Development and characterization of Analytical methods for

Stability study of some Herbal formulations

Synopsis

Prepared by -
Pooja Deshpande
Research Scholar
University Institute of Pharmacy
Pandit Ravishankar Shukla University
Raipur (C.G)

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 CONTENTS

S. CONTENTS Slide no.

NO.

1 Introduction 03-06

2 Regulatory basis of herbal drug stability testing 07

3 Recommendations for stability study 08

4 Scientific basis of herbal drug stability testing 09

Current scenario in stability testing of herbal drugs 10

5 and products

6 Analytical methods for determination of stablity 11

7 Review of Literature 12 – 19

8 References 20-23

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 STABILITY

The capacity of drug or product to remain within the established

specifications of identity, purity in a specific period of time
OR
USP defines stability of pharmaceutical product as, ‘extent to which a product
retains within specified limits and throughout its period of storage and use (
shelf life ).1

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 HERB

• Herbs and products containing herb(s) have been in trade and commerce and are currently used for a variety
of purposes. As a country, India has a rich history of use of herbs, processed herbs and formulations
containing herbs both from traditional wisdom as well as cultural usage.

• Crude Herbs -Means, mainly whole, fragmented or cut, plants, parts of plants, algae, fungi, and lichen
in a form which is not processed.

• Processed Herbs - Processed herbs means preparations obtained by subjecting herbs to treatment such as
extraction, distillation, expression, fractionation, purification, concentration and partial or full fermentation.
tincture, preparing extract, distilling to get essential oils, fatty oils (either expressed or solvent extracted or a
blend of both) expressed juices, extracted exudates, gums and oleo resins, liquid extract where the solvent
is evaporated to yield concentrated semi solid mass or dried mass. 6

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 HERBAL FORMULATIONS

• Herbal formulation shall mean a dosage form consisting of one or more herbs or processed
herb(s) in specified quantities to provide specific nutritional, cosmetic benefits, and/or other
benefits meant for use to diagnose treat, mitigate diseases of human beings or animals and/or
to alter the structure or physiology human beings or animals. Dosage forms known in
traditional medicines may also be adopted for preparing herbal formulations, either for
external use or for internal administration. 6

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 STABILITY STUDY OF HERBAL DRUGS

• Herbal drugs are traditionally safe for treatment of any ailment because of being
natural substances.
• Drug Regulatory Agencies across the globe have provided guidelines for
manufacturing and controlling Quality , Safety and Efficacy (QSE) of drugs .
• According to United State Food and Drug Administration ( USFDA), criteria for
herbal drug claimed to have specific therapeutic benefits. .
• According to European Medicine Agency EMEA requires herbal medicinal product
be identified in terms of therapeutic or analytical markers and by any analytical
methods . 2

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 REGULATORY BASIS OF HERBAL DRUG STABILITY TESTING 2

• Guidelines provided by drug regulatory agencies –

1. EMEA – European medical agency
2. ICH – International conference on harmonization
3. WHO – World health organisation
4. USFDA – United State food and Drug Administration

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• WHO’s Supplementary guidelines for the manufacture of herbal medicines specifically states under
its section 17.4 that-
If the expiry date is given for a herbal material or herbal preparation, some stability data which
supports the proposed shelf life under the specified storage conditions should be available. Stability
data is always required to support the shelf-life for the finished herbal products. 3

• EMEA has also issued specific set of guidelines on quality of HMPs, which clearly states that
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., by means of appropriate fingerprint
chromatograms. It should also be demonstrated that their proportional content remains comparable to
the initial fingerprint. 4

• Current Good Manufacturing Practice USFDA states that

Nevertheless, if you use an expiration date on a product, you should have data to support that date.
You should have a written testing program designed to assess the stability characteristics of the
dietary supplement, and you should use the results of the stability testing to determine appropriate
storage conditions and expiration dates (USFDA, 2003). 5

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 Recommended conditions for stability testing (WHO, 2009) 9

Study Storage condition Minimum time Testing frequency

period covered by (months)
data at submission
(months)

Long-term 25 ± 2 °C/60 ± 5% 12 0, 3, 6, 9, 12, 18, 24

RH or and then annually
30 ± 2 °C/65 ± 5%
RH or
30 ± 2 °C/75 ± 5%
RH
Intermediate 30 ± 2 °C/65 ± 5% 6 0, 6, 9 and 12
RH
Accelerated 40 ± 2 °C/75 ± 5% 6 0, 3 and 6
RH

