-III
Characteristic
Characteristic
Characteristic
Characteristic
Characteristic
Characteristic
Characteristic
Characteristic
Characteristic
No
change
3 Taste
BC-I
BC-II
BC-III
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
No
change
4
Moistur
e
Content
BC-I
BC-II
BC-III
8.23±0.472
8.22±0.662
8.16±0.863
8.14±0.745
8.15±0.539
8.12±0.658
8.21 ±0.499
8.20±0.389
8.16±0.978
Minor
change
5
Piperin
e
content
BC-I
BC-II
BC-III
0.44±0.23
0.43±0.26
0.43±0.09
0.44±0.43
0.43±0.32
0.43±0.39
0.43±0.10
0.43±0.19
0.42±0.41
Minor
change
n= 3 Average of three determinants, NC = No change
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 103
4.9 Shringyadi churna
Formulation was strictly prepared as prescribed in the official book of Ayurvedic
Formulary of India (2003). 50 grams of each ingredients which includes Krsna
(pippali), Aruna (ativisa) and Sringi (karkatasringi) were taken. All the ingredients
were weighed accurately and made fine powder by passing through sieve no. 80. Fine
powders were mixed geometrically in plastic tray and packed in plastic container.
Three sample batch of Shringyadi churna were prepared using above mentioned
methods and were named as SC-I, SC-II, SC-III. One Marketed formulations named
MF was purchased from local pharmacy store Raipur. These samples were stored at
optimized conditions of Temperature, light and moisture.
(i) Organoleptic profile and physical characteristics
Organoleptic profile of the formulations and each raw material were determined by
the method described earlier. Findings of the different organoleptic parameters is
summarized in (Table 4.21). Physical characteristics of all the three batches of
Shringyadi churna (SC-I, SC-II and SC-III), its marketed formulation (MF) and all
raw materials were established through the method described earlier and these data
are also recorded in (Table 4.21).
Discussion
Evaluation of organoleptic characters of raw materials Krsna (pippali), Aruna
(ativisa) and Sringi (karkatasringi) was performed and characters are recorded.
Laboratory batches of Shringyadi churna (SC-I, SC-II and SC-III) and one marketed
preparations were also evaluated for organoleptic characters. The results for the
marketed formulations (MF and Laboratory formulations are found comparable. The
above mentioned organoleptic data are very useful for the preliminary identification
of raw material and formulations.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 104
Table 4.21 Organoleptic and physical characteristics of Shringyadi churna and
its raw materials
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
The value of angle of repose for raw materials Pistacia integerrima, Piper longum
and Aconitum palmatum, lab formulation and marketed formulation were 24.34,
31.28, 29.86, 24.34, 26.68 (SC-I), and 25.42 respectively which shows good flow
properties of prepared lab formulation. The flow properties of lab (SC-I) and
marketed formulations were also confirmed by Hausner’s ratio and Carr’s index; it
was found 1.25, 20, and 1.23, 19 respectively and indicates good flow characteristics.
(ii) Determination of foreign matter and loss on drying
Raw materials, Laboratory batches of Shringyadi churna (SC-I, SC-II and SC-III)
and one marketed preparation were also evaluated for foreign matter and loss on
drying through the methods described earlier in this chapter. The results of both
parameters are tabulated in (Table 4.22).
SN Name Colour Odour Taste Tap
density
Bulk
density
Angle
of
repose
Hausner
ratio
Carr’s
index
1 PI Light
brown Pungent Bitter 0.52 0.41 24.34 1.26 21
2 PL Dark
Brown
Charact
eristic Pungent 0.66 0.52 31.28 1.26 21
3 AP Dark
Brown
Charact
eristic Pungent 0.60 0.50 29.86 1.20 17
4 SC-I Light
brown
Charact
eristic Pungent 0.50 0.40 26.68 1.25 20
5 SC-II Light
brown
Charact
eristic Pungent 0.52 0.41 24.34 1.26 21
6 SC-III Light
brown
Charact
eristic Pungent 0.50 0.40 27.36 1.25 20
7 MF Light
brown
Charact
eristic Pungent 0.64 0.52 25.42 1.23 19
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 105
Table 4.22 Loss on drying and foreign matter content in Shringyadi churna
and its raw materials
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC (Shringyadi churna),
MF (Marketed formulation)
Discussion
The amount of moisture in the crude drugs should be minimized in order to prevent
decomposition of either due to chemical changes or due to microbial contamination.
The percent moisture content for SC-I, SC-II and SC-III are 2.24±0.242, 2.46±0.143
and 2.78±0.621, while it is 2.32±0.282for MF. The moisture content of formulation
was within acceptable range (<8%) thus implying that the formulation can be stored
for a long period and would not easily be attacked by microbes. The percent of
foreign matter was found to be 1.38±0.121, 1.48±0.025 and 1.28±0.241 for Pistacia
integerrima, Piper longum and Aconitum palmatum respectively. Laboratory
formulations of Shringyadi churna were prepared after removal of foreign matter. In
examination no poisonous, dangerous and harmful foreign matter or residue was
found.
(iii) Ash values determination
Determination of ash values
The percent total ash and acid-insoluble ash was determined for each batch of
Shringyadi churna , its one marketed formulations and separately its raw materials as
per above mentioned method. The results are recorded in (Table 4.23).
SN Name %LOD
±S.D. (n=6)
%Foreign Matter
±S.D. (n=6)
1 PI 2.78±0.642 1.38±0.121
2 PL 2.29±0.382 1.48±0.025
3 AP 2.02±0.196 1.28±0.241
4 SC-I 2.24±0.242 NIL
5 SC-II 2.46±0.143 NIL
6 SC-III 2.78±0.621 NIL
7 MF 2.32±0.282 NIL
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 106
Total ash value of Pistacia integerrima, Piper longum, Aconitum palmatum, lab
formulation (SC-I) and marketed formulation were 5.032±0.624, 2.981 ±0.243, 4.621
±0.334, 7.224 ± 0.247 and 19.633 ± 0.552 respectively. The value of total ash in
marketed formulation is comparatively high in comparison to lab formulation may be
because of the higher amounts of inorganic components present in marketed
formulation. Acid-insoluble ash value of prepared lab formulations (SC-I) and
marketed formulation were 3.446 ± 0.268 and 5.041 ± 0.368 respectively shows that a
small amount of the inorganic component is insoluble in acid it indicates adulteration
of raw ingredients by substance like silica, rice husk is very less in both formulation.
Low acid-insoluble ash value may also affect amount of the component absorbed in
the gastrointestinal canal when taken orally.
Table 4.23 Percentage ash value of Shringyadi churna and its raw materials
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
SN Name
Total ash
(% w/w ± S.D.,
n=6)
Acid insoluble ash
(% w/w ± S.D., n=6)
Water Soluble ash
(% w/w ± S.D., n=6)
1 PI 5.032 ± 0.624 1.302 ± 0.346 23.163± 0.736
2 PL 2.981 ±0.243 1.324 ± 0.078 38.263±0.642
3 AP 4.621 ±0.334 2.418 ± 0.249 18.725±0.354
4 SC-I 7.224 ± 0.247 3.446 ± 0.268 43.422 ± 0.322
5 SC-II 7.345 ± 0.275 3.520 ± 0.231 43.455 ± 0.541
6 SC-
III 7.414 ± 0.214 3.412 ± 0.246 43.112 ± 0.421
7 MF 19.633 ± 0.552 5.041 ± 0.368 51.403 ± 0.223
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 107
(iv) Determination of extractive values
The extractive values are recorded in alcohol and water with a view to study the
distribution of various constituents of Shringyadi churna (SC-I, SC-II and SC-III),
Marketed formulation (MF) and its raw materials. The method for determination of
extractive value in different solvent are described earlier in this chapter. The findings
were recorded in terms of percentage extractive values in (Table 4.24).
Table 4.24 Extractive values of Shringyadi churna and its raw materials
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
Alcohol-soluble and water soluble extractive values of ingredients and formulation
are depicted in (Table 4.24), which shows 38.486±1.842 and 31.824±0.251 alcohol-
soluble extractive value for lab and marketed formulation respectively which is
higher than water soluble extractive value of both formulations. Higher ethanol-
soluble extractive value implies that ethanol is a better solvent of extraction for the
formulation than water.
SN Name Alcohol-soluble extractive Water-soluble extractive
1 PI 28.282 ± 0.368 12.246±2.638
2 PL 37.442±2.664 20.842±2.425
3 AP 48.726±1.263 12.856±1.424
4 SC-I 38.486±1.842 20.224±0.682
5 SC-II 38.128±1.221 20.981±0.226
6 SC-III 37.984±1.262 21.118±0.242
7 MF 31.824±0.251 19.331±1.552
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 108
(v) Qualitative phytochemical studies
Results of the phytochemical screening of the raw materials, lab formulation and
marketed formulation of Shringyadi churna are concluded in (Table 4.25). One
notable difference as a result of the methods of extraction is the possibility that the
alkaloids in Piper longum and Pistacia integerrima are more soluble in ethanol, the
reason why the presence of that group was not detectable in the aqueous extract.
Furthermore, where more than one test was conducted for the detection of a chemical
group such as the alkaloids, no differences in the results were observed for the
different tests.
Out of the nine phytochemical groups investigated, seven namely carbohydrate,
glycosides, tannins, flavonoids, fixed oil and proteins were detected in the ethanolic
extract of lab and marketed formulations however the aqueous extracts of both
formulations shows the presence of saponins with previously stated seven
phytochemical groups. Steroids were absent in all the ingredients and formulations
for both methods of extraction.
(vi) Development of thin layer chromatography
The TLC was performed for each batch of Shringyadi churna (SC-I, SC-II and SC-
III), marketed formulation (MF) and separately for raw materials (Table 4.26) and
(Table 4.27) . The experimental technique used for preparation and development of
TLC plate is given earlier.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal Formulations 109
Table 4.25 Phytochemical characterization of Shringyadi churna and its raw materials
Test
Ethanolic extract Aqueous extract
PI PL AP SC-I SC-II SC-III MF PI PL AP SC-I SC-II SC-
III MF
Alkaloids + + + + + + + - - + + + + +
Carbohydrates + + + + + + + + + + + + + +
Glycosides + + - + + + + + + + + + + +
Tannins + + + + + + + + + + + + + +
Flavonoids + - + + + + + + - + - - - +
Fixed oil - - + + + + + + - - + + + +
Saponins - - - - - - - - - + + + + +
Proteins and
Amino acids + - + + + + + + - + + + + +
Steroids - - - - - - - - - - - - -
- : Absent, + : Present, PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC (Shringyadi churna ), MF
(Marketed formulation)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 110
Table 4.26 Development of solvent system and TLC plate
Particulars Description
Solvent System (Best) Toluene: ethyl acetate (70:30v/v)
Adsorbent Silica gel
Detecting method Ultra Violet lamp at 366 nm
Table 4.27 TLC profile of Shringyadi churna and its raw material
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
SN Name No. of Spots Rf values
1 PI 03 Overlapping
2 PL 07 0.08,0.21,0.27,0.42,0.50, 0.53,0.57,
4 AP 05 0.17,0.23, 0.26,0.32, 0.53
5 SC-I 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.32,
0.42,0.50, 0.53,0.57,
6 SC-II 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.32,
0.42,0.50, 0.53,0.57,
7 SC-III 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.32,
0.42,0.50, 0.53,0.57,
8 MF 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.32,
0.42,0.50, 0.53,0.57,
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 111
(vii) Determination of swelling index
The swelling index was determined for each batch of each batch of Shringyadi churna
(SC-I, SC-II and SC-III), marketed formulation (MF) and separately for raw
materials. The findings are reported in experimental technique used for preparation
and development of TLC plate is given earlier (Table 4.28).
Table 4.28 Swelling index of Shringyadi churna and its raw materials
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
(viii) Determination of foaming index
The Foaming index was determined for all the formulations and its ingredients
include PI (Pistacia integerrima), PL (Piper longum), AP (Aconitum palmatum), The
method used is depicted earlier in this chapter.
(ix) Determination of microorganisms
The SC-I and SC-II and SC-III, Marketed formulation (MF) and its raw materials PI
(Pistacia integerrima), PL (Piper longum), AP (Aconitum palmatum) were tested for
presence of Escherichia coli, Salmonella spp., Staphylococcus aureus as per the
standard method described in WHO (1998). The results shows that all above
mentioned microorganism were absent in all formulation and ingredients (Table
4.29).
