APPLIED AND FORENSIC
PHARMACOGNOSY
(PG 706)
Prof. Dina Abou-Hussein;
Email: dina.abouhussein@pharma.cu.edu.eg
Protection or safeguard of a public health can be
established by ensuring appropriate standards of
safety, quality and efficacy for medicines
available on the market.
The safety and efficacy
of herbal medicines largely
depend on their quality.
Learning
Outcomes
➢ Define Quality management in Drug
Industry and determine its components.
➢ Highlight the importance of Quality
Assurance in the manufacture of herbal
medicines.
➢ Recognize Good Manufacturing practice for
herbal medicines.
➢ Understand the challenge in applying
Quality control in herbal drugs.
➢ Identify the Problems facing QC analysis of
herbal drugs.
Quality management (QM) in the Drug Industry
• QM: is defined as the aspect of management function that
determines and implements the “quality policy”.
• The concepts of Quality Assurance (QA), Good Manufacturing
Practice (GMP) and Quality Control (QC) are interrelated
aspects of quality management.
QA
GMP
QC
QA
GMP
QC
Quality Management for Herbal Medicines
1- Quality Assurance in the manufacture of herbal medicines:
• It is the totality of the arrangements made with the purpose of ensuring that
pharmaceutical products are of the quality required for their intended use.
• The system of quality assurance appropriate to the manufacture of herbal
medicine should ensure that:
a) Requirements of GMP and other associated codes such as of Good
Laboratory Practice (GLP) and Good Clinical Practice (GCP) are adopted.
b) Production and control operations are clearly specified in a written form.
c) Managerial responsibilities are clearly specified in job descriptions.
d) All necessary controls on starting materials, intermediate products and
bulk products and other in-process controls, calibrations and validations
are carried out.
2- Good manufacturing practice for herbal medicines:
• GMP is that part of quality assurance which ensures that products are
consistently produced and controlled to the quality standards appropriate
to their intended use and as required by the marketing authorization.
• In addition, there are supplementary guidelines specifically in relation to
the production and control of herbal medicines.
Collection/cultivation of medicinal plants should follow relevant guidance
such as the WHO guidelines on Good Agriculture and Collection Practices
(GACP) for medicinal plants.
2- Good manufacturing practice for herbal medicines:
• Under GMP:
a) All manufacturing practices are clearly defined, systematically
reviewed and comply with specifications of the required quality of the
pharmaceutical products.
b) Qualification and validation are performed: The reproducibility of the
production process is the main means for ensuring consistency of
quality, efficacy and safety between batches.
c) All necessary resources are provided.
d) Instructions and procedures are written in clear language.
e) Operators are trained to carry out procedures correctly.
f) Records are made during manufacture to show that the quality and
quantity of the product are as expected; any significant deviations are
investigated.
g) Records covering manufacture and distribution enable the complete
history of a batch to be traced.
2- Good manufacturing practice for herbal medicines:
• Under GMP:
h) A high level of sanitation and hygiene during manufacture is necessary.
i) A proper storage of products minimizes any risk to their quality.
j) Complaints about marketed products are examined, the causes of
quality defects investigated, and appropriate measures taken in respect
of the defective products to prevent recurrence. They are basically two
types of complaints 1) product quality complaints 2) adverse
reactions/events.
k) A system is available to recall any batch of product from sale or supply.
There should be a Standard Operating Procedure (SOP) for storage of
recalled herbal medicines in a secure segregated area.
l) Personnel: the release of herbal medicines should be authorized by a
person who has been trained in the specific features of the processing
and quality control of herbal materials, preparations and products.
Training records should be maintained and periodic assessments of the
effectiveness of training programmes should be made.
2- Good manufacturing practice for herbal medicines:
• Under GMP:
m) Premises should be designed, located, constructed, adapted and
maintained to suit the operations to be carried out according to
WHO good manufacturing practices for pharmaceutical products.
n) Reference samples and standards should be of a quality
appropriate to their purpose. They should be stored under
appropriate conditions to prevent degradation. Their expiry and/or
revalidation date should be determined and indicated.
o) Effective cleaning of the equipment is particularly important
specially because of the processing of herbal materials that may
generate dust or material which is susceptible to pest-infestation or
microbiological contamination and cross contamination.
