AU2021105036A4 - Dietary flavonoids as immunity enhancer against hiv - Google Patents

Abstract

The present invention relates to Natural formulation for the modulation of immune system of HIV infected patients and a process of preparation herewith are provided. The composition includes hydromethanolic extract of at least one plant or total listed plants selected from Beta vulgaris, Limonia acidissima, Couropita guianensis, Curcuma amada, Ammannia baccifera.

Description

DIETARY FLAVONOIDS AS IMMUNITY ENHANCER AGAINST HIV FIELD

[0001] The present invention relates to a to a composition of natural ingredients for the modulation of immune system of HIV infected patients and a process of preparation of the said composition.

BACKGROUND

[0002] The human immunodeficiency virus (HIV) infection has attracted maximum attention of the scientific community with an associated interest in finding remedial measures for the prevention and management of Acquired Immuno-Deficiency Syndrome (AIDS). The recent advancements in basic immunology aided the scientific community to examine functioning of the immune system and the causes of its failure. The development of monoclonal antibody technology led to the characterization of large number of surface receptor molecules on leukocytes which permit tracking disease progression. HIV (AIDS) is one of the most important clinical challenges for the medical sciences in terms of prevention and management.

[0003] The difficulties experienced because of the viral disease are complex including social, medicinal and ethical aspects. HIV is a genuine worldwide pandemic with contaminations reported from each side of the world. Without the moderate and available demonstrative offices, the genuine worldwide viral frequency is not known. In 2004, 52 million individuals were living with HIV/AIDS around the world, predominance rate was 1.4 %, around 5.1 million got to be tainted and almost 2.5 million individuals kicked the bucket of the contamination. The high rates of casualty together with the absence of suitable treatment or accessibility of a viable antibody by and large bargained the personal satisfaction making HIV/AIDS a genuine worldwide wellbeing issue. The human immunodeficiency infection (HIV) tainted people bit by bit show declining pattern of both cell and humeral safety, breaking down different elements of imperative organs especially reticulo-endothelial framework, sensory system and kidney function.

[0004] The prime cause of immune defect in AIDS is the disfunction of the thymus derived lymphocytes (T-cells), described by the vicinity of the CD4 surface atoms which are the cell receptors for HIV. AIDS is characterized by cellular immunodeficiency and the time from infection to onset of the disease progression is approximately 7-10 years.

[0005] HIV that has a place with the group of human retrovirus and the subfamily of Lenti-infections is the etiologic agents of AIDS. HIV is transmitted by sexual contact, blood, blood items or by contaminated mother to newborn child. In the beginning, the patients are asymptomatic. Due course of time, depending upon immunity of the individual subject and seriousness of the disease, a disorder of clinical side effects are showed including contaminations like tuberculosis, Cryptococcus, pneumonia, and so forth. In the dormant time of the viral disease, practical variations from the norm of the lymphocytes are showed. As an aftereffect of wide variety in host variables including HLA and differential anti viral immune response, the clinical aspects and the rate of disease goes significantly altogether.

[0006] Psycho-neuro-endocrine indication because of HIV infection is common among the people. Due course of time HIV could bring about peripheral neuropathy and sub-acut encephalitis including dementia complexity. Clinically sub-acute encephalitis is described by poor memory, inability to think, psychomotor disorder, focal motor abnormalities and behavioral changes.

[0007] At present accessible anti-retroviral treatment (ART) has a very limited role in expanding future and enhancing the personal satisfaction in serology subjects. The costliest therapy to anti-retroviral treatment, and unsatisfactory levels of side effects, the treatment couldn't get more wider acceptance by the medical scientist in the world. In the creating nations, reverse transcriptase inhibitors that repress the in vivo multiplication of spread of irresistible infection have been in wide utilize. Without a assured/concrete or effective medication, immunorestorative treatment is a meaningful and superior healing measure. It is in this way supported to propose a polynatural drug that is very much well tolerated and free from side effects and/or reactions.

