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Reproducibility of Dwarf Pea Shoot Growth Stimulation by Homeopathic Potencies - Baumgartner2008

Reproducibility of Dwarf Pea Shoot Growth Stimulation by Homeopathic Potencies - Baumgartner2008

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Complementary Therapies in Medicine (2008)
16
, 183191
available at www.sciencedirect.comjournal homepage: www.elsevierhealth.com/journals/ctim
Reproducibility of dwarf pea shoot growthstimulation by homeopathic potenciesof gibberellic acid
Stephan Baumgartner
a
,
b
,
, Devika Shah
a
,
b
, Johann Schaller
c
,Urs K¨ampfer
c
, Andr´e Thurneysen
a
, Peter Heusser
a
a
Institute of Complementary Medicine KIKOM, University of Bern, Bern, Switzerland 
b
Society for Cancer Research, Institute Hiscia, Kirschweg 9, Arlesheim, Switzerland 
c
Department of Chemistry and Biochemistry, University of Bern, Bern, Switzerland 
Available online 24 April 2008
Summary
Objectives:
Investigation of the conditions for reproducibility of dwarf pea shoot growth stim-ulation through homeopathic potencies of gibberellic acid.
Methods:
4 batches of pea seed (
Pisum sativum
L. cv. Fr¨uher Zwerg; harvests from 1997, 1998,1999, and 2000) were tested regarding their reaction to gibberellic acid 17x and 18x (comparedto unsuccussed and succussed water (1x) as controls) in 8 independent randomized and blindedexperiments. Pea seed was immersed for 24h in watery solutions of homeopathic potenciesor controls, and cultivated under controlled laboratory conditions. Pea shoot length was mea-sured after 14 days. Two systematic negative control experiments assessed the stability of theexperimental set-up.
Results:
The systematic negative control experiments yielded no significant effects and con-firmed the stability of the experimental set-up. 2 out of 4 seed batches reacted to thehomeopathic treatment (
p
<0.05). Seed batch 1997 showed a reproducible reaction to gib-berellic acid 17x (shoot length stimulation of +11.2%,
p
=0.007), and seed batch 1998 showeda significant varying response (increase/decrease). Seed batch 1997 differed from the other3 batches by an increased glucose and fructose content, and reduced 1000kernel weight.Meta-analysis with data of earlier experiments is in accordance with the results of the presentexperimental series.
Conclusions:
We identified ‘seed quality’ as a possible trigger factor for successful repro-ducibility in homeopathic basic research. Premature harvesting as a possible key factor forresponsivenessofdwarfpeastohomeopathicpotenciesofgibberellicacidisourcurrentworkinghypothesis to be tested in future experiments.© 2008 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +41 61 706 72 18; fax: +41 61 706 72 00.
E-mailaddress:
stephan.baumgartner@kikom.unibe.ch(S. Baumgartner).0965-2299/$  see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.ctim.2008.03.001
 
