Factors affecting Herb Quality, Determination of Arsenic and Heavy Metals, Microorganism and Aflatoxin

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Factors affecting Herb Quality, Determination of Arsenic and Heavy
Metals, Microorganism and Aflatoxin

Contents

• Factors
affecting herb quality

Ø Cultivation
factors

Ø Collection

Ø Drying

Ø Garbling

Ø Packaging

Ø Storage

Ø Preservation

• Limit
test for Arsenic

• Limit
test for Cadmium and lead

• Determination
of Microorganisms

• Determination
of Aflotoxins

Objective

At the end of the session, student will be able to

• Identify
factors affecting herb quality

• Evaluate
crude drugs for presence of microorganisms, heavy metals and Aflotoxins

Factors Affecting Herb Quality

1. Cultivation
factors

Soil
Environmental
factors

• Temperature

• Rainfall

• Climate

• Light

• Humidity

• Altitude

2. Collection

3. Drying

4. Garbling

5. Packaging

6. Storage

7. Preservation

Cultivation Factors – Soil

• Soil
content

• Soil
PH

• Different
plant species – varies – soil and
nutritive requirements

• Soil
– good – half of pores – water and rest with air

• Basic
characters of soil affecting growth and plant development

Physical
properties – particle size
Chemical
properties – Composition of nutrients
Microbial
properties – microorganisms present

• PH
– quality and content of secondary metabolites

• Acidic
soil – not suitable for Leguminous plants – due to poor development

• Ground
nut, sunflower seeds, cotton and rice –
grow better – alkaline soil

• Acidic
PH – disadvantages – solubilize – more
iron

• Tobacco,
cinchona, tea and potatoes – acidic soil

• Alkaline
soil – phosphorous – converted –
insoluble form – calcium phosphate – cannot be made available for plants

• PH
range – 6.5 -7.5

Environmental Factors – Temperature

• Major
factor – control – development and metabolism of plant

• Excessive
temperature or frost –quality

• Eg.
Cinchona – 60 -75˚C

• Coffee – 55 -70 ˚C

• Tea
– 70 -90 ˚C

• Annual
temperature variation – affects – plant cultivation

• Singapore
-1.5 ˚C, Moscow – 29.3 ˚C

Examples

• Nicotiana
rustica – Nicotine – 20 ˚C – decreases – 11-12 ˚C and 30 ˚C

• Quality
of cotton – temp ↑ – molecule – fatty material –cuticle – reoriented – water
cannot penetrate – extremely thin layer -volatile

• Cotton – absorbent – non absorbent

• Other
drug – volatile oil – Buchu, chamomile, ginger and asafoetida

Environmental Factors – Climate

• Each
plant species – specific climate condition – grow well – maximum concentration
of secondary metabolites

• Tropical
and subtropical plant will grow in temperate region

• Continuous
rain – loss of water soluble substances from leaves and roots by leaching

• Low
yield – wet seasons

• Cassia
angustifolia – short term drought – ↑ concentration of Sennosides A and B –
Long term – causes loss of leaf biomass

Environmental Factors – Light

• Amount
and intensity of light – plant – varies

• Wild
state – shade requirements –fulfilled – under cultivation – similar shade
should be provided

• Full
sunshine – higher content of alkaloids –
than shade – solanaceous drugs and
cinchona

• Datura
stramonium var tatula – long exposure – intense light – sharp ↑ – Hyoscine
content – flowering

• Peppermint
leaves – long day – menthone, menthol and traces of menthofuran

• Plants
grown – short day – menthofuran as major constituents – volatile oil

• Hirata
et al – Planta medica – irradiation – intact plants – near ultraviolet range – 290-380nm – synthesis of dimeric
alkaloid

• Flavonoids
and anthocyanin – uv – ß radiation

• Ocimum
basilicum – raised under glass – received no uv radiation – ↑level of both
phenyl propanoids and terpenoids – leaves

Environmental Factors – Altitude

• Important
factor – production of secondary metabolites

• Some
plants – altitudes – some – lower levels

• Coconut
palm – maritime climate – sugar cane – lower land plant

• Tea,
coffee, cocao, rhubarb, tragacanth and cinchona – elevation

• Cinchona
succirubra – grow well in low levels – no alkaloids production

• Bitter
principle – gentian – ↑ – altitude

• Alkaloids
of Aconitum napeullus and Lobelia inflata , oil content Thyme, peppermint
decreases

