STABILITY TESTING OF

PHYTOPHARMACEUTICALS

Under the guidance of presented by

Dr. SK.Mastanamma M. Pharm., Ph.D SHAIK SALAM
Assistant Professor Y20MPH03015
Pharmaceutical analysis UNIVERSITY COLLEGE OF PHARMACEUTICAL SCIENCES
ACHARYA NAGARJUNA UNIVERSITY
Contents
❖Introduction
❖Stability testing
❖Regulations
❖Recommended protocols
❖HPTLC Finger printing
❖Conclusions
❖References
INTRODUCTION
❖Phytopharmaceuticals or Herbal drugs referred as
plants materials or herbalism, involves the use of
whole plants or part of plants, to treat injury or
illness. Herbal drugs are the use of therapeutic herbs
to prevent and treat diseases.
❖There are many herbal products offered that assert
to treat the symptoms of a broad range of problems
from depression to cold and flu
 WHO estimates that 80% of the world populations currently use
herbal drugs for major healthcare.
 Exceptionally, in some countries herbal drugs may also enclose by
tradition.
 The herbal drug is a chief constituent in traditional medicine and a
common constituent in ayurvedic, homeopathic, naturopathic and
other medicine systems.
 Now a days, due to side effects of allopathic drugs, herbal drugs are
preferred
ADVANTAGES OF HERBAL DRUGS
 Low /minimum cost
 Potency and efficiency
 Enhanced tolerance
 Fewer side effects
 Recyclable
DISADVANTAGES OF HERBAL
DRUGS

 Not able to cure rapid sickness and accidents

 Risk with self dosing
STABILITY TESTING

 It is to provide evidence on how the quality of a compound varies with time under
the influence of a variety of environmental factors such as temperature, humidity,
and light, and to establish a retest period or shelf life for the compound
STABILITY TESTING OF HERBAL
PRODUCTS
 Stability testing is necessary to ensure the product is of acceptable
quality through out its entire storage period.
 An important part of quality control of herbal products is the
evaluation of the chemical stability of a finished product during
storage period.
 The objective of a stability testing is to provide evidence on how the
quality of the herbal product s with stability.
 Stability testing examines the quality and potency of drug at suitable
time intervals under the influence of environmental factors such as
temperature, light, oxygen, moisture, other ingredient or excipient in
the dosage form ,particle size of drug ,microbial contamination ,trace
metal contamination ,leaching from the container
STORAGE CONDITIONS AND SAMPLING
TIMES FOR STABILITY TESTING
Parameters studied at each
Conditions Sampling time conditions
Long term testing 25 2 0,3,6,12,18,24,36,48,60 Organoleptic properties
0C/60%RH 5%RH
Months

Accelerated testing
3,6 months PH viscosity
400 2 0c/75%RH 5%RH

3,6,9,12 months Particle size

Intermediate testing
300 2 0c/60%RH 5%RH
 TESTING FREQUENCY
 For long term study
year 1: every 3months
Year 2 : evey 6months
Subsequent years annually
 At accelerated storage conditions: {6months study}
minimum 3 points including t0 and t final
Eg ; o [initial] 3 6 {final}
 At intermediate storage conditions : {12 montha study}
four points including t0 and t final
eg . 0(intial) 6 9 12(final)
Different stability test conditions

Test Test conditions

 Elevated temperature  4 0C/ambient humidity;250C/60% 5% relative humidity
 Elevated humidity  450C/70% relative humidity
 Cycling tests  370c/80% RH, alternating 24hourly with 200c ambient
 Freez thaw test humidity
 Exposure to light
 -300C/80% , alternating 24 hourly with ambient
temperature
 Mechanical tests
 Continuous exposure in a light
 Stress testing
 In centrifuge or vibration apparatus
 Above 50-600C and 75% RH
 Types of stability and conditions required
throughout the shelf life

