Regulatory Requirements, Protocols, Interaction and complexity involved in stability testing of phytopharmaceuticals

1. STABILITY TESTING OF
PHYTOPHARMACEUTICALS
By
Dr Gana Manjusha Kondepudi
Associate Professor
Vignan Institute of Pharmaceutical Technology

2. INTRODUCTION
• About 70–95% of the population in developing countries
depends upon herbs and herbal medicinal products to meet
their primary healthcare needs. These products are
increasingly being used in developed countries as well.
• Production of herbal substances, preparations and products
in large volumes has raised questionson their quality, safety,
and efficacy (QSE).
• Drug regulatory authorities across the globe have laid down
guidelines for manufacture of herbal substances,
preparations and products to assure their QSE.

3. •The stability testing of herbal medicinal products is as important
as that of synthetic drug products.
•But it is much more difficult and challenging for the former.
•In a synthetic drug product, the active pharmaceutical ingredient
is well defined qualitatively as well as quantitatively, and its content
is directly related to the therapeutic effectiveness of the product.
•Therefore, the actives serve as direct markers for stability testing
of the product, to establish its shelf-life.

4. • In contrast to it, a herbal drug substance, preparation or
medicinal product is a very complex heterogeneous mixture of
chemicals belonging to different categories (such as alkaloids,
terpenoids, flavonoids, organic acids, carbohydrates and
glycosides, saponins, amino acids, and others).
• All these chemicals are also liable to degrade, similar to
synthetic drugs, under the influence of varied environmental
factors such as temperature, light, air, moisture, pH, and others.
• It implies that the contents of these phytochemicals are most
likely to change during the shelf-life of the product causing
possible negative effects on the QSE.

5. • Many studies are reported on stability of different herbal substances,
preparations and products under a variety of environmental factors
(see Table 1), which support this argument that herbal materials are
also chemically unstable.
• Furthermore, therapeutic actions of a herbal medicinal product are
usually a function of additive or synergistic actions of chemically diverse
phytoconstituents.
• So, any change in content of a specific marker or a set of specific
markers during stability testing of a herbal drug substance, preparation
or medicinal product may not transcend to similar change in its
therapeutic effectiveness.
• It entails that quantitative monitoring of specific marker(s) during
stability studies of herbal materials/products may not form a firm basis
of establishing their shelf-life, during which they are expected to elicit
consistent therapeutic effectiveness.

8. Global Regulations
•Drugs regulatory agencies across the globe such as World Health
Organization (WHO), European Medicines Agency (EMEA), and
International Conference on Harmonisation (ICH) recommend the
submission of stability data of any medicinal product prior to its
approval.
• WHO and EMEA have issued specific guidelines, i.e., “Stability
testing of active pharmaceutical ingredients and finished
pharmaceutical products”, and “Stability testing of herbal medicinal
products and traditional herbal medicinal products”, respectively,
for this purpose.

9. •WHO’s “Supplementary guidelines for the manufacture of herbal
medicines” explicitly require that any specifically proposed shelf-life
of a herbal material or herbal preparation should be supported by
stability data generated under the specified storage conditions

10. •In addition to these, EMEA has issued other guidelines too, for
assessment of quality of herbal medicinal products, which include
“Quality of herbal medicinal products/traditional herbal medicinal
products” , “Test procedures and acceptance criteria for herbal
substances, herbal preparations and herbal medicinal
products/traditional herbal medicinal products”, “Markers used for
quantitative and qualitative analysis of herbal medicinal products
and traditional herbal medicinal products”, and “Quality control of
combination herbal medicinal products/traditional herbal medicinal
products”

11. •USFDA also states in its guideline entitled “Stability Testing
Guidelines for Dietary Supplements” that an expiration date, if any
on a product, should be supported by appropriately generated
stability data.
•Many other countries individually or regional groups have issued
their own set of guidelines on stability testing to ensure that
patients get herbal medicinal products that are safe and efficacious

12. •The approaches in different guidelines to achieve QSE include
assay of markers (active or analytical), biological assays, and/or
chromatographic chemoprofiling or fingerprinting of control and
stability samples of a product stored under the defined stability test
conditions.
•Nevertheless, according to both EMEA and WHO, a herbal
medicinal substance or herbal preparation is regarded as an active
substance.
•Therefore, mere assay of marker compound(s) may not reflect the
true stability characteristics of the product.

