6.1 objective - shodhganga : a reservoir of indian...
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Chapter-6 Herbal formulations
101
6.1 OBJECTIVE
To develop simple herbal formulations using the selected plants extract.
6.2 INTRODUCTION
Herbal formulations many times fails to gain the patient acceptability, hence an
attempt was made to increase the same by developing some simple formulations. The
developed formulations were subjected to standardization by using analytical
methods. Standardization of herbal medicine is important and a challenging task, for
this the author selected some marker compounds and quantitative determination
methods were developed to determine the same in final formulation.
6.3 LITERATURE REVIEW
According to World Health Organization (WHO) more than 80% of the world’s
population relies on traditional medicine for their primary healthcare needs. Use of
herbal medicines in Asia indicates a long history of human interactions with the
environment. Plants used as traditional medicine contain a wide range of substances
that can be used to treat chronic as well as infectious diseases. A vast knowledge of
how to use the plants against different illnesses is expected to have accumulated in
areas where the use of plants is still of great importance (Diallo et al, 1999). The
medicinal value of plants lies in some chemical substances that have a definite
physiological action on the human body. The most important of these bioactive
compounds of plants are alkaloids, flavanoids, tannins and phenolic compounds
(Edeoga et al, 2005).
Scoparia dulcis Linn. is reported as a traditional antidiabetic plant (Latha M et
al, 2004). Scoparia acid D isolated from Scoparia dulcis Linn. is reported for
antidiabetic activity (Latha M et al, 2009). Scoparia dulcis Linn. reported to contain
antidiabetic activity (Pari L et al, 2004). Scoparia dulcis Linn. ethanolic extract
reported to contain antidiabetic and antioxidant activity when studied on alloxan
induced albino mice (Abu Hasanat Md. Zulfiker et al, 2010).
The usage of medicinal plants as traditional medicines is well known in rural
areas of many developing countries. Traditional healers claim that their medicine is
cheaper and more effective than the modern medicine. In developing countries,
Chapter-6 Herbal formulations
102
people of small isolated villages and native communities use folk medicine for the
treatment of common infections (Sandhu et al, 2005; Gupta et al, 2005).
Even though pharmaceutical industries have produced a number of new
antibiotics in the last three decades, resistance to these drugs by microorganisms has
increased. In general, bacteria have the genetic ability to transmit and acquire
resistance to drugs, which are utilized as therapeutic agents. Such a fact is cause for
concern, because of the number of patients in hospitals who have suppressed
immunity, due to new bacterial strains which are multi-resistant. Consequently, new
infections can occur in hospitals resulting in high mortality (Cohen, 1992).
Liquid oral formulations: The stability of the active ingredients in the final product
is of prime concern to the formulator. In general drug substance is less stable in
aqueous media than in the solid dosage form and it is important, therefore, to properly
stabilize and preserve, in particular those solutions, suspensions, emulsions that
contain water.
Liquid preparations for oral use are usually solutions, syrups, emulsions or
suspensions containing one or more active substances in a suitable vehicle. Some
preparations for oral use are prepared by dilution of concentrated liquid preparations,
or from powders or granules for the preparation of oral solutions or suspensions, for
oral drops or for syrups, using a suitable vehicle. The vehicle for any preparations for
oral use is chosen based on nature of the active substances and to provide organoleptic
characteristics appropriate to the intended use of the preparation.
Excipients may be defined as the constituents of a pharmaceutical dosage form
that are not the active substance; according to their function they are the stabilizers to
retain the physical and chemical property of dosage form (European Medicines
Agency 2006).
Liquid preparations for oral use may contain suitable excipients such as
antimicrobial preservatives, antioxidants, dispersing agent, suspending agent,
thickening agent, emulsifying agent, buffering agent, wetting agent, solublizing agent,
stabilizing agent, flavoring agent, sweetening agent and coloring matter those are
authorized by the competent authority. Several categories of preparations may be
Chapter-6 Herbal formulations
103
distinguished as oral solutions, emulsions, suspensions, powders and granules for oral
solutions and suspensions, oral drops, powders for oral drops, syrups, powders and
granules for syrups (British pharmacopoeia, 2007).
Syrups: Syrups are aqueous preparations characterized by a sweet taste and a viscous
consistency. They may contain sucrose at a concentration of at least 45 per cent m/m.
The sweet taste can also be obtained by using other polyols or sweetening agents.
Syrups usually contain aromatic or other flavoring agents (British pharmacopoeia,
2007).
In addition to sucrose, certain other polyols such as glycerin or sorbitol may be
added to retard crystallization of sucrose or to increase the solubility of added
ingredients. Alcohol often is included as a preservative and also as a solvent for
flavors; further resistance to microbial attack can be enhanced by incorporating
antimicrobial agents. When the aqueous preparations contain some added medicinal
substance, the syrup is called as medicated syrup. Syrups contain remarkable masking
properties for bitter or saline drugs. Gycerrhiza syrup has been recommended for
disguising the salty taste of bromides, iodides, and chlorides. This has been attributed
to its colloidal character and its double sweetness of the glycyrrhizin. This syrup is
also of value in masking bitterness in preparations containing B complex vitamins
(Alfonso & Gennaro, 1997).
Acacia syrup because of its colloidal character is of great value as a particular
vehicle for masking disagreeable taste of many medicaments. Raspberry syrup BP
1088 is one of the most efficient flavoring agents, and is especially useful in masking
the taste of bitter drugs. A series of papers by Schumacher deals with improving the
palatability of bulk-compounded products using flavoring and sweetening agents
(Schumacher, 1967).
It is important that the concentration of sucrose approach but may not reach
the saturation point. In dilute solutions sucrose provides an excellent nutrient for
molds, yeasts and other microorganisms. In contractions of 65% by weight or more,
the solution will retard the growth of such micro organisms. However, a saturated
solution may lead to crystallization of a part of the sucrose under conditions of
changing temperature (Alfonso & Gennaro, 1997).
Chapter-6 Herbal formulations
104
Sorbitol solution is the sweetening agent and contains 70% w/w of the total
solids, consisting mainly of d-sorbitol. It has about half the sweetening power of
syrup (Alfonso & Gennaro, 1997).
The effect of trace metals can be minimized by using citric acid or EDTA
which are sequestering agents. Antioxidants however, may retard or delay oxidation
by reacting with the free radicals formed in the product. Examples of antioxidants are
the propyl, octyl and dodecyl esters of Gallic acid, butylated hydroxyanisole (BHA)
and the tocopherols or vitamin E (Alfonso & Gennaro, 1997).
Preparation: Syrups are prepared in various ways; the choice of the proper method
depends on the physical and chemical characteristics of the substances entering into
the preparation.
Solution with heating: This is usual method for making syrups when the valuable
constituent is neither volatile nor injured by heat, and when it is desirable to make the
syrup rapidly. The sucrose usually is added to the purified water or aqueous solution
and heated until solution is affected, then it is strained and sufficient purified water
added to make the desired weight or volume (Alfonso & Gennaro, 1997).
Agitation without heating: This process is used in those cases where heat would
cause the loss of valuable, volatile constituents. Glass-lined tanks with mechanical
agitators, especially adapted to dissolving of sucrose, are used for making syrups in
large quantities (Alfonso & Gennaro, 1997).
