Article on homeopathic remedy Calcarea carbonica
This is an interesting article because unlike the typical homeopathy article, it analyzes a homeopathic remedy as if it were a conventional drug rather than resorting to supposed magical properties of highly diluted and potentized preparations. Skip to my comments toward the end if you are not interested in the details (though I have tried to condense to the most important points in my excerpts).
>>>>>>>>>>>>>>>>>>>>>>>>>>
J Integr Med. 2020 Sep;18(5):441-449.
Calcarea carbonica treatment rescues lipopolysaccharide-induced inflammatory response in human mononuclear cells via downregulation of inducible cyclooxygenase pathway
Swatantra Kumar (1), Vimal K Maurya (1), Debadatta Nayak (2), Anil Khurana (2), Raj K Manchanda (2), Srinivasulu Gadugu (3), Madan L B Bhatt (1), Shailendra K Saxena (4)
1 Centre for Advanced Research, Faculty of Medicine, King George's Medical University, Lucknow 226003, India.
2 Central Council for Research in Homoeopathy, Ministry of Ayush, Janakpuri, New Delhi 110058, India.
3 Department of Medicine, Jaisoorya and Potti Sreeramulu Government Medical College, Hyderabad 500013, India.
4 Centre for Advanced Research, Faculty of Medicine, King George's Medical University, Lucknow 226003, India.
Abstract
Objective: Prolonged use of nonsteroidal anti-inflammatory drugs is associated with severe side effects and toxicity. Therefore, we studied the anti-inflammatory role of Calcarea carbonica which had minimal toxicity at the low doses.
Methods: THP-1 human mononuclear cells were treated with C. carbonica to evaluate the 50% cytotoxicity concentration (CC50) and 50% effective concentration (EC50). Cell survival was evaluated in lipopolysaccharide-stimulated C. carbonica-treated cells. Nitric oxide (NO) and tumor necrosis factor-a (TNF-a) were measured to evaluate the anti-inflammatory activity of C. carbonica. Cyclooxygenase-2 (COX-2) protein expression was determined by Western blotting analysis, and the interaction of C. carbonica with the COX-2 protein was evaluated using molecular docking simulation.
Results: The CC50 and EC50 of C. carbonica were found to be 43.26 and 11.99 µg/mL, respectively. The cell survival assay showed a 1.192-fold (P = 0.0129), 1.443-fold (P = 0.0009) and 1.605-fold (P = 0.0004) increase in cell survival at 24, 48 and 72 h after initiating C. carbonica treatment, respectively. C. carbonica-treated cells showed a reduction in NO levels by 2.355 folds (P = 0.0001), 2.181 folds (P = 0.0001) and 2.071 folds (P = 0.0001) at 24, 48 and 72 h, respectively. The treated cells also showed a reduction in TNF-a levels by 1.395 folds (P = 0.0013), 1.541 folds (P = 0.0005) and 1.550 folds (P = 0.0005) at 24, 48 and 72 h, respectively. In addition, a 1.193-fold reduction (P = 0.0126) in COX-2 protein expression was found in C. carbonica-treated cells. The molecular docking showed interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.
Conclusion: C. carbonica exhibited anti-inflammatory properties in lipopolysaccharide-stimulated cells by significantly reducing NO production and TNF-a level through downregulation of the COX-2 protein. This effect is probably mediated through interaction of C. carbonica with the phenylalanine 367 residue present in active site of Cox-2.
https://www.sciencedirect.com/science/article/abs/pii/S2095496420300571Excerpts:
"Complementary and alternative medicine (CAM) can be used in conjunction with conventional medicines, and may include nutritional supplements, botanical products and others [11]. CAM provides an opportunity to develop an effective anti-inflammatory drug with few or no side effects [12], [13]. Among the available CAM therapies, Calcarea carbonica has been shown to exhibit anticancer properties [14], [15]. Cancer is a condition of chronic inflammation, yet the anti-inflammatory effects of C. carbonica have not yet been well established. C. carbonica is obtained from the pure middle layer of thick oyster shells; this material is comprised of calcium carbonate (CaCO3). C. carbonica is crystalline in nature and includes other trace elements. Several medicinal effects have been attributed to the use of C. carbonica, viz antipsoriatic effects, and as a remedy for tickling cough, fleeting chest pains, nausea and acid reflux, pituitary and thyroid dysfunctions, and increasing the ability of blood to coagulate [16], [17]. It is typically used in conjunction with belladonna in chronic disease conditions. Therefore, we have planned to test the anti-inflammatory effects of C. carbonica in the treatment of the lipopolysaccharide (LPS)-induced inflammatory response in human mononuclear cells and evaluate potential mechanisms of action."
