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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 13  |  Issue : 4  |  Page : 229-235

Preparation and standardisation of Plasmodium falciparum nosode


Life Force, Mumbai, Maharashtra, India

Date of Submission20-Jul-2017
Date of Acceptance08-Aug-2019
Date of Web Publication27-Dec-2019

Correspondence Address:
Dr. Rajesh Shah
Director and Research Head, Life Force, 411-Krushal Commercial Complex, GM Road, Chembur, Mumbai - 400 089, Maharashtra
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijrh.ijrh_38_17

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  Abstract 


Background: Malaria officinalis, a Homoeopathic preparation used against malaria, was prepared from decomposed vegetable matter from a marshy area by G. W. Bowen in 1862. No scientific published material is available on the source of any malaria-related nosode. There is a need to develop the new nosode using the organisms. The author has developed the nosode from Plasmodium falciparum parasites, and the detailed method of preparation is described in this manuscript. Objective: Preparation and standardisation of Homoeopathic nosode prepared from P. falciparum parasites, which could be explored for further research and therapeutic efficacy. Materials and Methods: The most widely cultivated strain of malaria parasite P. falciparum was collected from Indian Institute of Technology-B (Mumbai). This parasite was triturated with nine parts of Saccharum lactis powder to achieve 1X potency. One part of 1X potency and nine parts of Saccharum lactis powder were taken to make subsequent 2X potency. Likewise, potencies till 6X were prepared by trituration method, prescribed in Homoeopathy pharmacopeia of India (HPI). Results: Nosode from P. falciparum strain was prepared by trituration method as prescribed in HPI and standardised for future research and clinical exploration. Conclusion: The nosode sourced from P. falciparum parasites will be one such addition, which will call for further exploration for its effect against malaria (and other diseases) as a prophylactic and therapeutic measure.

Keywords: Homoeopathy, Malaria, Nosode, Parasites, Plasmodium falciparum, Potentization


How to cite this article:
Shah R. Preparation and standardisation of Plasmodium falciparum nosode. Indian J Res Homoeopathy 2019;13:229-35

How to cite this URL:
Shah R. Preparation and standardisation of Plasmodium falciparum nosode. Indian J Res Homoeopathy [serial online] 2019 [cited 2020 Feb 28];13:229-35. Available from: http://www.ijrh.org/text.asp?2019/13/4/229/274019




  Introduction Top


The nosodes are Homoeopathic preparations made from biological sources including organisms, diseased material or tissues, and prepared by the process of serial dilutions and rigorous succussions/trituration, called as potentization. The nosodes may resemble vaccines but they differ by the virtue of the preparation technique as well as by their therapeutic application.

The nosodes have demonstrated prophylactic role in Swine Flu,[1],[2] Helicobacter pylori ulcers [3] and Leptospirosis.[4] Certain nosodes have also shown anti-disease activities in vitro,[5],[6],[7] animal models [8] as well as against HIV [9] and Hepatitis C [10] infected individuals. The anti-Plasmodium activity of a Homoeopathic medicine in Bagg Albino (inbred research mouse strain)/c mice in an in vivo model against Plasmodium berghei infection has been documented.[8]

Interestingly, the nosodes have also exhibited anti-disease efficacy against the conditions other than the diseases the source organisms primarily cause. For example, Hepatitis C nosode and HIV nosode have demonstrated the anti-cancer effect in in vitro study.[11],[12]

Further therapeutic and prophylactic potential of nosodes needs to be scientifically investigated as this space has remained unexplored even after the use of nosodes for almost two centuries.

The older nosodes such as Psorinum[13] (1826–30), Tuberculinum[14] (1879), Medorrhinum[15] (1880–85), Syphilinum[16] (1880) and others were prepared in the era when microbiological techniques were not well established. No major development or updates happened in the area of nosodes for a long time. However, recent research by the author has already revamped and standardised the method of nosode preparation using the current technology [17] and has presented HIV nosode,[9] Hepatitis C nosode,[18] and  Mycobacterium tuberculosis Scientific Name Search sode.[19] The nosode category is certainly undergoing a revolution.

Plasmodium falciparum is a protozoan parasite causing malaria in humans. Plasmodium was first identified when Charles Louis Alphonse Laveran described parasites in the blood of malaria patients in 1880.[20] Malaria officinalis, a Homoeopathic medicine (not a nosode) used against malaria, was prepared from decomposed vegetable matter from a marshy area by G. W. Bowen in 1862.[21] It was based on the assumption that the decomposed matter might have contained some disease-causing vector or diseased mass, years before the discovery of the parasites. No scientific published material is available on the source of any malaria-related nosode. The author has communicated with three of the leading suppliers of Malaria officinalis and Malaria nosodes in Europe and has concluded that the source of the said nosodes is obscure and not scientifically defined.

