Title of Invention

HEPATOPROTECTIVE ACTIVITY OF 10-O-P-HYDROXYBENZYLAUCUBIN

Abstract A method for isolating 10-O-p-hydroxybenzoylaucubin compound of Formula 1, also called Agnuside and its use as an hepatoprotectant.
Full Text HEPATOPROTECTIVE ACTIVITY OF 10-O-PHYDROXYBENZOYIAUCUBIN
Field of the invention
This invention relates to hepatoprotective activity of an iridoid glycoside, 10-Op-
hydroxybenzoylaucubin (agnuside) of the formula 1 isolated from Vitex negundo by
extracting the aerial parts/whole plant with polar solvent like 95% ethanol, methanol,
aqueous ethanol or water, removing fatty non polar constituents by triturating the
extract with solvents such as ethylene chloride, methylene chloride, chloroform or
ethyl acetate to get a fraction from which agnuside is separated by column
chromatography. The hepatoprotective activity of agnuside has been confirmed by
evaluation of its protective action against CCl and galactosamine induced liver
damage models.
Background and Prior art references
Vitex negundo Linn (family Verbenaceae) is widely used in the indigenous system of
medicine in India. Various medicinal properties are ascribed to leaves and roots of
this plant. The leaves are aromatic, tonic and vermifuge and the roots are used as
expectorant, febrifuge and tonic.(Chopra, R.N., Nayar, S.L. and Chopra, I.C.,
Glossary of Indian Medicinal Plants, CSIR, New Delhi, 1956, p. 256; Wealth of India
Raw Material, CSIR, New Delhi, 1976, vol. X. p. 522 ) A number of compounds
have been isolated from various parts of this plant From the leaves Ghosh and Krishna
isolated a number of compounds viz. glucononitol, p-hydroxybenzoic acid, 5-
hydroxyisophthalic acid and' 3, 4 - dihydroxybenzoic acid along with two
glucosides and an amorphous alkaloid [Ghosh, T.P. and Krishna, S.,7. Indian Chem.
Soc. 1936, 13, 634]. Masilungan reported the presence of the terpenes, oc-pinene,
camphene, citral and P- caryophyllene in the essential oil of the leaves Masilungan,
V.A. Philip. J. Sci. 1955, 84, 275]. A large number of flavonoids have been isolated
from the leaves and twigs. These are casticin, orientin, isoorientin, luteolin, luteolin-
7-0-glucoside, corymbosin, gardenins A and B, 3-0-desmethylartemetin,5-0-
desmethylnobiletin,3',4',5,5',6,7,8-heptamethoxy-flavone, 3',5-dihydroxy-4',7,8-
trimethoxyflavanone and 3',5-dihydroxy-4',6,7-trimethoxy flavanone [Sirait.L.M.,
Rimpler, H. and Haensal, R., Experientia 1962, 18, 72; Haensal, R.et al.
Phytochemistry 1965,4 ,19; Banerji A. el al. Phytochemistry 1969, 8,511; Ferdous, A.
J. etal Bangladesh AcadSci. 1984, 8,23; Dayrit, FM.et al. Philipp. J.Sci. 1987, 116,
403; Banerji, J. et al. Indian J.Chem .1988; 27B, 597; Achari, B. et al.
Phytochemistry. 1984, 23, 703]. Stem-bark afforded five new flavone glycosides
along with luteolin and acerosin. The new flavone glycosides are 6p -
glucopyranosyl - 7 hydroxy-3',4',5',8-tetramethoxyflavone-5-O-a-Lrhamnopyranoside,
3', 7-dihydroxy-4',6, 8-trimethoxy flavone-5-O-(6"-O-acetyl-p-Dglucopyranoside),
3,3',4',6,7-pentamethoxy flavone 5'-O-(4"-O-|3-D-glucopyranosyl-arhamnopyranoside,
4',5, 7-tri-hydroxyflavone-8-(2"-caffeoyl-a-glucopyranoside) and
3', 5,5',7-tetrahydroxy-4-methoxyflavone -3'-O-(4"-O-a-D- galactopyranosyl)
galactopyranoside [Rao, V.K.et al. Indian J. Pharm. 1977,39, 41; Subramamian, P.M.