(Materials and Methods: Preparation) "C. carbonica was prepared from the precipitated CaCO3 mineral powder and contained no less than 98.5% of CaCO3 following the guidelines of the Pharmacopoeia of India, 1971 [18]. Briefly, to prepare 100 mg of 1× solution, 10 mg of C. carbonica precipitate was mixed with 90 mg of saccharum lactis using a mortar and pestle. The stock was further diluted in ethanol to achieve the desired dilution of C. carbonica. Similarly, placebo, a hydro-alcoholic solution, was prepared following the same procedure, but without C. carbonica precipitate."
(Biophysical characterization) "C. carbonica was characterized using various biophysical methods...For characterization, 11.99 µg/mL C. carbonica was used."
(Cell survival assay) "Differentiated THP-1 cells were treated with a range of concentrations (1–100 µg/mL) of C. carbonica to identify the 50% cytotoxicity concentration (CC50) after 24 h of incubation. Similarly, the differentiated cells were stimulated with 200 ng/mL of LPS and treated with various concentrations (below the CC50) of C. carbonica to elucidate the 50% effective concentration (EC50) after 24 h of incubation."
(Molecular docking) "The protein data bank (PDB) structure of the human COX-2 protein (ID: 5F1A) was retrieved from Research Collaboratory for Structural Bioinformatics PDB [25]. The three dimensional (3D) structure of C. carbonica (ID: 767) was downloaded in structure-data file format from PubChem...In order to determine the active pocket of the COX-2 protein, we used metaPocket 2.0...a meta server, that uses a consensus method to identify the active site of a modeled protein [26]. Molegro Virtual Docker was used to simulate the docking of C. carbonica with the target COX-2 protein [27] and the MolDock score was compared for different poses."
(Drug-likeness and pharmacokinetics property) "The prediction of the drug-likeness of C. carbonica was performed using the Lipinski filter of the admetSAR server...a comprehensive tool for predicting absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of drug candidates; it provides an interface to screen compounds for their drug-like properties. In the present study, admetSAR was used to identify orally active drug molecules based on molar refractive index, H-bond acceptor, H-bond donor, cLogP and molecular mass...The structure of C. carbonica was retrieved from PubChem to calculate the ADMET properties [29], [30]."
(Results: Biophysical characterization) "FTIR spectroscopy indicated the presence of CaCO3 and ethanol in samples. Peaks were present around 1399.49 cm-1, characteristic of the CaCO3 spectrum. Further, peaks indicating C–H stretch, around 2917.32 cm-1, and peaks indicating O–H stretch, around 3372.87 cm-1, demonstrated the presence of ethanol (Fig. 1A). In addition, TEM images of C. carbonica showed the presence of CaCO3 nanoparticles, captured in a layer that might be acting as the stabilizing material (Fig. 1B)."
(Cytotoxicity and effective concentration, cell survival, NO production, TNF-a level, COX-2 expression: see Abstract for summary of results)
(Interaction of C. carbonica with human COX-2 protein) "The significant reduction of C. carbonica-mediated COX-2 protein expression led us to explore the interaction of the COX-2 protein with C. carbonica...Docking simulation showed that the C. carbonica preparation could have significant interaction with the human COX-2 protein. The molecular docking simulation showed strong binding affinity in the active site of the protein..."
