Title of Invention

AYURVEDIC IMMUNO-MODULATOR COMPOSITION FOR TREATMENT OF ACQUIRED IMMUNO DEFICIENCY SYNDROME

Abstract

An ayurvedic immuno modulator composition for treatment of Acquired Immuno Deficiency Syndrome comprising Guduchi or Giloe (cordifolium) 5 mg - 2 gm Panash or ^Kathal (jack fruit) 2 mg - 5 gm Tulsi or Krishna Tulsi (Holy Basil) 5 mg - 5 gm Kuda or Kutaja (Kurchi) 2 mg - 2 gm Bhui Amla or Bahu Patra (Gooseberry) 5 mg - 2 gm Gingko Biloba 5 mg - 2 gm Shilajeet or Si la Ras (Asphaltam) 2 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm in combination with pharmaceutically acceptable excipients.

Full Text

ORIGINAL 1048/MUM/2003 Form 2 THE PATENTS ACT 1970, (39 of 1970) As amended by the Patents (Amendment) Act, 2002 COMPLETE SPECIFICATION (See Section 10; Rule 13) TITLE Ayurvedic immuno modulator composition for treatment of Acquired Immuno Deficiency Syndrome APPLICANT Bakul Jain, Shikhar Kunj, 6th Floor, 29-A, Carmichael Road, Mumbai 400026, Maharashtra, India, an Indian National The following specification particularly describes the nature of this invention and the manner in which it is to be performed: GRANTED FIELD OF INVENTION This invention relates to an ayurvedic immuno modulator composition for the treatment of Acquired Immuno Deficiency Syndrome. This invention also relates to a process for the preparation of the ayurvedic immuno-modulator composition. PRJORART Acquired Immuno Deficiency Syndrome (AIDS) was first diagnosed in the year 1981 and has since emerged as the greatest medical challenge. It is caused by infection with Human Immuno Deficiency Virus (HIV) and has achieved an epidemic form among the young adult population in most of the countries of the world. Nearly 13 million people the world over are already infected with the HIV virus and at least one third of these are expected to develop AIDS within the next few years. It is expected that by the year 2003, adults and children infected with HIV will rise to 50 - 70 million and the number of AIDS cases will increase to 20 - 25 million. India is classified by the WHO as a pattern II/III region. In pattern II areas the number of infected male and female is approximately equal and the primary mode of transmission is heterosible. Two forms of retroviruses have been demonstrated as the causative agents of AIDS in India. HIV-1 is essentially responsible for 82%, HIV-2 for 5-6% and HIV-1 and HIV-2 together for 11-12% of HIV-related infection. HIV is very liable and can only be transmitted viz direct exchange of body fluids. The major routes of spread are sexual intercourse, contact with infected blood (by transfusion or by contact with infected needles) or vertical transmission from infected mother to child. The virus consists of a core including viral RNA and the p24 antigen, surrounded by an envelope containing the glycoprotein g p 120. This virus glycoprotein binds to the CD4 antigen on the host cell allowing virus entry. Once entry has occurred the genetic material of the HIV particle is released. This consists of two copies of the virus RNA housed within the core in association with the virus enzyme reverse transcriptase (RT). Inside the cell the virus must convert its RNA genome to DNA which it does through a complex mechanism with the help of the enzyme RT. This DNA copy of the viral genome, termed proviral DNA, then integrates into the host cell genome. It is clear that the CD4 antigen is the major receptor for binding of the HIV particle, thus the predominant cell that the virus infects bear this antigen. The current information suggests that HIV - infection of CD4 cells results in severe impairment of the cellular system followed by cellular lysis and death, exemplified by the characteristic significant decline in the CD4 cell population. In addition to cell destruction of infected CD4 cells, these cells appear to be dysfunctional. Associated with CD4 cells lysis and dysfunction is a significant decline in immunological