WO2022085028A1

WO2022085028A1 - Nano curcumin homeopathic formulation for treatment of malaria

Info

Publication number: WO2022085028A1
Application number: PCT/IN2021/051000
Authority: WO
Inventors: Santosh Kar; Bhaskar Saha; Debadatta NAYAK; Anil KHURANA
Original assignee: Central Council For Research In Homoeopathy
Priority date: 2020-10-21
Filing date: 2021-10-21
Publication date: 2022-04-28

Abstract

The present invention relates to nano-curcumin and nano-curcumin based homeopathic formulation for prevention and treatment of malaria and other inflammatory diseases. The invention also relates to a process for the preparation of nano-curcumin and nano-curcumin based homeopathic formulation. In particular the invention relates to a curcumin nanoparticle of 200 nm average diameter and homeopathic formulation thereof for prevention and treatment of malaria and other inflammatory diseases.

Description

NANO CURCUMIN HOMEOPATHIC FORMULATION FOR TREATMENT OF MALARIA

FIELD OF THE INVENTION

The present invention relates to a homeopathic formulation for prevention of malaria. More particularly invention relates to nano-curcumin based formulation for treatment and prevention of malaria including cerebral malaria.

BACKGROUND OF THE INVENTION

Malaria is an infectious disease, transmitted by mosquitoes and caused by five species of Plasmodium parasites (P. falciparum, P. vivax, P. ovale, P. malariae and P. knowelsi). Symptoms include fever, chills, headache, muscle aches, tiredness, nausea and vomiting, diarrhoea, anaemia, and jaundice. Convulsions, coma, severe anaemia, and kidney failure can also occur. It remains a leading cause of death globally killing 409,000 persons in 2019 as per WHO report. While repeated chronic infections may leads to partial immunity in endemic areas, however people still suffer significant morbidity and loss of productivity.

Plasmodium falciparuminducedmaiaha continues to be the predominant infectious disease in tropical and sub-tropical countries. Cerebral malaria (CM) is a severe complication of P. falciparum infection. This complex and potentially reversible encephalopathy leads to coma and occurs with or without pathology in other organs.

Although the pathogenesis of CM is unclear, a simplified explanation is that the adherence to endothelial cells and the sequestration of parasitized erythrocytes and immune cells in brain capillaries cause an inflammatory process and release of other neurotoxic molecules. Antimalaria drugs have been used to treat CM cases.

IN670/DEL/2009 describes a process and method for preparation of curcumin nanoparticles from ethanol solution of commercially available curcumin. The curcumin nanoparticles have improved bioavailability.

IN201811011436 describes a process for potentization of homeopathic medicines. The process comprises of mixing together a homeopathic substance with a suitable solvent in a ratio of 1:9 to 50:50 followed by number of succussions to obtain a 1C potency and diluting 1 part of 1C potency obtained in step (i) with 99 parts of suitable solvent and dilution and succussions are repeated to obtain different formulations.

Curcuma longa rhizome powder (turmeric) is a well-known indigenous herbal medicine. It is known for its diverse biological actions and pharmacological activities including antiinflammatory, antioxidant, antiproliferative, antimicrobial, anticarcinogenic and antiangiogenic properties. Curcumin is one of the biologically active components of Curcuma longa rhizome powder (turmeric). However, the systemic bioavailability of curcumin is low, most of it being excreted in the faeces and only traces appeared in the urine (Wahlstrom B, Blennow G. A study on the fate of curcumin in the rat. Acta Pharmacol Toxicol (Copenh). 1978 Aug;43(2):86-92). Converting natural curcumin into pure nanoparticles improves its bioavailability significantly without altering its chemical properties. Homeopathic medicines have been used as alternative for the treatment of many diseases, including malaria. Homeopathic formulation of Curcuma longa rhizome powder is available but there is no homeopathic formulation of curcumin or pure nanoparticles of curcumin which can utilize the beneficial effects of this central compound in a homeopathic setup.

OBJECTIVES OF THE INVENTION

The primary object of the present invention is to provide a nano-curcumin based therapeutic homeopathic formulation for treatment of inflammatory diseases.

Another object of the present invention is to provide a nano-curcumin based homeopathic formulation for management of malaria.

Further, object of the present invention is to provide a homeopathic formulation of a defined potency prepared from pure nanoparticles of curcumin.

Yet another object of the present invention is to develop a potent nano-curcumin homeopathic formulation for cerebral malaria.

SUMMARY OF THE INVENTION

Accordingly, present invention aims to provide a nano particle based homeopathic formulation for treatment of chronic inflammatory diseases. Further, the present invention aims to provide a nano-curcumin based homeopathic formulation for prevention and therapy of malaria including cerebral malaria. In one of the aspects, the present invention provides a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, wherein said curcumin nanoparticles are of 200 nm average diameter.