In the manufacturing, packaging, storage and distribution of liquid
preparations for oral use, suitable measures are taken to ensure their microbial quality;
recommendations on this aspect are provided in the text on Microbiological quality of
pharmaceutical preparations (Alfonso & Gennaro, 1997).
Herbal drugs and herbal drug preparations present in the dosage form are not subject
to uniformity of dosage units (British pharmacopoeia, 2007).
Preservation: Syrups should be made in quantities that can be consumed within few
months, except in those cases where special facilities can be employed for their
preservation; a low temperature is the best method. Concentration with out
supersaturating is also a condition favorable to preservation. The USP states that the
Chapter-6 Herbal formulations
105
syrups may contain preservatives, glycerin, methyl paraben, benzoic acid and sodium
benzoate which may prevent bacterial and mold growth. Combination of alkyl esters
of p-hydroxy benzoic acid are effective inhibitors of yeast that have been implicated
in the contamination of commercial syrups (United States Pharmacopeia, 2007).
Syrups can be preserved by a) storage at low temperature, b) adding preservative such
as glycerin, benzoic acid, sodium benzoate, methyl paraben or alcohol in the
formulation. High sucrose concentrations will usually protect an oral liquid dosage
form from growth of most microorganisms. A problem arises, however, when
pharmacist must add other ingredients to syrups that can result in a decrease in the
sucrose concentration. This may cause loss of the preservative effectiveness of the
sucrose. This can be overcome however by calculating the quantity of a preservative
(such as alcohol) to be added to the formula to maintain the preservative effectiveness
of the final product (Loyd V Allen, 2004).
General preservatives used:
Alcohols: Ethanol is useful as a preservative when it is used as a solvent, however, it
does need a relatively high concentration, some what greater than 10%, to be
effective. Too high a concentration may result incompatibilities in suspension and
emulsion systems. Propylene glycol is also used as a solvent in oral solutions and
topical preparations, and it can function as a preservative in the range of 15% to 30%.
It is not volatile like ethanol and is used frequently not only in solution but also in
suspensions and emulsions.
Acids: Benzoic acid has low solubility in water, about 0.34% at 25ºC, the
concentration range used for inhibitory action varies from 0.01 to 0.5%. Only the
ionized form is effective, and therefore its use is restricted to preparations with a pH
below 4.5. Sorbic acids also have a low solubility in water, 0.3% at 30ºC. Suitable
concentrations for preservative action are in the range of 0.05 to 2%. Its preservative
action is due to the non ionized form; consequently, it is only effective in acid media,
because of double bonds in its structure, it is subjected to oxidation.
Esters: Parabens are the esters of Para hydroxyl benzoic acid and include the methyl,
ethyl, propyl and butyl derivatives. The solubility in the water decreases as the
molecular weight increases from 0.25% for the methyl esters to 0.02% for the butyl
Chapter-6 Herbal formulations
106
ester. These compounds are effective and stable over a pH range of 4-8; they are
applied at a concentration up to about 0.2%. Frequently two esters are used in
combinations.
Other excipients: Hydroxyethylcellulose (HEC) is nonionic cellulose ether that
readily dissolves in water to form viscous solutions. Chemically it is high purity
cellulose that has been etherified to form hydroxyethyl groups to give the desired
solubility in water. In solution the product acts as a thickener and rheology control
agent, as a protective colloid, binder, stabilizer and suspending agent (British
pharmacopoeia 2007).
Polymers such as hydroxypropylcellulose, hydroxyethylcellulose, sodium
carboxymethyl cellulose, hydroxypropyl methylcellulose, polyvinylpyrrolidone,
pectin, carrageenan and guar gum have wide application in the pharmaceutical
industry (Jian-Hwa Guo, 1998).
Sorbitol may have a mild laxative effect, Calorific value 2.6 kcal/g (European
commission; 2003), Sorbitol is a sugar which can be used in diabetic patients because
of its special properties. The slow absorption of sorbitol, and its conversion to fructose
and glycogen prior to its availability as glucose, has led to consideration of this
substance for use as an adjunct in diabetic diets (Charles R Shuman, 1956; Brian A.
Burt, 2006). Several reporters reported sorbitol also show side effects like diarrhoeia
(Chusid, 1981; Jain, 1985; Badiga, 1990).
Glycerol is used as humactant, it is a chemical compound that is also known as
glycerin. It is a neutral, sweet tasting, colorless, thick liquid. It can easily dissolve in
water or alcohol but not in oils. But, there are many substances which are more
soluble in glycerin than in water or alcohol. So, it is said that glycerin is a good
solvent. A very interesting property of glycerin is that it is highly hygroscopic, which
means that it absorbs water even from the air (Jain, 2006).
Saccharin sodium is used as sweetening agent (Cooney, 1977).
Comprehensive reviews had been done by WHO in 1993 on biological risk
assessment of sodium saccharin. The usage of saccharin sodium had no effect on
Chapter-6 Herbal formulations
107
bladder cancer (Chappel, 1992; Elcock and Morgan, 1993). Samuel M. Cohen et al,
1991 reported sodium saccharin having tumor promoting activity.
6.4 MATERIALS AND METHODS
Scoparia dulcis Linn. plant powder extract, Achyranthes aspera Linn. plant powder
extract, hydroxy ethyl cellulose (Natrosol 250 HX PHARM), sorbitol 70% solution,
Glycerol, Saccharin sodium, Sodium benzoate, Sorbic acid, Propylene glycol, Apricot
168153 flavour and peppermint troomint flavor.
Scoparia dulcis Linn. plant powder extract herbal formulation:
Sorbitol based liquid oral syrups: Finished product specification includes
description, identification, weight per ml, pH of the oral liquid dosage form, assay of
some active ingredients and microbiological purity. Specification for microbiological
purity is included and the specification covers total bacterial, fungi & yeast count and
some pathogens.
Table 6.1. Specifications for Scoparia dulcis Linn. syrup.
Code Test Specifications
T-01 Description Clear Green colored syrup
T-02 Identification
( By HPLC)
In the Assay test, the retention time of the peaks
corresponding to Scopadulcic acid B, Alpha amyrin in
the Sample solution matches with the retention time of the peaks corresponding to Scopadulcic acid B, Alpha
amyrin obtained with Standard solution
T-03 Weight per ml 1.10 to 1.20 gram per ml
T-04 pH 2.50 to 3.50
T-05 Viscosity Between 100 and 210 cps
T-06
Assay 90.0 % to 110.0 % of label claim
Assay of Scopadulcic Acid B,
Sod. Benzoate & Sorbic acid
90.0 % to 110.0 % of label claim
T-07 Heavy Metals (By AAS) Arsenic : Not more than 2 ppm
Mercury : Not more than 1 ppm
Lead : Not more than 10 ppm
Cadmium : Not more than 0.3ppm
T-08 Microbiological quality TAMC : NMT 105 (Maximum acceptable count: 500
000) CFU/g or CFU/mL
TYMC : NMT 104 (Maximum acceptable count: 50 000)
CFU/g or CFU/mL
Bile tolerant gram-negative bacteria : 104 CFU/g or
CFU/mL
Escherichia coli : Absence (1g or mL)
Salmonella : Absence (25g or 25mL)
Chapter-6 Herbal formulations
108
Pharmaceutical development:
Preparation of ethanol extract by sox let apparatus: 10 gram of Scoparia dulcis Linn.
plant powder was accurately weighed and packed in vertical tube, which is connected
to the round bottom flask (500ml volume) containing 100 ml of 90% ethanol. The
round bottom flask was connected on the heating mantle having auto control for the
temperature selected. The temperature was fixed as 90ºC and heated for 1 hour for
proper extraction of active constituents. After completion of one hour the apparatus
allowed to cool and the extract was collected aside. Again 50ml of fresh solvent
added to the round bottom flask and subjected for same process. The two extractives
were mixed and subjected for Rota evaporation until the residue remained 50ml.
