Introduction
Nanoparticles (NPs) of starting materials and contaminants were recently detected
in homeopathic high dilutions (HDs) above Avogadro's number.[1]
[2] Since homeopathic medicines are traditionally prepared using glass vials, it was
postulated that contaminant silica NPs detaching from vials during the step of agitation
might represent a critical component involved in the biological effects of these medicines
observed experimentally.[1]
[2]
[3]
[4]
[5]
[6] The understanding of their real effects (specific or non-specific) is crucial to
characterize the mechanisms involved.
It is known that nanosilica can form stable three-dimensional structures using DNA,
proteins or living cells as template (epitaxial growth).[7]
[8]
[9]
[10]
[11]
[12]
[13] This phenomenon led to a microstructural hypothesis to account for the action of
homeopathic medicines. Briefly, nanosilica would behave as non-specific amplifier
or vehicle for the specific (structural or electromagnetic) information contained
in starting materials, subsequently identified and processed by biological systems.[14]
[15]
[16] Hypothetically, initial interaction and adsorption of starting material with silica
NPs in low homeopathic dilutions would create “nanoseeds” that would be preserved
in the preparation of the subsequent dilutions.[6]
[17]
[18]
[19] Nanoparticles might also be associated with changes in electrical conductivity,
as was shown for the homeopathic medicine Ferrum metalicum,[20] although other factors can also alter the conductivity of homeopathic aqueous solutions.[21]
Few studies have sought to assess the influence of glass (silica) on the effects of
homeopathic HDs. In the pioneer study carried out by Boiron et al,[22] HDs prepared in paraffin-covered glass vials had smaller effect compared with the
ones prepared in standard glass vials. One single study investigated trace elements
in plastic and glass vials used to prepare and store homeopathic HDs.[23] The results showed absence of silica in the propylene vials and increase in the
concentration of trace elements, mainly silica, lithium and sodium, when bi-distilled
water was transferred from propylene to glass vials. The largest difference in trace
element concentration occurred in the step from mother tincture to the first centesimal
dilution (1cH); the later steps to 30cH were not associated with increase in trace
element concentration. To the best of our knowledge, no study investigated differences
in biological effects between HDs prepared in plastic and glass vials.
Therefore, the aim of the present study was to compare biological effects of homeopathic
HDs—Arsenicum album (Ars) 6cH and 200cH—prepared in glass and plastic vials to investigate the role of silica
microparticles in such effects. The presence of metal microparticles in the drug samples
and other relevant physical properties of the solution (electrical conductance) were
analyzed. The experimental design and choice of the medicine were based on the literature[24] and the model chosen, macrophages in co-culture with microorganisms, was previously
used in homeopathic fundamental research.[25] Macrophages are myeloid cells specialized for phagocytosis of micro-organisms, such
as yeasts, and stand out for their ability to respond epigenetically to small environmental
stimuli, mainly by production of cytokines/chemokines[26]
[27] which makes these cells adequate biological sensors in HD research.
Materials and Methods
High Dilutions
Homeopathic matrix samples were produced according to the Good Manufacturing Practices
at the Pharmacy Faculty of Federal University of Rio de Janeiro, following the Brazilian
Homeopathic Pharmacopoeia guidelines.[28] The starting materials, arsenic and lactose monohydrate, were purchased from Sigma
Aldrich (As2O3, batch number BCBD 1149V) and Pharma Nostra Comercial Ltda (Rio de Janeiro, Brazil), respectively.
Arsenic raw material containing 99.984% and 0.99984 mol/mol2 of arsenic trioxide, as declared by the supplier, was acquired as a fine powder.
The homeopathic manufacturing process followed the statements of Brazilian Homeopathic
Pharmacopoeia[28] and started with the separation of lactose powder in three equal parts. One-third
of the lactose was placed in a porcelain vessel to avoid As2O3 loss. Following this, As2O3 was added and triturated, meeting the centesimal scale (1 part in 99 parts of inert
vehicle), and the complex (Arsenicum album plus lactose) was submitted to two independent and vigorous cycles of crushing and
scraping for 20 minutes. After this first cycle, the second and third parts of lactose
were added to the porcelain vessel, and submitted to the second and third cycles of
crushing and scraping, for 20 minutes each, respectively. The trituration process
was concluded at the end of 60 minutes. Each sample received the designation of first
triturated 1/100, coded as Arsenicum album 1c. For the second triturated compounding, one part of the first one (1cH) was mixed
in 99 parts of lactose (centesimal scale), and all crushing and scraping procedure
repeated to obtain Arsenicum album 2cH. The last trituration process produced Arsenicum album 3cH, following the same protocol described above, using one part of Arsenicum album 2cH for 99 parts of lactose.
