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


       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


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







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


       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


       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


       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


       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


       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 – 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)


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


       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


       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


       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


       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


       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


       Develop and observe under UV 365nm

       Std shows blue fluorescence

       If residue shows  - +ve

       Estimation – comparing the intensity of spots with standard mixtures


       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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