SN Name
Swelling index ±S.D.(n=6)
1 PI 3.426±0.228
2 PL 3.662±0.664
3 AH 4.206±0.314
4 SC-I 3.678±0.612
5 SC-II 3.542±0.269
6 SC-III 3.661±0.296
7 MF 3.112±0.381
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 112
(x) Determination of arsenic and heavy metals
Limit test for arsenic
The limit test for arsenic was performed for each batch of Shringyadi churna (SC-I,
SC-II and SC-III), marketed formulation (MF) and ingredients as per the methods
described above. The findings are recorded in (Table 4.29).
Limit test for heavy metals
The amount of heavy metals for each batch of Shringyadi churna (SC-I, SC-II and
SC-III), marketed formulation (MF) and raw materials were estimated for lead by
matching the intensity of color with that of the standard stain using I.P. method. The
inferences are recorded in (Table 4.29).
Table 4.29 Limits of arsenic and heavy metals in Shringyadi churna and its
raw material
SN Name Standard
stain
Color intensity as
compared to
standard strain E. coli
Salmonel
la sp
S.
Aureus
Inference
as per
WHO Arsenic
(Lead)
1. PI
1 ppm Less Less absent absent absent Permissible
2. PL
1 ppm Less Less absent absent absent Permissible
3. AP
1 ppm Less Less absent absent absent Permissible
4. SC-I
1 ppm Less Less absent absent absent Permissible
5. SC-II
1 ppm Less Less absent absent absent Permissible
6.
SC-
III 1 ppm Less Less absent absent absent Permissible
7. MF
1 ppm Less Less absent absent absent Permissible
PL (Piper longum), AP (Aconitum palmatum), PI (Pistacia integerrima), SC
(Shringyadi churna), MF (Marketed formulation)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 113
Arsenic and heavy metals are below the specified limit in all ingredients, lab and
marketed formulations. Tests were also performed for specific pathogen, E. coli,
Salmonella species and S. aureus which are found absent. This ensures the level of
safety of formulation.
(xi) Development of fingerprinting method
The Fingerprinting method was developed for each batch of Shringyadi churna , its
one marketed formulation and for its raw materials by using sophisticated instrument
UV, HPLC and HPTLC.
Development of UV spectroscopy fingerprinting method
The UV spectroscopy fingerprinting method was developed via estimation of piperine
which is an important content in Shringyadi churna and its raw materials. All the
chemicals and solvents were used of A.R. Grade. Shringyadi churna and its
ingredients were estimated for their piperine contents against standard piperine
solution on UV-Visible Spectrophotometer (Shimadzu, UV-1700, Pharmaspec). In
this formulation again piperine constitutes the main active ingredient and hence it is
used as marker for standardization purpose.
Preparation of piperine extract of Shringyadi churna
The whole parameters for piperine extraction were same as the previous formulations
discussed in section (4.8 xi).This procedure was performed for each batch of
Shringyadi churna and separately powdered Piper longum (Pippali). As other
ingredients does not contain piperine was not included in present study.
Preparation of standard solution of piperine
The absorbance characteristics show that piperine follow Beer Lambert’s law within
the concentration range 2-10 µg/ml at the λ-max of 342.5 nm (Table 4.11) (Figure
4.2). The determination of formulations was carried out through UV
spectrophotometer at 342.5 nm for piperine.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 114
Preparation of standard curve of piperine
A series of calibrated 10 ml volumetric flask were taken and appropriate aliquots of
the working standard solution of piperine were withdrawn and diluted up to 10 ml
with ethanol. The absorbance was measured at absorption maxima 342.5 nm (Figure
4.2), against the reagent blank prepared in similar manner without the piperine. The
absorption maxima and Beer’s law limit were recorded and data that prove the
linearity and obey Beer’s law limit were noted.
The estimation of piperine content of the Shringyadi churna and powder of Piper
longum (Pippali) was carried out separately. The concentration of piperine content in
raw material was found to be 1.852 ± 0.241 w/w in Piper longum. The content of
piperine in different batches of Shringyadi churna was found to be 0.605 ± 0. 001 %,
0.599 ± 0.127 %, 0.602 ± 0.008 % and 0.535 ± 0.015 % w/w respectively for lab
formulation (SC-I, SC-II, SC-III) and marketed formulation (MF) (Table 4.30). The
developed method was found to be reliable, accurate, precise and sensitive.
Table 4.30 Spectrophotometric determination of piperine content
Name Piperine Content (% w/w ± SD)
Piper longum 1.852 ± 0.241
SC-I 0.605 ± 0. 001 %
SC-II 0.599 ± 0.127 %
SC-III 0.602 ± 0.008 %
MF 0.535 ± 0.015 %
P<0.001 (highly significant)
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 115
Precision and accuracy
The method was validated for precision and accuracy, by performing the recovery
studies at two levels by adding known amount of piperine extract of Shringyadi
churna, of which the piperine content have been estimated previously. The data were
obtained and recovery was calculated (Table 4.31).
Table 4.31 Recovery study
S.no. Amount of piperine ( g/ml)
Recovery% Sample Added Estimated SD
(n=6)
1
2
100
100
50
100
149.11 0.34
199.5 0.01
99. 40 0.29
99.75 0.23
Mean 99.55
(xii) HPLC fingerprinting method for formulations
The present study is an attempt to develop the chromatographic fingerprint method
for Shringyadi churna by High-performance liquid chromatographic method using
Piperine as a standard, which is as an important and major content in formulation.
RP- HPLC methods for determination of piperine from the fruits of Piper longum,
Shringyadi churna (SC-I, SC-II and SC-III) and one marketed formulation (MF) have
been developed by using following methods. A C18 LUNA (5 micron 25 cm×4.6
mm) column from Phenomenex a binary gradient high- pressure liquid
chromatograph (Shimadzu HPLC class VP series) with two LC–10 AT VP pumps,
variable wavelength programmable UV/Visible SPD 10 AVP were used. The mobile
phase consisted methanol. The flow rate was 1.0 mL/min. The wavelength of
detection was 343 nm and the injection volume was 10 μl.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 116
Extraction of piperine from Shringyadi churna
The whole parameters for piperine extraction was same as the previous formulations
discussed in section (4.8. xii). This procedure was performed for each batch of
Shringyadi churna and separately powdered Piper longum (Pippali). As other
ingredients does not contain piperine was not included in present study.
Calibration curve of standard piperine
The stock solution of piperine was prepared by dissolving 10.0 mg of piperine in
100.0 mL methanol, creating a 100 μg/mL solution of piperine. This solution was
diluted with the solvent as needed to prepare different standard solutions (2-
20μg/mL). All the parameters including linearity, area against concentration,
precision, reproducibility, limit of quantitation, limit of detection and validation
parameters was discussed in section (4.8. xii) (Figure 4.4, Table 4.14) as the main
active ingredients that is piperine is same as previous formulation.
Estimation of Piperine in Shringyadi churna
The appropriate aliquots from piperine extract of each batch of Shringyadi churna, its
marketed formulation and powdered Piper longum were withdrawn in 10 ml
volumetric flask separately. The corresponding concentrations of piperine against
respective peak areas value were determined using the piperine calibration curve
(Table 4.32).
Table 4.32 Estimation of Piperine Content in Shringyadi churna(HPLC)
S.N. Name Piperine content %w/w
1 Piper longum 1.852 ± 0.001
2
Shringyadi churna
SC-I 0.605 ± 0.012
SC-II 0.599 ± 0.119
SC-III 0.602 ± 0.106
MF 0.535 ± 0.549
Mean SD (n=3),
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 117
Recovery studies
The recovery studies performed at three levels by adding known amount of piperine
to extract of Shringyadi churna, of which the piperine content has been estimated
previously. The data were obtained and recovery calculated (Table 4.33).
Table 4.33 Recovery study
S.No
Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.17 0.41 0.275 0.24 99.45
2 100 100 199.24 0.74 0.371 0.43 99.62
3 100 150 249.12 0.05 0.020 0.03 99.65
Mean 0.222 0.23 99.57
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
(xiii) Development of HPTLC fingerprinting method
The HPTLC fingerprinting method was developed for each batch of Shringyadi
churna, its marketed formulation and powdered Piper longum via estimation of
piperine, which is an important content in Shringyadi churna. All the chemicals and
solvents used were of A.R. Grade.
Preparation of piperine extract of Shringyadi churna
The whole parameters for piperine extraction were same as the previous formulations
discussed in section (4.8.xiii). This procedure was performed for each batch of
Shringyadi churna and separately powdered Piper longum (Pippali). As other
ingredients does not contain piperine was not included in present study. All the
parameters including equipment details, chromatogram (Figure 4.6) method of
preparation of standard curve and validation details (Table 4.18) was remained same
as discussed in section (4.8.xiii) as the main active ingredients that is piperine is
same as previous formulation.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 118
Estimation of piperine
The aliquots from piperine extract of each batch of Shringyadi churna, marketed
formulation MF and Piper longum withdrawn in 10 ml volumetric flask separately.
The corresponding concentrations of piperine against respective peak area were
determined using calibration curve of piperine. The results are cited in (Table 4.34).
Table 4.34 Estimation of piperine content in Shringyadi churna (HPTLC)
S.no. Name Piperine content %w/w
1 Piper longum 1.920 ± 0.082
2
Shringyadi churna
SC-I 0.624 ± 0.005
SC-II 0.618 ± 0.046
SC -III 0.614 ± 0.032
MF 0.569 ± 0.225
Mean SD of six determinations
Recovery studies
The recovery studies performed at three levels were done by adding known amount of
piperine to extract of Shringyadi churna of which the piperine content have been
already estimated. The observations and recovery was recorded (Table 4.35).
Table 4.35 Recovery study
S.No Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.48 0.05 0.033 0.03 99.65
2 100 100 199.59 0.14 0.070 0.08 99.80
3 100 150 249.78 0.81 0.324 0.47 99.91
Mean 0.143 0.192 99.78
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 119
(xiv) Development of Method for Stability Testing
All the three batches SC-I, SC-II, SC-III of Shringyadi churna were subjected to
stability studies at accelerated condition at 45 0C at 75% Relative humidity for six
month. The sample were withdrawn at intervals of time (1,3 and 6 months) and
evaluated previously developed parameter colour, odour, taste, moisture content,
volatile oil content and piperine content. The changes in value of above parameters
with respect to developed parameters are recorded in (Table 4.36).
Table 4.36 Accelerated stability testing at 45oC Temperature of Shringyadi
churna
S.
No
.
Paramete
r
Formulatio
ns
Time duration (after months) Inferenc
e
1 3 6
1 Colour
SC-I
SC-II
SC-III
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
Dark
Brown
No
change
2 Odour
SC-I
SC-II
SC-III
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
Characteris
tic
No
change
3 Taste
SC-I
SC-II
SC-III
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
No
change
4 Moisture
Content
SC-I
SC-II
SC-III
8.14±0.212
8.22±0.232
8.13±0.228
8.15±0.242
8.23±0.121
8.13±0.269
8.15 ±0.287
8.23±0.564
8.13±0.491
Minor
change
5 Piperine
content
SC-I
SC-II
SC-III
0.60±0.54
0.59±0.21
0.60±0.12
0.60±0.24
0.59±0.34
0.60±0.28
0.60±0.45
0.59±0.41
0.59±0.62
Minor
change
n= 3 Average of three determinants, NC = No change
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 120
4.10 Thirikadu choornam
Formulation was strictly prepared as prescribed in the official book of Siddha
Formulary of India. 50 grams of each ingredient which includes Piper longum
(Pippali), Piper nigrum (Marica) and rhizomes of Zingiber officinale (Saunth) were
taken. All the ingredients were weighed accurately and made fine powder by passing
through sieve no. 80. Fine powders were mixed geometrically in plastic tray and
packed in plastic container.
Three sample batch of Thirikadu choornam were prepared using above mentioned
methods and were named as TC-I, TC-II, TC-III. One Marketed formulations named
MF was purchased from local pharmacy store Raipur. These samples were stored at
optimized conditions of Temperature, light and moisture.
(i) Organoleptic profile and physical characteristics
Organoleptic profile of the formulations and each raw material were determined by
the method described earlier. Findings of the different organoleptic parameters are
summarized in (Table 4.37). Physical characteristics of all the three batches of
Thirikadu choornam (TC-I, TC-II and TC-III), its marketed formulation (MF) and all
raw materials were established through the method described earlier and these data
are also recorded in (Table 4.37).
