Title of Invention

A PROCESS FOR ISOLATION OF ACTEOSIDE FROM PLANT COLERBROOKEA OPPOSITIFOLIA

Abstract A process for isolation of Acteoside from plant Colerbrookea oppositifolia , said process comprising steps of drying aerial parts of the plant, grounding the dried parts into powder, percolating the powder with water or ethanol for 3-4 times to obtain an extract, filtering the extract for clearing of suspended particles to obtain supernatant, drying the supernatant at about 45 to 55 C to obtain a residue, fractionating the residue with chloroform, ethyl acetate, and butanol successively, subjecting butanol fraction to adsoiption chromatography of SiO2 after adding methanol to the fraction, charging the adsorbed fraction to glass column, eluting the column with solvents of increasing polarity of methanol : chloroform to obtain further fractions and repeating the process one more time, subjecting the fractions to column chromatography to obtain fractions, concentrating the fractions under reduced pressure to obtain acteoside as residue; and a method of effectively hepatoprotecting a subject using pure Acteoside from plant Colerbrookea oppositifolia, said method comprising steps of administering appropriate low-dose of the acteoside to the subject.
Full Text A PROCESS FOR ISOLATION OF ACTEOSIDE FROM PLANT COLERBROOKEA OPPOSITIFOLIA
Field of the present invention
A process for isolation of Acteoside from plant Colerbrookea oppositifolia, said process comprising steps of drying aerial parts of the plant, grounding the dried parts into powder, percolating the powder with water or ethanol for 3-4 times to obtain an extract, filtering the extract for clearing of suspended particles to obtain supernatant, drying the supernatant at about 45 to 55°C to obtain a residue, fractionating the residue with chloroform, ethyl acetate, and butanol successively, subjecting butanol fraction to adsorption chromatography of SiCh after adding methanol to the fraction, charging the adsorbed fraction to glass column, eluting the column with solvents of increasing polarity of methanol : chloroform to obtain further fractions and repeating the process one more time, subjecting the fractions to column chromatography to obtain fractions, concentrating the fractions under reduced pressure to obtain acteoside as residue; and a method of effectively hepatoprotecting a subject using pure Acteoside from plant Colerbrookea oppositifolia, said method comprising steps of administering appropriate low-dose of the acteoside to the subject. Background of the present invention
Phenylethanoids are a class of compounds reported to be present in plants like Cistanche deserticola and Buddleja species ( Quanbo Xing, Koji Hase et al, Planta Median, 64. 120-125 (1998); Peter J. Houghton and Hiroshi Hikino. Planta Medial. Vol .55, 123-126(1989).
It is for the first time that a phenylethanoid - verbascoside (also called acteoside or kusaginin) has been isolated by the authors from Colebrookea oppositifolia and found to possess a very strong antihepatotoxic/ hepatoprotective activity at unusually very low doses (between 1.25 and 2.5 mg /kg) in rats and mice.
The presence of number of flavonoids and glycoflavonoids has already been reported in the literature from Colebrookea oppositifolia [S. Aziz Ahmed, S.A. Siddiqui and Asil" Zaman, Indian J. Chemistry 12 1327-28 (1974); S.A. Patwardhan and A.S.Gupta. Indian J. Chemistry. 20B, 627, (1981); Fan Yank, Xing-Li, HAN-Qing Wang and Chong-Ren Yang, Phytochemistry 42 , 867-69 (1996)]. However, the presence of Acteoside from this plant is being reported for the first time.