In another aspect, the present invention provides a process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, said process comprising of: a) dissolving natural curcumin in ethanol and filtering to obtain a clear solution; b) stirring the above obtained clear solution in a high-speed homogenizer along with simultaneous and slow addition of Milli Q water containing citric acid to obtain pure curcumin nanoparticles from the solution; and c) homogenizing the entire suspension over ice in a high-pressure homogenizer for > 20 cycles to obtain curcumin nanoparticles of 200 nm average diameter.

In one of the aspects, the process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed at the homogenizing speed of 12,000 - 15,000 RPM.

In one of the aspects, the process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed with the Milli Q water contains 0.1% citric acid.

In one of the aspects, the process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed with the Milli Q water containing citric acid in step b), which being added over a period of 60 minutes.

In one of the aspects, the process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed with the Milli Q water containing citric acid in step b), which being added until the ethanol concentration became 40% (V/V).

In one of the aspects, the process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed with the pressure of homogenizer at 30,000 PSI in step c). In another aspect, the present invention provides a homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, wherein said formulation comprises of a) a nano-curcumin; and (b) a solvent.

In one of the aspects, the homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is comprises a nano- curcumin in a potentized, ultra-low dilution of 100-30.

In one of the aspects, the homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is comprises solvent selected from a group consisting of water, ethanol, or a mixture thereof.

In one of the aspects, the homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is reduces the expression of PD1, PDL1, PDL2, CTLA4, CD 80, CD86, CD40, CD40L, IFN gamma, TNF alpha, IL 1 beta, IL 6, and IL 12, MIP 1 beta, CCR7, CXCL10, CXCR3 and ICAM 1 and increases the expression of GATA3, FOXP3, IL 4, and IL 10.

In another aspect, the present invention provides a process for preparation of a homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, said process comprising steps of: a) suspending the curcumin nanoparticles of 200 nm average diameter in water followed by homogenization and filtration; b) drying the filtered slurry to obtain nano-curcumin powder; and c) suspending the above obtained nano-curcumin powder in a solvent selected from the group consisting of water, ethanol, or a mixture thereof.

In one of the aspects, the process for preparation of a homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is performed with the water provided with 0.1% polysorbate 80 in step a), and wherein the homogenization is carried-out for 1 hour at 12,000 - 15,000 RPM, and wherein the drying temperature in step b) is 60°C.

In one of the aspects, the process for preparation of a homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is potentized to an ultra-low dilution (100-30) in step c). In one of the aspects, the process for preparation of a homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, as described in present invention is carried out by dissolving 4 gm of nano-curcumin in 1 L ethanol, followed by addition of 99 parts of alcohol/water to obtain formulation of a potency of 1c, and to the one part of a formulation of potency 1c is added 99 parts of alcohol/water and is succussed progressively for 30 times to produce final formulation (NC 30c) in step c).

DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1: Effect of different homeopathic drug treatments on the blood parasitemia of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 6 different groups (n=10 per group) after being infected with IX 10⁶ P. berghei ANKA infected erythrocytes. The first group was left untreated (infected-untreated). The second group (Infected-potentized EtOH treated) was orally administered with vehicle (potentized EtOH) given orally after dilution with 200 pl PBS, twice daily from day 2 to day 8 post-infection. The third, fourth, fifth and sixth groups were treated with Homeopathic Curcuma longa 30c (CL 30c), Nano-curcumin 6c (NC 6c), Nano-curcumin 18c (NC 18c), Nano-curcumin 30c (NC 30c) formulations respectively, diluted with 200pl PBS twice daily from day 2 to day 8 post infection. The trend in blood parasitemia has been depicted. Data is representative of three independent experiments.

Figure 2: Effect of different homeopathic drug treatments on the survival of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 6 different groups as mentioned in description to Figure 1. The survival curve of all the groups has been depicted. Data is representative of three independent experiments.

Figure 3: Effect of NC30ctreatment on the blood parasitemia of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group). The first group was left untreated (infected-untreated). The second group (Infected-potentized EtOH treated) was orally administered with vehicle, and the third group was orally treated with NC 30c (infected NC 30c treated). Fifty microliters of vehicle/NC 30c was given orally after dilution with 200 pl PBS, twice daily from day 2 to day 8 postinfection. The trend in blood parasitemia has been depicted. Data is representative of three independent experiments. Figure 4: Effect of NC30c treatment on the survival of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. The survival curve has been depicted. Data is representative of three independent experiments.

Figure 5: Effect of NC30c treatment on Evan’s Blue Dye extravasation in the brain of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. A group (n=10) of age-matched, healthy uninfected mice were kept separate as control group (Uninfected). Mice were injected with 0.2% Evan’s Blue Dye in PBS intravenously on day 12 post-infection and the brain was isolated from the different groups to assess Blood-Brain Barrier (BBB) damage after 2 hours.

