M.Pharm II sem

1. DETERMINATION OF FOREIGN MATTER, HEAVY METALS, PESTICIDE RESIDUES, PHOTO
TOXIN AND MICROBIAL CONTAMINATION IN HERBAL FORMULATIONS

2. Herbal drugs are derived from plants or their parts by converting them
into phytopharmaceuticals through simple process like harvesting, drying,
and storage.
What are Herbal drug ?

3. Herbal drugs
 Phytomedicines or Phytopharmaceuticals sold as Over The Counter ( OTC) products in
modern dosage forms such as Tablets, Capsules & Liquids for oral use.
 Dietary Supplements containing Herbal Products, also called Neutraceuticals
available in modern dosage forms.
 Herbal Medicines consisting of either Crude, Semi Processed or Processed
Medicinal Plants.

4.  Quality control of crude drugs material, plant preparations and finished
products
 Stability assessment and shelf life
 Safety assessment; documentation of safety based on experience or
toxicological studies
 Assessment of efficacy by pharmacological informations and biological
activity evaluations
Guidelines for Quality Control of Herbal formulation

6.  Foreign matter,
 Heavy metals,
 Pesticide residues,
 Photo toxin
 Microbial contamination in herbal formulations

7.  Parts of the medicinal plant material or materials other than those named
with the limits specified for the plant material concerned,
 Any organism, part or product of an organism, other than that
in the specification and description of the plant material concerned
 Mineral admixtures that is adhering to the medicinal plant materials, such as
soil, stones, sand, and dust.
Foreign matter: NMT 2%w/w
Foreign matter

8. Procedure
100-500 gm of Sample
Spread out as a thin layer Detected
by inspection eye / lens
Separate & weight
Calculate the percentage

9.  Contamination by heavy metals like cadmium, arsenic, lead, copper, and mercury .
 These metals can be determined by colour reactions with special reagents.
 Then these are compared with standards
 Instrumental analysis have to be employed when the metals are in trace amounts.
Determination of heavy metals

10. Procedure:
Take 5g of drug powder(dried at 150°c)
Incinerate the drug
T
o the ash add con.H2S04
Incinerate at 600°c for 2-3 hour Ash obtained
Dissolve the ash in 100ml of 5% HCL Subjected to Atomic
absorption spectroscopy.

11. Determination of pesticide residues
 Pesticide residue - WHO and FAO (Food and Agricultural Organization) set limits for
pesticides, which are usually present in the herbs. These pesticides are mixed with the herbs
during the time of cultivation. Mainly pesticides like DDT, BHC, toxaphene, aldrin cause
serious side-effects in human beings if the crude drugs are mixed with these agents.
 Chromatography (mostly column and gas) is recommended as the principal method for the
determination of pesticide residues.
 Samples are extracted by a standard procedure, impurities are removed by partition and/or
adsorption, and the presence of a moderately broad spectrum of pesticides is measured in a
single determination.
Determination of total chlorine and phosphorus
Most pesticides contain organically bound to chlorine or phosphorus.

12. Preparation of the column
Prepare a Florisil column (external diameter 22 mm) which contains, after settling 10
cm of activated Florisil topped with about 1 cm of anhydrous sodium sulfate. Pre-wet the
column with 40-50 ml of light petroleum. Place a graduated flask under the
to receive the eluate.

13. Preparation of samples
Grind the material to allow it to pass through a sieve no. 710 or 840 and mix thoroughly. Place 20-50 g of the ground
sample into a blender, add 350 ml of acetonitrile with a water content of 35% (to 350 ml of water add sufficient acetonitrile
to produce 1000 ml).
Blend for 5 minutes at a high speed. Filier under vacuum through an appropriate funnel, fitted with filter paper, into a 500-ml
suction flask.
Transfer the filtrate to a 250-ml measuring cylinder and record the volume.
Transfer the measured filtrate to a 1-litre separating funnel and carefully add 100 ml of light petroleum. Shake vigorously
for 1-2 minutes, add 10 ml of sodium chloride (400 g/l) and 600 ml of water.
Hold the separating funnel in a horizontal position and mix vigorously for 30-45 seconds. Allow to separate, discard the
aqueous layer and gently wash the solvent layer with two 100-ml portions of water.