Antifertiiity activity of this plant in male rats with special reference to testicular cell
population dynamics have earlier been reported (R.S. Gupta, R.J. Yadav, V.P. Dixit
and M.P. Dobhal, Fitoterapia, 72, 236-45 (2001).
Though the Hepatoprotective/Antihepatotoxic activity of Acteoside (Verbascoside)
isolated from other two plants viz. Cistanche species and Buddleja species has been
reported. ( Quanbo Xing, Koji Hase et al, Planta Medica, 64, 120-125 (1998); Peter
J. Houghton and Hiroshi Hikino. Planta Medica, Vol .55, 123-126 (1989), however
the present invention not only reports a distinct source namely Colebrookea
oppositifolia but the desired activity is accomplished at unusually very low dosage
between 1.5 to 2.5 mg/kg in rats.
As already stated above the Hepatoprotective/Antihepatotoxic activity of acteoside
has been reported both in vitro and in vivo (Quanbo Xing, Koji Hase et al, Planta
Medica, 64, 120-125 (1998). But the present invention describes the isolation of
acteoside from a hitherto unreported source in such a way that the compound shows
antihepatotoxic/hepatoprotective activity at doses nearly 12 to 25 times lower than
those already reported in the prior art (Quanbo Xing, Koji Hase et al, Planta
Medica, 64, 120-125(1998); Peter J. Houghton and Hiroshi Hikino. Planta Medica,
Vol .55, 123-126 (1989). Moreover the parameters evaluated in the prior reports are
limited to ALT (in vivo) and AST & MDA (in vitro) whereas the present invention
describes the influence of acteoside practically in all important parameters leading to
for a clear decision on the efficacy of any hepatoprotective or antihepatotoxic
product.
The reason for the activity of acteoside at such low doses compared to the one
reported in the prior art may be due to the specific method of its isolation being
distinct to yield a product of higher purity. Contaminants in phytochemicals are well
documented to seriously jeopardize the activity of pure active compound though
there could be synergistic action also of which, however, there in no mention or
indication in the prior art.
Objects of the present invention
The main object of the present invention is to isolate a hepatoprotective from plant
Colerbrookea oppositifolia.
Another object of the present invention is to develop a process for the isolation of
acteoside from plant Colerbrookea oppositifolia.
Yet another object of the present invention is to develop a method of hepatoprotecting
a subject using Colerbrookea oppositifolia.
Summary of the present invention
A process of isolation of pure Acteoside of high hepatoprotection from plant
Colerbrookea oppositifolia , said process comprising steps of drying aerial parts of
the plant, grounding the dried parts into powder, percolating the powder with water or
ethanol for 3-4 times to obtain an extract, filtering the extract for clearing of
suspended particles to obtain supernatant, drying the supernatant at about 45 to 55°C
to obtain a residue, fractionating the residue with chloroform, ethyl acetate, and
butanol successively, subjecting butanol fraction to adsorption chromatography of
SiC>2 after adding methanol to the fraction, charging the adsorbed fraction to glass
column, eluting the column with solvents of increasing polarity of methanol :
chloroform to obtain further fractions and repeating the process one more time,
subjecting the fractions to column chromatography to obtain fractions, concentrating
the fractions under reduced pressure to obtain acteoside as residue; and a method of
effectively hepatoprotecting a subject using pure Acteoside from plant Colerbrookea
oppositifolia, said method comprising steps of administering appropriate low-dose of
the acteoside to the subject.
Detailed description of the present invention
A process of isolation of pure Acteoside of high hepatoprotection from plant
Colerbrookea oppositifolia , said process comprising steps of drying aerial parts of
the plant, grounding the dried parts into powder, percolating the powder with water or
ethanol for 3-4 times to obtain an extract, filtering the extract for clearing of
suspended particles to obtain supernatant, drying the supernatant at about 45 to 55°C
to obtain a residue, fractionating the residue with chloroform, ethyl acetate, and
butanol successively, subjecting butanol fraction to adsorption chromatography of
SiO2 after adding methanol to the fraction, charging the adsorbed fraction to glass
column, eluting the column with solvents of increasing polarity of methanol :
chloroform to obtain further fractions and repeating the process one more time,
subjecting the fractions to column chromatography to obtain fractions, concentrating
the fractions under reduced pressure to obtain acteoside as residue; and a method of
effectively hepatoprotecting a subject using pure Acteoside from plant Colerbrookea
