Formulation and Evaluation of Anticough Herbal Preparation

Introduction

A cough is a sudden and often repetitively occurring defence reflex which helps to clear the large breathing passages from excess secretions, irritants, foreign particles and microbes. The cough reflex consists of three phases: an inhalation, a forced exhalation against a closed glottis, and a violent release of air from the lungs following opening of the glottis, usually accompanied by a distinctive sound.¹ Frequent or severe coughing usually indicates the presence of a disease. Many viruses and bacteria benefit by causing their host to cough, which helps to spread the disease to new hosts.

A cough can be dry or productive, depending on whether sputum is coughed up. Most of the time, coughing is acute and caused by a respiratory tract infection. Coughing can be triggered by food entering the windpipe rather than the oesophagus due to a failure of the epiglottis in patients who have difficulties in swallowing. Smoking and air pollution are common causes of coughing.² The three most common causes of chronic cough are asthma, gastroesophageal reflux disease and post-nasal drip. Other causes of chronic cough include chronic bronchitis, heart failure and medications such as ACE inhibitors.³ Besides recent upper airways infections, such as the common cold and flu, other common causes of coughs include: Allergies and asthma, Lung infections such as pneumonia or acute bronchitis, Gastroesophageal reflux disease (GERD) and ACE inhibitors (Medications used to control blood pressure) Hydration of respiratory tract by steam inhalation, demulcents are effective in reducing symptoms in majority of cases but, for uncontrolled cough, opiodergic central cough suppressants like codeine, pholcodeine, noscapine, dextro methorphan are used. These drugs have certain inherent side effects like sedation, constipation and also some addiction liability.

Furthermore, their use in severe cough conditions like asthma is contraindicated, as they are known to further compromise the respiratory function. Cough suppressant and antiasthmatic activities have been claimed for many medicinal plants in traditional systems of medicine. Plants like Ocimum sanctum⁴ , Ficus racemosa, ⁵ and Asperagus racemosus⁶ have screened for anti-tussive activity in animal models. An anti-cough, antitussive, and throat soothing synergistic herbal formulation comprising of an extract of piper cubeba, Glycyrrhiza and pharmaceutically acceptable additives as syrup preventing cracking of voice, dryness of mouth and toning of voice,vocal cord;the present invention also provides a methods of preparation of this formulation. Anti-tussives are substances that specifically inhibit or suppress the act of coughing.⁷ Till now no work has been reported regarding the anticough herbal syrup formulations using standardized extracts. Hence in our present work, we have prepared anticough herbal syrup formulations and were subjected to stability studies and analysed by TLC, HPTLC, and HPLC methods.

Materials and Methods

Pre-formulation Studies^7-18

The preformulation study like characterization of drug sample which includes physical characterization and evaluation of herbal syrup including determination of solubility, pH were performed for drug as shown (Table 1, 2, and 3).

Physical Characterization of Drug Sample

The drug was physically characterized according to following methods

Description

The sample of Glycyrrhiza glabra extract, Curcuma longa extract, Zingiber officinale and Piper longum extract was subjected to the following tests for its characterization.

Nature of the drug sample

The drug sample was observed visually and viewed under the compound microscope for the determination of its nature.

Color of the drug sample

The drug sample was viewed visually for the determination of its color using the black and white backgrounds.

Melting point of the drug sample

The melting point is one of the important methods for the identification of the drug sample.The melting point of the given drug sample was carried out by using Liquid paraffin as a bath liquid.

Solubility

The solubility of the drug sample was carried out in different solvents using aqueous and organic phase.

Analysis of drug samples

Analysis was carried out by thin-layer chromatography, high performance thin layer chromatography and high performance liquid chromatography method.

Manufacturing procedure

Step 1 Checking of weights: Once again check the weights of all the ingredients and herbal extracts. Observed the cleanliness of the homogenizer and stainless steel container, area and other precautions, etc.

Step 2 Preparation of sugar syrup: In a suitable two litter stainless steel container fitted with a homogenizer, 300 mL purified water taken and is heated to 80 ºC. Dissolve in it weighed quantity of methyl paraben and Propyl Paraben. Add sugar (commercial grade) to it under continues stirring till sugar gets completely dissolved. Filter the syrup through nylon cloth and cool to room temperature.