[0008] Rasayana treatment is one of the major clinical aspects in Natural system of Medicine. The primary object of rasayana treatment is to advance general body immunity which is useful in anticipation of ailment and early rot of the body. In all traditional Natural messages a few medications are endorsed demonstrating rasayana property. The rasayana is not particular treatment for ailment conditions rather it has a helpful property fit for increasing so as to keep the movement of disease the body resistance. Taking lead from ancient world we have screened expansive number of restorative medicinal plants and chosen five plants indicating rasayana property to keep the movement of T lymphocytes pulverization created by HIV disease.

[0009] Hence, we felt a need to provide a natural formulation for the prevention and management of abnormal immune profile in HIV infected patients with the following objectives.

[00010] The following prior art is being reported:

[00011] IN202041018869 The invention relates to a plant based antiviral drug or composition for the treatment of HIV disease and HIV related Acquired Immunodeficiency Syndrome (AIDS), Severe respiratory distress associated Severe Acute Respiratory Syndrome (SARS) & Corona viral diseases (COVID-19/ SARS-CoV 2). The invention particularly relates to a process for preparing an injectable antiviral composition or drug which is plant based for the treatment of HIV disease and HIV related Acquired Immuno Deficiency Syndrome (AIDS), Severe respiratory distress associated Severe Acute Respiratory Syndrome (SARS) & Corona viral diseases (COVID-19). It specifically relates to the herbal composition prepared from the purified extract of holy basil for the treatment of HIV disease and HIV related Acquired Immunodeficiency Syndrome (AIDS), Severe respiratory distress associated Severe Acute Respiratory Syndrome (SARS) & Corona viral diseases (COVID-19). It also relates to the treatment of the infections caused by HIV, SARS virus & Corona virus. Further, the composition of the present invention is effective in relieving symptoms, curing and eliminating entire HIV from the body of the HIV of AIDS affected person as well as SARS & COVID-19 affected person.

[00012] KR1020090125879 An anti-HIV composition containing flavonoid compound is provided to ensure suppression activities of HIV reverse transcriptase and HIV protease. An anti-HIV(human immunodeficiency virus) composition contains apigenin, scutellarein, kaempferol or myricetin as an active ingredient. The apigenin is isolated from a plant such as parsley, artichoke, basil, salary, chamomile or citrus. The scutellarein is isolated from a plant such as Scutellaria L. or Sorbifolia. The Kaempferol is isolated from a plant such as apple, Allium tuberosum, Polygonatum odoratum var., Artemisia princeps var., onion, grape or citrus.

[00013] AU2002013528 This invention relates to compositions derived from Chinese herbal medicines, medicinal plants and extracts thereof, and to their use for the treatment of animals infected with viruses, especially with hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV). More specifically, the compositions of the present invention are derived from various Chinese herbal medicines or medicinal plants which have a long history of human consumption.

[00014] WO2012176215The invention relates to a plant based antiviral composition and more particularly to a process for preparing an injectable antiviral composition comprising holy basil for the treatment of HIV disease and HIV related Acquired Immunodeficiency Syndrome (AIDS). The process comprising boiling the holy basil leaves in distilled water, filtering the boiled mixture, adding strong sulfuric acid to precipitate the vegetable matter, siphoning off the clear solution, adding ammonium carbonate for crystallization of the clear solution, adding spirit of ammonia to decolorize the crystals, and dissolving the decolorized crystals in boiling ethyl alcohol for purification to obtain the final composition. The final composition of the present invention destroys surface antigens as well as HIV Genome by invading into the virus and there by killing HIV

SUMMARY

[00015] The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the present invention. It is not intended to identify the key/critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some concept of the invention in a simplified form as a prelude to a more detailed description of the invention presented later

[00016] The present invention is generally directed to According to providing a novel natural formulation for the modulation of immune system of HIV infected patients and a process of preparation thereof, comprising

[00017] An embodiment of the present invention is preparing a hydromethanolic extract of at least one plant selected from Beta vulgaris, Limonia acidissima, Couropita guianensis, Curcuma amada, Ammannia Baccifera at 80-90° C., maintaining the pH of the solution between 6-7

[00018] It is an embodiment of the present invention is isolating the active compounds chromatographically and subjecting the active separated compounds to the step of molecular characterization.

