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

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 analyte and reduction of - Folin-Denis reagents to form chromophore - Prussian blue 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 Acetone-free Plant extract Plant extract Pink Alkaline it Rhodanine solution + colour, Acidify to make in methanol with KOH A520nm 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 HPLC method (Makkar and Becker, Unpublished) Free gallic acid (A) Bound plus free gallic acid (B) Column: Nucleosil 120-5 C18 (250 mm x 4.6 mm) Solvents: Buffer A, water-methanol-phosphoric acid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 280 nm Flow rate: 1.2 ml/min 0 100 0 15 100 0 Abs 22 0 100 25 0 100 30 100 0 14 - 15.5 33 100 0 Min

3. KIO 3 method for gallotannins Hartzfeld et al (2002) • Haslam (1965), Bate-Smith (1977) – Transient colored species when KIO 3 is reacted with galloyl esters at low temperature – Variable reaction times – Variable reaction products – Interference from other phenolics

3. KIO 3 method for gallotannins Chemistry of reaction between galloyl esters with KIO 3 O R OH O KIO 3 + A B OH λ max 525 nm yellow OH galloyl esters -KIO 3 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 O G O O O O G G G O Methanol H 2 SO 4 H 3 C OH O G 85 C O OH 11 hours O G 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 1 Abs = 0.0132 * ug + 0.0701 Limit of detection 1.5 ug methyl gallate 0.5 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) Porter et al., 1986 Heat at 95 - 100 C (60 min) 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 d 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 (1 ml) Alkaline hydrolysis + SDS-TEA reagent Amino acids by ninhydrin assay (3 ml) FeCl 3 TA equivalent = A 510 nm * 125-I labelled BSA Gamma counter

Simplified radiolabelled BSA precipitation assay Application of tannin Extract on the disks Filter paper disks Filter paper disks Transfer to Petri dish To Petri dish 125-I BSA 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

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 + PEG Feed Gas Gas Tannin + PEG = Tannin-PEG Complex Microbial protein (MP) Tannin effects Purines 15-N DAPA 32-P 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 53 % Increase 100 % 25 15 20 NDF + PEG 10 15 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 � 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 Butanol-HCl-iron reagent tannins: (0 to 26.3 % as leucocyan. eq.)

� PPC: BSA precipitation (Makkar et (0 to 1.07 g BSA pptd./g) al., 1988) � Tannin bioassay Percent increase in gas on addition of PEG (Makkar et al., 1995) (0 to 457 % increase) (CP : 5.4 to 27 %)

Correlations (r) TP TT % Inc. gas PPC 0.22NS CT 0.52** 0.38* 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 PPC is 0, When GV’ is 0, TT = 1.9 % TT = 2.1 %

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 R 2 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 2 h + 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 + 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