184 S. Baumgartner et al.
Introduction
Specific effects of homeopathic remedies (as used in home-opathy and anthroposophical medicine) have been observedin several randomized clinical trials.
Incompatibility ofhomeopathic remedy production standards
5
with currentstandard pharmacological theories
6
can lead to the con-clusion that these clinical results must be false-positiveartefacts.
7
The opposite consequence  the actual stateof knowledge of physics and chemistry is incomplete  isalso discussed.
8
However, any such conclusion would requireconvincing empirical evidence. The latter may arise fromclinical as well as from preclinical research.Basic research into homeopathic potentization can begrouped into four main areas: animal research, plantbioassays (including plant-pathogen interactions),
in vitro
models (fungi, bacteria, cell cultures and biochemicaltests), and physicochemical research. Recent reviews
of these four areas all ended up with a similar con-clusion: there is some evidence for substance specificeffects of homeopathic potencies, but only very few modelscould be independently reproduced. Several reproductionsfailed
or resulted in inverted or altered effects.
Even internal reproduction seems to be a non-trivialtask,
giving rise to speculations about irreproducibil-ity as a possibly inherent feature of homeopathic potencyeffects.
The aim of the present study was to investigate condi-tions for internal reproducibility of the dwarf pea model.
We had observed that gibberellic acid 17x induced a repro-ducible shoot growth increase, which albeit seemed toweaken in the course of the experiments. In this follow-up study we wanted to answer two questions: 1. Cananother experimenter reproduce the effects observed? 2.Has the factor ‘seed batcha relevant influence on theresults?
Methods
General experimental design
We replaced the person in charge for potentization, seedtreatment, plant care and final measurement (S.B. by D.S.)and investigated the effects of homeopathic potencies ofgibberellic acid on four batches of dwarf pea seed (harvestsfrom 1997, 1998, 1999, and 2000). We tested the effect ofwater, succussed water (1x), gibberellic acid 17x and 18xon pea shoot growth in 8 independent experiments, all fullyrandomized and blinded. In every experiment, three seedbatches were used (2000, 1999, 1998 or 1997  the lattertwo alternating). A typical experiment involving homeo-pathic potencies comprises 200 seeds (4 parameters
×
50seeds) per pea batch (harvest).In order to investigate the stability of the experimen-tal set-up, we conducted two systematic negative controlexperiments. These are full-size experiments with 800 seedseach (50 seeds
×
4 harvests
×
4 treatment parameters),using unsuccussed water as identical cultivation parameter.Furthermore, seed batches were characterized regardingmajorchemicalconstituents(sugar,starch,aminoacids)and1000kernel weight.
Preparation of homeopathic potencies and controlsolutions
Full details concerning the preparation of the investigatedsolutions (homeopathic potencies of gibberellic acid andcontrols) are given in an earlier publication.
In short,gibberellic acid (Sigma, Switzerland) was dissolved and suc-cussed in acetone (Merck, Switzerland) to obtain 50mMgibberellic acid (1x). All further potency levels (2x18x)were obtained by potentization in distilled water accord-ingtothemultipleglassmethod(HahnemannHpotencies,decimal steps). In contrast to the former protocol,
poten-tization vessels were reused in successive experimentsafter thorough rinsing once with deionized and twice withdistilled water. Control solutions were unsuccussed and suc-cussed (1x) water, according to the reasoning publishedelsewhere.
Potencies and controls  using the same batchof distilled water  were prepared freshly for each experi-ment. The test solutions were coded with a letter code byanother person not being involved in the experiments.
Plant cultivation
Plant cultivation also was described in detail earlier.
Incontrast to the former protocol,
15
±
0.2g of dwarf peaseed (
Pisum sativum
L., cv. Fr¨uher Zwerg) were immersedin 100ml homeopathic or control solution for 24h in dupli-cate. Seed of four different harvests (1997, 1998, 1999,2000) was used. Due to a limited residual amount, seed of1997 and 1998 was used in alternating experiments. 2
×
25well-swollen grains were selected at random and plantedinto 2
×
5 pots, filled with a standard cultivation substrate,a 1:1 mixture (v/v) of TKS1 (peat, Floragard, Germany) andVermex F (expanded vermiculite, Vermica AG, Switzerland).Pots were watered with tap water from beneath (400mlper pot during the entire cultivation). Plants were grown inartificial lighting of 60
±
10
mol/m
2
s photosynthetic activeradiation (PAR) in a 14h/10h light/dark cycle at controlledtemperature (24
±
2
C). After two weeks, plants were har-vested and shoot length was measured by stretching theplants on a sheet of millimeter graph paper. Trays werecleaned by hand with a brush and hot tap water. In contrastto the former protocol,
pots were cleaned in a dishwasher.Both pots and trays were reused in random assignment insubsequent experiments.
Analysis of pea seed carbohydrates
Dry pea seeds (approx. 5g) were ground to a fine pow-der with a mechanical grinder. Samples of 100mg wereextracted four times with 1ml 80% aqueous ethanol toremove soluble sugars. Insoluble material containing thestarch was resuspended in 1ml water and homogenisedfurther with a pestle and mortar to obtain a homogeneoussuspension. Aliquots of 200
l were autoclaved for 20minat 121
C to solubilise the starch and 200
l of Na Acetate,pH 4.8 containing 10 units of
-amylase and 1.25 units ofamyloglucosidase was added to digest the starch to glucose(37
C, 16h). Liberated glucose was measured as describedelsewhere.
Control samples treated in the same waybut incubated without enzymes contained no glucose. To
 