• Pyrethrum – best yield
– flower heads and pyrethrin – high altitude

Collection

• Drugs
– collected – wild or cultivated plants

• Task
– casual, unskilled – Ipecac

• Skilled
labor – Highly scientific manner

• Season – drug collected – importance – amount and nature of Phyto
constituents

• Rhubarb
– no anthraquinone derivatives – winter

• Warmer
climate – by oxidation – anthranols –
converted – anthraquinones

• Age – not only the total quantity of active
constituents – relative proportions – components in active mixture

• Mentha
piperita – Young leaves – pulegone as leaves mature – menthone and menthol

• Digitalis
pupurea – glycoside content varies – age – Pupurea glycoside A – formed last
but reaches – 50 % total glycoside
content

• Papaver
somniferum – morphine content – highest after 2-3 weeks of flowering

• Ammi
visnaga – unripe fruit – rich in Khellin and visnagin

• Leaves
– flowers begin to open

• Flowers
– just before – fully expanded

• Underground
part – aerial parts die down

• Leaves,
flowers and fruits – not collected –
covered dew or rain

• Discolored,
attacked by insects – rejected

• Hand
picking – difficult – make leaves, flowers – entirely free from other parts

• Barks
– after damp weather – separate more readily from wood

• Gums
gum resin – dry weather – exclude vegetable debris

• Underground
organs – shaking the drug –before, after and during drying or brushing – sufficient to remove sandy soil

• Clay
or heavy soil – washing is essential

• Valerian
– washed in streams – in which they grow – wormy or diseased rejected

• Small
size – replanted

• Larger
roots and rhizomes – sliced before drying

• Gentian
roots – before drying – made in to heaps – ferment

• Seeds
– nuxvomica and cocoa – mucilaginous fruits – washed free from pulp before
drying

Drying

• Duration
– few hours to weeks

• Open
air drying and shade drying

• Artificial
drying – spray dryer, Tray dryers

• Open
air drying – cardamom, cinnamon, clove

• Drying – artificial – rapid – open air drying

• Often
necessary in tropical countries – humid is high

• Europe
– continuous belt dryers – large crops – Digitalis

• Rapid
drying – leaves and flowers to retain aroma – temp – constituents and physical
property

• Leaves,
herbs, flowers – 20 -40 ˚C

• Barks
– 30 -65 ˚C

• Digitalis
– BPC &BP –temp NMT 65 ˚C

• Solar
dryers – advantages- conventional

• Unorganized
drug – spray drying

• Length
of drying – affects the quality

• TAXOL
from Taxus species – length of drying extended – 15 days –recovery of Taxol is
affected

Garbling

• Preparation
of drug to market

• After
collection and drying – drug – scrutinized – to remove unwanted materials

• Dried
crude drug – checked – foreign organic and extraneous matter

• Foreign
organic matter – other parts of plant than the official part

• Extraneous
matter – sand, silica, animal excreta, moulds, insects

• Example
– stems – Senna leaves

• Stalks
and blown clove – clove bud

• Wood
– bark

• Fine
clay, sand, finer crude drugs – removed – shake seivers

• More
specific treatments – product –more appealing

• Yellow
bees wax – sunlight – slow bleaching

• Ginger
– liming / dusting with calcium carbonate – whitening

Packaging

• Dried
drugs –packed- characters and quantity

• Sacks
–containers

• Crude
drugs – moisture – plastic containers/ bags

• Ergot
–paper bags/cardboard containers

• Opium
– wrapped in sheets

• Poisonous
drugs – dried separately – containers –well labelled

Storage

• Storage
– prevent detoriation of drugs – enzymatic hydrolysis

• Drugs
stored – usual containers – sacks, bales, wooden cases, cardboard boxes and
paper bags – tend to absorb -10 -12% moisture

• Digitalis,
Indian hemp – stored

• Large
quantities – sealed containers – dehydrating agent – bottom – quick lime
separated from drug –perforated grid

