|Year : 2019 | Volume
| Issue : 4 | Page : 209-218
Effect of Cuprum metallicum potentised through both serial dilution and succussion in comparison to succussion alone on Escherichia coli bacterial system and electrical properties of poly (vinylidene fluoride-co-hexafluoropropylene) polymer
AL Gayen1, D Mondal2, D Bera1, P Biswas2, BK Paul2, DS Bhar1, S Das3, R Narula4, AK Khurana4, RK Manchanda4, P Nandy1
1 Centre for Interdisciplinary Research and Education, Kolkata, West Bengal, India
2 Physics Department, Jadavpur University, Kolkata, West Bengal, India
3 Centre for Interdisciplinary Research and Education; Physics Department, Jadavpur University, Kolkata, West Bengal, India
4 Central Council for Research in Homeopathy, New Delhi, India
|Date of Submission||02-Aug-2019|
|Date of Acceptance||28-Nov-2019|
|Date of Web Publication||27-Dec-2019|
Dr. P Nandy
Centre for Interdisciplinary Research and Education, Kolkata - 700 068, West Bengal
Source of Support: None, Conflict of Interest: None
Background: Homoeopathic medicines are traditionally potentized by serial dilution followed by succussion. Respective roles of these two components need to be assessed and explored for which the present study was undertaken. Objective: To compare the effect of the medicine Cuprum metallicum (Cup. met.) potentised through both serial dilution and succussion with succussion alone on selected biological and physical systems. Method: Starting with the medicine Cup. met. at 6C, we potentized it further to 30C and 200 C by serial dilution, followed by succussion (Set A). The same medicine at 6C was also potentized to 30C and 200C by using succussion alone (Set B). The antibacterial property of these two sets was compared on E. coli, a biological system and electrical properties on polymer matrix PVDF-HFP (widely used as charge separator) a physical system. Results: Field Emission Scanning Electron Microscopy shows that the particles get more agglomerated at higher potency in Set B. Antibacterial effect of Cup. met. in Set B at 30C and 200C was observed to be more significant as compared to Set A. Effect of Cup. met. on polymer matrix in Set A varied significantly with the potency as compared to Set B wherein less beta phase crystallization was produced followed by no significant change in electrical properties. Conclusion: Comparison of results using the medicine Cup. met. in two experimental set ups shows that serial dilution with succussion makes an important difference between the two sets.
Keywords: Cuprum metallicum, Escherichia coli, Polymer film, Potentisation, Serial dilution, Succussion
|How to cite this article:|
Gayen A L, Mondal D, Bera D, Biswas P, Paul B K, Bhar D S, Das S, Narula R, Khurana A K, Manchanda R K, Nandy P. Effect of Cuprum metallicum potentised through both serial dilution and succussion in comparison to succussion alone on Escherichia coli bacterial system and electrical properties of poly (vinylidene fluoride-co-hexafluoropropylene) polymer. Indian J Res Homoeopathy 2019;13:209-18
|How to cite this URL:|
Gayen A L, Mondal D, Bera D, Biswas P, Paul B K, Bhar D S, Das S, Narula R, Khurana A K, Manchanda R K, Nandy P. Effect of Cuprum metallicum potentised through both serial dilution and succussion in comparison to succussion alone on Escherichia coli bacterial system and electrical properties of poly (vinylidene fluoride-co-hexafluoropropylene) polymer. Indian J Res Homoeopathy [serial online] 2019 [cited 2020 Apr 6];13:209-18. Available from: http://www.ijrh.org/text.asp?2019/13/4/209/274021
| Introduction|| |
Homoeopathic potentisation of Hahnemannian era constituted of serial dilution followed by either trituration or succussion or both depending on the type of drug substances. In the preparation of potencies from solid drug substances, decimal and centesimal scales are used. In the preparation of liquid drug substances, manufacturing of homoeopathic medicines is from the mother solution (tincture) by serial dilution and succussion (mechanical agitations) in one of the three potency ranges, decimal (1:9), centesimal (1:99), or fifty-millesimal. Solid insoluble materials (e.g., Cuprum metallicum [Cup. met.]) are triturated with lactose in the ratio of 1:10 till the 6X potency, after which, the liquid dilutions are carried out. The liquid dilutions are prepared by the Hahnemannian method  till the 200C potency and the Korsakovian method for potencies beyond 200C.