14. •It is for this reason, regulatory guidelines recommend that stability
of herbal medicinal product should be assessed in terms of stability
of not only the known therapeutic marker(s), but also other
constituents (as maximum as possible) by appropriate techniques,
such as chromatographic fingerprint analysis in addition to the other
tests like the study of physical, chemical, and microbiological
parameters.

15. •The content of known therapeutic constituent(s) during the
proposed shelf-life should not vary by +/-5% of the declared assay
value, unless justified. However, the content of marker in a herbal
medicinal product having unknown therapeutic constituent(s) should
not vary by > +/- 10% of the initial assay value during the proposed
shelf-life.

16. Issues in Stability Testing of Herbal Materials and Medicinal Product
•Despite the specific regulatory recommendations and scientific expertise
available across the globe for conducting stability studies, stability
assesments in dossier submissions of herbal medicinal products are usually
not based on sound and systematic stability studies.
•Various issues that may be considered responsible for this noncompliance
in stability testing are related to quality of herbal raw materials, marker
selection for analytical studies, possible inter-constituent interactions, and
selection of monitoring parameters during stability testing.

17. 1. Quality of Herbal Raw Material
• In synthetic drug products, the raw materials are of definite
chemical composition and purity, and variation in quality or purity of
these raw materials in different batches of any synthetic drug
product is negligible.
• The stability data generated through systematic studies on select
batches act as supporting data for other batches of that product.
• The shelf-life determined by stability testing of initial batches of a
product also applies to subsequent batches of the product, provided
there is no change in master formula and formulation process.

18. • However, the same cannot be said about a herbal material and
product because they are a complex heterogeneous mixture of
varied phytoconstituents.
• Due to such chemical complexity, the composition of a herbal
medicinal product is not fully characterized.
• Moreover, the phytoconstituents in a drug substance, preparation or
product often work synergistically in delivering therapeutic effects.
• These facts entail that all constituents in a herbal medicinal product
responsible for its particular therapeutic activity are not identified
and/or quantified for the want of reference standards.

19. • Further, batch-to-batch consistency in a herbal medicinal product is
dependent upon the quality of raw material.
• A herb or herbal substance from different cultivators of different
geographical location or harvested at different times during a year
cannot have same concentrations of phytoconstituents in it.
• It implies that different batches of a herbal raw material are most
likely to have variable quality attributes, and shelf-life of a product
determined through stability testing of one set of batches cannot
necessarily extrapolate to other batches.

20. Various determinants, directly or indirectly, govern the content and
nature of phytoconstituents in a herbal raw material, as discussed
below.
 Polyonymous Drugs
 Processing Methods
 Contamination of Raw Material
 Adulteration and Substitution
 Improper Agricultural Practices

21. 2. Selection of Marker(s)
The selection of markers for assessment of shelf-life of a herbal
material/product is the most challenging task during rational
stability testing.
A critical study of the available physicochemical stability reports on
herbal materials and products reveals that different research groups
have used different markers for stability testing of a herbal
substance, preparation or the medicinal product.

22. • For stability studies on Actaea racemosa, Budukh et al. used
cimiracemosideA, actein, and 27-deoxyactein (triterpenes) as
markers, whereas Jiang et al. used cimiracemoside-F, 3-epi-26-
deoxyactein, actein (triterpenes), and polyphenols as markers.
• Comparative analysis of these two studies reveals that polyphenols
are unstable compared to triterpenes at higher temperatures.
Therefore, shelf life of A. racemosa with respect to polyphenolic
content is shorter than that with respect to triterpene content.

23. • Similarly, stability studies on Echinacea purpurea were conducted
using alkamide and cichoric acid as well as using alkamides. Cichoric
acid is very unstable compared to alkamides, which again implies
two different shelf-lives for the same herb.
• These literature reports suggest that there is wide variability in (i)
susceptibility of different classes of makers to chemical change
during shelf-life, and (ii) selection of markers for monitoring of
stability of herbal materials/products.