The Arsenicum album 3cH was used as matrix to the following dynamizations, always according to Brazilian
Homeopathic Pharmacopoeia.[28] HDs were prepared using standard type II amber glass or PET (polyethylene terephthalate,
R18/400, Apace, São Paulo, Brazil) vials. The plastic vials were sterilized by microwave
heating, three 5-minute shocks at high intensity. The glass vials were sterilized
in an autoclave at 121°C for 15 minutes. The PET material was silica free, according
to the manufacturer.
High dilutions were prepared as follows: centesimal serial dilutions (6 for 6cH and
200 for 200cH) were made using different flasks and performing 100 strong vertical
motions (agitation) for each dilution. All dilutions were prepared from Ars 3cH using 70% hydro-alcoholic solution (p/p)—which is equivalent to 77% (v/v)—in
the following steps. A minor deviation of the pharmacopeia guidelines was included
to prepare the final working homeopathic dilution, in which sterile water was used:
this was necessary to treat single cell cultures without inducing biases related to
alcohol concentration of the vehicle and to the possibility of culture contamination.
Vertical agitation (“potentization”) was performed in each step by means of a mechanical
arm used specifically for this purpose (Denise, AUTIC, São Paulo, Brazil). Theoretically,
the concentration of Ars 6cH and Ars 200cH was 1.3 × 10−11 M and Ars 1.3 × 10−366 M, respectively.
Ars 200cH prepared in glass vials was ultra-centrifuged at 5,000 rpm for 30 minutes to
separate heavier particles, and the supernatant alone was analyzed. The tested HDs
were designated Ars 6cHP (prepared in plastic vials), Ars 6cHG (prepared in glass vials), Ars 200cHP (prepared in plastic vials), Ars 200cHG (prepared in glass vials), and Ars 200cHGC (prepared in glass vials and centrifuged after). The control was untreated
macrophage-yeast co-culture (Control + yeast).
The vials were wrapped with aluminum foil and labeled with a code by a laboratory
technician who did not participate in the study (to make the procedures blinded).
The investigators were blinded to the vials' content until statistical analysis was
completed.
Macrophage–Yeast Culture and Treatments
RAW 264.7 macrophages were thawed and sown into culture bottles containing RPMI-1640
medium (Cutilab, Brazil) supplemented with 20% fetal bovine serum and 200 U mL−1 penicillin, 100 U mL−1 streptomycin, and 2.6 µg mL−1 amphotericin B (Cutilab, Campinas, Brazil). Plating was performed in triplicate on
six-well plates with 25 × 105 cells per well. Glass slides were not used during plating to keep the cell environment
silica-free. Yeast cells (Saccharomyces cerevisiae) were added onto macrophages adhered to the well bottom in proportion 1:10 (25 × 106 yeast cells per well).
The treatment solutions were prepared with pure water filtered in a 0.22 µm Millipore
filter. The test solutions were added in proportion 20% to the well volume. A second
application of treatments was performed 24 hours later, in proportion 1%. Forty-eight
hours after the onset of treatments, the macrophages were fixed with absolute methanol
for 15 minutes and subjected to Giemsa staining. This staining method is useful for
clear visualization of the yeasts inside the macrophages.
Histomorphometry
Ten digital photographs were taken per field (NIKON E 200 microscope coupled to Coolpix
digital camera, Tokyo, Japan) and 200 cells/well were counted through oil-immersion
objective with 100× magnification. The mean area (pixels) of cells, representing cell
spreading, and the percentage of phagocytozed yeast cells were counted automatically
using the software Metamorph (Molecular Devices; Saint Jose, California, United States).