Potentisation, the most vital part of preparation of homoeopathic medicine, transforms the starting material to a therapeutically active one. It has got two components: (a) Serial dilution and (b) vigorous vertical jerking, known as succussion.
The paradox created debate between practicing homoeopaths and rationalists, as they felt that there is no medicine present at very high dilution. However, as the effectiveness of high potencies is experienced by the practicing homoeopaths and countless patients, more positive opinions started accumulating, backed by different models, in favour of the therapeutic effect of these medicines at high potency.
The question naturally arises, why do, in the preparation of homoeopathic medicine, we dilute instead of concentrating. We have, for the first time, shown that there is a difference between the effects of two kinds of potentisation: serial dilution, followed by succussion, and only succussion.
Recently, it has been realised that it is not only the dilution, but the succussion followed after each dilution, which is responsible for these effects at high potency. By means of vigorous shaking, a large amount of mechanical energy is transferred, This energy breaks the drug associates and reduces the size to nanodimension.,, This reduction in size increases membrane permeability.
The empirical relation between potency (X) and the size of the drug aggregates (Y) is given by the following equation:
Y = aX -n
Where a and n are characteristic constants of different medicine.
It has been experimentally proven that extreme homoeopathic dilutions retain starting materials. To understand the extreme dilutions from a biological perspective, it has been shown that metal concentration as low as fg/ml increases the intracellular protein synthesis. It has also been shown that the particles develop a coat of silica, and a hypothesis has been proposed that all types of metal and inorganic salt-based homoeopathic medicines consist of silicate-coated nanostructures dispersed in the solvent.
Hence, the term potentisation indicates the qualitative and quantitative increase in medicinal power as compared to mere dilution. And thus, Homoeopathy is seen as nanomedicine. The increase of the activity of the drug with potentisation arises as the surface area increases manifold and the number of points of contact with the living fibre increases. At the same time, the process of succussion is responsible for inducing electrical nature due to domain formation as predicted by quantum electrodynamics (QED). Due to this, the physicochemical properties of the vehicle medium change drastically, as the effect of QED starts playing a major role.,,,,,
According to Hahnemann, a small dose of medicine, when potentised, is also very powerful. This not only reduces the toxic effect of overdose but also forms intimate mixture of the medicine with the vehicle due to vigorous shaking. This is intended to avoid aggravation of the disease and increase the activity of the medicine as the medicine acts 'not atomically but dynamically'. Dilution is essential for getting the dynamising effect of succussion and that 'all the shaking in the world will not dynamize an undiluted substance'.
Thus, potentisation (dilution, followed by succussion) affects both the drug material of a homoeopathic medicine and the medium as follows:
- From classical point of view, due to succussion, the drug material achieves nanodimension and nanoparticles are formed. This reduces the toxic effect and increases the activity due to the increase in aspect ratio ,,,,
- From quantum mechanical point of view, it changes the electrical nature of the polar medium through the formation of coherent domains of the solvent molecules, which provides quasi-free electrons and increases the stored electrical energy of the medium.,,,,,
This establishes the electrical nature and the mode of action of homoeopathic medicine. Perhaps, this electrical energy which is stored in the system is the so-called dynamic power of the homoeopathic medicine as envisaged by Dr. Hahnemann.
The question then naturally arises is, as to how to compare the role of dilution and succussion in potentising a medicine. In the present study, the experiment is conducted to observe and compare the effect of serial dilution and succussion to succussion alone using homoeopathic medicine, Cup. met. on the biological and physical systems, whereas in our earlier study, the standard potentised Cup. met. has been used. This medicine is used because of its good dispersion in different polymers and good antibacterial and conductive properties which can be utilised as a dielectric charge separator in high charge storage system. In addition, the formation of the film using this polymer is an easy, low-cost and simple solution casting technique.
| Methods|| |
For both the biological and physical systems, we have used control, where Cup. met. was not used. That the presence of the drug is responsible for the observed effect is justified as the vehicle medium of 91% ethanol had been evaporated in both cases within a very short span of time. Hence, the presence of drug only is responsible for the observed effects.