[00019] An embodiment of the present disclosure is Moreover, as per this invention there is a process for the preparation of novel plant based Natural formulation as proclaimed in claim-I comprising of preparing aqueous adding methanolic extract of Beta vulgaris(whole tuber), Limonia acidissima(pulp), Couropita guianensis (leaves), Curcuma amada (rhizome), Ammannia Baccifera(leaves), by using aqueous and methanol (50:50)ratio at 80°-90° C. temp. and maintaining the pH of solution between 6-7, separating the active compound (bioactive guided fractionation system) chromatographically of each plant material (extract) by using TLC, UPLC and HPTLC separation of the molecules of plant extract by using GCMS, LCMS and 2D NMR.

BRIEF DESCRIPTION OF DRAWING

[00020] Figure 1. flow diagram of the whole process.

DESCRIPTION OF EMBODIMENTS

[00021] The following description is of exemplary embodiments only and is not intended to limit the scope, applicability or configuration of the invention in any way. Rather, the following description provides a convenient illustration for implementing exemplary embodiments of the invention. Various changes to the described embodiments may be made in the function and arrangement of the elements described without departing from the scope of the invention.

[00022] Beta vulgaris (beet) is a plant in the Amaranthaceae family has many cultivated varieties, the most well known of which is known as the beetroot or garden beet used as vegetables. Among the cultivated varieties which include the leaf vegetable chard;the sugar beet, used to produce table sugar; which is known as fodder crop. Three subspecies are typically recognised. Almost every cultivated varieties or species fall into the subspecies Beta vulgaris. The roots are most commonly deep red-purple in colour, red and-white striped roots.Beta vulgaris is a herbal tuborrarely, perennial plant with leafy stems growing to 1-2 m tall. The leaves are greenish about 5-20 cm long on wild plants (often much larger in cultivated plants). The flowers are very small, 3-4 mm diameter, green or tinged reddish, with five petals; they are wind pollinated. The tuber have shown medicinal property. Beta vulgaris is a rich source of flavonoids, vitamins, proteins, amino acids, folic acid, phytosterol, alpha-tocopherol and phenolic compounds. There are at least 21 chemical elements present in this planteg. anthocyanin, isobetaninand neobetanin, betacyani,indicaxanthin,vulgaxanthins,probetanin,betaxanthins,Indicaxanthin,thalassemia, alpha-tocopherol ,nitrogen, phosphorous, Quercetinand others. It has shown anti-oxidant, immuno-modulatory, anti-inflammatory effects and uplifts the mental function.

[00023] Limonia acidissimafamilybelongs to Rutaceae is a large tree growing to 9 metres (32 ft) tall, with rough, spiny bark. The leaves are pinnate, with 5-8 leaflets, each leaflet 25-37 mm long and 10-23 mm broad, with a citrus-scent when crushed. The fruit is a berry 5-8 cm diameter, and may be sweet or sour. It has a very hard carp which can be difficult to crack open, and contains sticky brown pulp and small white seeds. Flavonoids mainly are isolated from Limonia acidissima. This has shown a significant immuno- potentiating activity both in experimental as well as in in-vitro and in-vivo studies. This plant has been included due to potent anti-modulatory activity found useful in enhancing both cellular and humeral immunity. Quercetin Pachypodol, Rhamnazin are the main dietary flavonoids chemically present in the pulp of Limonia acidissima.

[00024] Couroupitaguianensis ,Family belongs to Lecythidaceae grows up to 37 m (meters) in height. The designed fleshy leaves vary in length, generally from 7 to 32centimetres, but reaching up to 59. The flowers are born in large bunches up to 82m(meters) long. Some trees ara covered with flowers profusely, until the entire trunk is hided with the flowers. One tree can bear 900-1000 flowers per day. They are very much strongly scented, especially at night and in the early morning. They are large, up to 5 7centimetres wide, and often brightly coloured, the six petals in shades of pink and red near the bases and yellowish toward the tips. Kaempferol-3-glucoside,a-amirin, p amirin,p-sitosterol,tannins, ketosteroids,kaempferol, 3,4-dihydroxycinnamic acid are the major active chemical constituents found in this plant showing pharmacological activity. This plant has shown Anti-anxiety, anti-depressant, hypotensive, immuno-modulatory, anti-oxidant and anti-inflammatory activity. It enhances mental competence.

