var. scopulorum (2-3 needles)

var. ponderosa (3 needles)

var. arizonica (4-5 needles)

TimberForage and coverWatershed protectionHorticulture and lanscape useRecreation

Cows will eat pine needles

Fresh green needles

Needles from windfall

Dry older needles (litter) on theground

Weather changesHungerChanges in FeedAccess to pine needlesBoredomAccidental ingestion

Abortion may occur within 48 hrs, but usually occurbetween 6-10 days.

Weak uterine contractions, incomplete cervical dilation and uterine hemorrhage.

Abortion may occur within 48 hrs, but usually occurbetween 6-10 days.

Weak uterine contractions, incomplete cervical dilation and uterine hemorrhage.

Abortions include stillbirths or the birth of small weak calves.

Abortion may occur within 48 hrs, but usually occurbetween 6-10 days.

Weak uterine contractions, incomplete cervical dilation and uterine hemorrhage.

Abortions include stillbirths or the birth of small weak calves.

Postpartum complications include retained placentas, uterine infection, agalactia, rumen stasis and sometimes death.

Pine Needle Abortion Losses = $5 - $20 million/annually

Abortifacient Compound in Ponderosa Pine Needles

CO2H

CH2OH

Isocupressic Acid

Feeding Trials with Isocupressic Acid

Days to Parturition

Dos

e m

g/kg

(2 x

dai

ly)

50

70

90

110

130

150

170

0 5 10 15 20 25 30 35

Diterpene Acid of Ponderosa Pine

Tricyclic Abietane Acids Labdane Acids

CO2H

CH2OH

CO2H

OO

CO2H

OO

OHO

CO2H

O OH

CO2H

CH2OH

ISOCUPRESSIC ACID ACETYLISOCUPRESSIC ACID

IMBRICATOLOIC ACID

SUCCINYLISOCUPRESSIC ACID

DIHYDROAGATHIC ACID

ABIETIC ACID NEOABIETIC ACID PALUSTRIC ACID

DEHYDROABIETIC ACID LEVOPIMARIC ACID ISOPIMARIC ACID

CO2HCO2H CO2H

CO2H CO2H CO2H

5. Isocupressic acid is metabolized in both the rumen and the hepatic systems

oxidation

reductaseLiver

ICA, Acetyl-ICA, Succinyl-ICA

CH2OH

CO2H

CH2OH

CO2H

CO2H

O

OH

CO2H

O

OH

CO2H

O

OH

esterase

1

4

CO2H

O

OH2CO2H

O

OH 3

3

2

5

(soluble enzyme oxidation)

Detected SerumMetabolites

Isocupressic Acid Content of Selected Species and Locations

Species Common Name Locations ICA % DWPinus ponderosa Ponderosa Pine OR, AZ, CA, UT, CO, WY 0.42 – 1.35Pins contorta Lodgepole Pine OR, ID, CO, UT, BC, AR 0.00 – 0.66Pinus jeffreyi Jeffrey Pine CA 0.04 – 0.54Pinus arizonica Arizona Pine CA, AZ n.d. – 0.49Pinus edulis Pinyon Pine AZ, CO, NM, UT n.d. – 0.45Pinus monophylla White Pine NV n.d. – 0.32Pinus taeda Loblolly Pine AR, AZ n.d. – tracePinus flexilis Limber Pine CO, UT n.d. – 0.06Pinus echinata Short leaf pine AR n.d.Pinus elliottii Slash Pine AR n.d.Pinus palustris Longleaf Pine AR n.d.Pinus montezumae Montezuma Pine CC n.d.Abies concolor White Fir CA, CO, UT n.d. – 0.04Abies lasiocarpa Subalpine Fir OR, ID 0.01 – 0.04Pseudotsuga menziesii Douglas Fir UT, CA, ID, AZ, OR n.d. – 0.04Juniperus communis Mountain common

juniperCO, UT 2.05 – 4.5

Juniperus scopulorum Rocky Mt. juniper UT, NM, AZ 0.25 – 0.84Juniperus osteosperma Utah juniper UT, NV, n.d. – 0.07Juniperus californicum California juniper CA 0.05 – 1.4Juniperus monosperma One Seed Juniper AZ, NM n.d. – 0.14Thuja plicata Western Red Cedar AZ, NM, UT 0.33 – 0.84Pinus koraiensis Korean Pine UT, Korea n.d. – tracePicea engelmannii Engelmann Spruce CA, CO, ID, MT, OR, UT n.d. – 0.31Picea pungens Blue Spruce UT, CO n.d. – 0.17Cupressus macrocarpa Monterey Cypress CA, New Zealand n.d. – 1.24Pinus longaeva Bristle cone pine CO 0.01 – 0.05Abies grandis Grand Fir OR n.d.Tsuga mertensiana Mt. Hemlock OR n.d.Larix occidentalis Wester Larch OR n.d.Libocedrus decurrens Incense cedar OR 0.07Abies procera Red fir CA 0.05

