Diversity of the 3,429 vascular plant species, of which 1,605 (46.8%) are endemic . Apicultural season in Central Chile starts at end of winter (middle August) and ends at the beginning of autumn (April). Honey harvesting takes place from December to April. Geographical and Botanical Origin. Certification
• WHAT WE DO In vitro In Vivo
13 UNIFLORAL NATIVE HONEYS ARE PRODUCED IN CHILE
EscalloniaEscallonia pulverulentapulverulenta
CORONTILLO GevuinaGevuina avellana
AVELLANO
EucryphiaEucryphia cordifolia
ULMO
Inhibition Halo Zone of Bacterial Growth
Experimental Procedure : Antibacterial activity of honeys and eextractsxtracts was performed by microdilution of trypticase/soy broth technique (37°x 24 h) Statistical analysis ANOVA a=0,05. in vitro
Unifloral Honeys Escherichia Enterobacter Pseudomonas Salmonella Staphylococcus colicolicoli aerogenesaerogenesaerogenes aeruginosaaeruginosaaeruginosa typhityphityphi aureusaureusaureus aureusaureusaureus Ulmo (Eucryphia 19,13 a 22,45 a 17,98 a 18,26 a 8,13 a cordifolia) Quillay (Quillaja 15,79 ab 17,66 b 13,15 bc 16,01 b 10,82 b saponaria) Corontilla (Escallonia 17,31 ab 13,54 ab 4,83 c No No pulverulenta) inhibition inhibition
Yuyo ( Brassica rapa) 6,12 cd s. inhibición 3,93 c No No inhibition inhibition Alfalfa chilota (Lotus 6,47 c 6,78 b 21,73 b No No uliginosus) inhibition inhibition
Maqui (Aristotelia 7,98 cd 13,62 ab 24,67 b No No chilensis) inhibition inhibition
Avellano (Gevuina 13,11 bc s. inhibición s. inhibición 15,90 b 10,82 b avellana) IINHIBITIONIINHIBITION OFOF ERWINIAERWINIA CAROTOVORACAROTOVORA GROWTHGROWTH
Honey Mean area of inhibition (cm2) Bacteria Culture Control 0,00a X – 042003-M182 25,81b ± 3,75 b
X- 032004-M335 Ulmo 22,21b ± 3,49 b X- 122002- M085 Honey 21,61b ± 2,52 b X-42002-M084 20,41b ± 3,06 b X-2004-M296 16,20bc ± 3,25 c V- 2004- M294 13,80c ± 3,75 c V- 112001-M007 13,20c ± 2,28 c RM- 1120002M121 9,00c ± 7,20 d V- 092002-M017 5,40c ± 6,23d
Statistic Analisis : 1. Andeva ( α=0.05); F 2,57> 2,510 probability = 0,04 2. Tukey Test (α= 0,05) Honeys of Different Origin but of the same Beekeeper showed different biological activity (Alberto Poch)
Multiple Range Tests ------Method: 95,0 percent Tukey HSD Count Mean Homogeneous Groups ------Honey 338 10 3,0 X Quillay False Honey 10 3,18 X Artificial Honey 343 10 3,36 X Polifloral Honey 340 10 4,59 X Polifloral Miel 342 10 15,3 X Ulmo ------Contrast Difference +/- Limits ------Miel 338 - Miel 340 -1,59 1,83602 Miel 338 - Miel 342 *-12,3 1,83602 Miel 338 - Miel 343 -0,36 1,83602 Miel 338 - Miel Falsa -0,18 1,83602 Miel 340 - Miel 342 *-10,71 1,83602 Miel 340 - Miel 343 1,23 1,83602 Miel 340 - Miel Falsa 1,41 1,83602 Miel 342 - Miel 343 *11,94 1,83602 Miel 342 - Miel Falsa *12,12 1,83602 Miel 343 - Miel Falsa 0,18 1,83602 ------Certificación de Origen Botánico Muestra Miel X-2005-M342 Apicultor: Alberto Poch Beekeeper Eucryphia cordifolia Mr. Alberto Poch Lotus uliginosus Blepharocalyx cruckshanksii Weinmannia trichosperma Tristerix tetrandrus Luma apiculata Trifolium repens Hypochaeris radicata Amomyrtus luma Eucalyptus globulus M elilotus indicus Rubus idaeus Plantago sp.