[00025] Curcuma amada (mango ginger) is a plant of the ginger (family Zingiberaceae)and is closely related to turmeric or curcumin. The rhizomes are very similar to ginger but have a raw mango taste. They are used in making pickles in south India. The structure and texture of the species is a subject of some confusion as some authorities have considered the name C. mangga as identical while others describe it as a distinct species with C. mangga being found in southern India while C. amada is of east Indian origin. Curcuma mangga extracts have shown cytotoxic activities on the human cancer cell lines MCF-7 (a hormone-dependent breast cell line), KB (a nasopharyngeal epidermoid cell line), A-549 ( lung cell line), cervical cell line, and HT-29 (colon cell line). The extracts showed no cytotoxicity against the non-cancerous human fibroblast cell line MRC-5. The whole plant contains sitosterol and carotene. Other compounds like Vitamin C, cartone, palmitic acid, triterpenoides, Myricetin, alkaloids, ergonovine and ergonovinine etc. are also present. Both HA litre DTH response indicated the Curcuma amada potentiates humoral as well as the cellular immunity. The presence of humoral response was evidenced by an enhancement of antibody in response to SRBC in mice of consequence of both pre and post Immunization protein treatment indicates the enhanced responsiveness of macrophages and B-lymphocytes, subsets involved in antibody syntheses.

[00026] Ammannia baccifera, belongs to the family Lythraceae is widespread in the tropical regions of Asia, America and Africa. It has been naturalized in Spain. It is an annual plant and herbaceous, and can be found in marshes, swamps, rice fields, water courses at low elevations. It is plentily available in India but considered endangered species in Israel, but because it is widespread and common elsewhere, the IUCN considers it to be 'Least Concern'. The plant Ammannia baccifera Linn. is erect, branched, smooth, slender, annual, more or less purplish herb 12 to 52centimeters in height. The stems are somewhat 4-angled. The leaves are oblong or narrowly elliptic, about 3.7centimeters long - those on the branches very numerous, small, and 1 to 1.7centimeters long - with narrowed base and pointed or somewhat rounded tip. The flowers are very small, about 1.2 millimeters long, greenish or purplish, and bome in dense axillary clusters. The capsules are nearly spherical, depressed, about 1.2 millimeters in diameter, purple, and irregularly circumscribes above the middle.Aqueous extract from leaves showed both humeral and cell mediated immune-response in rats and mice, it is an immunomodulator. Fisetin and Pyranoflavonols are the main chemical constituents present in its leaves.

[00027] The hydro-methanolic extract of five Natural plants i.e. Beta vulgaris, Limonia acidissima, Couropita guianensis, Curcuma amada and Ammannia Baccifera by using 50% water and 50% methanol was prepared for the development of present natural formulation by conducting various tests and clinical studies. The water used for extraction was disinfected for any type of bacterial or unusual growth by using reverse osmosis plant. After finishing extraction procedure the final extracted materials was taken to determine the presence of percentage of active molecules in all the five selected plants and were identified by UPLC, HPTLC, GCMS, LCMS and 2DNMR procedures.

[00028] The biological activity was evaluated by conducting various experimental animal models of immune injury of single plant extract as well as combined formulation acting on various targets responsible for immune deficiency through different mode of actions.

[00029] The interaction between chemical constituents and biological markers like Ig, IgM, IgA, CD4, CD8, Total white cell count, neutrophils, lymphocytes, eosinophils and haemoprotein together with inflammatory cytokines and oxidative stress markers were evaluated and mode of drug action was established through such studies. Before victimisation the formulation for human consumption the pre-clinical acute, sub-acute and chronic toxicity studies were allotted to attain safety profile. Further, the immunomodulatory activity, CD4 amelioratory and stabilizing effects were determined in animal studies. The mechanism of action of single and combined formulation was established through numerous mechanism based mostly studies. equally the effective dose of every plant extract material made up our minds through action on totally different targets (bio-markers)involved the disease condition.

[00030] Extraction Procedure: The dried tuber of Beta vulgaris, pulp of Limonia acidissima, dried leaves of Couropitaguianensis, dried rhizome of Curcuma amada, dried leaves of Ammannia baccifera, were utilized to obtain extracted material of the plants. The water and methanol 50:50 ratio was utilized for the extraction. After extraction the extracted material was taken for identification and separation of active compound present in the extract of the plants by using TLC, HPLC, HPTLC, GCMS, and LCMS. Afterwards the molecular characterization was carried out by using IR and NMR.