Figure 6: Quantitation of Evan’s Blue Dye extravasation in the brain of mice infected with P. berghei ANKA and treated with NC 30c. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) and injected with 0.2% Evan’s Blue Dye as mentioned in description to Figure 5. The Evan’s blue dye was extracted out from the brain tissue after 2 hours using 100% formamide and the absorbance was measured at 620 nm to quantify the extent of BBB damage by Evan’s Blue Dye Extravasation. Data is expressed as mean ± S.D and is representative of three independent experiments.

Figure 7: FACS analysis of splenic T cell population of mice infected with P. berghei ANKA and treated with NC 30c. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. A group (n=10) of age-matched, healthy uninfected mice were kept separate as control group (Uninfected). Percentage of CD3+CD4+Tbet+ T cells, CD3+CD4+GATA3+ T cells and CD3+CD4+CD25+FOXP3+ T cells in the spleen of mice was determined by FACS analysis on day 12 post infection. Data is representative of three independent experiments.

Figure 8: Effect of NC 30c treatment on expression of pro-inflammatory markers in the brain of mice infected with P. berghei ANKA. 6-8 week old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. A group (n=10) of age-matched, healthy uninfected mice were kept separate as control group (Uninfected). qPCR analysis was performed on day 12 post-infection using mRNA isolated from the brain of different groups and checked for the expression of PD1, PDL1, PDL2, CTLA4, CD80, CD86, CD40, CD40L, IFN gamma, IL 12, IL 1 beta, MIP 1 beta, CCR7, CXCL10, CXCR3, ICAM in the brain. Data has been normalized to GAPDH and expressed as mean ± S.D of relative fold change in mRNA expression and is representative of three independent experiments (* P<0.05, **P<0.01, ***P<0.001, n.s: not significant).

Figure 9: Effect of NC 30c treatment on gene expression of pro-inflammatory markers in the spleen and level of different cytokines in the serum of mice infected with P. berghei ANKA. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. A group (n=10) of age-matched, healthy uninfected mice were kept separate as control group (Uninfected). Quantitative PCR analysis of Tbet, GATA3, FOXP3, IFN gamma, IL 6 and IL 10 was performed on day 12 post-infection using mRNA isolated from the spleen cells for determining changes in cytokine expression. Data has been normalized to GAPDH and expressed as mean ± S.D of relative fold change in mRNA expression and is representative of three independent experiments. Serum from different groups was collected on day 12 post-infection and ELISA was performed to determine the levels of IFN gamma, TNF alpha, IL 12, IL6, IL4, and IL 10 in the blood. Data are expressed as mean ± S.D of serum cytokine concentration (pg/ml) and are representative of three independent experiments (* P<0.05, **P<0.01, ***P<0.001, n.s: not significant).

Figure 10: Histological analysis of brain, spleen and liver of mice infected with P. berghei ANKA and treated with NC 30c. 6-8-week-old female C57BL/6 mice were divided into 3 different groups (n=10 per group) as mentioned in description to Figure 3. Mice were sacrificed on day 12 post-infection and tissues from the brain, spleen and liver were collected and stored in 10% formaldehyde and then subjected to a tissue processor using an automated tissue processor (Leica, Germany) to remove the water from the tissues. The specimens were embedded into melted paraffin wax using a histoembedder (Leica, Germany), sectioned into a 4.0pm thick slice with a microtome (Leica, Germany) and stained with hematoxylin and eosin (H&E) using an auto Stainer. A) Brain, B) spleen, and C) liver from different groups were analyzed after H&E staining under 60x magnification using a light microscope.

DETAILED DESCRIPTION OF THE INVENTION

The present invention aims to provide a nano particle based homeopathic formulation for treatment of inflammatory diseases. Homeopathic medicines have been used as an alternative for the treatment of inflammatory diseases, including malaria. Further, the present invention aims to provide a nano-curcumin based homeopathic formulation for treatment and prevention of malaria including cerebral malaria. The nano-curcumin based formulation is prepared from pure nanoparticles of curcumin by potentization and succuction.

While the invention is susceptible to variations and modifications other than those specifically described herein by specific embodiments and examples. It is to be understood that the present disclosure includes all modifications, equivalents, and alternative falling within the spirit and the scope of the invention as defined by the appended claims.

The figures and protocols have been represented for only showing the specific details that are pertinent to understanding of the embodiments of the present invention and not to obscure the disclosure with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the disclosure.

The present invention provides a homeopathic formulation comprising: (a) a nano-curcumin; and (b) a solvent.

The invention describes a homeopathic formulation comprising a nano-curcumin and a solvent selected from a group consisting of water, ethanol (98%) or a mixture thereof.

The nano-curcumin based homeopathic formulation described in present invention is prepared by potentization and succussions.