Preparation of the two sets of homoeopathic medicine
For making potencies by trituration with lactose in porcelain mortar and pestle, a mechanical device manufactured by F. Kurt Retsch KG from Germany was used. During potentisation in centesimal scale with ethyl alcohol, each step of potentisation was done manually in neutral (USP III) glass bottles using new bottle in each step. The whole operation was done following the Good Manufacturing Practice.
Potencies of Cup. met. were prepared from pure copper powder, first by trituration with lactose up to 6X potency, converting the same into liquid 8X (=4C) potency and potentising the 4C potency by dilution (with 91% ethanol) followed by succussion up to 6C potency. This 6C potency was taken as the starting material for our study.
Set A: The potencies of Cup. met. were prepared in the method described in the Homoeopathic Pharmacopoeia of India (HPI). The method may be elaborated as one part of copper metal powder (99.6% pure) was triturated mechanically with 9 parts of 80 mesh lactose powder of pharmacopoeial (HPI, British Homoeopathic Pharmacopeia [BHP] and United States Homoeopathic Pharmacopeia [HPUS]) quality for 1 h to produce 1X potency. The same 1X potency of Cup. met. was again triturated with lactose in the same way and proportion to yield 2X potency. This method of trituration was continued till we got 6X potency of Cup. met.
One part by weight of this Cup. met. 6X was dissolved in 50 parts by volume of distilled water (purified water of HPI/BHP/HPUS standard) to which 50 parts by volume of ethyl alcohol (91%) was added, and the mixture was given ten succussions to get 8X (=4C) potency. Henceforth, potentisation was done by diluting one part of the previous potency with 99 parts of ethyl alcohol and giving the mixture ten succussions (shaking strongly with downwards stroke) always in a new glass bottle, one-third volume of which was kept empty for proper shaking, to get the next potency in centesimal scale. This process of serial dilution and succussion was carried out till 6C initially, as 6C is taken as the starting material in both the groups (Set A and Set B).
- Set A: Serial dilution and succussion was carried out as stated above till 200C as per the standard method of potentisation
- Set B: Here starting from 6C potency, further potentisation was done without further dilution by succussing the 6C potency, making it equivalent to 30 and then further succussion to make it equivalent to 200C potency.
The schematic diagram for the preparation of these two sets of medicine is shown in [Figure 1].
Effects of these two sets of potentised medicines were compared for the antibacterial property on Gram-negative bacteria Escherichia More Details coli and on the electrical properties of a polymer matrix poly (vinylidene fluoride-co-hexafluoropropylene) (PVDF-HFP).,,,
Antibacterial effect of Cuprum metallicum
Fresh culture of E. coli in nutrient broth was treated with Cup. met. at required potency, prepared as in Set A and Set B in 91% alcohol and left overnight. The alcohol was allowed to evaporate gradually. There was a possibility of the bacteria being affected by the alcohol present in the medicine. However, this effect should be same for all the potencies of the medicine used. And hence, it was inferred that the final outcome was the effect of the potency of the medicine.
The vehicle control was 91% alcohol. In order to make sure about the reproducibility, all the experiments with Set A and Set B at the potencies 6C, 30C and 200C were repeated at least 3–4 times.
Cuprum metallicum – poly (vinylidene fluoride-co-hexafluoropropylene)composite film preparation technique
The Cuprum metallicum-doped composite films were synthesised by low-cost and simple solution-casting fabrication technique. In a typical synthesis process, 100 mg of PVDF-HFP (Sigma Aldrich, USA, 3050 Spruce St., St. Louis, MO 63103, 400 Summit Drive, Burlington, MA 01803) was dissolved into 2 ml of dimethyl sulfoxide (Merck, India, 8th Floor, Godrej One, Pirojshanagar, Eastern Express Highway, Vikhroli (E) Mumbai - 400 079, India) and mixed together under vigorous stirring at 50°C for 4 h. Measured amount of freshly prepared Cup. met. at a specific potency was obtained from Hahnemann Publishing Company, India (Near Sealdah Fly-Over & Koley Mrkt, Kolkata, West Bengal 700012), and was added to the solution and stirred for another 2 h at 50°C. Afterwards, the whole solution was sonicated three times for 10 min each at 50°C with 30 min of time interval for the complete removal of the air bubbles from the solutions. Finally, films were obtained by casting the whole mixture in clean dry Petri dish More Detailses and solvent was evaporated in an oven at 80°C for 24 h. The films were then coated by silver paste on both sides for electrical measurements.,,, The synthesised films had the thickness in the range of 50–55 μm as measured using a digital micrometer. The schematic diagram for the preparation of Cup. met. PVDF composite films is shown in [Figure 2].