[00031] The extraction was done at the temperature of 80-90°C. The pH of the solution was maintained between 6-7. The steps which were adopted and carried out to isolate the active compound, preparation of test drug as well as to develop a final new drug have been illustrated in FIG. 1.

[00032] As per this invention there is an Natural formulation provided for the prevention and management of deficiency of immune profile among diagnosed HIV infected patients, with the object to improve their quality of life, to prolong the longevity and to prevent them from opportunistic infections particularly tuberculosis and pneumonia. The present test formulation comprising of the following five ingredients

[00033] Name of the Plants Parts used

[00034] Beta vulgaris (whole tuber)

[00035] Limonia acidissima (pulp)

[00036] Couropitaguianensis (leaves)

[00037] Curcuma amada (rhizome)

[00038] Ammannia Baccifera (leaves)

[00039] Preferably the aforesaid plants are present in the test drug in the following range of doses

[00040] Beta vulgaris 100-300 mg/day

[00041] Limonia acidissima 100-400 mg/day

[00042] Couropitaguianensis 150-400 mg/day

[00043] Curcuma amada 125-400 mg/day

[00044] Ammannia Baccifera 75-300 mg/day

[00045] The formulation may also comprise known additives such as minerals, vitamins, salts filler (for capsulation or to prepare syrup) and binders, if required to present in trace amount.

[00046] Thus any known additive or supplement is added to prepare the final formulation as required and present in trace amount. Reference is made here in capsule form. However, it would be apparent that the preparation may also be in the form of syrup/tablet.

[00047] Preferably but without implying any limitation the preparation (formulation) comprises:

Name of the plants Dose

1. Beta vulgaris 175 mg/day

2. Limonia acidissima 150 mg/day

3. Couropitaguianensis 250 mg/day

4. Curcuma amada 225 mg/day

5. Ammannia baccifera 150 mg/day

[00048] The present Natural formulation is prepared out of five plant extracts namelyBeta vulgaris, Limonia acidissima,Couropitaguianensis, Curcuma amada, Ammannia baccifera that are mixed in effective doses. The beneficial role of present test formulation is through its immunomodulatory activity as it enhances immunity against a pathogen by activating the immune system. HIV belongs to the Lentivirinae subfamily of retrovirus which has an RNA genome. The RNA genome is encapsulated with a core which in turn is enwrapped by an envelope. The virus gains entry to the target cells by binding to the CD4 receptor using the viral surface membrane glycoprotein GP120. The CD4 receptor is present predominantly on T-helper lymphocytes which are the major target for the virus. Thus HIV principally infects CD4 helper T-lymphocytes. The cells are responsible for the initiation and maintenance of the immune responses to pathogens. Following the viral infection there is attrition in the CD4 cell population resulting in gradual dysfunction of the cellular immunity. Further, HIV also affects cells of the monocyte/macrophage lineage, and dendritic cells.

[00049] During the course of HIV infection there is gradual reduction in the number of CD4 cells and this phenomenon serves as a prognostic marker indicating the progression as well as classification of the disease state. CD4 cell count is also used to determine when the anti-retroviral or a microbial therapy should be instituted. The estimation of the serum immunoglobulin levels is a direct measure to detect thehumoral immunity. Serum immunoglobulin refers to a group of serum molecules produced by plasma cells, they are soluble and counter the invasion of a pathogen. It has been demonstrated that the active constituents of the plant extracts could improve prognosis in the viral infection by means of immunomodulatory activities as well as anti-microbial, anti-inflammatory, anti-viral and anti-oxidant properties. Further, it is proposed that the present polynatural formulation caused specific activation of T-lymphocytes, phagocytic cells as well as elevation in cytokine levels including gamma-interferon and tumor necrosis factor (TNF). Thus the mechanism of action of the present polynatural formulation seems to be through activating the cell mediated immune system.