In the present invention, therapeutic efficacy of different nano-curcumin based formulation in preventing the disease severity brought about by Plasmodium berghei (P. berghei) ANKA infection in a C57BL/6 mice modelwasevaluated. Treatment with homeopathic formulation of Nano-curcumin 30c (NC 30c) potency delayed the death of the infected animals by 10 days in comparison to infected untreated animals. Oral feeding of Homeopathic Nano- curcumin formulation 30c to mice protected against damage to the blood brain barrier and haemorrhages in the brain and prevented the development of Experimental Cerebral Malaria (ECM) in the infected mice by reducing excessive inflammation, thereby improving the period of survival in the P. berghei ANKA infected mice.

Preparation of Curcumin Nano particles

Four grams of natural curcumin (Sigma, USA) dissolved in 1 L of distilled ethanol at room temperature and filtered to obtain a clear solution. This solution was then stirred in a high- speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM and a required volume of Milli Q water containing 0.1% citric acid (Merck, India) was added to it slowly over a period of 60 min until the ethanol concentration became 40% (V/V) and pure curcumin nanoparticles started to precipitate from the solution. Then the entire suspension was homogenized over ice in a high-pressure homogenizer (Avestin C5 High Pressure Homogeniser BPS, UK) at 30,000 PSI for 20 cycles or more till uniform particles of 200 nm average diameter were obtained. The aqueous suspension was then made to 0.1% polysorbate 80 (Sigma, USA) and homogenized at 12,000-15,000 RPM (T 25 digital ULTRA-TURRAX, IKA, USA) again for 1 hr and filtered. The filtered slurry was dried at 60°C in an oven to get nano-curcumin powder. Particle size was determined by a high-resolution transmission electron microscope (JEM 21 OOF, JEOL, USA).

Following the preparation of pure nanoparticles of curcumin, the material was taken and converted into a homeopathic formulation.

In the present embodiment the therapeutic efficacy of homeopathic formulation of Nano- curcumin in preventing the disease severity brought about by Plasmodium berghei (P. berghei) ANKA infection in a C57BL/6 mice model. C57BL/6 mice were infected with P. berghei ANKA and infected mice were orally fed with different potencies of homeopathic Nano-curcumin formulation to assess the efficacy of the homeopathic formulation in controlling the infection observed over a 30-day period.

Homeopathic formulations of nano-curcumin were prepared in three different potencies viz 6c, 18c, and 30c.

Nano-curcumin 6c (NC 6c): Nano-curcumin mother tincture was prepared by dissolving 4 gm nano-curcumin powder in 1 L ethanol. Next, to one part of mother tincture in a glass container, was added 99 parts of alcohol/water and the mixture was succussed giving it a potency of 1c. To one part of a solution of potency 1c in a fresh container, was further added 99 parts of alcohol/water, and the mixture was similarly succussed progressively for 6 times to produce NC 6c.

Nano-curcumin 18c (NC 18c): Nano-curcumin mother tincture was prepared by dissolving 4 gm Nano-curcumin powder in 1 L ethanol. Next, to one part of mother tincture in a glass container, was added 99 parts of alcohol/water and the mixture was succussed giving it a potency of 1c. To one part of a solution of potency 1c in a fresh container, was further added 99 parts of alcohol/water, and the mixture was similarly succussed progressively for 18 times to produce NC 18c.

Nano-curcumin 30c (NC 30c): Nano-curcumin mother tincture was prepared by dissolving 4 gm Nano-curcumin powder in 1 L ethanol. Next, to one part of mother tincture in a glass container, was added 99 parts of alcohol/water and the mixture was succussed giving it a potency of 1c. To one part of a solution of potency 1c in a fresh container, was further added 99 parts of alcohol/water, and the mixture was similarly succussed progressively for 30 times to produce NC 30c.

EXAMPLE 1

Determination of LD₅₀ of NC 6c homeopathic formulations when fed orally to 8-12 weeks old female BALB/c mice

Three groups of 8-12 weeks old female BALB/c mice were orally fed with three different doses of nano-curcumin homeopathic formulation 6c for determination of LD50 as follows:

Group 1- 300pl 6c + 300pl PBS (pH 7.2) Given orally once.

Group 2- 200pl 6c + 200pl PBS (pH 7.2) Given orally once.

Group 3- lOOpl 6c + lOOpl PBS (pH 7.2) Given orally once.

Results: Table 1 depicts the results after feeding nano-curcumin homeopathic formulation NC 6c for the determination of LD50. The LD50 of NC 6c when delivered orally in BALB/c mice was found to be 200pl (200ul of 6c diluted in 200pl of PBS (pH 7.2).

Table 1

EXAMPLE 2

Determination of LD₅₀ of NC 30c homeopathic formulations when fed orally to 8-12 weeks old female BALB/c mice Three groups of 8-12 weeks old female BALB/c mice were orally fed with three different doses of nano-curcumin homeopathic formulation 30c for determination of LD50 as follows:

Group 1- 300pl 30c + 300pl PBS (pH 7.2) Given orally once.