 Chemical
 Every active constituent retains its chemical integrity and potency within
specified time
 Physical
 The original physical properties like appearance, palatability, uniformity,
dissolution are retained
 Microbiological  Sterility or resistance to microbial growth is retained
 Therapeutic  Therapeutic effect remains unchanged
 toxicological  No significantincrease in toxicity occurs
REGULATIONS
GLOBAL SCENARIO OF HERBAL
MEDICINE
 Globally, herbal medicine has been considered an important alternative to modern
Allopathic medicine.
 The safety of herbal medicines remains a major concern. In the United States, the
FDA has estimated that over 50,000 adverse effects are caused by botanical and
other dietary supplements.
 In addition, for most herbal drugs, the efficacy is not proved and the quality is not
assured.
 To address these issues, US-FDA has created a separate guidance document for
botanical products for NDAs and IND submissions - 2004 (first revision Aug 2015)
 Similarly, in India, CDSCO (DCGI) has created a separate guidelines for botanical
products called ‘phytopharmaceutical guidelines’– 2013 (first revision – Nov
2015).
Global regulations
USA
 US FDA has issued way back in 2000 a document “Guidance to
industry for Botanical Drugs”.
 As per this, detailed guidelines have been provided for
chemistry, manufacture, quality, safety and efficacy of botanical
origin leads for approval as drug.
 Such approvals are given by the same FDA which regulates
synthetic drugs
INDIAN SENERIO
 In India, ASU drugs have been under the purview of Ministry of
Ayush.
 In contrast, 2015 regulatory requirements for
phytopharmaceutical are under the purview of the Central
Drugs Standards Control Organization (CDSCO).
 The gazette notification defines regulatory provisions for
phytopharmaceuticals and regulatory submission requirements
for scientific data on quality, safety and efficacy to evaluate and
permit marketing for an herbal drug on similar lines to
synthetic, chemical moieties.
EUROPE
 In Europe, except in Germany where “FITO therapy regulations
permit botanicals as drugs”, the situation is very similar to the UK.
 However, since the TMP directive has elapsed it is understood that
medicine control agency of Europe has begun exercise for
regulations, for botanical drugs.
 In fact, this has caused a kind of trade barrier preventing India
Ayurvedic products to be approved in EU nations.
 In Indian regulations, the major class of Ayurveda, Siddha, or Unani
(ASU) drugs
RECOMMENDED PROTOCOLS
 In order to assure a consistent and acceptable quality herbal
product ,care should be taken right from the identification and
authentication of herbal raw materials to the verification
process of final product.
 It is desirable to establish a document database containing
information on each approved medicinal herb.
 A central digital document database which is regulatory updated
and which contains this information with linkages to references
in other databases like NAPRALERT should be established for
easy access by all beneficiaries, producers and stake holders
 The knowledge base for an herb or herbal medicine promoted for wider use
should be strengthened and expanded so that there is a scientific basis for each
use. This would require the presentation of data for each herb, used in herbal
medicine including,Plant identification, including cultivar with voucher specimen

❖Plant, preferably voucher stored in a herbarium ,for future reference

❖Age of the herb(maturity/flowering plant etc….)
❖Location of cultivation, including altitude ,longitude
❖Fertilizers /pesticides used ,if any
❖Drying process
❖Storage conditions of the plant ,before sal
 Certificate ,confirming the above
 Apart from details on each herb ,details regarding the Time of harvest, including
time after application of pesticides (if applicable)manufacture of the herbal
medicine including the following data should be made available:
 Protocol or pharmacopoeia or method used for producing the medicine
 Plant or plants used
 Part of plant used in medicine
 Vehicle used for producing drug /medicine ,e.g., alcohol or water, type of
preservative used, documents maintained by the supplier regarding the herb
should be made available by the manufacturer
 Prepare monographs for traditionally used herbal medicines in a suitable
format.
 Legislate to ensure that manufacturers provide relevant data on herbs and
manufacturing processess.
 Work toward amending TRIPS to include protection of IPR contained in
indigenous knowledge and to make the development of herbal medicines
attractive to pharmaceutical companies.
 Legislate to establish standards in herbal medicine manufacturing and ensure
their implementation.
 Patents involving biological resources should be granted only if the source of
material is specified and reference made to the material transfer agreement
 ❖As most herbal medicines prepared from more than one plant material,
it is imperative the documentation should be made both on single
medicinal plant and on composite herbal preparations.
 ❖Single plants ❖Plant identification: Family, genus, species with
synonyms and older names where applicable; english (common) name(s);
local name ❖Herbarium voucher; specimen number and date with
collectors name and identity
 ❖Age of the herb (maturity/flowering plant, etc.,) ❖Local of
cultivation/collection including altitude and longitude/latitude
❖Fertilizers/pesticides used(if any)
 ❖Time of harvest including time after application of pesticides ❖Storage
conditions of the plant before sale.
 Drying process
Composite herbal medicine
❖ Apart from details on each herb as outlined for single plant, details
regarding the manufacture of the composite herbal medicine, including the
following data, should made available:
❖Protocol/pharmacopoeia/method used for producing the medicine
❖Plant/plants (for multi-plant herbal medicine) used
❖Part of plant plants used in the medicine
❖Vehicle use for producing the drug/medicine, eg., alcohol or water (with
composition with if a mixed solvent); type of preservative used, if any,
amount ❖Excipients (if any) used, amount Techniques use
Techniques used