2- Good manufacturing practice for herbal medicines:
• Under GMP:
p) Documentation: Essential part of the QA and should exist for all aspects
of GMP
To ensure that:
1. The specifications and procedures for all materials and methods of
manufacture and control are defined.
2. All personnel concerned with manufacture know what to do and when to
do it.
3. Authorized persons have all the information necessary to decide whether
or not to release a batch of a drug for sale.
4. Documented evidence, records and audit track that will permit
investigation exists.
5. The data needed for validation; reviews of statistical analysis are
available.
3- Quality Control
of Herbal Drugs
Introduction
Is it worthy to apply QC
on herbal medicinal
products?
• Herbal medicinal products play an important role not only in self-medication but also in
medical practice.
• Many physicians believe that herbal medicinal products are able to beneficially
complement or even replace chemical medicines and that they have less or even no side
effects.
• There is also a preference consumers for natural therapies.
• Manufacturers should accept the challenge to fulfill the expectations related to quality,
safety and efficacy of these products.
• Herbal medicinal products are always mixtures of a number of substances.
• Quality control is applied on starting raw materials, as well as on finished products.
• The quality of finished products is influenced by the quality of the raw materials used.
Quality control is applied in two stages.
A) QC of starting raw material used in manufacture:
e.g. Dried mentha leaves, mentha oil, mentha extract
To enusre identity (authentic), pure (not cantaminated or adulterated), content
(yield of oil, % of menthol).
B) QC of finished product:
e.g. Mentha herbal tea, products containing oil (capsule, cream, syrup, etc.)
products containing mentha extract (capsule, syrup, etc.)
Mentha extract
Mentha dry
leaves
Mentha oil
Learning
Outcomes
➢ Compare between the specifications of
herbal materials and those of herbal
finished products.
➢ Define herbal drugs, herbal preparations
and herbal extracts.
➢ Classify and Differentiate between different
types of herbal extracts .
➢ Identify the Problems facing QC analysis of
herbal drugs.
What are the specifications of Herbal materials?
The specifications of finished herbal products?
Specifications of Herbal Materials:
• The family and botanical name of the plant used according to
the binomial system.
• Details of the source of the plant.
• Details of which part of the plant is used.
• For dried materials, the drying system should be specified.
• A description of the plant material based on macroscopic and
microscopic examination.
• Suitable identity tests.
• Details of the assay.
• Limit tests such as dry residue of liquids.
• Suitable methods for the determination of possible pesticide,
toxic material and microbial contamination.
Specifications of Finished Herbal Products:
• Tests for microbiological contamination and tests for other
toxicants.
• Uniformity of weight, disintegration time, hardness and
friability, viscosity, consistency and dissolution, if applicable.
• Physical appearance such as color, odor, form , shape, size and
texture.
• Loss on drying, or water content.
• Identity tests, qualitative determination of relevant substances
of the plants (e.g. fingerprint chromatogram).
• Quantification of relevant active ingredients, if they have been
identified, and the analytical methods that are available.
• Limit tests for residual solvents.
Finished herbal products may consist of herbal preparations made
from one or more herbs.
They may also contain excipients in addition to the active ingredients.
Define:
•Herbal drugs
•Herbal drug preparations
•Herbal extracts (classify herbal extracts)
Definitions:
• Herbal drugs: are mainly whole, fragmented or cut, plants, parts of plants,
algae, fungi, lichen in an unprocessed state, usually in a dried form, but
sometimes fresh. Certain exudates that have not subjected to a specific
treatment are also considered to be herbal drugs.
• Herbal drug preparations: are obtained by subjecting herbal drugs to
treatments such as extraction, distillation, expression, fractionation, purification,
concentration and fermentation. These include comminuted or powdered herbal
drugs, tinctures, extracts, essential oils, fatty acids, expressed juices and
processed exudates.