Reproducibility of growth stimulation by homeopathic gibberellic acid 185determine soluble sugars, the four 80% ethanol extracts foreach sample were combined and 2ml evaporated to a smallvolume (400
l on average) under an air stream. Glucose andfructose content were determined spectrophotometricallyas described previously.
To measure sucrose, 100
l ofthe evaporated soluble extract was added to 100
l of NaAcetate, pH 4.8 containing 150 units of
-fructosidase tohydrolyse sucrose to glucose and fructose (37
C, 2h). Totalglucose and fructose was measured and the sucrose contentcalculated by subtracting the values for the free hexosesugars measured in the undigested extract. All sampleswere coded and analyzed in 6-fold replication.
Amino acid analysis of pea seed
Dry pea seeds (approx. 5g) were ground to a fine powderwith a mechanical grinder. Samples were hydrolysed in thegas phase with 6M hydrochloric acid containing 0.1% (by vol-ume) phenol for 24h at 115
C under N
2
vacuum according toChang and Knecht.
The liberated amino acids were reactedwith phenylisothiocyanate and the resulting phenylthiocar-bamoylaminoacidswereanalyzedbyRP-HPLConaNovaPakODS column (3.9mm
×
150mm, 4
m; Waters) in a DionexSummit liquid chromatograph with an automatic injectionsystem according to Bidlingmeyer et al.
and Cohen andStrydom.
All samples were coded and analyzed in tripli-cate.
Data evaluation and statistical analysis
All data analysis was performed with the statistics software‘Statistica4.1forMac(Statsoft,Inc.,Tulsa,OK74104,USA).Effects of the treatments on mean shoot length were sta-tistically analyzed by a 2-way analysis of variance (ANOVA),comprising treatment and experiment number (date) as thetwo independent variables (factors) and pea shoot length
Table 1
Pea shoot length (mean
±
standard deviation [mm]) for all seed batches and treatment groups in eight independentexperimentsExperiment N
o
Seed batch TreatmentUnsuccussed water Succussed water GA
3
17x GA
3
18x33199779.5
±
38.9 71.7
±
21.4 85.9
±
28.1 73.3
±
25.534 35 66.6
±
22.7 67.7
±
21.0 86.5
±
25.9 74.6
±
31.536 37 81.0
±
27.0 74.6
±
24.4 71.4
±
23.4 61.6
±
28.438 39 67.4
±
30.4 69.8
±
31.4 77.8
±
19.4 73.7
±
22.040 33199834 88.7
±
23.6 85.0
±
24.6 83.1
±
25.3 86.7
±
24.035 36 83.9
±
22.3 94.1
±
21.6 79.7
±
23.0 86.5
±
24.737 38 76.6
±
21.6 76.3
±
24.3 90.7
±
22.6 81.3
±
28.439 40 81.2
±
26.5 75.4
±
16.4 73.2
±
25.5 73.3
±
19.6331999108.3
±
23.6 104.6
±
24.6 103.9
±
25.3 107.3
±
24.034 103.8
±
22.3 105.6
±
21.6 108.9
±
23.0 107.4
±
24.735 104.5
±
21.6 105.1
±
24.3 104.0
±
22.6 101.9
±
28.436 103.8
±
26.5 107.6
±
16.4 100.4
±
25.5 102.5
±
19.637 97.4
±
23.6 97.5
±
24.6 96.1
±
25.3 103.4
±
24.038 93.2
±
22.3 94.8
±
21.6 90.3
±
23.0 96.7
±
24.739 100.4
±
21.6 91.9
±
24.3 97.9
±
22.6 100.0
±
28.440 95.6
±
26.5 99.1
±
16.4 96.5
±
25.5 95.3
±
19.6332000105.5
±
23.2 104.9
±
25.5 98.6
±
24.4 102.0
±
21.834 105.3
±
21.9 101.6
±
20.8 109.0
±
27.3 108.7
±
21.135 99.5
±
24.4 96.3
±
19.4 98.8
±
19.3 101.3
±
22.636 103.3
±
28.0 101
±
19.6 99.6
±
28.4 101.1
±
23.837 100.3
±
19.3 99.4
±
22.1 94.8
±
19.5 107.0
±
22.038 92.8
±
22.0 92.1
±
20.4 94.1
±
23.5 101.0
±
21.939 100.2
±
21.1 96.9
±
22.3 93.9
±
17.9 97.4
±
21.940 93.8
±
23.6 95.7
±
19.6 95.7
±
22.2 93.6
±
24.7
Experiment N
o
=internal experiment numbering.

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