The phagocytic index was calculated as percentage of internalized yeasts to the number
of macrophages in each field.
Acridine Orange Test
The co-cultured cells were incubated after 48 hours of treatment with acridine orange
(5 µg/mL) under a 5% CO2 hood for 20 minutes. Next, the wells were rinsed with phosphate buffered saline (PBS);
samples were collected by touching the cells with a slide covered with one drop of
PBS, and then examined under fluorescence microscope (Olympus-BX60, Tokyo, Japan)
to investigate the lysosome activity. Images were taken with a digital camera coupled
to the microscope (Dino-Eye AM7023, New Taipei, Taiwan). In this test, DNA-containing
structures emit green fluorescence, and acid structures, such as lysosomes and phagolysosomes,
emit red/orange fluorescence. Qualitative analysis of cell morphology was performed
on serial photomicrographs.
Nitric Oxide Production
The supernatant from cells kept in co-culture for 48 hours was frozen at –80°C for
later measurement of nitrite and nitrate production. Measurement was performed in
duplicate by means of the colorimetric method on 96-well plates: to each plate 100
µL of supernatant and 100 µL of Griess reagent (0.1% N-alpha-naphthyl-ethylenediamine
(NED); 1% sulfanilamide; 2.5% of phosphoric acid; Sigma-Aldrich, São Paulo, Brazil)
were added. A standard curve was plotted starting with the mother solution (140 mg
of sodium nitrite [Sigma-Aldrich, São Paulo, Brazil] + 10 mL of distilled water).
The plates were left at room temperature for 10 minutes and then read in enzyme-linked
immunosorbent essay spectrophotometer (Thermoplate, TP reader basic, Beijing, China)
at 540 nm. The results were assessed by comparing the optical density to the nitric
oxide (NO) standard curve and expressed as µg of nitrite per 105 cells.
Immunoenzymatic Assay for Cytokine Measurement
Supernatant of the yeast-infected macrophage cultures was collected 48 hours after
the onset of treatments, centrifuged at 15,000 rpm for 5 minutes and frozen at –80°C
for later cytokine measurement. Cytokines were measured by means to the MAGPIX-Luminex
method (kit MILLIPLEX e-Bioscience mouse cytokine Magnet; Darmstadt, Germany) following
the manufacturer's instructions. This kit includes detection systems for interferon-γ,
interleukin (IL)-1α, IL-1β, IL-6, IL-10, IL-12 (P240), IL-12 (P70), monocyte chemoattractant
protein 1 (MCP-1), macrophage inflammatory protein-1β (MIP-1 beta), granulocyte macrophage
colony-stimulating factor (GM-CSF), regulated on activation, normal T cell expressed
and secreted chemokine (RANTES), vascular endothelial growth factor (VEGF), and tumor
necrosis factor-α (TNF alpha). The samples were analyzed in triplicate and the results
expressed as pg/mL. Samples with values below the limit of detection were recorded
as 0 (zero).
Scanning Electron Microscopy/Energy-Dispersive X-Ray Spectroscopy
The identification of silicon content in suspended microparticles probably contained
in the tested solutions was performed by means of the micro-evaporation method, followed
by scanning electron microscopy (SEM) under 10-kV working conditions and by energy-dispersive
X-ray spectroscopy (EDX) (JSM 6510; JEOL Ltd, Tokyo, Japan).
Microtubes containing samples of each tested solution were centrifuged at 5,000 rpm
for 30 minutes for the microparticles to sediment. A single drop of 10 µL was harvested
from the bottom of each tube and placed on a stub. The stubs were previously cleansed
with 70% acetone and dried in a closed recipient. The stubs were then kept within
a closed Petri dish to ensure that only particles obtained through centrifugation
of the tested solutions were analyzed. The drops were left to dry naturally on the
stubs up to the time of microscopic analysis. Images were taken with a 10-µm scale
(see example in [Fig. 1]). The samples were harvested only once from each tube, because in a pilot study
we found that the second and third drops were almost particle free.
Fig. 1 Scanning electron microscopy (SEM) showing numerous micro-particles deposited on
the stub surface. Note that the stub surface is irregular. SEM was used only to identify
the presence of silica in the microparticles. Their morphology and size were not evaluated.