|Figure 2: Field emission scanning electron microscope images of poly (vinylidene fluoride-co-hexafluoropropylene) doped 200C Cup. met. medicine of (a) Set A and (b) Set B|
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Field emission scanning electron microscope analysis of pure Cuprum metallicum medicine of Set A and Set B potentisation
To examine the microstructure, 20 drops of each medicine was dried in a cover slip and finally made to pass through the experiment under the microscope namely FEI-F50 (Netherlands).
Measurement of electrical properties
The dielectric properties were investigated using a LCR meter (HP model 4274A, Hewlett Packard, USA, 99 Washington Street, Melrose, MA 02176-6024). The dielectric constant (εr), tangent loss (tanδ) and AC conductivity (σac) were recorded in the frequency range of 20 Hz–2 MHz at room temperature.
| Results|| |
Field emission scanning electron microscope analysis of pure Cuprum metallicum medicine of Set A and Set B potentisation
The field emission scanning electron microscope (FESEM) microstructural overview of pure Cup. met. at 200C as obtained from two different potentisation processes of Set A and Set B is shown in [Figure 3]a and [Figure 3]b, respectively. [Figure 3]a shows the morphology and microstructure of pristine 200C Cup. met. of Set A. The microstructure of the sample confirmed a very good and homogeneous distribution of the nanoparticles on the surface of the glass cover slip. The particles are more scattered, are well separated and also homogeneously distributed, maintaining an intermolecular distance. This is due to the very high dilution at 200C.
|Figure 3: Antibacterial effect of Cup. met. for Set A (a) 6C, (b) 30C and (c) 200C and Set B (d) 6C, (e) 30C and (f) 200C|
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On the other hand, [Figure 3]b shows the microstructure and morphology of the pristine 200C Cup. met. of Set B, where only succussion is done without dilution. The microstructure of the sample confirmed an evidence of large number of densely packed agglomerated particles having very low dimension embedded in the surface of the glass cover slip.
In the dilute medium, particles are scattered [Figure 3]a. However, in the absence of dilution, nanoparticles created due to succussion get agglomerated [Figure 3]b.
Antibacterial effect of Cuprum metallicum
The antibacterial effect shown in [Figure 4]a (6C), [Figure 4]b (30C) and [Figure 4]c (200C) is for the potentised medicine Cup. met. namely as in Set A, whereas [Figure 4]d (6C), [Figure 4]e (30C) and [Figure 4]f (200C) are for Set B of Cup. met. From the figure, we realise that for Set A, the antibacterial effect is more for drug at 30C, compared to that for 6C. The effect becomes more for drug at 200C. The reason for this is perhaps as at higher potency, the size of the drug particles becomes smaller  and the penetration through membrane barrier is more, giving rise to higher antibacterial effect.
|Figure 4: The schematic diagram for the preparation of Cup. met.–poly (vinylidene fluoride-co-hexafluoropropylene) composite films|
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In Set B, as there is no dilution, the amount of drug available is same for all potencies from 6C to 200C, a larger number of nanoparticles are produced by succussion and the antibacterial effect is more here for 30C and 200C compared to that in Set A.
For Set B, at 200C, the figure indicates that in the presence of significant number of drug molecules, a large number of nanoparticles are created through succussion, which agglomerate, as is evident from the FESEM, and are incapable to penetrate the bacterial membrane.
