|Year : 2017 | Volume
| Issue : 1 | Page : 41-47
Characterization and haemocompatibility of Aurum metallicum for its potential therapeutic application
Subrata Kar1, Monalisa Chakraborty1, Papiya Nandy2, Ruma Basu3, Sukhen Dasa2, Durga Sankar Bhar2, Raj K Manchanda4, Anil Khurana4, Debadatta Nayak4
1 Department of Physics, Jadavpur University; Centre for Interdisciplinary Research and Education, Kolkata, West Bengal, India
2 Centre for Interdisciplinary Research and Education, Kolkata, West Bengal, India
3 Centre for Interdisciplinary Research and Education; Department of Physics, Jogamaya Devi College, Kolkata, West Bengal, India
4 Central Council for Research in Homoeopathy, New Delhi, India
|Date of Web Publication||23-Feb-2017|
Centre for Interdisciplinary Research and Education, Kolkata - 700 068, West Bengal
Source of Support: None, Conflict of Interest: None
Background: The objective of the study was to characterize homoeopathic nanomedicine Aurum metallicum and evaluate its biocompatibility, to explore its possible application as injectables. Metal-based homoeopathic medicine, Aurum metallicum, was chosen as a model drug and the haemocompatibility of the drug at three different potencies 6C, 30C, and 200C were studied to find the justification of the drug as an injectable candidate for clinical application.
Methods: The model drug Aurum metallicum at the three potencies was characterized by dynamic light scattering (DLS), zeta potential, field emission scanning electron microscopy (FESEM), and energy dispersive X-ray analysis. Hemocompatibility of the homoeopathic medicine was performed by haemolysis assay. Red blood cell obtained from fresh human blood by centrifugation was incubated with Aurum metallicum. Haemoglobin release was measured using UV-vis spectrophotometer at 540 nm. Results: The DLS and FESEM studies show a decrease of particle size with increasing potency. The zeta potential values show a fairly constant value measured at an interval of 10 days. The haemolysis percentage for 6C, 30C, and 200C was 9.73%, 8.16%, and 0.73%, respectively. Conclusion: The nanomedicine Aurum metallicum was nontoxic at all doses of 6C, 30C, and 200C. The haemolytic percentage also shows that 200C is nonhemolytic, showing haemolysis <2% as per the American Society for Testing and Materials guidelines. The undertaking of larger controlled and in-depth qualitative studies is warranted.
Keywords: Haemocompatibility, Homoeopathy, nanomedicine
|How to cite this article:|
Kar S, Chakraborty M, Nandy P, Basu R, Dasa S, Bhar DS, Manchanda RK, Khurana A, Nayak D. Characterization and haemocompatibility of Aurum metallicum for its potential therapeutic application. Indian J Res Homoeopathy 2017;11:41-7
|How to cite this URL:|
Kar S, Chakraborty M, Nandy P, Basu R, Dasa S, Bhar DS, Manchanda RK, Khurana A, Nayak D. Characterization and haemocompatibility of Aurum metallicum for its potential therapeutic application. Indian J Res Homoeopathy [serial online] 2017 [cited 2019 Nov 19];11:41-7. Available from: http://www.ijrh.org/text.asp?2017/11/1/41/200847
| Introduction|| |
Inordinate use of antibacterial agents such as chemically modified natural compounds (penicillins and cephalosporins), pure natural products (aminoglycosides), and purely synthetic antibiotics (sulfonamides) lead to the development of drug-resistant microbes. These resistant pathogens cause the emergence of diseases, which are difficult to diagnose and control. The causes of antibiotic resistance mainly include overuse, inappropriate prescribing, extensive agricultural use, availability of few new antibiotics, and regulatory barriers. The Centers for Disease Control and Prevention has classified a number of bacteria as having urgent, serious, and concerning threats. Many of which are already responsible for placing a substantial clinical and financial burden on the US health care system, patients, and their families.,,, In 2010, India was the world's largest consumer of antibiotics for human health. The combination of factors such as poor public health infrastructure, rising incomes, a high burden of disease, and cheap, unregulated sales of antibiotics is causing such effect.,,
Use of Homoeopathy has been looked upon as the potential answer for alternative therapy to microbes that are resistant to antibiotics. Studies suggested that homoeopathic remedies contain nanoparticles  which are heterogeneously dispersed in colloidal solution and act by modulating biological functions of cytokines, heat shock proteins and immune, endocrine, metabolic, autonomic, central nervous system functions, etc.,,,,,
In general, there are several methods of introducing homoeopathic drugs into the body for remedial purposes. According to Bergmann et al., 1917, it is advantageous for the homoeopathic remedy if it does not have to pass through the gastrointestinal tract where it is severely altered by the gastric and intestinal juices. The potentized homoeopathic remedies work best when applied unchanged to the mucosa or - even better -injected under the skin or into the bloodstream. Subcutaneous and intravenous administrations were the first parenteral forms of administrations used in Homoeopathy. Others forms of administrations are intramuscular, intracutaneous, intra-articular, and periarticular. Baars et al., 2003, discussed about the risks of the subcutaneous form of administration of homoeopathic medicine. Bell et al., 2011, discussed about the short-term effects of repeated olfactory administration of homoeopathic Sulphur or Pulsatilla. Cazin et al., 1987, studied the intraperitoneal injection of decimal and centesimal dilutions of Arsenicum album on the retention and mobilization of arsenic in rat and concluded that arsenic excretion is primarily fecal for the period of 12–20 h and among the dilutions studied most active dilution was 14dH and 7cH.,, Hahnemann instructed that the tongue, mouth, and stomach are the most effective routes of administration for this purpose. Recently, medicinal solutions, manufactured in accordance with the German Homoeopathic Pharmacopoeia, are therapeutically applied as injections for better results for a wide range of conditions, especially in the treatment of acute and chronic diseases.,
It is necessary that the selected homoeopathic drug should be compatible with blood cells and must not cause any damage or haemolysis of blood cells. We have reported here our study on the effect of a homoeopathic medicine Aurum metallicum at various potencies (6C, 30C, and 200C) on haemolysis. Our result indicates that the medicine is haemocompatible and the magnitude of this effect depends on the potency of the medicine.