The extract obtained was subjected to analysis of the Scopadulcic acid B and Alpha
amyrin content.
Development objective: The development objective was to formulate a liquid oral
preparation of plant extract with acceptable taste and flavor.
Developmental pharmaceutics:
Conventional vehicle for medicated syrup is sugar syrup. However it is associated with
crystallization and cap locking problem. It can be replaced with other vehicles either
alone or in combination to give properties similar to sugar syrup. Such preparations
can be called as sugar free syrups and can be administered by diabetic patients
especially.
In developing antidiabetic syrup this approached was used, combination of
35% w/v sorbitol solution (70% Non crystallizing) with 15% w/v glycerol produced
excellent vehicle. It also imparted sweetness to the syrup. However this vehicle did not
have suitable viscosity. Hence, in order to impart a viscosity to the formulation,
hydroxyethylcellulose which is commonly used as a viscosity-builder was used.
Natrosol 250 HX PHARM is the commercially available grade of
hydroxyethylcellulose. It readily dissolves in cold or hot water and gives clear, smooth
suspension. When used in 0.2% w/v concentration it gave a clear solution without
formation of lumps.
Some of the components are sparingly soluble in water. However it possesses
adequate solubility in propylene glycol, warm water and in acidic environment. Hence
Chapter-6 Herbal formulations
109
the pH of formulation was aimed at 2.5-3.5 where it possesses optimum stability
(Loyd V Allen, 2004).
Liquid oral dosage forms such as syrups and suspensions are susceptible to
microbiological growth during its shelf life. Addition of antimicrobial preservative is
essential for such dosage forms. But this liquid oral dosage form consisting of plant
extract will assign antimicrobial activity to the formulation and hence preservatives are
not necessary. Ethanol present in the formulation will also apart the same and prevents
the growth of microbes during its stability (Guidance on Development Pharmaceutics
1996).
The ethanol may tend to evaporate and can leads to the growth of microbes; hence
sorbic acid and sodium benzoate are used as preservatives in the present study.
For medicated syrup organoleptic properties such as taste and flavor are important to
make it palatable to patients. Plant extract possesses bitter taste which remains
persistent after administration. To mask this bitter taste saccharin sodium was used as
a sweetener since it is approximately 300 times sweeter than sugar and possesses
excellent solubility in water. The concentration of saccharin sodium was optimized to
0.03 %w/v. Apricot flavor was used as flavoring agent. When only apricot flavor was
used in the concentration of 0.264%v/v it did not mask the bitter taste. Hence, a
combination of Apricot flavor and menthol was tried. However, the bitter taste still
persisted. On replacing menthol with peppermint troomint flavor in the concentration
of 0.1325%v/v along with Apricot, the bitter taste was successfully masked and a
pleasantly flavored syrup formulation was obtained.
Chapter-6 Herbal formulations
110
Table 6.2. Formulae for Scoparia dulcis Linn. syrup
SN Ingredient Per 5 ml Function Manufacturer
1 Scoparia dulcis Linn. Liquid
extract 500 mg
Active
ingredient ----
2 Hydroxy ethyl cellulose
(Natrosol 250 HX PHARM) 10.0 mg
Viscosity
building agent Hercules
3 Sorbitol 70% solution 1750.0 mg Vehicle Noble Drugs
Ltd.
4 Glycerol 750.0 mg Vehicle Godrej
Industries
5 Saccharin sodium 1.5 mg Sweetener Tiansin
Changhe
6 Propylene glycol 150.0 mg Solvent for
preservative
Regent
Chemicals
7 Apricot 168153 flavour 0.0132 ml flavor Symrise
8 Peppermint troomint flavour 0.0066 ml flavor Quest
international
9 Sodium Benzoate 1.125 mg Preservative Across
10 Sorbic acid 1.25 mg Preservative S.D fine
11 Purified water q.s. 5 ml Solvent In house
Table 6.3. Manufacturing Formulae for Scoparia dulcis Linn. Syrup (Batch 1
liter)
Ingredient Typical Quantities
Plant extract 100ml
Hydroxy ethyl cellulose ( Natrosol 250 HX PHARM) 2g
Sorbitol 70% solution 350g
Glycerol 150g
Saccharin sodium 0.3g
Propylene glycol 30g
Apricot 168153 Flavor 0.25 ml
Peppermint Troomint Flavor 0.15 ml
Sodium Benzoate 0.225g
Sorbic acid 0.25g
Purified water q.s. 1000 ml
Chapter-6 Herbal formulations
111
Manufacturing process:
Stage 1) Preparation of active solution:
a) Dispersed 2.0 g Hydroxyethyl cellulose in 300 ml purified water and allowed
to swell at room temperature for 30 minutes. Added Sodium benzoate.
b) Heated the solution to 800C under stirring for 1 hour.
c) Allowed the solution to cool to room temperature and added 100ml of plant
extract and Sorbic acid under stirring.
Step 2) Preparation of flavor solution:
Mixture of 0.25 ml Apricot 168153 Flavor and 5 g propylene glycol, 0.15 ml
Peppermint troomint flavor in additional 5 g propylene glycol were prepared
separately.
Stage 3) Preparation of vehicle solution:
a) To a mixing vessel added 350 grams of Sorbitol 70% solution.
b) Further added 150 grams glycerol to mixing vessel under stirring, rinsed the
container with 50 ml purified water and added it to mixing vessel under
stirring.
c) Dissolved 0.3 grams saccharin sodium in 10 ml purified water and added to
mixing vessel, rinsed the containers with 10 ml purified water and added to
mixing vessel under stirring.
Stage 4) Preparation of final syrup:
a) Added herbal extract to mixing vessel under stirring. Rinsed the containers
with 10 ml purified water and added the rinsing to the mixing vessel under
stirring. Give such 5 consecutive rinsing.
b) Cooled the solution to 35-400
C.
c) Added solution of Apricot 168153 flavor to mixing vessel under stirring.
Rinsed the container with 20 ml purified water and added to mixing vessel
under stirring.
d) Added solution of peppermint troomint flavour to mixing vessel under stirring,
rinsed each container separately with 10 ml purified water each and added to
the mixing vessel.
Chapter-6 Herbal formulations
112
Figure: 6.1. Process flow chart for Scoparia dulcis Linn. herbal formulation
Disperse
Hydroxyethylcellulose
(Natrosol 250HX PHARM)
in water and allow swelling
for 30 minutes. Add sod.
Benzoate, Heat to 800C for
60 minutes under stirring.
Cool to room temperature
and add Plant extract
,Sorbic acid, maintain at 40-
45 0 C for 30 minutes
Dissolve Apricot 168153
flavour in part of propylene
glycol under stirring.