oppositifolia, said method comprising steps of administering appropriate low-dose of
the acteoside to the subject.
Accordingly, the present invention relates to a process for isolation of Acteoside from plant Colerbrookea oppositifolia characterized in that the said processs comprising steps of:
a. drying aerial parts of the plant,
b. grounding the dried parts into powder,
c. percolating the powder with water or ethanol for 3-4 times to obtain an
extract,
d. filtering the extract for clearing of suspended particles to obtain
supernatant,
e. drying the supernatant at about 45 to 55 C to obtain a residue,
f. fractionating the residue with chloroform, ethyl acetate, and butanol
successively,
g. subjecting butanol fraction to adsorption chromatography of Si02 after
adding methanol to the fraction,
h. charging the adsorbed fraction to glass column,
i. eluting the column with solvents of increasing polarity of methanol :
chloroform to obtain further fractions and repeating the process one
more time, j. subjecting the fractions to column chromatography to obtain fractions, k. concentrating the fractions under reduced pressure to obtain acteoside as
residue.
In another embodiment of the present invention, wherein the acteoside. obtained by the
process shows hepatoprotective activity at 12 to 25 times lesser dosage as compared to
the one obtained from other sources.
In yet another embodiment of the present invention, wherein the extracts are aqueous
and alcoholic extracts.
In still another embodiment of the present invention, wherein acteoside is about 1.0" o
(wt.) of the total extract.
In still another embodiment of the present invention, wherein a method of effectively
hepatoprotecting a subject using pure Acteoside from plant Colerbrookea oppositifolia.
said method comprising steps of administering appropriate low-dose of the acteoside to
the subject.
In still another embodiment of the present invention, wherein the dosage is ranging
between 0.5 to 10.0 mg/kg body weight.
In still another embodiment of the present invention, wherein acteoside is administered
through P.O. routes.
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of serum glutamine transferase (GPT).
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of serum glutamine transferase (GOT).
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of serum alkaline phosphatase (ALP).
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of serum Bilirubin.
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of serum triglycerides (TG).
In still another embodiment of the present invention, wherein acteoside reduces the
abnormally elevated levels of lipid peroxidase (LP).
In still another embodiment of the present invention, wherein acteoside increases the
abnormally reduced levels of albumin.
In still another embodiment of the present invention, wherein acteoside increases the
abnormally decreased levels of reduced-glutathione.
In still another embodiment of the present invention, wherein acteoside is about 10 to
20 times more effective as compared to commercially available hepatoprotectants.
In still another embodiment of the present invention, wherein acteoside provides
about 40-85% protection against hepatotoxicity.
The present invention relates to unusually very low dosage antihepatotoxic /
hepatoprotective activity, of a phenylethanoid named as Verbascoside-also termed as
Acteoside or Kusaginin isolated and reported for the first time from a plant named
Colerbrookea oppositifolia. The invention describes the isolation of verbascoside in a
specific manner and demonstrates antihepatotoxic / hepatoprotective activity at very low
doses against different toxins.
The active compound acteoside (verbascoside) is being reported for the first time by
the authors of invention from the plant Colebrookea oppositifolia. The authenticity of
the acteoside isolated by the authors has been confirmed by *H and 13C NMR data
similar to reported in literature (Phytochemistry 21, 1123-1127 (1982).
Brief description of the accompanying drawings
Fig 1 shows HPLC graph confirming purity.
Fig 2 shows comparative hepatoprotective activity (%) of acteoside, silymarin, and
glycyrrhizia.
The process of isolation of acteoside from different extracts ( 95% ale., 50% ale. and
aqueous) of the plant is described in the examples appended herewith as Annexure-1.
The HPLC graph confirming purity is enclosed as fig-1.
The compound has been bioevaluated for hepatoprotective/antihepatotoxic activity by
using a comprehensive study design annexed herewith as Annexure-2. The
parameters studied during the bioevaluation to establish the bioefficacy and optimum
dose were preferred using the kits ( Clonital, Italy and Accurex Biomedical Pvt. Ltd
Thane). The list of the parameters is given in Annexure-2 appended herewith. The