• Herbal extracts: are preparations of liquid, semi-solid or solid consistency,
obtained from herbal drugs.
Herbal extracts are classified
I- in terms of their physical state: liquid, semi-solid (soft) and dry extracts.
II- in terms of containing
1- only herbal extractable matter (never contain excipients or residual
solvents)= Native extracts.
2- additional excipients (technical excipients needed for adjustment or
extraction solvent)= Not-native extracts.
III- in terms of knowledge relating to therapeutically active constituents:
1- Adjusted/ standardized extracts
2- Quantified extracts
3- Other extracts.
Definitions:
• Standardized extracts: are adjusted within a narrow tolerance to a given content
of constituents with known therapeutic activity. (Adjustment is carried out with inert
excipients or by blending of production batches with a higher or lower content of
therapeutically active constituents). E.g. Belladonna leaf dry extract, tincture
0.95% - 1.05% of total alkaloids, expressed as hyoscyamine.
• Quantified extracts: are adjusted to a defined range of constituents which are
accepted to contribute clinical or pharmacological efficacy. (Adjustment can only
be achieved by blending of batches of extracts with the same specifications). E.g.
Ginkgo dry extract (22-27% flavonoids, 2.6-3.2% bilobalide, 2.8-3.4%
ginkgolides).
Marker substances are chemically defined constituents of a herbal drug that
are important for the quality of the finished product whether it is
responsible for pharmacological activity or not.
Definitions:
• Other extracts: are extracts in which constituents with therapeutic activity or
active markers are unknown. They are characterized by a constant amount of
native herbal drug preparation and of inert excipients.
Problems facing QC analysis of herbal drugs (phytomedicines):
Related to the biological variability
▪ Chemo-varieties and chemo cultivars exist.
▪ The source and quality of the raw material are variable.
▪ The methods of harvesting, drying, storage, transportation, and
processing have an effect.
Related to the marker selection:
▪ Herbal drugs are usually mixtures of many constituents.
▪ The active principle(s) is (are), in most cases unknown.
▪ Selective analytical methods or reference compounds may not be
available commercially.
▪ Sometimes chemical markers used for QC purposes, have no therapeutic
activity.
Therefore strict guidelines have to be followed for the
successful production of a quality herbal drug.
Among them are :
✓ proper botanical identification
✓ phytochemical screening
✓ standardization
Standardization of Herbal Drugs
Standardization is the basic prerequisite for consistent
efficacy of herbal products.
It is the body of information and controls that are necessary
to guarantee the constancy of composition, and
consequently the constancy of activity, of an herbal medicine.
Is it easy to obtain
standardized herbal
medicinal products?
Standardization is a hard task and involves:
1- Standards relating to the herbal drug:
- Cultivating the plant according to Good Agricultural Practice (GAP).
- Setting a detailed specification/monograph of the herbal drug.
2- Standards related to the herbal drug preparations:
- The extraction solvent (type and concentration).
- Manufacturing process.
- chemical isolation and characterization of active reference substances.
- Setting up validated analytical methods.
- In-process-control.
3- Standards related to the finished herbal products:
- Good Manufacturing Practice (GMP).
- Setting detailed specifications of the final product.
Marker Substances:
• What does it mean?
• What is the purpose of its presence in an herbal
preparation?
• How to select the marker?
• Should the marker be stated in the label?
Learning
Outcomes
➢ Define Marker substance and criteria of its
selection.
➢ Differentiate the categories of marker
substances.
➢ Highlight the importance of marker
substances.
➢ Use
of
Fingerprint
chromatograms
producing full spectrum standardized
herbal
extracts
for
authentication,
identification and quality control purposes
of herbal products.
➢ Distinguish plants by DNA fingerprinting.
Marker Substances:
?????????????????
• What does it mean?
• What is the purpose of its presence in an herbal
preparation?
• How to select the marker?
• Should the marker be stated in the label?
30
Marker Substances:
Definition:
Marker substances or groups of marker
substances are chemically defined constituents
of herbal drugs, herbal drug preparations and
herbal medicinal products which, according to
the state of scientific knowledge, do not
contribute to therapeutic activity.