status as mainfested by the appearance of clinical signs, symptoms, opportunistic infections and neoplasms. HIV is also known to infect cells of the nonocyte/macrophage cell line and further compromise immune function. HIV also affects dendritic cell line and some cells in the central nervous system which is believed to be responsible for the neurologic manifestations. Common clinical features of HIV- in order of prevalence are weight loss, persistent generalized lymphadenopathy, chronic cough, herpes zoster, diarrhoea, recurrent fever, tuberculosis and orophyngeal candidasis. These clinical features, however, are not all diagnostic stages of HIV. At present these is no cure for HIV infection. However, with the help of drugs such as azidothymidine (AZT), dideoxyinoisine (ddl) and dideoxycytidine (ddC) it has been possible to prolong the life of an infected individual and postpone the onset of AIDS symptoms. Non-nucleoside RT inhibitors and benzodiazepines (also called TIBO compounds) are undergoing clinical trials. Drugs acting on virus replication at other stages such as TAT (a regulatory gene) inhibitors and protease inhibitors are also undergoing preliminary clinical trials. Combination therapies to improve the efficiency are also being tried out eg (AZT + ddl) or (AZT + ddC). These drugs, are however, very toxic and develop adverse reactions. They are also not well tolerated by most patients. It has been found that most patients develop resistance to the drugs. They also do not show effective vivo activity. These drugs are also very expensive. Effors are, therefore, underway to develop safe, effective and economical anti-HIV drugs. Medical opinion varies on the prospects of a vaccine against HIV infection. Some think it is in the realm of reality, others feel that the technical barriers may never be fully overcome. Even if, effective vaccine is developed within the next couple of years, technical and financial obstacles probably will limits its use. Ayurveda is a traditional Indian system of medicine based on medicinal plants, herbs and minerals. One of the therapeutic strategies in Ayurvedic medicine is to increase body's natural resistance to the disease causing agent(s). In practise, this is achieved by using extracts of various plants, materials called rasayanas (Charak Samhita 1000 BC). This concept in modern scientific understanding would mean enhancement of immune responsiveness of an organism against a pathogen by non-specifically activating the immune system using immunomodulatory agents of plant origin. It is now being recognized that immunomodulation could provide an alternative to conventional chemotherapy for a variety of diseased conditions, especially when host defence mechanisms have to be activated under the conditions of impaired immune responsiveness. Since HIV infection is characterized by progressive immuno-deficiency, there is a clear rationale for developing strategies using combination of immunomodulators and anti-retroviral, which would inhibit viral replication as well as regulate immune functions to defend against opportunistic infections. In my Indian Patent No 182157 (355/BOM/96), there is described an ayurvedic composition for the treatment of AIDS comprising the following ingredients : Guduchi (Tinospora cordifolia) (5 mg to 2 gm) Yashthimadhu (Glycrrhiza glabre) (5 mg to 2 gm) Panash (Atrocarpus integrifolia) (2 mg to 5 gm) Neem (Melia azadirachchata) (5 mg to 2 gm) Sunth (Zingiber officinale) (5 mg to 2 gm) Tulsi (Occimum sanctum) (5 mg to 5 gm) Karavella (Momordica charantia) (5 mg to 2 gm) Kutaki (Picorhiza kurroa) (2 mg to 2 gm) Kuda (Holarrhena antidysenterica) (2 mg to 2 gm) OBJECTS OF INVENTION An object of the invention is to provide an ayurvedic immuno-modulator composition for treatment of AIDS having improved immuno modulation activities. Another object of the invention is to provide an ayurvedic immuno-modulator composition for treatment of AIDS which is safe. Another object of the invention is to provide an ayurvedic immuno-modulator composition for treatment of AIDS which shows effective invitro activities in the treatment of AIDS. Another object of the invention is to provide an ayurvedic immuno-modulator composition for treatment of AIDS which is inexpensive. Another object of the invention is to provide a process for the preparation of an ayurvedic immuno-modulator composition for treatment of AIDS having improved immuno modulation activities. Another object of the invention is to provide a process for the preparation of an ayurvedic immuno-modulator composition for treatment of AIDS which is safe. Another object of the invention is to provide a process for the preparation of an ayurvedic immuno-modulator composition for treatment of AIDS which shows effective invitro activities in the treatment of AIDS. Another object of the invention is to provide a process for the preparation of an ayurvedic immuno-modulator composition which is inexpensive. iDETAILED DESCRIPTION OF INVENTION According to the invention there is provided an ayurvedic immuno-modulator composition for treatment of Acquired Immuno Deficiency Syndrome comprising Guduchi or Giloe (cordifolium) -5 mg -2 gm Panash or Kathal (jack fruit) 2 mg -5 gm Tulsi or Krishna Tulsi (Holy Basil) 5mg • -5 gm Kuda or Kutaja (Kurchi) 2mg • -2gm Bhui Amla or Bahu Patra (Gooseberry) 5 mg -2gm Gingko Biloba 5mg -2 gm Shilajeet or SilaRas (Asphaltam) 2mg -2gm Karavella or Karela (bitter gourd) 5mg- -2gm in combination with pharmaceutically acceptable excipients. Preferably, the composition comprises Guduchi or Giloe (cordifolium) 100 mg - 600 mg Panash or Kathal (jack fruit) 100 mg - 700 mg Tulsi or Krishna Tulsi (Holy Basil) 20 mg - 300 mg Kuda or Kutaja (Kurchi) 20 mg - 200 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg - 600 mg Gingko Biloba 5 mg - 2 gm Shilajeet or Sila Ras (Asphaltant) 50 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm Still preferably, the composition comprises Guduchi or Giloe (cordifolium) 250 mg Panash or Kathal (jack fruit) 75 mg Tulsi or Krishna Tulsi (Holy Basil) 60 mg Kuda or Kutaja (Kurchi) 100 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg Gingko Biloba 100 mg Shilajeet or Sila Ras (Asphaltant) 100 mg Karavella or Karela (bitter gourd) 60 mg According to the invention there is also provided a process for the preparation of an ayurvedic immuno modulator composition for treatment of Acquired Immuno Deficiency Syndrome comprising mixing Guduchi or Giloe (cordifolium) 5 mg - 2 gm Panash or Kathal (jack fruit) 2 mg - 5 gm Tulsi or Krishna Tulsi (Holy Basil) 5 mg - 5 gm Kuda or Kutaja (Kurchi) 2 mg - 2 gm Bhui Amla or Bahu Patra (Gooseberry) 5 mg - 2 gm Gingko Biloba 5 mg - 2 gm Shilajeet or Sila Ras (Asphaltam) 2 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm with pharmaceutically acceptable excipients. Preferably the process comprises mixing Guduchi or Giloe (cordifolium) 100 mg - 600 mg Panash or Kathal (jack fruit) 100 mg - 700 mg Tulsi or Krishna Tulsi (Holy Basil) 20 mg - 300 mg Kuda or Kutaja (Kurchi) 20 mg - 200 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg - 600 mg Gingko Biloba 5 mg - 2 gm Shilajeet or Sila Ras (Asphaltam) 50 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm Still preferably, the process comprises Guduchi or Giloe (cordifolium) 250 mg Panash or Kathal (jack fruit) 75 mg Tulsi or Krishna Tulsi (Holy Basil) 60 mg Kuda or Kutaja (Kurchi) 100 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg Gingko Biloba 100 mg Shilajeet or Sila Ras (Asphaltant) 100 mg Karavella or Karela (bitter gourd) 60 mg The botanical names of the ingredients are as under: Guduchi (Tinospora Cordifolia) Panash (Atrocarpus Integrifolia) Tulsi (Occimum Sanctum) Kuda (Holarrhena antidyscentrica) Bhui Ami a (Phylanthus Niruri) Gingko Biloba (Gingko Biloba) Shilajeet (Asphaltam) Karavella (Momordica charantia) The pharmaceutically acceptable excipients are, for example, magnesium