Stability testing

1. Stability testing of herbal
medicinal products

2. FACTORS AFFECTING STABILITY OF HERBAL FORMULATIONS
• The phytochemical stability of herbs and herbal products are affected by
many variables, including pH, oxygen, temperature, time of processing,
light, water activity (AW), enzymes, structure, self-association,
concentration, metallic ions, atmospheric composition, co-pigments, the
presence of antioxidants, and storage conditions,
• few phyto-molecules are unstable and highly susceptible to degradation and
decomposition.

4. 1] Effect of pH: Phytomolecules, such as anthocyanins, are sensitive to pH, and show faded color above a
value of 2. β-carotene is stable to pH change and was reported to be stable in foods over the range pH 2–7.
• Catechins as a mixture are extremely unstable in neutral or alkaline solutions (pH >8), whereas in acidic
solutions (pH< 4) they are stable. In alkaline solution, green tea catechins change their color from light
brown to dark brown due to degradation.
2] Concentration: The color and pigment stability of strawberry and blackcurrant syrups, which were
processed and stored under identical conditions, has been reported. The study revealed that colour stability
was dependent on total anthocyanin level rather than qualitative pigment composition.
• At high concentrations, anthocyanins may self-arrange, resulting in reduced hydrolytic attack, resulting
in colour intensification in red raspberry.
• Betalain concentration plays a crucial role in stabilizing betalain during food processing, because
betacyanin stability appears to increase with pigment concentration.

5. 3] Light: The effect of fluorescent light on the degradation rates of the major cranberry
anthocyanins was assayed in model systems in the presence of oxygen in the temperature range
of 25-55°C. The study revealed that light degraded most of the anthocyanins at 40°C.
• Light exhibited a significant effect on anthocyanin degradation in the presence of molecular
oxygen.
4] Temperature: A lower storage temperature can extend the shelf life of catechins and for
ready-to-drink tea beverages. A low temperature (4°C) and acidic pH (4.0) were found to be the
optimal storage conditions for catechin.
5] Relative Humidity (RH):
• To maintain the postharvest quality of herbs, fruits, and vegetables, RH plays a major role.

6. 6] Water Activity (AW): AW plays a crucial role in the stability profiling of several phytocomponents.
• Amaranthus pigment powders showed higher stability than the respective aqueous solutions, this being
ascribed to varying AW values.
• Additionally, some stabilizers, such as pectin and guar gum, appeared to improve the storage stability
by lowering the AW value.
7] Atmosphere: In the presence of oxygen, both betanidin and betanin were found to be unstable. The
stability of betanin was negatively correlated with oxygen concentration, indicating the involvement of
oxygen in betanin degradation. Conversely, betanin stability was observed to be improved in nitrogen
environment.

7. • Since the herbal substance or herbal preparation in its entirety is regarded as the active
substance, a mere determination of the stability of the constituents with known therapeutic
activity will not suffice. The stability of other substances present in the herbal substance or in
the herbal preparation, should, as far as possible, also be demonstrated, e.g., using appropriate
fingerprint chromatograms. It should also be demonstrated that their proportional content
remains comparable to the initial fingerprint.
• If a herbal medicinal product contains combinations of several herbal substances or herbal
preparations, and if it is not possible to determine the stability of each active substance, the
stability of the medicinal product should be determined by appropriate fingerprint
chromatograms, appropriate overall methods of assay and physical and sensory tests or
other appropriate tests. The appropriateness of the tests shall be justified by the applicant.

8. Predictable changes in Herbal medicinal Product:
Hydrolysis: Reaction with water takes place results in degradation of product.
Oxidation: Due to addition of electro negative atom (o), Removal of electro
positive atom, radicals formation results in decomposition of natural products.
Racemization: Racemization is the process in which one enantiomer of a
compound, such as an L-amino acid, converts to the other enantiomer. The
compound then alternates between each form while the ratio between the (+) and
(–) groups approaches 1:1, at which point it becomes optically inactive.
Geometric isomerization: Products can be change in trans or cis form. One
form may be more therapeutically active.