Malaria parasites are microorganisms belonging to the genus Plasmodium. Out of more than 100 species of Plasmodium, four species (P. falciparum, Plasmodium vivax, Plasmodium ovale and Plasmodium malariae) have been reported to infect the humans. Fifth species Plasmodium knowlesi has recently been shown to be a significant cause of Zoonotic malaria, particularly in Malaysia.[22]

P. falciparum is found worldwide in tropical and subtropical areas and responsible for most human deaths. It has the highest complication and mortality rate. P. falciparum is the most prevalent malaria parasite on the African continent. It is responsible for most malaria-related deaths globally.[23]

The 2015 World Health Organization report found 212 million new cases of malaria worldwide resulting in an estimated 429,000 deaths (range 235,000–639,000).[24] In 2016, there were an estimated 216 million cases of malaria in India, an increase of about 5 million cases over 2015. Deaths reached up to 445,000. As per world malaria report 2017, weak malaria surveillance systems are reported in India.[25]

The most undeveloped, poor and remote parts of the country contribute mainly to the burden of malaria in India, with >90%–95% cases reported from rural areas and <5%–10% from urban areas. The state of Orissa contributes nearly 20%–30% of deaths caused by malaria in India, followed by Meghalaya, Mizoram, Maharashtra, Rajasthan, Gujarat, Karnataka, Goa, southern Madhya Pradesh, Chhattisgarh and Jharkhand.[26]

Since P. falciparum parasites multiply rapidly in blood, malaria can cause severe blood loss leading to anaemia. They make the surface of the cell sticky, which could form clots within small blood vessels, thus causing severe organ damage. Complications can be fatal in cases of cerebral malaria.[27]

Cultivation of the Plasmodium species in vitro has been achieved successfully. Life cycle stages of the four species have been established in vitro, of which P. falciparum remains the only species for which all stages have been cultured in vitro.[28] Hence, the author has chosen to prepare a nosode using P. falciparum strain.

Objectives

Preparation and standardisation of Homoeopathic nosode prepared from P. falciparum parasites, which could be explored for further research and therapeutic efficacy.


  Materials and Methods Top


The most widely cultivated strain of malaria parasite P. falciparum was collected from Bioscience and Bio-engineering Department of Indian Institute of Technology, Mumbai (IIT-B). Saccharum lactis powder, which was used as a vehicle, procured from a commercially available source.

P. falciparum (3D7 strain) was grown on culture (red blood cells [RBCs]) as per the in vitro cultivation parasite protocol (Trager and Jensen protocol). RBCs (O positive) for culturing were collected from healthy volunteers using 10% citrate-phosphate-dextrose-adenine at final concentration.

P. falciparum parasites were cultured in Roswell Park Memorial Institute 1640 (Gibco ®) with an additional 2 mg/ml sodium bicarbonate (Sigma ®), supplemented with 0.5% Albumax (Gibco ®), 48 mg/L hypoxanthine (Sigma ®), 2 mg/ml glucose (Sigma ®) and 50 μg/ml gentamicin (Abbott). A haematocrit of 3% was maintained using human O-positive RBCs.

Parasites, predominantly in late rings and trophozoite stage [Figure 1] - parasite image observed under oil immersion lens at 100x (total magnification 1000x)], were released from the RBCs by treatment with 0.02% saponin which lyses the RBCs leaving the parasites intact. This was followed by three washes with phosphate-buffered saline to remove the debris and other chemical components from the preparation.
Figure 1: Parasite image

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Parasites were isolated from 40 ml culture containing 3% haematocrit (1.2 ml packed blood cells). Parasitemia was 3% (means 3 parasites in 100 RBCs). The number of parasites was calculated based on percentage of parasitemia and the total volume of packed blood cells, which is 1.8 × 108.

Concentration 3% parasitaemia (4% is indicator of high risk)[29] was practically possible to achieve at the laboratory, and hence, it was taken at start. The parasites were isolated using centrifuge Rota 4R/VFm made by Plasto Craft. The conditions for pelleting the parasites are 10,000 rpm (9000 g) for 5 min.