(Drug likeness and pharmacokinetics property) "According to the Lipinski rule for drug likeness, any drug molecule having MW > 500 g/mol, H-bond-donating atoms > 5, H-bond-accepting atoms > 10 or cLogP > 5 is not considered to be a good orally active pharmaceutical agent. The Lipinski analysis showed that C. carbonica had a MW of 100.09 g/mol, a cLogP value of -2.83 and three H-bond donors, suggesting that C. carbonica met the drug-likeness threshold of the Lipinski rule. Further, the ADMET analysis identified various other drug-likeness properties of C. carbonica, including that it may be absorbed in human intestine, can cross the blood–brain barrier and has oral bioavailability (Table 1)."
(Discussion)
“Among the inflammatory mediators implicated in a wide range of disease conditions, COX plays an important role. COX is an enzyme involved in the synthesis of prostanoids—a major target of NSAIDs...Therefore, in the present work we studied the anti-inflammatory activity of C. carbonica to see if it could provide a suitable alternative for the selective inhibition of COX-2.
“For the first time, we have shown the anti-inflammatory effects of C. carbonica in the treatment of LPS-stimulated cells, and identified a potential mechanism of action...In the present study, we have shown that treatment with C. carbonica improves survival of LPS-stimulated cells by reducing the expression of pro-inflammatory mediators.”
“Our data showed a reduction in NO production in C. carbonica-treated LPS-stimulated cells. The inhibition of NO production in an activated macrophage-like cell line via iNOS has also been reported in response to treatment with established anti-inflammatory drugs...”
“TNF-a is a strong pro-inflammatory cytokine that plays a crucial role in inflammation, cell death, cell proliferation and cell differentiation...We found a reduction in TNF-a levels in C. carbonica-treated LPS-stimulated cells...Thus, downregulation of inflamed macrophage-derived TNF-a could be a novel strategy to treat macrophage-mediated chronic inflammation.”
“COX-2 is an inducible form that is activated during inflammation. We have shown that C. carbonica treatment in LPS-stimulated cells inhibits expression of COX-2 (Fig. 6)...We have modeled C. carbonica interacting with the COX-2 protein in the same active site and thereby reducing its expression. In our simulation, C. carbonica interacted with the phenylalanine 367 residue, which has been shown as one of the residues to be targeted by successful COX-2 inhibitors. Moreover, C. carbonica has been found to be safe and absorable [sic] in the human intestine, cross the blood-brain barrier and have oral bioavailability. Considering the potential role of C. carbonica and its safety, it should be suitable for use as an anti-inflammatory medicine in chronic conditions. However, clinical trials should be performed in order to test its safety and efficacy.”
(Conclusions)
“Collectively, our novel data show that treatment with C. carbonica has anti-inflammatory properties in LPS-stimulated cells by significantly reducing the NO production and TNF-a level through downregulation of the COX-2 protein, which is probably mediated by its interaction with phenylalanine 367. These findings suggest various opportunities for the development of novel anti-inflammatory therapeutics that could be effective at low doses.”
“Funding
“The study was not supported by any specific funding. Saxena SK is supported by Central Council for Research in Homoeopathy, Ministry of Ayush, Government of India.”
>>>>>>>>>>>>>>>>>>>>>>>>>
Comments:
As I note at the start, this paper treats the homeopathic remedy as if it were a conventional drug with measurable quantities of active materials. The active ingredient is calcium carbonate. A typical dose used in the studies (which were done with cultured cells) was 12 micrograms per milliliter. Since the remedy was diluted 10 times by mass with "saccarum lactis" (lactose), the concentration of calcium carbonate would be 1.2 micrograms per milliliter, or 1.2 mg per liter, which is 12 micromolar (molar mass of calcium carbonate = 100 g).
The cells were cultured in RPMI 1640 medium, which contains 2 g per liter sodium bicarbonate and is designed to give a pH of 8 when used with a 5% CO2 incubator (which was the case here). The authors indicated that they also added 2 g per liter sodium bicarbonate. Together, this gives a concentration of 47.6 millimolar bicarbonate ion.