An illustrative SEM profile showing particles on the stub from a sample of Ars 6cHP is represented. Magnification 2,000x.
The results of EDX are expressed as the frequency of silicon among all analyzed microparticles.
Thirty-four microparticles deposited on the stubs were randomly chosen for EDX analysis;
for samples that did not reach this number, all the deposited particles were analyzed.
As the stubs were made of copper, this element was blocked in the reader control to
avoid interference. Considering the characteristics of the microscope, to warrant
a minimum sensitivity threshold, for each identified element, the percentage of atoms
per particle below 1.5% was considered as zero (negative result). Thus, traces of
other elements (C, O, Na, Mg, Zn, Ca, Be, Nb, Pb, Cl, Tc, Rb, Al, S, Fe, P) were randomly
detected in all the analyzed dilutions, but, since they were present under the limit
of sensitivity of the device, they were not considered.
Electrical Current Flow (Conductance)
A micro-ammeter (Ryodoraku; São Paulo, Brazil), calibrated to 200 M/A, was used to
measure the capacity to allow electric current in each dilution. The device wires
were connected to two parallel electrodes (30 × 40 mm). The solutions were put into
flat cryotubes with parallel walls to avoid contact between the electrodes and the
tube wall.[29] For measurement, the electrodes were inserted 1 cm into the liquid. The test was
performed in three series, in triplicate, at different times of the day, under a closed
hood; electronic devices were kept at least 2 m away from the test site to avoid any
influence of magnetic fields. A thermo-hygrometer was placed inside the hood to monitor
temperature (24.8°C, on average) and humidity (57%). Conductivity was measured after
a 5-second interval to allow for stabilization of the device. Pure sterilized water
was used as standard for comparison, as its conductivity is quite stable and equal
to 100 µA. Sterilization included autoclaving for 20 minutes followed by filtering
through a 22-µ Millipore filter. The vehicle (filtered and agitated 70% alcohol) was
used as experimental control. A sample of water subjected to 100 vertical motions
(potentization) was used as supplementary control.
Statistical Analysis
Statistical analysis was performed using IBM SPSS, version 21.0, and GraphPad Prism,
version 6.0, for Windows. Normality was evaluated by means of the Shapiro–Wilk test
and assessed by inspection of quartile–quartile plots (Q–Q plots). Homogeneity of
variances was evaluated by Levene's test and the Welch correction was applied to the
ANOVA in cases of non-linearity. Outliers were assessed by inspection of Q–Q plots
and were removed when necessary.
Outcomes were evaluated by means of two-way ANOVA and Dunnett's post-test. Partial
eta-squared (η
2), a measure of effect size, was also reported. This parameter was particularly useful
for variables presenting large deviation values and analyses with p-values that could lead to type I (rejection of a true null hypothesis) or II (retaining
a false null hypothesis) errors.
Results are presented as mean ± standard error and p-values < 0.05 were considered significant.
The profile of the atomic composition of microparticles detected in samples of arsenic
dilutions was represented by dot plot graphics with mean and 95% confidence interval.
Results
Assessment of In Vitro Spreading, Phagocytosis and Nitric Oxide Production
On the orange acridine test, Ars 200cHP and Ars 200cHG induced greater lysosomal activity compared with the controls, manifested
as patent fluorescent acid vacuoles ([Fig. 2]).
Fig. 2 Photomicrograph of cells stained by means of the acridine orange method under fluorescence
microscope. (A) Macrophages co-cultured with yeast. (B) Arsenicum album 200cH prepared in plastic vials. (C) Arsenicum album 200cH prepared in glass vials. (D) Supernatant of centrifuged Arsenicum album 200cH prepared in glass vials. The arrow indicates a phagolysosome with acid content.
Magnification 40x.
Macrophages treated with Ars 200cHP exhibited increased phagocytic index (p = 0.001, F(2,81) = 5.638, η
2 = 0.173) compared with the other groups. Similarly, NO production after 48-hour incubation
was higher in Ars 200cHP (p = 0.006, F(7,8) = 9.199, η
2 = 0.775). However, Ars 200cHG and Ars 200cHGC presented significant reduction in spreading (p = 0.001, F(3,60) = 11.893, η
2 = 0.373) compared with the control and Ars 200cHP ([Figs. 3] and [4]).
