COMPOUNDS AND PHARMACEUTICAL FORMULATION THEREOF

FIELD OF THE INVENTION:

The present invention deals with a novel method of preparation of pharmaceutical compounds in highly pure, bioactive and bioavailable form anda novel pharmaceutical composition thereof comprising of strong antimalarial agents, followed by immunomodulatory and anti-inflammatory and neuroprotective compounds of the present invention used for the treatment diseases of parasitic or viral origin like malaria particularly cerebral malaria, Covid- 19, its variants etc. and its administration.

BACKGROUND OF THE INVENTION:

Cerebral malaria (CM) is one of the major complications arising out of Plasmodium falciparum infection in humans. It is associated with severe disturbances of the consciousness (deep coma), respiratory distress and other neurological abnormalities. The pathogenesis of human cerebral malaria stems from a dys -regulated, hyper-inflammatory immune response that leads to the breach of the blood brain barrier and entry of parasitized erythrocytes and other toxic agents into the brain causing extensive neuronal damage.

The World Health Organization (WHO) has recommended intravenous administration of artesunate as the first line of treatment for severe malaria. However, artesunate and other artemisinin derivatives at high concentrations are toxic and despite their effectiveness, malaria results in 4,05,000 deaths each year all over the world and a significant percentage of individuals’ present neurological and memory deficits even after cure.

Because of the difficulty in observing the cellular mechanisms associated with cerebral malaria directly in the human brain, it is imperative to use an animal model that closely reflects the clinical symptoms, immune mechanisms and histopathology associated with human cerebral malaria. P. berg he i ANKA infected CBA, Swiss Webster, or CB57B1/6 mice represent a model that is well- characterized and widely used for studying experimental cerebral malaria (ECM) that shares several similarities with P. falciparum induced cerebral malaria in humans. Both ECM and human cerebral malaria are characterized by severe vasculopathy, i.e. endothelial activation and dysfunction with increased expression of adhesion molecules, upregulation of pro inflammatory cytokines, reduced blood flow, vascular leakage, acute edema of both vasogenic and cytotoxic origin, and microhemorrhages leading to neurological impairment.

Artemesinin is a very potent plant derived antimalarial. But it is not soluble in water and therefore has been derivatised and has to be injected. It controls parasitemia very rapidly but does not reduce inflammation and repair neuronal damage. Curcumin is an anti-inflammatory molecule and can prevent neuronal damage. Therefore a synergisticcombination therapy of Artemesin derivatives which has to be injected along with oral delivery of natural curcumin as such or after embedding onto PLGA nano particles to improve bioavailability has been used. This combination has made the animals to survive upto90 days. Nanotized curcumin holds a considerable promise in therapeutics as demonstrated in the work done till date for treating malaria as a test system.

There exist a demand for an improved formulation for the treatment of cerebral malaria, other parasitic diseases, viral diseases and other diseases those can be treated by the identified actives and a huge scope for the improvement to utilize these actives to their full potential.

SUMMARY OF THE INVENTION

The present invention deals with a novel method of preparation of pharmaceutical compounds in highly pure, bioactive and bioavailable form and a novel pharmaceutical composition used for the treatment of diseases of parasitic or viral origin like malaria particularly cerebral malaria, Covid- 19, its variants and other viral infections, etc.

BRIEF DESCRIPTION OF THE DRAWINGS

1. Figure 1. FTIR profile of allylatedchalcone 9, soluble allylatedchalcone 9, andrographolide, soluble andrographolide, artemisinin, soluble artemisinin, resveratrol, soluble resveratrol, natural curcumin and soluble natural curcumin.

2. Figure 2. 6-8 week old female C57BL/6 mice were divided into different groups Gl, G2, G3 followed by intraperitoneal injection of 1X10 ^A 6 P berghei ANKAparasitised RBCs to mice belonging to G2 & G3 groups. Gl was left uninfected. G2 mice were treated with 200 ul of PBS orally from day 3 to day 10 post infection. G3 were treated with combinations A-F a) Survival data of Gl, G2 and G3 (Combinations A-F) groups b) Parasitemia data of Gl, G2 and G3 (Combination F) groups c) Differential staining of brain tissues from Gl, G2 and G3 (Combination F) groups with Evan’s Blue Dye on day 10-12 post infection. d) Quantitation of Evan’s Blue Dye extravasation into the brains of mice from Gl, G2 and G3 (Combination F) groups

Data is expressed as mean +_SD and is representative of three independent experiments.