With the help of modern analytical techniques like HPLC , HPTLC

and by employing proper guidelines it is possible to establish stability
data for herbal products and predict their shelf life which will help in
global acceptability of herbal products.
HPTLC FINGER PRINTING
 Finger printing:
 a pattern or impression which is highly specific enough to
become useful as characteristic identifier for that particular
entity.
 HPTLC Fingerprinting:
 It is a pattern on TLC plate (or a densitogram of that pattern) of separated
compounds, generated according to their highly specific RF values, presented
in sample, specific enough to become an authentic statement or bio-chemical
marker( in the case of biological samples) for that sample and capable
enough to provide unique, specific and characteristic identity
Importance of HPTLC Fingerprinting:
❖HPTLC provide a deep inside into the plants compound profile and their
chemistry.
❖There is no substitution of qualitative visual results of HPTLC for botanicals.
❖HPTLC fingerprinting is cheap, fast and solvent saving as compared to
HPLC/GC and other chromatographic techniques.
❖Sensitivity and accuracy is always a matter ( as compared with HPLC) but
HPTLC is advanced enough for providing a reliable authentic biochemical
integrity statement for botanicals
Basic steps:
❖Firstly, plate is checked before using it for sample applicator under 254nm,
whether it was giving fluorescence or not and mark the limit of run at 80mm
and direction by a HB-pencil.
❖Cut all 20x20 cm plate, in to the 20x10cm or 10x10cm plate with the use of
scissor.
❖Working programme is generated with the use of software in the computer.
❖Specific volume of sample is taken by the use of syringe and applied on plate
by the means of sample applicator.
❖ After the completion of the sample applicator program the plate is
subjected for drying with the use of a dryer and then placed on the TLC plate,
heat for 10 minutes to remove any water or moisture content from the plate.
❖ An optimised mobile phase is used for analytical demand of experiment and
that mobile phase is subjected to the development chamber.
❖ A filter paper rinsed with mobile phase is also subjected in the chamber for a
uniform vapour saturation of the chamber, prior adding of the sample applied
plate.
❖Chamber saturation time is now optimised(basically20-25mins)
❖The plate is placed in it, till the solvent front reached up to the distance of
80mm(previously marked).
❖After it densitometry evaluations can be carried out under 254nm(deuterium
lamp) and 366nm (Hg lamp), peak height and peak areas can be calculated with
the use of scanner.
❖In the last step, the plate is derivatized with specific derivatizing agent for
visual identification in the derivatizing chamber(specific time) and air dried.
❖Final images are quickly captured by the plate visualizer under visible white
light and fluorescence (366nm). Then densitometry scanning was performed at
540nm (W lamp) and 366nm(HG lamp).
❖After it, spectral analysis, RF value calculations, peak area calculations are
analyzed for final interpretation and HPTLC fingerprinting profile generation.
CONCLUSION
❖Provide evidence on how the quality of the compound varies with
time
❖Predict shelf life
❖Determine the recommended storage conditions
REFERENCE
❖For HPTLC fingerprinting experimental and analytical purpose-
(reich,E.., and Schibli , A.(2007).High performance thin layer
chromatography for the analysis of medicinal plants; published by Thieme
medical publishers,)
❖Quantitative and densitometric al analysis –(Monica, W.H.,joseph , S.,
and Teresa, K (2008). Thin layer chromatography in phytochemistry.