Indian Patents. 218499:PREPARATION OF OLIGOSACCHARIDE BIONANOPARTICLES FROM MORINGA OLEIFERA LAM

Title of Invention	PREPARATION OF OLIGOSACCHARIDE BIONANOPARTICLES FROM MORINGA OLEIFERA LAM
Abstract	A process of preparation of bionanoparticles from Moringa oleifera Lam gum is described. The bionanoparticles were between 60nm to 100nm. They protected plasmid DNA and RNA from degradation, inhibit the growth of onion roots and induce membrane blebbing leading to apoptosis and death of human epidermoid carcinoma HEp2 cells

Full Text	Moringa oleifera Lam is a tree yielding edible fruits. Traditionally extracts of leaves of Moringa oleifera has been used against microbial infections. Glucosinolates and phenolics extracted from vegetative and reproductive tissues inhibited the growth of Helicobacter sp. (US Patent 6737441). Nitrile glycosides isolated from Moringa oleifera has enhanced the bioactivity of antibiotics against gram positive and negative bacteria (US Patent 6858588). Herbal formulations where Moringa oleifera is also a component has been shown to be active against skin disorders (US Patent 6383495). And thiocarbamates like Niazimin isolated from the leaves of Moringa oleifera has been shown to inhibit tumor induction by compounds like teleocidin in Raji cells. (Murakami et al 1998 ; Guevara et al, 1999 ). Also polysaccharides extracted from edible fungi like Flammulina velutipes, Leutinus edodes and Agaricus bisporus inhibited mitosis of hepatoma cells from 51.9 % to 56.6% (Jiang, 1999 ). The large scale preparation of oligosaccharide phosphate (predominantly a penta maiman) from the fungus Pichia holstii using acid hydrolysis has been reported (Ferro et al 2001). J.P-06-329514 informs the preparation of leachates or extraction of various plants including Moringa towards the use as pest controlling agent. J.P-07-304685 describes the preparation of active ingredients as a methanol extract from M oleifera which gives thiocarbamates and its glycosides. Though a variety of phenolic compounds are being used as anticancer drugs, many tumor cells have already developed resistance. Therefore there is a need to invent new drugs from natural sources. The composition of polysaccharides from different plants and fimgi are different in their ability to form bioactive secondary structure which not only depends on its monomer composition but also the freedom of their movement which eventually determine their bioactive structure. The structure activity relationship (SAR) plays an important role in their therapeutic value. Summary of the Prior art: Extraction of different compounds like thiocarbamates and their derivatives, phenols, glycosides especially rhanmosyl glycosides from Moringa oleifera has been reported earlier. A variety of solvents like water, methanol, ethanol and lipid solvent were used to extract compounds and their use for a variety of ailments were reported. Objectives of the present invention To prepare bionanoparticles of Moringa oleifera measuring less than 150 nm and an ability to protect DNA, RNA, to inhibit cell division in Onion root and human Carcinoma cells. Summary of the invention Bionanoparticles of Moringa oleifera measuring less than 150nm were prepared from the soft fibres through controlled hydrolysis, selective size fractionation by ultrafiltration and particle preparation by spray drying. The bioactivity of bionanoparticles was elucidated. 4. Description of the invention • 10 grams of Moringa gum powder was washed with 20ml of diethyl either, 20ml of chloroform : Methanol (2:1) 20ml of 50% Methanol with 20ml of 100% ethanol and dried • Extract of purified Moringa gum of step 1 made aqueous (100mg/ml) was taken in a Sahxlet apparatus and treated with HCl to give a final concentration of 0.5N and 60 minutes at 100°C. • The acid hydrolysed polysaccharides were brought to 25°C and IM NaHCO3 solution was added to give a final concentration of 0.1 M. • The cooled suspension was filtered through either 0.22 micron filters or through a ultra filtration membranes with a cut off molecular weight of 3000 daltons. • The bionanoparticles measuring 60nm to 100nm were prepared by spray drying at 100°C. Moringa bionanoparticle prepared was viewed under the atomic force microscope and the image is presented in Fig 1. The nanoparticles were between 60nm to 100 nm. The bioactivity of the bionanoparticles was ascertained. Advantages of the