Discussion
Evaluation of organoleptic characters of raw materials Piper longum (Pippali), Piper
nigrum (Marica) and rhizomes of Zingiber officinale (Saunth) were performed and
characters are recorded. Laboratory batches of Thirikadu choornam (TC-I, TC-II and
TC-III) and one marketed preparations were also evaluated for organoleptic
characters. The results for the marketed formulations (MF and Laboratory
formulations are found comparable. The above mentioned organoleptic data are very
useful for the preliminary identification of raw material and formulations.
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 121
Table 4.37 Organoleptic and physical characteristics of Thirikadu choornam
and its raw materials
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
The value of angle of repose for raw materials Piper longum (Pippali), Piper nigrum
(Marica), Zingiber officinale (Saunth), lab formulation (TC-I) and marketed
formulation were 25.34, 25.43, 26.68, 26.61 (TC-I), 23.54, and 26.47 respectively
which shows good flow properties of prepared lab formulation. The flow properties
S
N
Nam
e
Colo
ur Odour Taste
Tap
densi
ty
Bulk
dens
ity
Angle
of
repose
Haus
ner
ratio
Carr
’s
inde
x
1 PL
Dark
Brow
n
Characteristi
c
Pungen
t 0.51 0.44 25.34 1.16
13.7
3
2 PN
Light
yello
w
Characteristi
c
Pungen
t 0.53 0.45 25.43 1.18
15.0
9
3 ZO Brow
n characteristic Bitter 0.61 0.5 26.68 1.22
18.0
3
4 TC-I Brow
n
Characteristi
c
Pungen
t 0.59 0.49 23.54 1.20
16.9
5
5 TC-
II
Brow
n
Characteristi
c
Pungen
t 0.57 0.48 26.61 1.19
15.7
9
6 TC-
III
Brow
n
Characteristi
c
Pungen
t 0.52 0.41 26.96 1.27
21.1
5
7 MF Brow
n
Characteristi
c
Pungen
t 0.58 0.45 26.47 1.29
22.4
1
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 122
of lab (TC-I) and marketed formulations were also confirmed by Hausner’s ratio and
Carr’s index; it was found 1.20, 16.95, and 1.29, 22.41 respectively and indicates
good flow characteristics.
(ii) Determination of foreign matter and loss on drying
Raw materials, Laboratory batches of Thirikadu choornam (TC-I, TC-II and TC-III)
and one marketed preparation were also evaluated for foreign matter and loss on
drying through the methods described earlier in this chapter. The results of both
parameters are tabulated in (Table 4.38).
Table 4.38 Loss on drying and foreign matter content in Thirikadu choornam
and its raw materials
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
Discussion
The amount of moisture in the crude drugs should be minimized in order to prevent
decomposition of either due to chemical changes or due to microbial contamination.
The percent moisture content for TC-I, TC-II and TC-III are 2.82±0.124, 2.93±0.442
and 2.76±0.125, while it is 2.93±0.323 for MF. The moisture content of formulation
was within acceptable range (<8%) thus implying that the formulation can be stored
SN Name %LOD
±S.D. (n=6)
%Foreign Matter
±S.D. (n=6)
1 PL 2.89±0.243 1.29±0.084
2 PN 2.20±0.239 1.08±0.129
3 ZO 2.86±0.216 1.46±0.125
4 TC-I 2.82±0.124 NIL
5 TC-II 2.93±0.442 NIL
6 TC-III 2.76±0.125 NIL
7 MF 2.93±0.323 NIL
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 123
for a long period and would not easily be attacked by microbes. The percent of
foreign matter was found to be 1.29±0.084, 1.08±0.129 and 1.46±0.125 for PL (Piper
longum), PN (Piper nigrum), ZO (Zingiber officinalis) respectively. Laboratory
formulations of Thirikadu choornam were prepared after removal of foreign matter.
In examination no poisonous, dangerous and harmful foreign matter or residue was
found.
(iii) Ash values determination
Determination of ash values
The percent total ash and acid-insoluble ash was determined for each batch of
Thirikadu choornam , its one marketed formulations and separately its raw materials
as per above mentioned method. The results are recorded in (Table 4.39).
Total ash value of PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis),
lab formulation (TC-I) and marketed formulation were 3.281 ± 0.548, 4.469 ± 0.276,
5.237 ± 0.342, 4.232 ± 0.321 and 5.235 ± 0.732 respectively. The value of total ash in
marketed formulation is comparatively high in comparison to lab formulation may be
because of the higher amounts of inorganic components present in marketed
formulation. Acid-insoluble ash value of prepared lab formulations (TC-I) and
marketed formulation were 0.681 ± 0.043 and 0.636 ± 0.056 respectively shows that a
small amount of the inorganic component is insoluble in acid it indicates adulteration
of raw ingredients by substance like silica, rice husk is very less in both formulation.
Low acid-insoluble ash value may also affect amount of the component absorbed in
the gastrointestinal canal when taken orally.
(iv) Determination of extractive values
The extractive values are recorded in alcohol and water with a view to study the
distribution of various constituents of Thirikadu choornam (TC-I, TC-II and TC-III),
Marketed formulation (MF) and its raw materials. The method for determination of
extractive value in different solvent are described earlier in this chapter. The findings
were recorded in terms of percentage extractive values in (Table 4.40).
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 124
Table 4.39 Percentage ash value of Thirikadu choornam and its raw materials
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
Alcohol-soluble and water soluble extractive values of ingredients and formulation
are depicted in (Table 4.40), which shows 39.478±1.546 and 31.014±0.321 alcohol-
soluble extractive value for lab and marketed formulation respectively which is
higher than water soluble extractive value of both formulations. Higher ethanol-
soluble extractive value implies that ethanol is a better solvent of extraction for the
formulation than water.
SN Name
Total ash
(% w/w ± S.D.,
n=6)
Acid insoluble ash
(% w/w ± S.D., n=6)
Water Soluble ash
(% w/w ± S.D., n=6)
1 PL 3.281 ± 0.548 0. 566 ± 0.061 3.922 ± 0.511
2 PN 4.469 ± 0.276 0.743 ± 0.045 3.731 ± 0.422
3 ZO 5.237 ± 0.342 0.399 ± 0.021 5.203 ± 0.213
4 TC-I 4.232 ± 0.321 0.681 ± 0.043 4.214 ± 0.856
5 TC-II 4.441 ± 0.749 0.675 ± 0.011 4.745 ± 0.356
6 TC-
III 4.219 ± 0.228 0.692 ± 0.009 4.241 ± 0.856
7 MF 5.235 ± 0.732 0.636 ± 0.056 5.235 ± 0.732
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal
Formulations 125
Table 4.40 Extractive values of Thirikadu choornam and its raw materials
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
(v) Qualitative phytochemical studies
Results of the phytochemical screening of the raw materials, lab formulation and
marketed formulation of Thirikadu choornam are concluded in (Table 4.41). One
notable difference as a result of the methods of extraction is the possibility that the
alkaloids in Piper longum and Piper nigrum are more soluble in ethanol, the reason
why the presence of that group was not detectable in the aqueous extract.
Furthermore, where more than one test was conducted for the detection of a chemical
group such as the alkaloids, no differences in the results were observed for the
different tests.
Out of the nine phytochemical groups investigated, seven namely carbohydrate,
glycosides, tannins, flavonoids, fixed oil and proteins were detected in the ethanolic
extract of lab and marketed formulations however the aqueous extracts of both
formulations shows the presence of saponins with previously stated seven
phytochemical groups. Steroids were absent in all the ingredients and formulations
for both methods of extraction.
SN Name Alcohol-soluble extractive Water-soluble extractive
1 PL 28.282 ± 0.358 14.144±2.614
2 PN 48.112±1.985 22.752±2.424
3 AP 49.126±1.045 12.856±1.124
4 TC-I 39.478±1.546 21.221±0742
5 TC-II 38.899±1.045 20.995±0.001
6 TC-III 38.124±1.124 21.001±0.014
7 MF 31.014±0.321 19.212±1.654
Standardization of Formulations
Development and Standardization of Modern Dosage Form for Indigenous Medicinal Formulations 126
Table 4.41 Phytochemical characterization of Thirikadu choornam and its raw materials
Test
Ethanolic extract Aqueous extract
PL PN ZO TC-I TC-II TC-III MF PL PN ZO TC-I TC-II TC-
III MF
Alkaloids + + - + + + + - - + + + + +
Carbohydrates + + + + + + + + + + + + + +
Glycosides + + - + + + + + + + + + + +
Tannins + - + + + + + + + + + + + +
Flavonoids - + + + + + + - + - - - +
Fixed oil - + + + + + + + - - + + + +
Saponins - - - - - - - - - + + + + +
Proteins and
Amino acids - + + + + + + + - + + + + +
Steroids - - - - - - - - - - - - -
- : Absent, + : Present, PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu choornam), MF
(Marketed formulation)
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 127
(vi) Development of thin layer chromatography
The TLC was performed for each batch of Thirikadu choornam (TC-I, TC-II and TC-
III), marketed formulation (MF) and separately for raw materials The experimental
technique used for preparation and development of TLC plate is given earlier (Table
4.42).
Table 4.42 Development of solvent system and TLC plate
Table 4.43 TLC profile of Thirikadu choornam and its raw material
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
Particulars Description
Solvent System (Best) Toluene: ethyl acetate (70:30v/v)
Adsorbent Silica gel
Detecting method Ultra Violet lamp at 366 nm
SN Name No. of Spots Rf values
1 PL 05 0.27,0.42,0.53,0.59,0.64
2 PN 07 0.08,0.21,0.42,0.50,0.57,0.64,0.80
4 ZO 03 0.17,0.23, 0.26
5 TC-I 12 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.32,
0.42,0.50, 0.53,0.57, 0.60
6 TC-II 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.42,0.50,
0.53,0.57,0.80
7 TC-III 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.42,0.50,
0.53,0.57,0.80
8 MF 11 0.08, 0.17, 0.21, 0.23, 0.26, 0.27, 0.42,0.50,
0.53,0.57,0.80
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 128
(vii) Determination of swelling index
The swelling index was determined for each batch of each batch of Thirikadu
choornam (TC-I, TC-II and TC-III), marketed formulation (MF) and separately for
raw materials. The findings are reported in experimental technique used for
preparation and development of TLC plate is given earlier (Table 4.44).
Table 4.44 Swelling index of Thirikadu choornam and its raw materials
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation)
(viii) Determination of foaming index
The Foaming index was determined for all the formulations and its ingredients
include PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), the method
used is depicted earlier in this chapter.
(ix) Determination of microorganisms
The TC-I and TC-II and TC-III, Marketed formulation (MF) and its raw materials PL
(Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis) were tested for
presence of Escherichia coli, Salmonella spp., Staphylococcus aureus as per the
standard method described in WHO (1998). The results shows that all above
mentioned microorganism were absent in all formulation and ingredients (Table
4.45).
SN Name Swelling index ±S.D.(n=6)
1 PL 3.662±0.664
2 PN 3.426±0.228
3 ZO 3.124±0.314
4 TC-I 3.545±0.451
5 TC-II 3.452±0.568
6 TC-III 3.245±0.234
7 MF 3.123±0.568
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 129
(x) Determination of arsenic and heavy metals
Limit test for arsenic
The limit test for arsenic was performed for each batch of Thirikadu choornam (TC-
I, TC-II and TC-III), marketed formulation (MF) and ingredients as per the methods
described above. The findings are recorded in (Table 4.45).
Limit test for heavy metals
The amount of heavy metals for each batch of Thirikadu choornam (TC-I, TC-II and
TC-III), marketed formulation (MF) and raw materials were estimated for lead by
matching the intensity of color with that of the standard stain using I.P. method. The
inferences are recorded in (Table 4.45).
Table 4.45 Limits of arsenic and heavy metals in Thirikadu choornam and its
raw material
SN Name Standard
stain
Color intensity as
compared to
standard strain E. coli
Salmonel
la sp
S.
Aureus
Inference
as per
WHO Arsenic
(Lead)
8. PL 1 ppm Less Less absent absent absent Permissible
9. PN 1 ppm Less Less absent absent absent Permissible
10. ZO 1 ppm Less Less absent absent absent Permissible
11. TC-I 1 ppm Less Less absent absent absent Permissible
12. TC-II 1 ppm Less Less absent absent absent Permissible
13. TC-III 1 ppm Less Less absent absent absent Permissible
14. MF 1 ppm Less Less absent absent absent Permissible
PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), TC (Thirikadu
choornam), MF (Marketed formulation),
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 130
Arsenic and heavy metals are below the specified limit in all ingredients, lab and
marketed formulations. Tests were also performed for specific pathogen, E. coli,
Salmonella species and S. aureus which are found absent. This ensures the level of
safety of formulation.