24. • Therefore, the major question, which needs to be answered in the
very first place, in assessment of shelf-life of a herbal drug
substance, preparation or medicinal product, is that “which
phytoconstituent is to be selected for monitoring during stability
testing, so that a reliable shelf-life of the herbal material/product
can be established”?

25. • The sole purpose of consuming any herbal medicinal product is to
get specific therapeutic and/or nutritional benefits, and these
benefits are functions of individual, additive, or synergistic actions
of its different phytoconstituents.
• Therefore, an ideal stability studies protocol for such a product
should involve quantitative analysis of marker(s), whose levels can
be extrapolated to its intended purpose(s).

26. • For instance, withanolides in Withania somnifera are related to
immunomodulatory activity; rebaudiside and stevioside are related
to sweetness of Stevia rebaudiana; curcumin is responsible for anti-
inflammatory activity of Curcuma longa; and hyperforin is chiefly
responsible for antidepressant activity of H. perforatum.
• Nonetheless, knowing the biologically active constituent in a herbal
material may not be sufficient, because most herbal medicinal
products are composed of a cocktail of herbal substances or
preparations.

27. • In such cases, it is usually not possible to ascribe a particular
biological activity to a set of active markers. In this regard, the
WHO’s Supplementary guidelines on good manufacturing practices
for the manufacture of herbal medicines states that “...it is often not
feasible to determine the stability of each active ingredient.
• Moreover, because the herbal material, in its entirety, is regarded as
the active ingredient, a mere determination of the stability of the
constituents with known therapeutic activity will not usually be
sufficient.

28. • Chromatography allows tracing of changes, which may occur during
storage of a complex mixture of biologically active substances
contained in herbal materials.
• It should be shown, as far as possible, e.g. by comparisons of
appropriate characteristic/fingerprint chromatograms, that the
identified active ingredient (if any) and other substances present in
the herbal material or finished herbal product are likewise stable
and that their content as a proportion of the whole remains within
the defined limits.”

29. • The US FDA has also included biological assay as one of the quality
control parameters.
• In light of these regulatory recommendations, it becomes
imperative to assess shelf-life of a herbal material and product in
terms of chemical stability as well as biological activity.
• But out of the huge pool of reports on stability studies on herbal
substances, preparations and products, only a few studies are
conducted with respect to both marker compound and biological
activity.

30. • For instance, immunomodulatory activity of W. somnifera is found
to vary proportionally to the concentration of withanolide; decrease
in free radical scavenging activity of Bacopa monnieri corresponds
well with decrease in concentration of bacopaside I; free radical
scavenging activity of Orthosiphon stamineus extract is proportional
to the content of polyphenols; and antiangiogenic activity of
Matricaria chamomilla extract is directly related to flavonoids and
apigenin-7-O-glucoside content.

31. • However, contrary to these correlating reports, some studies have
defied a correlation between stability of a selected marker and a
particular biological activity tested during the stability studies.
Exposure of Olea europaea to high temperature causes decrease in
pheophytin (marker), but the antioxidant activity is increased.
Change in azadirachtin A content in Azadirachta indica formulations
does not conform to similar changes in antibacterial and anti-
diabetic activities.
• Determination of biological half-life (in terms of antibacterial
activity) and chemical half-life (in terms of allicin) of garlic extracts
indicates that allicin alone is not responsible for antibacterial activity
of the extract

32. • Therefore, shelf-life assignment to a herbal medicinal product to
ensure consistent therapeutic efficacy and safety should be based
on systematic stability testing that include evaluation of physical and
chemical stabilities as well as the intended biological activity of the
stability samples by appropriate in vitro and/or in vivo methods.