3. Figure 3. 6-8 week old female C57BL/6 mice were divided into different groups Gl, G2, G3 (Combination F) as described in Figure 2. mRNAwas isolated from the brains of mice belonging to Gl, G2 and G3 groups on day 10-12 post infection to compare the changes in the level of gene expresssion of a) IFN gamma, b) MIP 1 beta, c) IL 12, d) IL 1 beta, e) CCR7, f) CXCR3, g) CXCL10, h) ICAM 1, i) PD1, j) PDL1, k) PDL2, 1) CTLA4, m) CD40, o) CD80, p) CD86 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 two independent experiments. [*P<0.05, **P<0.01, ***P<0.001, n.s: not significant].

4. Figure 4. 6-8 week old female C57BL/6 mice were divided into different groups Gl, G2, G3 (Combination F) as described in Figure 2. Serum was collected from mice belonging to Gl, G2 and G3 groups on day 10-12 post infection to measure the levels of a) IFN gamma, b) TNF alpha, c) IL 12, d) IL 6, e) IL 4 and f) IL 10

Data is expressed as mean + S.D and is representative of two independent experiments. [*P<0.05, **P<0.01, ***P<0.001, n.s: not significant].

5. Fig 5. 6-8 week old female C57BL/6 mice were infected with 1X1O ^A 6 P. berghei ANKA parasitised RBCs and divided into different groups Gl, G2, G3 (Combination F) as described in Figure 2. Spleens were collected on day 10-12 post infection from Gl, G2 and G3 groups.

A) Splenocytes were isolated and surface stained with anti-mouse CD3 FITC conjugated antibodies and anti-mouse CD4 PB conjugated antibodies. Intracellular staining with Anti mouse Tbet APC conjugated antibodies was performed to determine the percentages of CD3+ CD4+ Tbet+ T cells in the spleen of Gl, G2 and G3 groups on day 10-12 post infection.

B) Splenocytes were isolated and surface stained with anti-mouse CD3 FITC conjugated antibodies and anti-mouse CD4 PB conjugated antibodies. Intracellular staining with Anti mouse GATA3 APC conjugated antibodies was performed to determine the percentages of CD3+ CD4+ GATA 3+ T cells in the spleen of Gl, G2 and G3 groups on day 10-12 post infection

C) Splenocytes were isolated and surface stained with anti-mouse CD3 FITC conjugated antibodies, anti mouse CD4 PB conjugated antibodies and anti mouse CD25_APC-Cy7 conjugated antibodies. Intracellular staining with Anti mouse FOXP 3_ Alexa Fluor® 647 conjugated antibodies was performed to determine the percentages of CD3+ CD4+ CD25+ FOXP 3+ T cells in the spleen of Gl, G2 and G3 groups on day 10-12 post infection.

