Chemical and biological assays for quantification of major plant - - PowerPoint PPT Presentation

Chemical and biological assays for quantification

• f major plant secondary metabolites

Harinder P.S. Makkar International Atomic Energy Agency Vienna, Austria

Presentation

• Tannins
• Saponins
• Cyanogens
• Mimosine
• Alkaloids

Analytical procedures

a) Chemical assays

Total tannins HT: Rhodanine assay, HPLC, KIO3 method CT: Butanol-HCl assay

b) Protein precipitation assays

Radio-labelled protein precipitation assay Protein precipitable phenolic assay Radial diffusion assay

c) Tannin bioassay

Vital steps before the assay….

• 1. Collection of plant material
• 2. Drying of plant material
• 3. Storage of plant material
• 4. Grinding of sample
• 5. Extraction of tannins

Determination of Total Phenols

Reagents :

• Folin-Ciocalteu
• Folin-Denis
• Prussian blue

Oxidation of phenolic analyte and reduction of reagents to form chromophore

Standards :

• Tannic acid or Gallic acid

Determination of Tannins

Tannin phenolics Nontannin phenolics (y) Phenolics (x)

(which bind proteins) (which do not bind proteins)

Treatment using PVPP: PVPP + plant extract, centrifugation Plant extract (x)

(Polyvinylpolypyrrolidone (PVPP; insoluble matrix binds to tannins)

Supernatant (non-tannin phenols) (y)

x and y by Folin Ciocalteu method

Determination of gallotannins

• 1. Rhodanine assay (Inoue & Hagerman, 1988)

Bound plus free gallic acid (A) 2M Sulphuric acid Gallotanins Gallic acid + Sugar 100 C, minus oxygen Measure gallic acid (as below) Free gallic acid (B) Remove acetone Acetone-free Plant extract Plant extract Pink colour, A520nm Alkaline it Rhodanine solution + in methanol Acidify to make 0.2 N sulphuric acid with KOH A minus B = gallotannins as gallic acid equivalent

Determination of gallotannins

• 2. HPLC method (Makkar and Becker, Unpublished)

Free gallic acid (A) Bound plus free gallic acid (B) Column: Nucleosil 120-5 C18 (250 mm x 4.6 mm) Solvents: Buffer A, water-methanol-phosphoric acid (975.5:19.5:1); Buffer B, methanol-water (700:300) Gradient elution Time (min) A B Flow rate: 1.2 ml/min Min 280 nm Abs

14 - 15.5

100 15 100 22 100 25 100 30 100 33 100

• 3. KIO3 method for gallotannins

Hartzfeld et al (2002)

• Haslam (1965), Bate-Smith (1977)

– Transient colored species when KIO3 is reacted with galloyl esters at low temperature – Variable reaction times – Variable reaction products – Interference from other phenolics

Chemistry of reaction between galloyl esters with KIO3

• 3. KIO3 method for gallotannins

B O OH OH OH O R KIO3 A λmax 525 nm yellow

+

galloyl esters

• KIO3 is an oxidizing agent
• A is a transient species with characteristic spectrum in visible wavelengths
• Rate of formation & decay of A varies with R, solvent, pH and temperature
• Gallic acid does not form the colored product A

Hartzfeld et al (2002)

Methanolysis

O O O O O O G G G G G O O O O O O G G G G G O OH OH OH O C H3 Methanol H2SO4 85 C 11 hours

All hydrolyzable tannins yield a single galloyl ester—methyl gallate

Hartzfeld et al (2002)

Color yield as a function

• f pH or time

0.1 0.2 0.3 0.4 3.5 4.5 5.5 6.5 7.5

pH absorbance (525 nm)

0.1 0.2 0.3 0.4 30 60 90 120

time (min) absorbance (525 nm)

Hartzfeld et al (2002)

Analytical parameters

0.5 1 1.5 50 100

ug methyl gallate abs 525 nm

Abs = 0.0132 * ug + 0.0701 Limit of detection 1.5 ug methyl gallate Linearity through 120 ug methyl gallate

Hartzfeld et al (2002)