They only serve analytical purposes.
31
Marker Substances:
Categories of Markers:
1. Markers which are characteristic for the
respective genus or family of the plant
(suitable for identification tests and assay).
2. Markers which occur universally in plants
(quantification).
32
Marker Substances:
Importance of the Marker:
1. Identification testing .
2. Evidence
for
the
consistency
of
the
manufacturing process (batch – to batch
control).
3. Stability
testing
(batch
–
to
batch
conformity over the whole shelf-life).
33
Marker Substances:
Criteria of Marker Selection:
A constituent of an herbal drug is eligible as a
marker
1. If it is specific for the respective test
2. If selective determination among all other
constituents is possible.
3. Other criteria : stability and availability
34
Marker Substances:
N.B.: As marker substances serve only analytical
purposes:
▪ Adjustment to a defined content of these
substances is not allowed.
▪ The content of marker substances must not be
stated in the label .
Because this would evoke expectations regarding
the therapeutic quality of the product which have
no scientific background.
35
There is often disagreement among
researchers as to which compound is
considered to be the “active” one.
36
A compound that may be “inert” today,
may be considered “active” tomorrow
37
It does not suffice to identify the
respective zone in the chromatogram of
the test solution
38
Explain how herbal fingerprints can be developed
by a multitude of chemical analytical methods.
Full Spectrum Standardized
Herbal Extracts
The most current approach in herbal validation
and standardization is herbal analysis of the
whole herb instead of its component parts.
As far as the standardization of extracts is
concerned, the aim has to be the
reproducibility
of
all
the
chemical
components contained in an extract, including
the unknown ones.
Fingerprint chromatograms must conform to
the total spectrum of constituents.
40
Full Spectrum Standardized
Herbal Extracts
I- Chromatographic Fingerprint
▪ Thin Layer Chromatography
▪ Gas Chromatography
▪ High Performance Liquid Chromatography
▪ Hyphenated Procedures
II- DNA Fingerprint
41
Full Spectrum Standardized
Herbal Extracts
I- Chromatographic Fingerprint
▪ Strongly recommended for the purpose of
quality control of herbal medicines.
▪ They represent appropriately the “chemical
integrities” of the herbal medicines.
▪ Used for authentication and identification of
the herbal products.
42
Full Spectrum Standardized
Herbal Extracts
I- Chromatographic Fingerprint
▪ A
chromatographic
pattern
of
pharmacologically active and or chemically
characteristic constituents present in the
extract.
▪ Demonstrates
both
“sameness”
“differences” between various samples.
and
▪ Evaluates the quality of herbal medicines
globally considering multiple constituents
present in the herbal medicines.
43
Chromatographic Fingerprint:
High Performance Liquid Chromatography (HPLC)
HPLC Fingerprint chromatogram of the spectrum of constituents of five batches of
Hypericum extract (extraction solvent: ethanol 60%) – conformity of batches
44
HPTLC Analysis of
Valerian Species
1: Valeriana officinalis ( authenticated )
2: Valeriana officinalis (Pacific source)
3: Valeriana officinalis (Dutch source)
4: Valeriana radix (PHH8)
5: Valerenic acid
6: Valeriana stichensis
7: Valeriana wallichi (Indian Valerian)
HPLC Chromatographic fingerprint of
different DGL extracts (DGL-Ev, DGL-Ge & DGL-Ph)
140
UV
DGL (EVA)
051DGL (EVA).dat
UV
DGL (Gemini)
049DGL (Gemini).dat
140
UV
DGL (phyto)
050DGL (phyto).dat
120
120
Liquiritigenin
100
80
80
60
60
40
40
DGL-Ev
20
20
DGL-Ge
DGL-Ph
0
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Minutes
17.5
20.0
22.5
0
25.0
27.5
30.0
mAU
mAU
100
Fingerprints of EtOAc & But. Extract of Passiflora
Stability Study by Fingerprint Chromatogram
Chromatographic Fingerprint:
Hyphenated Procedures
Combining
▪ A chromatographic separation system
with
▪ A spectroscopic separation system
with
▪ A spectroscopic detector
In order to obtain structural information
on the analytes present in a sample
49
Chromatographic Fingerprint:
Difficulties in Development of Chromatographic
Fingerprints for Herbal Medicines:
1. When herbal drugs are considered for analysis, a
large number of chemical components are
involved and many of them are in low
concentration
2. Chromatographic instruments and experimental
conditions are difficult to reproduce during real
analysis.
