Title of Invention	PROCESS FOR ISOLATING GLYCOPROTEIN FROM NEEM LEAF AND ITS CHARACTERIZATION TO DEFINE THE IMUNOMODULATORY AND CANCER PREVENTIVE FUNCTIONS OF THIS GLYCOPROTEIN
Abstract	The invention provides a process for isolating and characterization of glycoprotein from neem leaf for immunomodulatory and cancer preventive functions of neem leaf glycoprotein (NLGP) thus obtained, said process comprising the steps of: a) preparartion of crude neem leaf preparation (NLP) by soaking neem leaf powder in PBS for a period of 24 hours, b) extensive dialysis of crude NLP thus obtained against PBS, followed by subsequent concentration by membrane filtration, bioactivity of NLGP was tested by: c) exposing said NLGP to temperature gradient of 0°C to 100°C d) exposing the NLGP to different proteolytic enzymes e) exposing the NLGP to various ions, Wherein the said NLGP is exposed to a temperature of 56°C and pH of PBS solution is maintained at 7.4 Important figures are given in next page. Fig. 1. illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured and denatured conditions. Fig. 2. illustrates mouse tumor growth restricting activity of NLP exposed to various proteolytic enzymes and survivability of treated mice.

Title of Invention

PROCESS FOR ISOLATING GLYCOPROTEIN FROM NEEM LEAF AND ITS CHARACTERIZATION TO DEFINE THE IMUNOMODULATORY AND CANCER PREVENTIVE FUNCTIONS OF THIS GLYCOPROTEIN

Abstract

The invention provides a process for isolating and characterization of glycoprotein from neem leaf for immunomodulatory and cancer preventive functions of neem leaf glycoprotein (NLGP) thus obtained, said process comprising the steps of: a) preparartion of crude neem leaf preparation (NLP) by soaking neem leaf powder in PBS for a period of 24 hours, b) extensive dialysis of crude NLP thus obtained against PBS, followed by subsequent concentration by membrane filtration, bioactivity of NLGP was tested by: c) exposing said NLGP to temperature gradient of 0°C to 100°C d) exposing the NLGP to different proteolytic enzymes e) exposing the NLGP to various ions, Wherein the said NLGP is exposed to a temperature of 56°C and pH of PBS solution is maintained at 7.4 Important figures are given in next page. Fig. 1. illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured and denatured conditions. Fig. 2. illustrates mouse tumor growth restricting activity of NLP exposed to various proteolytic enzymes and survivability of treated mice.