0,0 20,0 40,0 60,0 80,0 100,0 P o rc e n ta je Muestra Miel X-032005-M342 Rango 95% confianza Especie Nombre Común N° granos Porcentaje +/- Min Max Eucryphia cordifolia Ulmo 1120 70,088 2,245 67,843 72,333 Lotus uliginosus Alfalfa chilota 225 14,080 1,705 12,375 15,785 Blepharocalyx cruckshanksii Temu 126 7,885 1,321 6,563 9,206 Weinmannia trichosperma Tineo 105 6,571 1,215 5,356 7,786 Otras 9 especies (<1%) 1,377 0,571 0,806 1,948 M uestra M iel Río San Pedro Código X-032006-M511
E ucryphia cordifolia Lotus uliginosus Castanea sativa W einm annia trichosperm a R u b u s s p . Amomyrtus luma A extoxicon punctatum C aldcluvia paniculata H ypochaeris radicata Lum a apiculata Beekeeper Trifolium repens C issus striata Mr. Ian Staples Taraxacum officinalis Gevuina avellana Raphanus sativus
0 ,0 2 0 ,0 4 0 ,0 6 0 ,0 8 0 ,0 1 0 0 ,0 P o r c e n t a je
E s p e c i e Nombre Común n ° p ó le n e s P o r c e n t a je EMV M in M a x Eucryphia cordifolia U lm o 1 0 5 3 89,389 1,759 8 7 ,6 3 0 9 1 ,1 4 8 Lotus uliginosus A lfalfa chilota, lotera 4 8 4 ,0 7 5 1 , 1 2 9 2 ,9 4 6 5 ,2 0 4 Castanea sativa C a s ta ñ o 2 0 1 ,6 9 8 0 , 7 3 8 0 ,9 6 0 2 ,4 3 6 W einmannia trichosperma T in e o 1 8 1 ,5 2 8 0 , 7 0 0 0 ,8 2 8 2 ,2 2 9 R u b u s s p . 1 3 1 ,1 0 4 0 , 5 9 7 0 ,5 0 7 1 ,7 0 0 Amomyrtus luma L u m a 6 0 ,5 0 9 0 , 4 0 7 0 ,1 0 3 0 ,9 1 6 Aextoxicon punctatum O liv illo 5 0 ,4 2 4 0 , 3 7 1 0 ,0 5 3 0 ,7 9 6 Caldcluvia paniculata T ia c a 4 0 ,3 4 0 0 , 3 3 2 0 ,0 0 7 0 ,6 7 2 Hypochaeris radicata H ierba del chancho 3 0 ,2 5 5 0 , 2 8 8 - 0 , 0 3 3 0 ,5 4 2 Trifolium repens T rébol blanco 2 0 ,1 7 0 0 , 2 3 5 - 0 , 0 6 5 0 ,4 0 5 Luma apiculata A r r a y á n 2 0 ,1 7 0 0 , 2 3 5 - 0 , 0 6 5 0 ,4 0 5 Raphanus sativus R ábano silvestre 1 0 ,0 8 5 0 , 1 6 6 - 0 , 0 8 1 0 ,2 5 1 Gevuina avellana A v e lla n o 1 0 ,0 8 5 0 , 1 6 6 - 0 , 0 8 1 0 ,2 5 1 Taraxacum officinalis D iente de león 1 0 ,0 8 5 0 , 1 6 6 - 0 , 0 8 1 0 ,2 5 1 Cissus striata V o q u i 1 0 ,0 8 5 0 , 1 6 6 - 0 , 0 8 1 0 ,2 5 1 Water Solution of Ulmo Honey 342 in different concentrations tested against de Escherichia coli
45000000
40000000 y = 2E+09x4 - 3E+09x3 + 2E+09x2 - 5E+08x + 4E+07 R 2 = 0,8838 35000000
30000000
25000000 MIC = Minimal Inhibitory Concentration 20000000
15000000
10000000 UFC(Colonias Bactaerianas/ml)
5000000
0
0% 10% 15%20% 30% 40% 50% 60% 70% 80% Concentracción % (v/v)
Lineal reggression between the concentration of ulmo honey versus the concentration of bacteria UFC (unit former colonies per ml) Escherichia coli. Levels of honey concentration greater than 15% showed to be optimun for killing the bacteria culture Separation of Solvent using Rotoevaporator at 45 ºC
Extraction of Phenolic and Flavonoids with Ethanol Obtention of the Honey Water in Amberlite Column Extractand filtering of the last product Solution of honey
Amberlite Columns
ANALYTICAL CONDITIONS
Temperature 25 °C
Column Rp C-18 Lichrospher