Fig. 6 TEM images of A) ART B) AC9 C) AND D) CUR E) GIN and F) RES showing spherical shaped nanoparticles. Scale bar 100 nm. DLS profiles of ART, AC9, AND, CUR, GIN, and RES showing (hydrodynamic radius) of G) ART, H) AC9, 1) AND, J) CUR K) GIN and L) RES determined to be 327 ± 180.7 nm, 331 ± 221.9 nm, 335.2 ± 215.8 nm, 230.5 ± 137.7 nm, 315.6 ± 223.2 nm and 287.2 ± 209.6 respectively; with a polydispersity index (Pdl) of 0.197, 0.219, 0.227, 0.180, 0.446 and 0.244 respectively by dynamic light scattering analysis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of manufacture of pure, bioactive and bioavailable water soluble forms of active ingredient(s) and a pharmaceutical formulations comprising of synergistic formulation ofstrongantimalarials to first reduce the parasite load in the blood, followed by the use of potent immunomodulators of natural origin to reduce inflammation, and finally used antiinflammatory and neuroprotective compounds that can prevent/repair the damage to the blood brain barrier and promote neuronal survival and development. The inventors of the present invention found that the compound Artemesinin and allylated chalcone9 are synergestic to each other and was givenorally at a specific stoichometric combination from the 3 ^rd day to 8 ^th day to P berghei ANKA infected mice for controlling parasitemia.Then a combination of plant derived compounds with immunomodulatory potential like andrgrapholide and gingerol (both of which also possess moderate antimalarial activities) has been given orallyfrom 5 ^th day till 8 ^lh day.Then a combination of NfKb inhibitors like curcumin which is anti-inflammatory andneuro protective, was given orally from 7 ^th day till 10 ^th day.As a result of this overlapping administration of the formulation for 10 daysthe animals controlled the parasite in the blood, reduced inflammation and protected the blood brain barrier.After 4weeks when the parasite specific 18srRNA appeared in the blood of the animal the administration of the pharmaceutical formulation was repeated again which kept the animals healthy till 120 days. After that the natural immune response took care of the parasite recrudescence.

The inventors have developed water soluble forms of all the identified active compounds, namely artemisinin, allylated chalcone9, andrographolide, gingerol, resveratrol and curcumin that can be given orally for the treatment of a diseases to provide therapeutic efficacy. Artemisinin is a strong and fast acting antimalarial but is not water soluble in its natural state. Therefore the use of artemisinin as an antimalarial is mediated by making synthetic derivatives of artemisinin that makes it more water soluble and stable. Allylated chalcone9 is a water insoluble synthetic chaicone derivative that has mild antimalarialactivity and has been shown to be synergistic with artemisinin against chloroquine resistant strains of Plasmodium falciparum in vitro. Likewise, andrographolide, gingerol, resveratrol and curcumin are all water insoluble in their natural occurrences, which greatly limits their therapeutic use. By converting these compounds into water soluble forms, without the need for derivatizing them and altering them chemically or embedding them into synthetic polymeric nanoparticles for enhancing their water solubility, the inventors utilized their inherent biological properties and combine them in a defined ratio used for the treatment of a disease in order to maximize the therapeutic efficacy againstcomplicated diseases like cerebral malaria, flu including flu caused by Covid- 19. The compounds prepared by method disclosed in the present invention would be significantly be more effective for treating all the disorders for which they are already known to have therapeutic effect. The present invention provides sufficient data to demonstrate and establishes that oral administration of water soluble artemisinin at Img/mice, and water soluble allylated chalcone9 at 2mg/mice to P.berghei ANKA infected mice for 6 days from 3 ^rd to 8 ^th day post infection followed by feeding of water soluble andrographolide and gingerol at Img/mice for 4 days from 5 ^th day till 8 ^th day post infection , which was followed by oral administration of water soluble forms of potent NFkB inhibitors like curcumin and resveratrol at 3mg/mice for 4 days from 7 ^th day till 10 ^th day post infection prevented cerebral malaria pathology, delayed recrudescence and prolonged survival of C57BL/6 mice infected with P. berghei ANKA.

As an embodiment of the present invention water solubility of insoluble pharmaceutical compounds has significantly been increased when manufactures by the method disclosed herein after. The pharmaceutical compounds thus prepared exhibit significantly increased bioavailability and efficacy. Thus a pharmaceutical formulation prepared using the actives as prepared by aforementioned method and pharmaceutical acceptable excipients would be highly effective. The water soluble pharmaceutical compounds like Nanocurcumin (CUR), Nanoartemisinin (ART), Nano allylated chalcone9 (AC9) Nanoandrographolide (AND), Nanogingerol (GIN) and Nanoresveratrol (RES) were prepared according to the following procedure. Briefly, a clear solution of 1-50 g of curcumin, or artemisinin, or gingerol, or resveratrol, or andrographolide, or allylatedchalcone 9or any other water insoluble pharmaceutical compound wasprepared by dissolving the compoundsindividually in 0.5-5 L of hot distilled ethanol.The solution was sprayed into equal volume ofMilliQ water containing 0.1-5.0 % polysorbate 80, and kept at 60 degree C and stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12, GOO- 15, 000 RPM over a period of 1 hour until the dissolved material precipitates. The precipitated material was filtered from the slurry and homogenized after suspending in 1 -5 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain nanoparticles of 10-500 nm range which makes the compounds soluble in water.

