Chemical and biological assays for quantification of major plant secondary metabolites

Harinder P.S. MakkarInternational Atomic Energy Agency

Vienna, Austria

Presentation

• Tannins

• Saponins

• Cyanogens

• Mimosine

• Alkaloids

Analytical proceduresa) Chemical assays

Total tannins

HT: Rhodanine assay, HPLC, KIO3 method

CT: Butanol-HCl assay

b) Protein precipitation assaysRadio-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 ofreagents 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 acidGallotanins Gallic acid + Sugar

100 C, minus oxygen

Measure gallic acid(as below)

Free gallic acid (B)Remove acetone Acetone-free

Plant extractPlant extract

Pinkcolour,A520nm

Alkaline itRhodanine solution +in methanol Acidify to make

0.2 N sulphuric acidwith KOH

A minus B = gallotannins as gallic acid equivalent

Determination of gallotannins2. HPLC method (Makkar and Becker, Unpublished)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 elutionTime (min) A BFlow rate: 1.2 ml/min

Min

280

nmA

bs

14 - 15.5

0 100 015 100 022 0 10025 0 10030 100 033 100 0

3. KIO3 method for gallotanninsHartzfeld 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

BO

OHOH

OHO

RKIO3 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

OO

O

O OG G

G

G

G

O

OO

O

O OG G

G

G

G

O

OHOH

OHO

CH3

MethanolH2SO4

85 C11 hours

All hydrolyzable tannins yield a single galloyl ester—methyl gallate

Hartzfeld et al (2002)

Color yield as a function of pH or time

0

0.1

0.2

0.3

0.4

3.5 4.5 5.5 6.5 7.5

pH

abso

rban

ce (5

25 n

m)

0

0.1

0.2

0.3

0.4

0 30 60 90 120

time (min)

abso

rban

ce (5

25 n

m)

Hartzfeld et al (2002)

Analytical parameters

0

0.5

1

1.5

0 50 100

ug methyl gallate

abs

525

nm

Abs = 0.0132 * ug + 0.0701Limit of detection 1.5 ug methyl gallateLinearity 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 complexDissolve 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 disksFilter paper disks

Application of tanninExtract on the disks

Transfer to Petri dish

To

125-I BSAPetri dish

Gamma counterHenson et al. (2003)

TANNIN BIOASSAYMakkar 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

12

3

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

15-N32-P35-SRNA probes

PurinesDAPA

Evaluation of tannin-containing byproducts and forage

Gas

Microbial protein (MP)

GasFeed 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

0

5

10

15Increase 53 %

ND

F

ND

F +

PEG

05

10152025

ND

F

ND

F +

PEG

Increase 100 %

Acacia saligna Acacia salicina

Gallotannin determination by rhodanine and HPLC methods

Rhodanine method HPLC method(mg/100g DM) (mg/100g DM)

Bro

swe

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 = 465. N.D. 250.1 29.9 194

6. N.D. 92.1, yellow 9.1 47.67. 150.3 432.4 28.3 453.8

Browses: 37 from ILRI

Freeze driedTotal 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 gason 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 of PEG in the in vitro gas method

Tannin %/ Nitrogen %

R2 did not increase

Saponins = Aglycone + sugar

Triterpenoidal saponinsSteroidal 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 of 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 mLin last dilution giving +ve haemolysis

Determination of Saponins – a spectrophotometric assay

Dissolve saponin-rich residue in 80 % methanol

+60 C, 10 minVanillin 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 PBS2 – 3 min

White spots on red background – haemolytic spot

Spray TLC plate with Vanillin-perchloric acid or sulphuric acid100 C, 5 min

Violet or blue spots

Sapindus rarak saponins

Cattle blood spray

Sulphuric acid spray

Cyanogens Glycosides of sugar & -CN containingaglycon (generally taste bitter)

R - C - O - Glucose

H

CN

Glucosidases

Cytochromoxidaseinhibits

ATP

Glucose + HCN

HCN Thiosulfat-Sulphurtransferase

SCN (Isothiocyanate)

Urine

Enhance nitrosamine formation;related to tumour incidence)Energy deprivation

Death(peripheral numbness,convulsions, terminalcoma)

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 waterin 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

SymptomsMimosine

Tyrosine

Present in Leucena leucocephala (CP in leaves ca 25%)

very young leaves: up to 12 %young leaves: 3 - 5 %old leaves: 1 - 2 %stems: 3 - 5 %seeds: 4 - 5 %

Zn, Mg, Cu++Mode of action:

OH

NH2

Pyridoxalphosphate

OHC

MimosinN

O

TransaminasesandCarboxylasesinhibits

CH - CH2

COOH

Cystathionsynthetase

CystathionaseCystineMethionine

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 mLdiazotised 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 inEthanol/chloroform

TLC plateDeveloping solvent: chloroform:methanol (3:1)

butanol:acetone:water (4:5:1)Spray reagent: Dragendorff (bismuth subnitrate, HCl, KI)

Structural, mass determination and identificationUsing Mass spectrophotometric approaches

1. Electron Ionization (EI)

2. Chemical isonization (CI)electrospray (ESI)

3. Atmospheric pressure chemical ionization (APCI)

4. Continuous flow fast atombombardment (CF-FAB)

•GC/MS

•LC/MS

•Collision induced dissociation tandem/MS

Capillary electrophoresis/MSNMR

Quantitation of Antinutritional Factors in Feed and Food ---a book under preparation1. 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