Title of Invention

"A PROCESS FOR THE ISOLATION OF NEW PROTEIN (INDIN-SAA) FROM ROOTS OF BOERHAAVIA DIFFUSA LINN.(SANTH)"

Abstract

A process for the isolation of protein (INDIN-SAA) from the roots of Boerhaavia diffusa Linn (Santh) characterized in that:- (a) Taking roots of plant such as herein described and sterilizing it by sterilization media (1.5 ml Dettol + 1 liter distilled water) for 30 min. (b) Homogenizing the sterilized roots with extraction buffer (1M NaCI, 1M Sucrose, 0.2M Acetate Buffer pH 5.0, 0.1M EDTA ph 8.0, ß-mercaptoethanol 200 µl and 50 ml of distilled water), such as herein described, then crushing the roots with acid wash sand. Then filtering them through four layer of cheese cloth to obtain the root extract. (c) Centrifuge the roots extract at 8500-9000 rpm for 30 min. discard the pellet so obtained and purify the supernatant by 0-80% Ammonium Sulphate precipitation. (d) Allow to this to stand overnight and again centrifuge at 9000-95000 rpm for 1 hrs, discard the supernatant and dissolve the pellet containing INDIN-SAA in Sodium acetate buffer (Ph=5) such as herein described. (e) The protein INDIN-SAA is estimated and identified by standard method.