Movil Phase H3PO4 (0,01 M) / ACN (used in gradient)
Wave lengh 314 nm
Injection Vol 20 µL
Time 0 Assigned to p-coummaric acid´s peak
Total running time 16 m DAD1 A, Sig=314,4 Ref =off (GZN\GZN00000.D) mAU 200 Beekeeper Mrs. Andrea Flaño. 175
150
125
100 Acido salicilico
75
50 ACIDO GALICO kaempherol Acido p-coumarico Acido ferulico 25 Rutina Naringenina
0
0 2 4 6 8 10 12 14 min Compounds isolated under non-polar movil phase Following compounds were determined under polar movil phase
H3PO4 (0,01 M)/ ACN 40:60
H3PO4 (0,01 M)/ ACN 84:16 Compound Ammount (µg/mL) Rt (m)
Ferulic Acid 0,235 + 0,05 7,29
Compound Ammount (µg/mL) Rt (m)
Salicilic Acid 0,435 + 0,05 9,17
Naringenina 0,456 + 0,05 10,43 p-Coummaric Acid 0,221 + 0,05 0
Kaempherol 0,174 + 0,05 10,84
Galic Acid 0,236 + 0,05 2,86 Rt: Retention Time PhenolicPhenolic CompoundsCompounds detecteddetected inin UlmoUlmo HoneysHoneys Micrograms/25 grams of honey
Compounds H342 H349 H430 H456 H515
Ac clorogenico 1,16 2,20 1,70 0,797 0,515
Esculetina 1,36 2,60 4,05 4,2 1,478 Ac cafeico 4,07 5,81 2,53 2,845 3,008 ac siringico 0 0 0 0 0 Rutina 0 0,05 0 0,007 0 Escopoletina 0,06 2,02 1,72 0,08 0,06
Ac Pcoumarico 1,25 0,06 0,07 1,572 1,248 Ac Vanillico 0,00 0 0 0 0 Ac Salicilico 4,23 5,65 7,62 17,994 3,432
Quercitina 0,04 0,04 0,06 0,03 0,03
Naringenina 0 0 0 0 0,09 Staphylococcus aureus Staphylococcus aureus et al Botritys cinerea Micrococcus luteus et al Staphylococcus epidermidis DAD1 A, Sig=314,4 Ref=off (GZN\MIEL0002.D) Chemical mAU 2.814
500 Fingerprint Rutine
400 of Flavonoids 6.919 2.896 4.119
Comparing 7.534 Ferulic 300 HONEY 3.563 Acid 3.186 Quercetine 7.141 6.238 7.224 7.952
200 6.611 6.444 8.165 8.559 3.762 9.667 8.872 9.058 6.093 Kaempferol 4.561 9.447 4.416 5.464 9.827 100 10.030 11.842 10.899 10.610 10.255 10.423 11.578 12.696 12.028 11.328 11.233 12.797 12.945 12.387 13.108 2.051 2.124 13.617 13.360 13.841 14.043 13.497 14.438 14.726 1.541 0 1.677
0 2 4 6 8 10 12 14 min
DAD1 A, Sig=314,4 Ref=off (MIELES\Z0000003.D) mAU
35 1.952
30 1.592
25
NECTAR20
15 Quercetine Rutine
10 3.010
1.757 Ferulic Kaempferol 6.711 2.498 5 2.185 Acid 7.015 7.111 7.672 2.758 7.424 6.287 8.972 7.568 8.690 7.288 8.093 8.381 9.180 9.587 3.637 12.716 9.443 9.806 4.775 5.862 13.366 1.438 14.116 12.341 10.953 0 11.992
-5
-10 0 2 4 6 8 10 12 14 min TERPENES in CHILEAN HONEYS Ulmo 335, Corontillo 329 y Quillay 337
MIEL 335 MIEL329 MIEL 337 Terpenos mg/Kg miel mg/Kg miel mg/Kg miel ααα-felandreno 0,02 Iso-terpinoleno 0,03 0,84 ααα-pineno Resinous 0,19 βββ-pineno 0,38 Limoneno 0,12 0,32 Sabineno 0,05 Camfeno 0,10 Linalool oxido 0,21 0,36 0,25 Linalool 0,15 SearchingSearching Hotrienol 0,56 0,85 0,09 for the Mirtenol 0,26 0,09 for the Neril acetona 0,14 0,37 ChemicalChemical Eucarvona 0,46 Liliac alcohol 34,54 Floral = jazmín MarkerMarker Liliac aldehido 36,13 trans-anetol 1,11 8-hidroxi-6,7-dihidrolinalool 2,03 p-cimeno 0,04 0,10 cis-rose oxido 0,19 0,11 Nerol 0,06 ααα-calacoreno 0,07 Cumeno 0,05 0,20 Cadaleno 0,10 TOTAL 75,79 3,39 1,37 Norisoprenoides Ulmo 335, Corontillo 329 y Quillay 337