Step 3 Addition of sodium benzoate and disodium EDTA: Dissolve sodium benzoate and Disodium EDTA in 30 mL Purified water in a stainless steel container and transfer this solution in to step no.2 under continuous stirring while passing through muslin cloth

Step 4 Addition of sorbitol 70 % solution: Add sorbitol 70 % solution directly to step no.2 under continuous stirring.

Step 5 Addition of xanthum gum: Add Xantham Gum slowly in filtered sugar syrup of step 2 under continuous stirring. Precaution must be taken during addition of Xantham Gum to avoid lump formation. Allow the sugar syrup to cool to room temperature and soaking of Xantham Gum.

Step 6 Addition of bronopol: Dissolve Bronopol in 30 mL purified water in a stainlesssteel container and transfer this solution in to step no.2. Rinse the container with purified water.

Step 7 Addition of glycyrrhiza extract: Disperse 4g Glycyrrhiza extract in 10 g warm (50^o C) Propylene glycol in a stainless steel container under continuous stirring then transfer this solution in to step 2. Rinse the container with the solution of step 2.

Step 8 Addition of Curcuma longa extract: Disperse 2 g Curcuma longa extract in 10 g warm (50^o C) Propylene glycol in a stainless steel container under continuous stirring then transfer this solution in to step 2. Rinse the container with the solution of step 2

Step 9 Addition of Zingiber officinale extract: Disperse 2 g Zingiber officinale Extract in 10g warm (50^o C). Propylene glycol in a stainless steel container under continuous stirring then transfer this solution in to step no.2. Rinse the container with the solution of step 2.

Step 10 Addition of Piper longum extract: Disperse 1g Piper longum extract in 10 g warm (50^o C) Propylene glycol in a stainless steel container under continuous stirring thentransfer this solution in to step no. 2. Rinse the container with the solution of step 2.

Step 11 Addition of menthol: Dissolve menthol in 5 mL warm Propylene glycol (50^o C) in a stainless steel container and transfer this solution in to step 2. Rinse the above container with this solution. Transfer the rinsed solution into step 2.

Step 12 Addition of citric acid monohydrate: Dissolve Citric acid monohydrate in 0.1 liter purified water in a stainless steel container and transfer this solution into step 2.

Step 13 Addition of caramel colour: Dissolve caramel in 5 mL Propylene glycol in aglass beaker and transfer this solution in to step 2.

Step 14 Addition of Ponceau 4 R (WS) colour: Dissolve Ponceau 4R WS colour in 0.05liter purified water in a stainless steel container and transfer this solution in to step 2 and rinse the container with purified water.

Step 15 Addition of flavors: Add Fennel flavor (sweet oil) and Raspberry sweet flavour directly in to step 2 and adjust the pH between 4.0 and 6.0. Make up the volume up to 1000 mL by using purified water, stir the batch continuously for 60 min.

Step 16 Filtration: Filter the syrup using the 60# no. mesh and collect the filtered syrup in a clean 1000 mL SS container.

In process check of sample for analysis: Collect final filtered sample of syrup for analysis.

Bottle filling, sealing, and packing: After Analysis of prepared herbal syrup filled in amber colour glass bottle and sealed with ROPP cap by ROPP cap sealing machine. Self adhesive label is placed on each bottle. Temperature of the filling area should be not more than 25±2 °C and humidity should be not more than 45% RH. (Targeted fill Volume: 100 ±2 mL).

Storage: Store in dry place and protect from light with the temperature below25^o С.

Stability Studies

The purpose of stability testing is to provide evidence on quality of a drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and to establish a shelf life for the drug product and recommended storage conditions. Long term, accelerated, and intermediate storage conditions for drug products were given in below table. At the accelerated storage condition, a minimum of three time points, including the initial and final time points (e.g., 0, 3, and 6 months), from a 6-month study is recommended.

Description

Syrup of dark brown colour with characteristic odour.

Identification

Test is held by thin-layer chromatography method.

Glycyrrhiza glabra Extract

Test solution: 1.0 g of test sample of the extract is extracted with 10 mL of methanol and filtered.