Fig. 3 Spreading (in pixels) (A) and phagocytic index (B) of RAW 264.7 macrophages treated with Arsenicum album 200cH. Values represent individual points, mean and standard error. Control + yeast:
macrophage-yeast co-culture. Ars 200cHG: Arsenicum album 200cH prepared in glass vials. Ars 200cHGC: Arsenicum album 200cH prepared in glass vials and centrifuged. Ars 200cHP: Arsenicum album 200cH prepared in plastic vials. (A) *ANOVA, p < 0.001, F(3,60) = 11.893, η
2 = 0.373, compared with Ars 200cHP and control; (B) *ANOVA, p < 0.001, F(2,81) = 5.638, η
2 = 0.173, compared with the other treatments. ANOVA, analysis of variance.
Fig. 4 Nitric oxide (µg/105 cells) in RAW 264.7 macrophage-yeast co-culture supernatant after 48-hour incubation.
Values represent individual points, mean and standard error. Control + yeast: macrophage-yeast
co-culture. Ars 200cHG: Arsenicum album 200cH prepared in glass vials. Ars 200cHGC: Arsenicum album 200cH prepared in glass vials and centrifuged. Ars 200cHP: Arsenicum album 200cH prepared in plastic vials. *ANOVA, p < 0.006, F(7,8) = 9.199, η
2 = 0.775, compared with the other treatments. ANOVA, analysis of variance.
Neither Ars 6cHP nor Ars 6cHG induced significant changes in macrophage spreading, phagocytic index or NO
production after 48-hour incubation. On the acridine orange test, no significant differences
were detected between Ars 6cH preparations and control ([Figs. 5] and [6]).
Fig. 5 Spreading (in pixels) (A), phagocytic index (B) and NO production (µg in 105 cells) (C) of RAW 264.7 macrophages treated with Arsenicum album 6cH. Values represent mean and standard error. Control + yeast: macrophage-yeast co-culture. Ars 6cHG: Arsenicum album 200cH prepared in glass vials. Ars 6cHP: Arsenicum album 6cH prepared in plastic vials. ANOVA, analysis of variance.
Fig. 6 Photomicrograph of cells stained by means of the acridine orange method under fluorescence
microscope. (A) macrophages co-cultured with yeast. (B) Arsenicum album 6cH prepared in glass vials. (C) Arsenicum album 6cH prepared in plastic vials. 40x magnification.
Cytokine Measurement
Interleukin-1β and GM-CSF were not analyzed because the measured values were below
the minimum limit of detection as declared by the manufacturer.
Ars 6cHP induced statistically significant increases in IL-1α (F(2,6) = 15.501; p = 0.04; η
2 = 0.838), IL-6 (F(2,6) = 10.894; p = 0.010; η
2 = 0.784), MCP-1 (F(2,6) = 14.165; p = 0.005; η
2 = 0.825), and VEGF (F(2,6) = 44.335; p = 0.01; η2 = 0.947) ([Fig. 7]). In turn, treatment with Ars 200cHP, Ars 200cHG, and Ars 200cHGC was associated with statistically significant reduction in IL-6 (F(3,8) = 4.722; p = 0.035; η2 = 0.639) compared with the non-treated control ([Fig. 8]).
Fig. 7 Concentration (pg/mL) of cytokines and chemokines in the supernatant collected from
RAW 264.7-yeast co-culture. IL-1α (A), IL-6 (B), MCP-1 (C), VEGF (D) produced by cells treated with Arsenicum album 6cH prepared in glass and plastic vials. Values represent mean and standard error.
*
p < 0.05 compared with the untreated control, two-way ANOVA. **
p < 0.005 compared with the untreated control, two-way ANOVA. See text for η
2 values. ANOVA, analysis of variance; IL-1α, interleukin-1α; MCP-1, monocyte chemoattractant
protein-1; VEGF, vascular endothelial growth factor.