stearate, dibasic calcium phosphate, methyl paraben, propyl paraben, gum acacia, maize starch, gelatine or talcum powder. The composition of the invention has been found to have improved immuno-modulation activities. Besides it is non-toxic and safe. It shows good invitro activites in the treatment of AIDS. It is also inexpensive as the ingredients are available naturally in abundance. It is to be understood that the composition of the invention is also useful in the treatment of other immune compromising conditions such as Hepatitis or degenerative diseases. The following experimental example is illustrative of the invention but not limitative of the scope thereof: Example 1 Whole plant of Guduchi (Tinospora Cordifolia), ripe fruits of panash (Atrocarpus Integrifolia), whole plant of Tulsi (Occimum Sanctum), barks of Kuda (Holarrhena antidyscentrica), fruits of Bhui Amla (Phylanthus Niruri), leaves of Gingko biloba (gingko biloba) and whole fruit of Karavella (Momordica Charantia) were extracted separately with steam, acetone, ethanol, petroleum and benzene sequentially at room temperature. The combined extracts were sun dried and powdered. Shudh shilajeet (asphaltam) was dissolved in hot water and the paste was mixed with the powder of the combined extracts. The mixture was further mixed with magnesium stearate (60 mg), dibasic calcium phosphate (60 mg), propyl paraben (60 mg), gum acacia paste (1.240 mg) and gelatine (100 mg). The mixture was granulated and the granules were dried at 50°C in drying oven for 2Vi hours. The dried granules were tabletted with talcum powder (25 mg). Each of the tablets contained the active ingredients in the following quantities: Guduchi (Tinospora Cordifolia) 250-mg Panash (Atrocarpus Integrifolia) 75 mg Tulsi (Occimum Sanctum) 60 mg Kuda (Holarrhena antidyscentrica) 100 mg Bhui Amla (Phylanthus Niruri) 100 mg Gingko Biloba (Gingko Biloba) 100 mg Shilajeet (Asphaltam) 100 mg Karavella (Momordica charantia) 60 mg Studies were carried out with the product of Example 1 as follows: Solubility and stability studies. Tablets of Example 1 were kept in a dessicator at room temperature with calcium chloride as desiccant for 10 minutes followed by dissolution in prefiltered distilled water. The tablets were found to dissolve in water completely. Tablets of Example 1 were powdered and dissolved in 50 ml of de-ionized water under stirring with a magnetic stirrer for one hour. The solution was filtered with the help of whatman-3 filter paper, followed by whatman-1 filter paper and sintered glass filter G-3. The filtrate was subjected to steam sterilization (120 °C at 15 pounds pressure) for 15 minutes and stored at 2 - 8°C. Samples of the filtrate were scanned with the help of UV spectrometer at two weeks intervals. The absorbance profiles of the samples were found to be the same without any significant divergence of wave length. This shows that the product is stable over a period of time. (Toxicity Studies, 8-10 ml of blood was collected from healthy voluntary participants in a EDTA (Ethylene Diamine tetreacetic Acid) vacutainer tube. Lymphocytes were separated by density gradient centrifugation on Hystopaque - 1077 (Sigma USA). The lymphocytes collected at the interphase were separated and washed with cold RPMI (Tissue Culture Medium for Lab studies) 1640 medium and finally suspended in RPMI - 1640 containing 10% heat inactivated fetal calf serum. The lymphocytes were adjusted at a concentration of 3 x 106/milliliter of RPMI medium. Tablets of Example 1 and tablets of Example 1 of Patent No 182157 were powdered separately and dissolved in de-ionized water in separate containers under stirring with magnetic stirrers for one hour. The solutions were filtered with whatman - 3 filter paper followed by whatman - 1 filter paper and sintered glass filter G-3. The filtrate obtained from the product of Example 1 and the filtrate obtained from the product of Example 1 of Patent No 182157 (hereinafter referred to as conventional product) was steam sterilized for 15 minutes and stored at 2 - 8°C. In microtire plate (Corning Plastic), 100 micro litres of RPMI medium was dispensed in each well except in first well. The filtrate of Example 1 was double diluted in RPMI medium by mixing equal volume of the filtrates in RPMI and transferred to the second to the last wells without frothing. 