Field emission scanning electron microscope morphological investigation of poly (vinylidene fluoride-co-hexafluoropropylene)-Cuprum metallicum nanocomposite films
[Figure 5]a, [Figure 5]b, [Figure 5]c shows the evidence of densely packed and good dispersion of nanoparticles embedded in the polymer matrix. From the critical observation, it can be seen that with increasing potentisation, the polymer matrix had an increasing crystallisation and reached maximum enhancement of electroactive β-polymorph at 200C potency for the medicine of Set A.
|Figure 5: Field emission scanning electron microscope microstructural overview for Set A of poly (vinylidene fluoride-co-hexafluoropropylene) doped Cuprum metallicum(a) 6C, (b) 30C and (c) 200C|
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[Figure 6]a and [Figure 6]b shows the evidence of significant number of nanoparticles which got agglomerated at higher potency of Set B and embedded in the PVDF-HFP matrix. This is due to the high number of succussions at 30C and 200C.
|Figure 6: Field emission scanning electron microscope microstructural overview for Set B of poly (vinylidene fluoride-co-hexafluoropropylene) doped Cup. met.(a) 30C, (b) 200C and (c) microstructure of poly (vinylidene fluoride-co-hexafluoropropylene) doped Cup. met. of 200C and the crystal structure of α-polymorph (inset graph) and (d) microstructure of Cup. met. of 200C embedded poly (vinylidene fluoride-co-hexafluoropropylene) and the crystal structure of β-polymorph (inset graph). 6C is the starting material both for Set A and Set B and is not shown separately|
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Thus, from the FESEM investigation, it can be clearly concluded that, for the Set A medicine [dilution followed by succussion – [Figure 5]a, [Figure 5]b, [Figure 5]c, there is no significant agglomeration with very good dispersion and distribution due to high dilution of the medicine, whereas for the Set B medicine [without dilution, only succussion – [Figure 6](a and b)], there is agglomeration of embedded nanoparticles in the PVDF matrix, which results in less β-phase crystallisation. The inset graph of [Figure 6]c shows the crystalline α-polymorph where the hydrogen and fluorine dipoles are packed in antiparallel way with the carbon atoms, whereas the inset graph of [Figure 6]d shows the positively charged CH2 dipoles of PVDF-HFP interacted with the negatively charged nanoparticles, leading to the alignment of stabilised β-chains which have been shown in the inset graph of [Figure 6]d and [Figure 7]. From chemistry point of view, a clear electrostatic interaction mechanism was also explained by the theory of β-phase nucleation in our previous publications.,, Based on this theory, when the positively or negatively charged nanoparticles are added to the host solution, the opposite (partially negative CF2 or positive CH2) dipoles are oriented towards the surface of the nanoparticles, which perform as substrates for β-phase nucleation. This mechanism leading to the alignment of stabilised PVDF-HFP chains in longer all trans-conformation, results in electroactive β-phase.,
|Figure 7: The schematic representation for nucleation of β-polymorph in poly (vinylidene fluoride-co-hexafluoropropylene)|
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Measurement of electrical properties of polymer matrix: effect of potentised Cuprum metallicum in Set A and Set B
For Set A, Cup. met. enhances the more electroactive β-phase of the polymer matrix, enhancing the conductivity and dielectric constant and reducing the tangent loss of the medium.,,,,
For the polymer matrix, when many more agglomerated nanoparticles are available as in the case of Set B, the arrangement of the β-polymorph in the matrix gets destroyed, which becomes less electroactive and reaches a stable nature. Hence, the electrical properties cannot be further enhanced.
[Figure 8] shows the variation of the dielectric constant, tangent loss and electrical conductivity of the composite material as a function of frequency. All the details regarding measurement procedure have been reported by us earlier in various reputed journals.,,,,, In Set A, the number of drug particles decreases with dilution and the size decreases with succussion. Hence, the particles are more scattered and well separated and also homogeneously distributed, maintaining an intermolecular distance between the particles, which enhances the nucleation of β-polymorph, resulting in very good dielectric performance.,,,, In Set B, the number of drug associates remains constant, but the size decreases with succussion, enhancing the agglomeration in the polymer matrix. Thus, the increasing agglomeration and the encapsulation of drug material by silicates will increase and the mobility of the particles will be inhibited, keeping the electrical properties unaltered.