Isocupressic Acid Content of Selected Species and Locations

Species Common Name Locations ICA % DWPinus ponderosa Ponderosa Pine OR, AZ, CA, UT, CO, WY 0.42 – 1.35Pins contorta Lodgepole Pine OR, ID, CO, UT, BC, AR 0.00 – 0.66Pinus jeffreyi Jeffrey Pine CA 0.04 – 0.54Pinus arizonica Arizona Pine CA, AZ n.d. – 0.49Pinus edulis Pinyon Pine AZ, CO, NM, UT n.d. – 0.45Pinus monophylla White Pine NV n.d. – 0.32Pinus taeda Loblolly Pine AR, AZ n.d. – tracePinus flexilis Limber Pine CO, UT n.d. – 0.06Pinus echinata Short leaf pine AR n.d.Pinus elliottii Slash Pine AR n.d.Pinus palustris Longleaf Pine AR n.d.Pinus montezumae Montezuma Pine CC n.d.Abies concolor White Fir CA, CO, UT n.d. – 0.04Abies lasiocarpa Subalpine Fir OR, ID 0.01 – 0.04Pseudotsuga menziesii Douglas Fir UT, CA, ID, AZ, OR n.d. – 0.04Juniperus communis Mountain common

juniperCO, UT 2.05 – 4.5

Juniperus scopulorum Rocky Mt. juniper UT, NM, AZ 0.25 – 0.84Juniperus osteosperma Utah juniper UT, NV, n.d. – 0.07Juniperus californicum California juniper CA 0.05 – 1.4Juniperus monosperma One Seed Juniper AZ, NM n.d. – 0.14Thuja plicata Western Red Cedar AZ, NM, UT 0.33 – 0.84Pinus koraiensis Korean Pine UT, Korea n.d. – tracePicea engelmannii Engelmann Spruce CA, CO, ID, MT, OR, UT n.d. – 0.31Picea pungens Blue Spruce UT, CO n.d. – 0.17Cupressus macrocarpa Monterey Cypress CA, New Zealand n.d. – 1.24Pinus longaeva Bristle cone pine CO 0.01 – 0.05Abies grandis Grand Fir OR n.d.Tsuga mertensiana Mt. Hemlock OR n.d.Larix occidentalis Wester Larch OR n.d.Libocedrus decurrens Incense cedar OR 0.07Abies procera Red fir CA 0.05

Isocupressic Acid Content of Selected Species and Locations

Species Common Name Locations ICA % DWPinus ponderosa Ponderosa Pine OR, AZ, CA, UT, CO, WY 0.42 – 1.35Pins contorta Lodgepole Pine OR, ID, CO, UT, BC, AR 0.00 – 0.66Pinus jeffreyi Jeffrey Pine CA 0.04 – 0.54Pinus arizonica Arizona Pine CA, AZ n.d. – 0.49Pinus edulis Pinyon Pine AZ, CO, NM, UT n.d. – 0.45Pinus monophylla White Pine NV n.d. – 0.32Pinus taeda Loblolly Pine AR, AZ n.d. – tracePinus flexilis Limber Pine CO, UT n.d. – 0.06Pinus echinata Short leaf pine AR n.d.Pinus elliottii Slash Pine AR n.d.Pinus palustris Longleaf Pine AR n.d.Pinus montezumae Montezuma Pine CC n.d.Abies concolor White Fir CA, CO, UT n.d. – 0.04Abies lasiocarpa Subalpine Fir OR, ID 0.01 – 0.04Pseudotsuga menziesii Douglas Fir UT, CA, ID, AZ, OR n.d. – 0.04Juniperus communis Mountain common

juniperCO, UT 2.05 – 4.5

Juniperus scopulorum Rocky Mt. juniper UT, NM, AZ 0.25 – 0.84Juniperus osteosperma Utah juniper UT, NV, n.d. – 0.07Juniperus californicum California juniper CA 0.05 – 1.4Juniperus monosperma One Seed Juniper AZ, NM n.d. – 0.14Thuja plicata Western Red Cedar AZ, NM, UT 0.33 – 0.84Pinus koraiensis Korean Pine UT, Korea n.d. – tracePicea engelmannii Engelmann Spruce CA, CO, ID, MT, OR, UT n.d. – 0.31Picea pungens Blue Spruce UT, CO n.d. – 0.17Cupressus macrocarpa Monterey Cypress CA, New Zealand n.d. – 1.24Pinus longaeva Bristle cone pine CO 0.01 – 0.05Abies grandis Grand Fir OR n.d.Tsuga mertensiana Mt. Hemlock OR n.d.Larix occidentalis Wester Larch OR n.d.Libocedrus decurrens Incense cedar OR 0.07Abies procera Red fir CA 0.05

