Indian Patents. 237613:"NOVEL PROTOCOL FOR ISOLATION AND PURIFICATION OF NUCLEIC ACIDS FROM TERMINALIA ARJUNA"

Title of Invention	"NOVEL PROTOCOL FOR ISOLATION AND PURIFICATION OF NUCLEIC ACIDS FROM TERMINALIA ARJUNA"
Abstract	The present invention relates to the method of isolation of nucleic acid from Terminalia arjuna, which would be of immense use in the construction of genomic libraries, cDNA libraries, subtractive libraries and other molecular biology techniques.

Full Text	(arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocynadins (OPCs), phytosterols, calcium, magnesium, zinc and copper. The mechanism of action involves improvement of cardiac muscle function and the subsequent improved pumping activity of the heart and this seems to be the primary benefit of Terminalia arjuna. Saponins Glycosides are considered to be responsible for the inotropic effects of Terminalia arjuna, whereas the flavonoids and OPCs provide free radical antioxidant activity and vascular strengthening. The constituents of Terminalia arjuna have not been extensively studied. A study of this would be beneficial in understanding this botanical plant. The bark is astringent, sweet, acrid, cooling, and acts as an aprhodisiac, demulcent, cardiac tonic, styptic, anti dysenteric, urinary, astringent, expectorant, alexiteric, lithontriptic tonic. It is useful in the treatment of fractures, ulcers, leucorrhoea, diabetes, anaemia, caridopathy etc. The bark contains a crystalline compound, arjunine, a lactone arjunetin, essential oils, tannin, reducing sugar and colouring matter (Nayar & Chopra, 1956). Besides Terminalia arjuna's four active constituents, Arjunic acid, a trihydroxyrtiterpene, along with P-sitosterol ellagic acid, and the glucoside-Arjunetin is isolated from the bark and sapogenin arjungenin is isolated and characterised as 2a, 23, 19a, 23 tetrahydroxyolean-12-en-28-oic acid; two saponins, arjunglucoside-I and arjunglucoside-II, have been isolated and characterised (Rastogi and Mehrotra, 1990). The genus is chemically characterised by thepresence of tannins and related compounds (Tanaka et al., 1986, Okuda et al., 1981, Haseam et al., 1967, Lin et al, 1990, Tanaka et al, 1991). In a highly competitive environment of contemporary pharmaceutical research, natural products (secondary metabolites) provide a unique element of molecular diversity and biological functionality, which is indispensable for drug discovery. Plants have evolved through an immense variety of biochemical pathways. The plasticity of plant metabolic activity is most evident in the variety of secondary metabolites accumulated by plants in their leaves, roots and other organs. The use of the whole plant preparation or the extracts thereof for medicinal purposes goes a long way back, in fact much before history was recorded. In recent times many plant derived products have reached the market as useful drugs for treating human disorders including atropine, hyoscyamine, scopolamine, taxol (anticancer), artemisin, quinine (antimalarial). Plants are now considered as metabolite factories for production of a variety of compounds that are of medicinal, nutritional and industrial values. On a more functional level, the application of molecular techniques has permitted the manipulation of biosynthetic pathways for a generation of novel chemical species. The role of secondary metabolites has been much debated upon. Terpenoids are the largest family of natural products and they play a vital role in plants. They are widespread in nature, the building block of terpene is hydrocarbon isoprene CH = C(CH3)-CH = CH2. What is common in these substances are the isoprene units of the 5-carbon atoms, which, after the subsequent polymerisation reaction, give rise to numerous isoprenoids (Chappell, 1995; Weissenborn et al., 1995). Terpene hydrocarbons have molecular formula (C5 H8)n. They are classified according to the number of isoprene units. Monoterpenes (Camphor, menthol, limonene), diterpene (phytol and vitamin A), triterpene (squalene) and tetraterpene (carotene) are also widely prevalent in plants. Compounds like alcohol, aldehydes or ketoses group containing oxygen are called terpenoids. The enzymes involved in their biosynthesis are hard to isolate. Modem methods for recombinant DNA technology bye pass this difficulty and allow direct isolation of the genes. A further study of these genes will