Standard solution: 1.0 g of standard sample of the extract is extracted with 10 mL of methanol and filtered.

Chromatographic conditions:

Plate: Silica gel 60F 254, 5cm ×20 cm (Merck or equivalent).

Mobile phase: Mixture of toluene, ethyl acetate, formic acid 50: 15: 5(by volume)

Applied volume: 5 mL

Front of solution: 15 cm

Assessment – sprinkle’s reagent vaniline- sulphuric acid, heat for 5-10 min at 120°C andvisually examine.

Specifications : In the position, size and intensity of coloration of spot on the chromatogram of test solutionmust correspond to the spot on the chromatogram of standard solution

Zingiber officinale Extract (Ginger)

2 to 8°C (Fridge) ------------- ------------ 5/10/2014 5/01/2015

Room Temperature 5/7/2014 5/08/2014 5/10/2014 5/01/2015

Test solution : 1.0 g of test sample of extract is extracted with 10 mL of methanol and filtered.

Standard solution : 1.0 g of standard sample of the extract is extracted with 10 mL of methanol and filtered.

Chromatographic conditions:

Plate: Silica gel 60F 254, 5cm ×20 cm (merk or equivalent).

Mobile phase: Mixture of ethanol, concentrated ammonia solution, ethyl acetate 13:27:60 (byvolume)

Front of solution: 15 cm

Assessment – sprinkle reagent Anisaldehydesulphuric acid, heat for 5- 10 min at 120° C andvisually examine.

Specifications: In the position, size and intensity of coloration of spot on the chromatogram of test solutionmust correspond to the spot on the chromatogram of standard solution.

Curcuma longa Extract

Test solution: 1.0 g of test sample of the extract is extracted with 10 mL of methanol and filtered.

Standard solution: 1.0 g of standard sample of the extract is extracted with 10 mL of methanol and filtered.

Chromatographic conditions:

Plate: Silica gel 60F 254, 5 cm ×20 cm (Merk or equivalent).

Mobile phase: Mixture of toluene, Diethyl ether, Dioxane 62.5:21.5:16 (volume)

Applied volume: 5 mL

Front of solution: 15cm

Assessment: Sprinkle reagent Vaniline- Sulphuric acid, heat for 5-10 min at 120°C and visually examined.

Specifications: In the position, size and intensity of coloration of spot on the chromatogram of test solutionmust correspond to the spot on the chromatogram of standard solution.

Density : Take clean and dry Pycnometer and weigh it (W₁ ). Fill the Pycnometer with boiled and cooled water at 25°C. Weigh the content (W₂ ). (The weight of 1 mL water at 25°C in air is (0.99602 g.) Adjust the temperature of the liquid to about 25°C and fill Pycnometer with it.Remove excess of the liquid and weigh (W₃ ). Calculate the weight per mL of the sample using following formulaW₃ – W ₁ . Weight per mL of the sample = -------W₂ – W₁ pH

Transfer the syrup into clean dry beaker. Dip the electrode into it and observe the pH that is displayed on the screen of previously standardized pH meter

Results

Preformulation Studies

The preformulation study like qualitative analysis of raw materials and herbal extract drug which includes physical characterization and found no changes after 30 days at acceleratory condition. As shown (Table 1, 2, and 3)

Physical Characterization of Drug Sample

The results of physical characterization of the herbal extracts are as follows -

Description

The description of the drug was found to be identical with the reference sample of herbal extract. It is dark brown coloured clear viscous syrup having sweet taste and characteristic odour and were represented in Table 4.

Identification of formulation

Identification of herbal syrup for the formulation were given in Table 5 and 7 by using HPLC/TLC method.

Fill volume :100 mL of syrup into dark glass bottle sealed with a screw metal cover with padding and sealing line control. Self-adhesive label is placed on each bottle.

pH at 25 °C: By Potentiometric method (pH limit between 2.5 to 4.5) were considered

Weight/mL :1.30 ± 0.05 gm/mL as shown (Table 7)

Evaluation of Herbal Syrup

The evaluation results of herbal syrup with three month stability data for the formulation were given in Table 8.

Stability studies

The stability studies were performed for formulations from F1 to F4 for 1 to 3 months at various ambient temperatures at 25^o C / 60% RH, 30^o C /65% RH and 40^o C / 75% RH and results were represented in Table 8.