Group 2- 200pl 30c + 200pl PBS (pH 7.2) Given orally once.

Group 3- lOOpl 30c + lOOpl PBS (pH 7.2) Given orally once. Results: Table 2 depicts the results for LD50 for NC 30c. There was no death observed in Groups 1-3 and therefore the LD50 of NC 30c in female BALB/c mice is higher than 300ul. The oral toxic dose of NC 30c in BALB/c mice was taken to be 200pl (200ul of30c diluted with 200pl of PBS (pH 7.2).

Table 2 EXAMPLE 3 Determination of Acute toxicity of 6c and 30c nano-curcumin homeopathic formulation given orally for 28 days at less than LD₅₀ dose to 8-12 weeks old Female/ Male BALB/c mice

8-12-week-old BALB/c mice were divided into different groups each consisting of 3 male and 3 female mice as described below in Table 3:

Table 3

Results: The results for acute toxicity of NC 6c and NC 30c in BALB/c mice have been tabulated in Tables 4-6 as given below.

Blood Report for the control BALB/c mice group (A)

Table 4

Blood Report for the BALB/c mice fed with NC 6c group (B)

Table 5

Blood Report for the control BALB/c mice fed with NC 30c group (C)

Table 6

EXAMPLE 4

Determination of LD₅₀ of NC 6c homeopathic formulations when fed orally to 12-14 weeks old female Wistar rats Three groups of 12-14 weeks old female Wistar rats were orally fed with three different doses of nano-curcumin homeopathic formulation 6c for determination of LD50 as follows:

Group 1- 300pl 6c + 300pl PBS (pH 7.2) Given orally once.

Group 2- 200pl 6c + 200pl PBS (pH 7.2) Given orally once.

Group 3- lOOpl 6c + lOOpl PBS (pH 7.2) Given orally once. Results: Table 7 depicts the results for determination of LD50 for NC 6c. The LD50 of NC 6c when delivered orally in Wistar rats was taken to be 200pl (200ul of 6c diluted in 200pl of PBS (pH 7.2).

Table 7

EXAMPLE 5

Determination of LD₅₀ of NC 30c homeopathic formulations when fed orally to 12-14 weeks old female Wistar rats Three groups of 12-14 weeks old female Wistar rats were orally fed with three different doses of nano-curcumin homeopathic formulation 30c for determination of LD50 as follows:

Group 1- 300pl 30c + 300pl PBS (pH 7.2) Given orally once.

Group 2- 200pl 30c + 200pl PBS (pH 7.2) Given orally once.

Group 3- lOOpl 30c + lOOpl PBS (pH 7.2) Given orally once. Results: Table 8 depicts the results for determination of LD50 for NC 30c. The LD50 of NC 30c when delivered orally in Wistar rats was taken to be 200pl (200ul of 6c diluted in 200pl of PBS (pH 7.2).

Table 8

EXAMPLE 6 Determination of Acute toxicity of 6c and 30c nano-curcumin homeopathic formulation given orally for 28 days at less than LD₅₀ dose to 12-14 weeks old Female/ Male Wistar rats 12-14-week-old Wistar rats were divided into 3 groups each consisting of 3 male and 3 female mice as described below in Table 9:

Table 9

Results: The results for acute toxicity of NC 6c and NC 30c in Wistar rats have been tabulated in Tables 10-12 as given below:

Blood Report for the control group of Wistar rats fed with PBS pH 7.2 (A)

Blood Report for the Wistar rat group (B) fed with 6c

Table 11

Blood Report for the Wistar Rat group (C) fed with 30c

Table 12

EXAMPLE 7

Infection and drug treatment

P. berghei ANKA parasites were maintained in 6-8 weeks old female C57BL/6 mice. The animals were injected with 1X10⁶ parasitized Red Blood Cells (pRBCs) intraperitoneally and monitored daily for symptoms of infection and determining the survival period. Each mouse in the treatment group was orally fed with 50pl of Homeopathic nano-curcumin 30c (NC30c) formulation diluted with 200pl PBS to evaluate the efficacy of the formulation to protect against infection. The vehicle control (infected potentized EtOH treated) group received only 50pl of potentized ethanol (vehicle) diluted with 200pl PBS. Animals were treated twice daily for 7 days starting from day 2 post-infection up to days 8 post-infection. The untreated control (Infected untreated) received no treatment. Parasitemia was determined by taking blood smears from the tail of the mice and staining with Acridine Orange (Sigma, St Louis, MO) followed by observation in a fluorescent microscope.