lead to an understanding of the various metabolic steps and regulation of this complex but economically significant metabolic pathway and this knowledge will assist in manipulating the genes in enhancing the production of these metabolites. The first step in the synthesis of the triterpenoid starts from the mevalonic acid (MVA) which is catalysed by the 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), Mevalonate, is a six carbon unit sequentially phosphorylated and decarboxylated to produce isopentylpyrophosphate (IPP) by mevalonate kinase and mevalonate 5-diphosphate decarboxylase enzyme. The IPP and Dimethylallyl Pyrophosphate (DMAPP), isomers of IPP from the conversion reaction catalysed by the IPP isomerase are the building blocks for the synthesis of all other isoprenoid compounds. The condensation of DMAPP with IPP produces Geranyl Pyrophosphate (GPP) and the addition of another IPP molecule will give rise to Famesyl Pyrophosphate (FPP) and the subsequent addition of IPP to this produces Geranylgeranyl Pyrophosphate (GGPP) (Chappell, 1995). Poulter and Rilling, 1981, reported that the isoprenoid metabolism is catalysed by Prenyl Transferases. Famesyl Diphosphate Synthases (E.G. 2.5.1.1) which is the control enzyme in the V-4 chain elongation process. It catalyzes the sequential condensation of DMAPP and Geranyl Diphosphate with IPP. The product. Farnesyl Diphosphate, gives rise to several branches in the pathway which supply C15 precursors for several classes of essential metabolites including sterols, dolichols, ubiquinone and triterpenoids. Squalene monxygenase (Squalene epoxidase) (E.G. 1.14.99.7) is involved in sterol and triterpenoid biosynthesis in almost all the eukaryotes (Gaik et al., 1983). The properties and corresponding DNA sequences of squalene monoxygenase from mammal and fungi have been investigated into (Favre & Ryder, 1997; Jandrosite et al., 1991, Kosuga et al., 1995: Sakakibara et al, 1995; Satoh et al, 1993). Three sequences of Arabidopsis thaliana and two of Brassica napus cDNAs encoding Squalene Monoxygenase homolog (Sqp 1 and Sqp2) have been reported by Schafer et al, 1999. The invention would be of immense use by aiding in the constmction of genomic libraries, cDNA libraries, subtractive libraries and other molecular techniques. Procedure I. DNA isolation: 1. One gram of mature leaves were mixed with Polyvinylpolypyriliodine and ground with liquid nitrogen. 2. The mixture was added to 10 ml extraction buffer with 5% Na Lauryl sulphate, pre-warmed at 65^C, shaken vigorously and centrifuged. 3. The supernatant was extracted twice with 7 ml of Chloroform: Isoamylalcohol (24:1), mixed thoroughly and centrifuged at 11,000 rpm at 4 ° C for 10 minutes. 4. To this aqueous phase was added, 6 ml of Buffer -2 (DNA Ext. Protocol -2) and incubated at 65 ° C for 20 minutes. 5. 7 ml Cholroform : Isoamylalcohol (24:1) was added to the mixture and then it was spun at 11,(X)0 rpm at 4 ° C for 10 minutes. 6. The aqueous supernatant was dispensed in micro-centrifuge tubes (500 µl) and to each micro-centrifuge tube was added, 700 µl of 95% Ethanol. 7. The tubes were spun at 14,000rpm at 4 ° C for 10 minutes 8. The pellet was washed with 70% Ethanol and then, air-dried. 9. Subsequently the pellet was dissolved in TE. II. RNA Extraction : 1. One gram of mature leaves were mixed with PVPP and ground with liquid nitrogen. 2. The mixture was added to an extraction buffer, pre-warmed at 65oC, shaken vigorously and centrifuged. 3. The supernatant was extracted twice with an equal volume of chloroform: isomyl alcohol. 4. To this aqueous phase, was added, 0.25 volumes of lOM LiCl and mixed well. 5. The RNA was precipitated at 4o C over night and then pelleted by spinning at 10,000 rpm at 4oC for 20 minutes. 6. The RNA was then dissolved in 50 µl of DEPC treated water. 7. Next, the RNA was re-precipitated with 0.1 Volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 96% ethanol by incubating for 4 hours at -70oC. 8. The RNA was pelleted by spinning it at 10,000 rpm at 4oC for 20 minutes. 9. The RNA was then dissolved in 21µol of DEPC treated water. Claims 1. The method of isolation of nucleic acid from Terminalia arjuna. 2. The claim as in claim 1, wherein, the nucleic acids isolated, thereof have immense use in the construction of genomic libraries, cDNA libraries, subtractive libraries and other molecular biology techniques.