invention: Bionanoparticles from Moringa oleifera oligosaccharides measuring less than 150 nm were prepared. These bionanoparticles have the utility of protecting plasmid DNA and bacterial RNA from degradation; inhibits cell division of Onion root tips and also malignant cell lines like HEp-2 cells. Example 1: Effect of Moringa bionanoparticles on plasmid DNA and RNA. • 1.5 ml of bacterial culture was taken in two eppendorffs and centrifuged for 1 minute, • the pellet was suspended in 350 μl of STET (O.IM NaCl, 10mM Tris.Cl (PH 8), 5% Triton x 100, ImM EDTA), • 25 μl of Lysozyme was added to each tube and inverted once and then kept in boiling water bath for 40 seconds, • immediately centrifuged at 10,000xg at room temperature for 15 minutes, • saved the supernatant, • added 40μl of sodium acetate (3 M pH 5.2) and 420 μl isopropanol and 100 μl of nanoparticle (Img/ml concentration) and mixed well, • centrifuged at 10,000 xg for 15 minutes at room temperature, • the pellet was saved and washed with 70% ethanol and the • pellet was suspended in 25μl of TE (10mM Tris.Cl (pH8) and ImM EDTA) and incubated at room temperature. • 0.7% agarose gel was used to check the presence of DNA and RNA for various time periods upto 44 hours. The bionanoparticles protected plasmid DNA and bacterial RNA (Fig. 2). Explanation to Fig. 2 : Lanel shows the Hindlll cut lambda DNA used as marker. Lane 2 shows the degradation of plasmid DNA and E.coli RNA in room temperature after 44 hrs of incubation. Lane3 shows the protection of plasmid DNA and E.coli RKA by bionanoparticles for a period of 44 hrs. Example 2: Influence of Moringa bionanoparticles on H£p2 cells. • HEp-2 cells were subcultured (2x104 cells/ml) and allowed to attain a confluent growth. • Before the addition of bionanoparticles the medium was changed and fresh medium was added (3ml). • 50μl of bionanoparticles (30mg/ml) was added to the plate and incubated at 37°C and after 12 hours the cells were observed under the microscope. The normal Hep2 cells are shown in Fig. 3 . The Hep2 cells treated with bionanoparticles are shown in Fig 4. The treated cells, show blebbing which eventually leads to apoptosis and death. This indicates the ability of Moringa bionanoparticles to arrest the growth of human epidermoid carcinoma cells. References: 1. Niaziminin, a thiocarbomate from the leaves of Moringa oleifera, holds a strict structural requirement for inhibition of tumor promoter induced Epstein-Bar virus activation. Murakami A, Kitazono Y, Jiwajinda S, Koshiniza K and Ohigashi H. Plantamed, 1998,4,319-23. 2. An antitumor promoter from Moringa oleifera Lam. Guevara AP, Vargas C, Sakurai N, Fujiwara Y, Hashimoto K, Maoka T, Kozuka M, Ito Y, Tokuda H, Nishino H. MutatRes, 1999,440, 181-8. 3. Inhibitory activity of polysaccharide extract from three kinds of edible fungi on proliferation of human hepatoma SMMC - 7721 cell and mouse implanted S180 tumor. Jiang SM, Xiao ZM and Xu ZH. World J. Gastroenterol, 1999, 5,404 - 407. 4. Large scale preparation of the oligosaccharide phosphate fraction of Pichia holstii NRRL Y-2448 phosphomannan for use in the manufacture of PI - 88. Vito Ferro, Kym Fewings, Maria C. Palermo and caiping Li Carbohydrate research, 2001,332,183-189 We claim: 1] A process to prepare bionanoparticles of 60nm to 120nm from Moringa oleifera gum comprising the steps of, i. 10 grams of Moringa oleifera gum powder was washed with 20ml of diethyl ether, 20ml of chloroform : methanol (2:1), 20ml of 50% methanol and with 20ml of 100% ethanol and dried, ii. extract of purified moringa gum of step (i) made aqueous (100mg/ml) was taken in Sahxlet apparatus and treated with HCl to give a final concentration of 0,5N and hydrolysed for 60 minutes at 100°C, iii. the acid hydrolysed polysaccharides were brought to 25°C and IM NaHCO3 solution was added to give a final concentration of 0.1 M, iv. the cooled suspension was filtered through either 0.22 micron filters or through a ultrafiltration membrane with a cut off molecular weight of 3000 daltons and V. the bionanoparticles of 60mn to 120 nm were prepared by spray drying at 100oC. 2] A process as claimed in claiml where in, the bionanoparticle of the size 60nm - 120nm protected plasmid DNA and bacterial RNA, inhibited onion root growth and induced blebbing of human epidermoid carcinoma HEp2 cells. Dated 5 May, 2003 To The Controller of Patents The Patent office at Chennai