Therefore several data treatments would
be needed during fingerprint analysis.
50
Chromatographic Fingerprint:
Chemometric Approaches and Data Processing
for Chromatographic Fingerprints of Herbal
Medicines:
Chemometrics is the science of extracting
information from chemical systems by datadriven means.
This can be used to compare common pattern of the
chromatographic fingerprints obtained.
Due to complexity of the chromatographic
fingerprint and the irreproducibility of
chromatographic instruments and experimental
conditions
51
DNA Fingerprinting:
DNA Fingerprinting is being applied in:
1. Authentication of medical herbs
2. Quality Control of medical herbs
3. Protection of property rights for new
plant
52
DNA Fingerprinting:
▪ DNA fingerprint is generally independent of
environment
▪ It is consistent throughout different parts and
developmental stages
53
DNA Fingerprinting:
▪ Similarity of DNA fingerprints depends on
genetic closeness of tested samples
▪ DNA fingerprinting can distinguish plants from
different families, genera, species, or cultivars
(cultivated variety)
▪ Clones have the same DNA fingerprint as their
mother plant
54
55
DNA Fingerprinting:
56
Combining
the use of
DNA Fingerprinting
& Chemical Fingerprinting
will be an effective tool in
Authentication and Quality Control
of Herbs
57
Stability
By means of appropriate fingerprint chromatogram, it must be shown that all
ingredients in the preparation are stable and that their proportional content
remains constant.
Example of a Certificate of Analysis
▪
▪
Name of the product:
Content of the product:
▪
Batch number:
▪
Manufacturing date:
▪
Expiry date:
1. Organoleptic properties:
Color:
Odor:
Taste:
2. Determination of certain Pharmacopoeial constants:
1. Determination of extractable matter
3. Macroscopic Investigation:
4. Microscopic Examination:
5. Chromatographic analysis:
TLC (2D fingerprint)
No. of the spots-Rf values-Intensity of the spots
HPLC analysis
No. of peaks-Rt of peaks-Area of peaks-Concentration of the
markers
GC analysis of volatile compounds
2. Determination of water and volatile matter
No. of peaks-Rt of peaks-Area of peaks-Concentration of the
markers
3. Determination of volatile oils
6. Use of NMR Spectroscopy and Multivariate Analysis.
4. Determination of bitterness value
7. Test for pesticide residue.
5. Determination of haemolytic activity
8. Determination of arsenic and heavy metals.
6. Determination of tannins
9. Determination of microorganisms.
7. Determination of swelling index
8. Determination of foaming index
10. Radioactive contamination.
References:
Pharmacognostic Methods for Analysis of Herbal Drugs, According to European
Pharmacopoeia: www.intechopen.com
Identification, evaluation and standardization of hebal drugs: A review: Der
Pharmacia Lettre, 2010, 2(6): 302-315
Facts about standardization of herbal medicine: a review: Journal of Chinese
Intergrative Medicine, October 2012, Vol.10, No. 10.
Quality Control of Herbal Medicines: From Traditional Techniques to State-ofthe-art Approaches: Planta Med 2021; 87: 964–988
References:
Egyptian Guidelines For Registration of Herbal Medicines: Arab Republic
of Egypt, Egyptian drug authority (EDA), Central Administration for
Pharmaceutical products (2021)
Egyptian Herbal Monograph: Egyptian Drug Authority (EDA) 2022
Guideline on quality of herbal medicinal products/traditional herbal
medicinal products: European Medicines Agency, 2022