MICROBIAL CONTAMINATION IN HERBS AND THEIR FORMULATIONSVK VIKRAM VARMA
INTRODUCTION
SOURCES OF CONTAMINATION
RAW MATERIALS
PACKAGING MATERIALS
LIMITS FOR MICROBIAL CONTAMINATION
LIMITS AS PER WHO
TYPES OF CONTAMINATION
DIRECT CONTAMINATION
CROSS CONTAMINATION
DETERMINATION OF CONTAMINATION
TOTAL VIABLE COUNT
PRETREATMENT OF HERBAL MATERIALS
REFRENCES
Adulteration is the debasement of Genuine materials3.
It may be deliberated or accidentally done.
In crude drugs, this practice includes the substitution of the original crude drug, fully or partially with sub-stranded substances.
Sub-stranded substances include- Inferiority and spoilage.
Inferiority- Natural sub-stranded condition whose natural constituent is below the minimum standard.
Spoilage- sub-stranded condition produced by microbial or pest infestation.
What is Efficacy
Why Efficacy is required for Herbal products
How Efficacy of Herbal products ensured
What are the various avenues of determining efficacy
Regulatory requirement for setting herbal drug industryRAGHAV DOGRA
The World Health Organization (WHO) estimates that 80 percent of the population of some Asian and African countries presently use herbal medicine for some aspect of primary health care.Pharmaceuticals are prohibitively expensive for most of the world's population, half of whom lived on less than $2 U.S. per day in 2002. In comparison, herbal medicines can be grown from seed or gathered from nature for little or no cost
patent (/ˈpætənt/ or /ˈpeɪtənt/) is a set of exclusive rights granted by a sovereign state to an inventor or assignee for a limited period of time in exchange for detailed public disclosure of an invention. An invention is a solution to a specific technological problem and is a product or a process. Patents are a form of intellectual property.
MICROBIAL CONTAMINATION IN HERBS AND THEIR FORMULATIONSVK VIKRAM VARMA
INTRODUCTION
SOURCES OF CONTAMINATION
RAW MATERIALS
PACKAGING MATERIALS
LIMITS FOR MICROBIAL CONTAMINATION
LIMITS AS PER WHO
TYPES OF CONTAMINATION
DIRECT CONTAMINATION
CROSS CONTAMINATION
DETERMINATION OF CONTAMINATION
TOTAL VIABLE COUNT
PRETREATMENT OF HERBAL MATERIALS
REFRENCES
Adulteration is the debasement of Genuine materials3.
It may be deliberated or accidentally done.
In crude drugs, this practice includes the substitution of the original crude drug, fully or partially with sub-stranded substances.
Sub-stranded substances include- Inferiority and spoilage.
Inferiority- Natural sub-stranded condition whose natural constituent is below the minimum standard.
Spoilage- sub-stranded condition produced by microbial or pest infestation.
What is Efficacy
Why Efficacy is required for Herbal products
How Efficacy of Herbal products ensured
What are the various avenues of determining efficacy
Regulatory requirement for setting herbal drug industryRAGHAV DOGRA
The World Health Organization (WHO) estimates that 80 percent of the population of some Asian and African countries presently use herbal medicine for some aspect of primary health care.Pharmaceuticals are prohibitively expensive for most of the world's population, half of whom lived on less than $2 U.S. per day in 2002. In comparison, herbal medicines can be grown from seed or gathered from nature for little or no cost
patent (/ˈpætənt/ or /ˈpeɪtənt/) is a set of exclusive rights granted by a sovereign state to an inventor or assignee for a limited period of time in exchange for detailed public disclosure of an invention. An invention is a solution to a specific technological problem and is a product or a process. Patents are a form of intellectual property.
In this slide contains Introductionnof Indian pharmacopeia, ayurvedic, unani pharmacopeia and monographs of herbal drugs.
Presented by: P.SUDHEER KUMAR (Department of pharmaceutical analysis ).RIPER, anantapur
Herbal drugs are usually considered safe but when taken along with other drugs of chemical origin (allopathic drugs), they do interact with them and cause Bio-drug interaction
INDIAN AND INTERNATIONAL PATENT LAW AS APPLICABLE TO HERBAL AND NATURAL PRODUCTSTejaswini Chandra
ITS A USEFUL TOPIS FOR LAW STUDNETS REGARDING THE HERBAL DRUGS AND NATURAL PRODUCTS .
ALSO USEFUL FOR THE MPHARM STUDENTS OF ALL DOMAIN TO STUDY REGARDING THE PATENT LAW
HOPE YOU ALL MAKE USE OF IT TO FULLEST AND SUCCEED.
Herbal remedies are plants used as medicine.
People use herbal remedies to help prevent or cure disease.