Discussion

In the present investigation, we have prepared a herbal anticough formulations F1.F2, F3 and F4 were prepared with a varying excipients in combination of standardized plant extracts like Glycyrrhiza glabra, Curcuma longa, Zingiber officinale and Piper longum which are used in treatment of cough. All the ingredients were studied for interactions amongst themselves. Physical properties like pH, density, Solubility, Weight per mL and viscosity of all the ingredients were analysed. Stability studies of formulations F1.F2, F3 and F4 were also carried out to check the physical and chemical nature of formulations under variant temperature conditions and relative humidity.

The preformulation study like characterization of drug sample which includes physical characteri -zation and evaluation of herbal syrup including determination of solubility, pH were performed for drug. The drug was physically characterized by taking the sample of Glycyrrhiza glabra extract, Curcuma longa extract, Zingiber officinale and Piper longum extract was subjected to the quantitative tests for its characterization.To know the Nature of the drug sample, It was observed visually and viewed under the compound microscope for the determination of its nature. In order to identify the Colour of the drug sample, It was viewed visually for the determination of its color using the black and white backgrounds.The Melting point of the drug samplewas carried out by using Liquid paraffin as a bath liquid. In view to determine Solubility, The solubility of the drug sample was carried out in different solvents using aqueous and organic phase. Identification and estimation of formulated syrup was done by TLC, HPTLC, and HPLC methods.

A stage of development of suitable and stable dosage form is an art of good manufacturing practice, during which the physicochemical properties of drug substance are characterized. Thorough preformulation work is the foundation of developing good formulations. Preformulation is the work of learning before doing and good science is always the right thing to do. An investigation of physical and chemical properties of a drug substance alone and whencombined with excipients was carried out to generate information for the development ofstable and quality product physiochemically. Physico -chemical characterization of the drugsubstance was done in order to define the conditions under which the product should beformulated.^7-18

Excipients, API compatibility studies or preformulation studies were conducted during development of product and were observed physically and chemically. Drug was mixed intimately with various excipients such as sweetener, solubilizing agents, anti-microbial preservatives, flavors and colour. It was mixed in the same ratio most likely to be encountered in the liquid dosage formulation as shown in Table 1, 2, and 3. Samples were kept for 30 days at 25^o C / 60% RH, 30^o C / 65% RH and 40^o C / 75% RH conditions for physical and chemical observation in glass vials USP type III. Based on the physical and chemical observation, the lists of selected excipients have been prepared to be used during formulation development activity. List of excipients selected includes Sugar, Sorbitol (70%), Menthol, Propylene glycol, Citric acid, Sodiumbenzoate, Methyl paraben, Guar gum, Sodium propyl paraben, Bronopol, Glycerin, Liquid glucose, Caramel, Fennel flavor and Raspberry sweet flavor. The different formulations F1, F2, F3, and F4 of an anti-cough herbal syrup was prepared as shown (Table 1, 2, and 3) by using standardized extracts and suitable excipients and were further subjected to stability studies. The drug and all the excipients were studied for solubility properties, pH, density and stability studies of formulations were carried out for 1 to 3 months under ambient temperature conditions of all the formulations F1 to F4 and further analysed by TLC/ HPTLC/HPLC methods.^19-28 Formulation F4 was found stable under variant temperature conditions as shown (Table 8) and HPTLC and HPLC were found to be suitable method for simultaneous estimation of standardized extracts of anticough herbal formulations as represented (Fig. F1-F4).

Conclusion

The developed anti-cough herbal syrup formulation-F4 was stable as compared to other formulation F1, F2 and F3 and can be analysed by HPTLC and HPLC Method which is found to be accurate, precise, and selective and can be employed successfully for the estimation of Glycyrrhiza glabra, Curcuma longa, Zingiber officinale and Piper longum standardized extracts in anti-cough herbal formulation.

Conflict of Interest

The authors declare no conflict of interest.

Acknowledgements

Authors are thankful to President Dr H.V. Dambal and Principal Dr V.H. Kulkarni SET’s College of Pharmacy for their encouragement and timely help during the experimental work.