Full Text	INTRODUCTION This invention relates to a process for isolating glycoprotein from neem leaf and its characterization to define immunomodulatory and cancer preventive functions. Background of invention: Neem (Azadirachta indica) is a widely prevalent and esteemed wonder tree, mainly cultivated in Indian subcontinent (1). This unique plant is well known in India and its neighbouring countries for more than 2000 years as one of the most versatile medicinal plants having a wide spectrum of biological activity (2). As per recommendation of the Indian Ayurvadic literature, eight parts of the neem tree (leaf, bark,flower, twig, gum, seed, pulp and oil) have shown to possess several biologically active components having a wide variety of activities against different ailments (2). This SARBAROGAMBAPINI neem generously contributes each and every parts of its body as suitable for human consumption. This valuable ancient knowledge surprisingly has been neglected in India and its recognition was chiefly limited within poor rural Indian population, as a village dispensary (3). Neem products never considered as a prime factor towards Indian scientists for proper exploration of its therapeutic values and also towards commercial agencies for proper marketing of the economic values hidden within neem products. Neem products hit the popular media headlines only after it became the subject of legal controversy when the European Patent office rejected a patent application from the WR Grace Company and the United States Department of Agriculture. Although, neem remained neglected for a long time in the Western countries, interest on this particular medicinal plant is gradually growing and as a consequence, US National Academy of Science published a report on 1992 with a title, 'Neem-a tree for solving global problem' (National Academic Press, USA, 1992). Neem can be exploited in modern biological research, using toxicological, molecular biological, immunological techniques. These effort may disclose some new applications of neem, those are not described in Indian traditional medicine. Immune boost up by neem was not considered as a potential field of medical research, but the research associated with present invention will disclose several applications of neem leaf in the prevention of cancerous growth by stimulation of the body's defense (immune) mechanism. Prior Art: Several pharmacological activities and medicinal applications of various parts of neem tree are known. Biological activities of neem tree are reported where crude extracts and their different fractions from leaf, bark, root, seed and oil were used. These reports have exhibited a wide diversity in biological functions from a group of compounds present in the various parts of the plant (4). Some of these are as follows: i. General Health: Neem is known to be useful remedy in some chronic skin diseases, ulcers, rheumatism, leprosy, dental troubles, intestinal helminthiasis, constipation, asthma and also as a general health promoter (5-7). Bark, leaf, root, flower and fruit together cure blood morbidity, biliary afflictions, itching, skin ulcers and burning sensations (4). Gastroprotective antiulcer effect of neem bark extract has been reported recently (8). ii. Reproduction: It also has wide range of effects on the reproductive system. Neem oil is used as a spermicidal (9) and early abortificient agent (10). It has contraceptive and antifertility properties (11). For long term contraceptive, single intrauterine application of neem extract has been reported (12). Oral administration of aqueous extract of neem leaf shows antifertility effect in mice (4). Neem seed preparations have been shown to be effective to abrogate pregnancy in rodents, monkeys and baboons (4). This antifertility effects are possibly mediated by activation of the cell mediated immune reaction, thus can overcome the side effects of steroidal contraceptives. iii. Immunomodulation: Various products of neem are known to have immunomodulatory properties. These may act by stimulating cell-mediated and/or humoral immunity. However, studies are incomplete and preliminary. Some of these published reports are encouraging. Neem kernel oil has been shown to induce the secretion of gamma interferon (13) and tumor necrotic factor-alpha (14). Dry neem leaf could enhance the antibody titre against new castle disease virus (15), bovine serun albumin and sheep erythrocytes (16). Intraperitoneal treatment of mice with neem oil reported to be increase the number of leukocytic cells in peritoneal lavage. Peritoneal macrophages exhibited enhanced phagocytic activity and expression of MHC class-II antigens after treatment with neem oil (13). Enhancement in macrophage migration inhibition and foot thickness in mice after neem treatment (17) was also observed. Neem (NIM-76) can enhance macrophage activity and lymphocyte proliferation response, while humoral component of immunity was unaffected 18). An aquaous extract from stem bark of neem has been shown to have an inhibitory influence on complement activation and superoxide ion production by polymorphonuclear (PMN) leukocytes while having a stimulatory effect on the production of migration inhibition factor (MIF) by PMN leukocytes (19). The aqueous extract of stem bark possesses anticomplement activity, acting both on the alternative as well as classical pathway of complement activation in human serum. iv. Cancer: Based on some preliminary in vitro studies and its known effects on immune system, it can be hypothesized that neem components may have some effects on the restriction of tumor growth. Neem seed preparation contains a variety of limonoids, some of which are cytotoxic to N1E-115 mouse neuroblastoma, 143B.TK-human osteosarcoma and sf9 insect cells (20). Water soluble polysaccharides isolated from the bark of Melia azadirachta, demonstrated antitumor effect with complete regression of tumors (21). Cytotoxicity of the neem toxin, azadirachtin A, in human glioblastoma cell lines is also reported (22). Recently, it is reported that the bark of the giant neem tree Melia dubia contains euphane type triterpenes, some of them can inhibit the growth of P388 lymphocytic leukemia cell line (23). Effects of these neem compounds and mechanism of inhibition of tumor cell growth are completely unknown in in vivo. Neem leaf extracts enhance hepatic glutathione and glutathione dependent enzymes, which may offer a degree of chemoprevention during N-methyl-N-mtro-N-nitrosoguanidine induced gastric carcinogenesis in rats (24) and on 7, 12-dimethylbenzanthracene induced buccal pouch carcinogenesis (25). However, none of these reports was focused on the involvement of immune system in neem induced tumor growth inhibition. The mechanism of immunopotentiation is still unknown. Various parts of neem (Azadirachta indica) is used to cure different human diseases from the dawn of civilization. In inspiration with the ancient experiences, we are working on an aquaous preparation of neem leaf (NLP), which was proved to be nontoxic, hematostimulatory and immunostimulatory. This preparation has immunoprophylactic function to prevent the growth of murine Ehrlich's carcinoma and B16 melanoma. Studies utilizing adoptive cell transfer technology suggest that activation of various immunocompetent cells by neem leaf component(s) may be responsible for tumor growth restriction. NLP also acts as an adjuvant by inducing an active antitumor immunity in murine model against tumor antigens. Moreover, NLP mediated immune activation protects mice from leukepenia, caused by cancer chemotherapy. Constituents present in the neem leaf responsible for the immune functions are not known. Our preliminary findings, suggest that NLP has an instructive role in the secretion of Thl cytokines, IFNy, IL-12 and TNFa. However, active principal(s) present in neem leaf responsible for such immunobiological activity and physiological condition required for optimum demonstration of these biologic activities are not disclosed earlier. Object of invention The object of the present invention is to disclose the chemical nature of active principal responsible for immunobiological activity of the neem leaf preparation. Another object of the invention is to provide a process for isolating glycoprotein from neem leaf and its characterization to define immunomodulatory and cancer preventive functions of this glycoprotein. Yet another object of the invention is for the extraction of this active ingredient and conditions required for its maximum biological activity, which may have use as a medicine to prevent cancerous growth. Summary of invention: The invention provides that a glycoprotein component present in neem leaf is responsible for the restriction of tumor growth after prior vaccination. This glycoprotein mediates its action through immunomodulation of the host's physiological system. Optimum biological activity is obtained upon exposure of this glycoprotein into a buffered solution with pH 7.4at a temperature 56°C. Unique immunomodulatory functions of aqueous preparation of neem leaf (NLP) in relation to\|cancer are disclosed. In an objective to find out the active principle present in NLP a glycoprotein was detected in NLP, designated as neem leaf glycoprotein (NLGP). NLGP appeafed in nondenatured PAGE as a single band, whereas, three bands in SDS- PAGE. This glycoprotein constitutes the carbohydrate moiety of about 33%, consisting of arabinose, galactose and glucose. Protein moiety of the glycoprotein consists of sixteen amino acids, except arginin. Scanning electron microscopy reveals the protein like structure of the NLP constituents. Immunogenecity of this protein was defined by strong reaction of the anti-NLP sera with NLP by ELISA and immunoblot analysis. To understand the association of the NLGP with tumor growth restriction, it was exposed to a gradient of temperature (0°C, 37°C, 56°C and 100°C), pH (5.7, 6.2, 7.4 and 8.2), enzymes (trypsin, papain and neuraminidase) and ions (phosphate, tris etc.). Exposure of NLP to the condition not favorable to maintain the protein conformation caused significant reduction or abolition of the tumor growth restricting function of NLP. Such exposure also caused diminished immune reactivity of NLP. Overall results demonstrated the immense potential of newly identified, NLGP, present in NLP as an immunoprophylactic agent for tumor growth restriction in a clinical setup. Brief description of accompanying drawing The scientific features underlying this invention as well as many of the advantages of the invention will be more readily appreciable when explained with conjunction to accompanying drawings wherein, Fig 1 illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured and denatured conditions. Fig 2 illustrates GLC-MS analysis of the carbohydrate moiety of the glycoprotein (NLGP). Fig 3 illustrates scanning electron micrographs of the glycoprotein (NLGP). Fig 4 illustrates immune reactivity of the NLP generated sera with NLP by ELISA and immunoblotting. Fig 5 illustrates mouse tumor growth restricting activity of dialysed and nondialysed NLP and survivability of treated mice. Fig 6 illustrates mouse tumor growth restricting activity of NLP exposed to various temperatures and survivability of treated mice. Fig 7 illustrates mouse tumor growth restricting activity of NLP exposed to various pH and survivability of treated mice. Fig 8 illustrates mouse tumor growth restricting activity of "NLP exposed to various proteolytic enzymes and survivability of treated mice. Fig 9 illustrates, immune reactivity of the NLP generated sera with NLP exposed to different temperatures, pH and enzymes by ELISA. Electrophoresis of NLP exposed to different temperatures and pH. Detailed description of the invention with reference to drawing/example Detailed descriptions of the accompanying figures are illustrated below- FIGURE 1 - Analysis of dialyzed NLP by PAGE. NLP was dialyzed against PBS and 20 µg of NLP was analyzed by 12% nondenatured (A) and 20% denatured (C) PAGE. Gel was stained with silver nitrate method. Molecular weight markers were electrophorsed with each run. FIGURE 2 - GLC-MS analysis of carbohydrate moiety of NLGP. NLP was hydrolysed with 2M Trifluroacetic acid and GLC-MS was performed as described in 'materials and methods'. FIGURE 3 - Scanning electron microscopy of NLGP. Freeze dried NLGP was mounted on circular aluminium stubs with double sticky tape and coated with 20 nm gold using I- B2 Ion coater. The sample was examined and photographed. Structures of NLP in different magnifications are presented in A, B, C, as indicated on the photographs. FIGURE 4 - Immune reactivity of anti-NLP sera with NLP. Mice were immunized with either NLP or PBS once weekly for four weeks. Immune sera were absorbed with NLP and absorbed/nonabsorbed sera were reacted with NLP coated on plates by ELISA (A) or electrophorsed on PAGE by immunoblotting (B). FIGURE 5 - In vivo tumor growth restriction by total and dialyzed NLP. Three groups of mice were