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

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

  1. Soil

  2. 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

  1. Physical
    properties – particle size

  2. Chemical
    properties – Composition of nutrients

  3. 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

â

Arsenic acid

Reduced reducing agent  â  KI, Stannated acid, Zn

â

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

â

20 ml of sulphuric
acid

â

                  HNO3 added drop by
drop

â

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

â

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

â

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

â

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

â

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

â

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

â

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

â

Dry inorganic residue
+ 2.5ml nitric acid

â

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)

â

Adjust the volume to
100 ml

â

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)

â

Adjust the volume to
100 ml + polysarbate 80R

â

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)

â

Adjust the volume to
100 ml

â

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

â

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

â

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

â

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

â

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

â

Discard first 50 ml,
collect 80 ml – C

â

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

â

Allow layers to
separate-lower layer – another separating funnel

â

Extract twice 25 ml
dichloromethane shake for 1 min

â

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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