Mix Peppermint troomint
flavour separately in part %
of propylene glycol.
1
Mixing Vessel
Sorbitol 70%
solution and
Glycerol under
stirring
Appearance
(Uniform
solution free
Dilute to volume with purified
water and mix for 10 minutes
Appearance,
pH, Specific
gravity, assay
Filtration
Machine /Manual Filling
Bottle filling
Labeling and Packaging
Fill
Volume
2
3
4
5
6
7
8
9
10
200 mesh.
Dissolve Saccharin sodium
in purified water.
Chapter-6 Herbal formulations
113
e) Made up the volume of syrup with purified water and continued stirring for 15
minutes to get syrup free from lumps, adjusted pH with ortho-phosphoric acid
to get required pH.
Stage 5) Filtration and packing of final syrup:
a) Filtered the syrup through 200 mesh.
b) Filled the syrup into bottles.
Proposed dosage for administration:
Table 6.4 Dosage of Administration for Scoparia dulcis Linn. herbal formulation
Dosage
Adult 30ml / day in 3 divided doses
Children 15ml / day in 3 divided doses
Table 6.5. Dosage of administration for formulation of Scoparia.
Number of teaspoonfuls (5 ml) of suspension
Adult 2 Teaspoonfuls of liquid syrup 3 times daily
Children 1/2-1 Teaspoonful of liquid 1-3 times daily
Proposed usage: Anti diabetic and anti viral syrup
Adverse effects (Undesirable effects): Not studied
Contra-indications: Not studied
Interactions: Not studied
Pregnancy and lactation: Not recommended
Side effects: Not studied
Pharmacokinetics: Currently not available
Chapter-6 Herbal formulations
114
Table 6.6. Pharmaceutical label for Scoparia dulcis Linn. sugar free syrup
Scoparia dulcis Linn. sugar free syrup
Label claim:
Each 10ml. contains
Scopadulcic acid B ……………………5.8 mg
Scoparia dulcis Linn. plant ethanol extract….994 mg
Sorbitol (70%).…………………………3.5 ml
Sodium Benzoate……………………….2.25 mg
Sorbic acid……………………………….2.5 mg
Dosage: 30ml per day in 3 divided doses.
Batch No: Scop003 Mfg. Lic. No: xxxxxx
Manufacturing date:Dec.2006 Expiry date: xxxxxx
Mfg.by: xxxxx
Achyranthes aspera Linn. plant powder extracts herbal formulation:
Table 6.7. Specification for Achyranthes aspera Linn. Syrup.
Code Test Specifications
T-01 Description Clear green colored syrup
T-02 Identification
(By HPLC)
In the Assay test, the retention time of the peaks
corresponding to Oleanolic acid solution matches with
the retention time of the peaks corresponding to
Oleanolic acid with Standard solution.
T-03 Weight per ml 1.10 to 1.20 gram per ml
T-04 pH 3.50 to 4.50
T-05 Viscosity Between 100 and 210 cps
T-06
Assay 95.0 % to 105.0 % of label claim
Assay of
Oleanolic acid
Sod. Benzoate & Sorbic
acid
95.0 % to 105.0 % of label claim
T-07 Heavy Metals
(By AAS)
Arsenic : Not more than 2 ppm
Mercury : Not more than 1 ppm
Lead : Not more than 10 ppm
Cadmium : Not more than 0.3ppm
T-08 Microbiological quality TAMC : NMT 105 (Maximum acceptable count: 500
000) CFU/g or CFU/mL
TYMC : NMT 104 (Maximum acceptable count: 50 000)
CFU/g or CFU/mL
Bile tolerant gram-negative bacteria : 104 CFU/g or
CFU/mL
Escherichia coli : Absence (1g or mL)
Salmonella : Absence (25g or 25mL)
Chapter-6 Herbal formulations
115
Pharmaceutical development: Preparation of ethanol extract by Sox let apparatus:
10 gram of Achyranthes aspera Linn. plant powder was accurately weighed and
packed in vertical tube, which is connected to the round bottom flask (500ml volume)
containing 100 ml of 90% ethanol. The round bottom flask was connected on the
heating mantle having auto control for the temperature selection. The temperature was
fixed 90ºC and allowed for 1 hour for proper extraction of active constituents. After
completion of one hour, allowed the apparatus to cool and the extract was collected
aside. Again added 50ml of fresh solvent to the round bottom flask and subjected for
same process. The two extractives were mixed and subjected for rota evaporation
until the residue remained 50ml.
The extract obtained was subjected to analysis for the content oleanolic acid.
Formulation development:
Development objective: The development objective was to formulate a liquid oral
preparation of plant extract with acceptable taste and flavor.
Developmental pharmaceutics (IP, BP, USP): Sugar free syrups can be administered
by diabetic patients especially, hence the advantage applied in developing antidiabetic
syrup. Combination of 35% w/v sorbitol solution 70% (non crystallizing) with 15%
w/v glycerol produced excellent vehicle. It also imparted sweetness to the syrup.
However this vehicle did not have suitable viscosity. Hence, in order to impart a
viscosity to the formulation, Hydroxyethylcellulose which is commonly used as a
viscosity-builder was used. Natrosol 250 HX PHARM is the commercially available
grade of Hydroxyethylcellulose. It readily dissolves in cold or hot water and gives
clear, smooth suspension. When used in 0.2% w/v concentration it gave a clear
solution without formation of lumps.
Some of the components are sparingly soluble in water. However it possesses
adequate solubility in propylene glycol, warm water and in acidic environment.
Hence the pH of formulation was aimed at 2.5-3.5 where it possesses optimum
stability.
The ethanol may tend to evaporate and lead to the growth of microbes, hence
sorbic acid and sodium benzoate are being used as preservatives in the present study.
For medicated syrups, organoleptic properties such as taste and flavor are important
to make it palatable to patients. Plant extracts possesses bitter taste which remains
Chapter-6 Herbal formulations
116
persistent even after administration. To mask this bitter taste saccharin sodium was
used as a sweetener since it is approximately 300 times sweeter than sugar and
possesses excellent solubility in water. The concentration of saccharin sodium was
optimized to 0.03 %w/v. Apricot flavor was used as flavoring agent. When only
apricot flavor was used in the concentration of 0.264% v/v it did not mask the bitter
taste successfully. Hence, a combination of Apricot flavor and menthol was tried.
However, the bitter taste still persisted. On replacing menthol with Peppermint
Troomint flavor in the concentration of 0.1325% v/v along with Apricot, the bitter
taste was successfully masked and a pleasantly flavored syrup formulation was
obtained.
Table 6.8. Formulae for Achyranthes aspera Linn. Syrup
SN. Ingredient Per 5 ml Function Manufacturer
1 Achyranthes aspera
Linn. Liquid extract 500mg Active ingredient -----
2
Hydroxy ethyl
cellulose
(Natrosol 250 HX
PHARM)
10.0 mg Viscosity
building agent Hercules
3 Sorbitol 70% solution 1750.0 mg Vehicle Noble Drugs
Ltd.