As yet another embodiment of the present invention, pharmaceutical formulations prepared are on the basis of the ability of the formulation to reduce inflammation and improve survivability without pathology generally associated with cerebral malaria infection, we prefer the formulation F in which (1 to 3 mg ART + 2 to 5 mg AC9) was given per mice orally in 200 pl PBS from day 3 post infection till day 8 post infection overlapped with a combination of (1 to 3 mg GIN + 0.5 to 3 mg AND) per mice from day 5 post infection till day 8 post infection and a combination of ( 1 to 3 mg RES + 3 to 5mg CUR) per mice from day 7 post infection till day 10 post infection.

The enhanced effect achieved on the brain tissues by the administration of pharmaceutical composition of the present invention has been discussed in tables 1 and 2:

Table 1. 6-8 week old female C57BL/6 mice were divided into different groups Gl, G2, G3 followed by intraperitoneal injection of 1X10 ^A 6 P berghei ANKAparasitised RBCs to mice belonging to G2 & G3 groups. Gl was left uninfected. G2 mice were treated with 200 ul of PBS orally from day 3 to day 10 post infection. G3 were treated with the combinations A-F. The histological findings between the groups in comparison to uninfected (control) group in the spleen of mice (3 mice per group) were tabulated. + is used to denote the severity and extent of the parameters at the site according to the following scale:- 1+ means < 25 % RBCs are parasitized, 2+ means 25-50 % RBCs are parasitized, 3+ means 50-75 % RBCs are parasitized, 4+ means > 75 % RBCs are parasitized, Absent - 0% RBCs parasitized.

Table 2. 6-8 week old female C57BL/6 mice were divided into different groups Gl, G2, G3 followed by intraperitoneal injection of 1X10 ^A 6 P berghei ANKAparasitised RBCs to mice belonging to G2 & G3 groups. Gl was left uninfected. G2 mice were treated with 200 ul of PBS orally from day 3 to day 10 post infection. G3 were treated with the combinations A-F. The histological findings between the groups in comparison to uninfected (control) group in the brain of mice (3 mice per group) were tabulated. + is useto denote the severity and extent of the parameters at the site according to the following scale:- 1+ means < 25 % RBCs are parasitized, + means 25-50 % RBCs are parasitized, 3+ means 50-75 % RBCs are parasitized, 4+ means > 5 % RBCs are parasitized, Absent - 0% RBCs parasitized.

Furthermore, the formulations would be highly effective in the treatment of Uncomplicated and complicated malaria including Cerebral Malaria, and MA-ARDS, Severe COVID 19, Viral encephalitis, Inflammatory bowel disease and other inflammatory responses as a result of any other diseases.

EXAMPLES:

The present invention is further being explained by the way of representative working examples. The scope of the invention is not limited to the working examples.

Example 1: Experimental Cerebral Malaria model and drug administration

P. berghei ANKA was revived from cryopreserved samples and maintained in 6-8 weeks old female C57BL/6 mice, weighing 18-20 g (de Oca et al., 2013). Blood was collected from infected donor C57BL/6 mice at parasitemia>l% and IxlO ⁶ parasitized RBCs (pRBCs) were passaged rapidly into naive C57BL/6 mice every fourth to fifth day by i.p injection. Briefly, mice are euthanized by carbon dioxide asphyxiation and 500 pl (optimal) blood is collected by cardiac puncture and transferred to 4.5 ml of RPMI/PS media (RPMI+1% PenStrep) containing 5 pl heparin (5,000 lU/ml) in a 10 ml polypropylene tube. The volume was then made upto 10 ml by adding 5 ml of RPMI/PS media. The cells were then centrifuged at 1,127 x g for 7 minutes at room temperature. The supernatant was then carefully aspirated and the pellet was resuspended in 1 ml RPMI/PS media. An accurate count of parasitized RBC (pRBC) in the suspension was then made using a hemocytometer and an inoculum of 1X10 ⁶ parasite infected RBCs, per 200 pl of RPMI/PS media was intraperitoneally injected into desired number of naive mice (de Oca et al., 2013). After 3 times passage in donor C57BL/6 mice, 1X1O ⁶ P. berghei ANKA parasites were intraperitoneally injected into experimental C57BL/6 mice as described, for the induction of ECM(100%) in infected mice.