Dynamic light scattering (DLS) and field emission scanning electron microscopy (FESEM) studies for characterization of the drug at three potencies including the potentized and unpotentized alcohol Aurum metallicum were performed to establish its role as potential nanomedicine.
| Materials and Methods|| |
The drug Aurum metallicum was obtained from Hahnemann Publishing Company, India and was used without further processing. Sodium chloride and Na2-EDTA were purchased from E. Merck (India).
Field emission scanning electron microscopy and energy dispersive X-ray analysis
Morphological characteristics of Aurum metallicum at different potencies (6C, 30C, and 200C) were observed by FESEM (Model FEI Quanta 250, USA). A small amount of sample was cast on a clean glass coverslip and was directly placed on carbon-coated grid, which was then sputter coated with gold and then observed by FESEM. The particle sizes were calculated using ImageJ software, NIH, USA.
For energy dispersive X-ray analysis (EDAX), the Aurum metallicum samples (6C, 30C, and 200C) including their succussed and unsuccussed control were placed on carbon grid and placed directly without gold coating.
Dynamic light scattering and zeta potential
DLS measurements of Aurum metallicum samples at different potencies (6C, 30C, and 200C) were performed using a Nano-ZS 90-Malvern instrument (Model DLS-nano ZS, Zetasizer, Nano series, USA) employing a 4 mW helium–neon laser (l¼ 632.8 nm) equipped with a thermostatic sample chamber. The zeta potential measurement of Aurum metallicum (6C, 30C, 200C) and their succussed and unsuccussed control was repeated thrice in an interval of 10 days, and the data were presented by average value ± standard error of the mean (SEM).
Five milliliters of venous blood was drawn from three healthy human volunteers, under the proper medical supervision and collected in a Na-EDTA vial (1.8 mg/ml). Blood was mixed by gentle inversion of the tube and centrifuged at 1200 ×g for 10 min. The plasma supernatant was discarded, and the red blood cells (RBCs) were washed three times by suspending them in normal saline (0.9%). The final suspension consisted of 5% by volume RBC in saline.
Fifty microliters of each of the Aurum metallicum potencies (6C, 30C, and 200C) was dried in 2 ml microcentrifuge tube in a vacuum desiccator. Fifty microliters of normal saline (0.9%) was added to all the tubes. Finally, 200 μl of RBC solution was added. Deionized water and 0.9% saline in the same volume served as negative and positive control, respectively. Samples were incubated at 37°C for 1 h. After incubation, the tubes were centrifuged at 1200 g for 10 min, and absorbance was taken at 540 nm. Haemolysis percentage was calculated from the formulae:
A substance is considered to be haemocompatible if the haemolysis percentage is < 10%. The results are represented as the mean ± standard deviation of the three independent experiments.
| Results and Discussion|| |
The FESEM images of Aurum metallicum are shown in [Figure 1]a,[Figure 1]b,[Figure 1]c. As seen in the figure, particles in nano dimensions are embedded in lactose matrix, which is used for the preparation of homoeopathic drugs. Further, with a gradual increase of dilution from 6C to 30C to highly diluted 200C, the size of the nanoparticles decrease.
|Figure 1: Field emission scanning electron microscopy images of Aurum metallicum prepared by drop casting followed by drying (a) 6C (b) 30C (c) 200C. Scale bar represents 1 μm|
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Particles are also seen to be agglomerated possibly due to the drying process during sample preparation. The average particle sizes of the homoeopathic medicine are represented in [Table 1]. The approximate size calculated from ten particles for 6C, 30C, and 200C are 268.15, 171, and 100 nm, respectively. The presence of nanoparticles of the gold, copper, tin, zinc, silver, and platinum present in Aurum metallicum, Cuprum metallicum, Stannum metallicum, Zincum metallicum, Argentum metallicum, and Platinum metallicum, respectively, at 6C, 30C, and 200C potency was demonstrated by Chikramane et al., 2010. Their group reported the presence of nanoparticle of the starting material in the extreme dilutions of 6C, 30C, and 200C. The work of Chikramane et al., 2012, does not show any dependence of size of nanoparticle on potency. However, our results (both FESEM and DLS) categorically show this dependence. Using four other homoeopathic medicines, we have been able to derive an empirical relation between the size and potency of drug particles.