(xi) Development of fingerprinting method
The Fingerprinting method was developed for each batch of Thirikadu choornam , its
one marketed formulation and for its raw materials by using sophisticated instrument
UV, HPLC and HPTLC.
Development of UV spectroscopy fingerprinting method
The UV spectroscopy fingerprinting method was developed via estimation of piperine
which is an important content in Thirikadu choornam and its raw materials. All the
chemicals and solvents were used of A.R. Grade. Thirikadu choornam and its
ingredients were estimated for their piperine contents against standard piperine
solution on UV-Visible Spectrophotometer (Shimadzu, UV-1700, Pharmaspec). In
this formulation again piperine constitutes the main active ingredient and hence it is
used as marker for standardization purpose.
Preparation of piperine extract of Thirikadu choornam
The whole parameters for piperine extraction were same as the previous formulations
discussed in section (4.8 xi). This procedure was performed for each batch of
Thirikadu choornam and separately powdered PL (Piper longum) and PN (Piper
nigrum). As other ingredients does not contain piperine was not included in present
study.
Preparation of standard solution of piperine
The absorbance characteristics show that piperine follow Beer Lambert’s law within
the concentration range 2-10 µg/ml at the λ-max of 342.5 nm (Table 4.46) (Figure
4.2).The determination of formulations was carried out through UV
spectrophotometer at 342.5 nm for piperine.
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 131
Preparation of standard curve of piperine
A series of calibrated 10 ml volumetric flask were taken and appropriate aliquots of
the working standard solution of piperine were withdrawn and diluted up to 10 ml
with ethanol. The absorbance was measured at absorption maxima 342.5 nm (Figure
4.2), against the reagent blank prepared in similar manner without the piperine. The
absorption maxima and Beer’s law limit were recorded and data that prove the
linearity and obey Beer’s law limit were noted.
Table 4.46 Standard curve parameter of piperine
The estimation of piperine content of the Thirikadu choornam and powder of PL
(Piper longum) and PN (Piper nigrum) were carried out separately. The concentration
of piperine content in raw material was found to be 1.852 ± 0.241 and 3.685 ± 0.164
w/w in Piper longum and Piper nigrum respectively . The content of piperine in
different batches of Thirikadu choornam was found to be 1.829 ± 0. 011, 1.823 ±
0.145, 1.826 ± 0.123 and 1.635 ± 0.156 % w/w respectively for lab formulation (TC-
I, TC-II, TC-III) and marketed formulation (MF) (Table 4.47). The developed method
was found to be reliable, accurate, precise and sensitive.
S.No. Parameter Value
1
2
3
4
5
Absorption Maxima
Beer’s Law limit
Regression equation (y= bx+a)
Correlation coefficients (r2)
Accuracy (%)
342.5 nm
2-10 g/ml
y= 0.174x -0.318
r2 = 0.962
99.14
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 132
Table 4.47 Spectrophotometric determination of piperine content
Name Piperine Content (% w/w ± SD)
Piper longum 1.852 ± 0.241
Piper nigrum 3.685 ± 0.164
TC-I 1.829 ± 0. 011
TC-II 1.823 ± 0.145
TC-III 1.826 ± 0.123
MF 1.635 ± 0.156
P<0.001 (highly significant)
Precision and accuracy
The method was validated for precision and accuracy, by performing the recovery
studies at two levels by adding known amount of piperine extract of Thirikadu
choornam, of which the piperine content have been estimated previously. The data
were obtained and recovery was calculated (Table 4.48).
Table 4.48 Recovery study
S.no.
Amount of piperine ( g/ml)
Recovery% Sample Added Estimated
SD (n=6) RSD% SE
1
2
100
100
50
100
149.66 0.74
199.43 0.12
0.494
0.060
0.43
0.07
99.77
99.72
Mean 99.72
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 133
(xii) HPLC fingerprinting method for formulations
The present study is an attempt to develop the chromatographic fingerprint method
for Thirikadu choornam by High-performance liquid chromatographic method using
Piperine as a standard, which is as an important and major content in formulation.
RP- HPLC methods for determination of piperine from the fruits of Piper longum,
Piper nigrum, Thirikadu choornam (TC-I, TC-II and TC-III) and one marketed
formulation (MF) have been developed by using following methods. A C18 LUNA
(5 micron 25 cm×4.6 mm) column from Phenomenex a binary gradient high-
pressure liquid chromatograph (Shimadzu HPLC class VP series) with two LC–10
AT VP pumps, variable wavelength programmable UV/Visible SPD 10 AVP were
used. The mobile phase consisted methanol. The flow rate was 1.0 mL/min. The
wavelength of detection was 343 nm and the injection volume was 10 μl.
Extraction of piperine from Thirikadu choornam
The whole parameters for piperine extraction were same as the previous formulations
discussed in section (4.8 xii). This procedure was performed for each batch of
Thirikadu choornam and separately powdered Piper longum (Pippali). As other
ingredients does not contain piperine was not included in present study.
Calibration curve of standard piperine
The stock solution of piperine was prepared by dissolving 10.0 mg of piperine in
100.0 mL methanol, creating a 100 μg/mL solution of piperine. This solution was
diluted with the solvent as needed to prepare different standard solutions (2-
20μg/mL). All the parameters including linearity, area against concentration,
precision, reproducibility, limit of quantitation, limit of detection and validation
parameters was discussed in section (4.8. xii) (Figure 4.4, Table 4.14) as the main
active ingredients that is piperine is same as previous formulation.
Estimation of Piperine in Thirikadu choornam
The appropriate aliquots from piperine extract of each batch of Thirikadu choornam,
its marketed formulation and powdered Piper longum and Piper nigrum were
withdrawn in 10 ml volumetric flask separately. The corresponding concentrations of
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 134
piperine against respective peak areas value were determined using the piperine
calibration curve (Table 4.49).
Table 4.49 Estimation of Piperine Content in Thirikadu choornam (HPLC)
S.no. Name Piperine content %w/w
1 Piper longum 1.852 ± 0.001
2 Piper nigrum 3.685 ± 0.164
3
Thirikadu choornam
TC-I 1.841 ± 0.005
TC-II 1.837 ± 0.009
TC-III 1.832 ± 0.111
MF 1.645 ± 0.741
Mean SD (n=3),
Recovery studies
The recovery studies performed at three levels by adding known amount of piperine
to extract of Thirikadu choornam, of which the piperine content has been estimated
previously. The data were obtained and recovery calculated (Table 4.50).
Table 4.50 Recovery study
S.No
Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.41 0.01 0.007 0.01 99.61
2 100 100 199.85 0.04 0.020 0.02 99.93
3 100 150 249.24 0.14 0.056 0.08 99.70
Mean 0.028 0.03 99.743
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 135
(xiii) Development of HPTLC fingerprinting method
The HPTLC fingerprinting method was developed for each batch of Thirikadu
choornam (TC), its marketed formulation, powdered Piper longum and Piper nigrum
via estimation of piperine, which is an important content in Thirikadu choornam. All
the chemicals and solvents used were of A.R. Grade.
Preparation of piperine extract of Thirikadu choornam
The whole parameters for piperine extraction were same as the previous formulations
discussed in section (4.8 xiii). This procedure was performed for each batch of
Thirikadu choornam and separately powdered Piper longum and Piper nigrum. As
other ingredients does not contain piperine was not included in present study. All the
parameters including equipment details, chromatogram (Figure 4.6) method of
preparation of standard curve and validation details (Table 4.18) was remained same
as discussed in (4.8.xiii).
Estimation of piperine
The aliquots from piperine extract of each batch of TC, marketed formulation MF,
Piper longum and Piper nigrum withdrawn in 10 ml volumetric flask separately. The
corresponding concentrations of piperine against respective peak area were
determined using calibration curve of piperine. The results are cited in Table 4.51.
Table 4.51 Estimation of piperine content in Thirikadu choornam (HPTLC)
S.N. Name Piperine content %w/w
1 Piper longum 1.889 ± 0.011
2 Piper nigrum 3.699 ± 0.064
3
Thirikadu choornam
TC-I 1.852 ± 0.005
TC-II 1.843 ± 0.009
TC-III 1.831 ± 0.121
MF 1.639 ± 0.852
Mean SD of six determinations
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 136
Recovery studies
The recovery studies performed at three levels were done by adding known amount of
piperine to extract of Thirikadu choornam of which the piperine content have been
already estimated. The observations recorded and recovery was calculated (Table
4.52).
Table 4.52 Recovery study
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
(xiv) Development of Method for Stability Testing
All the three batches TC-I, TC-II, TC-III of Thirikadu choornam were subjected to
stability studies at accelerated condition at 45 0C at 75% Relative humidity for six
month. The sample were withdrawn at intervals of time (1,3 and 6 months) and
evaluated previously developed parameter colour, odour, taste, moisture content,
volatile oil content and piperine content. The changes in value of above parameters
with respect to developed parameters are recorded in (Table 4.53).
S.No
Amount of Piperine ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.01 0.78 0.523 0.45 99.34
2 100 100 199.08 0.96 0.482 0.55 99.54
3 100 150 249.91 0.85 0.340 0.49 99.96
Mean 0.449 0.498 99.61
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 137
Table 4.53 Accelerated stability testing at 45oC Temperature of Thirikadu
choornam
S.
No
.
Paramete
r
Formulation
s
Time duration (after months) Inferenc
e
1 3 6
1 Colour
TC-I
TC-II
TC-III
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
Dark Brown
No
change
2 Odour
TC-I
TC-II
TC-III
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
No
change
3 Taste
TC-I
TC-II
TC-III
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
No
change
4 Moisture
Content
TC-I
TC-II
TC-III
7.24±0.241
7.22±0.232
7.13±0.228
7.26±0.242
7.23±0.121
7.15±0.269
7.30 ±0.287
7.26±0.564
7.17±0.491
Minor
change
5 Piperine
content
TC-I
TC-II
TC-III
1.83±0.21
1.83±0.42
1.83±0.15
1.83±0.24
1.82±0.25
1.82±0.27
1.82±0.43
1.82±0.38
1.82±0.34
Minor
change
n= 3 Average of three determinants, NC = No change
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 138
4.11 Safoof-E-Sana
Formulation was strictly prepared as prescribed in the official book of Unani
Pharmacopoeia of India. 50 grams of each ingredient which includes senna leaves
(Cassia angustifolia), dry ginger (Zingiber officinale), haritakee (Terminalia
chebula), and balck salt (Vit lavana or vidam) were taken. All the ingredients were
weighed accurately and made fine powder by passing through sieve no. 80. Fine
powders were mixed geometrically in plastic tray and packed in plastic container.
Three sample batch of Safoof-E-Sana were prepared using above mentioned methods
and were named as SS-I, SS-II, SS-III. One Marketed formulations named MF was
purchased from local pharmacy store Raipur. These samples were stored at optimized
conditions of Temperature, light and moisture.
(i) Organoleptic profile and physical characteristics
Organoleptic profile of the formulations and each raw material were determined by
the method described earlier. Findings of the different organoleptic parameters are
summarized in (Table 4.54). Physical characteristics of all the three batches of
Safoof-E-Sana (SS-I, SS-II and SS-III), its marketed formulation (MF) and all raw
materials were established through the method described earlier and these data are
also recorded in (Table 4.54).
Discussion
Evaluation of organoleptic characters of raw materials senna leaves (Cassia
angustifolia), dry ginger (Zingiber officinale), haritakee (Terminalia chebula), and
balck salt were performed and characters are recorded. Laboratory batches of Safoof-
E-Sana (SS-I, SS-II and SS-III) and one marketed preparations were also evaluated
for organoleptic characters. The results for the marketed formulations (MF and
Laboratory formulations are found comparable. The above mentioned organoleptic
data are very useful for the preliminary identification of raw material and
formulations.