[00050] The humeral immunity is mediated by antibodies produced by plasma cells aided by CD4+ T-helper 2 (Th2) cells. Th2 activation and cytokine production, germinal centre formation and isotype switching affinity, maturation and memory cell generation come under T-help. Antibodies mediate pathogen or toxin neutralization, complement activation and opsonin promotion all contributing to pathogen elimination. The present polynatural formulation has shown improvement in T-lymphocyte functioning and stalling of the kinetics of CD4+ cell reduction. Immune dysfunction leads to disease progression in HIV seropositive subjects. Through immunomodulatory properties, the polynatural formulation enhanced the concentrations of IgG, IgM and IgA as well as the numbers of total white blood cells, neutrophils, and lymphocytes, further increased the levels of hemoglobin and total serum protein.

[00051] The viral infection may produce a variety of neurologic manifestations due to opportunistic infections. Monocyte macrophage lineage is predominantly infected. HIV infected individual may manifest both white matter legion as well as neuronal loss. A series of changes take place due to neurotoxicity of gp120, TNF-a, IL-1, IL-2, IL-6, TGF p and endotheline. We concur that polynatural formulation could delay the process of cell apoptosis in the neurons. The loss of cholinergic and glutamatergic receptors have shown that early action may prevent the deterioration of cognitive function due to prevention of decline in glutamatergic and cholinergic neurons.

[00052] During the latency period the protection of vital organs like brain, kidney and liver is essential. The structural damage of brain particularly limbic system can be prevented by the drugs which can increase the neural capability to combat T-cell and B cell deficiency in HIV infected cases. The present test formulation has potentiality to fulfil above objects to a great extent.

EXAMPLES

[00053] The innovations/invention will be described but not herewith limited/constrained by the the given samples. Those talented work or invention perceive that different modifications can be made to the creation without leaving from the spirit and scope thereof.

EXAMPLE 1

[00054] When the hydro-methanolic extract of Beta vulgaris in the dose of 45 mg/kg, Limonia acidissima40 mg/kg, Couropitaguianensis 50 mg/kg and Ammania baccifera 50 mg/kg mixed and given to experimental animals showed activated T-cell response indicating an enhancement of antigenic potency and stimulated lymphocyte proliferative response. Thus the above combined formulation exerted cell mediated immune responsiveness.

EXAMPLE 2

[00055] When the hydro-methanolic extract of Limonia acidissimain the dose of 45 mg/kg, Couropitaguianensis 60mg/kg and Beta vulgaris 70 mg/kg mixed and given to immobilized stressed model animals the IgG, IgM and IgA immune responsive markers modulated in treated group than non-treatment group. A significant increase in the humeral immunity was noticed.

EXAMPLE 3

[00056] When the hydro-methanolic extract of Limonia acidissimain the dose of 30 mg/kg, Couropitaguianensis 45 mg/kg, Beta Vulgaris60 mg/kg mixed and given to sleep deprivation stressed animals showing poor learning with deteriorated immunity, a reduced number of error in completing learning task were recorded in treated group of animals in comparison to non treatment control group where animals were induced only stress and no treatment. Thus the test drug has neuromodulatory potentials as it enhanced protein synthesis of neurons, modulated the cholinergic, glutamatergic, GABAergic, nor adrenergic and dopaminergic receptors.

EXAMPLE 4

[00057] After determination of safety and efficacy profile of single plant candidate as well as combined formulation in pre-clinical studies the formulation containing hydro methanolic extract of selected ingredients in effective doses were evaluated in HIV infected patients for modulation of immune profile. When the hydro-methanolic extract of Beta vulgaris in the dose of 325 mg/day, Ammania baccifera 200 mg/day, Couropita guianensis 250 mg/day and Curcuma amada 175 mg/day mixed and given to diagnosed HIV infected patients, improvement in decline in T-lymphocytes are observed. Further, reduced rate of decline of CD4 cell count and decrease in CD8 is also recorded. The continuous administration of drug indicated the stabilizing of CD4 cell count with decrease CD8 cell indicated arrest of progression of disease process.

EXAMPLE 5

[00058] When the hydro-methanolic extract of Beta vulgaris in the dose of 325 mg/day, Ammania baccifera 275 mg/day,Curcumaamada 325 mg/day mixed and given to HIV infected patients a significant increase in RBC, platelet count and hemoglobin level. The test formulation has shown potentiality to prevent neutropenia and thrombocytopenia in HIV infected patients. Thus the test formulation is an effective immuno-stimulant as IgG, IgM levels increased to a significant extent in treated group.