Results: All the infected-untreated and infected-potentized EtOH treated animals remained asymptomatic till day 4 post infections and started displaying symptoms of infection like ruffled hair and hunching by day 5 to 6 post-infection. The infected animals further developed the neurological symptoms associated with the development of ECM like ataxia, convulsions and hind limb paralysis which rapidly progressed to coma and resulted in the death of the infected mice between days 8-12post-infection when the parasitemia reached around 20% in the blood. In a preliminary experiment, each mice in the treatment group was orally fed with 50pl of Homeopathic Curcuma longa 30c (CL 30c) or nano-curcumin 6c (NC 6c), or nano-curcumin 18c (NC 18c), or nano-curcumin 30c (NC 30c) formulation diluted with 200pl PBS to evaluate the efficacy of different formulations to protect against infection. The median survival period of P. berghei ANKA infected C57BL/6 mice, in the preliminary study was around 10 days (Fig. 1). There was no difference in the median survival period of infected mice that were treated with the homeopathic formulation of CL 30c, whereas the median survival period for the infected mice treated with 6c, 18c, and 30c potencies of nano- curcumin homeopathic formulation were 10, 12, and 24 days respectively (Fig. 1). No significant differences were discernible among CL 30c formulation and the NC 6c potency of nano-curcumin formulation used, with respect to parasitemia, whereas the parasitemia in NC 18C and NC 30c were lower (Fig. 2). Overall, NC 30c treated animals appeared to be consistently more active than the rest and hence for all further studies NC 30c was chosen as the drug candidate to be used for evaluation of efficacy and mechanism of protection. When the experiment was separately repeated with NC 30c alone, it was observed that while all the mice in the infected untreated group developed ECM and died by days 8-12 post-infection, around 40 % of the NC 30c treated animals developed neurological symptoms and succumbed due to ECM and the remaining animals controlled parasitemia better than infected control groups and survived till day 24 post-infection without any cerebral malaria symptoms but succumbed due to heavy parasite burden which reached around 55-60% at the time of death (Fig. 3, 4).

EXAMPLE 8

Blood-Brain Barrier permeability

Blood-Brain Barrier permeability was checked by Evan’s Blue Dye Extravasation. On day 12 post-infection, experimental mice were anesthetized and given an intravenous injection of 0.2% Evans Blue Dye (Sigma, St Louis, MO) in PBS and the dye was extracted from brain tissue after 2 hours in 100% formamide and the absorbance was measured at 620 nm to determine the extent of extravasations.

Results: One of the characteristic features of the pathological conditions elicited by ECM is the breakdown of the Blood-Brain Barrier (BBB) integrity. Under normal circumstances the BBB only selectively allows certain small molecules and gases to exchange into the brain tissue while preventing entry of the rest. Disruption of the BBB integrity allows foreign substances to enter into the brain tissues which can result in neuronal toxicity. The integrity of the BBB is assessed by its ability to prevent Evan’s blue, a vital dye to enter the surrounding brain tissue. Evan’s Blue has a high affinity to bind to albumin in the blood. When the integrity of the BBB is compromised, albumin can freely cross the endothelial barrier into the brain tissue and since Evan’s blue dye binds with a high affinity to albumin it is carried into the surrounding brain tissue. The integrity of the BBB is assessed by its ability to prevent Evan’s blue, a vital dye to enter the surrounding brain tissue. The brain isolated from the P. Berghei ANKA infected-untreated and infected potentized EtOH treated C57BL/6 mice showed the disruption of the BBB as evidenced by the percolation of Evan’s Blue dye into the brain tissue (Fig. 5), and treatment of the infected mice with NC 30c prevented the BBB breakdown (Fig. 5) thereby preventing the onset of ECM in the treated mice. The extent of extravasation of the dye can be determined by spectrophotometrically after extracting the dye and measuring O.D at 620 nm. The extent of Evan’s Blue Dye Extravasation in infected-untreated and infected potentized EtOH treatedwas significantly higher than untreated group. There was no significant change in NC30c treated group as compared to the untreated group (Fig. 6).

EXAMPLE 9

FACS analysis of splenocytes

Spleens were collected from the experimental animals on the 12^th day post-infection. Splenocytes were isolated from the spleen and subjected to RBC lysis and blocked with blocking buffer (30% FBS in PBS) for 30 minutes at 4 °C. The cells were then washed with 2 ml PBS, counted, and split into respective tubes keeping the count to 1X10⁶ cells per tube. Surface staining for each sample from each group was done with a cocktail of anti-mouse CD3 FITC conjugated antibodies (Biogene India, CAT no. 100203) and anti-mouse CD4 PB conjugated antibodies (Biogene India, CAT no. 100427). For Treg set, the samples were additionally stained with anti-mouse CD25_APC-Cy7 conjugated antibodies (Biogene India, Cat no. 102025). Following surface staining, the samples were then permeabilized and intracellular staining for the different sets was done using anti-mouse Tbet_APC conjugated antibodies (Biogene India, Cat no. 644813), anti-mouse GATA3 APC conjugated antibodies (Biogene India, Cat no. 653805), and anti-mouse FoxP3_Alexa Fluor® 647 conjugated antibodies (Biogene India, Cat no. 320013) respectively. Following intracellular staining, the cells were fixed with 1% paraformaldehyde and stored in dark at 4 degree C till FACS was done. FACS analysis was done using a FACS Canto II machine.