injected with either PBS or total or dialyzed NLP for 4 weeks. Seven days after the last injection mice were inoculated with EC tumor cells subcutaneously (1 X106) (n=6 in each group). Tumor growth was monitored and survivability was noted. Growth of EC tumor in PBS, total and dialyzed NLP treated mice. p PBS injected mice. +p of each group of mice (B). FIGURE 6 - In vivo tumor growth restriction by total NLP exposed to different temperatures. Five groups of mice were injected with either PBS or total NLP exposed to 100°C, 56°C, 37°C and 0°C weekly for four weeks. Seven days after the last injection mice were inoculated with EC tumor cells subcutaneously (1 X106) (n = 6 in each group). Tumor growth was monitored and survivability was noted. Growth of EC tumor in PBS and NLP (exposed to different temperature) treated mice. p PBS injected mice. p (A). Survival of each group of mice (B). FIGURE 7 - In vivo tumor growth restriction by total NLP exposed to different pHs. Four groups of mice were injected with total NLP exposed to pH 8.0, pH 7.4, pH 6.2 and pH 5.7 weekly for four weeks. Seven days after the last injection mice were inoculated with EC tumor cells subcutaneously (1 X106) (n = 6 in each group). Tumor growth was monitored and survivability was noted. Growth of EC tumor in NLP (exposed to different pH) treated mice. *p injected with NLP, exposed to pH 8 (A). Survival of each group of mice (B). FIGURE 8 - In vivo tumor growth restriction by total NLP exposed to different enzymes. Four groups of mice were injected with either total NLP or NLP exposed to trypsin, papain and neuraminidase weekly for four weeks. Seven days after the last injection mice were inoculated with EC tumor cells subcutaneously (1 X106) (n=6 in each group). Tumor growth was monitored and survivability was noted. Growth of EC tumor in NLP (exposed to various enzymes) treated mice. p injected with trypsin, papain and neuraminidase exposed NLP (A). Survival of each group of mice (B). FIGURE 9 - Alterations in NLP by differential exposure to enzymes, pHs and temperatures. NLP was exposed to different enzymes (trypsin, papain and neuraminidase), temperatures (100°C, 56°C, 37°C and 0°C) and'pHs (pH 8.0, pH 7.4, pH 6.2 and pH 5.7). A. These differentially treated NLPs were coated on microtitre plates. Anti-NLP sera (generated by immunization of Swiss mice with NLP) was tested for its immune reactivity with coated NLPs by ELISA. p reaction with enzyme treated NLP; p NLP exposed to pH 8 and pH 5.7; +p NLP, exposed to 0°C and 100°C. B. Differentially treated NLPs were electrophorsed on 20% SDS-PAGE and stained with silver nitrate. a) Methods involved in the invention. Neem Leaf Preparation (NLP) Matured neem leaves of same size and color (indicative of same age), procured from a standard source were shed-dried to pulverize. Neem powder was soaked overnight in phosphate buffered saline (PBS), pH 7.4 just one day, before the experiment. Supernatant was collected by centrifugation at 1500 rpm and was membrane (0.22 µm) filtered for in vivo or in vitro uses. Extraction condition was changed in studies for characterization of the active component. Crude NLP was then extensively dialyzed against PBS, pH 7.4 and concentrated by Centricon Membrane Filter (Millipore Corporation, Bedford, MA, USA) with 10 Kda molecular weight cut off. Endotoxin content of the freshly prepared NLP was determined by Limulus Amebocyte Lysate (LAL) test as per manufacturer's (Salesworth India, Bangalore) instruction. The endotoxin content of all the batches of NLP was found to be less than 6 pg/ml. Animals and tumor cells Female Swiss mice (age, 6-8 weeks, body weight, 24-27 gms) were obtained from Institutional Animal Care and Maintenance Department. Dry pellet diet and water were given ad libitum. Maintenance and treatment of animals were given according to the guidelines established by the Institutional Animal Care and Ethics Committee. Ehrlich's carcinoma (EC) was maintained in Swiss mice by intraperitoneal passage of viable tumor cells (1 X 107), as determined by trypan blue dye exclusion assay. These EC cells were used for in vivo tumor development. Protein estimation Protein concentration of NLP was measured by Lowry's method using Folin Phenol reagent (Lowry et al, 1951) and by UV absorption. For these two methods of protein estimation, optical density was measured at 660 nm and 280 nm respectively. Analysis of carbohydrate moiety Amount of carbohydrate moiety in protein, present in NLP was estimated using phenol- sulphuric acid method (Dubois et al, 1956). In brief, 80% phenol (lOOul) was mixed with NLP (1 ml) and then concentrated sulphuric acid (1 ml) was added slowly. After 10 mins, absorbance was measured at 490 nm using microplate reader (Tecan Spectra, Grodig, Austria). Analysis of sugar moiety by GLC-MS NLP was hydrolysed with 2M Trifluroacetic acid for 3 hrs at 100°C in a boiling water bath (Ghosh et al, 2005; Ghosh et al, 2005). Myo-inositol was used as internal standard. Hydrolytic losses were accounted for by using standard sugar mixtures containing all the sugars usually present in the cell wall of a plant. After acid hydrolysis, the solution was neutralized with 25% ammonia solution (0.7 ml) and the monosaccharides were reduced with 0.1 ml sodium borohydride in 3M ammonia solution at room temperature for one hr. the excess borohydride after reduction was decomposed by addition with acetic acid. Ultimately, the free alditols were obtained as a mixture in dried condition by lyophilizing the above solution. Acetylation of dried sample was first carried out by acetic anhydride and L-Methylimidazole as catalyst (Blakeney et al, 1983). Derived alditol acetates were then analyzed and identified by GLC-MS. GLC-MS was carried on a Shimadzu GC-MS (QP-5050) instrument (Shimadzu, Japan) with a Flame ionization Detector and DB-225 (JW) column using helium as carrier gas. The temperature of the injection port was 250°C and the column oven temperature was maintained at 210°C (isothermal). Polyacrylamide gel electroporesis (PAGE) Crude and dialyzed NLP, after concentration by membrane filtration, were analyzed by native-PAGE and SDS-PAGE with different acrylamide concentrations. Molecular weight marker (SC-2035; Santacruz, USA) was electrophorsed in each time to know the molecular weights of protein bands. In some cases, NLP was exposed to different temperature and pH before electrophoretic analysis. Antibody production and ELISA Swiss mice (n=6) were injected with NLP (extract obtained from 0.25mg of dry neem powder) weekly for 4 weeks in total. Blood was collected after completion of the fourth injection from anaesthesized mice by retro-orbital puncture and serum separated. Serum antibodies were assessed by ELISA (Baral et al, 2001). Briefly, the microtiter plates were coated with NLP (10µg/50µl/well) for overnight and blocked with 5% BSA. Anti-NLP sera (1:50 diluted in 1% BSA) were added in the wells in triplicate and incubated for 2 hours. The plates were washed with PBS containing Tween-20 and goat anti-mouse Ig labeled with peroxidase (Sigma, St. Louis) was added at a dilution of 1: 1000. Color was developed with TMB substrate solution (OptEIA™, BD-Pharmingen). Reaction was stopped with 2N H2SO4 solution and absorbance was measured at 450 nm using microplate reader (Tecan Spectra, Grodig, Austria). Immunoblotting NLP was electrophorsed on 10% SDS-PAGE, transferred on nitrocellulose membrane, blocked with POD blocking substrate (Roche Diagnostics, Mannheim, Germany) and incubated with anti-NLP sera, before and after absorption with NLP, for overnight at 4°C. After washing, blots were incubated with horseradish peroxidase conjugated secondary antibody for two hours at room temperature. Bands were detected using chemiluminescence (Roche Diagnostics, Mannheim, Germany) according to the manufacturer's manual. Absorption of immune sera with NLP NLP generated immune sera was absorbed with NLP (5 µg/ml of 1:50 diluted sera) for 1 hr at 37°C and centrifuged at 5000g for 10 mins. Absorbed sera were then tested by ELISA and immunoblotting for their reactivity. Scanning electron microscopy Structural morphology of protein fraction present in NLP was studied using Scanning Electron Microscope (Chavan et al, 2001). Freeze dried neem protein was mounted on circular aluminium stubs with double sticky tape and coated with 20 nm gold using I-B2 Ion coater. The sample was examined and photographed in a scanning electron microscope (Hitachi S-530 SEM, Hitachi Ltd., Tokyo, Japan). Amino acid analysis Amino acid analysis was performed in a PICO.TAG system according to the PICO.TAG operation manual (Waters, USA). NLP was extensively dialyzed against deionised water for 24 hrs, dried and the protein (20µg) was hydrolysed by 6N HC1 containing 1% phenol for 24 hrs at 105°C in the PICO.TAG work station. Hydrolysed sample and standard amino acid mixture, 'Standard S' (0.0005 ml) were taken in respective'tubes, introduced into the reaction vial and were dried completely. These were then derivatized by phenyl isothiocyanate (PITC) solution (ethanol: triethylamine : water : PITC = 7:1:1:1 by volume) for 20 min at 25°C (33). The vials were then dried and samples were reconstituted in diluent solution (Na2HPO4. 0.071%, w/w, pH 7.4 containing acetonitrile 5%, v/v). The samples were analysed at 38°C as per PICO.TAG manual using C18 column (Ghosh et al, 1997). Detector setting, chart speed and run time were AT 128 at 254 nm, 2 cm/min and 32 min respectively. Amino acids present in unknown sample (NLP) were determined quantitatively by comparing the peak areas (745b data module print out) of amino acids present in 'Standard S'. Exposure of NLP in different temperature, pH and enzymes Crude and dialyzed NLP were exposed to different temperatures (100°C for 5 mins, 56°C, 37°C and 0°C for 1h) and phosphate buffers with pH 5.7, 6.2, 7.4 and 8.0 for overnight. NLP was also digested with proteolytic enzymes, like, trypsin (SRL) (20 µg/mg NLP), papain (SRL) (50 µg/mg NLP) and neuraminidase (Sigma) (0.6 U with 2 mM PMSF/mg of NLP) for one hour at 37 C. Trypsin and papain were inactivated by rapid chilling and neuraminidase was removed by centrifugation. Exposure of NLP to various ions NLP was prepared by soaking neem leaf powder into water with pH 6.0 and pH 7.4, PBS, pH 7.4, Tris buffered saline (TBS), pH 7.4, 10mM disodium hydrogen phosphate solution, 1.4mM potassium dihydrogen phosphate solution individually. Except variation in the ions, other conditions were identical, as described in the section, 'Neem Leaf Preparation'. Tumor growth restriction assay Swiss mice were injected with NLP either exposed to various temperatures, pH or digested with different enzymes or soaked with solutions containing various ions. Extracts obtained from 0.25 mg of neem powder were used for treatment of each week and NLP treatment was given for 4 weeks in total. After completion of the treatment, each mouse was inoculated with EC cells (1X107) subcutaneously on right hind leg quarter. Growth of solid tumor (in mm3) was monitored weekly by caliper measurement using the formula: (width2 x length)/2. Survival of mice was monitored regularly, till tumor size reached to 25 mm in either direction. Statistical analysis All results from in vivo experiments represent the average of two separate experiments involving eight mice in each group. Statistical significance was established by using INST AT 3 Software. Features of the invention NLP contains protein Protein estimation suggests the presence of protein in NLP NLP is soaked in PBS, pH 7.4 for overnight, extract was collected and dialyzed extensively against PBS. Protein concentration of the dialyzed NLP was measured by Lowry's method using Folin's Phenol reagent. Determination of the optical density confirms the presence of protein and quantitative estimation of NLP revealed the presence of protein and yield of it is 1.4 µg/mg of dry neem leaf (Table 1). Polyacrylamide gel electrophoresis identifies proteins in NLP Dialyzed NLP was analyzed by 12% nondenatured PAGE and 20% denatured SDS- PAGE. Nondenatured PAGE and SDS-PAGE demonstrated one and three major protein bands respectively (Fig. 1). Molecular weight of three bands appeared in 20% SDS-PAGE was 65Kda, 36Kda and 22Kda. Identified NLP protein is a glycoprotein Biochemical estimation reveals the presence of 33.3% carbohydrate in the protein present in NLP, thus, may be glycoprotein. After dialysis, concentration of this glycoprotein was increased significantly (Table 2). Carbohydrate moiety in NLP was analyzed by GLC-MS and results suggest the presence of 53% arabinose, 36% galactose and 11% glucose (Fig. 2). Henceforth, the protein present in NLP will be termed as neem leaf glycoprotein (NLGP). NLGP consists of various amino acids, except arginin Amino acid analysis of protein part of the NLP revealed the presence of sixteen amino acids of which eight were essential. This protein is rich in Glutamic acid, Glycine, Threonine and Valine but low in Methionine, Cysteine