They use them to get relief from symptoms, boost energy, relax, or lose weight.
Herbal remedies are a type of dietary supplement. They are not medicines.
Herbals are not regulated like medicines.
Herbals do not need to be rigidly tested before they are sold.
Herbals may not work as claimed.
Labels do not need to be approved.
It may not list the correct amount of an ingredient.
Stability testing of natural products.docxKipaPape
Stability is defined as the capacity of drug to remain within established specification limits to maintain its identity, strength, quality and purity throughout the retest or expiration dating period.
It is the ability of formulations to retain its physical, chemical, microbiological and toxicological parameters same that time of manufacturer.
As we all know chromatographic fingerprinting of botanicals is a quite recent concept. This presentation will help to the beginners to understand basic thories and fundamantals of thin layer chomatography. The presentation will also provide basic experiemental understanding to perfrom HPTLC fingerprinting of samples/extracts/formulations.
In this slide contains Introductionnof Indian pharmacopeia, ayurvedic, unani pharmacopeia and monographs of herbal drugs.
Presented by: P.SUDHEER KUMAR (Department of pharmaceutical analysis ).RIPER, anantapur
Herbal drugs are usually considered safe but when taken along with other drugs of chemical origin (allopathic drugs), they do interact with them and cause Bio-drug interaction
INDIAN AND INTERNATIONAL PATENT LAW AS APPLICABLE TO HERBAL AND NATURAL PRODUCTSTejaswini Chandra
ITS A USEFUL TOPIS FOR LAW STUDNETS REGARDING THE HERBAL DRUGS AND NATURAL PRODUCTS .
ALSO USEFUL FOR THE MPHARM STUDENTS OF ALL DOMAIN TO STUDY REGARDING THE PATENT LAW
HOPE YOU ALL MAKE USE OF IT TO FULLEST AND SUCCEED.
Herbal remedies are plants used as medicine.
People use herbal remedies to help prevent or cure disease.
They use them to get relief from symptoms, boost energy, relax, or lose weight.
Herbal remedies are a type of dietary supplement. They are not medicines.
Herbals are not regulated like medicines.
Herbals do not need to be rigidly tested before they are sold.
Herbals may not work as claimed.
Labels do not need to be approved.
It may not list the correct amount of an ingredient.
Stability testing of natural products.docxKipaPape
Stability is defined as the capacity of drug to remain within established specification limits to maintain its identity, strength, quality and purity throughout the retest or expiration dating period.
It is the ability of formulations to retain its physical, chemical, microbiological and toxicological parameters same that time of manufacturer.
As we all know chromatographic fingerprinting of botanicals is a quite recent concept. This presentation will help to the beginners to understand basic thories and fundamantals of thin layer chomatography. The presentation will also provide basic experiemental understanding to perfrom HPTLC fingerprinting of samples/extracts/formulations.
Phytochemical and acute toxicity study of leaves of artocarpus heterophyllus lampharmaindexing
Artocarpus heterophyllus Lam commonly known as jack fruit widely distributed in north east India, West Bengal and south Karnataka. In the present study intended with various phytochemical screening and toxicity studies were carried out on the leaves of Artocarpus Heterophyllus. The phytochemical study shows the presences of flavonoids, tannins, saponins and carbohydrates in methanolic and aqueous extracts. In acute toxicity study both the extract were found safe on a dose of 2000 mg/Kg.
Similar to Determination of foreign matter, heavy metals, pesticide residues, photo toxin and microbial contamination in herbal formulations (20)
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Determination of foreign matter, heavy metals, pesticide residues, photo toxin and microbial contamination in herbal formulations
1. Determination of foreign matter, heavy metals,
pesticide residues, photo toxin and microbial
contamination in herbal formulations
Presented by
selim akhtar
Roll No: 20HMPA04
M.pharm (Pharmaceutical Analysis)
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
5.
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
To 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).
Combustion of the organic matter
Combustion of the organic matter 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).
Procedure:
Extract 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
18. Determination of pesticide residues
Not more than 1%
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
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
Prakashan,Pune, p. 14-19.
2. Mukherjee P.K. 2010. Quality control of herbal drugs. 4 ed. Business
Horizones, New Delhi,P. 184-219.
3. Ansari S.H. 2006. Essentials of Pharmacognosy. l" ed. Birla Publications, New
Delhi, p. 581596.
4. http://www.ncbi.nlm.nih.gov/pubmed/18396809.
5. WHO guidelines ISBN 978 92 4 1547 16 1.
6. Anonymous, 2010. Indian Pharmacopoeia. Vol-3, Government of India,
Ministry of Health and Family Welfare, New Delhi p. 2467-2472.
7. https://www.researchgate.net/publication/336115407_Microbial_Contaminati
on_of_Herbal_Medicine