Full Text

(arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocynadins (OPCs), phytosterols, calcium, magnesium, zinc and copper. The mechanism of action involves improvement of cardiac muscle function and the subsequent improved pumping activity of the heart and this seems to be the primary benefit of Terminalia arjuna. Saponins Glycosides are considered to be responsible for the inotropic effects of Terminalia arjuna, whereas the flavonoids and OPCs provide free radical antioxidant activity and vascular strengthening. The constituents of Terminalia arjuna have not been extensively studied. A study of this would be beneficial in understanding this botanical plant.
The bark is astringent, sweet, acrid, cooling, and acts as an aprhodisiac, demulcent, cardiac tonic, styptic, anti dysenteric, urinary, astringent, expectorant, alexiteric, lithontriptic tonic. It is useful in the treatment of fractures, ulcers, leucorrhoea, diabetes, anaemia, caridopathy etc. The bark contains a crystalline compound, arjunine, a lactone arjunetin, essential oils, tannin, reducing sugar and colouring matter (Nayar & Chopra, 1956). Besides Terminalia arjuna's four active constituents, Arjunic acid, a trihydroxyrtiterpene, along with P-sitosterol ellagic acid, and the glucoside-Arjunetin is isolated from the bark and sapogenin arjungenin is isolated and characterised as 2a, 23, 19a, 23 tetrahydroxyolean-12-en-28-oic acid; two saponins, arjunglucoside-I and arjunglucoside-II, have been isolated and characterised (Rastogi and Mehrotra, 1990). The genus is chemically characterised by thepresence of tannins and related compounds (Tanaka et al., 1986, Okuda et al., 1981, Haseam et al., 1967, Lin et al, 1990, Tanaka et al, 1991).
In a highly competitive environment of contemporary pharmaceutical research, natural products (secondary metabolites) provide a unique element of molecular diversity and biological functionality, which is indispensable for drug discovery. Plants have evolved through an immense variety of biochemical pathways. The plasticity of plant metabolic activity is most evident in the variety of secondary metabolites accumulated by plants in their leaves, roots and other organs. The use of the whole plant preparation or the extracts thereof for medicinal purposes goes a long way back, in fact much before history was recorded. In recent times many plant derived products have reached the market as useful drugs for treating human disorders including atropine, hyoscyamine, scopolamine, taxol (anticancer), artemisin, quinine (antimalarial). Plants are now considered as metabolite factories for production of a variety of compounds that are of medicinal, nutritional and industrial values. On a more functional level, the application of molecular techniques has permitted the manipulation of biosynthetic pathways for a generation of novel chemical species.
The role of secondary metabolites has been much debated upon. Terpenoids are the largest family of natural products and they play a vital role in plants. They are widespread in nature, the building block of terpene is hydrocarbon isoprene CH = C(CH3)-CH = CH2. What is common in these substances are the isoprene units of the 5-carbon atoms, which, after the subsequent polymerisation reaction, give rise to numerous isoprenoids (Chappell, 1995; Weissenborn et al., 1995).
Terpene hydrocarbons have molecular formula (C5 H8)n. They are classified according to the number of isoprene units. Monoterpenes (Camphor, menthol, limonene), diterpene (phytol and vitamin A), triterpene (squalene) and tetraterpene (carotene) are also widely prevalent in plants. Compounds like alcohol, aldehydes or

ketoses group containing oxygen are called terpenoids. The enzymes involved in their biosynthesis are hard to isolate. Modem methods for recombinant DNA technology bye pass this difficulty and allow direct isolation of the genes. A further study of these genes will lead to an understanding of the various metabolic steps and regulation of this complex but economically significant metabolic pathway and this knowledge will assist in manipulating the genes in enhancing the production of these metabolites.
The first step in the synthesis of the triterpenoid starts from the mevalonic acid (MVA) which is catalysed by the 3-hydroxy 3-methylglutaryl coenzyme A reductase (HMGR), Mevalonate, is a six carbon unit sequentially phosphorylated and decarboxylated to produce isopentylpyrophosphate (IPP) by mevalonate kinase and mevalonate 5-diphosphate decarboxylase enzyme. The IPP and Dimethylallyl Pyrophosphate (DMAPP), isomers of IPP from the conversion reaction catalysed by the IPP isomerase are the building blocks for the synthesis of all other isoprenoid compounds. The condensation of DMAPP with IPP produces Geranyl Pyrophosphate (GPP) and the addition of another IPP molecule will give rise to Famesyl Pyrophosphate (FPP) and the subsequent addition of IPP to this produces Geranylgeranyl Pyrophosphate (GGPP) (Chappell, 1995). Poulter and Rilling, 1981, reported that the isoprenoid metabolism is catalysed by Prenyl Transferases. Famesyl Diphosphate Synthases (E.G. 2.5.1.1) which is the control enzyme in the V-4 chain elongation process. It catalyzes the sequential condensation of DMAPP and Geranyl Diphosphate with IPP. The product. Farnesyl Diphosphate, gives rise to several branches in the pathway which supply C15 precursors for several classes of essential metabolites including sterols, dolichols, ubiquinone and triterpenoids. Squalene monxygenase (Squalene epoxidase) (E.G. 1.14.99.7) is involved in sterol and triterpenoid biosynthesis in almost all the eukaryotes (Gaik et al., 1983).
The properties and corresponding DNA sequences of squalene monoxygenase from mammal and fungi have been investigated into (Favre & Ryder, 1997; Jandrosite et al., 1991, Kosuga et al., 1995: Sakakibara et al, 1995; Satoh et al, 1993). Three sequences of Arabidopsis thaliana and two of Brassica napus cDNAs encoding Squalene Monoxygenase homolog (Sqp 1 and Sqp2) have been reported by Schafer et al, 1999.
The invention would be of immense use by aiding in the constmction of genomic libraries, cDNA libraries, subtractive libraries and other molecular techniques.
Procedure
I. DNA isolation:
1. One gram of mature leaves were mixed with Polyvinylpolypyriliodine and ground with liquid nitrogen.
2. The mixture was added to 10 ml extraction buffer with 5% Na Lauryl sulphate, pre-warmed at 65^C, shaken vigorously and centrifuged.
3. The supernatant was extracted twice with 7 ml of Chloroform: Isoamylalcohol (24:1), mixed thoroughly and centrifuged at 11,000 rpm at 4 ° C for 10 minutes.
4. To this aqueous phase was added, 6 ml of Buffer -2 (DNA Ext. Protocol -2) and incubated at 65 ° C for 20 minutes.