To determine the efficacy of the specified combination regimen, 6-8 week old female C57BL/6 mice were randomly divided into 3 groups (G1-G3) followed by infection of lxlO ⁶ P. berghei ANKA parasitized RBCs by i.p injection into each mice from Groups G2 and G3. Group 1 (Gl) mice served as the uninfected normal control. The mice belonging to Group G2 were given 200 ul PBS per mice orally from day 3 to day 10 post infection and served as the infected untreated control.

The experimental animals from Group G3 were treated with any of the following combination regimens

A) (0.5 to 2 mg ART + 1 to 3 mg AC9 + 0.5 to 2 mg AND + 1 to 3 mg CUR) per mice orally in 200 pl PBS from day 6 post infection till day 12 post infection

B) (0.5 to 2 mg ART + 1 to 3 mg AC9 + 0.5 to 2 mg AND + 1 to 3 mg CUR) per mice orally in 200 pl PBS from day 6 post infection till day 9 post infection and again from day 24 post infection to day 27 post infection.

C) (1 to 3 mg AC9 + 3 to 5 mg CUR) per mice orally in 200 pl PBS, twice daily from day 6 post infection till day 9 post infection

D) (0.5 to 3 mg ART + 1 to 3 mg CUR +0.5 to 1 mg GIN) per mice orally in 200 pl PBS, twice daily, from day 3 post infection till day 8 post infection

E) (1 to 3 mg ART + 2 to 5 mg RES) per mice orally in 200 pl PBS overlapped with a combination of (2 to 5 mg AC9 +1 to 3 mg CUR) at 6 hour intervals from day 6 post infection till day 9 post infection

F) (1 to 3 mg ART + 2 to 5 mg AC9) per mice orally in 200 pl PBS from day 3 post infection till day 8 post infection overlapped with a combination of (1 to 3 mg GIN + 0.5 to 3 mg AND) per mice orally in 200 pl PBS from day 5 post infection till day 8 post infection and a combination of (1 to 3 mg RES + 3 to 5mg CUR) per mice orally in 200 pl PBS from day 7 post infection till day 10 post infection

Preferred pharmaceutical forma latioa

On the basis of the ability of the formulation to reduce inflammation and improve survivability without pathology generally associated with cerebral malaria infection, we prefer the formulation F in which ( 1 to 3 mg ART + 2 to 5 mg AC9) was given per mice orally in 200 pl PBS from day 3 post infection till day 8 post infection overlapped with a combination of (1 to 3 mg GIN + 0.5 to 3 mg AND) per mice from day 5 post infection till day 8 post infection and a combination of ( 1 to 3 mg RES + 3 to 5mg CUR) per mice from day 7 post infection till day 10 post infection.

Example 2: Preparation of soluble curcumin

1-10 grams of curcumin (Sigma, USA) were dissolved in 0.8-1 L of distilled ethanol at 60 degree C and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 0.5- 3% polysorbate 80 over a period of 30 min to 1 hour until the ethanol concentration became 50-40% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 1-2 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain curcumin nanoparticles (CUR) of 10-500 nm range which makes the compounds soluble in water.

Example 3: Preparation of soluble artemisinin

10-20 grams of artemisinin (Sigma, USA) were dissolved in 1-2 L of distilled ethanol at 60 degree C and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 1-3% polysorbate 80 over a period of 45 min to 1 hour until the ethanol concentration became 20-30% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 2-5 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain artemisinin nanoparticles (ART) of 10-500 nm range which makes the compounds soluble in water.