Full Text

Moringa oleifera Lam is a tree yielding edible fruits. Traditionally extracts of leaves of Moringa oleifera has been used against microbial infections. Glucosinolates and phenolics extracted from vegetative and reproductive tissues inhibited the growth of Helicobacter sp. (US Patent 6737441). Nitrile glycosides isolated from Moringa oleifera has enhanced the bioactivity of antibiotics against gram positive and negative bacteria (US Patent 6858588). Herbal formulations where Moringa oleifera is also a component has been shown to be active against skin disorders (US Patent 6383495). And thiocarbamates like Niazimin isolated from the leaves of Moringa oleifera has been shown to inhibit tumor induction by compounds like teleocidin in Raji cells. (Murakami et al 1998 ; Guevara et al, 1999 ). Also polysaccharides extracted from edible fungi like Flammulina velutipes, Leutinus edodes and Agaricus bisporus inhibited mitosis of hepatoma cells from 51.9 % to 56.6% (Jiang, 1999 ). The large scale preparation of oligosaccharide phosphate (predominantly a penta maiman) from the fungus Pichia holstii using acid hydrolysis has been reported (Ferro et al 2001). J.P-06-329514 informs the preparation of leachates or extraction of various plants including Moringa towards the use as pest controlling agent. J.P-07-304685 describes the preparation of active ingredients as a methanol extract from M oleifera which gives thiocarbamates and its glycosides.
Though a variety of phenolic compounds are being used as anticancer drugs, many tumor cells have already developed resistance. Therefore there is a need to invent new drugs from natural sources. The composition of polysaccharides from different plants and fimgi are different in their ability to form bioactive secondary structure which not only depends on its monomer composition but also the freedom of their movement which eventually determine their bioactive structure. The structure activity relationship (SAR) plays an important role in their therapeutic value.

Summary of the Prior art:
Extraction of different compounds like thiocarbamates and their derivatives, phenols, glycosides especially rhanmosyl glycosides from Moringa oleifera has been reported earlier. A variety of solvents like water, methanol, ethanol and lipid solvent were used to extract compounds and their use for a variety of ailments were reported.
Objectives of the present invention
To prepare bionanoparticles of Moringa oleifera measuring less than 150 nm and an ability to protect DNA, RNA, to inhibit cell division in Onion root and human Carcinoma cells.
Summary of the invention
Bionanoparticles of Moringa oleifera measuring less than 150nm were prepared from the soft fibres through controlled hydrolysis, selective size fractionation by ultrafiltration and particle preparation by spray drying. The bioactivity of bionanoparticles was elucidated.
4. Description of the invention
• 10 grams of Moringa gum powder was washed with 20ml of diethyl either, 20ml of chloroform : Methanol (2:1) 20ml of 50% Methanol with 20ml of 100% ethanol and dried
• Extract of purified Moringa gum of step 1 made aqueous (100mg/ml) was taken in a Sahxlet apparatus and treated with HCl to give a final concentration of 0.5N and 60 minutes at 100°C.
• The acid hydrolysed polysaccharides were brought to 25°C and IM NaHCO3 solution was added to give a final concentration of 0.1 M.
• The cooled suspension was filtered through either 0.22 micron filters or through a ultra filtration membranes with a cut off molecular weight of 3000 daltons.
• The bionanoparticles measuring 60nm to 100nm were prepared by spray drying at 100°C.

Moringa bionanoparticle prepared was viewed under the atomic force microscope and the image is presented in Fig 1. The nanoparticles were between 60nm to 100 nm.
The bioactivity of the bionanoparticles was ascertained.
Advantages of the invention:
Bionanoparticles from Moringa oleifera oligosaccharides measuring less than 150 nm were prepared. These bionanoparticles have the utility of protecting plasmid DNA and bacterial RNA from degradation; inhibits cell division of Onion root tips and also malignant cell lines like HEp-2 cells. Example 1:
Effect of Moringa bionanoparticles on plasmid DNA and RNA.
• 1.5 ml of bacterial culture was taken in two eppendorffs and centrifuged for 1 minute,
• the pellet was suspended in 350 μl of STET (O.IM NaCl, 10mM Tris.Cl (PH 8), 5% Triton x 100, ImM EDTA),
• 25 μl of Lysozyme was added to each tube and inverted once and then kept in boiling water bath for 40 seconds,
• immediately centrifuged at 10,000xg at room temperature for 15 minutes,
• saved the supernatant,
• added 40μl of sodium acetate (3 M pH 5.2) and 420 μl isopropanol and 100 μl of nanoparticle (Img/ml concentration) and mixed well,
• centrifuged at 10,000 xg for 15 minutes at room temperature,
• the pellet was saved and washed with 70% ethanol and the
• pellet was suspended in 25μl of TE (10mM Tris.Cl (pH8) and ImM EDTA) and incubated at room temperature.
• 0.7% agarose gel was used to check the presence of DNA and RNA for various time periods upto 44 hours.