5. 7 ml Cholroform : Isoamylalcohol (24:1) was added to the mixture and then it was spun at 11,(X)0 rpm at 4 ° C for 10 minutes.
6. The aqueous supernatant was dispensed in micro-centrifuge tubes (500 µl) and to each micro-centrifuge tube was added, 700 µl of 95% Ethanol.
7. The tubes were spun at 14,000rpm at 4 ° C for 10 minutes
8. The pellet was washed with 70% Ethanol and then, air-dried.
9. Subsequently the pellet was dissolved in TE.
II. RNA Extraction :
1. One gram of mature leaves were mixed with PVPP and ground with liquid
nitrogen.
2. The mixture was added to an extraction buffer, pre-warmed at 65oC, shaken vigorously and centrifuged.
3. The supernatant was extracted twice with an equal volume of chloroform: isomyl alcohol.
4. To this aqueous phase, was added, 0.25 volumes of lOM LiCl and mixed well.
5. The RNA was precipitated at 4o C over night and then pelleted by spinning at 10,000 rpm at 4oC for 20 minutes.
6. The RNA was then dissolved in 50 µl of DEPC treated water.
7. Next, the RNA was re-precipitated with 0.1 Volume of 3 M sodium acetate (pH 5.2) and 2.5 volumes of 96% ethanol by incubating for 4 hours at -70oC.
8. The RNA was pelleted by spinning it at 10,000 rpm at 4oC for 20 minutes.
9. The RNA was then dissolved in 21µol of DEPC treated water.

Claims
1. The method of isolation of nucleic acid from Terminalia arjuna.
2. The claim as in claim 1, wherein, the nucleic acids isolated, thereof have
immense use in the construction of genomic libraries, cDNA libraries,
subtractive libraries and other molecular biology techniques.

Documents:

781-che-2003 complete specification as granted.pdf

781-che-2003 correspondence others.pdf

781-che-2003 correspondence po.pdf

781-che-2003-abstract.pdf

781-che-2003-claims.pdf

781-che-2003-description(complete).pdf

781-che-2003-form 1.pdf

EXAMINATION REPORT REPLY.PDF

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Patent Number

237613

Indian Patent Application Number

781/CHE/2003

PG Journal Number

2/2010

Publication Date

08-Jan-2010

Grant Date

29-Dec-2009

Date of Filing

24-Sep-2003

Name of Patentee

M/S AVESTHA GENGRAINE TECHNOLOGIES PVT LTD

Applicant Address

"DISCOVERER", 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK, WHITEFIELD MAIN ROAD, BANGALORE-560 066, KARNATAKA, INDIA.

Inventors:

#	Inventor's Name	Inventor's Address
1	DR. VILLOO MORAWALA-PATELL	"DISCOVERER", 9TH FLOOR, UNIT 3, INTERNATIONAL TECH PARK, WHITEFIELD MAIN ROAD, BANGALORE-560 066, KARNATAKA, INDIA.

PCT International Classification Number

C12N15/10

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA