Basics of standerdisation by Dr.U.Srinivasa, Professor and Head, Srinivas college of pharmacy, Mangalore
Dr.U.SRINIVASA, M.Pharm, Ph.D
STANDARDIZATION ?Standardization involves adjusting the herbaldrug preparation to a defined content of aconstituent or a group of substances with knowntherapeutic activity by adding excipients or bymixing herbal drugs or herbal drug preparations. Botanical extracts made directly from crude plantmaterial show substantial variation incomposition, quality, and therapeutic effects.
In general, quality control is basedon three important pharmacopoeialdefinitions:1. Identity: Is the herb the one it should be?2. Purity: Are there contaminants, e.g., in theform of other herbs which should not bethere?3. Content or assay: Is the content of activeconstituents within the defined limits?
Identity can be achieved by macro- andmicroscopical examinations. Voucher specimens are reliable referencesources. Outbreaks of diseases among plantsmay result in changes to the physicalappearance of the plant and lead toincorrect identification. At times an incorrectbotanical quality with respect to the
Marker substances are chemically definedconstituents of a herbal drug that areimportant for the quality of the finishedproduct. IdeallyIdeally, the chemical markerschosen are responsible for the botanical’seffects in the body.
Purity is closely linked with the safe use ofdrugs and deals with factors such ash values,contaminants (e.g. foreign matter in theform of other herbs), and heavy metals. However, due to the application ofimproved analytical methods, modernpurity evaluation also includes microbialcontamination, radioactivity, and pesticideresidues.
Analytical methods such as photometric analysis,thin layer chromatography (TLC), highperformance liquid chromatography (HPLC),and gas chromatography (GC) can be employedin order to establish the constant composition ofherbal preparations.
Content or assay Is the most difficult area of quality control toperform, since in most herbal drugs the activeconstituents are not known. Sometimes markerscan be used. In all other cases, where no activeconstituent or marker can be defined for theherbal drug, the percentage extractable matterwith a solvent may be used as a form of assay, anapproach often seen in pharmacopeias.
Importance of standardization: Vegetable drugs are invariably inconsistent incomposition. Seasonal changes, geographicalchanges, genetic factors, edaphic factors i.e ageof the plant, season of the harvest, method ofdrying etc., In any system of medicine , the medicines must be
In some cases, when bioactive ingredient is notknown, assay is not possible. In many cases,preparations contain complex heterogeneousmixtures. Only in few cases, the drug activity is due to asingle compound. Often many activeconstituents contribute the activity. Even inertconstituents may influence the bioavailabilityand elimination of active constituents.
Occurrence – Dried roots with 2-3 lateral roots Size – 20-30 cms and 6-12mm (diameter) Shape – Straight, unbranched Outer surface – Buff to grey yellow, longitudinalwrinkles , at centre soft solid mass with scatteredpores Odour – Characteristic Taste – Bitter
As a root the following characters are seen 1. Cork – Cells are isodiametric and non lignified 2.Starch – In cortex as well as in vascular region,simple, oval in shape 3. Secondary xylem – Xylem fibres are present 4. Xylem vessels – Reticulate thickened 5. Tracheids
Marker compound is Withaferin – A, which isestimated by HPLC SYNOPSIS : Column – Poracil A coiled column (12feet X 1/8 thinch) Mobile phase – n-hexane : isopropanol (9:1) Flow rate – 0.2 ml/minute Detector – 225nm (UV)
F.O.M – Not more than 2% Total ash – Not more than 7% Acid insoluble ash - Not more than 1.2% Alcohol soluble extractive – Not less than20% Alcohol (25%) soluble matter – Not lessthan 16%
1.Standards specifications ; Total ash: Not more than 4% Loss on dryinig : Not more than 6% Solubility : In water – Minimum 80% andIn 50% alcohol – 80% Heavy metals : Not more than 20ppm Aflatoxin : Negative
Microbial tests: a. Total plate count - < 1000 cfu/gm b. Yeast and moulds - < 100 cfu/gm c. Coliform : Negative d. Salmonella and Shigella - Negative
SYNOPSIS FOR TLC Adsorbent – Silica gel G Solvent system - For withanolides : n.butanol:acetic acid: water(4:1:5) For alkaloids: Toluene: ethyl acetate: diethylamine ( 70:20:10) Detection: Withanolides : Vanillin-sulphuric acid reagentAlkaloids : Drogendroffs reagent
Sample preparation: Dissolve 0.25g of the extract in 25 ml of methanol .Filter and concentrate the filtrate to 5ml. Identification: Withanolides: 2 bluish violet spots with identical Rfvalues in both extract and standard withania root Alkalods: 3 orange spots with identical Rf values inboth extract and standard withania root
Alkaloids : A minimum of 0.75% Withanolides : A minimum of 1.5% Glyco-withanolides : A minimum of 2.5%
A. MacroscopyLeaves –Type – Simple and petiolatedShape - orbicular - reniformMargin- Crenate - denateApex- round with 5-7 nervedBase – cordateFlowers – smallFruit – seeded and indehiscentOdour – Charecteristic, Taste - Bitter
Mesophyll – Fragments are seen Stomata – both Paracytic and Diacytic Trichomes – absent Calcium oxalate – rosette crytals Mid rib – 3-5 vascular bundles, strips ofcollenchyma bellow the upper epidermis and above lowerepidermis
Adsorbent – Silica gel G Solvent system n-butanol: ethyl acetate: water (4:1:5 ) Detection:Development of chromatogram - 12cmReference standard –Rf value – cassoside (0.26), asiaticoside(0.38) and brahmoside (0.58)
Column- C18- µ Bondapack (10cmX8mm) Mobile phase- Acetonitrile: water (1:3) Flow rate – 1.5 ml/minute Detector- 205mm ( UV ) Standard : Known concentration madecassoside (0.2-4mg/ml) and asiaticoside in methanol ( 0.02- 0.4 mg/ml)Sample preparation – 2 gm of drug powder reflux with90% of methanol for 1hrs in boiling water bath. cool andfilter, collect the filtrate, concentrate the extract andmake up the volume to 50ml with methanol.
Procedure – Known volume of both test and standardsare subjected to HPLC and the respectivepeak areas for madecassoside andasiaticoside are recorded. Estimation – Calculate their percentages by comparingthe recorded peak areas.
F.O.M – Not more than 2% Total ash- Not more than 26% Acid insoluble ash- Not more than 7% Saponin content – Not less than 1%
1.Standards specifications ; Total ash: Not more than 15% Loss on dryinig : Not more than 10% Heavy metals – Not more than 20ppm Microbial tests: a. Total plate count - < 1000 cfu/gm b. Yeast and moulds - < 100 cfu/gm c. Salmonella and Shigella – Negative
C. Identification by TLC : Adsorbent – Silica gel G Solvent system – Chloroform : Methanol (95:5) Detection – Anisaldehyde – sulphuric acidreagent and heating at 120 for 10 minutes Test sample – Dissolve 0.25 g extract in 25mlpure methanol on a water bath , filter andconcentrate to 5ml Identification - Rf values 0.42 & 0.56
D. Estimation : By HPLC Marker compound – Cordifolioside A Column – C-18 Symmetry (30cmsX15mm) Mobile phase – Gradient elution Time – 0-15 mts – 95- 60% (Solvent A)5- 40% (Solvent B)- 15-30 mts- 60-0% (Solvent A)40- 100% (Solvent B)- 30-35 mts – 0% (Solvent A)100% (Solvent B)
Flow rate – 1ml/minute Reference solution – Cordifolioside A inmethanol ( 1mg in 2ml) Test sample – Defat the powdered drug in asoxhlet apparatus using petroleum ether for2hrs . Air dry the marc and extract withmethanol for 4hrs .Filter and concentrate thefiltrate to dryness under vaccum and dissolvethe residue in 10ml of methanol.
Procedure : Subject 10µl of reference solution and testsample to HPLC and record the peaks.Identify Cordifolioside A and calculatethe amount by comparing the retentiontime and peak areas respectively.
Quantitative standards : F.O.M – Not more than 2% Total ash- Not more than 11% Acid insoluble ash- Not more than 2% Alcohol soluble extractive- Not less than3% Water soluble extractive- Not less than11%
Total ash: Not more than 10% Loss on dryinig : Not more than 5% Solubility : In water – Minimum 80% and In 50%alcohol – 70% Heavy metals – Not more than 20ppm Microbial tests: a. Total plate count - < 1000 cfu/gm b. Yeast and moulds - < 100 cfu/gm c. Salmonella and Shigella - Negative Coliform – Negative Pesticidal residue – Nil Aflatoxin - Nil
The amount of total bitters andCordifolioside A are estimated. The amount of Cordifolioside A in the dryextract determined by HPLC method.
SYNOPSIS FOR TLC Adsorbent – Silica gel G Solvent system Toluene: Methanol : Dioxane : Ammonia(1:1:2:5:0.5) Detection:Modified Dragendroffs reagentDevelopment of chromatogram - 12cm
Standard :Known concentration in methanol ( 50-80 µg/ml) .Sample preparation – 1 gm of drugpowder reflux with methanol for 2hrs ,filter, collect the filtrate, concentrate andacidified with dil Hcl. Follow the generalmethod of extraction of alkaloids
Procedure – Apply 5µl of reference and sample Scanning – At 298nm and finger printprofiles can be recorded Identification –Saffron colored spot with a Rf value of0.71( vasicine) , both in sample and standard.
Reference standard –vasicine in methanol (1mg/ml)Test sample –1gm of drug is refluxed with 10ml ofmethanol, filter and collect the filtrate ,use for sampling.
E.STANDARDS FOR CRUDE DRUG F.O.M – Not more than 2% Total ash - Not more than 17% Acid insoluble ash - Not more than 1.0% Water soluble extractive - Not less than 27% Alcohol soluble extractive – Not more than 6% Stomatal index – 10.8 – 18.2 Palisade ratio – 5.0- 8.5 Vasicine content – 0.4% by HPLC