|Table 1: Comparative of dynamic light scattering and field emission scanning electron microscopy of Aurum metallicum for different potencies|
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The preparation of homoeopathic nanomedicine mainly consists of two steps. The trituration with lactose followed by succession step, in which the triturated preparations are potentized with alcohol in glass container whereby the particles develop a coat of silica. These particles were seen embedded in a meso-microporous silicate layer through interfacial encapsulation. Thus, metal and inorganic salt-based homoeopathic medicines retain the starting material as nanoparticles encapsulated within a silicate coating.,,, Our study also shows the presence of silica as well as gold shown by EDAX analysis [Table 2].
|Table 2: Elemental analysis by energy dispersive X-ray analysis represented by mass percentage of Aurum metallicum (6C, 30C, 200C) and potentized and unpotentized alchohol|
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Dynamic light scattering studies
DLS is one of the most established particle-sizing techniques that use the light scattered by particles to determine their size, in terms of the hydrodynamic diameter. Drawbacks of DLS include its intrinsic propensity to detect larger particles. It is considered less suitable for characterizing heterogeneous systems. The particle size distribution of the Aurum metallicum shows a decrease in particle size with increase in potency [Figure 2]a, [Figure 2]b, [Figure 2]c. The particle size for 6C, 30C, and 200C is, respectively, 295.3 nm, 88.98 nm, and 10.3 nm. The DLS data validate the trend observed in the FESEM images [Table 1]. It is also noted that the size observed in SEM is slightly more than that as seen in the DLS size distribution.
|Figure 2: Hydrodynamic diameter distribution plots as determined by DLS measurements of Aurum metallicum (a) 6C (b) 30C (c) 200C|
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This may perhaps be due to the fact that preparation for SEM causes agglomeration with respect to the dispersed form, causing the increase in the particle size.
The zeta potential values are represented in [Table 3]. As seen in the table, the values were fairly constant when recorded at an interval of 10 days representing the stable nature of the particle. Moreover, it is to be noted that the value of zeta potential increases after succession and further increases after the addition of metal. The data support that homoeopathic medicines Aurum metallicum were more stable than their succussed and unsuccussed controls.
|Table 3: Zeta potential of Aurum metallicum (6C, 30C, 200C), unpotentized and potentized alcohol|
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The haemolysis percentage of 6C, 30C, and 200C was found to be 9.73%, 8.16%, 0.73%, respectively [Figure 3]. There are several views available in the literature regarding the limit percentage of haemolysis and toxicity. Some authors opine limit of nonhemolytic percentage as 10%., Accordingly, our result signifies that Aurum metallicum is nontoxic toward RBC at 6C, 30C, and 200C potencies. While according to the American Society for Testing and Materials guidelines, a material is classified as nonhemolytic, slightly hemolytic if the haemolytic index is <2%, 2%–5%, and haemolytic if >5%, respectively. As per their guidelines, homoeopathic nanomedicine Aurum metallicum is nonhemolytic at the 200C potency (0.73%).
|Figure 3: Haemolysis percentage and potency of Aurum metallicum (values ± standard error of the mean)|
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| Conclusion|| |
The size of the drug particles of Aurum metallicum used in this study at three potencies are found to be in the nanometer range where the particle size decreases with increase in potency. All the samples at different potencies are found to be nontoxic, causing minimum harm to the RBCs. However, out of these three potencies, hemolysis is maximum for the drug at 6C potency. This result can be justified from our previous study of measurement of fluidity of artificial lipid membrane of dipalmitoylphosphatidylcholine with different potencies of another metal-derived homoeopathic medicine Cuprum metallicum where it has been observed that the drug at 6C potency fluidizes the membrane most followed by 30C and 200C. The decrease in membrane anisotropy with increasing potency was further explained by the fact that with an increase in potency, not only the particle size reduces but also the number of drug particle decreases. Therefore, the possibility of interaction of the drugs of higher potency (200C) with the RBC membrane reduces, causing less damage to the membrane than the drug at higher potency (6C and 30C). Hence, from the study, we may conclude that homoeopathy drug Aurum metallicum has a potential scope in future medicine in the injectable form. More in-depth studies are required to understand the interaction of homoeopathic medicine with RBC and other blood cells. Further, in vivo studies will throw more light toward the development of nanomedicine as injectables.
We thank Dr. D. S. Bhar for donating the drug Aurum metallicum without any condition.
Financial support and sponsorship
The authors are thankful to the Central Council for Research in Homoeopathy (CCRH), Ministry of AYUSH, Government of India, for providing the financial assistance. The study was undertaken in joint collaboration between Centre for Interdisciplinary Research and Education, Kolkata and CCRH, New Delhi.
Conflict of interest
There are neither any financial nor any personal conflict of interest with respect to the work carried out for this article.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]