MIEL 335 MIEL329 MIEL 337 Norisoprenoides mg/Kg miel mg/Kg miel mg/Kg miel Isoforona 6,99 0,30 Anís Cetoisoforona 5,10 1,07 Safranal 1,05 Azafrán trans-βββ-damascenona 1,09 3,72 0,19 3-hidroxi-5.6-epoxi-b-ionona 0,27 0,50 3,4-dehidro-b-ionona 0,14 3,5-dehidro-βββ-ionona 0,33 Megastigmatrienona 0,53 Megastigma-5,7,9-trienona 0,24 TOTAL 13,59 6,97 0,96 Microplates Dishes by Microdilution Methodology in order to detect the Minimal Concentration of the active ingredient that inhibit pathogens growth
Ps a S a E c C a S t S pn E a Ps a Pseudomonas aeruginosa. Lysoform mas caldo de soya S a Staphylococcus aureus. Ps a E c Escherichia coli .
C a Candida albicans S a
S t Salmonella typhi Pocillos con bacterias, Lysoform y E c caldo de soya S pn Streptococcus pneumoniae
50,0 25,00 12,50 6,25 3,13 1,56 0,78 0,39 0,20 0,10 0,05 0,02 E a Enterobacter aerogenes Percentage of Dilution C a Ps a Pseudomonas aeruginosa.
S a Staphylococcus S t aureus.
E c Escherichia coli . S pn C a Candida albicans
S t Salmonella typhi E a Pocillos con bacterias, producto final y caldo de soya S pn Streptococcus pneumoniae 50,0 25,00 12,50 6,25 3,13 1,56 0,78 0,39 0,20 0,10 0,05 0,02
E a Enterobacter Percentage of Dilution aerogenes
Determination of the Minimal Concentration of the active ingredient that inhibit pathogens growth Control positivo pocillos con caldo Control negativo pocillos de soya y bacterias con caldo de soya
Ps a S a E c C a S t S pn E a Ps a Pseudomonas aeruginosa. Pructo final Fundacion Copec Puc S a Staphylococcus aureus. Ps a E c Escherichia coli .
C a Candida albicans S a
S t Salmonella typhi Pocillos con bacterias, Producto final E c y caldo de soya S pn Streptococcus pneumoniae
50,0 25,00 12,50 6,25 3,13 1,56 0,78 0,39 0,20 0,10 0,05 0,02 E a Enterobacter aerogenes Percentage of Dilution
Determination of the Minimal Concentration of the active ingredient that inhibit pathogens growth µ µµµ Crecimiento micelial Botrytis cinerea
100
80
60 DÍA 4 DÍA 6 40
Diámetro (mm) 20 0 75 50 25 Testigo Concentración extracto M349 (%)
Figura 9. Crecimiento micelial de Botrytis cinerea sobre medio Figura 10. Crecimiento micelial de B.cinerea PDA + extracto miel de Ulmo M349. a los 4 (A) y 6 días (B), de la segunda repetición. µ µµµ Perdidas debidas a Erwinia carotovora en postcosecha de papas
In Chile the production loss is between 20 to 25% (Acuña, 2000) equivalent to 500.000 tons ( ODEPA 2005).
Soft Rot caused by Bacteria Erwinia carotovora Potatoes tubers infected with Erwinia carotovora pv. carotovora
Infected Potataoes storage for 50 days at 30ºC. Left tubers infected but not tretated with ulmo extract Right tubers infected with Erwinia carotovora pv. carotovora treated with ulmo extract at 12,5% v/v INOCULATION OF PATHOGENS AT THE INIA CAMPUS Dr Richard Cahoon, Cornell University