|Figure 8: Effect of Cup. met. on the electrical properties of polymer matrix (a), (b), (c) for Set A and (d), (e), (f) for Set B|
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Hence, the outcome of these two experiments using Set A and Set B on E. coli and polymer matrix has been quite interesting. The generation of larger number of nanoparticles in Set B plays a major role and changes the usual pattern obtained with Set A. As in both the cases the solvent had been evaporated to dryness to make films, we can rule out the effect of vehicle medium on the observed phenomena. The presence of copper in the film had been verified earlier.,
| Discussion|| |
The results using Set A and Set B have been summarised as follows:
Antibacterial effect of the medicine Cuprum metallicum on Escherichia coli
In Set A, the number of drug particles decreases with dilution and the size decreases with succussion, enhancing the membrane permeability, and the growth is inhibited, as expected. However, for Set B, when there is no dilution, the amount of drug available is same for all potencies from 6C to 200C and a larger number of nanoparticles are produced by succussion and the antibacterial effect is more here for 30C and 200C compared to that in Set A.
Effect of Cuprum metallicum on the electrical properties of poly (vinylidene fluoride-co-hexafluoropropylene) matrix
- For Set A, the effect varied significantly with potency (i.e., with dilution). Here, the number of drug associates decreased with dilution and the size decreased with succussion, enhancing the mobility of the particles, which enhances the electroactive beta phase of the polymer matrix, enhancing the conductivity and good dielectric performance
- For Set B, no significant change in electrical properties with change in potency is observed. Here, the number of drug associates remains constant, but the size decreases with succussion, enhancing the surface area. Thus, the effect will be twofold: the number of nanoparticles will increase, increasing the possibility of agglomeration and restricting the movement of charged particles, and also due to the encapsulation of drug material by silicates, the mobility of the particles will be inhibited. Both these factors will keep the electrical properties unaltered.
The difference between the two results gives an indication about the role of dilution and succussion in the process of potentisation of homoeopathic medicines and their impact on the biological and physical systems.
From the conducted experiments, it is observed that in case of Set B, when there is no serial dilution and only succussion, the total number of drug associates remains the same at all potencies, and a large number of nanoparticles are created due to succussion, which produced more significant antibacterial effect in both 30C and 200C of Set B, as compared to Set A where the maximum antibacterial effect was exhibited only at 200C. In Set B, the electrical properties of the polymer matrix remained unaltered with potency of Cup. met. as compared to Set A, where serial dilution with succussion was carried out. Here, the particles were more scattered and well separated and also homogeneously distributed, maintaining an intermolecular distance between the particles. This enhanced the nucleation of β-polymorph results in very good dielectric performance, which signifies the role of dilution as in Set A.
We agree with the principle of Homoeopathy, where it is always advocated to use the minimum dose, which means that the lower the concentration of the drug, better is the result. For higher concentration of the drug, the functioning of the medicine will be inhibited, which may be due to the re-agglomeration of nanoparticles of the drug material or due to saturation of the holding/absorption points in the system.
Higher number of succussions would ensure proper formation of the drug nanoparticles of different degree while bringing these remedies into the range of hormetic biological action.
| Conclusion|| |
Our experimental results show that the drug Cup. met., potentised by serial dilution, followed by succussion, can produce significant effect on both bacterial system and polymer matrix. However, by increasing the potency by succussion only, a large number of nanoparticles are created which produce significant change in lower potencies, monitoring the effect of the medicine in both biological and physical systems chosen. Due to proximity, these nanoparticles may agglomerate again to make bigger particles. Thus, while succussions are necessary for the potentisation, the role of dilution seems equally important.
Further experimentations are required using different drugs to identify the appropriate and most effective way of the degree of dilutions i.e., the critical level of dilution.
We would like to mention here that to the best of our knowledge, this experiment is the first of its kind to find out the role of succussion and dilution to potentise the starting material to a therapeutically active one.
The study was undertaken as a joint collaboration between the Centre for Interdisciplinary Research and Education (CIRE), Kolkata, and the Central Council for Research in Homoeopathy (CCRH), the Ministry of AYUSH, Government of India, New Delhi, and was funded by the CCRH (till 31 March 2018).
The results from References 22 and 24 were quoted here for the sake of comparison, and the authors express their thanks for the permission received from Springer Nature as follows:
Financial support and sponsorship
Centre for Interdisciplinary Research and Education, Kolkata.
Conflicts of interest
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8]