• Lime – moist –renewed

• Volatile
oil and fixed oils –sealed well filled containers – dark, cool place

• Air
– container – inert gas

• Air
dried drugs – susceptible – attack – insects and other pests – examined
periodically during storage

• Any
mould or worminess – rejected or treated

• Avoid
– microbial contamination – some drugs require –sterilization

• Ethylene
oxide or methyl chloride

• Drugs
so treated – comply acceptable limit for toxic residues

• Senna
pods – 50 ppm – ethylene oxide

Limit Test for Arsenic

• Toxic
and cumulative

The plant materials may contain Arsenic traces due to

• Application
of pesticides

• Environmental
pollution

• Manufacturing
process

• Process
equipment and storage container( due to solvent action the metals may leach
into the final product)

Principle

Sample dissolved in acid

Arsenic acid

Reduced reducing agent â KI, Stannated acid, Zn

arsenious acid

(H) â Nascent hydrogen

Arsine gas (ASH₃)

â React with mercuric chloride paper

Yellow stain

• Intensity
of stain directly proportional – amount
of arsenic present

• The
rate of evolution of gas – maintained by using a particular size of Zinc

• Any
impurity coming along with ASH₃
(like H₂S) – trapped by lead acetate soaked cotton plug

• All reagents used should be arsenic free and
mentioned as AsT

Procedure

• Limit
test is performed by matching the depth of color with that of a standard stain

• Preparation
of sample by acid digestion method:

35 to 70g of coarse
plant material

â Kjeldahl flask

10-25ml of water and
25ml of nitric acid

20 ml of sulphuric
acid

HNO₃ added drop by
drop

Organic matter
destroys (indicated by darkening of solution Vapours of SO₃
evolves

Cool, add 75 ml of
water & 25 ml of ammonium oxalate Until SO₃ vapours develop

Transfer and makeup
to 250 ml with water

Method: Gutzeit test

• Moisten
some cotton wool – lead acetate – allow
to dry- pack it in to a tube – which fits in to the wide mouthed bottle

• Between
the flat surfaces of the tubes – place a
piece of mercuric bromide paper that is large enough to cover their openings

• The
mercuric bromide paper can be fitted by any means provided that

Procedure

• The
whole of the evolved gas passes through the paper

• The
portion of the paper in contact with the gas is a circle( 6.5mm in diameter)

• The
paper is protected from sunlight during the test

25 – 50 ml sample
aliquot

â Wide mouthed bottle

1 g – KI and 10 g –
granulated zinc, 5 ml – stannous chloride

â Keep the assembly in
position

Allow the reaction
for 40 min

Compare yellow stain
on the mercuric chloride paper with standard stain – known quantity of dilute
arsenic AsTS

Preparation of Standard Stain

10 ml stannated
hydrochloride + 1 ml dilute arsenic

â 50 ml water

Resulting solution
exposed to same condition

Yellow stain –
mercuric chloride paper AsR – Standard stain

Limit Test for Cadmium and Lead

Apparatus used

• The
apparatus consist of a digestion vessel, consisting of a silica crucible (62mm
height, 50mm diameter) of capacity 75ml, with a silica cover or lid

Reagents required

• Digestion
mixture for up to 3 hrs

• 2
parts by weight of nitric acid and 1 part by weight of perchloric acid

Standard reference material

• Olive
leaves (Olea europaea) and hay powder

Wet digestion
method

200-250mg – finely
cut air dried plant material – clean silica crucible

1.0ml of the
digestion mixture

â Cover the crucible – oven – heat slowly

100˚C – maintain
temperature – up to 3 hrs – 120˚C and maintain at this temperature for 2 hrs

Raise the temperature
– very slowly to 240˚C, avoiding losses

Dry inorganic residue
+ 2.5ml nitric acid

Atomic absorption
Spectrophotometry

• The
maximum amounts in dried plant materials, which are based on ADI values are

• Lead
– 10mg/kg

• Cadmium
– 0.3 mg/kg.