4 Glycerol 750.0 mg Vehicle Godrej
Industries
5 Saccharin sodium 1.5 mg Sweetener Tiansin
Changhe
6 Propylene glycol 150.0 mg Solvent for
preservative
Regent
Chemicals
7 Apricot 168153
flavour 0.0132 ml flavor Symrise
8 Peppermint troomint
flavor 0.0066 ml flavor
Quest
international
9 Sodium Benzoate 1.125mg Preservative Across
10 Sorbic acid 1.25mg Preservative S.D fine
11 Purified water q.s. 5 ml Solvent In house
Chapter-6 Herbal formulations
117
Table 6.9. Manufacturing Formulae for Achyranthes aspera Linn. Syrup (Batch
Size: 1 Liter)
Ingredient Typical Quantities
Plant extract 100ml
Hydroxy ethyl cellulose ( Natrosol
250 HX PHARM) 2g
Sorbitol 70% solution 350g
Glycerol 150g
Saccharin sodium 0.3g
Propylene glycol 30g
Apricot 168153 Flavor 0.25 ml
Peppermint Troomint Flavor 0.15 ml
Sodium Benzoate 0.225g
Sorbic acid 0.25g
Purified water q.s. 1000 ml
Manufacturing process: The process was same as mentioned above for Scoparia
dulcis Linn. formulation that herbal extract added was Achyranthes aspera Linn..
Table 6.10. Pharmaceutical label for Achyranthes aspera Linn. sugar free syrup
Achyranthes aspera Linn. sugar free syrup
Label claim:
Each 10ml contains
Oleanolic acid ……………………………….2.96 mg
Achyranthes aspera Linn. plant ethanol extract……995 mg
Sorbitol (70%).………………………………3.5 ml
Sodium Benzoate…………………………....2.25 mg
Sorbic acid………………………………..… 2.5 mg
Dosage: 30ml per day in 3 divided doses.
Batch No: Vsp004 Mfg. Lic. No: xxxxxx
Manufacturing date:Nov.2006 Expiry date: xxxxxx
Mfg.by: xxxxx
Chapter-6 Herbal formulations
118
Proposed dosage for administration:
Table 6.11. Dosage for formulation of Achyranthes aspera Linn. sugar free syrup
Dosage
Adult 30ml / day in 3 divided doses
Children 15ml / day in 3 divided doses
Table 6.12. Dosage Administration for formulation of Achyranthes aspera Linn. sugar
free syrup
Number of teaspoonfuls (5 ml) of suspension
Adult 2 Teaspoonfuls of liquid syrup 3 times daily
Children 1/2-1 Teaspoonful of liquid 1-3 times daily
Proposed usage: Hepatoprotective and antiviral
Adverse effects (Undesirable effects): Not studied
Contra-indications: Not studied
Interactions: Not studied
Pregnancy and lactation: Not recommended
Side effects: Not studied
Pharmacokinetics: Currently not available
Chapter-6 Herbal formulations
119
Formulation development for poly herbal formulation:
Table 6.13. Specifications & test procedure for strong herbal cough syrup.
TAMC: Total aerobic microbial count
TYMC: Total yeasts and moulds count
Components of drug product:
Extracts
Glycyrrhiza glabra (Yashtimadhu) (Aqueous Extract 5:1)
Adhatoda vasica (Vasaka) (Aqueous Extract 4:1)
Terminalia belerica (Vibhitaka) (Aqueous Extract 5:1)
The above all extracts are ready made, procured from chemiloids & Plantex
Zingiber officinale (Alcohol Extract)
S. No. Tests Specifications
1 Description Dark green colored syrupy liquid with
characteristic flavor of pineapple, taste sweet.
2 pH Between 4.0 and 6.0
3 Weight per ml Between 1.15 and 1.35 g / ml
4 Viscosity Between 100 and 210 cps
5 Assay of Gallic Acid NLT 65.0 mg per 100 ml
6 Assay of
Glycyrrhizic Acid NLT 120.0 mg per 100 ml
7 Assay of Vasicine NLT 2.0 mg per 100 ml
8
Assay of 6-Gingerol
Sod. Benzoate &
Sorbic acid
NLT 2.0 mg per 100 ml
9 Heavy Metals (By
AAS)
Arsenic : Not more than 2 ppm
Mercury : Not more than 1 ppm
Lead : Not more than 10 ppm
Cadmium : Not more than 0.3ppm
10
Microbiological
quality
TAMC : NMT 105 (Maximum acceptable count:
500 000) CFU/g or CFU/mL
TYMC : NMT 104 (Maximum acceptable count:
50 000) CFU/g or CFU/mL
Bile tolerant gram-negative bacteria : 104 CFU/g
or CFU/mL
Escherichia coli : Absence (1g or mL)
Salmonella : Absence (25g or 25mL)
Chapter-6 Herbal formulations
120
Table 6.14. Manufacturing Formulae for poly herbal formulation (Batch Size:1
Liter)
S. No Ingredients Manufacturer Qty. per 1000
ml
1. Glycyrrhiza glabra
(Yashtimadhu)
(Aqueous Extract 5:1)
Chemiloids 20.00g
2. Adhatoda vasica (Vasaka)
(Aqueous Extract 4:1)
Chemiloids 10.00g
3. Terminalia belerica
(Vibhitaka)
(Aqueous Extract 5:1)
Plantex 12.00g
4. Zingiber officinale.
(Alcohol Extract)
Self generated 10.00g
5. Sucrose USP/NF 750.0g
6. Glycerol Noble Drugs
Ltd.
50.00g
7. Propylene Glycol Regent
Chemicals
50.00g
8. Citric Acid Monohydrate S.D fine
chemicals
0.65g
9. Sodium Benzoate Across 0.45g
10. Sorbic Acid S.D fine
chemicals
0.50g
11. Color BQ Supra
Amerind Colors
& Chemicals
0.45g
12. Essence Pineapple Symrise 0.002 ml
13. Purified Water In house Q.s. to
1000 ml
Chapter-6 Herbal formulations
121
Figure: 6.2. Process flow chart for poly herbal formulation
1
Extract Glycyrrhiza glabra,
Adhatoda vasica, Terminalia
belerica, into purified water
Extract the Zingiber
officinale into Ethanol
Add
Colour &Flavour
Filter the
Macerated Herbals
Add glycerol,
citric acid
Dilute to volume with purified
water and mix (10 minutes)
Appearance,
pH, Specific
gravity, assay
Filtration
Machine /Manual Filling
Bottle filling
Labeling and Packaging
Fill Volume
2 3
4
5
6
7
8
9
10
200 mesh
Mixing Vessel
PREPARATION OF
SUGAR SYRUP
Chapter-6 Herbal formulations
122
Step-1: In beaker 100ml of purified water was boiled and added the specified quantity
of sodium benzoate and sorbic acid and mixed well to dissolve the contents. To the
above added the following duly weighed herbal extracts dry powder one by one under
continuous stirring of the contents in suitable capacity.
Glycyrrhiza glabra (Yashtimadhu) extract dry powder [5 : 1]
Adhatoda vasica (Vasaka) extract dry powder [4: 1]
Terminalia belerica (Vibhitaka) extract dry powder [5: 1]
After addition of dry herbal extracts powder the contents stirred for 1 hour and kept
the solution for maceration for a period of not less than 12 hours.
Weighed specified amount of propylene glycol BP in a cleaned suitable capacity of
vessel and heated to 70°C to 80°C.