and Histidine. Arginine is not present in NLGP. Result of amino acid analysis of NLGP is presented in Table 3. Electron microscopy identifies protein structure Fig. 3 reveals the structure of NLGP after detection in scanning electron microscope using different magnifications. SEM micrographs show 3-dimensional structures through 2-dimensional images. The image demonstrates the heterogeneous surface with void spaces of NLGP. NLGP is efficient to generate antibody Mice were immunized with NLP weekly for four weeks. Blood was collected on seventh day after last injection to separate the serum. Immune sera were strongly reactive with NLGP, as detected by ELISA and immunoblotting, demonstrating its nature like a protein antigen (Fig. 4). This reactivity of the immune sera was significantly decreased after its absorption with NLP. NLGP of NLP is responsible for tumor growth restriction Dialysis increases tumor growth restricting activity of NLP Two groups of Swiss mice were injected with either total NLP or dialyzed NLP weekly for four weeks. Three days after the last injection both groups of mice were inoculated with Ehrlich carcinoma cells. Fig. 5 a documented the tumor growth pattern in dialyzed and nondialyzed NLP injected mice, which clearly shows that dialyzed NLP gave better protection to the growth of Ehrlich's carcinoma. Mice received dialyzed NLP before tumor inoculation also survived longer than the group received total NLP (Fig. 5b). Nondialyzed NLP also caused tumor growth restriction in comparison to PBS control. Exposure to 56° C increases the tumor growth restricting activity of NLP than 0oC,3rC and 100oC Four groups of Swiss mice were injected with total NLP exposed to temperatures, like, 100°C, 56°C, 37°C and 0°C weekly for four weeks in total. Three days after the last injection mice of all groups were inoculated with Ehrlich carcinoma cells. Minimum tumor growth was demonstrated in mice treated with NLP exposed to 56°C, than those mice immunized with NLP exposed to 0°C, 37°C and 100°C (Fig. 6). Identical experiment was performed with dialyzed NLP, and almost similar result was obtained (data not shown). pH of extracting buffer influences tumor growth restricting activity of NLP Four groups of Swiss mice were injected with total NLP soaked in phosphate buffer with different pH, like, pH 5.7, 6.2, 7.4 and 8.2 weekly for four weeks. Three days after the last injection mice of all groups were inoculated with Ehrlich carcinoma cells. Maximum tumor growth was observed in mice injected with NLP prepared with pH 5.7 buffer and significant tumor growth restriction was demonstrated when NLP prepared with pH 7.4 and pH 6.2 buffers. Moderate tumor growth restriction was observed during injection with NLP, pH 8.0 (Fig. 7). Identical experiment was performed with dialyzed NLP, demonstrated almost similar result (data not shown). Exposure of NLP to proteolytic enzymes, trypsin, papain and neuraminidase abolishes tumor growth restricting activity Four groups of Swiss mice were injected with either total NLP or NLP digested with trypsin, papain and neuraminidase weekly for four weeks. Three days after the last injection mice of all groups were inoculated with Ehrlich carcinoma cells. No significant restriction of the tumor growth was noticed when NLP was exposed to all three enzymes, in comparison to mice injected With NLP only (Fig. 8). Similar experiment was performed with dialyzed NLP and also demonstrated abolition of the tumor growth restricting activity of NLP (data not shown). Antibody against NLP reacts differentially with NLP exposed to different temperatures, pH and enzymes Swiss mice were injected with NLP weekly for four weeks. Immune sera obtained after fourth injection were reacted with NLP before and after exposure to various temperatures, pH, enzymes or soaked with solutions containing various ions by ELISA (Fig. 9a). Maximum immune reactivity was noticed with those treated NLP, showed significant tumor protection. For example, injection with NLP exposed to 56°C demonstrated maximum tumor protection (Fig. 6) and this NLP reacted maximally with anti-NLP sera. Differentially treated NLPs were electrophorsed and partial degradation of the NLP was detected, in those cases where minimum tumor protection was observed (Fig. 9b). References: 1. Schmutterer H (ed), in The neem tree: Source of unique natural products for integrated pest management, medicine, industry and other purposes, (VCH, Weinhrim: Hiedelberg) 1995,1. 2. Devasagayam T P A & Sainis K B, Immune system and antioxidants, especially those derived from Indian medicinal plants, Indian J Exp Biol, 40, 2002, 639. 3. Satyavati G V, Raina M K, Sharma M, in Medicinal plants of India, (Indian Council of Medical Research, New Delhi) 1976, 363. 4. Biswas K, Chattopadhyay I, Banerjee R K and Bandopadhyay U, Biological activities and medicinal properties of Neem (Azadirachta indica). Curr Sci, 82, 2002,1336. 5. Ross I A, Azadirachta indica, A. Juss, in Medicinal plants of the World, Vol 2: Chemical Constituents, Traditional and Modern Uses, (umana Press Inc.Totowa, NJ), 2001,81. 6. Singh P P, Junnarkar A Y and Reddi G S, Azadirachta indica: Neuro-Psycho- Pharmacological and Antimicrobiological studies, Fitoterapia, 58, 1987, 235. 7. Meisner J and Azcher K R S, Insect growth regulating (IGR) effects of neem products on Spodoptera littoralis, Schriftner Gtz, 161, 1984, 345. 8. Bandopadhyay U, Biswas K, Chatterjee R, Bandopadhyay D, Chattopadhyay I, Ganguly C K, Chakraaborty T, Bhattacharya K, Banerjee R K, Gastroprotective effect of neem {Azadirachta indica) bark extract; possible involvement of H9+0- K(+)-ATPase inhibition and scavenging of hydroxyl radical, Life Sci, 71,2002,2845. 9. Shaikh P D, Manivannam B and Pathan K M, Antispermatic activity of. Azadirachta indica leaves in albino rats, curr Sci, 64,1993, 688. 10. Sinha K C, Riar S s and Bardhan P, Anti-implantation effect of neem oil. Ind J Med Res, 80, 1984, 708. 11. Choudhury D N, Singh J N and Verma S K, Antifertility effects of leaf extracts of some plants in male rats, Ind J Exp Biol, 28,1990, 714. 12. Upadhyay S N, Kaushic C and Talwar G P, Antifertility effects of neem {Azadirachta indica) oil by single intra-uterine administration: A novel method for contraception, (Proc Royal Soc Lond) 242, 1990, 175. 13. Upadhyay S N, dhawan S, Garg S and Talwar G P, immunomodulatory effects of neem {Azadirachta indica) oil, Int J Immunopharmac, 14,1992, 1187. 14. Thatte U and Dahanukar S, Rasayana concept: clues fro immunomodulatory therapy, in Immunomodulation, edited by S N Upadhyay, narosa Publishing House, New Delhi, 1997,141. 15. Sadekar R D, Kolte A Y, Barmase B S and Desai V F, Immunopotentiating effects of Azadirachta indica (Neem) dry leaves powder in broilers, naturally infected with IBD virus, Ind j Exp Biol, 36,1998,1151. 16. Logamble S M and Michael R D, Immunostimulatory effect of azadirachtin in Oreochromis mossambicus (Peters), Ind J Exp Biol, 38, 2000, 1092. 17. Ray A, Banerjee B D and Sen P, Modulation of humoral and cell mediated immune responses by Azadirachta indica (neem) in mice, Ind J Exp Biol, 34,1996, 698. 18. Sai Ram M, Sharma S K and Ilavazhagan G, Immunomodulatory effects of NIM-76, avolatile fraction from neem oil, J Ethnopharmacol, 55,1997,133. 19. Labadie R P, Nat J M and Simons V D, Advances in immunomodulatory agents from medicinal plants and traditional preparations. In Proceedings of XI Asian Symposium on Medicinal Plants and Species, Bandung, Indonesia, 1989,407. 20. Cohen E, Quistad G B & Casida J E, Cytotoxcity of nimbolide, epoxyazadiradione and other limonoids from neem insecticide. Life Sci, 58,1996,1057. 21. Shimizu M, Takai M, Inone K, takeda T and Ogiwara Y, Antitumor polysaccharides from Melia azadirachta bark extracts and their purification. (Patent-Japan Kokai Tokkyo Koho-01 275, 602,1989), 9. 22. Akudugu J Gade G and Bohm L, Cytotoxicity of azadirachtin a in human glioblastoma cel lines, Life Sci, 68, 2001, 1153. 23. Pettit G R, Numata A, Iwamoto C, Morito H, Yamada T Goswami A, Clewlow P J, Cragg G M and Schmidt J M, Antineoplastic agents. 489. Isolation and structures of meliastatins 1-5 and related euphane triterpenes from the tree Melia dubia, J Nat Prod, 65, 2002, 1886. 24. Arivazhagan S, Balasenthil S, Nagini S, Garlic and neem leaf extracts enhance hepatic glutathione dependent enzymes during N-methyl-N'-nitro-nitrosoguanidine (MNNG)-induced gastric carcinogenesis in rats, Phytother Res, 14, 2000, 291. 25. Balasenthil S, Arivazhagan S, Ramchandran C R and Nagini S, Chemopreventive potential of neem {Azadirachta indica) on 7, 12-dimethylbenz[a]nthracene (DMBA) induced hamster buccal pouch carcinogenesis, J Ethnopharmacol, 67,1999,189. 26. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with folin phenol reagent, J Biol Chem, 193,1951, 265. 27. Dubois M, Gilles KA, Hamilton JK, Rubers PA and Smith F, Colorimetric method for determination of sugars and related substances, Anal Chem, 28,1956, 350. 28. Ghosh P, Ghoshal S, Thakur P, Lerouge CL, Driouich A and Ray B, Cell wall polysaccharides from Brasica compestis seed cace: Isolation and structural features, Carbohydrate Polym, 57, 2004, 7. 29. Ghosh P, Ghoshal S, Thakur P, Lerouge CL, Driouich A and Ray B, Non-starch polysaccharides frm Sesamum indocum: Isolation and structural features, Food Chemistry, 90, 2005, 719. 30. Blakeney AB, Harris PJ, Henry RJ and Stone BA, A simple and rapid preparation of alditol acetates for monosaccharides analysis. Carbohydrate Res, 113,1983, 291. 31.Baral RN, Sherrat A, Das R, Foon KA and Bhattacharya-Chatterjee M, Murine Monoclonal Anti-Idiotype Antibody as a surrogate Antigen for human Her2/neu. Int J Carter, 92, 2001, 88. 32. Chavan UD, Mackenzie DB and Sahidi F, Protein classification of beach pea (Latyrus maritimus L). Food Chemistry, 75, 2001, 145. 33. Ghosh AK, Naskar AK and Sengupta S, Characterisation of a xylanolytic amyloglucosidase of Termitomyces clypeatus. Biochem Biophys Acta, 139, 1997, 289. We Claim: 1. A process for isolating and characterization of glycoprotein from neem leaf for immunomodulatory and cancer preventive functions of neem leaf glycoprotein (NLGP) thus obtained, said process comprising the steps of: a) Preparation of crude neem leaf preparation (NLP) by soaking shed dried neem leaf powder in Phosphate Buffer Saline (PBS) for a period of 24 hours, b) Extensive dialysis of crude NLP thus obtained against PBS, followed by subsequent concentration by membrane filtration with 10KD molecular weight cut-off. c) Biochemical estimation reveals the presence of glycoprotein (NLGP) in membrane concentrate. d) Exposing said NLGP to temperature gradient of 0°C to 100°C. e) Exposing the NLGP to different proteolytic enzymes. f) Exposing the NLGP to various ions. 2. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said NLGP is exposed to various temperature gradiant of 0°C to 100°C. 3. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said NLGP is exposed to temperature of 56°C for obtaining maximum activity. 4. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the pH of PBS solution is maintained within the range of 5.7 to 8.2 for effectiveness of the NLGP. 5. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said NLGP is exposed to PBS with pH 7.4 for obtaining maximum activity. 6. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the enzymes are selected from the group of proteolytic enzymes, trypsin, papain, and neuraminidase individually for confirming the glycoprotein nature of active component of neem leaf by observation of NLGP degrading activity. 7. A process for isolating and characterization of glycoprotein as claimed in claim 1, where NLGP is also obtained by soaking neem leaf powder into water with pH 6.0 and 7.4, PBS with pH 7.4, Tris buffer saline (TBS) with pH 7.4 individually. ABSTRACT Title: Process for isolating glycoprotein from neem leaf and its characterization to define the imunomodulatory and cancer preventive functions of this glycoprotein The invention provides a process for isolating and characterization of glycoprotein from neem leaf for immunomodulatory and cancer preventive functions of neem leaf glycoprotein (NLGP) thus obtained, said process comprising the steps of: a) preparartion of crude neem leaf preparation (NLP) by soaking neem leaf powder in PBS for a period of 24 hours, b) extensive dialysis of crude NLP thus obtained against PBS, followed by subsequent concentration by membrane filtration, bioactivity of NLGP was tested by: c) exposing said NLGP to temperature gradient of 0°C to 100°C d) exposing the NLGP to different proteolytic enzymes e) exposing the NLGP to various ions, Wherein the said NLGP is exposed to a temperature of 56°C and pH of PBS solution is maintained at 7.4 Important figures are given in next page. Fig. 1. illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured and denatured conditions. Fig. 2. illustrates mouse tumor growth restricting activity of NLP exposed to various proteolytic enzymes and survivability of treated mice.