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 SCIENTIFIC BASIS OF HERBAL DRUG STABILITY TESTING

• Constituents belonging to the different chemical classes may undergoes variated intramoleular and
intermolecular changes under the influence of heat , light , temperature , humdity . For Eg. Polyphenols
form reversible complex with protein and polysaccharides via H- bonding that leads to decrease in
levels of polyphenols ,
• Alkaloids are precipitated in the presence of tannins .
• These interactions are liable to change in the chemical profile of an herbal drug during its storage and
effect or alter its therapeutic profile .
• Therefore, it is necessary to show , through chromatographic fingerprints generated by sophisticated
analytical methods, that the overall chemical composition of a drug remains unchanged during its
shelf life . 2

• Herbal drugs are commonly formulated and dispensed as tablets , capsules , topical products and oral
solutions .
• Other parameters such as disintegration , dissolution , hardness , pH, viscosity , clarity , suspendebility ,
homogeneity and microbial contaimination are also affected during storage , these tests are also should be
able to justify the overall stability study. 2

10
CURRENT SCENARIO IN STABILITY TESTING OF HERBAL DRUGS AND
PRODUCTS

The analysis of various reports by literature review reveals that there is a wide variability in conditions
employed and duration of stability testing and parameters evaluated for shelf life , based on the parameters
employed to assess the stability testing conditions are classfied in to four categories-
1. Physical characteristics
2. Physico – chemical / chemical studies
3. Biological studies
4. Physico – chemico – biological studies 2

These activities presents an ideal stability testing protocol for herbal products , because evaluation of
physical , chemical as well as therapeutic qualities of these products is the key to scientifically established
shelf life . 2

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 Reported Methods in Literature

• Verma et al ( 2019 ) studied Shelf-life evaluation of Rasayana Churna(RC) , RC was levigated with
decoction of Amalaki, Guduchi and Gokshura to prepare Bhavita Rasayana Churna (BRC). Both the
samples were subjected to accelerated stability studies by following standard guidelines. Both the samples
are bitter and astringent with characteristic odor. RC is creamish brown in colour, while BRC is dark
blackish brown. These Organoleptic characters were unchanged till the 6th month of study. On
comparision, BRC is found to be more stable than RC. 26
• Sachan et al (2015) studied Stability testing of herbal products The stability testing of herbal products
check the quality of herbal products which varies with the time under the influence of environmental
factors, such as temperature, humidity, light, oxygen, moisture, other ingredient or excipient in the dosage
form, particle size of drug, microbial contamination, trace metal contamination, leaching from the
container, etc. and also provide statistics for the determination of shelf lives. Therefore evaluation of the
parameters based upon chemical, physical, microbiological, therapeutic andtoxicological studies can serve
as an important tool in stability studies. 7
• Vaclavik et al (2014) discussed Mass spectrometric analysis of pharmaceutical adulterants in products
labeled as botanical dietary supplements or herbal remedies, critically assesses the role and summarizes the
applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and
herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known
pharmaceutical adulterants as well as in screening and structure elucidation of unexpected adulterants and
novel designer drugs. 11

12
• Kumari et al (2012) studied on the Standardization of herbal medicine, Herbal drug technology is
used for converting botanical materials into medicines, where standardization and quality control
with proper integration of modern scientific techniques and traditional knowledge is important. For
global harmonization WHO specific guidelines for the assessment of the safety, efficacy and quality
of herbal medicines are of utmost importance. Standardization of drug means confirmation of its
identity, quality and purity throughout all phases of its cycle. 25

• Nikam et al (2012) studied Future Trends in Standardization of Herbal Drugs Herbal drug
technology is used for converting botanicals materials into medicines, where standardization and
quality control with proper integration of modern scientific techniques and traditional knowledge is
important to prove constant composition of herbal preparations, adequate analytical methods the
have to be applied such as photometric analysis, thin layer chromatography [TLC], high performance
liquid chromatography [HPLC], and gas chromatography [GC], DNA Fingerprinting. 28

• Atmakuri and Dathi ( 2010) et al discussed current Trends in Herbal Medicines, Herbal medicines
make up an important component of the trend toward alternative medicine. Herbal medicine is
becoming ever more popular in today’s world as people seek out natural remedies. Herbal medicines
have been used since the dawn of civilization to maintain health and to treat various diseases. To
compete with the growing pharmaceutical market, there is an urgency to utilize and scientifically
validate more medicinally useful herbal products. The article provides an overview of herbal
medicines and aimed to explain the therapeutic efficacy of various herbal medicines, adverse drug
reactions, drug interactions, standardization and stability testing of herbal medicines,
pharmacovigilance and regulatory status of herbal medicines. 31

13
• Sawant et al (2016) studied formulation and evaluation of herbal ointment containing Neem and
Turmeric extract , Plant derived substances and herbal medicines have recently attracted the great
interest towards their versatile application, as medicinal plants are the richest source of bioactive
compounds used in traditional and modern medicine. The present work is to formulate and evaluate
the ointment of Neem (Azadirachta indica) and Turmeric (Curcuma longa) extract, After completion
of formulation it was evaluated for its physicochemical parameters like colour, odour, pH,
spreadability, extrudability, consistency, diffusion study, solubility, washability. Also the formulation
was evaluated for its stability at various temperature conditions which shows no change in the
irritancy, spreadability and diffusion study. 32