Results: Flow cytometric analysis indicated a change in the Thl/Th2 ratio of infected untreated animals on day 12 post-infection in comparison to uninfected animals with the response in the case of the control animals being more Thl skewed as evidenced by an increase in the percentage of CD3+CD4+Tbet+ T cells and reduction in the percentage of CD3+CD4+GATA3+ T cells and CD4+CD25+Foxp3+ T cells in the spleen (Fig. 7). In contrast, the percentage of CD3+CD4+Tbet+ T cells was reduced and CD3+CD4+GATA3+ T cells and CD4+CD25+Foxp3+ T cells was increased in the spleen of infected NC30c treated mice (Fig. 7).

EXAMPLE 10

Quantitative PCR analysis & Detection of serum cytokines by ELISA

RNA was extracted from the mouse whole brain and spleen by TRI reagent on 12^th day postinfection. Ipg of RNA was taken for reverse transcription reaction using random hexamers. The prepared cDNA was diluted 1:20 times and used for qPCR in Applied Biosystems Step One Plus Real-Time PCR System (Applied Biosystems Inc., Foster City, CA). GAPDH was used as the endogenous control and relative fold expression was calculated by 2'^AACT method.

Cytokine levels were measured in serum samples collected on day 12 post-infection from the experimental groups using DuoSet mouse ELISA kits (R&D systems) as per the manufacturer’s protocol. Biotinylated Detection Antibody and strept avidin-HRP conjugate were used with TMB liquid substrate (R&D systems) to give a colored product. Reaction was stopped using 2N H2SO4 and absorbance at 450 nm was measured to detect and quantify the levels of different cytokines in the serum.

Results: ECM is characterized by an uncontrolled increase in the level of inflammation in the brain which subsequently leads to disruption of the specialized endothelial cells that help regulate the flow of substances into and out of the brain. The pathophysiology associated with ECM as a consequence of heightened, prolonged inflammatory responses is orchestrated by a multitude of factors that collectively mediate the process of disruption of the Blood- Brain Barrier which subsequently leads to neuronal damage. Key factors that play a role in the process involve the expression of cell adhesion molecules, chemokines and their receptors, pro-inflammatory cytokines, co-stimulatory, and co-inhibitory molecules which collectively determine the extent and magnitude of the immune responses elicited as a result of the infection. qPCR analysis indicated that there was a significant increase in the expression levels of PD1, PDL1, PDL2 and CTLA4 in the infected control group (Fig. 8). The expression of co-stimulatory molecules CD80, CD86, CD40 and CD40L also increased in the brain of the infected control group (Fig. 8). There was a significant increase in the expression levels of the pro-inflammatory cytokines namely, IFN gamma, IL 12, IL 1 beta, MIP 1 beta, the chemokine receptor CCR7, the chemokine CXCL10, its receptor CXCR3 and cell adhesion molecule ICAM 1 in the brain of infected control groups (Fig. 8). Treatment with NC 30c lowered the expression of PD1, PDL1, PDL2, CTLA4, CD 80, CD86, CD40, CD40L, IFN gamma, IL 12, IL 1 beta, MIP 1 beta, CCR7, CXCL10, CXCR3 and ICAM 1 in the brain of infected animals (Fig. 8).

In the spleen of infected control groups, the expression of Tbet was significantly increased along with the increase in expression of IFN gamma and IL6 whereas the expression of GATA3 and FOXP3 were significantly decreased indicating that the immune response was highly Thl skewed (Fig. 9). Treatment with NC30c reduced the expression of Tbet, IFN gamma and IL 6 while increasing the expression of GATA3, FOXP3, and IL 10 significantly thereby preventing an overly skewed Thl response (Fig. 9). Data has been normalized to GAPDH and expressed as mean ±S.D of relative fold change in mRNA expression and is representative of three independent experiments. (* P<0.05, **P<0.01, ***P<0.001, n.s: not significant)

The serum levels of pro-inflammatory cytokines like IFN gamma, TNF alpha, IL 12 and IL 6, was lesser in NC 30c treated C57BL/6 mice as compared to that infected control groups whereas the levels of anti-inflammatory cytokines like IL 4 and IL 10 was higher in the NC 30c treated animals as compared to infected control groups (Figure 9). Data are expressed as mean ±S.D and are representative of three independent experiments. (* P<0.05, **P<0.01, ***P<0.001, n.s: not significant.