Full Text

BRIEF DESCRIPTION OF INVENTION:- UA PROCESS FOR THE ISOLATION OF PROTEIN (INDIN-SAA) FROM ROOTS OF Boerhaavia diffusa Linn. (SANTH). Disease- Anjeer Bal- Lymph node swelling (Scrofula adenitis), Tumor cancer and AIDS (HIV) PRIOR ART:- Boerhaavia diffusa Linn (Santh) is an herb and distributed throughout India, the plant is mentioned as a very useful drug in standard books on "Ayurveda". Botanical Name: Boertiaavia diffusa Linn. Family: Nyctaginaceae Genus: Boertiaavia Species: diffusa, hirsuta Synonyms: B. repens Linn. Common English Names: Hog Weed, Horse Purslane Common Indian Names: Hindi: Snathikari, Sant, Santh Sanskrit: Punamava, Raktakanda, Shothaghni, Varshabhu Gujarati: Dholia-saturdo, Moto-satoda. Canarese: Kommegida Marathi: Tambadivasu Bengali: Punurnava Tamil: Mukaratee-Kirei Telugu: Punernava Other Names: Pig Weed, Atikamaamidi, Biskhapra, Djambo, Etiponia, Fowl's Lice, Ganda'dar, Ghetuli, Katkatud, Mahenshi, Mamauri, Ndandalida, Oulouni Niabo, Paanbalibis, Patal-jarh, Pitasudu-pala, Purnoi, Samdelma, San, Santi, Satadi Thikedi, Satodi, Spreading Hog Weed, Tellaaku, Thazhuthama, Thikri, Touri-touri, Tshrana, Yoegbe. Santh helps maintain efficient kidney and urinary functions with its diuretic, anti¬spasmodic and anti-inflammatory action. (1) Habitat: It occurs abundantly as a weed throughout India, up to an altitude of 2,000 m in the Himalayas. It is also cultivated to some extent in West Bengal. (2) Morphology Description (Habit): A diffusely branched, pubescent or glabrous, prostrate herb. The rootstock is stout, fusiform and woody; the stems are creeping, often purplish, swollen at the nodes; the leaves are long-petioled, ovate or oblong-cordate, entire or sinuate; the flowers are red, pink or white, in small umbels; the fruits are ovate, oblong, pubescent, five-ribbed, glandular anthocarps. (3) Useful Parts: Leaves, Seeds (It may be used but not more effective) and Roots (More effective part against Scrofula adenitis and tumor cancer). (4) Chemical Constituents: Santh contains b-Sitosterol, a-2-sitosterol, palmitic acid, ester of b-sitosterol, tetracosanoic, hexacosonoic, stearic, arachidic acid, urosilic acid, Hentriacontane, b-Ecdysone, triacontanol etc. (5) Medicinal Uses: According to Ayurveda, Santh is bitter, cooling, astringent to bowels, useful in biliousness, blood impurities, leucorrhoea, anaemia, inflammations, asthma, alternatives etc. The leaves are useful in dyspepsia, tumors, spleen enlargement and abdominal pains. According to Unani system of medicine, the leaves are appetizer, alexiteric, useful in opthalmia, in joint pains. Seeds are tonic expectorant, carminative, useful in lumbago, scabies. The seeds are considered as promising blood purifier. This plant worldwide used for the disease treatment as a crude form (Study in Ethanbotany Branch). India, Brazil, Elsewhere, Guatemala, Iran, Nigeria, West Africa, Japan are mostly used this plant. The roots are employed for many purposes including liver, Gallbladder, Kidney, Renal and Urinary disorder. DETAIL INVENTION:- First we isolate protein from roots of Santh. This protein show low molecular weight (SDS-PAGE) and this protein is new, so we called by new name INDIN-SAA (taken from origin country India) has anti disease activity against Scrofula adenitis and tumor cancer. We show the experiment and results on Guiny pig/White rat and man, this results show that this protein more effective action against Scrofula adenitis (Anjeer Bal) and Tumor cancer. ♦ In Scrofula adenitis diseases, swelling of lymph node in neck or lymph node swells up. In this disease several tumors take growth in neck from which several fibers generate. Fever is caused. After all this these tumors ruptures and secretion start. Patient becomes weak. Operation is only a treatment of this disease. In operation the tumor is taken out but fibers remains there fixed which after some time again form new tumors. After this entire patient will die. These fibers are also found in Cancer. When cancer tumor is removed from the body of Patient but fibers remains fixed there, which again cause Cancer. A tuberculosis infection of the of the skin of the neck most often caused by Mycobacteria (including Mycobacterium tuberculosis). In adults and children it is usually caused by Mycobacterium scrofulaceum and M. avium. INFECTION. SPREDING & TUMOUR FORMATION Infection with Mycobacteria is usually caused by inhallingair, contaminated by These organisms. The bacteria spread through out the body and may cause rubbery enlargement of the lymph nodes in the neck (cervical lymph nodes) as well as elsewhere. If they are not treated the lymph nodes may become ulcer, producing draining sores. FUNCTION OF LYMPH CELL & TUMOUR FORMATION Cellular immunity, in particular the T-Cell population, is instrumental in controlling infection. Activated T-Cell generates cytokines that enable tissue macro phases and monocytes to destroy the Mycobacteria and form a tubercle or granuloma. Therefore in the population with HIV, the incidence of tuberculosis infection is 500 times greater then in general population. The skin become adherent to the under line mass. This stage may be progress to rupture, hence sinus formation. Extra pulmonary T.B., such as scrofula (in modem times surgery has played a pivotal role in the diagnosis and treatment of scrofula. PATHOPHYSIOLOGY Humans are only its reservoir. Transmission is from person to person via respiratory route by inhalation of small aerosols. After a short period of replication (DNA dependent bacteria but not RNA polymerase) in the lungs silent dissemination occurs through the lymphohematogenous system to extra pulmonary sites including cervical lymph nodes. Scrofula is the Latin word for brood sow, & it is termed applied to T.B. of the neck. Cervical T.B. is usually a result of an infection in the lymph nodes known as Lymphadenitis. SYMPTOMS OF DISEASES > Painless swelling of cervical (neck) lymph nodes. > Ulceration. > Lymph node may be enlarged elsewhere. > Fever and chills, sweats and weight loss can occur in 20% of individuals. > Histology (examination under a micro scope) shows granulomas. LOCAL TREATMENT Treatment is usually with 9-12 months of antibiotics. Some antibiotics are- > INH > RIFAMPIN > PYRAZINAMIDE > ETHEMBUTOL etc. > Santh (Boerhaavia diffusa) plants were obtained from local rural areas. Flowchart:- (Diagram Removed) We take plant (Boertiaavia diffusa Linn) roots weight 320 gm; sterilize by sterilization media (1.5 ml Dettol + 1 liter distilled water) for 30 min. We homogenize sterilize root with Extraction buffer (1M Nacl, 1M Sucrose, 0.2M Acetate Buffer pH 5.0, 0.1M EDTA ph 8.0, ß- mercaptoethanol 200 µl and 50 ml of distilled water), then crush the root with acid wash sand. Now filters this through four layers of cheese cloth. By this we get crude protein. This crude protein is centrifuged at 8500 rpm for 30 min, discard the pellet & use supernatant. Now purify the protein by 0-80% Ammonium Sulphate precipitation. Again centrifugation for 1 hrs at 9000 rpm. Again discard supernatant & dissolve Protein pellet in Sodium Acetate buffer [Ph =5], Estimation of protein is done by Lowry Method. Protein Identified through SDS-PAGE (Gel Electrophoresis method), & a band is obtained. Low molecular weight protein called INDIN-SAA. This protein is of used against Anjeer bal.Tumor cancer and AIDS. This Protein can be take orally within a period of about 3 months of symptoms of above mention diseases. The raw material is extensively available in this country for animal and human use. No complicate procedures are required because protein extract can be used orally. Accordingly, A process for the isolation of protein (INDIN-SAA) from the roots of Boertiaavia diffusa Linn (Santh) characterized in that:- (a) Taking roots of plant such