100 microlitres of lymphocytes suspension was added in each well except the first. The plate was covered and incubated for a period of 30 minutes. 20 micro litres of Trypan blue dye was added in each well except first one followed by addition of 4% normal saline. Unstained and stained lymphocytes were counted using a hemocytometer. The percentages of live cells were calculated for diluted and undiluted filtrate and the results were as shown in the following Table 1 [Table 1 Dilution of the filtrate Pecentage of live cells 1:2 14% 1:4 25% 1:8 30% 1:16 40% 1:32 60% 1:64 93% Percentage of the live cells with undiluted filtrate was 10%. The product of Example 1 was found to be non-toxic at 1:64 dilution as it shows more than 90% live lymphocytes. The product of the invention is thus safe. IMMUNOMODULATION ACTIVITY 'Non-specific response: Plant lectiun such as Phyto-hemagglutinin (PHA) has a property to induce mitusis in resting lymphocytes. This characteristic feature is often used to identify the in-vitro responsiveness of lymphocytes in normal as well as in experimental situations. This approach is still regarded as a standard parameter to assess deviations in experimental situations. RESPONSE TO PHA INTRODUCTION) Studying responses to non-specific stimulus such as with lectinson on the lymphocyte population can assess the effect on Cell Mediated immunity (within the Lymphocytes). The response variability can be checked directly in the presence of test agents i.e. filtrates of product of Example 1 and conventional product. These can also be used indirectly using lymphocytes from subjects under treatment with these filtrates. Phytohemaggultinin - P was obtained from DIFCO USA. It was dissolved in de-ionized water to yield 1 mg/ml concentration. A serial double dilution of this stock was prepared using RPMI medium supplemented with 10% heat inactivated FCS (Fetal Calf Serum) and numbered as indicated below. Number Dilution Stock RPMI PHA-1 1.2 dil 250.0 250.0 PHA-2 1.10 dil 25.0 225.0 PHA-3 1.100 dil 25.0 of (a) 225.0 PHA-4 1.1000 dil 25.0of(b) 225.0 PHA-5 1.10000 dil 25.0 of (c) 225.0 PHA-6 1.100000 dil 25.0 of (d) 225.0 Blood samples were collected from healthy volunteers in a EDTA. The cells were adjusted at a concentration of 10 million cells/ml in RPMI 1640 supplemented with 10% heat inactivated FCS (Fetal Calf Serum) (SIGMA, India). The cultures were initiated with flat bottom microtitre wells (Corning, USA) as described later. In brief, control wells received 0.050 ul of cell suspension and 0.150 ml of RPMI 1640 supplemented with 10% heat inactivated FCS. Experimental wells received 0.025 ml of different concentrations of PHA (DIFCO, USA) 0.001 to 0.150 ug/ml). The filtrates were diluted at various dilutions previously determined to be non-toxic to cells in 0.225 ml of medium. The wells were incubated at 37° C in 5% C02 atmosphere for a period of 48 hours. Thymidine in 0.225 ml of RPMI medium was added and the wells were further incubated for a period of 24 hours. At the end of 72 hours, the cultures were harvested by collecting lymphocytes onto glass fibre filter discs by using automated cell Harvester (Skatron INC USA). The incorporation of label was counted in LS 100, using PPO / POPOP (Flourescent Agents) as fluorescence. The results were expressed in CPM (Count Per Minute) and stimulation indices compared to controls and under experimental conditions. Lymphocytes cultures in absence of PHA served as negative controls and with various concentrations of PHA as positive control. The response in positive controls were compared