EXAMPLE 11

Tissue histology of brain, spleen, and liver

6-8 week old female C57BL/6 mice were divided into 3 different groups (n=10 per group). The first group was left untreated (infected-untreated). The second group (Infected-potentized EtOH treated) was orally administered with vehicle, and the third group was orally treated with NC30c (infected NC 30c treated). Fifty microliters of vehicle/NC30c were given orally after dilution with 200 pl PBS, twice daily from day 2 to day 8 post-infection. Experimental animals were sacrificed on day 12 post-infection and tissues from the brain, spleen and liver were collected for histological analysis.

Results: Histological analysis of the brain showed sequestration of infected RBCs and vascular occlusion in the brain vasculature of infected untreated and infected potentized EtOH treated groups that developed cerebral malaria symptoms (Fig. 10A). Occlusion was absent in the brain of infected animals treated with NC30c (Fig. 10A). Compared to uninfected spleen, the spleen of infected untreated and infected potentized EtOH treated groups showed heavy deposition of hemozoin. In the infected NC30c treated group hemozoin deposition was present but in lesser magnitude (Fig. 10B). Similarly, in comparison to uninfected, the liver of infected untreated and infected potentized EtOH treated groups showed heavy deposition of hemozoin (Fig. 10C). The deposition was present in lesser magnitude in the group treated with NC30cindicating that NC30c treatment did not lead to the elimination of the infection but prevented the pathological features in the brain that is associated with the development of ECM.

Statistical analyses

Statistical analysis was performed by using the GraphPad Prism software (GraphPad Prism v 5). Statistical difference between untreated and treated groups was determined by Bonferroni’s multiple comparison test. p<0.05 was considered significant.

Claims

WE CLAIM:

1. A therapeutic curcumin nanoparticles for the treatment and prevention of malaria and other inflammatory diseases, wherein said curcumin nanoparticles are of 200 nm average diameter.

2. A process for preparation of a therapeutic curcumin nanoparticles for the treatment and prevention of malaria and inflammatory diseases, said process comprising of: a) dissolving natural curcumin in ethanol and filtering to obtain a clear solution; b) stirring the above obtained clear solution in a high-speed homogenizer along with simultaneous and slow addition of Milli Q water containing citric acid to obtain pure curcumin nanoparticles from the solution; and c) homogenizing the entire suspension over ice in a high-pressure homogenizer for > 20 cycles to obtain curcumin nanoparticles of 200 nm average diameter.

3. The process as claimed in claim 2, wherein the speed of homogenizing in step b) is 12,000 - 15,000 RPM.

4. The process as claimed in claim 2, wherein the Milli Q water contains 0. 1% citric acid.

5. The process as claimed in claim 2, wherein the Milli Q water containing citric acid in step b) is added over a period of 60 minutes.

6. The process as claimed in claim 2, wherein the Milli Q water containing citric acid in step b) is added until the ethanol concentration became 40% (V/V).

7. The process as claimed in claim 2, wherein the pressure of homogenizer in step c) is 30,000 PSI.

8. A homeopathic formulation for the treatment and prevention of malaria and inflammatory diseases, wherein said formulation comprises of a) a nano-curcumin; and (b) a solvent.

9. The homeopathic formulation as claimed in claim 8, wherein the nano-curcumin is in potentized, ultra-low dilution of 100'³⁰. The homeopathic formulation as claimed in claim 8, wherein the solvent is selected from a group consisting of water, ethanol, or a mixture thereof. The homeopathic formulation as claimed in claim 8, wherein the formulation reduces the expression of PD1, PDL1, PDL2, CTLA4, CD 80, CD86, CD40, CD40L, IFN gamma, TNF alpha, IL 1 beta, IL 6, and IL 12, MIP 1 beta, CCR7, CXCL10, CXCR3 and ICAM 1 and increases the expression of GATA3, FOXP3, IL 4, and IL 10. A process for preparation of a homeopathic formulation for the treatment and prevention of malaria and other inflammatory diseases, said process comprising steps of: a) suspending the curcumin nanoparticles of 200 nm average diameter in water followed by homogenization and filtration; b) drying the fdtered slurry to obtain nano-curcumin powder; and c) suspending the above obtained nano-curcumin powder in a solvent selected from the group consisting of water, ethanol, or a mixture thereof. The process as claimed in claim 11, wherein the water in step a) is provided with 0.1% polysorbate 80, and wherein the homogenization is for 1 hour at 12,000 - 15,000 RPM, and wherein the drying temperature in step b) is 60°C. The process as claimed in claim 11, wherein the nano-curcumin in the formulation is potentized to an ultra-low dilution (100'³°). The process as claimed in claim 11, wherein in step c) 4 gm of nano-curcumin is dissolved in in 1 L ethanol, followed by addition of 99 parts of alcohol/water to obtain formulation of a potency of 1c, and to the one part of a formulation of potency 1c is added 99 parts of alcohol/water and is succussed progressively for 30 times to produce final formulation (NC 30c).