EXAMPLE 6

[00059] When the hydro-methanolic extract of Beta vulgaris 350 mg/day, Curcuma amada 225 mg/day and Ammania baccifera in the dose of 375 mg/day mixed and given to HIV infected patients liver function and renal function improved and continuous application of the drug protected the HIV patients from the development of hepatitis and renal diseases particularly improved the micro-albuminuria, and reduced SGOT, SGPT along with alkaline phosphatase.

EXAMPLE 7

[00060] The hydro-methanolic extract of Beta vulgaris in the dose of 275 mg/day, Limonia acidissima 225 mg/day,Couropitaguianensis 325 mg/day mixed and given in the form of combined formulation to HIV patients, improvement in cognitive function including memory performance was observed. Improvement in depression level and sleep pattern of HIV patients was also determined.

EXAMPLE 8

[00061] The HIV infection causes persistence chronic inflammation. Severe oxidative stress has been reported in HIV infected patients. The hydro-methanolic extract of Beta vulgaris in the dose of 350 mg/day, Couropitaguianensis 325 mg/day, Curcuma amada 225 mg/day mixed and given to HIV infected patients the elevated homocystein and inflammatory cytokines IL- 6 and TNF-a reduced significantly. The plant Beta vulgaris is rich in containing folic acid, B1, B 12 and other micronutrients therefore improved body resistance and feeling of well being is reported by HIV patients. It is proposed that the risk of CHD and neurodegenerative disorders was minimized by test formulation administration in HIV infected patients.

EXAMPLE 9

[00062] The hydro-methanolic extract of Beta vulgaris in the dose of 250 mg/day, Limonia acidissima 125 mg/day,Couropitaguianensis 175 mg/day, Curcuma amada 225 mg/day, Ammania baccifera 150 mg/day mixed and administered to HIV infected patients showed increase of WBC and platelet count, T-lymphocyte cells count and hemoglobin also increased to a great extent, prevented neutropenia and thrombocytopenia. The most important result obtained out of the present clinical trial is the stabilization of cluster of differentiation (CD4), cell count. Further, the retarded level of IgG, IgM and IgA also enhanced. A neuromodulatory activity of test formulation is also reported as the present test drug checked the loss of cholinergic neurons, ameliorated the nor-adrenergic, GABAergic, dompaminergic pathways, particularly the gulatmatergic receptors. The drug also exerted anti-anxiety and anti-depression effects. The test formulation has potential role in the protection from kidney and liver diseases. As the test drug stabilized the loss of CD4 cell count the onset of opportunistic infections particularly tuberculosis, pneumonia including frequent cold and cough was prevented/the duration of onset was enhanced significantly, thus the longevity of HIV patients is increased and quality of life also improved.

[00063] Continuous use of test formulation did not show any adverse reaction even after its prolonged application.

Conventional treatment includes:

[00064] Becosule (1 capsule once in a day) ascorbic acid (VIT C)+biotin+ Calcium Pantothenate + Cyanocobalamin (Vit B12) + Folic Acid + Niacinamide (Nicotinamide/

Vit B3)+ Pyridoxine Hydrochloride (VitB6)+ Riboflavin(VIT B2)+Thiamine Hydrochloride (Vit BI)

[00065] Ferrochelate-Z (1 capsule once in a day) elemental iron 100 mg and folic acid 1.5 mg.

[00066] Kombina (Once Tablet twice in a day) Sulfamethoxazole (400mg)

+ Trimethoprim (80mg)

[00067] Supplement of protein according to the serum protein level

[00068] The conventional treatment was given as per standard schedule. The test drug was given continuously. The long-term follow-up study following test formulation treatment among HIV infected patients shows that the CD4 cell count following test formulation treatment considerably stabilized.

[00069] TABLE 1: Slowing down of the process of decline of CD4 cell count following test formulation treatment in HIV positive patients.