as herein described and sterilizing it by sterilization media (1.5 ml Dettol + 1 liter distilled water) for 30 min. (b) Homogenizing the sterilized roots with extraction buffer (1M NaCI, 1M Sucrose, 0.2M Acetate Buffer phi 5.0, 0.1M EDTA ph 8.0, ß-mercaptoethanol 200 µl and 50 ml of distilled water), such as herein described, then crushing the roots with acid wash sand. Then filtering them through four layer of cheese cloth to obtain the root extract. (c) Centrifuge the roots extract at 8500-9000 rpm for 30 min. discard the pellet so obtained and purify the supernatant by 0-80% Ammonium Sulphate precipitation. (d) Allow to this to stand overnight and again centrifuging for at 9000-95000 rpm for 1 hrs, discard the supernatant and dissolve the pellet containing INDIN-SAA in Sodium acetate buffer (Ph=5) such as herein described. (e) The protein INDIN-SAA is estimated and identified by standard method. The following examples are illustrative of the present invention but do not limiting the invention: Example 1 Isolation of the protein from roots- (a) Preparation of sodium acetate buffer (1m) In 50 ml of 2 M Glacial acetic acid, 6 M NaOH was added until the pH was adjusted to 5.0 and then the volume was made up to 100 ml with distilled water. (b) Preparation of crude protein extract 320 gm of Boertiaavia Diffusa linn roots were chopped into small pieces sterilized with one liter of sterilizing media (1.5 ml Dettol + 1 liter distilled water) for 30 minutes, roots were crushed in 500ml of extraction buffer containing 1M NaCI, 1M Sucrose, 0.2M Acetate Buffer pH 5.0, 0.1M EDTA ph 8.0, ß-mercaptoethanol 200 µl and 50 ml of distilled water mixed together. The tissue homogenate was filtered through four layer of cheese cloth and centrifuged at 8500 rpm for 30 minutes. The clear supernatant was referred as crude protein, unless stated otherwise the supernatant was stored at 4°C. Example 2 Maintenance of the Protein activity - Composition of extraction buffer For crushing the sterilized roots 500 ml extraction buffer was prepared, compositions of extraction buffer is as follows: 1 M NaCI = 50 ml 1 M Sucrose = 125 ml 0.2 M Acetate Buffer (pH-5.0) = 250 ml 0.1 M EDTA (pH-8.0) = 25 ml ß-mercaptoethanol = 200 µl Distilled water = 50 ml Example 3 Purification and Estimation of Protein-(a) Antidisease protein purification Proteins present in the crude extract of roots were fractionated by (NH4)2SO4 precipitation. The proteins precipitating at 0-80 % saturations of (NH4)2SO4 were separated from the supernatant by cenfrifugation at 9000 rpm for 1 hour. The pellets obtained were dissolved in 1-2 ml of 0.1 M Sodium acetate buffer pH-5.0 and all operations were carried out at 4°C. (b) Estimation of protein Protein was determined by the method of lowery et al. (1951) using Bovine serum albumin as standard. Example 4 Protein identification by SDS-PAGE Through SDS-PAGE (Gel Electrophoresis method) we get band of this protein, we get new low molecular protein called as INDIN-SAA (taken from origin country India) Figure-1. Chemical composition & structure of Protein Chemical composition is C18H14O6 & Protein structure sees on the Figure-2. Results Example 1 Testing of the antidisease activity-(a) By Agar well diffusion method The proteins extracted were then tested for their potential for inhibiting bacterial growth (Mycobacterium) by agar well diffusion method. The plates were observed for the formation of inhibition zone around the wells indicated by the relatively clear areas on a lawn of bacterial growing on agar. The diameter of the inhibition zone in mm was the measure of the antibacterial activity of protein fractions. The unit of antibacterial activity was defined as the amount of protein in mg which produced an inhibition zone of 10 mm diameter around the well on agar gel in 36 hour at 30°C. Specific activity was defined as the number of units per mg protein under assay conditions. (b) By Spectrophotometer The protein was tested for bacterial growth Mycobacterium scrofulacium to spectrophotometer on 600nm. After some hours, we found that protein decreased this disease & decreasing order (Figure-3). Example 2 Protein effect on infected cancer cells & tumor cancer cells We used EAGLE-Medium for the experiment We culture cancer & tumor cancer ceils on culture media, we load the protein sample in wells and the plates were incubated for 37ºC. After some day's, we see the inhibition zone around the wells. This clear inhibition zone proved that the protein has an antidisease protein. Specific activity was defined as the number of units per mg protein under assay conditions. Example 3 Experiment on research animals Guinea-pig/White rat infected with Scrofula adenitis and cancer, tumor cancer, after 22 day's- 1 month Guinea-pig/White rat infected. Then Discovered INPIN-SAA protein is fed through orally to the Guinea-pig/White rat day by day. After 3 to 4 months, we found normal Guinea-pig/White rat, so this protein cures disease. So this protein is antidisease protein for Scrofula adenitis (Anjeer bal),and tumor cancer. > Same results are seen on Human beings. Example 4 Experiment on AIDS (HIV) Patients- The protein was tested for viral disease AIDS (HIV). This protein was used orally day by day. After some day's, we found positive results for AIDS (HIV) patient. Specific activity was defined as the number of units per mg protein under assay conditions. Mechanism of curing by this protein (INDIN-SAA) (Diagram Removed) EXPRIMENT ON GUINEA-PIG/WHITE RAT & MAN (Diagram Removed) This invention has the following advantages 1- The process is simple straight-forward & economical. 2- The raw material is extensively available in this country INDIA & all over world. 3- The product prepared by total anti Scrofula adenitis (Anjeer bal), anticancer & anti AIDS (HIV) activity of the constituents of the plant roots protein named INDIN-SAA used. 4- That the product is stable & does not deposit crystalline materials. 5- The product is suitable for incorporation into cosmetic & other pharmaceutical preparations. 6- It is (INDIN-SAA) a natural product, so it have not side effect on humanbeings. WE CLAIM:- 1. A process for the isolation of protein (INDIN-SAA) from the roots of Boerhaavia diffusa Linn (Santh) characterized in that:- (a) Taking roots of plant such as herein described and sterilizing it by sterilization media (1.5 ml Dettol + 1 liter distilled water) for 30 min. (b) Homogenizing the sterilized roots with extraction buffer (1M NaCI, 1M Sucrose, 0.2M Acetate Buffer pH 5.0, 0.1M EDTA ph 8.0, ß-mercaptoethanol 200 µl and 50 ml of distilled water), such as herein described, then crushing the roots with acid wash sand. Then filtering them through four layer of cheese cloth to obtain the root extract. (c) Centrifuge the roots extract at 8500-9000 rpm for 30 min. discard the pellet so obtained and purify the supernatant by 0-80% Ammonium Sulphate precipitation. (d) Allow to this to stand overnight and again centrifuge at 9000-95000 rpm for 1 firs, discard the supernatant and dissolve the pellet containing INDIN-SAA in Sodium acetate buffer (Ph=5) such as herein described. (e) The protein INDIN-SAA is estimated and identified by standard method. 2. A process as claimed in claim 1 wherein the protein is identified by standard following lowery method. 3. A process as claimed in claims 1 wherein the protein identified through SDS-PAGE method. 4. A process as claimed in claim 1 as and when used for the preparation of a pharmaceutical composition along with the pharmaceutical acceptable carrier in the form of powdered husk, tablets, capsule and liquid syrup. 5. A process for the isolation of protem (INDIN-SAA) from roots of Boerhaavia diffusa Linn (santh) substantially as here in described with reference to examples accompanying this specification.

