6.1 OBJECTIVE To develop simple herbal formulations using the selected plants extract.

Transcription

1 6.1 OBJECTIVE To develop simple herbal formulations using the selected plants extract. 6.2 INTRODUCTION 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, 101

2 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 102

3 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). 103

4 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 104

5 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 105

6 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 106

7 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 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) 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 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-06 Assay 90.0 % to % of label claim Assay of Scopadulcic Acid B, 90.0 % to % of label claim Sod. Benzoate & Sorbic acid 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 10 5 (Maximum acceptable count: ) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: ) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 107

8 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 108

9 the ph of formulation was aimed at 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 %v/v along with Apricot, the bitter taste was successfully masked and a pleasantly flavored syrup formulation was obtained. 109

10 Table 6.2. Formulae for Scoparia dulcis Linn. syrup SN Ingredient Per 5 ml Function Manufacturer 1 2 Scoparia dulcis Linn. Liquid extract Hydroxy ethyl cellulose (Natrosol 250 HX PHARM) 500 mg 10.0 mg Active ingredient Viscosity building agent 3 Sorbitol 70% solution mg Vehicle 4 Glycerol mg Vehicle 5 Saccharin sodium 1.5 mg Sweetener 6 Propylene glycol mg Solvent for preservative ---- Hercules Noble Drugs Ltd. Godrej Industries Tiansin Changhe Regent Chemicals 7 Apricot flavour ml flavor Symrise 8 Peppermint troomint flavour ml flavor Quest international 9 Sodium Benzoate 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 Flavor 0.25 ml Peppermint Troomint Flavor 0.15 ml Sodium Benzoate 0.225g Sorbic acid 0.25g Purified water q.s ml 110

11 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 80 0 C 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 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 C. c) Added solution of Apricot 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. 111

12 Figure: 6.1. Process flow chart for Scoparia dulcis Linn. herbal formulation Dissolve Saccharin sodium in purified water. Disperse Hydroxyethylcellulose (Natrosol 250HX PHARM) in water and allow swelling for 30 minutes. Add sod. Benzoate, Heat to 80 0 C for 60 minutes under stirring. Cool to room temperature and add Plant extract,sorbic acid, maintain at C for 30 minutes Dissolve Apricot flavour in part of propylene glycol under stirring. Mix Peppermint troomint flavour separately in part % of propylene glycol Mixing Vessel Sorbitol 70% solution and Glycerol under stirring 6 Dilute to volume with purified water and mix for 10 minutes 7 Filtration Appearance (Uniform solution free Appearance, ph, Specific gravity, assay 200 mesh. 8 Machine /Manual Filling 9 Bottle filling Fill Volume 10 Labeling and Packaging 112

13 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 Children 30ml / day in 3 divided doses 15ml / day in 3 divided doses Table 6.5. Dosage of administration for formulation of Scoparia. Number of teaspoonfuls (5 ml) of suspension Adult Children 2 Teaspoonfuls of liquid syrup 3 times daily 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 113

14 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 Manufacturing date:dec.2006 Mfg.by: xxxxx Mfg. Lic. No: xxxxxx Expiry date: xxxxxx 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) T-03 Weight per ml 1.10 to 1.20 gram per ml 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-04 ph 3.50 to 4.50 T-05 Viscosity Between 100 and 210 cps T-06 Assay 95.0 % to % of label claim Assay of Oleanolic acid Sod. Benzoate & Sorbic acid T-07 Heavy Metals (By AAS) 95.0 % to % of label claim 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 10 5 (Maximum acceptable count: ) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: ) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 114

15 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 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 115

16 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 % 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 2 Achyranthes aspera Linn. Liquid extract Hydroxy ethyl cellulose (Natrosol 250 HX PHARM) 500mg Active ingredient mg Viscosity building agent 3 Sorbitol 70% solution mg Vehicle 4 Glycerol mg Vehicle 5 Saccharin sodium 1.5 mg Sweetener 6 Propylene glycol mg 7 8 Apricot flavour Peppermint troomint flavor Solvent for preservative Hercules Noble Drugs Ltd. Godrej Industries Tiansin Changhe Regent Chemicals ml flavor Symrise 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 116

17 Table 6.9. Manufacturing Formulae for Achyranthes aspera Linn. Syrup (Batch Size: 1 Liter) Plant extract Ingredient Hydroxy ethyl cellulose ( Natrosol 250 HX PHARM) Sorbitol 70% solution Glycerol Typical Quantities 100ml 2g 350g 150g Saccharin sodium 0.3g Propylene glycol Apricot Flavor Peppermint Troomint Flavor 30g 0.25 ml 0.15 ml Sodium Benzoate 0.225g Sorbic acid 0.25g Purified water q.s ml Manufacturing process: The process was same as mentioned above for Scoparia dulcis Linn. formulation that herbal extract added was Achyranthes aspera Linn.. Table 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 mg Sorbic acid 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 117