14.  Discard the washings, transfer the solvent layer to a 100-ml glass- stoppered cylinder, and record the volume.
Add about 15 g of anhydrous sodium sulfate and shake vigorously
 Transfer the extract directly to a Florisil column.
 Allow it to pass through the column at a rate of not more than 5 ml per minute.
 Carefully rinse the cylinder with two portions, each of 5 ml, of light petroleum, transfer them to the column,
rinse with further small portions of light petroleum if necessary, and then elute at the same rate with 200 ml of
ether/light petroleum.
 Change the receiver and elute with 200 ml of ether/light petroleum.Again change the receiver and elute with
200 ml of ether/light petroleumn.
 Evaporate each eluate to a suitable volume.

15.  The first eluate contains chlorinated pesticides (aldrin, DDE,), heptachlor, heptachlor
epoxide, lindane, methoxychlor), polychlorinated biphenyls (PCB), and phosphated
pesticides (carbophenothion, ethion and fenchlorphos).
 The second eluate contains chlorinated pesticides (dieldrin and endrin) i phosphated
pesticides (methyl parathion and parathion).
 The third eluate contains phosphated pesticide (malathion).
Combusion of Organic Compounds in oxygen is the preparatory step for the
determination of chlorine and phosphorus. The pesticide is extracted from the sample and
purified if necessary. The extract is concentrated, evaporated to dryness, transferred to a
sample holder, and burned in a suitable conical flask flushed with oxygen. The gases
produced during combustion are then absorbed in a suitable solution. The absorbed chlorine
is determined as chloride and the absorbed phosphorus as orthophosphate, both using
colorimetry.

16. Determination of Pesticide residues by GC
Materials
Fruits of Emblica officinalis (amla), Terminalia belerica (bahera), roots of With in a
somnifera (ashwaganda).
P

ro
E
c
xt
e
r
d
a
u
ct
re
2
:
g of each sample in Soxhlet apparatus with 150 ml hexane.
 Remove traces of water and oil from hexane extract.
 After oil removal, concentrate this extract on rotary evaporator under reduced
pressure and transfer this concentrated extract to clean-up column.
 Collect the elute carefully and make up to 5 ml with hexane.
 Inject Aliquots of above concentrate into precalibrated GC machine equipped with
electron capture detector.
 Programmed operation temperature at 195°C, 200°C, 220°C for column, injector,
and detector respectively.
 Use Purified nitrogen gas as carrier gas at flow rate of 60 ml/min

17. .
ND: not detected, MDL: minimum detected limit

18. Not more than 1%
Determination of pesticide residues
An Acceptable Residue Level (ARL) (in mg of pesticide per kg of plant
material) can be calculated on the basis of the maximum acceptable daily
intake of the pesticide for humans (ADI), as, recommended WHO, and the
mean daily intake (MDI) of the medicinal plant material
ARL=𝐀𝐃𝐈 ×𝐄×𝟔𝟎
𝐌𝐃𝐈×𝟏𝟎𝟎
• ADI = maximum acceptable daily intake of pesticide (mg/kg of body
weight):
• E = extraction factor, which determines the transition rate of the pesticide
from the plant material into the dosage form;
• MDI = mean daily intake of medicinal plant product.

19. Phototoxins are toxins that can cause allergic reactions in particularly susceptible individuals and which ca
cause dangerous photosensitivity in a much broader range of subjects.
Phototoxins are common in a variety of plants like:
many citruses contain essential oils that are photosensitizers;
some herbal remedies;
the carrot family of Apiaceae.
Toxic plants are of 2 types
i. Plant containing toxic ingredients & are known to be toxic to animals
ii.Plants which are normally not toxic to animals but becomes so under
unfavorable conditions.
Determination of phototoxins
 Celery (Apium graveolens) is a marshland plant in the family Apiaceae.
 Extracts of Celery seed are used in herbal medicine.
 Celery seed contains furanocoumarins which are phototoxic.
PHOTOTOXINS

20. Analytical methods of furanocoumarins isolation
Extraction from plant material:
Furanocoumarins are usually isolated from plants by extraction with
solvents such as ethanol, methanol, benzene, chloroform, diethyl and
petroleum ethers, or their combinations.
The most exhaustive extraction of furanocoumarins is achieved with
ethanol and its aqueous solutions, either in cold or on heating. The dense
extract obtained after the evaporation of extract is purified by treatment
with chloroform and diethyl or petroleum ethers.