At pH 8, there will be an equilibrium between dissolved carbon dioxide, carbonic acid, bicarbonate ion, and carbonate ion. Using the equilibrium constant for bicarbonate, carbonate, and hydroxide ion, and specifying a pH of 8, I calculate that 47.6 millimolar bicarbonate will be in equilibrium with 267 micromolar carbonate in the medium. If 12 micromolar carbonate were added with in the form of homeopathic calcium carbonate, most of that would be converted to bicarbonate according to the equilibrium, and the change in dissolved carbonate concentration would likely be trivial.
Thus, I find it hard to believe that biological effects such as those reported would be produced by adding carbonate ions with the homeopathic remedy. They are also unlikely to be due to the added calcium, since the medium contains 610 micromolar calcium already (compared to an added 12 micromolar from the Calcarea at the typical dose discussed above).
If the reported effects are genuine, perhaps they are due to the "nanoparticles" which were observed (see Biophysical characterization). These particles would consist of lactose, undissolved particles of calcium carbonate, and perhaps other contaminants introduced during the grinding process. The original mineral was also stated to have "other trace elements." (There is also ethanol, but the same concentration of ethanol was used in controls.) Maybe the interaction of these particles with cell membranes produced some effects, such as toxicity at high concentrations and induction of enzymes at lower levels.
However, the authors do not invoke nanoparticles or any other special property of homeopathic remedies to explain the effects; their proposal is based on the simple inorganic compound calcium carbonate acting in a conventional chemical manner. They focus on the production of pro-inflammatory mediators nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha), and on the activity of the enzyme cyclooxygenase-2 (COX-2). Their explanation of how this happens is confusing. On the one hand, they proposed that "C. carbonica inhibits LPS[lipopolysaccharide]-stimulated TLR4 [toll-like receptor 4] signaling, which downregulates the expression of COX-2 and iNOS [inducible nitric oxide synthase]..." Thus, they are talking about inhibition of the expression of genes to make new proteins (the enzymes COX-2 and iNOS). How this happens is not specified, but a diagram shows balls representing Calcarea carbonica on the outside of the cell next to TLR4, so presumably there is a direct interaction of the compound with the external domain of TLR4 (which spans the membrane and carries the signal from LPS to the inside of the cell). Western blots were used to show reduced expression of the COX-2 protein (levels of iNOS apparently were not measured).
On the other hand, they perform a molecular docking analysis to proposed that calcium carbonate interacts directly with the active site of the COX-2 enzyme, presumably to inhibit its activity. This is an entirely different effect (since it concerns proteins that have already been synthesized), yet the authors incorrectly refer to this as "reducing its [COX-2] expression." Such an inhibitory effect would require that the calcium carbonate enter the cell. Changes in the concentration of carbonate intracellularly would be expected to be even less than those in the medium, which are negligible. Also, calcium ions are extremely low in the cell and carbonate ions, if they should get in, would be more likely complexed with magnesium ions, yet the authors model calcium carbonate as the entity that is binding to COX-2.
They also analyze the likelihood of calcium carbonate acting like a drug, using Lipinski analysis and ADMET analysis (see above, "Drug likeness and pharmacokinetics property"). They concluded that " it may be absorbed in human intestine, can cross the blood–brain barrier and has oral bioavailability." Yet if this is taken as a homeopathic drug, the amount of calcium carbonate actually reaching any tissue would be infinitesimal. The preparation studied by the authors was diluted 10-fold, but it appears that calcarea carbonica used in real life is much more dilute (e.g., Boiron's 30C) and would essentially have no calcium carbonate at all. (Compare to a typical antacid tablet providing 750 mg of calcium carbonate.) Again, note that the authors are not proposing actions based on magical homeopathic properties, but ones arising from the actual chemical, which could not possibly be delivered in significant amounts to body tissues if you start with a homeopathic remedy. It should be noted that in the Introduction, the authors cite several studies in support of the use of Calcarea carbonica (references 13-17), but these all employed highly dilute (1C to 200C) preparations.
In summary, the studies show effects of Calcarea carbonica, at concentrations that may actually include some of the mineral, with cultured cells, but the proposed mechanisms appear implausible and are irrelevant to the actual use of the homeopathic remedy.