The bionanoparticles protected plasmid DNA and bacterial RNA (Fig. 2).
Explanation to Fig. 2 : Lanel shows the Hindlll cut lambda DNA used as marker. Lane 2 shows the degradation of plasmid DNA and E.coli RNA in room temperature after 44 hrs of incubation. Lane3 shows the protection of plasmid DNA and E.coli RKA by bionanoparticles for a period of 44 hrs.
Example 2:
Influence of Moringa bionanoparticles on H£p2 cells.
• HEp-2 cells were subcultured (2x104 cells/ml) and allowed to attain a confluent growth.
• Before the addition of bionanoparticles the medium was changed and fresh medium was added (3ml).
• 50μl of bionanoparticles (30mg/ml) was added to the plate and incubated at 37°C and after 12 hours the cells were observed under the microscope.
The normal Hep2 cells are shown in Fig. 3 . The Hep2 cells treated with bionanoparticles are shown in Fig 4. The treated cells, show blebbing which eventually leads to apoptosis and death. This indicates the ability of Moringa bionanoparticles to arrest the growth of human epidermoid carcinoma cells.

References:
1. Niaziminin, a thiocarbomate from the leaves of Moringa oleifera, holds a strict structural requirement for inhibition of tumor promoter induced Epstein-Bar virus activation. Murakami A, Kitazono Y, Jiwajinda S, Koshiniza K and Ohigashi H. Plantamed, 1998,4,319-23.
2. An antitumor promoter from Moringa oleifera Lam.
Guevara AP, Vargas C, Sakurai N, Fujiwara Y, Hashimoto K, Maoka T, Kozuka M, Ito Y, Tokuda H, Nishino H. MutatRes, 1999,440, 181-8.
3. Inhibitory activity of polysaccharide extract from three kinds of edible fungi on
proliferation of human hepatoma SMMC - 7721 cell and mouse implanted S180
tumor.
Jiang SM, Xiao ZM and Xu ZH. World J. Gastroenterol, 1999, 5,404 - 407.
4. Large scale preparation of the oligosaccharide phosphate fraction of Pichia holstii
NRRL Y-2448 phosphomannan for use in the manufacture of PI - 88.
Vito Ferro, Kym Fewings, Maria C. Palermo and caiping Li Carbohydrate research, 2001,332,183-189

We claim:
1] A process to prepare bionanoparticles of 60nm to 120nm from Moringa oleifera
gum comprising the steps of,
i. 10 grams of Moringa oleifera gum powder was washed with 20ml of diethyl ether, 20ml of chloroform : methanol (2:1), 20ml of 50% methanol and with 20ml of 100% ethanol and dried,
ii. extract of purified moringa gum of step (i) made aqueous (100mg/ml) was taken in Sahxlet apparatus and treated with HCl to give a final concentration of 0,5N and hydrolysed for 60 minutes at 100°C,
iii. the acid hydrolysed polysaccharides were brought to 25°C and IM NaHCO3 solution was added to give a final concentration of 0.1 M,
iv. the cooled suspension was filtered through either 0.22 micron filters or through a ultrafiltration membrane with a cut off molecular weight of 3000 daltons and
V. the bionanoparticles of 60mn to 120 nm were prepared by spray drying at 100oC. 2] A process as claimed in claiml where in,
the bionanoparticle of the size 60nm - 120nm protected plasmid DNA and bacterial RNA, inhibited onion root growth and induced blebbing of human epidermoid carcinoma HEp2 cells.
Dated 5 May, 2003
To
The Controller of Patents
The Patent office at Chennai

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Patent Number

218499

Indian Patent Application Number

375/CHE/2003

PG Journal Number

21/2008

Publication Date

23-May-2008

Grant Date

02-Apr-2008

Date of Filing

05-May-2003

Name of Patentee

DR (MRS) SUGUNA SHANMUGASUNDARAM

Applicant Address

SENIOR LECTURER, DEPARTMENT OF MICROBIAL TECHNOLOGY, SCHOOL OF BIOLOGICAL SCIENCES MADURAI KAMARAJ UNIVERSITY, MADURAI 625 021,

Inventors:

#	Inventor's Name	Inventor's Address
1	MR. PARTHASARATHY KRUPAKAR	DEPARTMENT OF MICROBIAL TECHNOLOGY, SCHOOL OF BIOLOGICAL SCIENCES MADURAI KAMARAJ UNIVERSITY, MADURAI 625 021,
2	DR. SAMBANDAM SHANMUGASUNDARAM	SENIOR PROFESSOR AND HEAD, DEPARTMENT OF MICROBIAL TECHNOLOGY, SCHOOL OF BIOLOGICAL SCIENCES MADURAI KAMARAJ UNIVERSITY, MADURAI 625 021,
3	DR (MRS) SUGUNA SHANMUGASUNDARAM	SENIOR LECTURER, DEPARTMENT OF MICROBIAL TECHNOLOGY, SCHOOL OF BIOLOGICAL SCIENCES MADURAI KAMARAJ UNIVERSITY, MADURAI 625 021,

PCT International Classification Number

A61K38/47

PCT International Application Number

N/A

PCT International Filing date

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1			NA