Treatment No. of CD4 1" Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases (cells/pL) Initials Conventional 45 467.85 ± 406.85 373.65 321.45 283.55 235.82 Treatment 130.67 92.25 73.46 52.43 44.85 120.32 Conventional 56 447.45 ± 418.65 396.65 382.90 365.32 342.85 Treatment + 128.42 93.90 62.75 71.76 55.80 63.35 Test Formulation

[00070] TABLE 2: Table shows the less increase in elevation of CD8 following test drug treatment in HIV positive patients.

Treatment No. of CD8 14Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases (cells/pL) Initials Conventional 45 538.95 ± 575.85 727.39 915.85 985.45 1010.38 Treatment 58.47 ±65.44 ±57.89 ±77.47 ±85.35 ±115.45 Conventional 56 519.76 ± 545.45 617.36 646.75 675.04 715.45 Treatment + 61.38 85.75 75.75 67.93 85.04 94.78 Test Formulation

[00071] TABLE 3: Effect of test formulation on immunologic markers among HIV infected patients

Treatment No. of IgG(mg/dl) 14Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases Initials Conventional 45 838.75 + 730.75 625.35 615.45 582.35 522.86 Treatment 110.45 92.45 83.35 62.75 102.35 112.35 Conventional 56 812.35 + 742.35 671.45 645.35 612.47 582.45 Treatment + 116.35 ± 77.55 67.85 Test 162.38 145.35 103.45 Formulation

[00072] TABLE 4: Effect of test formulation on immunologic markers among HIV infected patients

Treatment No. of IgM 1" Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases (mg/dl) Initials Conventional 45 192.53 ± 162.57 132.65 110.55 87.35 ± 35.65± Treatment 63.35 ±62.35 ±25.83 ±30.45 25.25 12.65 Conventional 56 211.52 ± 182.87 162.35 142.52 125.65 70.54± Treatment + 85.55 25.55 23.38 36.25 37.45 22.85 Test Formulation

[00073] TABLE 5: Effect of test formulation on immunologic markers among HIV infected patients.

Treatment No. of IgA 1" Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases (mg/dl) Initials Conventional 45 207.35 ± 218.65 152.65 125.42 115.25 272.36 Treatment 50.32 ±43.34 ±46.32 ±65.70 ±32.82 ±18.85 Conventional 56 201.56 ± 165.45 166.65 142.55 115.76 97.86 Treatment + 24.85 22.65 15.55 21.45 32.38 23.34 Test Formulation

[00074] TABLE-6: Level of depression in HIV infected patients following test formulation

Treatment No. of IgA 1" Year 2 "dYear 3 rdYear 4 th Year 5 th Year Groups Cases (mg/dl) Initials Conventional 45 28.72 ± 29.45 29.25 ± 31.32 28.45 35.95 ± Treatment 6.35 5.58 5.65 6.25 5.35 7.35 Conventional 56 28.45 ± 25.35 26.35 ± 26.34 26.25 24.65 ± Treatment + 5.35 6.85 4.35 3.35 3.65 2.32 Test Formulation

[00075] In other words disease process is significantly slowed down under influence of test formulation treatment.

[00076] While considerable emphasis has been placed herein on the specific features of the preferred embodiment, it will be appreciated that many additional features can be added and that many changes can be made in the preferred embodiment without departing from the principles of the disclosure. These and other changes in the preferred embodiment of the disclosure will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.

Claims (4)

1. An immune modulator composition for HIV patients comprising of extract of: i. Beta vulgaris tuber; ii. Limonia acidissimapulp; iii. Couropitaguianensis flower petals; iv. Curcuma amada rhizome; and v. Ammannia baccifera leaf.

2. The immune modulator composition as claimed in claim 1, wherein the extract is hydro methanolic extract in ratio of 50:50.

3. The immune modulator composition as claimed in claim 1, wherein the dose required to produce immune modulation is: i. 100 to 300 mg/day for Beta vulgaris; ii. 100 to 400 mg/day for Limonia acidissima; iii. 150 to 400 mg/day for Couropita guianensis; iv. 125 to 400 mg/day for Curcuma amada; and v. 75 to 300 mg/day for Ammannia baccifera.

4. The immune modulator composition as claimed in claim 1, wherein it is combined with: i. Becosule one capsule once in a day; ii. Ferrochelate-Z one capsule once in a day; iii. Kombina One Tablet twice in a day; and iv. Supplement of protein according to the serum protein level.