INTRODUCTION:

Ayurvedic medicines are in great demand in the all countries of the world for primary health care because of their wide range of biological activities, higher safety margins and lesser costs. Public and government interest in herbal medicines is growing exponentially due to increased incidence of the adverse drug reactions and economic burden of the modern system of medicine. Herbal therapy is one of the best practices to overcome the illness. In order to have a good coordination among the quality of raw herb material, in process materials and in final product, it has become essential to develop reliable, specific quality control methods using combination of classical and modern instrumental method of analysis. Standardization is an essential factor for polyherbal formulation in order to assess the quality of the drugs based on the concentration of their active principle. The medicine prepared using traditional methods may not have the desired quality and batch to batch consistency.

Hence this formulation required standardization of following scientific parameters including organoleptic evaluation, chemical analysis, chromatographic pattern and microbial screening. The objective of present study was to standardize URIFLUX Syrup and to prove it’s superiority such as safety by various guidelines according World Health Organization.

MATERIALS AND METHOD^[1]:

Materials:

Marketed polyherbal “URIFLUX’’ syrup procured from Bewell Pharmaceuticals Kagal MIDC, Kolhapur, Maharashtra, India. All the chemicals and reagents used were of analytical grade For heavy metal analysis Atomic Absorption Spectroscopy Chemito used. Specific rotation (Digital Polarometer- Equip Tronics), pH (Digital pH meter- Equip Tronics), colony count (Digital Colony Counter AE Max Electronics), Laminar air flow system (Suprashesh Aditi Associate, Mumbai-68), stability testing apparatus (REMI) such materials and models used.

Methods:

As per World Health Organization guidelines and guidelines given in different traditional system of medicines all standardization were performed. Standardization was carried out by using applicable parameters like odour, general appearance, taste, pH, viscosity, specific gravity, optical activity, Thin Layer Chromatography profile, microbial contamination study, short term stability study, heavy metal analysis and pesticide residues analysis.

Determination of pH^[1]:

The pH of marketed polyherbal formulation was obtained by Digital pH meter. Calibration was done by using distilled water, buffer (at pH 5 and7) pH till constant reading.

Determination of viscosity:

Ostwald viscometer was used to detect viscosity of syrup. The method followed by standard operating procedure.

Determination of density^[1]:

Pycnometer used to detect density of syrup.

Specific gravity^[1]:

Pcynometer was used to detect specific gravity at 25⁰C.By dividing sample weight in grams to weight of water in ml.

Determination of Refractive index^[2]:

Abbe’s refractometer was used to determine Refractive Index of marketed syrup. Method was followed as per standard procedure described.

Determination of Viscosity ^[2]:

Ostawald’s viscometer was used to determine viscosity of marketed syrup. Method was followed as per standard procedure described.

Determination of Optical activity ^[6]:

Optical activity was detected by using Digital polarometer. Sucrose was used as reference standard .

Heavy metal and toxic metal analysis^[5]:

Identification test performed for lead, Arsenic, Mercury and Cadmium as per standard procedure given in Indian Pharmacopeia 2007. The standards of Arsenic (As), Lead (Pb), Mercury (Hg) and Cadmium (Cd) were prepared as per the protocol in the manual and the calibration curve was developed for each of them. Then samples were analyzed for the presence of As, Pb, Hg and Cd, using Atomic absorbance spectrophotometer (AAS) Chemito.

Determination of Pesticide residue:

Thin layer chromatography method used for detection of organophosphoric and organochlorine pesticide, using cyclohexane and chloroform as solvent system in 1:1 proportion.

Phytochemical studies ^[8]:

Various chemical test performed according to conventional method. Thin layer chromatographic studies were performed for R_f values and to detect presence of active chemical for various water extracts of herbs used in marked polyherbal diuretic syrup. Detection was obtained by spraying iodine fumes.

Microbiological Analysis ^[2,3,7]:

Total Viable Count ^[2]:

Add 1ml marketed polyherbal syrup to 15 ml liquefied casein soyabean digest agar medium at temperature no exciding 45⁰C in to 9-10cm petridishes. Alternatively spread pretreated syrup on surface of solidified medium in petridish. Prepared two dishes using the same dilution and incubate them at 30-35^oC for 48-72 hours. Colonies developed were counted by colony counter.

Yeast and mold count was determined by as per World Health Organization guidelines. petridishes used of 9-10 cm in diameter. To one dish added mixture of 1ml of pretreated syrup and about 15ml sabroud and glucose agar and incubated at 30-35°C for 24-72 hours.