Full Text

INTRODUCTION
This invention relates to a process for isolating glycoprotein from neem leaf and its
characterization to define immunomodulatory and cancer preventive functions.
Background of invention:
Neem (Azadirachta indica) is a widely prevalent and esteemed wonder tree, mainly
cultivated in Indian subcontinent (1). This unique plant is well known in India and its
neighbouring countries for more than 2000 years as one of the most versatile medicinal
plants having a wide spectrum of biological activity (2). As per recommendation of the
Indian Ayurvadic literature, eight parts of the neem tree (leaf, bark,flower, twig, gum,
seed, pulp and oil) have shown to possess several biologically active components having
a wide variety of activities against different ailments (2). This SARBAROGAMBAPINI
neem generously contributes each and every parts of its body as suitable for human
consumption. This valuable ancient knowledge surprisingly has been neglected in India
and its recognition was chiefly limited within poor rural Indian population, as a village
dispensary (3). Neem products never considered as a prime factor towards Indian
scientists for proper exploration of its therapeutic values and also towards commercial
agencies for proper marketing of the economic values hidden within neem products.
Neem products hit the popular media headlines only after it became the subject of legal
controversy when the European Patent office rejected a patent application from the WR
Grace Company and the United States Department of Agriculture. Although, neem
remained neglected for a long time in the Western countries, interest on this particular
medicinal plant is gradually growing and as a consequence, US National Academy of
Science published a report on 1992 with a title, 'Neem-a tree for solving global problem'
(National Academic Press, USA, 1992).
Neem can be exploited in modern biological research, using toxicological, molecular
biological, immunological techniques. These effort may disclose some new applications
of neem, those are not described in Indian traditional medicine. Immune boost up by
neem was not considered as a potential field of medical research, but the research
associated with present invention will disclose several applications of neem leaf in the
prevention of cancerous growth by stimulation of the body's defense (immune)
mechanism.
Prior Art:
Several pharmacological activities and medicinal applications of various parts of
neem tree are known. Biological activities of neem tree are reported where crude extracts
and their different fractions from leaf, bark, root, seed and oil were used. These reports
have exhibited a wide diversity in biological functions from a group of compounds
present in the various parts of the plant (4). Some of these are as follows:
i. General Health: Neem is known to be useful remedy in some chronic skin diseases,
ulcers, rheumatism, leprosy, dental troubles, intestinal helminthiasis, constipation, asthma

and also as a general health promoter (5-7). Bark, leaf, root, flower and fruit together cure
blood morbidity, biliary afflictions, itching, skin ulcers and burning sensations (4).
Gastroprotective antiulcer effect of neem bark extract has been reported recently (8).
ii. Reproduction: It also has wide range of effects on the reproductive system. Neem oil
is used as a spermicidal (9) and early abortificient agent (10). It has contraceptive and
antifertility properties (11). For long term contraceptive, single intrauterine application
of neem extract has been reported (12). Oral administration of aqueous extract of neem
leaf shows antifertility effect in mice (4). Neem seed preparations have been shown to be
effective to abrogate pregnancy in rodents, monkeys and baboons (4). This antifertility
effects are possibly mediated by activation of the cell mediated immune reaction, thus
can overcome the side effects of steroidal contraceptives.
iii. Immunomodulation: Various products of neem are known to have
immunomodulatory properties. These may act by stimulating cell-mediated and/or
humoral immunity. However, studies are incomplete and preliminary. Some of these
published reports are encouraging. Neem kernel oil has been shown to induce the
secretion of gamma interferon (13) and tumor necrotic factor-alpha (14). Dry neem leaf
could enhance the antibody titre against new castle disease virus (15), bovine serun
albumin and sheep erythrocytes (16). Intraperitoneal treatment of mice with neem oil
reported to be increase the number of leukocytic cells in peritoneal lavage. Peritoneal
macrophages exhibited enhanced phagocytic activity and expression of MHC class-II
antigens after treatment with neem oil (13). Enhancement in macrophage migration
inhibition and foot thickness in mice after neem treatment (17) was also observed. Neem
(NIM-76) can enhance macrophage activity and lymphocyte proliferation response, while
humoral component of immunity was unaffected 18). An aquaous extract from stem bark
of neem has been shown to have an inhibitory influence on complement activation and
superoxide ion production by polymorphonuclear (PMN) leukocytes while having a
stimulatory effect on the production of migration inhibition factor (MIF) by PMN
leukocytes (19). The aqueous extract of stem bark possesses anticomplement activity,
acting both on the alternative as well as classical pathway of complement activation in
human serum.
iv. Cancer: Based on some preliminary in vitro studies and its known effects on immune
system, it can be hypothesized that neem components may have some effects on the
restriction of tumor growth. Neem seed preparation contains a variety of limonoids, some
of which are cytotoxic to N1E-115 mouse neuroblastoma, 143B.TK-human osteosarcoma
and sf9 insect cells (20). Water soluble polysaccharides isolated from the bark of Melia
azadirachta, demonstrated antitumor effect with complete regression of tumors (21).
Cytotoxicity of the neem toxin, azadirachtin A, in human glioblastoma cell lines is also
reported (22). Recently, it is reported that the bark of the giant neem tree Melia dubia
contains euphane type triterpenes, some of them can inhibit the growth of P388
lymphocytic leukemia cell line (23). Effects of these neem compounds and mechanism of
inhibition of tumor cell growth are completely unknown in in vivo. Neem leaf extracts
enhance hepatic glutathione and glutathione dependent enzymes, which may offer a
degree of chemoprevention during N-methyl-N-mtro-N-nitrosoguanidine induced gastric
carcinogenesis in rats (24) and on 7, 12-dimethylbenzanthracene induced buccal pouch

carcinogenesis (25). However, none of these reports was focused on the involvement of
immune system in neem induced tumor growth inhibition. The mechanism of
immunopotentiation is still unknown.
Various parts of neem (Azadirachta indica) is used to cure different human diseases from
the dawn of civilization. In inspiration with the ancient experiences, we are working on
an aquaous preparation of neem leaf (NLP), which was proved to be nontoxic,
hematostimulatory and immunostimulatory. This preparation has immunoprophylactic
function to prevent the growth of murine Ehrlich's carcinoma and B16 melanoma.
Studies utilizing adoptive cell transfer technology suggest that activation of various
immunocompetent cells by neem leaf component(s) may be responsible for tumor growth
restriction. NLP also acts as an adjuvant by inducing an active antitumor immunity in
murine model against tumor antigens. Moreover, NLP mediated immune activation
protects mice from leukepenia, caused by cancer chemotherapy. Constituents present in
the neem leaf responsible for the immune functions are not known. Our preliminary
findings, suggest that NLP has an instructive role in the secretion of Thl cytokines,
IFNy, IL-12 and TNFa. However, active principal(s) present in neem leaf responsible for
such immunobiological activity and physiological condition required for optimum
demonstration of these biologic activities are not disclosed earlier.
Object of invention
The object of the present invention is to disclose the chemical nature of active principal
responsible for immunobiological activity of the neem leaf preparation.
Another object of the invention is to provide a process for isolating glycoprotein from
neem leaf and its characterization to define immunomodulatory and cancer preventive
functions of this glycoprotein.
Yet another object of the invention is for the extraction of this active ingredient and
conditions required for its maximum biological activity, which may have use as a
medicine to prevent cancerous growth.
Summary of invention:
The invention provides that a glycoprotein component present in neem leaf is responsible
for the restriction of tumor growth after prior vaccination. This glycoprotein mediates its
action through immunomodulation of the host's physiological system. Optimum
biological activity is obtained upon exposure of this glycoprotein into a buffered solution
with pH 7.4at a temperature 56°C.
Unique immunomodulatory functions of aqueous preparation of neem leaf (NLP) in
relation to|cancer are disclosed. In an objective to find out the active principle present in
NLP a glycoprotein was detected in NLP, designated as neem leaf glycoprotein (NLGP).
NLGP appeafed in nondenatured PAGE as a single band, whereas, three bands in SDS-
PAGE. This glycoprotein constitutes the carbohydrate moiety of about 33%, consisting of
arabinose, galactose and glucose. Protein moiety of the glycoprotein consists of sixteen

amino acids, except arginin. Scanning electron microscopy reveals the protein like
structure of the NLP constituents. Immunogenecity of this protein was defined by strong
reaction of the anti-NLP sera with NLP by ELISA and immunoblot analysis. To
understand the association of the NLGP with tumor growth restriction, it was exposed to
a gradient of temperature (0°C, 37°C, 56°C and 100°C), pH (5.7, 6.2, 7.4 and 8.2),
enzymes (trypsin, papain and neuraminidase) and ions (phosphate, tris etc.). Exposure of
NLP to the condition not favorable to maintain the protein conformation caused
significant reduction or abolition of the tumor growth restricting function of NLP. Such
exposure also caused diminished immune reactivity of NLP. Overall results demonstrated
the immense potential of newly identified, NLGP, present in NLP as an
immunoprophylactic agent for tumor growth restriction in a clinical setup.
Brief description of accompanying drawing
The scientific features underlying this invention as well as many of the advantages of the
invention will be more readily appreciable when explained with conjunction to
accompanying drawings wherein,
Fig 1 illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured
and denatured conditions.
Fig 2 illustrates GLC-MS analysis of the carbohydrate moiety of the glycoprotein
(NLGP).
Fig 3 illustrates scanning electron micrographs of the glycoprotein (NLGP).
Fig 4 illustrates immune reactivity of the NLP generated sera with NLP by ELISA and
immunoblotting.
Fig 5 illustrates mouse tumor growth restricting activity of dialysed and nondialysed NLP
and survivability of treated mice.
Fig 6 illustrates mouse tumor growth restricting activity of NLP exposed to various
temperatures and survivability of treated mice.
Fig 7 illustrates mouse tumor growth restricting activity of NLP exposed to various pH
and survivability of treated mice.
Fig 8 illustrates mouse tumor growth restricting activity of "NLP exposed to various
proteolytic enzymes and survivability of treated mice.
Fig 9 illustrates, immune reactivity of the NLP generated sera with NLP exposed to
different temperatures, pH and enzymes by ELISA. Electrophoresis of NLP exposed to
different temperatures and pH.

