Chemical and biological assays for quantification

of 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
Oxidation of phenolic
-Folin-Denis analyte and reduction of
reagents to form
- Prussian blue chromophore

Standards :

-Tannic acid or Gallic acid

 Determination of Tannins
Phenolics (x)

Tannin phenolics Nontannin phenolics (y)

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

Plant extract
(x)
Treatment using PVPP:
PVPP + plant extract,
centrifugation
(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
Plant extract Acetone-free
Plant extract
Pink Alkaline it
colour, Rhodanine solution +
Acidify to make
A520nm with KOH in methanol 0.2 N sulphuric acid

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 HPLC method (Makkar and Becker, Unpublished)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
Abs 280 nm

Flow rate: 1.2 ml/min

0 100 0
15 100 0
22 0 100
25 0 100
30 100 0
14 - 15.5
33 100 0
Min
 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
 3. KIO3 method for gallotannins
Chemistry of reaction between galloyl esters with
KIO3
O
R OH
O
KIO3 A B
+
OH
λmax 525 nm yellow
OH

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
G
O
G O
O
O
O O G
G G

Methanol O
H2SO4 H3C OH
O
G 85 C
O 11 hours OH
G O OH
O
O
O O G
G G

All hydrolyzable tannins yield a single galloyl ester—methyl gallate

Hartzfeld et al (2002)
 Color yield as a function
of pH or time
0.4 0.4
absorbance (525 nm)

absorbance (525 nm)

0.3 0.3

0.2 0.2

0.1 0.1

0 0
3.5 4.5 5.5 6.5 7.5 0 30 60 90 120
pH time (min)

Hartzfeld et al (2002)
 Analytical parameters

1.5
abs 525 nm

Abs = 0.0132 * ug + 0.0701

0.5 Limit of detection 1.5 ug methyl gallate
Linearity through 120 ug methyl gallate

0
0 50 100
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)

986 Heat at 95 - 100 C

. , 1
l (60 min)
ta e
ter
r
Po Pink colour, A 550 nm
Standard ? Tannic acid
Quebracho tannins (weak colour)
Leucocyanidin equivalent (E of 1%, 1 cm, 550nm) = 460
Radial diffusion assay (Hagerman, 1987)

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
*Protein determination Tannin determination
Alkaline hydrolysis (1 ml)
+
Amino acids by ninhydrin assay SDS-TEA reagent
(3 ml)
FeCl3
TA equivalent = A 510 nm

* 125-I labelled BSA Gamma counter

 Simplified radiolabelled BSA precipitation assay

Application of tannin
Filter paper disks Extract on the disks
Filter paper disks

Transfer to Petri dish

To
Petri dish
125-I BSA
Henson et al. (2003) Gamma counter
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

 In vitro incubation = Simulation of Rumen fermentation

500 mg
Feed

3
2
1 40 ml medium containing rumen
liquor (10 ml), bicarbonate buffer
(10 ml), 5 ml macro- and
micromineral (0.002 ml of the latter)
and 15 ml d. water
Evaluation of tannin-containing byproducts and forage
Feed Feed + PEG
Gas Gas

Tannin + PEG = Tannin-PEG Complex

Microbial protein (MP)

Purines Tannin effects
15-N
32-P DAPA
f( Gas, MP)
35-S
RNA probes
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

Increase 100 % Increase 53 %

25
15
20

NDF + PEG
15 10
NDF +
10
5

NDF
PEG
NDF

5
0 0

Acacia saligna Acacia salicina

 Gallotannin determination by rhodanine
and HPLC methods
Rhodanine method HPLC method
(mg/100g DM) (mg/100g DM)
Broswe

Free gallic Bound + Free gallic Bound +

acid free gallic acid free gallic
acid 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
¾Total phenols: Folin-Ciocalteu reagent
(1.8 to 25.3 % as TA eq.)

¾Total tannins: PVPP-bound phenols

(Folin-Ciocalteu reagent)
(0.2 to 21.4 % as TA eq.)

¾Condensed Butanol-HCl-iron reagent

tannins: (0 to 26.3 % as leucocyan. eq.)
 ¾PPC: BSA precipitation
(Makkar et (0 to 1.07 g BSA pptd./g)
al., 1988)

¾Tannin bioassay Percent increase in gas

(Makkar et on addition of PEG
al., 1995) (0 to 457 % increase)

(CP : 5.4 to 27 %)
 Correlations (r)
TP TT % Inc. gas PPC

CT 0.52** 0.38* 0.22NS 0.41*

TP 0.95*** 0.76*** 0.87***

TT 0.76*** 0.83***

% Inc. gas 0.72***

P < 0.05; ** P < 0.01 ***; P < 0.001; n = 37

 Linear regressions
% increase (GV’) in gas PPC & TP
& TP
GV’= 9.81*TP - 41.9
PPC= 27.1*TP-117.3

When GV’ is 0, When PPC is 0,

TP = 4.3 % TP = 4.3 %

% increase (GV’) in gas PPC & TT

& TT
GV’= 11.97*TT - 24.9 PPC=31.5*TT-58.3

When GV’ is 0, When PPC is 0,

TT = 2.1 % TT = 1.9 %
 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

correlated significant with

• total phenol
• total tannins
Tannin %/ Nitrogen %
• radiolabelled BSA method
R2 did not increase • percentage increase in gas on addition
of PEG in the in vitro gas method

None of these values was a good predictor of feed intake

 Saponins = Aglycone + sugar

Steroidal saponins Triterpenoidal 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 2h

+
A series of 2-fold diluted solution of saponin with PBS

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

+
Vanillin in ethanol 60 C, 10 min

+
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 3–4h

(Chloroform/methanol/water: 65:35:10)

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

Sulphuric acid spray

Cattle blood spray

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

CN inhibits
ATP Cytochromoxidase

Enhance nitrosamine formation;

Energy deprivation related to tumour incidence)

HCN Thiosulfat-
Death SCN
Sulphur
(Isothiocyanate)
(peripheral numbness, transferase
convulsions, terminal
coma) Urine
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

Mimosine --Toxic amino acid
Symptoms
O OH
• Alopecia : hair loss, wool loss
OH • Growth depression
• Goiter
N NH2 N NH2 • Reproductive problem
(long calving to conception interval &
CH - CH disturbed cyclicity)
CH - CH
2 2 • Excessive saliva production
COOH COOH
• Decrease in feed intake
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%
Mode of action: ++
Zn, Mg, Cu

O
Pyridoxalphosphate
OH Transaminases

C
and

H
Mimosin

O
Carboxylases
N NH 2 inhibits

CH - CH
2

COOH

Cystathionsynthetase
Methionine Cystine
Cystathionase
 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

Dissolve in
Precipitate
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)

•GC/MS
2. Chemical isonization (CI)
electrospray (ESI) •LC/MS
3. Atmospheric pressure •Collision induced
chemical ionization (APCI) dissociation
tandem/MS
4. Continuous flow fast atom
bombardment (CF-FAB)

9Capillary electrophoresis/MS
9NMR
Quantitation of Antinutritional Factors in Feed and Food
---a book under preparation
1. Saponins 9. Lectins

2. Cyanogens 10. Mimosine

3. Glucosinolates 11. Canavanine

4. Nitrate 12. DOPA

(L-3,4- dihydroxyphenylalanine)
5. Phytate
13. Gossypol
6. Oxylate
14. Chlorogenic acid
7. Protease inhibitors
15. Alkaloids
8. Amylase inhibitors