Example 4: Preparation of soluble gingerol 1-5 grams of gingerol (Sigma, USA) were dissolved in 3-5 L of distilled ethanol at 60 degree C and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 1- 3% polysorbate 80 over a period of 30 min to 1 hour until the ethanol concentration became 60-50% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 0.5-1 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain gingerolnanoparticles (GIN) of 10-500 nm range which makes the compounds soluble in water.

Example 5: Preparation of soluble resveratrol

20-30 grams of resveratrol (Sigma, USA) were dissolved in 1-3 L of distilled ethanol at 60 degree C and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 1-3% polysorbate 80 over a period of 30 min to 45 mins until the ethanol concentration became 20-30% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 0.5-1 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly dispersed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain resveratrol nanoparticles (RES) of 10-500 nm range which makes the compounds soluble in water.

Example 6: Preparation of soluble andrographolide

1-5 grams of Andrographolide (Sigma, USA) was dissolved in 0.5-1 L of a mixture of distilled ethanol and acetone in 9: 1 - 7:3 ratio at room temperature and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 2-3% polysorbate 80 over a period of 30 min to 1 hour until the ethanol: acetone concentration became 60-50% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 0.5-1 L of MilliQ water in an ultrasonic probe sonicator at 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain curcumin nanoparticles (CUR) of 10-500 nm range which makes the compounds soluble in water.

Example 7: Preparation of soluble allylatedchalcone 9

Allylatedchalcone9 was first synthesized in accordance to published literature (Sharma et al., 2014).

1-5 grams of curcumin (Sigma, USA) were dissolved in 1-3 L of distilled ethanol at 60 degree C and filtered to obtain a clear solution. This solution was then slowly sprayed over a required volume of Milli Q water containing 0.5- 3% polysorbate 80 over a period of 30 min to 1 hour until the ethanol concentration became 40-50% (V/V) while being stirred in a high-speed homogenizer (T 25 digital ULTRA-TURAX, IKA, USA) at 12,000-15,000 RPM. The precipitated material was filtered from the slurry and homogenized after suspending in 1-2 L of MilliQ water in an ultrasonic probe sonicatorat 30 KHz for 20 to 30 mins with 9 sec pulse till the material is uniformly disperesed. The suspension was then lyophilized at -55 degree C in Alpha 1-2LD plus Freeze dryer to obtain allylatedchalconenanoparticles (AC9) of 10-500 nm range which makes the compounds soluble in water.

Example 8: Parasitemia and survivability

Blood parasitemia was determined from smears prepared from tail blood followed by fixation in absolute methanol for 1 minute, Acridine Orange staining and observation in a fluorescent microscope under 60X magnification . Percentage parasitemia in the blood was calculated according to the formula:

[(Number of infected erythrocytes) / (Total number of erythrocytes) X 100%].

The mice were daily monitored for determining the period of survival. Example 9: Blood Brain Barrier permeability

Blood Brain Barrier permeability was checked by Evan’s Blue Dye extravasations. On day 10-12 post infection, all experimental mice were anesthetized and given intravenous injection with 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 .

Example 10: Quantitative real time PCR analysis

Brains were harvested from mice from G1-G3 on day 10-12 post infection. RNA was extracted from brain samples using TRI reagent and 1 pg of RNA was taken for reverse transription 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. GAPDH was kept as the endogenous control and relative fold expression was calculated by 2 ^-AACT method.

Example 11: Serum cytokine Analysis by ELISA

Cytokine levels were measured in serum samples collected on day 10-12 post infection from mice from Groups G1-G3, using DuoSet mouse ELISA kits (R&D systems) as per the manufacturer’s protocol. Biotinylated Detection Antibody and Avidin-HRP conjugate were used with TMB liquid substrate 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 IFN gamma, TNF alpha, IL 12, IL 6, IL 4 and IL 10 in the serum.