Step-2: The Zingiber officinale extract was prepared by process of percolation:
100g of Zingiber officinale wet material was weighed and subjected for size reduction
of coarse irregular pieces each of 3 mm using knife and packed in percolator (500ml
volume) as a dense bed. Loaded 100 ml of 90% ethanol; remove the locked air, from
the packed bed by allowing the ethanol to drain out from down side, the herbal drug to
be extracted is always covered with the remaining extraction solvent.
Allowed the whole setup to stand for an appropriate time of 24 hours and collected
the extracted solvent. The residue remained was pressed out and the expressed liquid
combined with percolate. The two extractives were mixed and subjected for rota
evaporation until the residue remained 50ml.
Step-3: Filtration of macerated herbals: The herbals after completion of maceration
were filtered through 200mesh. The collected filtrate was kept for storage under clean
air tight container.
Step-4: Preparation of sucrose syrup: 300ml of purified water was taken in a suitable
capacity of glass vessel & heated up to 55°C temperature, added sodium benzoate and
sorbic acid in sequence, stirred to dissolve for 5 minutes.
Specified amount of Sucrose was added under constant stirring. Boiled the sugar
syrup up to 90°C and maintain the temperature at 90°C for 30 minutes and stirred well
Chapter-6 Herbal formulations
123
until Sucrose gets completely dissolved. Then Glycerol BP was added under
continuous stirring for 10 minutes.
Step-5: Mixing of extracts solution with sucrose syrup: Filtered the supernatant
solution of both the extracts. Stirred the obtained extracts solution for 5 minutes,
boiled the extract solution up to 70°C. Transferred the boiled extract solution to
sucrose syrup. Then added the zingier extract after reaching temperature of 50ºC and
mixed, purified water added up to required volume.
Addition of citric acid: Specified amount of citric acid weighed and dissolved in 10ml
of previously boiled purified water in a separate vessel, citric acid solution slowly
added with constant stirring to syrup.
Addition of color: Specified amount of color BQ supra weighed and dissolved in
20ml of previously boiled purified water in a separate vessel, and added to bulk syrup.
Step-6: Dilution to volume: Measured the volume of syrup obtained, made up to the
required volume with water.
Step-7: Filtration: Filtered the syrup through 200 mesh to remove any unwanted
particles or lumps after attainment to room temperature.
Table 6.15. Pharmaceutical label for poly herbal formulation.
Poly herbal formulation
Label claim:
Each 5ml. contains
Gallic acid …………………… 1.9 mg
Glycyrrhizic acid…………..…. 8.4 mg
Vasicine...……………………… 0.19 mg
6-Gingerol………………………0.35 mg
Sodium Benzoate……………… 2.25 mg
Sorbic acid…………………….. 2.5 mg
Dosage: 15ml per day in 3 divided doses.
Batch No: VSP007 Mfg. Lic. No: xxxxxx
Manufacturing date:Dec.2006 Expiry date: xxxxxx
Mfg.by: xxxxx
Chapter-6 Herbal formulations
124
Proposed dosage for administration:
Table 6.16. Dosage of administration for poly herbal formulation
Dosage
Adult 15ml / day in 3 divided doses
Children 7.5 -15 ml / day in 3 divided doses
Table 6.17. Dosage of administration for poly herbal formulation
Number of teaspoonfuls (5 ml) of suspension
Adult 1 Teaspoonful of liquid syrup 3 times daily
Children 1/2-1 Teaspoonful of liquid 1-3 times daily
Proposed use: Cough suppressant
Adverse effects (Undesirable effects): Not studied
Contra-indications: Not studied
Interactions: Not studied
Pregnancy and lactation: Not recommended
Side effects: Not studied
Pharmacokinetics: Currently not available
General reference test procedure section:
1. Description: Placed the Syrup in a clean and dry Petri dish, and observed against
specification.
2. Identification: In the HPLC Assay test, the retention time of the peaks
corresponding to the active in the Sample solution matches with the retention time of
the peaks corresponding to active obtained with standard solution.
3. Weight per ml: Weighed a previously dried 10 ml Pycnometer (W1). Filled the
Pycnometer with sample and adjust the temperature of the sample to 25°C, removed any
excess of sample & weighed (W2).
Determined the weight per ml using the formula given below.
Weight per ml = W2 – W1 / 10
Chapter-6 Herbal formulations
125
4. pH of the solution: Measured the pH with about 30 ml of the Syrup in 50 ml glass
beaker. Allowed the equilibration time of 2 minutes, and noted the pH after
stabilization.
5. Viscosity (using the rotating viscometer):
a. Instrument : Brook field viscometer RVT
b. Spindle No. : 2
c. Factor : 4
d. Speed : 100 RPM
Sample preparation: Transfer 200 ml of the sample to 250 ml beaker and bring the
temperature of the sample to 25°C. Maintain the temperature throughout the test.
Procedure:
i. Fixed the spindle No. 2 on the instrument.
ii. Turned the speed knob to 100.
iii. Switched on the instrument and adjusted the reading to 0.000.
iv. Put off the instrument and placed the sample, so that the spindle dips inside the
sample to the described height.
v. Started the instrument and allowed to running till a constant reading is achieved on
the scale after pressing the switch.
vi. Recorded the reading and calculated the viscosity using the factor.
6. Assay: Individual methodology for the contents mentioned in HPLC chapter.
7. Heavy metals:
Preparation of 2M Sodium Hydroxide Solution: Dissolved 80g of Sodium Hydroxide
in 1000ml water.
Blank Preparations: 0.1M Hydrochloric Acid: Dissolved 8.5ml of Hydrochloric Acid
in 1000ml water.
Standard solution:
A) For Arsenic (As) :
Dissolved about 1.320 g of Arsenic trioxide in 20ml of 2M Sodium hydroxide
(NaOH) and diluted to 1000ml with water (1000 ppm). Diluted 10ml of this solution
to 100ml with 0.1M HCl which contains 100ppm of As.
Above stock diluted 2ml → 100ml (2ppm)
Chapter-6 Herbal formulations
126
Above stock diluted 10ml → 100ml (10ppm)
Above stock diluted 20ml → 100ml (20ppm)
Above stock diluted 30ml → 100ml (30ppm)
B) For Mercury (Hg):
Dissolved about 1.354 gm of Mercuric chloride(HgCl2) in 50ml of 2M nitric acid and
diluted to 1000ml with water (1000 ppm). Diluted 10ml of this solution to 100ml
with 0.1M HCl which contains 100ppm of Hg.
Above stock diluted 1ml → 100ml (1ppm)
Above stock diluted 2ml → 100ml (2ppm)
Above stock diluted 5ml → 100ml (5ppm)
Above stock diluted 10ml → 100ml (10ppm)
C) For Lead (Pb):
Dissolved about 0.400 g Lead nitrate in water containing 2ml of nitric acid and made
upto 250ml using water (1000 ppm) . Diluted 10ml of this solution to 100ml with
0.1M HCl which contains 100ppm of Pb.