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 139
Table 4.54 Organoleptic and physical characteristics of Safoof-E-Sana and its
raw materials
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), BS
(Black salt), SS (Safoof-E-Sana), MF (Marketed Formulation)
The value of angle of repose for raw materials Zingiber officinale, Cassia
angustifolia, Terminalia chebula, Balck salt, lab formulation (SS-I) and marketed
formulation were 34.48, 29.52, 28.92, 22.54, 27.68 and 26.68 respectively which
shows good flow properties of prepared lab formulation. The flow properties are also
confirmed by Hausner’s ratio and Carr’s index. Values of Hausner’s ratio less than
S
N Name Colour Odour Taste
Tap
densi
ty
Bulk
densi
ty
Angle
of
repose
Haus
ner
ratio
Carr
’s
index
1 ZO Brown Characterist
ic Pungent 0.57 0.36 34.48 1.58 37
2 CA Light
green Faint Bitter 0.47 0.30 29.52 1.56 37
3 TC Brown Characterist
ic
Astringe
nt 0.83 0.68 28.92 1.22 18
4 BS Black Characterist
ic Salty 0.42 0.38 22.54 1.10 10
5 SS-I Brown Characterist
ic Pungent 0.48 0.39 27.68 1.23 19
6 SS-II Brown Characterist
ic Pungent
0.48 0.38 28.42 1.26
20.8
3
7 SS-III Brown Characterist
ic Pungent
0.47 0.37 28.12 1.27
21.2
8
8 MF Brown Characterist
ic Pungent
0.52 0.41 26.68 1.27
21.1
5
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 140
1.25 indicate good flow (20% Carr Index) and the value greater then 1.25 indicates
poor flow (33% Carr Index). The flow properties of lab (SS-I) and marketed
formulations were also confirmed by Hausner’s ratio and Carr’s index; it was found
1.23, 19, and 1.27, 21.15 respectively and indicates good flow characteristics.
(ii) Determination of foreign matter and loss on drying
Raw materials, Laboratory batches of Safoof-E-Sana (SS-I, SS-II and SS-III) and one
marketed preparation were also evaluated for foreign matter and loss on drying
through the methods described earlier in this chapter. The results of both parameters
are tabulated in (Table 4.55).
Table 4.55 Loss on drying and foreign matter content in Safoof-E-Sana and its
raw materials
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS
(Safoof-E-Sana), MF (Marketed Formulation)
Discussion
The moisture content of formulation was within acceptable range (<8%) thus
implying that the formulation can be stored for a long period and would not easily be
attacked by microbes. Laboratory formulations of Safoof-E-Sana were prepared after
removal of foreign matter. In examination no poisonous, dangerous and harmful
foreign matter or residue was found.
SN Name %LOD
±S.D. (n=6)
%Foreign Matter
±S.D. (n=6)
1 ZO 3.13±0.682 1.42±0.245
2 CA 3.34±0.445 1.28±0.123
3 TC 3.82±0.474 1.43±0.456
4 SS-I 3.25±0.582 NIL
5 SS-II 3.93±0.442 NIL
6 SS-III 3.96±0.125 NIL
7 MF 3.93±0.323 NIL
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 141
(iii) Ash values determination
The percent total ash and acid-insoluble ash was determined for each batch of Safoof-
E-Sana , its one marketed formulations and separately its raw materials as per above
mentioned method. The results are recorded in (Table 4.56).
Total ash value of Zingiber officinale, Cassia angustifolia, Terminalia chebula, and
lab formulation were 5.023± 0.643, 7.023 ± 0.426, 3.891 ±0.423 and 19.146±0.237
respectively (Table 4.56). The value of total ash in formulation is comparatively high
because of the presence of black salt in lab formulation. Acid-insoluble ash value of
prepared formulation 2.351±0.223 shows that a very small amount of the inorganic
component is insoluble in acid it indicates adulteration of raw ingredients by
substance like silica, rice husk is very less and low Acid-insoluble ash value may also
affect amount of the component absorbed in the gastrointestinal canal when taken
orally.
Table 4.56 Percentage ash value of Safoof-E-Sana and its raw materials
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS
(Safoof-E-Sana), MF (Marketed Formulation)
SN Name
Total ash
(% w/w ± S.D.,
n=6)
Acid insoluble ash
(% w/w ± S.D., n=6)
Water Soluble ash
(% w/w ± S.D., n=6)
1 ZO 5.023± 0.643 0.567 ± 0.011 12.134± 0.883
2 CA 7.023 ± 0.426 1.023 ± 0.643 34.247± 0.648
3 TC 3.891 ±0.423 0.423 ± 0.008 50.362±0.424
4 SS-I 19.146±0.237 2.351 ± 0.223 49.216±0.634
5 SS-II 19.245 ± 0.749 2.315 ± 0.011 50.124 ± 0.356
6 SS-III 18.999 ± 0.228 2.145 ± 0.009 49.123 ± 0.856
7 MF 21.145 ± 0.732 2.541 ± 0.056 51.120 ± 0.732
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 142
Discussion
Total ash value of Zingiber officinale, Cassia angustifolia, Terminalia chebula, lab
formulation (SS-I) and marketed formulation were 5.023± 0.643, 7.023 ± 0.426,
3.891 ±0.423, 19.146±0.237 and 20.223±0.336 respectively. The value of total ash in
formulation is comparatively high because of the presence of black salt in
formulation. Acid-insoluble ash value of lab formulation (SS-I) and marketed
formulation were 2.351±0.223 and 2.541 ± 0.056 which shows that a very small
amount of the inorganic component is insoluble in acid it indicates adulteration of
raw ingredients by substance like silica, rice husk is very less.
(iv) Determination of extractive values
The extractive values are recorded in alcohol and water with a view to study the
distribution of various constituents of Safoof-E-Sana (SS-I, SS-II and SS-III),
Marketed formulation (MF) and its raw materials. The method for determination of
extractive value in different solvent is described earlier in this chapter. The findings
were recorded in terms of percentage extractive values in (Table 4.57).
Table 4.57 Extractive values of Safoof-E-Sana and its raw materials
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS
(Safoof-E-Sana), MF (Marketed Formulation)
SN Name Alcohol-soluble extractive Water-soluble extractive
1 ZO 10.248±0.981 14.232±0.883
2 CA 5.648 ± 0.228 28.226±3.268
3 TC 22.21±1.442 49.668±3.102
4 SS-I 19.12±1.724 45.784±0.876
5 SS-II 18.214±1.045 44.213±0.001
6 SS-III 18.214±1.124 45.123±0.014
7 MF 17.245±0.321 40.147±1.654
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 143
Alcohol-soluble and water soluble extractive values of ingredients and formulation
are depicted in (Table 4.57), which shows 19.12±1.724 alcohol-soluble extractive
value and 45.784±0.876 water soluble extractive value of lab formulation and
17.245±0.321 alcohol-soluble extractive value and 40.147±1.654 water soluble
extractive value of marketed formulation. Higher water-soluble extractive value of
both formulations implies that water is a better solvent of extraction for the
formulation than ethanol.
(v) Qualitative phytochemical studies
Results of the phytochemical screening of the raw materials and lab formulation of
Safoof-E-Sana are concluded in (Table 4.58). One notable difference as a result of the
methods of extraction is the possibility that the alkaloids in Cassia angustifolia are
more water soluble, the reason why the presence of that group was not detectable in
the ethanolic extract. Furthermore, where more than one test was conducted for the
detection of a chemical group such as the alkaloids, no differences in the results were
observed for the different tests. Out of the nine phytochemical groups investigated,
five namely carbohydrate, glycosides, tannins, and proteins were detected in the
ethanolic extract of lab formulation and seven were present in the aqueous extract of
lab formulation including alkaloids and saponins with previously stated five. Fixed oil
and steroids were absent in all the ingredients and lab formulation for both methods
of extraction.
(vi) Development of thin layer chromatography
The TLC was performed for each batch of Safoof-E-Sana (SS-I, SS-II and SS-III),
marketed formulation (MF) and separately for raw materials. The experimental
technique used for preparation and development of TLC plate is given earlier (Table
4.59).
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL FORMULATIONS 144
Table 4.58 Phytochemical characterization of Safoof-E-Sana and its raw materials
Test Ethanolic extract Aqueous extract
ZO CA TC SS-I SS-II SS-III MF ZO CA TC SS-I SS-II SS-III MF
Alkaloids + + - + + + + - - + + + + +
Carbohydrates + + + + + + + + + + + + + +
Glycosides + + - + + + + + + + + + + +
Tannins + - + + + + + + + + + + + +
Flavonoids - + + + + + + - + - - - -
Fixed oil - + + + + + + + - - + + + +
Saponins - - - - - - - - - + + + + +
Proteins and
Amino acids - + + + + + + + - + + + + +
Steroids - - - - - - - - - - - - -
- : Absent, + : Present, ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS (Safoof-E-Sana), MF
(Marketed formulation)
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 145
Table 4.59 Development of solvent system and TLC plate
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS
(Safoof-E-Sana), MF (Marketed Formulation)
Table 4.60 TLC profile of Safoof-E-Sana and its raw material
SN Name No. of Spots Rf values
1 ZO 04 0.27,0.42,0.50,0.59
2 CA 04 0.08, 0.21 ,0.50, 0.80
4 TC 05 0.17,0.21, 0.26, 0.64, 0.75
5 SS-I 11 0.08, 0.17,0.21, 0.26, 0.27,
0.42,0.50,0.59, 0.64, 0.75
6 SS-II 11 0.08, 0.17,0.21, 0.26, 0.27,
0.42,0.50,0.59, 0.64, 0.75
7 SS-III 11 0.08, 0.17,0.21, 0.26, 0.27,
0.42,0.50,0.59, 0.64, 0.75
8 MF 11 0.08, 0.17,0.21, 0.26, 0.27,
0.42,0.50,0.59, 0.64, 0.75
Particulars Description
Solvent System (Best) Chloroform: Ethyl acetate: Formic acid, 7.5 : 6: 0.5
Adsorbent Silica gel
Detecting method Ultra Violet lamp at 292 nm
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 146
(vii) Determination of swelling index
The swelling index was determined for each batch of each batch of Safoof-E-Sana
(SS-I, SS-II and SS-III), marketed formulation (MF) and separately for raw materials.
The findings are reported in experimental technique used for preparation and
development of TLC plate is given earlier (Table 4.61).
Table 4.61 Swelling index of Safoof-E-Sana and its raw materials
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS
(Safoof-E-Sana), MF (Marketed Formulation)
(viii) Determination of foaming index
The Foaming index was determined for all the formulations and its ingredients
include ZO (Zingiber officinale), CA (Cassia angustifolia), and TC (Terminalia
chebula) the method used is depicted earlier in this chapter.
(ix) Determination of microorganisms
The SS-I and SS-II and SS-III, Marketed formulation (MF) and its raw materials ZO
(Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula) were tested
for presence of Escherichia coli, Salmonella spp., Staphylococcus aureus as per the
standard method described in WHO (1998). The results shows that all above
SN Name Swelling index ±S.D.(n=6)
1 ZO 4.125±0.425
2 CA 4.654±0.278
3 TC 4.258±0.123
4 SS-I 4.369±0.345
5 SS-II 4.965±0.245
6 SS-III 4.752±0.248
7 MF 4.125±0.125
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 147
mentioned microorganism were absent in all formulation and ingredients (Table
4.62).
(x) Determination of arsenic and heavy metals
Limit test for arsenic
The limit test for arsenic was performed for each batch of Safoof-E-Sana (SS-I, SS-II
and SS-III), marketed formulation (MF) and ingredients as per the methods described
above. The findings are recorded in (Table 4.62).
Limit test for heavy metals
The amount of heavy metals for each batch of Safoof-E-Sana (SS-I, SS-II and SS-
III), marketed formulation (MF) and raw materials were estimated for lead by
matching the intensity of color with that of the standard stain using I.P. method. The
inferences are recorded in (Table 4.62).
Table 4.62 Limits of arsenic and heavy metals in Safoof-E-Sana and its raw
material
SN Name Standard
stain
Color intensity as
compared to
standard strain E. coli
Salmonel
la sp
S.
Aureus
Inference
as per
WHO Arsenic
(Lead)
1. ZO
1 ppm Less Less absent absent absent Permissible
2. CA
1 ppm Less Less absent absent absent Permissible
3. TC
1 ppm Less Less absent absent absent Permissible
4. SS-I
1 ppm Less Less absent absent absent Permissible
5. SS-II
1 ppm Less Less absent absent absent Permissible
6. SS-III
1 ppm Less Less absent absent absent Permissible
7. MF
1 ppm Less Less absent absent absent Permissible
ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS (Safoof-
E-Sana), MF (Marketed Formulation)
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 148
Arsenic and heavy metals are below the specified limit in all ingredients, lab and
marketed formulations. Tests were also performed for specific pathogen, E. coli,
Salmonella species and S. aureus which are found absent. This ensures the level of
safety of formulation.