Determination of Condensed tannins

• 1. Butanol-HCl method (Bate Smith, 1973)
• 2. Butanol-HCl-iron method (Porter et al., 1986)

Plane extract + butanol-HCl (95:5, v/v) + Ferric ions (0.5 ml) (3 ml) (0.1 ml) Heat at 95 - 100 C (60 min) Pink colour, A 550 nm Standard ? Tannic acid Quebracho tannins (weak colour) Porter et al., 1986 Leucocyanidin equivalent (E of 1%, 1 cm, 550nm) = 460

Radial diffusion assay (Hagerman, 1987)

d Agarose + BSA Tannins = (k) d.d Standard? TA equivalent

Protein precipitable phenolics and protein pption capacity

(original by Hagerman and Butler, 1978; modified by Makkar et al., 1988) *Protein-tannin complex Dissolve in 1.5 ml of 1 % SDS Dissolved complex Tannin determination *Protein determination (1 ml) + SDS-TEA reagent (3 ml) FeCl3 Alkaline hydrolysis Amino acids by ninhydrin assay TA equivalent A 510 nm = * 125-I labelled BSA Gamma counter

Simplified radiolabelled BSA precipitation assay

Filter paper disks Filter paper disks Application of tannin Extract on the disks Transfer to Petri dish To 125-I BSA Petri dish Gamma counter Henson et al. (2003)

TANNIN BIOASSAY

Makkar et al (1995)

Tannin + PEG = Tannin-PEG Complexes Tannin + Protein = Tannin-Protein complexes Tannin-Protein complexes + PEG = Tannin-PEG complexes + Protein Tannin + Protein + PEG = Tannin-PEG complexes + Protein

PEG, polyethylene glycol

500 mg Feed 40 ml medium containing rumen liquor (10 ml), bicarbonate buffer

In vitro incubation = Simulation of Rumen fermentation

1 2 3

(10 ml), 5 ml macro- and micromineral (0.002 ml of the latter) and 15 ml d. water

15-N 32-P 35-S RNA probes Purines DAPA Evaluation of tannin-containing byproducts and forage

Gas Microbial protein (MP) Gas Feed Feed + PEG Tannin effects f( Gas, MP) Tannin + PEG = Tannin-PEG Complex

Makkar et al (1995)

Nutritional implications of bound proanthocyanidins (condensed tannins) Condensed tannins Extractable Bound (Unextractable)

Increase in gas on incubation of NDF with PEG

5 10 15

Increase 53 % NDF NDF + PEG

5 10 15 20 25

NDF

NDF + PEG

Increase 100 %

Acacia saligna Acacia salicina

Gallotannin determination by rhodanine and HPLC methods

Rhodanine method HPLC method (mg/100g DM) (mg/100g DM) Broswe Bound + free gallic acid Free gallic acid Bound + free gallic acid Free gallic acid 1. N.D. 85.5 2.1 12.1 2. 127.7 658.2 38.9 463.0 3. 2397.4 15809 743.2 14544 4. 180.1 2578 158.9 2131 n = 46 5. N.D. 250.1 29.9 194 6. N.D. 92.1, yellow 9.1 47.6 7. 150.3 432.4 28.3 453.8

Browses: 37 from ILRI Freeze dried

Total phenols: Folin-Ciocalteu reagent

(1.8 to 25.3 % as TA eq.)

Total tannins: PVPP-bound phenols (Folin-Ciocalteu reagent)

(0.2 to 21.4 % as TA eq.)

Condensed tannins: Butanol-HCl-iron reagent

(0 to 26.3 % as leucocyan. eq.)

BSA precipitation (0 to 1.07 g BSA pptd./g) PPC: (Makkar et al., 1988) Percent increase in gas

• n addition of PEG

Tannin bioassay (Makkar et al., 1995) (0 to 457 % increase) (CP : 5.4 to 27 %)

0.22NS TP 0.95*** 0.76*** 0.87*** TT 0.76*** 0.83*** % Inc. gas 0.72*** TP TT % Inc. gas PPC CT 0.52** 0.38* 0.41* P < 0.05; ** P < 0.01 ***; P < 0.001; n = 37

Correlations (r)