Determination of Microorganism

• Indicate
the quality – production and harvesting practices

• Medicinal
plant – normally carry bacteria and moulds

• Current
practices – harvesting, handling and production – may cause additional
contamination and microbial growth

Pretreatment of material being examined

For water soluble materials

10 g or 10 ml plant
material

â Dissolve/dilute

Lactose broth or any
other media (not having any antimicrobial activity)

Adjust the volume to
100 ml

PH 7

For non – fatty materials insoluble in water

10 g or 10 ml of
plant material

â Dissolve/dilute

Lactose broth or any
other media (not having any antimicrobial activity)

Adjust the volume to
100 ml + polysarbate 80R

Adjust the PH – 7

For fatty materials

10 g or 10 ml of
plant material

â homogenised
with 5 gm polysorbate 80 –40˚C

85ml lactose broth or
any other media (not having any antimicrobial activity)

Adjust the volume to
100 ml

Adjust the PH – 7

Test for Specific Microorganisms

• Prepared
pretreated material – detection of
different bacterias – enterobacteria and certain gram –ve bacteria

• Homogenised/pretreated
material – incubated – 30-37˚C – 25hrs

Enterobacteria

• Anaerobic
bacteria – Septicemia, urinary tract infection, wound, burn and meningitis

1gm/ml pretreated material
– homogenized + Enterobacteria enriched broth mossel

â Incubated 37˚C 18-48
hrs

Prepare subculture –
plate- violet red bile agar with glucose and lactose

â Incubate

Red color colonies –
presence of enterobacteria

E.coli

• Anaerobic
bacteria – Diarrhea, urinary tract infection, wound, burn and meningitis

Pretreated material –
homogenized + Lactose broth

â Incubated 43 – 47˚C
18-24 hrs

1ml/gm – Macconkey’s
broth

Prepare subculture –
plate- Macconkey’s broth

â Incubate – same
condition

Red color colonies,
rod shaped with reddish zone – presence of E.coli

Salmonella Species

• Anaerobic
gram –ve bacteria – Typhoid, enteric fever, GIT infection and septicemia

Pretreated material –
homogenized + Nutrient broth

â
Incubate 35 – 37˚C 5 -24 hrs

10ml – 100 ml
Tetrathionate bile brilliant green broth

â Incubate 42- 43˚C 5
-24 hrs

Prepare subculture –
Nutrient agar

â Incubate 35 – 37˚C 5
-24 hrs

Colorless to pink-red
or black colonies – presence of Salmonella Species

Pseudomonus aeruginosa

• Aerobic
gram –ve bacteria – Respiratory tract infection

Pretreated material –
buffered Nacl peptone solution – PH 7

1gm/ml – Soyabean casein
digest broth

â Incubate
35 – 37˚C 5 -24 to 48 hrs

Subculture –
Cetrimide agar plate

â Incubate
35 – 37˚C 24 to 48 hrs

Green fluorescence –
presence of Pseudomonas aeruginosa

·
Biochemical test

·
2 or 3 drops – freshly prepared
N,N,N’,N’-tetramethyl-p-phenylenediamine dihydrochloride on a filter paper +
apply a smear of suspected colony – purple color 5 – 10 sec – presence of Pseudomonas

·
Material passes test – colonies do not appear or
confirmatory biochemical test is –ve

Staphylococcus spp

• Gram
+ve bacteria – extracellular toxins

Pretreated material –
buffered Nacl peptone solution – PH 7

1gm/ml – Soyabean
casein digest broth

â Incubate 35 – 37˚C 5
-24 to 48 hrs

Subculture – Baird –
parker agar media

â Incubate
35 – 37˚C 24 to 48 hrs

Black colonies–
presence of Staphylococcus species

Total Viable Count

Total viable count of the material being examined

• Membrane
filtration

• Plate
count

• Serial
dilution

Membrane Filtration

• Use
membrane filter – nominal pore size – not greater than 0.45µm – can effectively
retain bacteria

• Eg.
Cellulose nitrate filters – oily and weakly alcoholic solution

• Cellulose
acetate filters – strongly alcoholic solution

• Keep
filter paper – filtration apparatus – sterilize

• Filter
10ml – material soln – to be tested

• Filter
paper – washed – buffer – fatty
substances –wash with surfactants –
polysorbate 80R, 20R