21. Chromatographic methods in the analysis of furanocounmarins
Column Chromatography (CC):
 The good results for purification, separation of the total furanocoumarins
and the isolation of individual compounds give column chromatography
(CC) a significant advantage of the use of various sorbents and solvent
systems.
 Furanocoumarins can be fractionated on an aluminium oxide column eluted
with petroleum ether, petroleum ether-chloroform (2:1).chloroform, and
chloroform-ethanol (9:1; 4:1; 2:1) mixtures or on silica gel column eluted
sequentially with hexane-chloroform and chloroform-ethanol systems

22. Thin Layer Chromatography (TLC)
Several adsorbents have been applied for the chromatographie analysis of furanocoumarins, e.g. silica gel, C18
layers, alumina, polyamide, Florisil, etc. Analyzed fractions of studied compounds are eluted using several
solvent systems.
1. benzene-acetone (90:10, v/v);
2. toluene-acetone (95:5, V/v);
3. benzene-ethyl acetate (9:1, v/v);
4. benzene-ethylic ether-methanol-chloroform (20:1:1:1, v/v);
5. chloroform, and
6. ethyl acetate-hexane (25:75, v/v) for analysis of furanocoumarins.
The spots of furanocoumarins thin-layer and on paper chromatograms are usually revealed by UV
fluorescence at certain characteristic wavelengths, before or after the treatment with an aqueous- ethanol
solution of potassium hydroxide or with ammonia vapor, or using some other color reactions

23. Some example of Pesticides
 Chlorinated hydrocarbons and related pesticides: BHC, DDT
 Chlorinated phenoxyalkanoic acid herbicides: 2,4-D; 2,4,5-T
 Organophosphonus pesticides: malathion, methyl parathion, parathion
 Carbamate insecticides: carbaryl (carbaril)
 Dithiocarbonate fungicides: ferbam, maneb, nabam, thiram, zineb
 Inorganic pesticides: calcium arsenate, lead arsenate
 Miscellaneous: ethylene dibromide, ethylene oxide, methyl bromide
 Pesticides of plant origin: tobacco leaf and nicotine; pyrethrum flower, pyrethrum extract and
pyrethroids; derris root and rotenoids.

24. Medicinal plants may be associated with a broad variety of microbial contaminants,
represented by bacteria, fungi, and viruses. Inevitably, this microbiological background
depend on several environmental factors and exerts an important impact on the overall quality
of herbal products and preparations.
 Raw materials of herbal formulation are frequently carrier of numerous possibly
pathogenic microorganisms which may cause serious infection.
 The pharmaceuticals is influenced by The WHO has specified total microbial
contamination limits for contamination crude plant materials the limit adopted for
untreated plant material harvested under acceptable hygienic condition.

25.  Herbal drugs normally carry a number of bacteria and molds, often originating in the
soil. Poor methods of harvesting, cleaning, drying, handling, and storage may also cause
additional contamination, as may be the case with Escherichia coli or Salmonella spp.
While a large range of bacteria and fungi are from naturally occurring microflora,
aerobic spore-forming bacteria frequently predominate.
Pretreatment of the test herbal material
 Depending on the nature of the crude herbal material, grind, dissolve, dilute, suspend
or emulsify it using a suitable method and eliminate any antimicrobial properties by
dilution, neutralization or filtration.
 Either phosphate buffer pH 7.2; buffered sodium chloride-peptone solution, pH 7.0; or fluid
medium, used for the test, is used to suspend or dilute the test specimen.

26. Test procedures
Plate count:
For bacteria use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 1 ml of the pre-treated
herbal material and about 15ml of liquefied casein- soyabean digest agar at a temperature not exceeding 45°C.
Alternatively, spread the material on the surface of the solidified medium in a petri dish. If necessary, dilute the
material to obtain an expected colony count of not more than 300.Prepare at least two dishes using the same
dilution, invert them and incubate them at 30-35°C for 48-72 hours, unless a more reliable count is obtained in a
shorter period of time. Count the number of colonies formed and calculate the results using the plate with the
largest number of colonies, up to maximum of 300.
For fungi
Use Petri dishes 9-10 cm in diameter. To one dish add a mixture of 1 ml of the pretreated material and about 15 ml
of liquefied Sabouraud glucose agar with antibiotics (also used is potato dextrose agar with antibiotics) at a
temperature not exceeding 45°C. Alternatively, spread the pretreated material on the
surface of the solidified medium in a petri dish. If necessary, dilute the material as described above to obtain an
expected colony count of not more than 100. Prepare at least two dishes using the same dilution and incubate
them upright at 20-25°C for 5 days, unless a more reliable count is obtained in a shorter period of time. Count
the number of colonies formed and calculate the results using the dish with not more than 100 colonies.

