Background
Highly diluted substances and tinctures are usually intended to enhance the immune system resulting in reduced frequency of various diseases. The immune system is a complex network that integrates lymphoid organs, cells, humoral factors, and cytokines. The essential function of the immune system is host defense, but when it does not work properly the disease emerges. For instance, underactivity may result in severe infections and tumors of immunodeficiency, overactivity in allergic and autoimmune disease [
1]. When the body successfully fights any potential harmfully agent by itself it results in an enhancement of the immune system. Conventional treatments often are directed to terminate the symptoms rather than fighting the disease cause, and frequently present side effects. Adverse drug reactions inflict serious injury to million people around the world. It is known that all substances can produce harmful effects associated with its toxic properties, and quoting Paracelsus (1493-1541) "all substances are poisons; there is none which is not a poison. The right dose differentiates a poison". The search for effective treatments with low toxicity to patient is needed. Changes in the dose could be effective in order to diminish the harmful side effects. All drugs present a dose response curve, or a range of doses that result in a graded effect between the extremes of no effect and 100% response which is the toxic effect. If the dose is low enough even a highly toxic substance will cease to cause the harmful effect on biological systems. Thus in the case of using highly diluted compounds or tinctures you might avoid the harmful effects as it often present no risk of serious side-effects because of their low toxicity, and still obtain an effective response such as modulation of the immune system.
Over the past years our research group has been testing the action of highly diluted substances and tinctures on cells from the immune system [
2‐
4]. The development of new combinations was based on laboratorial tests and assays were performed independently and in a blind manner. These were crucial to determine which substances to use on each combination of highly diluted tinctures. For instance, one substance when used alone presents modification in biological responses, i.e. it increases cytokine production by cells, but when combined to another substance the effects on biological responses are annulled (data not published yet). Biological assays are intended to understand better what happens within a cell when a molecule interacts with its target. In order to determine efficacy and safety of new drugs there is a clear move within the pharmaceutical industry towards increased emphasis on cell-based assays. Well-structured methodological studies are useful to understand how highly diluted tinctures work. Thus we have developed and tested several new highly diluted tinctures, of which three of them are described here. As products were coded by an external observer, they were initially coded as M followed by a number. In the present study we describe the biological activity of M1, M2 and M8 both
in vitro in immune cells from mice and human, and
in vivo in mice.
Discussion
Plant extracts are common subjects of basic research. For instance, imunopharmacological potential of
Thuya occidentalis was widely demonstrated both
in vivo and
in vitro as reviewed by Naser and colleagues in 2005 [
12]. Highly diluted tinctures or substances are less studied, but there is an increased interest in these type of formulations due to its low cost and biological effectiveness. Bellavite and col. revised in 2006 [
13] articles containing basic research on cells of the immune system and inflammation. They have shown that there are few and rather small groups working on laboratory models, although evidences of the biological activity
in vitro of highly diluted-dynamized solutions is slowly accumulating. Our group has tested some highly diluted tinctures alone and we have found that they present different responses when different potencies are tested (data not published yet). Here we have investigated the effects of three combinations, namely M1, M2, and M8 on mice and human cells. M1, M2, and M8 are prepared from 15, 13, and 11 highly diluted components, respectively, in different dilutions as seen on table
1. We have observed that all these induced different effects on cells, as well as different immune response type, even though they have some similarity on its composition as seen on table
1.
In order to evaluate M1, M2 and M8 safety we have first determined cell viability and cell cycle after
in vitro treatment. We have used K562, HT-29 cell line and macrophages to determine if treatment could induce cell death and/or changes in cell cycle. We have observed that none of the tested combinations of highly diluted tinctures were cytotoxic or affected cell cycle. Next we sought to determine if there was any effect of these highly diluted tinctures on cytokine release by monocytes-derived macrophages. Treatment alone did not change cytokines production by cells, but when cells were stimulated with LPS and then treated IFN-γ and TNF-α production was decreased. TNF-α is an important inflammatory factor that acts as a master switch in establishing an intricate link between inflammation and cancer. TNF-α secretion can be induced by conserved structural elements common to microbial pathogens as well as by tumour cells. Several studies have focused on the transcriptional regulation of TNF-α looking at transcription factors that bind to the responsive element sites within the TNF-α promoter. NF-κB is a transcription factor that plays crucial roles in inflammation and immunity [
14]. Many proinflammatory stimuli can activate NF-κB, mainly through IKK-dependent phosphorylation and degradation of the IκB inhibitory proteins. When NF-κB translocates to the nucleus, it activates the transcription of target genes, including cytokines like TNF-α, chemokines, and antiapoptotic factors [
15]. We have used a reporter cell line to find out if M1, M2, and M8 in the presence or absence of TNF-α stimulus, have any effect on NF-κB activity. The reporter cell line HT29-pNF-κB-hrGFP is routinely used to screen natural or synthetic compounds that interfere and/or modulate NF-κB activity. We have observed that only M1 has decreased NF-κB activity but it was not downregulated by M2 and M8. However we have observed TNF-α reduction by these highly diluted tinctures. NF-κB, C/EBPβ, Ets, NF-AT, activating protein 1 (AP-1), cAMP response element-binding protein, signal transducers and activators of transcription (STAT1), and lipopolysaccharide (LPS)-induced TNF-α factor (LITAF) were all implicated on regulating TNF-α transcription [
16]. Regulatory elements in transcriptional activation of the TNF-α gene are not completely understood. Further investigation of others transcription factors that regulate TNF-α induction may be conducted in order to investigate the relative contributions of these various regulatory elements in transcriptional decrease of the TNF-α by M2 and M8. Improving the efficacies of anti-inflammatory mediators hold promises for the therapy of cancers and chronic inflammatory disorders. Thus after detecting that M1, M2, and M8 have
in vitro effects we have established the biological activity of those highly diluted tinctures after mice treatment (
in vivo).