18 Proposed dosage for administration: Table Dosage for formulation of Achyranthes aspera Linn. sugar free syrup Dosage Adult Children 30ml / day in 3 divided doses 15ml / day in 3 divided doses Table Dosage Administration for formulation of Achyranthes aspera Linn. sugar free syrup Number of teaspoonfuls (5 ml) of suspension Adult Children 2 Teaspoonfuls of liquid syrup 3 times daily 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 118

19 Formulation development for poly herbal formulation: Table Specifications & test procedure for strong herbal cough syrup. S. No. Tests Specifications 1 Description Dark green colored syrupy liquid with characteristic flavor of pineapple, taste sweet. 2 ph Between 4.0 and 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 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 Arsenic : Not more than 2 ppm 9 Heavy Metals (By Mercury : Not more than 1 ppm AAS) Lead : Not more than 10 ppm Cadmium : Not more than 0.3ppm 10 Microbiological quality TAMC : NMT 10 5 (Maximum acceptable count: ) CFU/g or CFU/mL TYMC : NMT 10 4 (Maximum acceptable count: ) CFU/g or CFU/mL Bile tolerant gram-negative bacteria : 10 4 CFU/g or CFU/mL Escherichia coli : Absence (1g or ml) Salmonella : Absence (25g or 25mL) 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) 119

20 Table Manufacturing Formulae for poly herbal formulation (Batch Size:1 Liter) S. No Ingredients Manufacturer Qty. per 1000 ml 1. Glycyrrhiza glabra Chemiloids 20.00g (Yashtimadhu) (Aqueous Extract 5:1) 2. Adhatoda vasica (Vasaka) Chemiloids 10.00g (Aqueous Extract 4:1) 3. Terminalia belerica Plantex 12.00g (Vibhitaka) (Aqueous Extract 5:1) 4. Zingiber officinale. Self generated 10.00g (Alcohol Extract) 5. Sucrose USP/NF 750.0g 6. Glycerol Noble Drugs 50.00g Ltd. 7. Propylene Glycol Regent 50.00g Chemicals 8. Citric Acid Monohydrate S.D fine 0.65g chemicals 9. Sodium Benzoate Across 0.45g 10. Sorbic Acid S.D fine 0.50g chemicals 11. Color BQ Supra Amerind Colors 0.45g & Chemicals 12. Essence Pineapple Symrise ml 13. Purified Water In house Q.s. to 1000 ml 120

21 Figure: 6.2. Process flow chart for poly herbal formulation 1 Extract Glycyrrhiza glabra, Adhatoda vasica, Terminalia belerica, into purified water 3 Filter the Macerated Herbals 2 Extract the Zingiber officinale into Ethanol Add Colour &Flavour 5 4 Mixing Vessel PREPARATION OF SUGAR SYRUP Add glycerol, citric acid 6 Dilute to volume with purified water and mix (10 minutes) 7 Filtration Appearance, ph, Specific gravity, assay 200 mesh 8 Machine /Manual Filling Fill Volume 9 Bottle filling 10 Labeling and Packaging 121

22 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 122

23 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 Pharmaceutical label for poly herbal formulation. Poly herbal formulation Label claim: Each 5ml. contains Gallic acid 1.9 mg Glycyrrhizic acid mg Vasicine mg 6-Gingerol 0.35 mg Sodium Benzoate 2.25 mg Sorbic acid mg Dosage: 15ml per day in 3 divided doses. Batch No: VSP007 Manufacturing date:dec.2006 Mfg.by: xxxxx Mfg. Lic. No: xxxxxx Expiry date: xxxxxx 123

24 Proposed dosage for administration: Table Dosage of administration for poly herbal formulation Dosage Adult Children 15ml / day in 3 divided doses ml / day in 3 divided doses Table Dosage of administration for poly herbal formulation Number of teaspoonfuls (5 ml) of suspension Adult Children 1 Teaspoonful of liquid syrup 3 times daily 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 (W 1 ). Filled the Pycnometer with sample and adjust the temperature of the sample to 25 C, removed any excess of sample & weighed (W 2 ). Determined the weight per ml using the formula given below. Weight per ml = W 2 W 1 /

25 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 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 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) 125

26 Above stock diluted 10ml 100ml (10ppm) Above stock diluted 20ml 100ml (20ppm) Above stock diluted 30ml 100ml (30ppm) B) For Mercury (Hg): Dissolved about gm of Mercuric chloride(hgcl 2 ) 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 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 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 126

27 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 nm and plotted calibration graph. Also taken reading of sample and found out arsenic content in ppm. 8.0 Microbiological purity (Ph. Eur. method ): 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. 127

28 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. 128

29 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. 129

30 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 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 lysinedeoxycholate agar medium Salmonella Red colonies with or without black centre Table Morphologic characteristics of Enterobacteriace. Selective medium Name of organism Characteristic colonial morphology Violet red bile glucose agar Enterobacteriace Pink color colonies group with bile precipitation. 130