dose response graphs with respect to different parameters in comparison with the
standard drug with silymarin and glycyrrhizin are given in Annexure-3 appended
herewith.
The bioevaluation data for each dose of the acteoside as compared to reference
materials viz. silymarin, glycyrrhizin, and required controls are given in the Table-1
which is appended herewith the invention. As is clear from the figure 2 and Table-1
all the parameters which were compromised after the administration of hepatotoxin
CCU have a very good tendency to revert back to normal levels from a dose of 1.25
mg upto 5 mg/kg of acteoside. The overall percent protection provided by acteoside
with reference to all the parameters studied in the invention is generally from 40-80%,
and is better than that rendered by silymarin even at 10-20 times the dosage of
acteoside.
The optimum dose to achieve this protection with acteoside of the invention lies
between 1.25 mg/kg and 2.5 mg/kg. Statistically, there is no significance observed
between the three doses viz. 1.25, 2.5 and 5 mg/kg except when 1.25 mg/kg dose data
is compared with that of 5mg/kg dose (Table-2). But no statistical significance is
observable between higher doses i.e. 2.5mg/kg and 5 mg/kg. At the same time, similar
observation holds fully well for dose effect between 1.25 and 2.5 mg/kg.
The above-stated invention is elaborated in the form of examples and should not be
construed to limit the scope of the invention
Example 1:
Dried aerial parts of plant material Colebrookea oppositifolia (500 gm) were ground
to a coarse powder. The powder was percolated with 95% ethanol for fourteen hours.
Extraction process was repeated four times using total of 9 litres of 95% ethanol (3.0
+2.0 + 2.0 + 2.0 Litre, four extractions) . All the four extracts were pooled and
filtered clear of suspended particles. The supernatant was evaporated to dryness on a
wiped film evaporator at 50±5°C. Residue obtained was 60 . 0 g , (coded as
RJM/0862/P08/A001) extractive value 12%.
The extract was fractionated with CHCh , EtOAc and «-BuOH (2 xl Litre each)
successively. 15.0 g w-BuOH extract was subjected to adsorption chromatography
after dissolving in minimum quantity of MeOH, and adsorbing on SiO2 gel, 100-200
mesh (100 gm.) Solvent was completely removed to get free flowing material. The
adsorbed extract was charged in a glass column of 37.5 mm 0. The column was
eluted with solvents by gradually increasing the %age of MeOH in CHCb 105
fractions of 100 ml each were collected and pooled on the bases of TLC patterns
checked by using EtOAc: HCOOH : I-bO : : 8:1:1 as mobile phase. Spots were
visualised by spraying with freshly prepared Borinate-PEG 4000 solution [1%
solution of 2-aminoethyldiphenylborinate in MeOH and 5% solution of polyethylene
glycol 4000 in EtOH (mixed 1:1 v/v before spraying)].
Seven of the fractions showing same TLC pattern were pooled, dried and subjected to
rechromatography using 100-200 mesh SiO2 gel column (1:20 ratio) and eluted with
CHCb : MeOH mixtures of increasing polarity. In all 60 fractions of 200 ml each
were collected. Eight of the fractions were pooled on the bases of TLC were dried and
again subjected to column chromatography. 30 fractions of 100 ml each were
collected. Six fractions were concentrated under reduced pressure. Residue was
crystallised from MeOH / CHC^ as a colourless amorphous powder, soluble in
MeOH . Compound found at Rf 0.42 , (solvent system EtOAc: HCOOH : H2O: :
8:1:1) was coded as 862-11. The purity of 862-11 was established on the basis of
HPLC using following operating conditions.
Column : RP-18e (E-Merck, SUJTI, 4.6 x 250 mm), at
Mobile Phase : Acetonitrile (B): 1.5% acetic acid in water (A)
Flow rate : Iml/min.
A,max : 335 nm
The compound was established as Verbascoside on the basis of NMR ('H and I3C)
and FABMS data.
Example 2
Dried aerial parts ( 500 gm) of Colebrookea oppositifolia were ground and
percolated with 50% aqueous ethanol four times (50% EtOH, 4 x 2.5 Litre) for 14
hrs each. All the four extracts were pooled. The pooled aqueous extracts were
centrifuged, clear supernatant was evaporated to dryness on a wiped film evaporator
at 50 ± 5°C. The residue (90 g) was coded as RJM/0862/P08/A002 (extractive value
18%) and fractionated with CHCh, EtOAc and «-BuOH successively.
The «-BuOH ext. was chromatographed on a column of silica gel (60-120 mesh)
eluted with a gradient of MeOH in CHC13. The CHC13 : MeOH (5:1) eluate was
rechromatographed on a silica gel (100-200 mesh) column using CHCh-MeOH :
F^O (6:1:0.1) as solvent. Fractions homogeneous on TLC were pooled, dried and
charged on a sephadex LH-20 column, eluted with MeOH to produce two fractions of
500 ml each. Second fraction containing mainly the target compound (862-11) was
subjected to further purification over a sephadex LH-20. Column was eluted with