Isocupressic Acid Content of Selected Species and Locations

Species Common Name Locations ICA % DWPinus ponderosa Ponderosa Pine OR, AZ, CA, UT, CO, WY 0.42 – 1.35Pins contorta Lodgepole Pine OR, ID, CO, UT, BC, AR 0.00 – 0.66Pinus jeffreyi Jeffrey Pine CA 0.04 – 0.54Pinus arizonica Arizona Pine CA, AZ n.d. – 0.49Pinus edulis Pinyon Pine AZ, CO, NM, UT n.d. – 0.45Pinus monophylla White Pine NV n.d. – 0.32Pinus taeda Loblolly Pine AR, AZ n.d. – tracePinus flexilis Limber Pine CO, UT n.d. – 0.06Pinus echinata Short leaf pine AR n.d.Pinus elliottii Slash Pine AR n.d.Pinus palustris Longleaf Pine AR n.d.Pinus montezumae Montezuma Pine CC n.d.Abies concolor White Fir CA, CO, UT n.d. – 0.04Abies lasiocarpa Subalpine Fir OR, ID 0.01 – 0.04Pseudotsuga menziesii Douglas Fir UT, CA, ID, AZ, OR n.d. – 0.04Juniperus communis Mountain common

juniperCO, UT 2.05 – 4.5

Juniperus scopulorum Rocky Mt. juniper UT, NM, AZ 0.25 – 0.84Juniperus osteosperma Utah juniper UT, NV, n.d. – 0.07Juniperus californicum California juniper CA 0.05 – 1.4Juniperus monosperma One Seed Juniper AZ, NM n.d. – 0.14Thuja plicata Western Red Cedar AZ, NM, UT 0.33 – 0.84Pinus koraiensis Korean Pine UT, Korea n.d. – tracePicea engelmannii Engelmann Spruce CA, CO, ID, MT, OR, UT n.d. – 0.31Picea pungens Blue Spruce UT, CO n.d. – 0.17Cupressus macrocarpa Monterey Cypress CA, New Zealand n.d. – 1.24Pinus longaeva Bristle cone pine CO 0.01 – 0.05Abies grandis Grand Fir OR n.d.Tsuga mertensiana Mt. Hemlock OR n.d.Larix occidentalis Wester Larch OR n.d.Libocedrus decurrens Incense cedar OR 0.07Abies procera Red fir CA 0.05

Utah Juniper

Juniperus osteosperma

Utah Juniper Bark

PN04.07.05_02 4/7/2005 1:12:36 PM

RT: 6.98 - 22.03

7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22Time (min)

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

17.6812.36

11.9812.42

11.85

16.7114.55 15.40 16.459.80 14.46 17.5216.1514.8812.66 17.12 18.1012.85 18.3511.63 18.919.23 11.198.70 19.85 20.527.12 21.638.56

NL:1.77E7TIC F: MS PN04.07.05_02

PN04.07.05_02 #1359-1363 RT: 17.68-17.71 AV: 5 SB: 8 17.59-17.65 NL: 6.77E5T: + c Full ms [ 50.00-650.00]

50 100 150 200 250 300 350 400 450 500 550 600 650m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

253.18

346.0873.04189.18

271.14229.15

121.08109.09 147.10

95.06 157.09 360.12213.14

307.10373.10 463.09

478.11421.13 549.15520.28 608.51 645.61581.48

O

HO

O

OH

Agathic acid ~ 1% dry weight

Bioassay for Utah Juniper Bark

Asssay Dose # Abortions/ #Tested

Time

Cows 106 – 143 3/3 4 – 9 days

Sheep 110 – 150 0/5 n.a.

Synthetic Preparation of Isocupressic Acid Metabolites

(1)(3) (13R,S)-4

(13S)-4 (8S,13S)-5 (8S,13R)-5

Scheme 1. Conversion of isocupressic acid (1) to (13R,S)-tetrahydroagathic acid (5).

Scheme 2. Conversion of (13S)-dihydroagathic acid (4) to (13S)-tetrahydroagathic acid (5).

(1a)

II. III.

IV.

IV.

Sodium chlorite oxidation

Sodium metal reduction

Tris(triphenylphosphine)chlororodiumhomogeneous hydrogenation

H

H

OH

HO

O

H

H

O

H

HO

O

H

H

O

OH

HO

O

O

OH

HO

O

O

OH

HO

O

H

H

O

OH

HO

O

H

H

O

OH

HO

O

Pr4N+RuO4-catalytic oxidation

Yes - Abortifacient

Broom snakeweed (Gutierrezia sarothrea)

Toxic and abortifacient to cattle

Broom snakeweed (Gutierrezia sarothrea)Toxic and abortifacient to cattle

Contains furano-diterpene acids

OH

CO2H

O

CO2H

Isocupressic Acid Polyalthic acid

O

CO2H CO2H

O

H

O

CO2H CO2H

O

H

CO2H

HO

H2

ICA ?

O

CO2H CO2H

O

H

CO2H

HO

H2

CO2H

O

OHO2

ICA ?

Agathic Acid ?