Detailed description of the invention with reference to drawing/example
Detailed descriptions of the accompanying figures are illustrated below-
FIGURE 1 - Analysis of dialyzed NLP by PAGE. NLP was dialyzed against PBS and 20
µg of NLP was analyzed by 12% nondenatured (A) and 20% denatured (C) PAGE. Gel
was stained with silver nitrate method. Molecular weight markers were electrophorsed
with each run.
FIGURE 2 - GLC-MS analysis of carbohydrate moiety of NLGP. NLP was hydrolysed
with 2M Trifluroacetic acid and GLC-MS was performed as described in 'materials and
methods'.
FIGURE 3 - Scanning electron microscopy of NLGP. Freeze dried NLGP was mounted
on circular aluminium stubs with double sticky tape and coated with 20 nm gold using I-
B2 Ion coater. The sample was examined and photographed. Structures of NLP in
different magnifications are presented in A, B, C, as indicated on the photographs.
FIGURE 4 - Immune reactivity of anti-NLP sera with NLP. Mice were immunized with
either NLP or PBS once weekly for four weeks. Immune sera were absorbed with NLP
and absorbed/nonabsorbed sera were reacted with NLP coated on plates by ELISA (A) or
electrophorsed on PAGE by immunoblotting (B).
FIGURE 5 - In vivo tumor growth restriction by total and dialyzed NLP. Three groups of
mice were injected with either PBS or total or dialyzed NLP for 4 weeks. Seven days
after the last injection mice were inoculated with EC tumor cells subcutaneously (1 X106)
(n=6 in each group). Tumor growth was monitored and survivability was noted. Growth
of EC tumor in PBS, total and dialyzed NLP treated mice. *p PBS injected mice. +p of each group of mice (B).
FIGURE 6 - In vivo tumor growth restriction by total NLP exposed to different
temperatures. Five groups of mice were injected with either PBS or total NLP exposed to
100°C, 56°C, 37°C and 0°C weekly for four weeks. Seven days after the last injection
mice were inoculated with EC tumor cells subcutaneously (1 X106) (n = 6 in each group).
Tumor growth was monitored and survivability was noted. Growth of EC tumor in PBS
and NLP (exposed to different temperature) treated mice. **p PBS injected mice. p (A). Survival of each group of mice (B).
FIGURE 7 - In vivo tumor growth restriction by total NLP exposed to different pHs.
Four groups of mice were injected with total NLP exposed to pH 8.0, pH 7.4, pH 6.2 and pH 5.7 weekly for four weeks. Seven days after the last injection mice were inoculated
with EC tumor cells subcutaneously (1 X106) (n = 6 in each group). Tumor

growth was monitored and survivability was noted. Growth of EC tumor in NLP
(exposed to different pH) treated mice. **p injected with NLP, exposed to pH 8 (A). Survival of each group of mice (B).
FIGURE 8 - In vivo tumor growth restriction by total NLP exposed to different
enzymes. Four groups of mice were injected with either total NLP or NLP exposed to
trypsin, papain and neuraminidase weekly for four weeks. Seven days after the last
injection mice were inoculated with EC tumor cells subcutaneously (1 X106) (n=6 in each
group). Tumor growth was monitored and survivability was noted. Growth of EC tumor
in NLP (exposed to various enzymes) treated mice. p injected with trypsin, papain and neuraminidase exposed NLP (A). Survival of each
group of mice (B).
FIGURE 9 - Alterations in NLP by differential exposure to enzymes, pHs and
temperatures. NLP was exposed to different enzymes (trypsin, papain and
neuraminidase), temperatures (100°C, 56°C, 37°C and 0°C) and'pHs (pH 8.0, pH 7.4, pH
6.2 and pH 5.7). A. These differentially treated NLPs were coated on microtitre plates.
Anti-NLP sera (generated by immunization of Swiss mice with NLP) was tested for its
immune reactivity with coated NLPs by ELISA. p reaction with enzyme treated NLP; p NLP exposed to pH 8 and pH 5.7; +p NLP, exposed to 0°C and 100°C. B. Differentially treated NLPs were electrophorsed on
20% SDS-PAGE and stained with silver nitrate.
a) Methods involved in the invention.
Neem Leaf Preparation (NLP)
Matured neem leaves of same size and color (indicative of same age), procured
from a standard source were shed-dried to pulverize. Neem powder was soaked overnight
in phosphate buffered saline (PBS), pH 7.4 just one day, before the experiment.
Supernatant was collected by centrifugation at 1500 rpm and was membrane (0.22 µm)
filtered for in vivo or in vitro uses. Extraction condition was changed in studies for
characterization of the active component. Crude NLP was then extensively dialyzed
against PBS, pH 7.4 and concentrated by Centricon Membrane Filter (Millipore
Corporation, Bedford, MA, USA) with 10 Kda molecular weight cut off. Endotoxin
content of the freshly prepared NLP was determined by Limulus Amebocyte Lysate
(LAL) test as per manufacturer's (Salesworth India, Bangalore) instruction. The
endotoxin content of all the batches of NLP was found to be less than 6 pg/ml.
Animals and tumor cells
Female Swiss mice (age, 6-8 weeks, body weight, 24-27 gms) were obtained from
Institutional Animal Care and Maintenance Department. Dry pellet diet and water were
given ad libitum. Maintenance and treatment of animals were given according to the
guidelines established by the Institutional Animal Care and Ethics Committee. Ehrlich's

carcinoma (EC) was maintained in Swiss mice by intraperitoneal passage of viable tumor
cells (1 X 107), as determined by trypan blue dye exclusion assay. These EC cells were
used for in vivo tumor development.
Protein estimation
Protein concentration of NLP was measured by Lowry's method using Folin
Phenol reagent (Lowry et al, 1951) and by UV absorption. For these two methods of
protein estimation, optical density was measured at 660 nm and 280 nm respectively.
Analysis of carbohydrate moiety
Amount of carbohydrate moiety in protein, present in NLP was estimated using
phenol- sulphuric acid method (Dubois et al, 1956). In brief, 80% phenol (lOOul) was
mixed with NLP (1 ml) and then concentrated sulphuric acid (1 ml) was added slowly.
After 10 mins, absorbance was measured at 490 nm using microplate reader (Tecan
Spectra, Grodig, Austria).
Analysis of sugar moiety by GLC-MS
NLP was hydrolysed with 2M Trifluroacetic acid for 3 hrs at 100°C in a boiling water
bath (Ghosh et al, 2005; Ghosh et al, 2005). Myo-inositol was used as internal standard.
Hydrolytic losses were accounted for by using standard sugar mixtures containing all the
sugars usually present in the cell wall of a plant. After acid hydrolysis, the solution was
neutralized with 25% ammonia solution (0.7 ml) and the monosaccharides were reduced
with 0.1 ml sodium borohydride in 3M ammonia solution at room temperature for one hr.
the excess borohydride after reduction was decomposed by addition with acetic acid.
Ultimately, the free alditols were obtained as a mixture in dried condition by lyophilizing
the above solution. Acetylation of dried sample was first carried out by acetic anhydride
and L-Methylimidazole as catalyst (Blakeney et al, 1983). Derived alditol acetates were
then analyzed and identified by GLC-MS. GLC-MS was carried on a Shimadzu GC-MS
(QP-5050) instrument (Shimadzu, Japan) with a Flame ionization Detector and DB-225
(JW) column using helium as carrier gas. The temperature of the injection port was
250°C and the column oven temperature was maintained at 210°C (isothermal).
Polyacrylamide gel electroporesis (PAGE)
Crude and dialyzed NLP, after concentration by membrane filtration, were
analyzed by native-PAGE and SDS-PAGE with different acrylamide concentrations.
Molecular weight marker (SC-2035; Santacruz, USA) was electrophorsed in each time to
know the molecular weights of protein bands. In some cases, NLP was exposed to
different temperature and pH before electrophoretic analysis.
Antibody production and ELISA
Swiss mice (n=6) were injected with NLP (extract obtained from 0.25mg of dry
neem powder) weekly for 4 weeks in total. Blood was collected after completion of the

fourth injection from anaesthesized mice by retro-orbital puncture and serum separated.
Serum antibodies were assessed by ELISA (Baral et al, 2001). Briefly, the microtiter
plates were coated with NLP (10µg/50µl/well) for overnight and blocked with 5% BSA.
Anti-NLP sera (1:50 diluted in 1% BSA) were added in the wells in triplicate and
incubated for 2 hours. The plates were washed with PBS containing Tween-20 and goat
anti-mouse Ig labeled with peroxidase (Sigma, St. Louis) was added at a dilution of 1:
1000. Color was developed with TMB substrate solution (OptEIA™, BD-Pharmingen).
Reaction was stopped with 2N H2SO4 solution and absorbance was measured at 450 nm
using microplate reader (Tecan Spectra, Grodig, Austria).
Immunoblotting
NLP was electrophorsed on 10% SDS-PAGE, transferred on nitrocellulose
membrane, blocked with POD blocking substrate (Roche Diagnostics, Mannheim,
Germany) and incubated with anti-NLP sera, before and after absorption with NLP, for
overnight at 4°C. After washing, blots were incubated with horseradish peroxidase
conjugated secondary antibody for two hours at room temperature. Bands were detected
using chemiluminescence (Roche Diagnostics, Mannheim, Germany) according to the
manufacturer's manual.
Absorption of immune sera with NLP
NLP generated immune sera was absorbed with NLP (5 µg/ml of 1:50 diluted
sera) for 1 hr at 37°C and centrifuged at 5000g for 10 mins. Absorbed sera were then
tested by ELISA and immunoblotting for their reactivity.
Scanning electron microscopy
Structural morphology of protein fraction present in NLP was studied using
Scanning Electron Microscope (Chavan et al, 2001). Freeze dried neem protein was
mounted on circular aluminium stubs with double sticky tape and coated with 20 nm gold
using I-B2 Ion coater. The sample was examined and photographed in a scanning
electron microscope (Hitachi S-530 SEM, Hitachi Ltd., Tokyo, Japan).
Amino acid analysis
Amino acid analysis was performed in a PICO.TAG system according to the
PICO.TAG operation manual (Waters, USA). NLP was extensively dialyzed against
deionised water for 24 hrs, dried and the protein (20µg) was hydrolysed by 6N HC1
containing 1% phenol for 24 hrs at 105°C in the PICO.TAG work station. Hydrolysed
sample and standard amino acid mixture, 'Standard S' (0.0005 ml) were taken in
respective'tubes, introduced into the reaction vial and were dried completely. These were
then derivatized by phenyl isothiocyanate (PITC) solution (ethanol: triethylamine : water
: PITC = 7:1:1:1 by volume) for 20 min at 25°C (33). The vials were then dried and
samples were reconstituted in diluent solution (Na2HPO4. 0.071%, w/w, pH 7.4
containing acetonitrile 5%, v/v). The samples were analysed at 38°C as per PICO.TAG