Example 12: FACS analysis of splenocytes

Spleens were collected on day 10-12 post infection from the Groups G1-G3. Splenocytes were isolated from the spleen and subjected to erythrocyte lysis and blocked with blocking buffer (30% FBS in PBS) for 30 minutes at 4 degree C. The cells were then washed with 2 ml PBS, counted and split into respective tubes keeping the count to IX 10 ⁶ cells per tube. Surface staining for each sample from each group was done with a cocktail of anti mouse CD3-FITC conjugated antibodies and anti mouse CD4-PB conjugated antibodies. For Treg set, the samples were additionally stained with anti mouse CD25-APC-Cy7 conjugated antibodies. Following surface staining, the samples were then permeabilized and intracellular staining for the different sets was done using anti mouse Tbet-APC conjugated antibodies, anti mouse GATA3-APC conjugated antibodies, and anti mouse FoxP3-APC conjugated antibodies respectively. Following intracellular staining, the cells were fixed with 1% paraformaldehyde and stored in dark at 4 degree C. FACS analysis was done in FACS Canto II machine.

Bioactivity analysis:

Example 13: Characterization of prepared compounds

The similarity ofartemesinin, allylatedchalcone 9, andrographolide, gingerol, resveratrol and natural curcumin with their corresponding soluble forms as determined by their bioactivity and FTIR spectra demonstrate that the soluble forms of the prepared compounds are unaltered in their chemical structure (Fig 1).

Example 14: Parasitemia and survivability

C57BL/6 mice that were infected with P. berghei ANKA and left untreated (G2) manifested neurological symptoms like ataxia, labored breathing, convulsions, hind leg paralysis and coma that ultimately resulted in death. The median survival period was 10 days and the average parasitemia at the time of death was 20 % (Fig 2a, 2b). Mice receiving combination treatment (G3, Combination F) did not develop any neurological symptoms and remained devoid of parasites till day 28 around when recrudescence was observed. A second dose at this time controlled the parasitemia completely and prevented further recrudescence (Fig 2b). All the treated animals went on to survive for 120 days, after which observation was discontinued (Fig 2a).

Example 15: Blood Brain Barrier permeability

One of the characteristic features of the inflammatory responses in ECM is the breakdown of the Blood Brain Barrier which is determined by the extent of Evans Blue percolation into the brain tissue. Evan’s Blue has a high affinity to bind to albumin in the serum. Under normal circumstances the BBB only selectively allows certain small molecules and gases to exchange into the brain tissue while preventing the rest. 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 crosses into the surrounding brain tissue. From the differential staining of Evans Blue staining of brains of untreated (G2) and Combination treated mice (G3, Combination F) that had been infected with P. berghei ANKA, it was evident that treatment protected the Blood Brain Barrier integrity and thus prevented the neurological damage elicited from the development of ECM (Fig 2c). The extent of extravasation as determined by quantitation of Evans Blue from the brain tissue showed that in infected untreated (G2) brains there was significant increase in the percolation of Evans Blue compared to uninfected brains (Gl) and Combination treated (G3, Combination F) brains were similar to uninfected brain in its profile (Fig 2d).

Example 16: Quantitative Real time PCR analysis

Experimental Cerebral Malaria 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. Key factors that play a role in the process involve 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. qPCR analysis indicated an increase in the expression of pro-inflammatory cytokines like IFN gamma (Fig 3a), MIP 1 beta (Fig 3b), IE 12 (Fig 3c) and IL 1 beta (Fig 3d) in the brain of infected untreated animals. The increase in the expression of pro-inflammatory cytokines was also accompanied with an increase in the level of expression the chemokine receptors CCR7 (Fig 3e) and CXCR3 (Fig 3f), of the chemokine CXCL10 (Fig 3g), and cell adhesion molecule ICAM 1 in brain (Fig. 3h). There was an increase in the expression of both co-inhibitory molecules PD1 (Fig 3i), PDL1 (Fig 3j), PDL2 (Fig 3k), and CTLA4 (Fig. 31) and co-stimulatory molecules CD40 (Fig 3m), CD80 (Fig 3o), CD86 (Fig. 3p) in the brain of untreated animals whereas there was no significant change in the expression levels of CD40L (Fig. 3n). Treatment of P. berghei ANKA infected mice with combination therapy F significantly decreased the expression of IFN gamma (Fig 3a), MIP 1 beta (Fig 3b), IL 12 (Fig 3c), IL 1 beta (Fig 3d), CCR7 (Fig 3e), CXCR3 (Fig 3f), CXCL10 (Fig 3g), ICAM 1 (Fig 3h), PD1 (Fig 3i), PDL1 (Fig 3j), PDL2 (Fig 3k), CTLA4 (Fig 31), CD40 (Fig 3m) , CD80 (Fig 3o), CD86 (Fig 3p), in the brain.

