Standardization of Formulations - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29448/9/09_chapter 4.pdf · Standardization of Formulations Development and Standardization

Standardization of Formulations

Development and Standardization of Modern Dosage Form for Indigenous Medicinal

Formulations 68

4


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



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.



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



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).



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



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-



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.



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



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.



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



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.



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



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



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.



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



Formulations 83

Table 4.4 Extractive values of Balacaturbhadrika churna and its raw

materials



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



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


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)



Formulations 86

Table 4.7 TLC profile of Balacaturbhadrika churna and its raw material



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,



Formulations 87

Table 4.8 Swelling index of Balacaturbhadrika churna and its raw materials



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



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


3. AH


4. PI


5. BC-I


6. BC-II


7.

BC-

III 1 ppm Less Less absent absent absent Permissible

8. MF




formulation)



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.



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



Formulations 91

Figure 4.2 UV scan of piperine in ethanol

Figure 4.3 Standard curve of piperine in ethanol at 342.5 nm



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



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).



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).



Formulations 95

Table 4.13 Validation parameters of piperine

Figure 4.5 HPLC chromatogram of piperine


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



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).



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).



Formulations 98


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.



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).



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


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).



Formulations 101


S.No


*RSD% *SE Recovery%


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


Figure 4.6 HPTLC chromatogram of piperine ( Rf = 0. 24)



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


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



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.



the method described earlier. Findings of the different organoleptic parameters is


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.



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.


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


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



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 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



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.


Determination of ash values


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



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



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



Formulations 107



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










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



Formulations 108



marketed formulation of Shringyadi churna are concluded in (Table 4.25). One


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.



different tests.








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.



Table 4.25 Phytochemical characterization of Shringyadi churna and its raw materials

Test


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)



Formulations 110






Table 4.27 TLC profile of Shringyadi churna and its raw material




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,



Formulations 111


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





include PI (Pistacia integerrima), PL (Piper longum), AP (Aconitum palmatum), The

method used is depicted earlier in this chapter.


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



Formulations 112



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).


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



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


Salmonel

la sp

S.

Aureus

Inference

as per

WHO Arsenic

(Lead)

1. PI


2. PL


3. AP


4. SC-I


5. SC-II


6.

SC-

III 1 ppm Less Less absent absent absent Permissible

7. MF






Formulations 113






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.



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.




4.2). The determination of formulations was carried out through UV




Formulations 114





4.2), against the reagent blank prepared in similar manner without the piperine. The



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.




SC-I 0.605 ± 0. 001 %

SC-II 0.599 ± 0.127 %

SC-III 0.602 ± 0.008 %

MF 0.535 ± 0.015 %




Formulations 115



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).


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



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.



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






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),



Formulations 117

Recovery studies


to extract of Shringyadi churna, of which the piperine content has been estimated

previously. The data were obtained and recovery calculated (Table 4.33).


S.No


*RSD% *SE Recovery%


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



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


discussed in section (4.8.xiii). This procedure was performed for each batch of


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.



Formulations 118


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)


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


Recovery studies


piperine to extract of Shringyadi churna of which the piperine content have been

already estimated. The observations and recovery was recorded (Table 4.35).


S.No Amount of Piperine ( g/ml)

*RSD% *SE Recovery%


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




Formulations 119


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





Table 4.36 Accelerated stability testing at 45oC Temperature of Shringyadi

churna

S.

No

.

Paramete

r

Formulatio

ns


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




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.



the method described earlier. Findings of the different organoleptic parameters are


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.



Formulations 121

Table 4.37 Organoleptic and physical characteristics of Thirikadu choornam


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



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.


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


Table 4.38 Loss on drying and foreign matter content in Thirikadu choornam




Discussion



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


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



Formulations 123


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.


Determination of ash values


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.



distribution of various constituents of Thirikadu choornam (TC-I, TC-II and TC-III),


extractive value in different solvent are described earlier in this chapter. The findings




Formulations 124

Table 4.39 Percentage ash value of Thirikadu choornam and its raw materials









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



Formulations 125

Table 4.40 Extractive values of Thirikadu choornam and its raw materials





marketed formulation of Thirikadu choornam are concluded in (Table 4.41). One


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.



different tests.