with those obtained in presence of the filtrates. Cultures were set in duplicates in microtitre wells with flat bottom (Corning, USA) as follows. Control wells received 150 ul of RPIMC and 50 ul of cells. In case of various doses of PHA of incubation, cultures received 1 uCi of tritated Thymidine (Chemical Agent for Clinical Studies) and were harvested 24 hours later using a cell harvester (Nunc Denmark). Incorporation of tritated Thymidine was determined using beta scintillation counter and the results were given as counts per minute (CPM). In order to determine the effect of various filtrates in the stimulation process individual filtrates were used. A typical microculture reaction contained in control 125 ul of RPMIC, 25 ul of drug/extract and 50 ul of lymphocytes. The experimental wells contained 125 ul of RPMIC, 25 ul of PHA, 50 ul lymphocytes and 25 ul filtrates. The concentration of filtrates was decided on the toxicity studies carried out earlier. In either case results are given as CPM means of the duplicate culture. The filtrates were subjected to spectrophotometric analysis. The scan revealed NO QUALITATIVE CHANGE in response profile for lymphocytes to PHA. Lymphocytes from normal healthy human volunteers were separated from whole blood collected in EDTA, using histopaque 1077 density gradient centrifugation. Purified lymphocytes were suspended in RPMI medium supplemented with 10% heat inactivated Fetal Calf Serum (FCS) at a concentration of 1 x 106 cells/ml. RESULTS The study results were as shown in the following Tables 2 and 3. Table 2 gives the results with the product of Example 1 of the invention, whereas Table 3 gives the results with the conventional product of Example 1 of Patent No 182157. [Table 2; Control Tablet % Dilution 1:32 PHA-1 4661.8 191409.2 +4005.90 PHA-2 4491.7 192223.7. +4197.50 PHA-3 81298.2 218063.5 +168.22 PHA-4 133726.9 117746.8 -13.57 PHA-5 120690.3 133299.4 +9.46 PHA-6 117415.2 171100.6 +31.38 [Table 3] Control Tablet % Dilution 1:32 PHA-1 35191 13313 -62.17 PHA-2 73957 37933 -48.71 PHA-3 98287 53054 -46.02 PHA-4 108570 35031 -67.73 PHA-5 7621 3553 -53.38 PHA-6 6904 3518 -49.04 CONCLUSION Table 2 shows that immunity level increased by over 4000% plus in dilution of 1:32 (at PHA 1 and 2) of the product of Example 1 of the invention whereas Table 3 shows that immunity level was suppressed by 62% in dilution of 1:32 (at PHA-l)ofthe conventional product. The product of the invention was thus found to have improved immuno-modulation properties. I claim 1. An ayurvedic immuno modulator composition for treatment of Acquired Immuno Deficiency Syndrome comprising Guduchi or Giloe (cordifolium) 5 mg - 2 gm Panash or ^Kathal (jack fruit) 2 mg - 5 gm Tulsi or Krishna Tulsi (Holy Basil) 5 mg - 5 gm Kuda or Kutaja (Kurchi) 2 mg - 2 gm Bhui Amla or Bahu Patra (Gooseberry) 5 mg - 2 gm Gingko Biloba 5 mg - 2 gm Shilajeet or Si la Ras (Asphaltam) 2 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm in combination with pharmaceutically acceptable excipients. 2. An Ayurvedic immuno modulator composition as claimed in claim 1, comprising Guduchi or Giloe (cordifolium) 100 mg - 600 mg Panash or Kathal (jack fruit) 100 mg - 700 mg Tulsi or Krishna Tulsi (Holy Basil) 20 mg - 300 mg Kuda or Kutaja (Kurchi) 20 mg - 200 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg -600mg Gingko Biloba 5 mg - 2 gm Shilajeet or Sila Ras (Asphaltam) 50 mg - 2 gm Karavella or Karela (bitter gourd) 5 mg - 2 gm 3. An Ayurvedic immuno modulator composition as claimed in claim 1, comprising Guduchi or Giloe (cordifolium) 250 mg Panash or Kathal (jack fruit) 75 mg Tulsi or Krishna Tulsi (Holy Basil) 60 mg Kuda or Kutaja (Kurchi) 100 mg Bhui Amla or Bahu Patra (Gooseberry) 100 mg Gingko Biloba 100 mg Shilajeet or SilaRas (Asphaltam) 100 mg Karavella or Karela (bitter gourd) 60 mg Dated this 6th day of October 2003 (M A Jose) of KHAITAN & CO Agent for the Applicant

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