GV’= 9.81*TP - 41.9 PPC= 27.1*TP-117.3 GV’= 11.97*TT - 24.9 PPC=31.5*TT-58.3 When GV’ is 0, TP = 4.3 % When PPC is 0, TP = 4.3 % When GV’ is 0, TT = 2.1 % When PPC is 0, TT = 1.9 %

Linear regressions

% increase (GV’) in gas & TP PPC & TP % increase (GV’) in gas & TT PPC & TT

Conclusions

Browses with.. Total phenol 4.3 % Total tannins 2.0 %

No significant adverse effects in ruminants

Tannin assays and biological significance

In vivo apparent digestibility coefficients of N None of these values was a good predictor of feed intake correlated significant with

• total phenol
• total tannins
• radiolabelled BSA method
• percentage increase in gas on addition
• f PEG in the in vitro gas method

Tannin %/ Nitrogen % R2 did not increase

Saponins = Aglycone + sugar

Triterpenoidal saponins

Steroidal saponins

Acacia auriculoformis saponins

Yucca saponins

Some major biological effects:

• 1. Haemolytic & piscicidal activity (toxicity towards fish)
• 2. Effect on palatability (bitter taste) & feed intake
• 3. Interaction with mucous membranes & influence on nutrient absorption
• 4. Bloat production
• 5. Photosensitization
• 6. Insecticidal & molluscicidal
• 7. Human health aspects: hypocholesterolemic, anticarcinogenic, immune

stimulating, antifungal, antibacterial and antiviral effects

Some other roles of Saponins

• 1. Effect on partitioning of nutrients in rumen

2. Concentrattion dependent effect on growth

• f E. coli
• 3. Growth stimulating effect on lambs & fish

Determination of Saponins – as haemolytic unit

• 1. Extract saponins in 50 to 80 % methanol
• 2. Remove methanol and lyophilize aqueous phase

(OR extract saponins from aqueous phase with butanol, remove butanol & then lyophilize/dry)

• 3. 10-20 mg saponin enriched fraction + 1 mL PBS
• 4. Three % suspension of red blood cells in PBS

Place 50 µl of the cell suspension in separate wells of microtitre plate + A series of 2-fold diluted solution of saponin with PBS

2 h

One haemolytic activity (HeU) = the least amount of saponins per mL in last dilution giving +ve haemolysis

Determination of Saponins – a spectrophotometric assay

Dissolve saponin-rich residue in 80 % methanol + 60 C, 10 min Vanillin in ethanol + Sulphuric acid (72 %) Absorbance at 544 nm Expression of values: Diosgenin equivalent; range 0 – 125 µg in the assay

Qualitative and quantitative evaluation of Saponins – a TLC approach

• 1. Dissolve saponin-rich residue in 50 - 80 % methanol
• 2. Apply 5 µl on TLC plate
• 3. TLC plate in Chromatography chamber

(Chloroform/methanol/water: 65:35:10) 3 – 4 h Dry TLC plate Spray TLC plate with 6 % erythrocyte in PBS 2 – 3 min White spots on red background – haemolytic spot Spray TLC plate with Vanillin-perchloric acid or sulphuric acid 100 C, 5 min Violet or blue spots

Sapindus rarak saponins

Cattle blood spray Sulphuric acid spray

Cyanogens

Glycosides of sugar & -CN containing aglycon (generally taste bitter) R - C - O - Glucose H CN Glucosidases Cytochromoxidase inhibits ATP Glucose + HCN

HCN Thiosulfat- Sulphur transferase SCN (Isothiocyanate) Urine Enhance nitrosamine formation; related to tumour incidence)

Energy deprivation Death

(peripheral numbness, convulsions, terminal coma)

Quantification of cyanogenic glucosides as total cyanide

Picric acid paper method

• 1. A screw capped bottle with Whatmann 3MM paper strip

dipped in picrate. Dried and fixed on the wall

• 2. Sample (20-100mg) + 1 mL Phosphate buffer (pH 8, 0.1 M) + 4 mL water

in a screw capped bottle 30 C, 16 h Remove picrate paper + 5 ml distilled water (extract for 30 min) Absorbance at 510nm Standard: 240 mg KCN/L = 100 µg HCN/mL (0.1 to 1 mL in screw-capped bottle)