• Filter
paper – plate – suitable media

• Incubate
– 30 – 35˚C – 5 days

• No
of colonies counted

• Calculate
no of microorganism per ml of solution

Plat Count Method

• 9
-10 cm plate

• 1
ml treated material + 15 ml liquified casein –soyabean digest agar

• Temp
≤ 45 ˚C

• Spread
pretreated material on the surface, solify

• Incubate 30 – 35˚C
– 5 days

• No of colonies counted

• Calculate
no of microorganism per ml of solution

Serial Dilution Method

• 12
tubes – 9-10 ml – soyabean casein digest agar medium

• First
3 tubes + 1 ml of 1:10 dilution pretreated material and media

• Next
3 tubes + 1 ml of 1:100 dilution pretreated material and media

• Next
3 tubes + 1 ml of 1:1000 dilution pretreated material and media

• Last
3 tubes + only diluent or media

• Incubate 30 – 35˚C
– 5 days

• Last
3 tubes – no microbial growth

• Calculate
no of microorganism per g or per ml of the material tested

Determination of Aflatoxins

• Aflatoxins –
poisonous cancer-causing chemicals –
certain molds (Aspergillus flavus and Aspergillus parasiticus)
– grow in soil, decaying vegetation, hay, and grains

• Regularly
found – improperly stored staple commodities

• Contaminated
poultry feed – high percentages of samples of aflatoxin – contaminated chicken meat and eggs in
Pakistan

• Childrens
– Stunded growth, delayed development

• Adults
– tolerance –risk

• Most
carcinogenic substance known

• Metabolized
– liver – reactive epoxide intermediate – aflaoxin M1

• Most
commonly ingested – Aflatoxin B1 – permeate through skin – most toxic

• FDA
– levels in food or feed – 20 to 300 ppb

• 14
types – nature

• Aflatoxin
B₁ and B₂, produced by Aspergillus flavus and A.
parasiticus

• Aflatoxin
G₁ and G₂, produced by Aspergillus
parasiticus

• Aflatoxin
M₁, metabolite of aflatoxin B₁ in humans and animals
(exposure in ng levels may come from a mother’s milk)

• Aflatoxin
M₂, metabolite of aflatoxin B₂ in milk of cattle fed
on contaminated foods

• Aflatoxicol

• Aflatoxin
Q₁ (AFQ₁), major metabolite of AFB₁ in in
vitro liver preparations of other higher vertebrates

Preparation of Sample

• Grind
or reduce NLT 100 g – crude drug

• Larger
the sample –chances of detecting greater

Weigh 50 g powdered
material – conical flask + 170 ml methanol R + 30 ml water

â Shake
vigorously – 30 min – filter

Collect 100 ml
filtrate A

Discard first 50 ml
and collect 40 ml of filtrate B

Eliminate pigments – special clean up procedures

100 ml filtrate A –
250 ML BEAKER + 20 ML Zinc acetate/aluminiumchloride + 80 ml water

â Stir
allow to stand for 5 min

Diatomaceous earth –
mix – filter

Discard first 50 ml,
collect 80 ml – C

Transfer B or C – Separating funnel + 40 ml sodium chloride +
25 ml light petroleum – shake 1 min

Allow layers to
separate-lower layer – another separating funnel

Extract twice 25 ml
dichloromethane shake for 1 min

Allow layers to
separate, combine both lower layers – 125 ml conical flask – boiling chips
–evaporate to dryness

Procedure – B1,B2,G1 & G2

• Residue
+ 0.2 ml of mixture (98 :2) chloroform : acetonitrile – close vial – shake
vigorously until residue dissolves

• TLC
– silica gel G

• Mobile
phase – chloroform : acetone :2-propanol (85:10:5)

• Standard
mixture – Aflatoxin

• Apply
standard and sample

Procedure

• Develop
and observe under UV 365nm

• Std
shows blue fluorescence

• If
residue shows – +ve

• Estimation
– comparing the intensity of spots with standard mixtures

Summary

• Determination
of microorganisms – Indicate the quality
– production and harvesting practices

• Medicinal
plant – normally carry bacteria and moulds

• Current
practices – harvesting, handling and production – may cause additional
contamination and microbial growth

• Aflotoxin – liver cancer

• Detected
by simple chromatography

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