Test for Escherichia coli:

Take 10 ml of syrup and the volume made up to 100 ml with lactose broth. This mixture was incubated at 35-37°C for 4 hrs. 1 ml sample from this to 100 ml Mac Conkey broth and incubated at 43-47°C for 24hrs. Subculture was prepared and inoculated on Mac Conkey agar media, and incubated at 43-47°C for 24 hrs. Growth of red, generally non mucoid colonies of Gram negative rods indicated the possible presence of E. coli

Test for Salmonella typhi:

Take 10 ml of syrup and the volume made up to 100 ml with lactose broth. This mixture was incubated at 35-37°C for 48 hrs. Further 10 ml of this sample was taken in 100 ml of tetrathionate bile brilliant green broth and incubated at 42-43°C for 18-24 hrs ml of sample was taken from it and plated on axylose lysine deoxycholate agar media and incubated at 35-37°C for 24 hrs. Well developed, red with or without black centre colonies indicated the presence of Salmonella typhi.

Table 1-: Macroscopic evaluation - stability study

Month No.	Temperature	Colour	Odour	Taste	General appearance
1	RT	Amber	Pleasant	Sweet	Clear
	30°C, 65%RH	Amber	Pleasant	Sweet	Clear
	40°C, 75%RH	Amber	Pleasant	Sweet	Clear
2	RT	Amber	Pleasant	Sweet	Clear
	30°C, 65%RH	Amber	Pleasant	Sweet	Clear
	40°C, 75%RH	Amber	Pleasant	Sweet	Clear
3	RT	Amber	Pleasant	Sweet	Clear
	30°C, 65%RH	Amber	Pleasant	Sweet	Clear
	40°C, 75%RH	Amber	Pleasant	Sweet	Clear

Test for Psuedomonas aeruginosa:

Take 10 ml of syrup and the volume made up to 100 ml with fluid soyabean-casein digest medium. This mixture was incubated at 35-37°C for24-48 hours. Examine the medium for growth and if growth is present, streak a portion of the medium on the surface of cetrimide agar medium, each plated on Petridishes. Cover and incubate at 35° to 37°C for 18 to 24 hours. Greenish colour colonies indicated the presence of Psudomonas aeruginosa.

Test for Staphylococcus aureus:

Take 10 ml of syrup and the volume made up to 100 ml with fluid soyabean-casein digest medium. This mixture was incubated at 35-37°Cfor 24-48 hours. Examine the medium for growth and if growth is present, streak portion of the medium on the surface of Mannitol salt agar, each plated on Petri dishes. Cover and incubate at 35º to 37º for 18 to 24 hours. Yellow colonies with yellow zones indicated the presence of Staphylococcus aureus.

Short term stability testing^[2]:

Short term stability study was done at temperature 30⁰C, 65% RH and 40⁰C, 75% RH and at room temperature. Microbiological and physical evaluation parameter detected for three months of time.

Table 2-: Physical evaluation –stability test

Month No.	Temperature	pH	Density in g/ml	Specific Gravity in g/ml	Viscosity in CPS	Refractive Index	Optical rotation
1	RT	5.6	1.389	1.633	1.9	1.438	-54.36
	30°C, 65%RH	5.5	1.387	1.621	2.03	1.432	-54.38
	40°C, 75%RH	5.4	1.380	1.622	1.9	1.436	-54.36
2	RT	5.6	1.39	1.623	2.04	1.431	-54.368
	30°C, 65%RH	5.6	1.37	1.620	2.04	1.438	-54.38
	40°C, 75%RH	5.6	1.37	1.620	2.03	1.436	-54.36
3	RT	5.5	1.32	1.621	1.9	1.437	-54.36
	30°C, 65%RH	5.4	1.39	1.621	2.1	1.440	-54.38
	40°C, 75%RH	5.4	1.387	1.620	2.5	1.439	-54.35

Table 3-: Microbial analysis -stability study

Month No.	Temperature	Total viable count	Yeast and mould count	*E. coli*	*S. typhi*	*P. aeruginosa*	*S. aureus*
1	RT	3440CFU/g	Absent	Absent	Absent	Absent	Absent
	30°C, 65%RH	3440CFU/g	Absent	Absent	Absent	Absent	Absent
	40°C, 75%RH	3432CFU/g	Absent	Absent	Absent	Absent	Absent
2	RT	3438CFU/g	Absent	Absent	Absent	Absent	Absent
	30°C, 65%RH	3432CFU/g	Absent	Absent	Absent	Absent	Absent
	40°C, 75%RH	3430CFU/g	Absent	Absent	Absent	Absent	Absent
3	RT	3440CFU/g	Absent	Absent	Absent	Absent	Absent
	30°C, 65%RH	3400CFU/g	Absent	Absent	Absent	Absent	Absent
	40°C, 75%RH	3428 CFU/g	Absent	Absent	Absent	Absent	Absent

CFU: Colony Forming Unit, g:gram, RT: Room Temperature, SD: Standard Deviation, E. coli: Escherichia coli, S. aureus: Staphylococcus aureus, S. typhi: Salmonella typhi, P. aeruginosa: Psuedomonas aeruginosa