MeOH: H2O (3:2) to afford a fraction, which on crystallisation from MeOH/CHCl3
yielded a colourless amorphous powder soluble in MeOH , Rf 0.42 (solvent system
EtOAc:HCOOH : H2O:: 8:1:1).
Standardisation of the extract RJM/0862/P08/A002 was carried out on the basis of
862-11 by HPLC using following operating conditions:
Column : RP-18e (E-Merck, Sum, 4.6 x 250 mm), at
Mobile Phase : Acetonitrile (B): 1.5% acetic acid in water (A)
Flow rate : Iml/min.
Xmax : 335 nm
The compound was established as Verbascoside on the basis of NMR (!H and I3C)
and FABMS data.
The %age of 862-11 in the extract RJM/0862/P08/A002 was found to be 0.86.
Example 3
Colebrookea oppositifolia aerial parts (500 g) were ground to a coarse powder and
then extracted with deionised water at 98± 1°C for 2 hrs. Extraction process was
repeated four times using total water (1 + 3 x 0.7 Litre, four extractions). All the four
extracts were pooled The pooled extract was centrifuged, the clear filtrate was
lyophilized to get light yellow amorphous powder (yield 95 gm). Aqueous extract
residue and coded as RJM/0862/P08/A003 (extractive value 19%) was fractionated
with CHClj, EtOAc and n-BuOH successively.
M-BuOH extract was subjected to adsorption chromatography on SiCh gel, 60-120
mesh (150 gm) . Solvent was completely removed to get free flowing material. A
glass column of 1.5 inch dia was packed with 100 gm SiC>2 gel, 60-120 mesh in
EtOAc. The adsorbed material was charged in the column. The column was eluted
with EtOAc and by gradually increasing the %age with MeOH . In all 120 fractions
of 100 ml each were collected and pooled on the basis of TLC pattern ( EtOAc:
HCOOH : H2O : : 8:1:1 as developing solvent). Spots were visualised by spraying
with freshly prepared Borinate-PEG 4000 solution [1% solution of 2-
aminoethyldiphenylborinate in MeOH and 5% solution of polyethylene glycol 4000 in
EtOH (mixed 1:1 v/v before spraying)].
Fractions eluted in EtOAc and 10% MeOH showed same TLC pattern. These
fractions were pooled, dried and then dissolved in minimum quantity of MeOH .
Crystallisation was carried out by the addition of CHCls in small portions to methanol
solution which yielded a colourless amorphous powder characterised as 862-11 .
Standardisation of the extract RJM/0862/P08/A003 was carried out on the basis of
862-11 by HPLC using following operating conditions :
Column : RP-18e (E-Merck, 5um, 4.6 x 250 mm), at
Mobile Phase : Acetonitrile (B): 1.5% acetic acid in water (A)
Flow rate : Iml/min.
A-max : 335 nm
The compound was established as Verbascoside on the basis of NMR ('H and 13C)
and FABMS data.
The %age of 862-11 in the extract RJM/0862/P08/A003 was found to be 0.22.
862-11, amorphous powder, mp 145-146°C,[a]21 -85.6 [C 0.5% MeOH], MS:
FABMS,
[M+Na]+ m/z 647 was found to be a known Phenyl propanoid i.e., Verbascoside
(Acteoside); fp-(3',4'-dihydroxyphenyl) ethyl -O-a-L-rhamnopyranosyl (1~>3)-P-D-
(4-O-caffeoyl)-glucopyranoside]. Structure was finally confirmed by *H and
I3CNMR data similar to that reported in literature (Andary C., Wylde, R. Laffite C.,
Privat G. and Winternitz F.; Laboratie de Botanique et Cryptogamie,
Faculte de Pharmacie. 34000 Montpellier. France; Phytochemistry, 21 (5), 1123-1127
(1982).
STUDY DESIGN
1. Animals : Albino rats (Wistar, 150-180 g) either sex
Albino mice (Swiss, 25-30 g) either sex
2. Hepatotoxin : Carbon tetrachloride (CC14)
3. Study : Prophylactic
4. Treatment Schedule : 48h, 24h, 02h, before and 06h after toxin; blood & liver
samples collection 18 h after last treatment of test material / reference standard.
5. Doses:
Acteoside : 0.625, 1.25, 2.5 & 5.0 mg/ kg, p.o.
Silymarin : 50 mg/kg, p.o.
Glycyrrhizin : 100 mg/kg, p.o.
CCI4 : 1 ml/kg p.o. in liquid paraffin (1:1)
6. Parameters
Serum
Bilirubin:
Triglycerides:
Albumin:
Transaminases
ALP:
Liver homogenate:
GSH:
Jendrassik Method by using Kit supplied by Clonital, 24030-
Carvico (BG)-Italy.
Enzymatic GPO-POD Method by the Kit supplied by
Accurex BiomedicalPvt. Ltd., Thane
Colorimetric B.C.G. Method by the Kit supplied by Accurex
Biomedical Pvt. Ltd., Thane
(ALT, & AST): Pyruvate formed by transamination
reaction was determined spectrophotometrically after
reaction with 2,4-dinitrophenylhydrazine (Reitman and
Frankel, 1957).
/»-nitrophenol formed in alkaline medium was
measured spectrophotometrically using p-nitrophenyl
phosphate as substrate (Waler and Schutt, 1974).
It was determined after deproteination by reaction with
DTNB (Ellman 1959 as modified by David 1987).
Lipid peroxidation :Thiobarbituric acid reacting substances were
determined spectrophotometrically at 535 nm. By the
method of Buege and Aust (1978).
Hepatoprotective activity :
Hepatoprotective activity (H) was calculated by the following equation :
H = [1-(TC- V/VC-V)]xlOO
Where TC, VC, and V are drug + toxin, vehicle + toxin and vehicle treated groups
of animals respectively.