Quantification of cyanogenic glucosides as total cyanide

Extraction and quantitation of total cyanide

• 1. Sample (4 g) + 125 mL water + 2.5 chloroform in Kjeldahl flask

Distill Absorb HCN in 2 % KOH (total volume after extraction = 20 ml) An aliquot 5 mL + 5 mL alkaline picrate Boiling water bath, 5 min Absorbance at 520nm Standard: 240 mg KCN/L = 100 µg HCN/mL

• -Toxic amino acid

N O OH CH - CH COOH NH2 2 N OH CH - CH COOH NH2 2

• Alopecia : hair loss, wool loss
• Growth depression
• Goiter
• Reproductive problem

(long calving to conception interval & disturbed cyclicity)

• Excessive saliva production
• Decrease in feed intake

Symptoms Mimosine

Tyrosine

Present in Leucena leucocephala (CP in leaves ca 25%) very young leaves: up to 12 % young leaves: 3 - 5 %

• ld leaves:

1 - 2 % stems: 3 - 5 % seeds: 4 - 5 %

Zn, Mg, Cu ++ Mode of action: OH NH2 Pyridoxalphosphate OHC Mimosin N O Transaminases and Carboxylases inhibits CH - CH 2 COOH Cystathionsynthetase Cystathionase Cystine Methionine

Mimosine determination (spectrophotometric method)

2 g leaves + 20 mL boiling water Boil for 5 min, then cool + 20 mL 0.2 M HCl Homogenize using ultraturrax, centrifuge to collect supernatant An aliquot 10 mL + 10 mg activated charcoal Boiling, 10 min Cool, filter, make volume 10 mL (A) An aliquot (3.5 mL) of A + 1 mL Phosphate buffer (pH 7, 0.25M) + 0.5 mL diazotised p-nitroaniline reagent (1 : 1, p-nitroaniline in MeOH & Na-nitrite) Room temp., 15 min Absorbance at 400 nm Standard curve: mimosine from 1.25 – 50 nmol/3.5 mL

Mimosine determination (HPLC method)

0.5 g leaves + 50 mL 0.1 M Citric acid or 0.1 M HCl Cation exchange resin- Na form Eluate containing mimosine An aliquot adjusted to pH 8.5 with 1 M diammomium hydrogen phosphate HPLC on Bondapak C-18 column

• Elute with 1% di-ammonium hydrogen phosphate

made to pH 2.4 with o-phosphoric acid

• Flow rate of 2 mL/min
• Detection at 282 nm

25 ppm mimosine (25 mg/litre of 0.1M citric acid) 25 ppm 2,3-DHP (25 mg/litre in 0.1M citric acid) 25 ppm 3,4-DHP (25 mg/litre in 0.1M citric acid)

TLC method for Alkaloids

Plant extract in methanol/ethanol with 10 % acetic acid Add ammonium hydroxide slowly Precipitate Dissolve in Ethanol/chloroform

TLC plate Developing solvent: chloroform:methanol (3:1) butanol:acetone:water (4:5:1) Spray reagent: Dragendorff (bismuth subnitrate, HCl, KI)

Structural, mass determination and identification Using Mass spectrophotometric approaches

• 1. Electron Ionization (EI)
• 2. Chemical isonization (CI)

electrospray (ESI)

• 3. Atmospheric pressure

chemical ionization (APCI)

• 4. Continuous flow fast atom

bombardment (CF-FAB)

• GC/MS
• LC/MS
• Collision induced

dissociation tandem/MS

Capillary electrophoresis/MS NMR

Quantitation of Antinutritional Factors in Feed and Food

• --a book under preparation
• 1. Saponins
• 2. Cyanogens
• 3. Glucosinolates
• 4. Nitrate
• 5. Phytate
• 6. Oxylate
• 7. Protease inhibitors
• 8. Amylase inhibitors
• 9. Lectins
• 10. Mimosine
• 11. Canavanine
• 12. DOPA

(L-3,4- dihydroxyphenylalanine)

• 13. Gossypol
• 14. Chlorogenic acid
• 15. Alkaloids