Above stock diluted 5ml → 100ml (5ppm)
Above stock diluted 10ml → 100ml (10ppm)
Above stock diluted 15ml → 100ml (15ppm Pb)
Above stock diluted 20ml → 100ml (20ppm Pb)
D) For Cadmium (Cd):
Dissolved about 0.100 g of Cadmium in minimum quantity of mixture of equal
volumes of hydrochloric acid and water and diluted to 100ml with 1% hydrochloric
acid (1000 ppm). Diluted 1ml of this solution to 100ml with 0.1M HCl which contains
10ppm of Cd.
Above stock diluted 3ml → 100ml (0.3ppm)
Above stock diluted 5ml → 100ml (0.5ppm)
Above stock diluted 10ml → 100ml (1.0ppm)
Above stock diluted 20ml → 100ml (2.0ppm)
Sample preparation:
Weighed about 10gm of sample in a suitable crucible. Slowly incinerated on
incinerator to near dryness. Added 2ml of Sulphuric acid to wet the sample. Ignited
carefully at a low temperature until thoroughly charred. Kept charred mass in muffle
Chapter-6 Herbal formulations
127
furnace at 700°C for 2hours. Cooled the crucible and dissolved the ash in 25ml of
0.1N Hydrochloric acid.
Taken the readings of standard on suitable AAS by using respective lamps and plotted
calibration graphs. 0.1N Hydrochloric acid used as blank. The reading of sample
noted and calculated content of respective heavy metals in ppm.
The above sample used for determination of the traces of arsenic, standard readings
noted on suitable AAS by using arsenic lamp at the wavelength 193.7 nm and plotted
calibration graph. Also taken reading of sample and found out arsenic content in ppm.
8.0 Microbiological purity (Ph. Eur. method 2.6.31):
Reagents and solution preparations:
Phosphate buffer stock solution pH 7.2: Dissolve 34g of monobasic potassium
phosphate in 1000ml of water, adjust pH with0.1M Hydrochloric acid or with 0.1M
Sodium hydroxide and sterilize in autoclave at 121°c for 20 minutes. Store at 2°C –
8°C.
Dilute Phosphate buffer solution pH 7.2: 1.25 mL of phosphate buffer stock solution
pH 7.2 and 20 g of polysorbate 80 mixed and dissolved in 500ml of purified water and
make up to 1000ml and sterilize in autoclave at 121°c for 20 minutes.
0.1M Hydrochloric acid: 8.5mL of hydrochloric acid make up to 1000ml with
purified water.
0.1M Sodium hydroxide: 4g of sodium hydroxide dissolved and make up to 1000ml
with purified water.
Culture media preparation:
Soybean–Casein Digest Medium broth pH 7.3±0.2: Dissolve pancreatic digest of
casein 17.0g, papaic digest of soyabean 3.0g, sodium chloride 5.0g, dipotassium
hydrogen phosphate 2.5g and glucose monohydrate 2.5g in 1000ml of purified water,
adjust pH with 0.1M Hydrochloric acid or with 0.1M Sodium hydroxide. Sterilize the
medium in autoclave at 121°c for 20 minutes.
Soybean–Casein Digest Medium agar pH 7.3±0.2: Dissolve pancreatic digest of
casein 15.0g, papaic digest of soyabean 5.0g, sodium chloride 5.0g, and agar 15g in
1000ml of purified water, adjust pH with0.1M Hydrochloric acid or with 0.1M
Sodium hydroxide. Sterilize the medium in autoclave at 121°c for 20 minutes.
Chapter-6 Herbal formulations
128
MacConkey broth pH 7.3±0.2: Dissolve pancreatic digest of gelatin 20.0 g, lactose
monohydrate 10.0g, dehydrated oxybile 5.0g and bromocresol purple 10mg in
1000mL of purified water, adjust pH with0.1M Hydrochloric acid or with 0.1M
Sodium hydroxide. Sterilize the medium in autoclave at 121°c for 20 minutes.
MacConkey agar pH 7.1±0.2: Dissolve pancreatic digest of gelatin 17.0 g, peptones
(Mean and casein) 3.0g, lactose monohydrate 10.0g, sodium chloride 5.0g, bile
salts1.5g, agar 13.5g, neutral red 30mg and crystal violet 1mg in 1000mL of purified
water, adjust pH with0.1M Hydrochloric acid or with 0.1M Sodium hydroxide.
Sterilize the medium in autoclave at 121°c for 20 minutes.
Rapport vassiliadis salmonella enrichment broth 5.2±0.2: Dissolve soya peptone
4.5g, sodium chloride 8.0g, potassium dihydrogen phosphate 0.6g, dipotassium
hydrogen phosphate 0.4g, magnesium chloride hexahydrate 29.0g and malachite green
36mg in 1000mL of purified water, adjust pH with0.1M Hydrochloric acid or with
0.1M Sodium hydroxide. Sterilize the medium in autoclave at 115°c for 15 minutes.
Xylulose – lysine-deoxycholate agar medium pH 7.4±0.2: Dissolve xylose 3.5g, L-
lysine 5.0g, lactose monohydrate 7.5g, sucrose 7.5g, sodium chloride 5.0g, yeast
extract 3.0g, phenol red 80mg, agar 13.5g, sodium deoxycholate 2.5g, sodium
thiosulphate 6.8g and ferric ammonium citrate 0.8g in 1000mL of purified water,
adjust pH with0.1M Hydrochloric acid or with 0.1M Sodium hydroxide. Heat to
boiling does not autoclave.
Enterobacteria enrichment broth-mossel pH 7.2±0.2: Dissolve pancreatic digest of
gelatin 10.0g, glucose monohydrate 5.0g, dehydrated ox bile 20.0g, potassium
dihydrogen phosphate 2.0g, disodium hydrogen phosphate dihydrate 8.0g, and brilliant
green 15mg in 1000ml of purified water, heat at 100°C for 30 minutes and cool
immediately.
Violet red bile glucose agar pH 7.4±0.2: Dissolve yeast extract 3.0g, pancreatic
digest of gelatin 7.0g, bile salts 1.5g, glucose monohydrate 10.0g, lactose monohydrate
10.0g, sodium chloride 5.0g and agar 15.0g in 1000ml of purified water, heat to
dissolve the contents and cool immediately.
Total aerobic microbial count (TAMC):
Diluted 10 ml of the sample to 100ml with sterile phosphate buffer and mixed well.
Chapter-6 Herbal formulations
129
Membrane filtration method: 1 ml of above sample was filtered through 0.45µ sterile
cellulose acetate filter paper under vacuum filtration unit, and then the filter paper
flushed with 4 x 100ml of phosphate buffer solution pH 7.2 containing polysorbate 80
as surface active agent and neutralizer for preservatives. Carefully transferred the filter
paper to surface of casein soyabean digest agar medium plate and incubated the plate
at 30°C – 35°C for 5 days. Then the membrane subjected for counting of colonies.
Total combined yeasts and mold count (TYMC):
Diluted 10 ml of the sample to 100ml with sterile phosphate buffer and mixed well.
Membrane filtration method: 1 ml of above sample was filtered through 0.45µ sterile
cellulose acetate filter paper under vacuum filtration unit, and then the filter paper
flushed with 4 x 100ml of phosphate buffer solution pH 7.2 containing polysorbate 80
as surface active agent and neutralizer for preservatives. Carefully transferred the filter
paper to surface of Saboraouds dextrose agar medium plate and incubated the plate at
20°C – 25°C for 7 days. Then the membrane subjected for counting of colonies.