Fig. 8 Concentration (pg/mL) of IL-6 in the supernatant collected from RAW 264.7-yeast co-culture
treated with Arsenicum album 200cH prepared in glass (centrifuged or not) and plastic vials. Values represent
mean and standard error. *
p = 0.035 compared with the untreated control, two-way ANOVA. See text for η2 values. ANOVA, analysis of variance; IL-6, interleukin-6.
No differences among groups were found regarding the other analyzed cytokines ([Table 1] and [2]).
Table 1
Cytokines, chemokines and VEGF-A measurements (pg/mL) in the supernatant of macrophages
treated with different protocols of Arsenicum albu
m. The supernatants were harvested after 48 hours of culture and treatment. Values
are expressed in mean and standard deviation. Experiment using Arsenicum album 6cH
|
Treatments
|
Untreated control
|
Ars 6cHG
|
Ars 6cHP
|
|
INF-γ
|
4.55 ± 1.19
|
4.69 ± 0.73
|
4.11 ± 0.97
|
|
IL-1α
|
56.64 ± 16.03
|
38.04 ± 1.15
|
92.34 ± 13.95[**]
|
|
IL-6
|
5.68 ± 0.95
|
4.16 ± 1.25
|
68.25 ± 33.20[*]
|
|
IL-10
|
3.61 ± 1.25
|
3.27 ± 0.39
|
3.98 ± 0.66
|
|
IL-12 p40
|
6.01 ± 2.30
|
4.63 ± 0.74
|
4.98 ± 0.95
|
|
IL-12 p70
|
10.86 ± 9.53
|
4.53 ± 1.22
|
5.42 ± 2.18
|
|
MCP-1
|
795.33 ± 193.08
|
638.66 ± 202.74
|
1576.33 ± 286.19[**]
|
|
MIP-1β
|
12727.00 ± 768.09
|
12647.33 ± 494.54
|
13796.33 ± 643.12
|
|
RANTES
|
261.33 ± 58.52
|
262.00 ± 75.43
|
352.66 ± 104.57
|
|
VEGF
|
4.48 ± 1.53
|
5.46 ± 3.28
|
66.08 ± 17.02[*]
|
|
TNF-α
|
890.66 ± 139.95
|
760.00 ± 113.84
|
714 ± 65.50
|
Abbreviations: IL-1α, interleukin-1α; INF-γ, interferon-γ; МCP-1, monocyte chemoattractant
protein-1; MIP-1β, macrophage inflammatory protein-1β; RANTES, regulated on activation,
normal T cell expressed and secreted chemokine; TNF-α, tumor necrosis factor-α; VEGF,
vascular endothelial growth factor.
*
p < 0.05 compared with the untreated control,
**
p = 0.005 compared with the untreated control, two-way ANOVA.
See text for η
2 values.
Table 2
Cytokines, chemokines and VEGF-A measurements (pg/mL) in the supernatant of macrophages
treated with different protocols of Arsenicum album. The supernatants were harvested after 48 hours of culture and treatment. Values
are expressed in mean and standard deviation. Experiment using Arsenicum album 200cH
|
Treatments
|
Untreated control
|
Ars 200cHG
|
Ars 200cHGC
|
Ars 200cHP
|
|
INF-γ
|
4.75 ± 1.36
|
3.96 ± 1.09
|
5.08 ± 1.47
|
4.75 ± 1.50
|
|
IL-1α
|
77.70 ± 71.27
|
82.06 ± 70.67
|
48.02 ± 42.20
|
71.71 ± 39.02
|
|
IL-6
|
78.48 ± 46.63
|
13.78 ± 5.22*
|
13.96 ± 35.77 *
|
19.56 ± 31.49*
|
|
IL-10
|
4.00 ± 0.94
|
4.36 ± 1.55
|
3.61 ± 1.27
|
3.81 ± 1.22
|
|
IL-12 p40
|
4.85 ± 1.42
|
4.89 ± 1.80
|
4.28 ± 1.46
|
4.22 ± 1.42
|
|
IL-12 p70
|
7.86 ± 3.63
|
6.06 ± 4.21
|
6.39 ± 2.55
|
6.84 ± 2.37
|
|
MCP-1
|
3237.00 ± 1574.47
|
1453.00 ± 940.16
|
1985.33 ± 1111.32
|
1742.66 ± 1053.59
|
|
MIP-1β
|
14675.33 ± 1302.98
|
13161.67 ± 1685.76
|
14763.33 ± 4743.15
|
13758.33 ± 4577.11
|
|
RANTES
|
230.00 ± 118.82
|
121.97 ± 84.36
|
114.12 ± 80.16
|
157.50 ± 82.97
|
|
VEGF
|
8.39 ± 6.61
|
8.31 ± 3.90
|
8.19 ± 3.30
|
5.88 ± 3.13
|
|
TNF-α
|
1765.00 ± 474.00
|
1345.66 ± 509.48
|
1448.33 ± 502.74
|
1536.33 ± 557.24
|
Abbreviations: ANOVA, analysis of variance; IL-1α, interleukin-1α; INF-γ, interferon-γ;
МCP-1, monocyte chemoattractant protein-1; MIP-1β, macrophage inflammatory protein-1β;
RANTES, regulated on activation, normal T cell expressed and secreted chemokine; TNF-α,
tumor necrosis factor-α; VEGF, vascular endothelial growth factor.
