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 SO₃evolves 

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

                                            âIncubate 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