We Claim:
1. A process for isolation of Acteoside from plant Colerbrookea oppositifolia
. characterized in that the said processs comprising steps of:
a. drying aerial parts of the plant,
b. grounding the dried parts into powder,
c. percolating the powder with water or ethanol for 3-4 times to obtain an
extract,
d. filtering the extract for clearing of suspended particles to obtain
supernatant,
e. drying the supernatant at about 45 to 55 C to obtain a residue,
f. fractionating the residue with chloroform, ethyl acetate, and butanol
successively,
g. subjecting butanol fraction to adsorption chromatography of SiO2 after
adding methanol to the fraction,
h. charging the adsorbed fraction to glass column,
i. eluting the column with solvents of increasing polarity of methanol :
chloroform to obtain further fractions and repeating the process one
more time, j. subjecting the fractions to column chromatography to obtain fractions, k. concentrating the fractions under reduced pressure to obtain acteoside as
residue.
2. A process as claimed in claim 1, wherein the extracts are aqueous and alcoholic extracts.
3. A process as claimed in claim 1, wherein acteoside is about 1.0% (\vt.) of the total extract.

Documents:

2976-DELNP-2004-Abstract-(11-02-2009).pdf

2976-DELNP-2004-Abstract-(29-12-2008).pdf

2976-delnp-2004-abstract.pdf

2976-DELNP-2004-Claims-(11-02-2009).pdf

2976-delnp-2004-claims.pdf

2976-delnp-2004-complete specification (granted).pdf

2976-DELNP-2004-Correspondence-Others-(11-02-2009).pdf

2976-DELNP-2004-Correspondence-Others-(29-12-2008).pdf

2976-delnp-2004-correspondence-others.pdf

2976-delnp-2004-description (complete)-11-02-2009.pdf

2976-delnp-2004-description (complete).pdf

2976-delnp-2004-drawings.pdf

2976-delnp-2004-form-1.pdf

2976-delnp-2004-form-18.pdf

2976-DELNP-2004-Form-2-(11-02-2009).pdf

2976-DELNP-2004-Form-2-(29-12-2008).pdf

2976-delnp-2004-form-2.pdf

2976-delnp-2004-form-3.pdf

2976-delnp-2004-form-5.pdf

2976-DELNP-2004-Others-Document-(29-12-2008).pdf

2976-DELNP-2004-Petition-137-(31-12-2008).pdf


Patent Number 229350
Indian Patent Application Number 2976/DELNP/2004
PG Journal Number 10/2009
Publication Date 06-Mar-2009
Grant Date 17-Feb-2009
Date of Filing 30-Sep-2004
Name of Patentee COUNCIL OF SCIENTIFIC AND INDUSTRIAL RESEARCH
Applicant Address RAFI MARG, NEW DELHI-110 001, INDIA.
Inventors:
# Inventor's Name Inventor's Address
1 GHULAM NABI QAZI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
2 KASTURI LAL BEDI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
3 OM PARKASH SURI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
4 KRISHAN AVTAR SURI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
5 GURDARSHAN SINGH REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
6 NARESH KUMAR SATTI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
7 MUSARAT AMINA REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
8 BAL KRISHAN KAPAHI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
9 BAL KRISHAN CHANDAN REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
10 NEELAM SHARMA REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
11 BISHAN DATT GUPTA REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
12 BUPINDER SINGH JAGGI REGIONAL RESEARCH LABORATORY (CSIR), JAMMU, INDIA.
PCT International Classification Number A61P 1/16
PCT International Application Number PCT/IB03/01180
PCT International Filing date 2003-03-31
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 10/403,700 2003-03-31 U.S.A.