(xi) Development of fingerprinting method
The Fingerprinting method was developed for each batch of Safoof-E-Sana , its one
marketed formulation and for its raw materials by using sophisticated instrument UV,
HPLC and HPTLC.
Development of UV spectroscopy fingerprinting method
The UV spectroscopy fingerprinting method was developed via estimation of gallic
acid which is an important content in Safoof-E-Sana and its raw materials. All the
chemicals and solvents were used of A.R. Grade. Safoof-E-Sana and its ingredients
were estimated for their gallic acid contents against standard Gallic acid solution on
UV-Visible Spectrophotometer (Shimadzu, UV-1700, Pharmaspec). In this
formulation again Gallic acid constitutes the main active ingredient and hence it is
used as marker for standardization purpose.
Preparation of Gallic acid extract of Safoof-E-Sana
The gallic acid extract of Safoof-E-Sana was obtained by refluxing the powdered
Safoof-E-Sana (1gm) with 60 ml methanol for 1 hour. Resulting extract filtered and
the marc was re-refluxed with 40 ml methanol for another one hour. Both the filtrates
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 149
were combined. The methanolic extract was concentrated under vacuum till the
semisolid mass obtained. The residue was dissolved in 100 ml methanol and filtered
through sintered glass funnel (G-2) by vacuum filtration assembly. The same
procedure was followed for each batch of Safoof-E-Sana, marketed formulation MF
and powdered Terminalia chebula and their gallic acid extract (100 ml) prepared. As
other ingredients does not contain gallic acid was not included in present study.
Preparation of standard solution of Gallic acid
The absorbance characteristics show that gallic acid follow Beer Lambert’s law
within the concentration range 2-10 µg/ml at the λ-max of 271 nm (Table 4.63)
(Figure 4.7). The determination of formulations was carried out through UV
spectrophotometer at 271 nm for gallic acid.
Figure 4.7 UV Spectrum of gallic acid in Methanol
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 150
Figure 4.8 Standard curve of gallic acid at 271 nm
Table 4.63 Standard curve parameter of Gallic acid
Preparation of standard curve of gallic acid
A series of calibrated 10 ml volumetric flask were taken and appropriate aliquots of
the working standard solution of gallic acid were withdrawn and diluted up to 10 ml
with methanol. The absorbance was measured at absorption maxima 271 nm (Figure
S.No. Parameter Value
1
2
3
4
5
Absorption Maxima
Beer’s Law limit
Regression equation (y= bx+a)
Correlation coefficients (r2)
Accuracy (%)
271 nm
2-10 g/ml
y = 0.141x - 0.226
0.999
99.75
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 151
4.8), against the reagent blank prepared in similar manner without the gallic acid. The
absorption maxima and Beer’s law limit were recorded and data that prove the
linearity and obey Beer’s law limit were noted.
The estimation of gallic acid content of the Safoof-E-Sana and powder of TC
(Terminalia chebula) were carried out separately. The concentration of gallic acid
content in raw material was found to be 2.952 ± 0. 124 in Terminalia chebula. The
content of gallic acid in different batches of Safoof-E-Sana was found to be 0.704 ±
0. 012, 0.699 ± 0.114, 0.702 ± 0.123 and 0.612 ± 0.151 % w/w respectively for lab
formulation (SS-I, SS-II, SS-III) and marketed formulation (MF) (Table 4.64). The
developed method was found to be reliable, accurate, precise and sensitive.
Precision and accuracy
The method was validated for precision and accuracy, by performing the recovery
studies at two levels by adding known amount of gallic acid extract of Safoof-E-Sana,
of which the gallic acid content have been estimated previously. The data were
obtained and recovery was calculated (Table 4.65).
Table 4.64 Spectrophotometric determination of Gallic acid content
P<0.001 (highly significant)
Name Gallic acid Content (% w/w ± SD)
Terminalia chebula 2.952 ± 0. 124
SS-I 0.704 ± 0. 012
SS-II 0.699 ± 0.114
SS-III 0.702 ± 0.123
MF 0.612 ± 0.151
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 152
Table 4.65 Recovery study
S.no.
Amount of Gallic acid ( g/ml)
Recovery% Sample Added Estimated
SD (n=6) RSD% SE
1
2
100
100
50
100
149.46 0.16
199.01 0.85
0.107
0.427
0.09
0.49
99.64
99.51
Mean 99.75
(xii) HPLC fingerprinting method for formulations
The present study is an attempt to develop the chromatographic fingerprint method
for Safoof-E-Sana by High-performance liquid chromatographic method using gallic
acid as a standard, which is as an important and major content in formulation. RP-
HPLC methods for determination of gallic acid from the fruits of Terminalia chebula,
Safoof-E-Sana (SS-I, SS-II and SS-III) and one marketed formulation (MF) have
been developed by using following methods. A C18 LUNA (5 micron 25 cm×4.6
mm) column from Phenomenex a binary gradient high- pressure liquid
chromatograph (Shimadzu HPLC class VP series) with two LC–10 AT VP pumps,
variable wavelength programmable UV/Visible SPD 10 AVP were used. The mobile
phase consisted methanol. The flow rate was 1.0 mL/min. The wavelength of
detection was 271 nm and the injection volume was 10 μl.
Extraction of Gallic acid from Safoof-E-Sana
The gallic acid extract of Safoof-E-Sana was obtained by refluxing the powdered
Safoof-E-Sana (1gm) with 60 ml methanol for 1 hour. Resulting extract filtered and
the marc was re-refluxed with 40 ml methanol for another one hour. Both the filtrates
were combined. The methanolic extract was concentrated under vacuum till the
semisolid mass obtained. The residue was dissolved in 100 ml methanol and filtered
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 153
through sintered glass funnel (G-2) by vacuum filtration assembly. The same
procedure was followed for each batch of Safoof-E-Sana, marketed formulation MF
and powdered Terminalia chebula and their gallic acid extract (100 ml) prepared. As
other ingredients does not contain gallic acid was not included in present study.
Calibration curve of standard gallic acid
The stock solution of gallic acid was prepared by dissolving 10.0 mg of gallic acid in
100.0 mL methanol, creating a 100 μg/mL solution of gallic acid. This solution was
diluted with the solvent as needed to prepare different standard solutions (2, 4, 6, 8,
10, 12, 14, 16, 18 and 20μg/mL) (Figure 4.9).
Linearity
Standard solutions (2-20μg/mL), each in three replicates, were injected into the
system. The method of linear regression was used for data evaluation. Peak area
ratios of standard compounds were plotted against theoretical concentrations of
standards. Linearity was expressed as a correlation coefficient (Table 4.66).
Mean ± S.D. (n = 3).
Figure 4.9 HPLC calibration curve of gallic acid
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 154
Figure 4.10 HPLC chromatogram of gallic acid
Table 4.66 Validation parameters of gallic acid
S.No. Parameter Value
1 Absorption Maxima 271 nm
1 Retention time 2.6 min
2 Beer’s Law limit 2-20 g/ml
3 Regression equation (y= bx+a) y = 62.45x - 26.97
4 Intercept (a) 26.97
5 Slope (b) 62.45
6 Correlation coefficients (r2) r
2 = 0.999
7 Precision (n=3 % RSD) 0.044
8 Accuracy (%) 99.49
9 Limit of quantification(LOQ) 0.338 g/ml
10 Limit of detection(LOD) 0.117 g/ml
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 155
Table 4.67 System repeatability
Mean ± S.D. (n = 3).
Reproducibility
Inter and intra-day variation was performed by injecting the standard solutions (2-
20μg/mL), each in three replicates, twice on the same day, and once on the next day
and Peak areas are determined and compared (Table 4.67).
Estimation of Gallic acid in Safoof-E-Sana
The appropriate aliquots from gallic acid extract of each batch of Safoof-E-Sana, its
marketed formulation and powdered Terminalia chebula were withdrawn in 10 ml
volumetric flask separately. The corresponding concentrations of gallic acid against
respective peak areas value were determined using the gallic acid calibration curve
(Table 4.68).
Concentration
(μg/mL)
Day 1 peak area
Day 1 peak area
Day 2 peak area
2 2294.57±0.45 2294.28±0.12 2293.95±0.43
4 4217.34±0.45 4217.41±0.34 4217.04±0.46
6 6346.45±0.12 6346.61±0.36 6346.12±0.15
8 8482.45±0.24 8482.73±0.13 8482.17±0.26
10 10617.56±0.67 10617.27±0.26 10617.90±0.68
12 12733.67±0.34 12733.30±0.36 12733.13±0.36
14 14852.67±0.24 14852.81±0.76 14852.21±0.28
16 16968.54±0.54 16968.37±0.85 16968.71±0.53
18 18108.34±0.32 18108.11±0.32 18108.10±0.36
20 20122.45±0.21 20122.10±0.54 20122.16±023
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 156
Table 4.68 Estimation of Gallic acid Content in Safoof-E-Sana (HPLC)
S.no. Name Gallic acid content
%w/w
1 Terminalia chebula 3.145 ± 0.013
2
Safoof-E-Sana
SS-I 0.735 ± 0.012
SS-II 0.738 ± 0.016
SS-III 0.730 ± 0.015
MF 0.646 ± 0.062
Mean SD (n=3),
Recovery studies
The recovery studies performed at three levels by adding known amount of gallic acid
to extract of Safoof-E-Sana, of which the gallic acid content has been estimated
previously. The data were obtained and recovery calculated (Table 4.69).
Table 4.69 Recovery study
S.No
Amount of Gallic acid ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.02 0.04 0.027 0.02 99.35
2 100 100 199.03 0.18 0.090 0.10 99.52
3 100 150 249.05 0.04 0.016 0.02 99.62
Mean 0.044 0.050 99.49
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 157
(xiii) Development of HPTLC fingerprinting method
The HPTLC fingerprinting method was developed for each batch of Safoof-E-Sana,
its marketed formulation and powdered Terminalia chebula via estimation of gallic
acid, which is an important content in Safoof-E-Sana. All the chemicals and solvents
used were of A.R. Grade.
Preparation of gallic acid extract of Safoof-E-Sana
The gallic acid extract of Safoof-E-Sana were prepared by refluxing the powdered
Safoof-E-Sana (1gm) with 60 ml methanol for 1 hour. The extract was filtered and
marc was re refluxed with 40 ml of methanol for another 1hours. Filter and combine
the filtrate. Concentrate the methanol extract under vacuum till the semisolid mass is
obtained. Dissolve the residue in 75 ml methanol and filter through sintered glass
funnel (G-2) by vacuum filtration assembly. The same procedure was performed for
each batch of Safoof-E-Sana, marketed formulation MF and separately powdered
Terminalia chebula solution (100 ml) for their gallic acid extract were prepared. The
instrument used for the estimation, was Camag Linomat V semi automatic sample
applicator, Camag TLC scanner 3, CATS V.4.06 software for interpretation of the
data, Hamilton syringe and Camag twin trough chamber. The pre coated silica gel G
60 F 254 was used as stationary phase, obtained from E. Merck. The gallic acid were
well resolved on the precoated silica gel G 60 F 254 on aluminum sheets, the mobile
phase was Chloroform: Ethyl acetate: Formic acid (7.5 : 6: 0.5) chamber saturation
time 20 min, migration distance 70 mm, wavelength scanning at 292 nm., band width
8 mm, slit dimension 5 * 0.45 mm, scanning speed 20 nm/sec, and the source of
radiation was a deuterium lamp. All the solvents used were of AR grade, obtained
from S. D. Fine Chemicals Ltd., Mumbai. Marketed formulation of Safoof-E-Sana
were purchased from a local pharmacy store.
Preparation of standard solution
Standard gallic acid (98%) was procured from Sigma Aldrich Pvt. Ltd. A standard
solution of gallic acid was prepared with accurately weighed 1mg into a 10 ml
volumetric flask. The content was dissolved in methanol, and volume was made up to
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 158
10 ml. The method was validated for linearity, accuracy, limit of detection, limit of
quantification, inter-day and intra - day assay precision, repeatability of measurement,
and repeatability of sample application (Table 4.70).
Estimation of gallic acid
The appropriate aliquots from gallic acid extract of each batch of Safoof-E-Sana,
marketed formulation MF and Terminalia chebula withdrawn in 10 ml volumetric
flask separately. The corresponding concentrations of gallic acid against respective
peak area were determined using calibration curve of gallic acid. The results are cited
in (Table 4.71).