manual using C18 column (Ghosh et al, 1997). Detector setting, chart speed and run time
were AT 128 at 254 nm, 2 cm/min and 32 min respectively. Amino acids present in
unknown sample (NLP) were determined quantitatively by comparing the peak areas
(745b data module print out) of amino acids present in 'Standard S'.
Exposure of NLP in different temperature, pH and enzymes
Crude and dialyzed NLP were exposed to different temperatures (100°C for 5
mins, 56°C, 37°C and 0°C for 1h) and phosphate buffers with pH 5.7, 6.2, 7.4 and 8.0 for
overnight. NLP was also digested with proteolytic enzymes, like, trypsin (SRL) (20
µg/mg NLP), papain (SRL) (50 µg/mg NLP) and neuraminidase (Sigma) (0.6 U with 2
mM PMSF/mg of NLP) for one hour at 37 C. Trypsin and papain were inactivated by
rapid chilling and neuraminidase was removed by centrifugation.
Exposure of NLP to various ions
NLP was prepared by soaking neem leaf powder into water with pH 6.0 and pH
7.4, PBS, pH 7.4, Tris buffered saline (TBS), pH 7.4, 10mM disodium hydrogen
phosphate solution, 1.4mM potassium dihydrogen phosphate solution individually.
Except variation in the ions, other conditions were identical, as described in the section,
'Neem Leaf Preparation'.
Tumor growth restriction assay
Swiss mice were injected with NLP either exposed to various temperatures, pH
or digested with different enzymes or soaked with solutions containing various ions.
Extracts obtained from 0.25 mg of neem powder were used for treatment of each week
and NLP treatment was given for 4 weeks in total. After completion of the treatment,
each mouse was inoculated with EC cells (1X107) subcutaneously on right hind leg
quarter. Growth of solid tumor (in mm3) was monitored weekly by caliper measurement
using the formula: (width2 x length)/2. Survival of mice was monitored regularly, till
tumor size reached to 25 mm in either direction.
Statistical analysis
All results from in vivo experiments represent the average of two separate
experiments involving eight mice in each group. Statistical significance was established
by using INST AT 3 Software.

Features of the invention
NLP contains protein
Protein estimation suggests the presence of protein in NLP
NLP is soaked in PBS, pH 7.4 for overnight, extract was collected and dialyzed
extensively against PBS. Protein concentration of the dialyzed NLP was measured by Lowry's
method using Folin's Phenol reagent. Determination of the optical density confirms the presence
of protein and quantitative estimation of NLP revealed the presence of protein and yield of it is
1.4 µg/mg of dry neem leaf (Table 1).

Polyacrylamide gel electrophoresis identifies proteins in NLP
Dialyzed NLP was analyzed by 12% nondenatured PAGE and 20% denatured SDS-
PAGE. Nondenatured PAGE and SDS-PAGE demonstrated one and three major protein bands
respectively (Fig. 1). Molecular weight of three bands appeared in 20% SDS-PAGE was 65Kda,
36Kda and 22Kda.
Identified NLP protein is a glycoprotein
Biochemical estimation reveals the presence of 33.3% carbohydrate in the protein
present in NLP, thus, may be glycoprotein. After dialysis, concentration of this
glycoprotein was increased significantly (Table 2). Carbohydrate moiety in NLP was
analyzed by GLC-MS and results suggest the presence of 53% arabinose, 36% galactose
and 11% glucose (Fig. 2). Henceforth, the protein present in NLP will be termed as neem
leaf glycoprotein (NLGP).
NLGP consists of various amino acids, except arginin
Amino acid analysis of protein part of the NLP revealed the presence of sixteen
amino acids of which eight were essential. This protein is rich in Glutamic acid, Glycine,
Threonine and Valine but low in Methionine, Cysteine and Histidine. Arginine is not
present in NLGP. Result of amino acid analysis of NLGP is presented in Table 3.
Electron microscopy identifies protein structure
Fig. 3 reveals the structure of NLGP after detection in scanning electron
microscope using different magnifications. SEM micrographs show 3-dimensional
structures through 2-dimensional images. The image demonstrates the heterogeneous
surface with void spaces of NLGP.
NLGP is efficient to generate antibody
Mice were immunized with NLP weekly for four weeks. Blood was collected on
seventh day after last injection to separate the serum. Immune sera were strongly reactive
with NLGP, as detected by ELISA and immunoblotting, demonstrating its nature like a

protein antigen (Fig. 4). This reactivity of the immune sera was significantly decreased
after its absorption with NLP.
NLGP of NLP is responsible for tumor growth restriction
Dialysis increases tumor growth restricting activity of NLP
Two groups of Swiss mice were injected with either total NLP or dialyzed NLP
weekly for four weeks. Three days after the last injection both groups of mice were
inoculated with Ehrlich carcinoma cells. Fig. 5 a documented the tumor growth pattern in
dialyzed and nondialyzed NLP injected mice, which clearly shows that dialyzed NLP
gave better protection to the growth of Ehrlich's carcinoma. Mice received dialyzed NLP
before tumor inoculation also survived longer than the group received total NLP (Fig.
5b). Nondialyzed NLP also caused tumor growth restriction in comparison to PBS
control.
Exposure to 56° C increases the tumor growth restricting activity of NLP than
0oC,3rC and 100oC
Four groups of Swiss mice were injected with total NLP exposed to temperatures,
like, 100°C, 56°C, 37°C and 0°C weekly for four weeks in total. Three days after the last
injection mice of all groups were inoculated with Ehrlich carcinoma cells. Minimum
tumor growth was demonstrated in mice treated with NLP exposed to 56°C, than those
mice immunized with NLP exposed to 0°C, 37°C and 100°C (Fig. 6). Identical experiment
was performed with dialyzed NLP, and almost similar result was obtained (data not
shown).
pH of extracting buffer influences tumor growth restricting activity of NLP
Four groups of Swiss mice were injected with total NLP soaked in phosphate
buffer with different pH, like, pH 5.7, 6.2, 7.4 and 8.2 weekly for four weeks. Three days
after the last injection mice of all groups were inoculated with Ehrlich carcinoma cells.
Maximum tumor growth was observed in mice injected with NLP prepared with pH 5.7
buffer and significant tumor growth restriction was demonstrated when NLP prepared
with pH 7.4 and pH 6.2 buffers. Moderate tumor growth restriction was observed during
injection with NLP, pH 8.0 (Fig. 7). Identical experiment was performed with dialyzed
NLP, demonstrated almost similar result (data not shown).
Exposure of NLP to proteolytic enzymes, trypsin, papain and neuraminidase abolishes
tumor growth restricting activity
Four groups of Swiss mice were injected with either total NLP or NLP digested
with trypsin, papain and neuraminidase weekly for four weeks. Three days after the last
injection mice of all groups were inoculated with Ehrlich carcinoma cells. No significant
restriction of the tumor growth was noticed when NLP was exposed to all three enzymes,
in comparison to mice injected With NLP only (Fig. 8). Similar experiment was

performed with dialyzed NLP and also demonstrated abolition of the tumor growth
restricting activity of NLP (data not shown).
Antibody against NLP reacts differentially with NLP exposed to different temperatures,
pH and enzymes
Swiss mice were injected with NLP weekly for four weeks. Immune sera obtained after
fourth injection were reacted with NLP before and after exposure to various
temperatures, pH, enzymes or soaked with solutions containing various ions by ELISA
(Fig. 9a). Maximum immune reactivity was noticed with those treated NLP, showed
significant tumor protection. For example, injection with NLP exposed to 56°C
demonstrated maximum tumor protection (Fig. 6) and this NLP reacted maximally with
anti-NLP sera. Differentially treated NLPs were electrophorsed and partial degradation of
the NLP was detected, in those cases where minimum tumor protection was observed
(Fig. 9b).