27. Serial dilution:
Prepare a series of 12 tubes each containing 9-10 ml of soyabean-casein digest medium. To each of
the:
 First group of three tubes, add1 ml of the 1:10 dilution of dissolved, homogenized material
(containing 0.1g or 0.Iml of specimen) prepared as described later in the section on Test procedure
for the Enterobacteriaceae and certain other Gram-negative bacteria", below);
 second group of three tubes, add 1 ml of a l:100 dilution of the material;
 third group of three tubes, add 1 ml of a 1:1000 dilution of the material;
 last three tubes, add I ml of the diluent.
 Incubate the tubes at 30-35°C for at least 5 days. No microbial growth should appear in the last three
tubes. If the reading of the results is difficult or uncertain, owing to the nature of the material being
examined, prepare a subculture in a liquid or solid medium, and evaluate the results after a further
period of incubation. Determine the most probable number of microorganisms per gram or per ml of the
material using table.
 If, for the first column, the number of tubes showing microbial growth is two or less, the most probable
number of microorganisms per g or per ml is less than100 table.

28. Determination of total viable aerobic count
Amounts in mg or ml are quantities of original plant material

29. Limits for microbial contaminations in finished products & raw materials

30. Microbial Contamination of Herbal Medicine
Herbal medicines are produced using plant parts; seeds, roots, barks, leaves, and flowers.
Some of the herbal medicine available in the markets worldwide include Pale catechu,
Olibanum and Hyoscyamus niger, and Dangshen.
Many herbalists manufacture herbal medicine without following good manufacturing
practice (GMP) creating alarming concerns of the herbal medicine being contaminated with
various Microbes.
Microbiological quality of herbal medicine produced in Nigeria, South Eastern Nigeria,
South western Nigeria, Pakistan and Malaysia has been investigated by many researchers

31. Methods
 Studies reviewed selected herbal medicines randomly from markets in
Nigeria, South eastern Nigeria, South western Nigeria, Kaduna metropolis
Nigeria, Pakistan and Malaysia.
 The microbiological quality was tested using standard methods
 Bacterial and fungal load were identified using petri plate and spread plate
technique. Characterization was carried out based on microscopic, colonial
and biochemical methods

32. Location of the
Herbal product
Bacteria
South western
Nigeria
Bacillus, Proteus, Staphylococcus, E.coli, Pseudomonas, Klebsiella,
Micrococcus, Corynebacterium, Streptococcus, Enterococcus,
Kaduna Metropolis Staphylococcus aureus, Escherichia coli, Salmonella typhi and Shigella
spp.
South East Nigeria Bacterial spp: E. coli, Klebsiella, Pseudomonas, Streptococcus,
Staphylococcus, Proteus Fungi spp:Candida, Microsporium and
curvularia
Nigeria Bacterial spp: E. coli, Klebsiella, Pseudomonas, Streptococcus,
Staphylococcus, Proteus Fungi spp:Candida, Microsporium and
curvularia
Malaysia E.coli, Salmonella, Streptococcus,
Bacteria found in Herbal medicine from different regions

33. Results
Some of the herbal medicines examined for microbial quality in Nigeria, South eastern Nigeria,
Southwestern Nigeria, Kaduna metropolis of Nigeria, and Malaysia exceeded the
international limit and may cause public health problems
Microbes isolated from the herbal medicines are described in the Table. Majority of the
identified microbes are residents of the air, water, vegetation and soil Among these microbes
Staphylococcus aureus, Bacillus spp. and fungal species (Rhizopus, Aspergillus and
Penicillium) produce enterotoxins, exotoxin and mycotoxins, respectively
High load of microbes in the herbal medicine investigated indicated inadequate sanitary
condition during herbal remedy manufacturing, packaging and storage. Thus, it is recommended
to monitor and maintain the quality and of herbal medicine to safe levels

34. Most of the herbal formulations contain pesticide residues,phototoxins and microbial
contaminants in their final finished products as a chemical and foreign matter. So there
is a need to evaluate the formulation and set the limits for control of contamination and
during their production maintaining of GMP,GLP regulations.
CONCLUSION

35. References
1. Kokate C.K., Gokhale, S.B. 2001. Practical Pharmacognosy. 2"ed. Nirali
2. Prakashan,Pune, p. 14-19.
Mukherjee P.K. 2010. Quality control of herbal drugs. 4 ed. Business
3. Horizones, New Delhi,P. 184-219.
Ansari S.H. 2006. Essentials of Pharmacognosy. l" ed. Birla Publications, New
4.
Delhi, p. 581596.
http://www.ncbi.nlm.nih.gov/pubmed/18396809.
5.
6. Anonymous, 2010. Indian Pharmacopoeia. Vol-3, Government of India,
Ministry of Health and Family Welfare, New Delhi p. 2467-2472.
7. on_of_Herbal_Medicine