Example 17: Serum cytokine Analysis by ELISA The mean levels of IFN gamma (Fig 4a), TNF alpha (Fig 4b), IL 12 (Fig 4c), IL 6 (Fig 4d), IL 4 (Fig 4e) and IL 10 (Fig 4f) in the serum of uninfected (Gl) mice were 71.5, 39.5, 91.0, 28.5, 11.5 and 79.0 pg/ml respectively. The mean levels of IFN gamma (Fig 4a), TNF alpha (Fig 4b), IL 12 (Fig 4c), IL 6 (Fig 4d), IL 4 (Fig 4e) and IL 10 (Fig 4f) in the serum of untreated (G2) mice were 1180.0, 474.5, 1118.5, 487.5, 27.0 and 260.5 pg/ml respectively. The mean levels of IFN gamma (Fig 4a), TNF alpha (Fig 4b), IL 12 (Fig 4c), IL 6 (Fig 4d), IL 4 (Fig 4e) and IL 10 (Fig 4f) in the serum of Combination treated (G3, Combination F) mice were 110.5, 41.0, 124.5, 28.5, 39.5 and 189.5 pg/ml respectively. Infection with P. berghei ANKA resulted in significant increase of the pro inflammatory cytokines IFN gamma, TNF alpha, IL 12, and IL 6 in the blood. There was also a significant increase in the anti inflammatory cytokine IL 10 however the increase in the levels of pro inflammatory cytokines were far greater than that of the anti inflammatory IL 10. Therefore, the slight increase observed in IL 10 levels of P. berghei ANKA infected untreated mice was possibly because of a measure to control the excessive inflammation. Combination treatment significantly lowered the levels of the pro inflammatory cytokines whereas it significantly increased the levels of IL 4 and IL 10 in the serum. Thus treatment with Combination lowered the extent of systemic inflammation in P. berghei ANKA infected C57BL/6 mice.

Example 18: FACS analysis of splenocytes

In the uninfected group (Gl), 23.3% of CD3+CD4+ T cells in the spleen expressed Tbet whereas the percentage of CD3+CD4+Tbet+ T cells increased to 38.6% in the infected untreated group (G2) on day 10-12 post infection (Fig. 5a). The percentage of CD3+CD4+GATA3+ T cells (Fig. 5b) in the spleen decreased to 20.8% in infected untreated group (G2) on day 10-12 post infection as compared to 30.4% in uninfected group (Gl). Therefore, Flow cytometric analysis indicated a change in the Thl/Th2 axis of infected untreated animals (G2) on day 10-12 post infection in comparison to uninfected animals (Gl) with the response in the case of infected untreated animals being more Thl skewed as evidenced by a increased expression of Tbet and lowered expression of GATA3 in CD3 ⁺ CD4 ⁺ T cells isolated from the spleen (Fig. 5B). The expression of CD3+CD4+CD25+Foxp3 T cells (Fig. 5c) in the spleen of infected untreated (G2) animals on day 10-12 post infection was 5.0% in comparison to 8.9% in the uninfected animals (Gl). In contrast, the percentage of CD3+CD4+Tbet+ T cells, CD3+CD4+GATA3+ T cells and CD3+CD4+CD25+Foxp3 T cells on day 10-12 post infection in the spleen was 23.9% (Fig 5a), 29.9% (Fig 5b) and 9.8% (Fig 5c) respectively in the Combination treated group (G3, Combination F).