*p = 0.035 compared with the untreated control, two-way ANOVA.
η2 = 0.639.
Scanning electron microscopy + energy-dispersive X-ray Particle Analysis
The presence of measurable silicon among identified particles was only found in Ars 6cHG and Ars 200cHG. No measurable silicon was detected in Ars 200cHP, Ars 200cHGC, and Ars 6cHP ([Table 3]).
Table 3
Frequency of the element silicon among the identified micro-particles found in different
Arsenicum album samples, using SEM/EDX system (number of particles “positive” to the presence of
silicon in relation to the total)
|
Frequency
|
Ars 6cHG
|
Ars 6cHP
|
Ars 200cHG
|
Ars 200cHGC
|
Ars 200cHP
|
|
Per particle
|
3/34
|
0/24
|
3/34
|
0/13
|
0/24
|
|
Percentage
|
8.8%
|
0%
|
12.5%
|
0%
|
0%
|
Abbreviations: EDX, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.
Electric Current Flow
The capacity for an electric current to flow in the fluid (conductance) of the pure
filtered water was used as a standard for comparison. Significant reduction in electric
flow in relation to water was found in 30% alcohol and Ars 200cHP, probably due to the presence of organic non-polar molecules. Ars 6cHP also presented low level of conductance, though without statistical significance
due to the dispersion of the datapoints. The greatest conductance was exhibited by
Ars 200cHGC and Ars 200cHG, in relation to the controls and Ars 200cHP (p = 0.001, F(6,95) = 134.342, η
2 = 0.895) ([Fig. 9]).
Fig. 9 Electrical current flow (conductance) of arsenic HDs (µA). Values represent individual
points, mean and standard error. W FILTR agitated: pure water filtered in a 22-µm
filter (Millipore) and vertically agitated (100 times). AL FILTR agitated: 70% alcohol
filtered in 22-µm filter (Millipore) and vertically agitated (100 times). Ars 6cHG: Arsenicum album 6cH prepared in glass vials. Ars 6cHP: Arsenicum album 6cH prepared in plastic vials. Ars 200cHG: Arsenicum album 200cH prepared in glass vials. Ars 200cHGC: Arsenicum album 200cH prepared in glass vials and centrifuged. Ars 200cHP: Arsenicum album 200cH prepared in plastic vials. ANOVA, F(6, 95) = 134.342, η2 = 0.895; *
p < 0.001 in relation to water; **
p < 0.001 in relation to the other treatments.
Discussion
The results of the present study show plastic-related non-specific effects and very
specific effects on IL production, which were independent of microparticles containing
silicon but probably dependent on the smallest nanostructures, which could include
water and the silica itself. In this sense, dilutions of arsenic prepared in plastic
vials exhibited pro-inflammatory effects attributed to putative organic residuals
detached from the vials' walls during disinfection and agitation, since these effects
were not seen in glass-prepared medicines ([Supplementary Fig. 1], available in the online version only).
Macrophage spreading, phagocytic activity and increase in IL-1α, IL-6, MCP-1, and
VEGF in the culture medium were found only in samples treated with medicines prepared
in plastic vials—Ars 6cHP and Ars 200cHP, but not in Ars 6cHG or Ars 200cHG. These findings suggest that plastic-derived soluble contaminants present
in higher potencies might be somehow related to the increase in macrophage activity.