• Chua et al (2016) studied interaction between warfarin and Chinese herbal medicines , traditional
Chinese medicine (TCM) not only remains a popular form of medical treatment among the Chinese,
but is also gaining popularity in the West. This phenomenon has brought along with it increasing
reports on herb-drug interactions, beckoning the attention of Western physicians, who will find it
increasingly difficult to ignore the impact of TCM on Western therapies. This paper aims to facilitate
the education of Western physicians on common Chinese herbs and raise awareness about potential
interactions between these herbs and warfarin, a drug that is especially susceptible to herb-drug
interactions due to its narrow therapeutic range. 34

• Shulammithi et al (2016) Standardization and quality evaluation of herbal drugs, Evaluation of

herbal drug is an important tool in the formulation of high quality herbal products herbal medicine
on the need to establish quality parameters with the help of advanced analytical tools and well
defined standardization methods in ensuring the safety of the global herbal market. The processes of
good quality assurance and standardization of herbal medicines and products using various
spectroscopic, chromatographic and electrophoretic methods. 36

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 ANALYTICAL METHODS FOR DETERMINATION OF STABILITY
STUDY OF HERBAL FORMULATIONS

• Physical methods
• Chemical methods
• Instrumentation methods
 Chromatographic techniques
1. UV spectrophotometer
2. High performance liquid chromatography
3. High performance thin layer chromatography
4. Gas chromatography
 DNA fingerprinting
• Hybridization based methods – Restriction fragment length
polymorphism(RFLP) , Variable Number Tandem Repeats (VNTR)
• PCR based techniques – Arbitarily Primed - PCR SimpleSequence
Repeats
• Sequence based techniques - Single nucleotide polymorphism

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 Reported Methods in Literature

S.NO HERBAL INTRUMENTATION REFERENCE

FORMULATION Method
1 Murmakki (Commiphora HPLC Kamal et al (2011) 14
mukul gum) Method
kundur (Boswellia
serrata gum),
2 Bu-yang-huan-wu-tang (HPLC-MS/MS) Shaw et al
(BYHWT) Chinese medicine (2012) 15

3 Trachyspermum ammi HPTLC ANALYSIS Haque et al ( 2012 ) 16

extract

4 A. marmelos fruit powder qNMR. Chauthe et al

W. somnifera leaves (2012) 17
E. jambolana fruit powder

5 Herbal capsules HPTLC

Ashwashila agandha, (Accelerated Bankoti et al (2012) 28
Shilajeet and Stability study )
Ashwashila) 16
S.NO HERBAL INTRUMENTATION REFERENCE
FORMULATION Method

5 Caturjata Churna HPLC method Saran et al (2013 )

18

6 Ginkgo biloba extract HPLC/DAD analysis Ronowicz et al

(2013) 19
7 Piper betle leaf extract HPTLC method Annegowda et al
(2013) 20

8 Rumex vesicarius L. Alam et al (2014) 21

(HPTLC)–densitometric method

9 6-gingerol in polyherbal RP-HPLC Kamal et al (2015) 8

formulations
10 Costunolide and Vadaparthi et al
Dehydrocostus Lactone HPLC-DAD (2015) 24
in Saussurea lappa
(Polyherbal
formulations)

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S.NO HERBAL FORMULATION INTRUMENTATION REFERENCE
Method

11 Silymarin in poly herbal UV/visible Gul et al (2015) 37

Spectrophotometric method
formulation
12 Turmeric(Curcuma longa L.), RP- HPLC and HPTLC Biswas et al (2016)
Chickpea(Cicer arietinum L.) 22
, sandalwood(Santalum
album L.).

13 Centella asiatica ( dried Accelerated Stability Kaur et al (2016) 30

extracts ) Study
HPLC, HPTLC

14 Banhasasim-tang decoction (HPLC) analysis Jin et al (2016) 33

15 Bacopa monnieri Thin layer Bhardwaj et al

chromatographic(TLC) (2016) 35

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 FINGERPRINTING TECHNIQUES IN HERBAL STANDARDIZATION

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Table - Fingerprinting techniques in herbal standardization

CHROMATOGRAPHIC FINGERPRINTING DNA FINGERPRINTING

TECHNIQUES
• Thin layer Chromatography (TLC) Hybridization based methods

• High performance thin layer PCR based techniques

chromatography (HPTLC)

• High performance Liquid Sequence based techniques

chromatography (HPLC)
• Gas Chromatography (GC)

• Hyphenated techniques

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