Documents:

2351-DEL-2004-Abstract-(10-09-2008).pdf

2351-DEL-2004-Abstract-(26-05-2008).pdf

2351-del-2004-abstract.pdf

2351-DEL-2004-Claims-(10-09-2008).pdf

2351-DEL-2004-Claims-(26-05-2008).pdf

2351-del-2004-claims.pdf

2351-DEL-2004-Correspondence-Others-(10-09-2008).pdf

2351-DEL-2004-Correspondence-Others-(26-05-2008).pdf

2351-del-2004-correspondence-others.pdf

2351-del-2004-correspondence-po.pdf

2351-DEL-2004-Description (Complete)-(10-09-2008).pdf

2351-DEL-2004-Description (Complete)-(26-05-2008).pdf

2351-del-2004-description (complete).pdf

2351-DEL-2004-Drawings-(10-09-2008).pdf

2351-DEL-2004-Drawings-(26-05-2008).pdf

2351-del-2004-drawings.pdf

2351-del-2004-form-1.pdf

2351-DEL-2004-Form-2-(10-09-2008).pdf

2351-DEL-2004-Form-2-(26-05-2008).pdf

2351-del-2004-form-2.pdf

2351-DEL-2004-Form-3-(10-09-2008).pdf

2351-DEL-2004-Form-3-(26-05-2008).pdf

2351-del-2004-form-9.pdf

2351-DEL-2004-PA-(10-09-2008).pdf