Table 4-: Preliminary phytochemical tests.TS AND DISCUSSION

Sr no.	Nature of product	Test performed	Result
1	Carbohydrate	a. Fehilng’s test b. Benedict’s test c. Molish’s test d. Selwinoff’s test	Positive(+)
2	Amino acid	Ninhydrin test	Positive(+)
3	Steroid	Salkowski test	Positive(+)
4	Alkaloides	a. Dragendorff’s test b. Mayer’s test c. Hager’s test d. Wagner’s test	Positive(+)
5	Volatile oil	a. Permant stain b. odour	Negative(-)
6	Cardiac glycoside	a. Baljet’s test b. Legal’s test c. Keller killani test	Positive(+)
7	Anthraquinone glycoside	a. Borntrager’s test b. Modified Borntrager’s test	Positive(+)
8	Saponin glycoside	Foam tet	Positive(+)
9	Cynogenetic glycoside	Sodium picrate test	Negative(-)
10	Coumarine glycoside	a. Odour b. Fluorescence	Negative(-)
11	Flavonide	Shinoda test	Positive(+)
12	Tanins and phenolic compounds	a. Test with acetic acid b. Test with dilute nitric acid c. Test with 5% ferric chloride solution	Positive(+)

A	B

Std L, V, S	Std Q, B, S

C	D

Syrup	S, B

S, C, B, V, W, G

G, S

Figure number 1-:Thin Layer Chromatography Profile

S-Syrup, B-Bakul/Moolsari phool, C-Chandan, G-Gokharu, V-Varun, W-Wala mool, Std L-Standard Lupeol, Std Q-Standard Quercein.

Table 7:Thin Layer Chromatography profile

Slide number	Mobile phase used	Proportion of solvent system
A	Tolune:Ethyl acetate:Formic acid	5:4:1
B	Ethyl acetate:Tolune	3:7
C	Butanol:Methanol:Dichloromethane:Water:Pet ether	5:3:2:1:1
D	Pet ether:acetone	7:3
E	Butanol:Methanol:Dichloromethane:Water:Pet ether	5:3:2:1:1
F	Acetonitrile:Water	8:2

Statistical analysis:

The data were expressed as Mean ± SD. and statistically analyzed using one way ANOVA followed by Tukey- Kramer’s Multiple comparison test, p>0.10 was considered significant.

RESULT AND DISCUSSIUON^[8]:

The primary and basic objective of this research is to evaluate or standardize marketed .palatability. Marketed syrup was standardized for the parameters like pH, viscosity, density, specific gravity, refractive index, optical rotation, microbiological analysis given in Table number 1, which also shows syrup passes stability test at different storage temperatures. Preliminary phytochemical screening and Thin Layer Chromatography profile have shown in Table number 7. Heavy and toxic metal analysis have given in Table number table no. 5.

CONCLUSION:

From above work findings, it is conclude that Uriflux polyherbal diuretic syrup is stable at different temperatures mentioned above and passes most of tests for standardizations and safe to use.

ACKNOWLEDGEMENT:

Authors are thankful to Principal, Vice Principal Dr. S. K. Mohite, Mr. Bhinge , Ms. Kakade and Management of Rajarambapu College of Pharmacy, Kasegaon, Sangli, Maharashtra, India, providing all the support and help to carry out the work in college.

REFERENCES:

1. Patel DA, Patel YK, Shah PB, Development and evaluation of herbal syrup from Neolamarckia, International Research Journal of Pharmacy, 2012, 3 (9), 201-202.

2. Salunkhe VR, Bhise SB, Formulation development and real time stability studies of herbal oral liquids containing natural sweetner, Journal of Pharmacy Research, 2009, 2 (6) 1055-1061.

3. Parmar S, Shah N, Shah N, Patel N, Standardization of zymodine syrup-A polyherbal formulation, International Journal of Pharmaceutical Sciences Review and Research, 10 (1) 2011, 95-98.

4. Patel HB, Patel AA, Patel NM, Rachh PR, Standardization and pharmacological screening of marketed digestive chewable tablet, International Journal of Pharmaceutical Research and bioscience, 2012, 1 (4).

5. Indian Pharmacopoeia 2007, volume (I), 67-76.

6. More HN, Hajare AA, Practical Physical Pharmacy, Nirali publication 3^rd edition 214.

7. Kokare C, Pharmaceutical Microbiology Experiments and Techniques, 3^rdedition, Career Publications, 79-224.

8. Khandelwal KR, Practical Pharmacognosy, Techniques and Experiments, Nirali Publication, 19^th edition; 2008: 149-157.

Received on 17.08.2013 Modified on 10.09.2013

Asian J. Research Chem. 6(10): October 2013; Page 977-982