Available evidence indicates that PET bottles subjected to high temperature (40–60°C)
do not exhibit cytotoxic activity,[30] but biomaterials containing PET might modify the macrophage activity in vitro.[31]
[32] In any case, plastic components have relevant biological actions, this fact being
of interest for homeopathic pharmaceutics and, thus, deserving of more thorough study.
For instance, one study compared homeopathic medicine Gelsemium sempervirens 6c, 30c, and 200c to agitated vehicle controls (6c, 30c, and 200c) and non-agitated
solvent prepared in vials with natural cork stoppers and with silicone stoppers. All
the vials with cork stoppers exhibited more than 4 × 108 NPs/mL, significantly more than with the non-agitated silicone stoppers. On ultraviolet–visible
spectroscopy, the agitated control 30c exhibited significantly higher absorbance,
while the non-agitated silicone stopper controls had significantly lower mean absorbance
than all the other samples.[33]
These findings suggest that the agitation inherent to homeopathic remedy manufacture
released trace materials from natural cork stoppers that could interfere with various
properties of HDs, and eventually also with their biological activity.[33] Also, potentized water exhibited a residual effect on macrophage spreading, which
might be attributed to a non-specific action of micro- and/or nano-bubbles.[34] A direct action of medicines on yeast is unlikely in the present case because, according
to the literature, treatment with Ars in decimal dilutions 8x and 16x did not induce any change in S. cerevisiae growth.[35]
The decrease in IL-6 production might be considered as the main specific biological
effect of Arsenicum album 200cH, since it was observed not only in this potency but in all situations: Ars 200cHG, Ars 200cHGC, and Ars 200cHP. Therefore, these specific effects were not related to the identification
of silicon-containing microparticles and agree with the effects of Ars 200cH on leukocytes previously described.[24] The decrease in macrophage spreading after treatment with Ars 200cHG and Ars 200cHGC corroborates this result; besides it is very specific, focused on the reduction
in IL-6 production itself. This specific feature is also seen in other studies involving
gene expression and homeopathy.[27]
[36]
[37]
The acridine orange test showed an interesting result. While Ars 200cHP and Ars 200cHG induced a similar morphological pattern, characterized by patent acid vacuoles
(lysosomes), Ars 200cHGC (superficial phase after centrifugation) induced a less active pattern. Maybe
the ultra-centrifugation of Ars 200cHG and the resulting changes in its microparticle composition can be related
to this result, since the biggest particles present before centrifugation could also
be phagocytosed, stimulating the cells.[38]
[39]
[40]
[41]
The correlation between electric current flow through the liquids and the type of
the vial used corroborates the hypothesis in which metal particles in suspension could
improve conductivity[1]
[2]
[21]
[41] and possible organic contaminants from plastic could impair it. The size of these
particles seems not to be important in this case, since Ars 200cHGC (only the supernatant of the medicine, after ultra-centrifugation) presented
the highest values of electric current flow.
In short, the results of the present study indicate lack of correlation between the
presence of silicon-containing microparticles and biological (specific or non-specific)
effects of the tested HDs. On these grounds, one might not infer that silicon-containing
microparticles could carry the information of homeopathic medicines to biological
systems. However, smaller particles, as NPs,[1]
[2]
[4]
[5]
[6]
[22]
[32]
[33] might explain the divergence. Moreover, in this model, NPs of the original substance
could not be observed, maybe due to the limitations of the microscope used. Some studies
showing these particles and their putative hormetic effects are well described in
the literature.[41]
[42]
[43]
Another hypothesis remains to be checked in the future: that the capacity of water
itself acts as information carrier, based on electromagnetic properties derived from
its dipole behavior.[44]
[45]
[46]
[47]
[48] Alternatively, the participation of both elements together—water and particles,
mixed in complex nano-structures that are almost impossible to separate in nature—could
elicit some biological effect.[49]
Many studies using refined methods are still needed to establish which hypotheses
account for the mechanism of action of homeopathy, since several rationales represent
reasonable possibilities.[50]
[51]