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



Table 4.41 Phytochemical characterization of Thirikadu choornam and its raw materials

Test


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



DEVELOPMENT AND STANDARDIZATION OF MODERN DOSAGE FORM FOR INDIGENOUS MEDICINAL

FORMULATIONS 127


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 4.43 TLC profile of Thirikadu choornam and its raw material








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



FORMULATIONS 128


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





include PL (Piper longum), PN (Piper nigrum), ZO (Zingiber officinalis), the method

used is depicted earlier in this chapter.


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



4.45).


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



FORMULATIONS 129



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).


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



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


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


choornam), MF (Marketed formulation),



FORMULATIONS 130






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.



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


Preparation of piperine extract of Thirikadu choornam


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.




4.2).The determination of formulations was carried out through UV




FORMULATIONS 131





4.2), against the reagent blank prepared in similar manner without the piperine. The



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.


1

2

3

4

5

Absorption Maxima

Beer’s Law limit



Accuracy (%)

342.5 nm

2-10 g/ml

y= 0.174x -0.318

r2 = 0.962

99.14



FORMULATIONS 132




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




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).


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



FORMULATIONS 133



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


discussed in section (4.8 xii). This procedure was performed for each batch of

Thirikadu choornam and separately powdered Piper longum (Pippali). As other





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



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)


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


to extract of Thirikadu choornam, of which the piperine content has been estimated



S.No


*RSD% *SE Recovery%


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




FORMULATIONS 135


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


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).


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




FORMULATIONS 136

Recovery studies


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).




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





S.No


*RSD% *SE Recovery%


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



FORMULATIONS 137

Table 4.53 Accelerated stability testing at 45oC Temperature of Thirikadu

choornam

S.

No

.

Paramete

r

Formulation

s


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




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.



the method described earlier. Findings of the different organoleptic parameters are


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.



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



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.


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



FORMULATIONS 141


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



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



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.



distribution of various constituents of Safoof-E-Sana (SS-I, SS-II and SS-III),


extractive value in different solvent is described earlier in this chapter. The findings


Table 4.57 Extractive values of Safoof-E-Sana and its raw materials




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



FORMULATIONS 143


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.


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.


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).


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




FORMULATIONS 145




Table 4.60 TLC profile of Safoof-E-Sana and its raw material


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


Solvent System (Best) Chloroform: Ethyl acetate: Formic acid, 7.5 : 6: 0.5





FORMULATIONS 146


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





include ZO (Zingiber officinale), CA (Cassia angustifolia), and TC (Terminalia

chebula) the method used is depicted earlier in this chapter.


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



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



FORMULATIONS 147


4.62).



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).


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



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


Salmonel

la sp

S.

Aureus

Inference

as per

WHO Arsenic

(Lead)

1. ZO


2. CA


3. TC


4. SS-I


5. SS-II


6. SS-III


7. MF


ZO (Zingiber officinale), CA (Cassia angustifolia), TC (Terminalia chebula), SS (Safoof-

E-Sana), MF (Marketed Formulation)



FORMULATIONS 148






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.


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


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



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



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


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


1

2

3

4

5

Absorption Maxima

Beer’s Law limit



Accuracy (%)

271 nm

2-10 g/ml

y = 0.141x - 0.226

0.999

99.75



FORMULATIONS 151

4.8), against the reagent blank prepared in similar manner without the gallic acid. The



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.



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


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



FORMULATIONS 152


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



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



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



FORMULATIONS 154

Figure 4.10 HPLC chromatogram of gallic acid

Table 4.66 Validation parameters of gallic acid


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



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



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



S.No

Amount of Gallic acid ( g/ml)

*RSD% *SE Recovery%


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




FORMULATIONS 157


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



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




FORMULATIONS 159

Recovery studies


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).


S.No Amount of Gallic acid ( g/ml)

*RSD% *SE Recovery%


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


Figure 4.11 HPTLC chromatogram of gallic acid ( Rf = 0. 29)



FORMULATIONS 160


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


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




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



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.



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



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



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



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



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



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



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



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.



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



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



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.


RH outside container after 6 months.



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



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.


RH inside container after 6 months.



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

Documents

Standardization of Formulations - Shodhgangashodhganga.inflibnet.ac.in/bitstream/10603/29448/9/09_chapter 4.pdf · Standardization of Formulations Development and Standardization