Isolated parasites P. falciparum were taken as original stock to start with making the Homoeopathy dilutions. 1.8 × 108 parasites (calculated based on % parasitemia) were triturated with nine parts of Saccharum lactis powder to achieve 1X potency. One part of 1X potency and nine parts of Saccharum lactis powder were taken to make subsequent 2X potency. Likewise, potencies till 6X were prepared by trituration method, prescribed in Homoeopathy pharmacopeia of India (HPI).[30] In HPI, the method of preparation of nosode sourced from organisms is described under Group IV category using triturations up to 6X potency.[31] Further, 8X potency (equivalent to 4c) was prepared using 50:50 proportion of water for injection and dispensing alcohol (92% sourced from Merck). Potencies 5c, 6c and further up to 30c were prepared using electromechanical potentizer developed by the author with well-defined force parameters 12,099 Nm.[32] All the potencies were labelled and stored appropriately [Figure 2].
Figure 2: Flow chart of Malaria nosode preparation

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The sterility testing for 30c potency was done by direct inoculation method to detect the presence of aerobic, anaerobic bacteria and fungi as per the Indian pharmacopeia method. Fluid thioglycollate medium was used for the culture of anaerobic bacteria, and Soyabean casein digest medium was used for the culture of both fungi and aerobic bacteria.

Biosafety laboratory guidance was followed during the cultivation and preparation. Clearance from the Institutional Biosafety Committee (IIT-B) was obtained. All the potencies were processed in a private laboratory setup using a laminar flow.


  Results Top


Nosode from P. falciparum (coded as PFA) strain was prepared and standardised for future research and clinical exploration. The nosode was tested negative for sterility as per HPI guidelines for nosodes. The sterility testing of P. falciparum nosode 30c potency was done as per the Indian Pharmacopoeia monograph.[33]

Scanning electron microscopy was performed for 1c, 6c and 30c using SEM-QUANTA 200 with EDS version 3.0.13 at ×300,000 magnification. Few particles of size measuring from 700 nm to 3 μm for 1x potency and 2–8 μm for 6c potency were seen. [Figure 3] and [Figure 4], which could be the particles of the vehicle (Saccharum lactis). There were no particulate matters seen in 30c potency at ×1000 magnification [Figure 5]. The Electron microscopy is a modern technique that can detect far smaller particles such as viruses, molecules and even individual atoms.[34]
Figure 3: Scanning electron microscope image: nosode made from Plasmodia falciparum (Malaria nosode) 1x. Particles of size measuring from 700 nm to 3 μm seen for 1x potency

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Figure 4: Scanning electron microscope image: nosode made from Plasmodia falciparum (Malaria nosode) 6c. Particles of size measuring from 2 to 8 μm seen for 6c potency

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Figure 5: 3. Scanning electron microscope image: nosode made from Plasmodia falciparum (Malaria nosode) 30c. No particulate matter seen in potency 30c

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Polymerase chain reaction (PCR) (PCR test-malaria differentiation Multiplex) for 30c was performed by ABI 7500 Dx real-time PCR. The testing was performed with an objective to trace whether any nucleic material is present in this dilution. This PCR test can detect parasitemia as low as one gene copy and allows differentiation of the common four Plasmodium species (P. falciparum, P. vivax and P. malariae and P. ovale). Test result shows that gene copy was not detected for P. falciparum, P. vivax and P. malariae and P. ovale.


  Discussion Top


The old nosodes in Homoeopathy are undergoing a revival, revamping; and new ones are getting introduced using current scientific techniques. Nosodes have already unveiled significant therapeutic potential. The anti-malarial potential of P. falciparum nosode has been demonstrated in the in vitro study,[35] the study was experimental study and more in vivo(and other) studies need to be explored in future evaluating the efficacy of P. falciparum nosode against malaria and other diseases.

Many of the nosodes in the past were prepared from diseased tissues as the techniques for isolation and culture of organisms were not well developed. It is difficult to standardise and reproduce nosodes prepared from diseased tissues.

Ideally, the P. falciparum nosode should have been prepared from the cell-free culture. However, it is not possible at this point to procure such a culture. The limitation noted during this preparation is total blood packed cells with 3% parasitemia. The nosode could be suitably updated in future overcoming the limitations. Due to technical limitations, it was not possible to prepare nosodes from other strains of Plasmodium such as P. vivax, P. ovale and P. malariae, which could be explored in future while developing the modified, next version of separate or polyvalent Malaria nosode, with higher parasite count.


  Conclusion Top


The P. falciparum nosode sourced from P. falciparum parasites is an addition to the existing nosodes list, which will call for further exploration for its efficacy against malaria (and other diseases) as prophylactic and therapeutic measures.

Acknowledgement

Dr. Swati Patankar, Ph.D., at the Department of Bio-sciences and Bio-engineering, at IIT, Mumbai, India, for providing the material (Malaria Strain) to prepare the nosode. Dr. N.O. Goel, MD, Head of the Department, Pharmacy, CMPH Medical College for guidance on conversion of potency from decimal to centesimal scale. Ankur Path-lab for allowing to use aseptic chamber.

Financial support and sponsorship

Life Force, Mumbai.

Conflicts of interest

The author has a pending patent for the nosode.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]



 

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