9. Polymerization: There is combination of two or more identical molecule to form much larger &
more complex molecule.
Temperature: The rate of most chemical reactions increase with increase in temperature. So that
‘Tropical’ area must be taken in consideration during preparation of the formula of the herbal
substance.
Moisture: Moisture absorbed on to the surface of solid drug will often increase the rate of
decomposition, if it is susceptible to the hydrolysis.
Light: Many type of chemical reactions are induced by exposure to light/ high energy.
Autoxidation of volatile oil / fixed oil takes place and substance becomes colored/rancid/
deteriorates.

10. • In the case of a herbal medicinal product containing a herbal substance or
herbal preparation with constituents of known therapeutic activity, the
variation in content during the proposed shelf-life should not exceed ± 5%
of the declared assay value, unless justified.
• In the case of a herbal medicinal product containing a herbal substance or
herbal preparation where constituents with known therapeutic activity are
unknown, a variation in marker content during the proposed shelf-life of
±10% of the initial assay value can be accepted if justified by the
applicant.

11. • Constituents belonging to different chemical classes in an herbal product may undergo varied
intra-molecular or inter-molecular reactions under the influence of heat, humidity and/or light
experienced during its manufacture, transportation and storage. The possible interactions
between different groups of constituents are liable to produce products that consequently may
render the product more or less active and/or toxic.
• For example, polyphenols form reversible complexes with proteins and polysaccharides via H-
bonding and hydrophobic interactions that lead to decreased levels of free polyphenols.
• Water solubility of tannins is increased in the presence of water soluble glycosides via non-
covalent interactions, as observed with paeniflorin and glycyrrhizin. Alkaloids are precipitated
in the presence of tannins.

12. • Polysaccharides, tannins and lignins form mono-dentate and bi-dentate complexes with
heavy metal ions. Cations form strong complexes with polysaccharides in alkaline medium
but weak complexes in the other media. Such interactions are liable to change the chemical
profile of an herbal drug during its storage, which may, consequently, affect or alter its
therapeutic profile.
• Therefore, it is necessary to show, through chromatographic fingerprints generated by
sophisticated analytical methods, that the over all chemical composition of a drug remains
unchanged during its shelf life.

13. In addition, herbal drugs are commonly formulated and dispensed as tablets, capsules, topical
products and oral solutions. Various parameters specific to these formulations such as
disintegration, dissolution, hardness, brittleness, pH, viscosity, clarity, suspendability,
homogeneity, extractables and microbial contamination do change with time during storage.
Hence, these parameters are also required to be evaluated during their stability testing as per
the Pharmacopoeia protocols. However, it is rather not necessary to undertake all the listed
tests for each type of product, but the tests undertaken should be able to justify the overall
stability studies. Therefore, stability studies of an herbal product constitute an crucial
component of its development process.

14. • An ideal stability testing protocol for herbal products, including evaluation of physical,
chemical as well as therapeutic qualities of these products is the key to the scientifically
established shelf life.
• Herbal drugs constituents are of different kind and have many constituents. The finished
products of herbal medicine generally have varied concentration of active constituent (s).
• Stability testing of herbal drugs is a challenging task, because the entire herb or herbal
product is regarded as the active matter, regardless of whether constituents with defined
therapeutic activity are known. The most important aspect in the evaluation of the stability
study of a product is its storage condition.

15. Role of markers
Markers are chemically known compounds, which may or may not have therapeutic effects,
used to calculate the quantity of herbal medicinal ingredients in herbal medicinal products.
The choice of the marker has to be justified. Finding the “right” analytical marker is a crucial
need for the stability testing of herbal medicinal products. Typical sources for finding markers
are:
1. Monographs and drafts
2. Experience
3. Literature research about known constituents.
4. Scientific research.

16. • Ideal chemical (analytical) markers should be the therapeutic components
of herbal medicines. However, for most herbal medicines, the therapeutic
components have not been fully elucidated or easily monitored.
• Bioactive, characteristic, molecular, main, synergistic, correlative, toxic, and
general components may be selected.
• Chemical fingerprinting has been demonstrated to be a powerful technique
for the quality control of herbal medicines. A chemical fingerprint is a
unique pattern that indicates the presence of multiple chemical markers
within a sample.

17. • The search for suitable and new marker substances is an important interface
between scientific research and the use of the results in the herbal industry´s
routine quality control.
• The isolation and structure elucidation of chemically defined substances in a
plant, drug and/or drug preparation helps to better understand the active
principle of an herb and boosts analytical quality control.