Controls in the analysis:
Media control: Culture media without sample are kept at same test conditions during
the analysis at each step.
Diluent control1ml of sterile diluent without sample are kept at same test conditions.
Test for Escherichia coli:
Diluted 10 ml of the sample to 100ml with sterile phosphate buffer and mixed well.
Primary incubation: Take 10 ml of above sample Soybean–Casein Digest Medium
broth and mix kept incubator at 30°C – 35°C for 24 hours.
Secondary incubation: Transfer 1 ml of above mixed broth sample in to 100ml
MacConkey broth and kept incubator at 42°C – 44°C for 48 hours.
Plating: Streaked loop full of culture from enriched MacConkey broth on MacConkey
agar medium and incubated at 30°C – 35°C for 72 hours.
Test for Salmonella species:
Primary incubation: Take 10 ml of above sample Soybean–Casein Digest Medium
broth and mix kept incubator at 30°C – 35°C for 24 hours.
Chapter-6 Herbal formulations
130
Secondary incubation: Transfer 0.1 ml of above mixed broth sample in to 10ml of
Rapport vassiliadis salmonella enrichment broth and kept incubator at 30°C – 35°C for
24 hours.
Plating: Streaked loop full of culture from enriched Rapport vassiliadis salmonella
broth on xylulose – lysine-deoxycholate agar medium and incubated at 30°C – 35°C
for 48 hours.
Test for bile-tolerant Gram-negative bacteria
Diluted 10 ml of the sample to 100ml with sterile phosphate buffer and mixed well.
Presence or absence test:
Pre incubation: Incubate the above prepared sample at 20°C – 25°C for 2 hours for
resuscitation.
Primary incubation: Take 10 ml of above 10:100 diluted samples in to 90ml of
Enterobacteria enrichment broth mossel and incubated at 30°C – 35°C for 48 hours.
Plating: Streaked loop full of culture from Enterobacteria enrichment broth mossel on
the violet red bile glucose agar. Incubated the plates at 30°C – 35°C for 24 hours.
Escherichia coli culture kept run simultaneously as reference.
Colony characters were compared with the reference for presence or absence.
Table 6.18. Morphologic characteristics of Escherichia coli and Salmonella species on
selective media.
Selective medium Name of organism Characteristic colonial
morphology
MacConkey agar medium Escherichia coli brick red colonies with or
without surrounding zone
of bile pigments
Rapport vassiliadis
salmonella broth on
xylulose – lysine-
deoxycholate agar medium
Salmonella Red colonies with or
without black centre
Table 6.19. Morphologic characteristics of Enterobacteriace.
Selective medium Name of organism Characteristic colonial
morphology
Violet red bile glucose agar Enterobacteriace
group
Pink color colonies
with bile precipitation.
Chapter-6 Herbal formulations
131
6.5 RESULTS
Table 6.20. Results obtained for Scoparia dulcis Linn. herbal formulation
S. No Name of the test Results
1. Description Clear green colored syrup
2. Identification Complies
3. Weight per ml 1.122
4. pH 3.21
5. Viscosity 130 cps
6. Assay
Scopadulcic acid B
Alpha amyrin
Sod. Benzoate
Sorbic acid
102.22 %
99.02 %
100.62 %
101.84 %
7. Microbial quality
TAMC
TYMC
Escherichia coli
Salmonella
Bile-tolerant Gram-negative bacteria
65 cfu/ml
33 cfu/ml
Absent
Absent
Absent
8. Heavy Metals (By AAS) Arsenic : Not detected
Mercury : Not detected
Lead : Not detected
Cadmium : Not detected
Table 6.21. Results obtained for Achyranthes aspera Linn. herbal formulation
S. No Name of the test Results
1. Description Clear green colored syrup
2. Identification Complies
3. Weight per ml 1.135
4. pH 3.81
5. Viscosity 151 cps
6. Assay
Oleanolic acid
Sod. Benzoate
Sorbic acid
101.25 %
99.91 %
102.24 %
7. Microbial quality
TAMC
TYMC
Escherichia coli
Salmonella
Bile-tolerant Gram-negative bacteria
48 cfu/ml
36 cfu/ml
Absent
Absent
Absent
Chapter-6 Herbal formulations
132
8. Heavy Metals (By AAS) Arsenic : Not detected
Mercury : Not detected
Lead : Not detected
Cadmium : Not detected
Table 6.22. Results obtained for poly herbal formulation
6.6 DISCUSSION OF RESULTS IN LIGHT OF OTHERS WORK
No herbal formulations were reported by using Scoparia dulcis Linn. plant extract
with pharmaceutical excipients.
There are some reports on methanol and ethanol extracts of leaves having anti viral
activity (Hayashi et al, 1988) Analgesic and antipyretic activity (Ahmed et al, 2001),
Anti oxidant properties (Latha & Pari, 2003), anti diabetic activity (Pari &
Venkateswaran, 2002), scavenging activity (Babincova & Sourivong, 2001).
S. No Name of the test Results
1. Description Dark green colored syrupy liquid
with characteristic flavor of
pineapple, taste sweet.
2. pH 4.53
3. Weight per ml 1.26 g / ml
4. Viscosity 181 cps
5. Assay of Gallic acid 101.53 %
6. Assay of Glycyrrhizic acid 100.10 %
7. Assay of Vasicine
6-Gingerol
Sodium Benzoate
Sorbic acid
99.47 %
100.86 %
102.44 %
103.68
8. Microbial quality
TAMC
TYMC
Escherichia coli
Salmonella
Bile-tolerant Gram-negative bacteria
92 cfu/ml
80 cfu/ml
Absent
Absent
Absent
9. Heavy Metals (By AAS) Arsenic : Not detected
Mercury : Not detected
Lead : Not detected
Cadmium : Not detected
Chapter-6 Herbal formulations
133
No herbal formulations were reported by using Achyranthes aspera Linn. plant extract
with pharmaceutical excipients. There are some reports on anti microbial activity
(Naidu et al, 2006), extracts showed moderate larvicidal effects (Bagavan et al, 2008),
antipyretic properties (Gokhale et al, 2002) due to presence of oleanolic acid it also
showed to contain antitumor and hepatoprotective properties (Liu, 1995), as well as
exhibiting antiviral properties, but these all studies were conducted on plant parts but
not as formulation.
Multi herbal formulations were reported for specific activities (Balaraman, 2004), but
there is no herbal formulations reported to contain these Adhathoda vasaka,
Glycyrrhiza glabra, Gingiber officinalis and Terminalia belerica plant extracts.
6.7 CONCLUSION
The herbal formulations developed for Scoparia dulcis Linn. and Achyranthes aspera
Linn. are sugar free hydro alcoholic based sweetened syrups; they can be
administrated by diabetic patients.
The herbal formulations developed for cough suppressant syrup was simple sugar
based syrup. It is contra indicated for diabetic patients because of the presence of
Glycyrrhizic acid and sugar based syrup.
6.8 FUTURE PROSPECTS
Other components need to be identified, quantified and their stability to be monitored
during their shelf life.
Bio assay need to be performed for checking the efficacy of the drug product.
6.9 REFERENCES
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Alfonso & Gennaro, Remington: The Science and Practice of Pharmacy., 1997; 721:
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134
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