33. 3. Drug–Drug Interactions
• As discussed above repeatedly, a herbal drug substance, preparation or
medicinal product contains numerous phytoconstituents of different
chemical classes.
• These constituents may undergo various inter- as well as intra-molecular
reactions under the influence of varied environmental conditions, such as
heat, humidity, air and/or light during processing, formulation and storage of
the material.
• These possible interactions between different groups of constituents (see
Fig. 1) may result in a product with altered therapeutic and toxicity profile

35. Drug–Drug Interactions
• Tannins cause precipitation of alkaloids.
• Proteins and polysaccharides form reversible complexes with
polyphenols via H-bonding and hydrophobic interactions causing a
decrease in the activity due to free polyphenols.
• Water-soluble glycosides increase the water solubility of tannins via
non-covalent interactions, as observed in paeniflorin and glycyrrhizin.
• Complex formation too has an effect on overall efficacy of the
product. Monoherbal products of Convolvulus pluricaulis can be
monitored for their anxiolytic activity using scopoletin as a marker,
but the presence of Centella asiatica interferes with this activity of
scopoletin.
• It has been suggested that phytoconstituents of Centella asiatica may
be involved in complex formation with scopoletin

36. • Herbal medicinal products are also exposed to varied stress
conditions before and during production process as well as during
storage.
• Phytoconstituents can degrade under the influence of these stress
conditions, which may, in turn, alter the therapeutic efficacy of the
products.
• Curcumin undergoes extensive degradation under alkaline
conditions to give furilic acid and vanillin.
• Phenolic compound like gallic acid are sensitive to alkaline and
acidic environment. Thus, the products containing gallic acid should
be maintained at neutral pH.
4. Stability of Phytoconstituents

37. • Flavonol glycosides (quercetin, kaempferol, and
isorhamnetin) in Ginkgo biloba are highly unstable to basic,
oxidative, and thermal stressors, but stable under acidic
conditions
• These chemical changes are not perceptible from any
possible alteration in physical attributes of the product, and
can be monitored by studying the changes in chemical
fingerprints using sophisticated analytical techniques.

38. 5. Selection of Analytical and Statistical
Techniques
• Chemical analysis can be performed for reliable prediction of shelflife
by only careful selection of analytical techniques and sample
preparation.
• The contents of markers in a herbal substance/preparation/product
vary from extremely low to appreciable amounts.
• Also, the chemical diversity of these markers range from being non-
chromophoric (such as sugars, steroids, and fatty acids) to strongly UV
absorbing (such as flavonoids and phenolics).

39. • Therefore, the analytical technique selected for
comprehensive chemical analysis or chemical profiling
should be able to detect maximum possible number of
markers as well as the biologically important markers present
in even trace amounts.
• Sophisticated hyphenated analytical techniques such as LC-
MS, LC-IR, LC-DAD, LC-NMR, GC-MS, LC-UV-MS, and LC-RI are
the methods of choice so far for the standardization of
herbal materials and products

40. 6. Storage Conditions for Control Samples
• Control samples are critically important in a stability testing
program.
• These are required for relative evaluation of varied
parameters of stability samples.
• Control samples have to be stored for long durations
(practically equal to the period of long-term stability testing)
under conditions in which these do not undergo any kind of
physical, chemical and biological change.
• Generally, the samples are stored at 4 C (refrigeration
temperature).

41. • But all constituents/ markers in a herbal material/product
may not be stable even at such low temperature. For
example, antibacterial activity of garlic extracts decreases
with decrease in temperature (ranging from -20 C to ambient
temperature).
• Though the herbal substances/preparations/products in solid
state can be stored at even sub-zero temperatures, but
storage of herbal liquids at such low temperature can cause
other complications related to thermodynamics, such as
crystallization and/or polymorphism of markers.
• Therefore, selection of appropriate storage temperature for
control samples can also be equally challenging.

42. TEST PARAMETERS (PROTOCOL FOR STABILITY TESTING)
• Herbal raw materials usually do not come with a certificate
of analysis.
• Therefore, it becomes imperative on the part of the product
manufacturer to conduct various tests on the raw material
assure its identity and quality prior to the production of
herbal medicinal products.
• Those tests become a guiding force for selecting appropriate
tests for monitoring of stability of the product

49. • With the help of modern analytical techniques like HPLC,
HPTLC and by employing proper guidelines it is possible to
establish sound stability data for herbal products and predict
their shelf life which will help in global acceptability of herbal
products.

50. Importance of Stability testing:
• It evaluates the efficacy of a drug.
•Stability studies are used to develop suitable
packaging information for quality, strength, purity &
integrity of product during its shelf life.
•It is used for determination of the shelf life.

51. THANK YOU