Table 4.70 Validation parameters
S. No. Parameter Value
1 Rf 0.29±0.03
2 Linearity (ng/spot) 100-500 ng
3 Correlation coeificients r2 0.999
4 LOD(ng /spot) 5 ng
5 LOQ(μg /spot) 5.7 ng
Rf : Retention factor, , LOD : Limit of detection, LOQ: Limit of quantification
Table 4.71 Estimation of gallic acid content in Safoof-E-Sana (HPTLC)
S.No. Name Gallic acid content
%w/w
1 Terminalia chebula 3.152 ± 0.002
2
Safoof-E-Sana
SS-I 0.739 ± 0.011
SS-II 0.738 ± 0.018
SS-III 0.733 ± 0.011
MF 0.649 ± 0.022
Mean SD of six determinations
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 159
Recovery studies
The recovery studies performed at three levels were done by adding known amount of
gallic acid to extract of Safoof-E-Sana of which the gallic acid content have been
already estimated. The observations recorded and recovery was calculated (Table
4.72).
Table 4.72 Recovery study
S.No Amount of Gallic acid ( g/ml)
*RSD% *SE Recovery%
In sample Added Estimated
1 100 50 149.87 0.09 0.060 0.05 99.91
2 100 100 199.30 0.39 0.196 0.23 99.65
3 100 150 249.82 0.08 0.032 0.05 99.93
Mean 0.096 0.108 99.83
Mean SD (n=3), RSD =Relative Standard Deviation, SE = Standard Error
Figure 4.11 HPTLC chromatogram of gallic acid ( Rf = 0. 29)
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 160
(xiv) Development of Method for Stability Testing
All the three batches SS-I, SS-II, SS-III of Safoof-E-Sana were subjected to stability
studies at accelerated condition at 45 0C at 75% Relative humidity for six month. The
sample were withdrawn at intervals of time (1,3 and 6 months) and evaluated
previously developed parameter colour, odour, taste, moisture content, volatile oil
content and gallic acid content. The changes in value of above parameters with
respect to developed parameters are recorded in (Table 4.73).
Table 4.73 Accelerated stability testing at 45oC Temperature of Safoof-E-Sana
S.
No
.
Paramete
r
Formulation
s
Time duration (after months) Inferenc
e
1 3 6
1 Colour
SS-I
SS-II
SS-III
Brown
Brown
Brown
Brown
Brown
Brown
Brown
Brown
Brown
No
change
2 Odour
SS-I
SS-II
SS-III
Characteris
tic
Characteris
tic
Characteris
tic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
Characterist
ic
No
change
3 Taste
SS-I
SS-II
SS-III
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
Pungent
No
change
4 Moisture
Content
SS-I
SS-II
SS-III
8.59±0.452
8.32±0.544
8.45±0.354
8.60±0.785
8.35±0.355
8.46±0.854
8.62 ±0.011
8.37±0.241
8.48±0.045
Minor
change
5
Gallic
acid
content
SS-I
SS-II
SS-III
0.73±0.01
0.72±0.25
0.73±0.35
0.73±0.43
0.72±0.45
0.73±0.04
0.73±0.63
0.72±0.01
0.72±0.45
Minor
change
n= 3 Average of three determinants, NC = No change
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 161
4.12 Development of Modern dosage form (Capsule) of selected Indigenous
formulations
(i) Selection of the capsule size
The volume of material that was to be filled into the capsule determined the size of
the capsule that was needed. Generally, capsules of sizes “0” to “4” were readily
available in the market and the relationship between the capsule size and related body
volume are shown in (Table 4.74) . For pharmaceutical products it is unusual to use a
size larger than “0” because of the difficulty in swallowing larger size capsules,
whilst size “5” is rarely used due to difficulties in the automatic filling process (Cole,
1987). In present study all the selected indigenous formulations namely BC
(Balacaturbhadrika churna), SC (Shringyadi churna), TC (Thirikadu choornam) and
SS (Safoof-E-Sana) have average dose of 500 mg and hence as per the below
mentioned table sizes “0” is best choice. All the empty capsule shells were purchased
from the local supplier of Raipur.
Table 4.74 Capsule size and body fill volumes
Capsule size Body volume (ml)
0 0.67
1 0.48
2 0.37
3 0.28
4 0.20
0 0.67
1 0.48
(ii) Selection of the filling method
It was also necessary to take into account the type of the filling machine that was
available and how each type of product is handled. For this study manual filling
equipment was used for the manufacture of the different capsule formulations. The
equipment used for this study consisted of sets of steel plates, which had predrilled
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 162
holes to take 50 capsules of size “0”. Empty “0” size capsules were placed into the
holes by hand with the bodies of the capsule fitting snugly into the plate. The caps of
the capsules were then removed, powder placed onto the surface of the body plate
(plate containing the capsule bodies) and spread with a spatula so that it flowed into
the empty capsule bodies. Because the uniformity of the fill weight was very
dependent upon the good flow properties of the powder, the capsule bodies were
filled using alternating gentle shaking of the plate, to remove any air pockets, and the
spreading of more material into the capsule body. Once filled to the brim, the cap of
each capsule was repositioned over the material filled body of the capsule and the two
capsule parts rejoined using gentle pressure. The quantity of the powder that could be
poured into the capsule was more or less established by a trial and error method i.e.
alternating shaking with filling until the desired result was obtained depending upon
the dose of respective formulations.
(iii) Determination of uniformity of weight and the amount of material in the
capsules
For the determination of the uniformity of weight of each selected formulations
includes BC (Balacaturbhadrika churna), SC (Shringyadi churna), TC (Thirikadu
choornam) and SS (Safoof-E-Sana) the Indian Pharmacopoeia (Indian
Pharmacopoeia, 2007) method was used. Twenty of each capsule formulations
prepared as described above were taken at random, their contents individually
weighed and the average weight (mass) of the content determined. Not more than two
of the individual weights (masses) had to deviate from the average weight (mass) by
more than 7.5% and none of the deviates by more than twice that percentage.
Method
Weigh an intact capsule. Open it without losing any part of the shell and remove the
contents as completely as possible. Weigh the shell, the difference between the
weighing gives the weight of the contents. Repeat the procedure with another 19
capsules.
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 163
(iv) Disintegration Test (Indian Pharmacopoeia, 2007)
This test determines whether capsules disintegrate within a prescribed time when
placed in a liquid medium under the prescribed experimental conditions. In present
study we have reported the disintegration time for each selected lab formulations of
BC (Balacaturbhadrika churna), SC (Shringyadi churna), TC (Thirikadu choornam)
and SS (Safoof-E-Sana). Detailed method for determination as per Indian
Pharmacopoeia, 2007 is discussed below.
Equipment
The apparatus consists of a basket-rack assembly, a 1-litre beaker, a thermostatic
arrangement for heating the fluid and a mechanical device for raising and lowering
the basket in the immersion fluid at a constant frequency rate (Figure 4.14). The time
required for the upward stroke was equal to the time required for the downward
stroke, and the change in stroke direction was smooth and not abrupt.
Medium
The assembly was suspended in the 0.1 N HCL in a 1-litre beaker. The volume of
liquid was maintained at 25 mm below the surface of the liquid, and at its lower point
was at least 25 mm above the bottom of the beaker. There was a thermostatic
arrangement for heating the liquid to maintain the temperature at 37º ± 2º.
Method
One capsule was introduced into each tube than a disc was added to each tube.
Assembly was introduced into the beaker containing the 0.1 N HCL and operate the
apparatus for the specified time. The capsules pass the test if all of them have
disintegrated.
(v) Moisture content
In modern pharmaceutical technology, the water content provides information
concerning the shelf life and the quality of the drugs. The most likely cause of drug
instability is hydrolysis, and water plays a dominant role in hydrolysis reaction
(Wells, 2002). In present study we have studied the percent of moisture content in
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 164
each selected lab formulation of BC (Balacaturbhadrika churna), SC (Shringyadi
churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana) at different storage
conditions as per in International Conference on Harmonization (ICH) guidelines.
Figure 4.12 Apparatus for Disintegration of Capsules
Method
For this study the European Pharmacopoeia (European Pharmacopoeia 4th edition
2002) method described below was used. About 500mg of the each formulation was
weighed in a flat-bottomed dish. The powder was then dried in an oven at 100-105℃
for 3 hours, allowed to cool (approximately 10 minutes) in a desiccator over
anhydrous silica gel, weighed and the weight recorded. The percent moisture content
as determined by this gravimetric method was then calculated using the following
equations.
Moisture weight = Initial weight (before drying) – Final weight (after drying)
Percent Moisture content = (Moisture weight / Initial weight)*100
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 165
(vi) Active ingredient content
The active ingredient was estimated for BC (Balacaturbhadrika churna), SC
(Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana) through
HPLC. As we know BC, SC and TC contain the active ingredient piperine and SS
have gallic acid as an active ingredient or standard. The complete method of
estimation of these active ingredients is discussed earlier (section 4.8 xii, 4.9 xii, 4.10
xii and 4.11 xii) in this chapter.
(vii) Stability studies
Degradation of active drug is the major and most important cause of the drug product
stability changes. Drug degradation occurs by four main processes: hydrolysis,
oxidation, photolysis, and trace metal catalysis. The most likely cause of drug
instability is hydrolysis. Water plays a dominant role and in many cases it is
implicated passively as a solvent vector between two reacting species in solution
(Wells, 2002). Thus storage of the product in atmospheres of high humidity will
typically accelerate decompositions that result from hydrolysis. Also, an increase in
temperature causes an increase in the rate of chemical reaction (Pugh, 2002).
Moisture content is an important parameter for capsule dosage form and it is also
important for herbal medicines, which are hygroscopic. Moisture content cannot
represent the hygroscopic property of the powder, but it can serve as a reference for
quality control (e.g. stability study).
4.13 Result and Discussion
(i) Determination of uniformity of weight and the amount of material in the
capsules
Uniformity of weight were determined for the each selected formulations as per the
method described above. Results shows that all the capsule formulations complies
with the Indian Pharmacopoeia range (<7.5%). The capsules produced were
physically elegant and acceptable and met the pharmacopoeia specifications for
content and weight uniformity (The average deviation in weight were 0.081±0.093,
0.108±0.093, 0.115±0.117 and 0.246±0.143 respectively for BC (Balacaturbhadrika
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 166
churna), SC (Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
.(Table 4.75,Table 4.76, Table 4.77 and Table 4.78).