References:
1. Schmutterer H (ed), in The neem tree: Source of unique natural products for
integrated pest management, medicine, industry and other purposes, (VCH,
Weinhrim: Hiedelberg) 1995,1.
2. Devasagayam T P A & Sainis K B, Immune system and antioxidants, especially those
derived from Indian medicinal plants, Indian J Exp Biol, 40, 2002, 639.
3. Satyavati G V, Raina M K, Sharma M, in Medicinal plants of India, (Indian Council
of Medical Research, New Delhi) 1976, 363.
4. Biswas K, Chattopadhyay I, Banerjee R K and Bandopadhyay U, Biological activities
and medicinal properties of Neem (Azadirachta indica). Curr Sci, 82, 2002,1336.
5. Ross I A, Azadirachta indica, A. Juss, in Medicinal plants of the World, Vol 2:
Chemical Constituents, Traditional and Modern Uses, (umana Press Inc.Totowa, NJ),
2001,81.
6. Singh P P, Junnarkar A Y and Reddi G S, Azadirachta indica: Neuro-Psycho-
Pharmacological and Antimicrobiological studies, Fitoterapia, 58, 1987, 235.
7. Meisner J and Azcher K R S, Insect growth regulating (IGR) effects of neem
products on Spodoptera littoralis, Schriftner Gtz, 161, 1984, 345.
8. Bandopadhyay U, Biswas K, Chatterjee R, Bandopadhyay D, Chattopadhyay I,
Ganguly C K, Chakraaborty T, Bhattacharya K, Banerjee R K, Gastroprotective
effect of neem {Azadirachta indica) bark extract; possible involvement of H9+0-
K(+)-ATPase inhibition and scavenging of hydroxyl radical, Life Sci, 71,2002,2845.
9. Shaikh P D, Manivannam B and Pathan K M, Antispermatic activity of. Azadirachta
indica leaves in albino rats, curr Sci, 64,1993, 688.
10. Sinha K C, Riar S s and Bardhan P, Anti-implantation effect of neem oil. Ind J Med
Res, 80, 1984, 708.
11. Choudhury D N, Singh J N and Verma S K, Antifertility effects of leaf extracts of
some plants in male rats, Ind J Exp Biol, 28,1990, 714.
12. Upadhyay S N, Kaushic C and Talwar G P, Antifertility effects of neem {Azadirachta
indica) oil by single intra-uterine administration: A novel method for contraception,
(Proc Royal Soc Lond) 242, 1990, 175.
13. Upadhyay S N, dhawan S, Garg S and Talwar G P, immunomodulatory effects of
neem {Azadirachta indica) oil, Int J Immunopharmac, 14,1992, 1187.
14. Thatte U and Dahanukar S, Rasayana concept: clues fro immunomodulatory therapy,
in Immunomodulation, edited by S N Upadhyay, narosa Publishing House, New
Delhi, 1997,141.
15. Sadekar R D, Kolte A Y, Barmase B S and Desai V F, Immunopotentiating effects of
Azadirachta indica (Neem) dry leaves powder in broilers, naturally infected with IBD
virus, Ind j Exp Biol, 36,1998,1151.
16. Logamble S M and Michael R D, Immunostimulatory effect of azadirachtin in
Oreochromis mossambicus (Peters), Ind J Exp Biol, 38, 2000, 1092.
17. Ray A, Banerjee B D and Sen P, Modulation of humoral and cell mediated immune
responses by Azadirachta indica (neem) in mice, Ind J Exp Biol, 34,1996, 698.
18. Sai Ram M, Sharma S K and Ilavazhagan G, Immunomodulatory effects of NIM-76,
avolatile fraction from neem oil, J Ethnopharmacol, 55,1997,133.
19. Labadie R P, Nat J M and Simons V D, Advances in immunomodulatory agents from
medicinal plants and traditional preparations. In Proceedings of XI Asian Symposium
on Medicinal Plants and Species, Bandung, Indonesia, 1989,407.

20. Cohen E, Quistad G B & Casida J E, Cytotoxcity of nimbolide, epoxyazadiradione
and other limonoids from neem insecticide. Life Sci, 58,1996,1057.
21. Shimizu M, Takai M, Inone K, takeda T and Ogiwara Y, Antitumor polysaccharides
from Melia azadirachta bark extracts and their purification. (Patent-Japan Kokai
Tokkyo Koho-01 275, 602,1989), 9.
22. Akudugu J Gade G and Bohm L, Cytotoxicity of azadirachtin a in human
glioblastoma cel lines, Life Sci, 68, 2001, 1153.
23. Pettit G R, Numata A, Iwamoto C, Morito H, Yamada T Goswami A, Clewlow P J,
Cragg G M and Schmidt J M, Antineoplastic agents. 489. Isolation and structures of
meliastatins 1-5 and related euphane triterpenes from the tree Melia dubia, J Nat
Prod, 65, 2002, 1886.
24. Arivazhagan S, Balasenthil S, Nagini S, Garlic and neem leaf extracts enhance
hepatic glutathione dependent enzymes during N-methyl-N'-nitro-nitrosoguanidine
(MNNG)-induced gastric carcinogenesis in rats, Phytother Res, 14, 2000, 291.
25. Balasenthil S, Arivazhagan S, Ramchandran C R and Nagini S, Chemopreventive
potential of neem {Azadirachta indica) on 7, 12-dimethylbenz[a]nthracene (DMBA)
induced hamster buccal pouch carcinogenesis, J Ethnopharmacol, 67,1999,189.
26. Lowry OH, Rosebrough NJ, Farr AL and Randall RJ, Protein measurement with folin
phenol reagent, J Biol Chem, 193,1951, 265.
27. Dubois M, Gilles KA, Hamilton JK, Rubers PA and Smith F, Colorimetric method
for determination of sugars and related substances, Anal Chem, 28,1956, 350.
28. Ghosh P, Ghoshal S, Thakur P, Lerouge CL, Driouich A and Ray B, Cell wall
polysaccharides from Brasica compestis seed cace: Isolation and structural features,
Carbohydrate Polym, 57, 2004, 7.
29. Ghosh P, Ghoshal S, Thakur P, Lerouge CL, Driouich A and Ray B, Non-starch
polysaccharides frm Sesamum indocum: Isolation and structural features, Food
Chemistry, 90, 2005, 719.
30. Blakeney AB, Harris PJ, Henry RJ and Stone BA, A simple and rapid preparation of
alditol acetates for monosaccharides analysis. Carbohydrate Res, 113,1983, 291.
31.Baral RN, Sherrat A, Das R, Foon KA and Bhattacharya-Chatterjee M, Murine
Monoclonal Anti-Idiotype Antibody as a surrogate Antigen for human Her2/neu. Int
J Carter, 92, 2001, 88.
32. Chavan UD, Mackenzie DB and Sahidi F, Protein classification of beach pea (Latyrus
maritimus L). Food Chemistry, 75, 2001, 145.
33. Ghosh AK, Naskar AK and Sengupta S, Characterisation of a xylanolytic
amyloglucosidase of Termitomyces clypeatus. Biochem Biophys Acta, 139, 1997,
289.

We Claim:
1. A process for isolating and characterization of glycoprotein from neem leaf for immunomodulatory and
cancer preventive functions of neem leaf glycoprotein (NLGP) thus obtained, said process comprising
the steps of:

a) Preparation of crude neem leaf preparation (NLP) by soaking shed dried neem leaf powder in
Phosphate Buffer Saline (PBS) for a period of 24 hours,
b) Extensive dialysis of crude NLP thus obtained against PBS, followed by subsequent
concentration by membrane filtration with 10KD molecular weight cut-off.
c) Biochemical estimation reveals the presence of glycoprotein (NLGP) in membrane
concentrate.
d) Exposing said NLGP to temperature gradient of 0°C to 100°C.
e) Exposing the NLGP to different proteolytic enzymes.
f) Exposing the NLGP to various ions.

2. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said
NLGP is exposed to various temperature gradiant of 0°C to 100°C.
3. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said
NLGP is exposed to temperature of 56°C for obtaining maximum activity.
4. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the pH of
PBS solution is maintained within the range of 5.7 to 8.2 for effectiveness of the NLGP.
5. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the said
NLGP is exposed to PBS with pH 7.4 for obtaining maximum activity.
6. A process for isolating and characterization of glycoprotein as claimed in claim 1, wherein the enzymes
are selected from the group of proteolytic enzymes, trypsin, papain, and neuraminidase individually for
confirming the glycoprotein nature of active component of neem leaf by observation of NLGP degrading
activity.
7. A process for isolating and characterization of glycoprotein as claimed in claim 1, where NLGP is also
obtained by soaking neem leaf powder into water with pH 6.0 and 7.4, PBS with pH 7.4, Tris buffer
saline (TBS) with pH 7.4 individually.

ABSTRACT

Title: Process for isolating glycoprotein from neem leaf and its
characterization to define the imunomodulatory and cancer preventive
functions of this glycoprotein
The invention provides a process for isolating and characterization of
glycoprotein from neem leaf for immunomodulatory and cancer preventive
functions of neem leaf glycoprotein (NLGP) thus obtained, said process
comprising the steps of: a) preparartion of crude neem leaf preparation (NLP) by
soaking neem leaf powder in PBS for a period of 24 hours, b) extensive dialysis
of crude NLP thus obtained against PBS, followed by subsequent concentration
by membrane filtration, bioactivity of NLGP was tested by: c) exposing said
NLGP to temperature gradient of 0°C to 100°C d) exposing the NLGP to
different proteolytic enzymes e) exposing the NLGP to various ions, Wherein the
said NLGP is exposed to a temperature of 56°C and pH of PBS solution is
maintained at 7.4
Important figures are given in next page.
Fig. 1. illustrates the electrophoretic profile of the glycoprotein (NLGP) in nondenatured
and denatured conditions.
Fig. 2. illustrates mouse tumor growth restricting activity of NLP exposed to various
proteolytic enzymes and survivability of treated mice.

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429-KOL-2007-(22-01-2014)-CORRESPONDENCE.pdf

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429-KOL-2007-GRANTED-DRAWINGS.pdf

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Patent Number

259434

Indian Patent Application Number

429/KOL/2007

PG Journal Number

11/2014

Publication Date

14-Mar-2014

Grant Date

12-Mar-2014

Date of Filing

21-Mar-2007

Name of Patentee

RATHINDRANATH BARAL, SUBRATA LASKAR, ANAMIKA BOSE, KOUSTAV SARKAR

Applicant Address

CHITTARANJAN NATIONAL CANCER INSTITUTE 37, S.P.MUKHERJEE ROAD, KOLKATA 700026,

Inventors:

#	Inventor's Name	Inventor's Address
1	SUBRATA LASKAR	DEPARTMENT OF CHEMISTRY, UNIVERSITY OF BURDWAN, BURDWAN,
2	RATHINDRANATH BARAL	CNCI,37,S.P.MUKHERJEE ROAD, KOLKATA 700026,
3	ANAMIKA BOSE	37, S.P.MUKHERJEE ROAD, KOLKATA 700026,
4	KOUSTAV SARKAR	37, S.P.MUKHERJEE ROAD, KOLKATA 700026,

PCT International Classification Number

A61K39/00

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA