About Authors:
Namdeo K.P. Dr.1, Bodhake S. H.1, Dwedi J. Dr.2, S. Shamim Dr.3 , Saifi Alimuddin3*
1Department of Pharmaceutical Sciences, Guru Ghasidas University, Bilaspur, CG.
2Department of Pharmacy, Banasthali University, Rajasthan.
3Translam Institute of Pharmaceutical Education & Research, Meerut.
Abstract
Ficus bengalensisis an indigenous plant belonging to family Moraceae possessing varied medicinal properties like antidiabetic, antimicrobial, antioxidant, antiseptic and also tender ends of hanging roots are prescribed to stop vomiting. Standardization is a method of assuring a minimum level of active ingredients in the extract and is becoming increasingly important as a means of ensuring a consistent supply of high quality phytopharmaceutical products. The World health Organization (WHO) emphasized the need to ensure quality control of medicinal plants products by using modern techniques and applying suitable standards. The following protocols for standardization of raw materials have been developedas authentication, foreign matter, macroscopic and microscopic examination, ash valueand extractive value, loss on drying, moisture content, Total flavonoid, total phenolic and total tannin contents determination of heavy metals, microbial contaminationand chromatographic profile(TLC &HPTLC).
Reference Id: PHARMATUTOR-ART-1154
Introduction
Herbal medicines have been used in medical practice for thousands of years and are recognised as a valuable and readily available resource of healthcare.1The process of evaluating the quality and purity of crude drugs by means of parameters like morphological, microscopical, physical, chemical and biological observation is called standardization.2The concept of standardization is relatively recent for phytomedcines, but it is rapidly becoming essential to ensure that patients are provided with high quality botanical products.3We planned our work as to evaluate the quality standards of stem bark of F. Bengalensis .
Material and Methods:
Plant material
The stem barks of F. bengalensis Linn. was collected from Sangam Vihar, New Delhi, in the month of May. The age of plant was found to be in the range of 25-30 years as enquired from local person. The specimen of collected bark was given for authantification in Raw Material and Laboratory of National Institute of Science Communication and Information Resources (NISCAIR), New Delhi (voucher no. NISCAIR/Consult/RHMD/2008-09/1010/41).
The stem barks of Ficus bengalrnsiswas washed and dried in an electric hot air oven at a temperature 40oC.
Macroscopic and Microscopic Examination
The plants was subjected to macroscopic studies which comprised of organoleptic characters of the drugs viz., colour, odour, appearance, taste, smell, texture, fracture, etc. Free hand TS of stem bark of Ficus bengalensis Linn. was taken and was cleared with chloral hydrate. The section was treated with phloroglucinol and a drop of concentrated hydrochloric acid to stain the lignified elements. Bark powder of Ficus bengalensis are oven dried at 40°C for 3-days to make it moisture free and grounded using electric grinder and 60# powder was prepared. The powdered slide was treated with phloroglucinol and a drop of concentrated hydrochloric acid to stain the lignified elements. The powdered slide was also treated with iodine to stain the starch grains.4
Physical Parameters:
Loss on drying 5 grams of accurately weighed drug powder was heated at 105?C in an oven to a constant weight. Weight loss after drying gave the moisture content of the material.4
Ash Values Total ashand Acid-insoluble ashwas calculated with reference to air dried drug.4
Extractive Values Extractive values in different solvents like alcohol (95%), water and chloroform was determined.5
Preparation of the extracts
Dried barks are coarsely powdered and defatted with petroleum ether by soxhlet apparatus. Defatted drug than exhaustively extracted with 95% ethanol in soxhlet apparatus. The extract was concentrated under reduced pressure to get dark brown mass. The viscous dark brown mass is than dried in air as dried powered extract.6, 7
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Preliminary Phytochemical Screening
The extracts was subjected to various qualitative chemical tests to determine the presence of various phytoconstituents like Alkaloids, Proteins & Amino acids, Carbohydrates, Flavonoids, Phenolic group, Glycosides, Saponins, Tannins, Steroids, Triterpinoidsusing reported methods.8-13
Total flavonoid, total phenolic and total tannin contents
Total Flavonoids of alcoholic extract of stem bark of Ficus bengalensis Linn. was estimated according to Cetkovic G method and total phenolic in the FB extract was determined using the Folin-Ciocalteu reagent.14 Total tannin content was determined by titrimetric method.15
Determination of Microbial Contamination
The count of viable aerobic bacteria is done by the technique of total aerobic bacterial count which is known as Plate Count Method.
Three different culture medias were used for this study were as follows
1. Fluid soyabean casein digest agar medium
2. Fluid soyabean casein digest medium
3. Sabouraud dextrose agar
Test for specific microorganism was done for Escherichia coli using Mac Conkey agar medium, Salmonella typhi using Brilliant green agar medium, Pseudomonas aeruginosa using Cetrimide agar medium and Staphylococcus aureus using Baird-Parker Agar Medium.16,17
Determination of heavy metal with respect to cadmium
The cadmium content was determined in the sample of stem bark of F.bengalensis.18
TLC finger-printing
Solvent system Toluene: ethyl acetate: formic acid (10: 6: 0.2) was developed for establishing the TLC patterns of alcoholic extract of drugs. Various visualizing techniques were used for best TLC fingerprinting, like UV 254, UV 366 nm, and plates are photographed. Rf value (s) were determined.
HPTLC finger-printing
The HPTLC was carried out using a Hemilton 100 µl HPTLC syringe, Camag Linomat V automatic spotting device, Camag twin trough chamber, Camag TLC Scanner-3, WINCAT integration software, aluminium sheet precoated with Silica Gel 60F254(Merck), 0.2 mm thickness. The sample concentration 5mg/ml was used for HPTLC finger-printing.
Result and discussion:
Macroscopic and microscopic studies
Macroscopic studies showed that colour of barks was gray to brown and had channelled curvature and astringent taste, thickness was 1.2-1.5 cm, fracture was fibrous and external surface was rough due to presence of furrows and lenticels.
TS of stem bark showed compressed cork tissues, stone cells. Parenchymatous cells were thin walled, cubical to oval and occur in between stone cells. Secondary phloem composed of sieve elements; parenchyma and calcium oxalate crystals were present. Medullary rays were 2-5 seriate.
The powder microscopy of all three stem barks of Ficus bengalensis was also done and it showed the presence of prismatic crystals of calcium oxalate, stone cells, starch grains, fragments of fibres, Laticiferous canal, medullary rays, Sclereids, and Parenchymatous cells.
Physical parameters
Physical parameters namely Loss on drying was 6.82% w/w. Total ash and Acid insoluble ash values were 5.0% w/w and 1.3% w/w respectively. Extractive values i.e. Alcohol soluble extractives, Chloroform soluble extractives and Water soluble extractives were 6.40% w/w were 1.68% w/w, and was 4.80% w/w respectively.
Phytochemical Screening
The percentage yield of ethanol extracts of stem bark of F. bengalensis was found to be 6.6 % w/w.
A qualitative chemical examination of alcoholic extract of stem bark of F.bengalensis showed the presence of proteins and amino acids, carbohydrates, flavonoids, Phenolic groups, glycosides, saponins, tannins, steroids and triterpenoids. Alkaloids were absent in the extract.
Total phenolic, total flavonoid and total tannin content
Total phenolic content in the alcoholic extract was determined using the Folin-Ciocalteu reagent and determined using calibration curve of standard Gallic acid and found to be 5.71% calculated in terms of Gallic acid.
Total flavonoid content in the alcoholic extract was found to be 1.24% w/w (expressed as equivalent to Rutin).
Total tannin content was determined by titrimetric method and it was found that 3.117% w/w tannic acid was present in alcoholic extract.
Microbial Contamination
From the results of determination of microbial contamination it was found that total bacterial count was >103 CFU/g which is less than themaximum number of microorganisms allowed is regulated in the European Pharmacopoeia:Up to 105 aerobic microorganism per g or ml. Including:Up to 103 yeast and fungi per g or ml andUp to 103 enterobacteria per g or ml.
Pathogenic microorganism namely Yeast/mould, E.coli. S. typhi, P. aeruginosa, and S. Aureus were foun to be absent.
Heavy metal analysis
In small quantities, certain heavy metals are nutritionally essential for a healthy life. Heavy metal analysis with respect to cadmium showed that cadmium was present as 0.63 ppm and is non toxic because it is less than toxic level of cadmium.
TLC and HPTLC Fingerprinting
TLC of alcoholic extract of stem bark of F.bengalensis showed 5 spots under UV 366. The Rf values of five compounds are 0.12, 0.24, 0.41, 0.58, 0.70 respectively.
HPTLC fingerprinting of alcoholic extract of stem bark of F.bengalensis showed the presence of 14 peaks indicating the presence of 14 different compounds in alcoholic extract. The Rf values of 14 compounds are 0.16, 0.22, 0.27, 0.34, 0.38, 0.44, 0.48, 0.59, 0.62, 0.67, 0.75, 0.87, 0.93, 0.97 respectively.
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