First we have detected changes on oxidative metabolism of macrophages after M1, M2, and M8
in vivo administration to mice. The phagocytic oxidative burst is a primary effector of innate immunity that protects against infection. When triggered by a phagocytic stimulus or certain membrane-active agents, macrophages activated
in vivo or
in vitro undergo a respiratory burst that is characterized by the production of high levels of oxygen metabolites. The NADPH-dependent oxidase (Phox), which produces superoxide, assembles in the phagosomal membrane [
17]. Activated macrophages also produce nitric oxide (NO), generated from arginine and oxygen by the inducible nitric oxide synthase (iNOS) [
18]. Nitric oxide is a cytotoxic product of activated macrophages, along with reactive oxygen species that have been shown to be involved in numerous regulatory functions [
19]. Reactive oxygen species have been increasingly implicated as playing a central role in the pathophysiology of clinical infections. More specifically superoxide, hydrogen peroxide, and recently, nitric oxide are thought to contribute to these processes. As these highly diluted tinctures exhibit the ability to modulate the respiratory burst and NO production they could be useful adjutants to host defence and to microbicidal activity [
20].
4-Hydroxynonenal (4-HNE) is a product derived from lipid peroxidation that can affect several mechanisms on the cell depending on its concentration. Low levels were shown to induce cell differentiation, even though the exact mechanism of action is still not known [
21,
22]. In fact, previous results from our lab have shown that highly diluted tinctures act on bone marrow cells stimulating proliferation and differentiation [
3]. Thus this might account as one the mechanism of action by how highly diluted tinctures stimulate bone marrow cells proliferation and differentiation, but further experiments are needed.
Bone marrow is a special, spongy, fatty tissue that houses stem cells, located inside a few large bones. These stem cells transform themselves into leukocytes, red blood cells and platelets, essential for immunity and circulation. A mouse lineage panel to bone marrow cells was used (CD11b (Mac-1)-monocytes/macrophages, Ly-6G-granulocytes, CD45R-B lymphocytes, CD11c-dendritic cells, CD3-T lymphocytes, and TER-119-erythrocytes), as well to lymph node cells (CD11b-Monocytes/macrophages, CD3-T lymphocytes, CD4-T helper lymphocytes, CD8-T cytotoxic lymphocytes, CD19-B lymphocytes, PanNK (DX5)-NK lymphocytes). We found that each highly diluted tincture presented a different pattern of host immune response. M1 has increased monocytes/macrophages and B lymphocytes in bone marrow and lymph nodes. CD3 lymphocytes and granulocytes were increased and erythrocytes were decreased in bone marrow. M2 has increased monocytes/macrophages and granulocytes and decreased B lymphocytes as well as erythrocytes in bone marrow. And M8 has increased B lymphocytes in bone marrow. Lymph nodes T cytotoxic lymphocytes were increased in lymph nodes by all treatments. Table
2 summarizes the biological activity analysis of M1, M2 and M8
in vivo.
The analysis of highly diluted tinctures after mice treatment showed that M1, M2, and M8 improved the innate (cellular) immunity. In addition, M1 also augmented acquired (humoral) immunity. M2 was the only highly diluted compound that diminished B lymphocytes precursors, which produce antibodies, responsible to humoral immunity. Decrease in antibody production by M2, but without causing immune suppression since it has increased the number of cytotoxic cells, turns out to be interesting to autoimmune diseases where this type of response is extremely important. The results from M8 treatment, as it increases innate immunity and does not alter acquired immunity, suggest a possible use in diseases where antibody production is irrelevant, i.e. viruses where the pathogenic agent is extremely mutable. As for M1 treatment we can conclude that it shows a possible general action on immune system, enhancing both innate and acquired immunity which are important in several diseases including some types of cancers.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
CCO conceived of the study, participated in all experiments from its design to its interpretation, and analyses, as well as draft the manuscript. APRA, SMO, ELOC have carried out the experiments with macrophages and bone marrow both in vivo and in vitro. FSFG, LFR carried out the in vivo experiments with mice, as well as lymphocytes analyses. RPB participated in the design of the study and helped on the statistical analysis. DK, EJL, JPG have designed and carried out the experiments using human cell. EST has provided critical help on experimental design and data analyses. DFB participated in experimental design and analyses, as well as coordination and helped to draft the manuscript. All authors read and approved the final manuscript.