Upload
vanbao
View
216
Download
1
Embed Size (px)
Citation preview
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 HPLC method (Makkar and Becker, Unpublished)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