Table 4.75 Weight variation test for Balacaturbhadrika churna (BC)
n=20 Wt. of
empty
capsule (mg)
Wt. of filled
capsule (mg)
Wt. of churna
(mg)
Deviation
in weight
from
average
(%)
Amount of
content of
capsule (%)
1 94.000 593.400 499.400 0.120 99.880
2 94.340 592.300 497.960 0.408 99.592
3 94.120 593.201 499.081 0.184 99.816
4 94.340 594.102 499.762 0.048 99.952
5 94.270 594.202 499.932 0.014 99.986
6 94.270 594.200 499.930 0.014 99.986
7 94.110 593.990 499.880 0.024 99.976
8 94.090 593.320 499.230 0.154 99.846
9 94.340 593.567 499.227 0.155 99.845
10 94.360 594.100 499.740 0.052 99.948
11 94.340 594.231 499.891 0.022 99.978
12 94.000 593.504 499.504 0.099 99.901
13 94.340 594.001 499.661 0.068 99.932
14 94.320 594.111 499.791 0.042 99.958
15 94.330 594.001 499.671 0.066 99.934
16 94.370 594.080 499.710 0.058 99.942
17 94.340 594.070 499.730 0.054 99.946
18 94.372 594.210 499.838 0.032 99.968
19 94.050 594.000 499.950 0.010 99.990
20 94.010 594.001 499.991 0.002 99.998
AVG
and
SD
94.010±0.142 593.830±0.483 499.594±0.466 0.081±0.093 99.919±0.093
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 167
Table 4.76 Weight variation test for Shringyadi churna (SC)
n=20 Wt. of
empty
capsule
(mg)
Wt. of filled
capsule (mg)
Wt. of
churna (mg)
Deviation in
weight from
average (%)
Amount of
content of
capsule (%)
1 93.988 593.619 499.631 0.074 99.926
2 94.328 592.519 498.191 0.362 99.638
3 94.108 593.420 499.312 0.138 99.862
4 94.328 594.321 499.993 0.001 99.999
5 94.258 593.348 499.09 0.182 99.818
6 94.234 594.104 499.87 0.026 99.974
7 94.098 593.988 499.89 0.022 99.978
8 94.078 593.539 499.461 0.108 99.892
9 94.123 593.581 499.458 0.109 99.892
10 94.765 594.736 499.971 0.006 99.994
11 94.328 592.328 498 0.400 99.600
12 93.988 593.723 499.735 0.053 99.947
13 94.328 594.220 499.892 0.022 99.978
14 94.654 593.744 499.09 0.182 99.818
15 94.318 594.220 499.902 0.020 99.980
16 94.358 594.299 499.941 0.012 99.988
17 94.444 594.405 499.961 0.008 99.992
18 94.360 593.480 499.12 0.176 99.824
19 94.038 593.348 499.31 0.138 99.862
20 93.998 593.428 499.43 0.114 99.886
AVG
and
SD
94.011±0.124 593.719±0.483 499.462±0.466 0.108±0.093 99.892±0.093
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 168
Table 4.77 Weight variation test for Thirikadu choornam (TC)
n=20 Wt. of
empty
capsule (mg)
Wt. of filled
capsule (mg)
Wt. of
churna (mg)
Deviation in
weight from
average (%)
Amount of
content of
capsule (%)
1 94.999 594.642 499.643 0.071 99.929
2 94.328 592.531 498.203 0.360 99.641
3 94.234 593.369 499.135 0.173 99.827
4 94.328 594.327 499.999 0.000 100.000
5 94.234 594.122 499.888 0.022 99.978
6 94.234 594.116 499.882 0.024 99.976
7 94.098 594.000 499.902 0.020 99.980
8 94.078 593.551 499.473 0.106 99.895
9 94.123 593.158 499.035 0.193 99.807
10 94.765 594.748 499.983 0.003 99.997
11 94.328 592.340 498.012 0.398 99.602
12 94.125 593.872 499.747 0.051 99.949
13 94.123 593.134 499.011 0.198 99.802
14 94.654 593.756 499.102 0.180 99.820
15 94.318 594.232 499.914 0.017 99.983
16 94.358 594.311 499.953 0.009 99.991
17 94.444 594.417 499.973 0.005 99.995
18 94.120 593.252 499.132 0.174 99.826
19 94.038 593.161 499.123 0.176 99.825
20 94.455 593.897 499.442 0.112 99.888
AVG
and
SD
94.010±0.248 593.747±0.668 499.428±0.582 0.115±0.117 99.886±0.116
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 169
Table 4.78 Weight variation test for Safoof-E-Sana (SS)
n=20 Wt. of
empty
capsule
(mg)
Wt. of filled
capsule (mg)
Wt. of
churna (mg)
Deviation in
weight from
average (%)
Amount of
content of
capsule (%)
1 94.765 594.066 499.301 0.140 99.860
2 94.094 591.955 497.861 0.429 99.572
3 94 592.793 498.793 0.242 99.759
4 93.586 592.000 498.414 0.318 99.683
5 94 591.781 497.781 0.445 99.556
6 94 593.540 499.54 0.092 99.908
7 92.356 591.916 499.56 0.088 99.912
8 93.844 592.975 499.131 0.174 99.826
9 93.889 591.877 497.988 0.403 99.598
10 94.531 594.172 499.641 0.072 99.928
11 94.094 591.764 497.67 0.467 99.534
12 93.891 593.296 499.405 0.119 99.881
13 93.889 592.558 498.669 0.267 99.734
14 94.42 593.180 498.76 0.249 99.752
15 94.084 591.975 497.891 0.423 99.578
16 93.475 593.086 499.611 0.078 99.922
17 94.21 593.841 499.631 0.074 99.926
18 93.886 592.676 498.79 0.243 99.758
19 93.804 591.696 497.892 0.423 99.578
20 94.221 593.321 499.1 0.180 99.820
AVG
and
SD
94.010±0.480 592.723±0.822 498.771±0.713 0.246±0.143 99.754±0.143
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 170
(ii) Disintegration Test
Disintegration time for each selected formulations BC (Balacaturbhadrika churna),
SC (Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana) were
estimated as per the method described earlier (Indian Pharmacopoeia, 2007). Finding
of disintegration time of above mentioned formulations were 5.20±0.480, 5.46±0.456,
5.10±0.431and 5.35±0.009 respectively (Table 4.79). Results were in the acceptable
range (< 7 min).
Table 4.79 Disintegration time for each selected lab formulation
Formulation Disintegration Time (Min.) (n=6)
BC 5.20±0.480
SC 5.46±0.456
TC 5.10±0.431
SS 5.35±0.009
BC (Balacaturbhadrika churna), SC (Shringyadi churna), TC (Thirikadu
choornam) and SS (Safoof-E-Sana)
(iii) Moisture content
After the capsules were filled the moisture level of its contents were tested as per the
above described method. The results of these tests are given in (Table 4.80) and
indicated that the moisture level of the contents of the each formulations BC
(Balacaturbhadrika churna), SC (Shringyadi churna), TC (Thirikadu choornam) and
SS (Safoof-E-Sana) were 3.02±0.34, 2.88±2.34, 2.70±3.32 and 3.96±3.45
respectively. All the freshly prepared capsule shows moisture content at acceptable
range (< 8%) as per WHO guidelines (WHO, 1998). Since the moisture absorbed may
speed up degradation, the humidity conditions during the manufacture of the capsules
can thus be a crucial factor and these capsules should preferably be manufactured
under more tightly controlled humidity conditions.
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 171
Table 4.80 Moisture content of each formulation from filled capsules before
storage.
(n=6), ±SD, BC (Balacaturbhadrika churna), SC (Shringyadi churna), TC
(Thirikadu choornam) and SS (Safoof-E-Sana)
(iv) Active ingredient content
The active ingredient was estimated for BC (Balacaturbhadrika churna), SC
(Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana) through
HPLC. As we know BC, SC and TC contain the active ingredient piperine and SS
have gallic acid as an active ingredient or standard. The complete method of
estimation of these active ingredients is discussed earlier (section 4.8 xii, 4.9 xii, 4.10
xii and 4.11 xii) in this chapter.
(v) Stability studies
Moisture content can serve as a reference for quality control to perform the stability
studies. Each formulation were kept in the different storage conditions as specified in
International Conference on Harmonization (ICH) guidelines.
Long term storage condition (25°C/60% relative humidity (RH) for 12 months),
accelerated conditions (40°C/75% RH) for 6 months and to reveal the effect of
container we were also determined the moisture content outside the container at
25°C/60% relative humidity (RH) for 12 months. Samples were charged in stability
chambers with humidity and temperature control.
Formulation Initial
Weight (mg)
Final Weight
(mg)
Percentage of
moisture (%)
Active ingredient
content (% w/w)
BC 499.4±0.19 484.34±4.42 3.02±0.34 0.435±0.025
SC 499.12±0.34 484.76±4.87 2.88±2.34 0.605±0.012
TC 498.23±0.76 484.78±5.43 2.70±3.32 1.841±0.005
SS 497.23±1.23 477.56±3.45 3.96±3.45 0.738±0.016
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 172
25°C/60% relative humidity (RH) inside container
Results shows that at 25°C/60% relative humidity (RH) inside container all the
selected lab formulations do not posses the significant moisture content which was
3.31±0.55, 3.18±0.66, 3.09±0.23 and 4.72±0.54 for BC (Balacaturbhadrika churna),
SC (Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
respectively. Hence it reflects that in this storage condition moisture content of each
capsule was <5 % w/w (Table 4.81), which indicates that there is less chances of
microbial growth and capsule will not become soft.
Percent active ingredient was estimated with respect to its initial concentration
present before storage in respective formulations (Table 4.80). It was found
98.88±0.29, 97.89±0.43 and 96.91±0.87% w/w of piperine in BC, SC and TC
respectively (Table 4.81). Similarly for SS gallic acid was found 98.68±0.22 % w/w
with respect to its concentration present in formulation before storage (Table 4.80).
The above results reveal that this storage condition is suitable for this capsule dosage
forms.
Table 4.81 Moisture content of each formulation from filled capsules 25ºC/60%
RH inside container after 12 months
(n=6), ±SD, RH (Relative humidity) BC (Balacaturbhadrika churna), SC
(Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
25°C/60% relative humidity (RH) outside the containers
At this condition all the formulations shows significant increase in the percent
moisture content (Table 4.82). It was 17.86±0.34, 17.51±0.43, 17.63±0.12 and
Formulation Initial Weight
(mg)
Final Weight
(mg)
Percentage of
moisture (%)
Active
ingredient
content (%)
BC 500.69±014 484.14±0.28 3.31±0.55 98.88±0.29
SC 500.58±0.23 484.65±0.32 3.18±0.66 97.89±0.43
TC 500.20±0.18 484.74±0.47 3.09±0.23 96.91±0.87
SS 502.14±0.21 478.42±0.36 4.72±0.54 98.68±0.22
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 173
18.88±0.43 for BC (Balacaturbhadrika churna), SC (Shringyadi churna), TC
(Thirikadu choornam) and SS (Safoof-E-Sana) respectively. Results at this condition
reflect that powder is hygroscopic in nature and it should be kept inside the container
immediately after the formulation. Percent active ingredient was estimated with
respect to its initial concentration present before storage in respective formulations
(Table 4.80). It was found 91.20±0.41, 90.62±0.34 and 89.48±0.73 % w/w of piperine
in BC, SC and TC respectively (Table 4.82). Similarly for SS gallic acid was found
92.22±0.28% w/w with respect to its concentration present in formulation before
storage (Table 4.82). The above results depict significant loss of active ingredients in
formulations.
Table 4.82 Moisture content of each formulation from filled capsules 25ºC/60%
RH outside container after 6 months.
(n=6), ±SD, RH (Relative humidity) BC (Balacaturbhadrika churna), SC
(Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
40ºC/75% relative humidity (RH) inside containers
The findings of moisture content of each formulation in this condition reveals that
even inside the container, storage of the capsule in atmospheres of high humidity will
increase the moisture content which typically accelerate decompositions that result
from hydrolysis. Also, an increase in temperature causes an increase in the rate of
chemical reaction. The results at this condition (Table 4.83) shows percent moisture
content 8.84±0.12, 8.67±0.32, 8.50±0.87 and 10.03±0.12 for BC (Balacaturbhadrika
churna), SC (Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
respectively. Percent active ingredient was estimated with respect to its initial
Formulation
Initial
Weight
(mg)
Final
Weight
(mg)
Percentage of
moisture (%)
Active ingredient
content (%)
BC 593.05±0.34 487.11±0.23 17.86±0.34 91.20±0.41
SC 589.30±0.45 486.14±0.98 17.51±0.43 90.62±0.34
TC 591.04±0.54 486.85±0.12 17.63±0.12 89.48±0.73
SS 590.74±0.56 479.21±0.43 18.88±0.43 92.22±0.28
Standardization of Formulations
DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL
FORMULATIONS 174
concentration present before storage in respective formulations (Table 4.80). It was
found 93.63±0.11, 92.28±0.85 and 90.61±0.32 % w/w of piperine in BC, SC and TC
respectively (Table 4.83). Similarly for SS gallic acid was found 91.11±0.48% w/w
with respect to its concentration present in formulation before storage (Table 4.83).
The above results again shows significant loss of active ingredients in comparison
with 25ºC/60% RH inside container storage condition.
Table 4.83 Moisture content of each formulation from filled capsules 40ºC/75%
RH inside container after 6 months.
(n=6), ±SD, RH (Relative humidity) BC (Balacaturbhadrika churna), SC
(Shringyadi churna), TC (Thirikadu choornam) and SS (Safoof-E-Sana)
Through these studies we can conclude that the capsule of each selected lab
formulations passed the test for uniformity of weight. All capsules disintegrated
within 7 minutes. Moisture content of capsule was <5 % w/w at 25°C/60% relative
humidity (RH) inside container which indicates that there is less chances of microbial
growth and capsule will not become soft and most important at this storage condition
(25°C/60% relative humidity (RH) inside container) the amount of active ingredients
was also found comparable with fresh formulation..
Formulation Initial
Weight
(mg)
Final
Weight
(mg)
Percentage of
moisture (%)
Active ingredient
content (%)
BC 532.16±0.76 485.12±0.56 8.84±0.12 93.63±0.11
SC 532.38±0.12 486.24±0.65 8.67±0.32 92.28±0.85
TC 532.41±0.23 487.15±0.12 8.50±0.87 90.61±0.32
SS 533.55±0.65 480.01±0.98 10.03±0.12 91.11±0.48