and Misra, G.S. Indian J. Chem. 1978,168, 615; Subramaniam, P.M. and Misra,
G.S.J. Nat. Prod. 1979,42, 540]. A diterpenoid, 5 |3-hydro-8,ll,13-abieta-trien-6a-ol
and three triterpenoids, 2a, 3a-dihydroxyoleana-5 12- dien-28-oic acid, 2a, 3adiacetoxyoleana-
5, 12-dien-28-oic acid, 2,3 cc-diacetoxy-18-hydroxyoleana-5,12-
dien-28-oic acid have been isolated from the seeds. These compounds exhibited anti
inflammatory activity [Chawla, A.S., Sharma, A.K., Handa, S.S. and Dhar, K.L.
Indian J. Chem. 1991, 30B, 773 and J.Nat. Prod. 1992,55,163]. Leaves, however, did
not yield any triterpenoids but from the roots acetyloleanolic acid was isolated
[Vishnoi, S.P.,Shoeb, A., Kapil, R.S. and Popli, S.P. Phytochemistry 1983, 22, 597].
Five Iridoid glycosides have been reported from the leaves of V. negundo. These are
aucubin, agnuside (Hansal et al. Phytochemistry 4, 1965, 9 negundoside 6'-phydroxybenzoyl
mussaenosidic acid (Sehgal et al. Phytochemistry, 21, 1982, 363) and
nishindaside (Datta et al. Tetrahedron 39,1983, 3067).
During our search for hepatoprotectives agents of plant origin, the aqueous alcoholic
extract of V. negundo and a fraction isolated from it exhibited strong immunostimulating
hepatoprotective activities. A process has been developed for the isolation
of an immuno-stimulating agent from the leaves of Vitex negundo for which a patent
has been granted to Regional Research Laboratory, Jammu [Suri, J.L. et. al Indian
Patent No. 78388 dt. 19-03.97). Another patent application has been submitted by
Regional Research Laboratory, Jammu for a process for isolation of a bioactive
composition possessing hepatoprotective and immuno-stimulating activity
(Application no. 16/DEL98 dt. 16.1.98) In view of the strong hepatoprotective
activity exhibited by the iridoid glycosides of Picrorhiza kurroa (Ansari, R.A. et al
Indian J. Med. Research, 1988, 87, 401) it was thought desirable to evaluate the
iridoid glycosides of V. negundo for hepatoprotective activity. Agnuside, an iridoid
glycoside, was isolated from V. negundo and evaluated for hepatoprotective activity
alongwith the aqueous alcoholic extract of the plant. Both aqueous alcoholic extract
(coded as 033) and agnuside (coded as 033 (1) showed marked hepatoprotective
activity in experimentally induced hepatic damage with CC14 and galactosamine
(GalN) in rats. A comparison with the known hepatoprotective agent silymarin
revealed that 033 and 033 (1) exhibited higher hepatoprotective potential in most of
the parameters with respect to their effect on elevated levels of serum and liver
homogenate parameters (Table 1 and 2).
Description of the invention
Thus the main objective of the present invention is to provide hepatopro tec live
activity of a defined bioactive molecule isolated from leaves of V. negundo viz.,
agnuside of formula 1, as shown in the diagram accompanying this specifications.
Accordingly, the present invention provides hepatoprotective activity of a compound
of formula 1 accompanying the specifications which comprises :
(a) powdering the plant material by known methods
(b) preparing the aqueous alcoholic extract by percolation
(c) concentrating the alcoholic extract by conventional method,
(d) removing fatty non polar constituenls by triturating the extract with solvents
such as ethylene chloride, methylene chloride, chloroform or ethyl acetate
(e) adsorbing the residue extract over silica gel,
(f) isolation of agnuside from the adsorbed extract by column chromatography
and
(g) evaluating for hepatoprotective activity
The solvent used for extraction in step (b) is ethanol, aqueous ethanol, methanol,
aqueous
methanol or water.
The hepatoprotective activity of the compound is expressed in 50 mg/kg"' oral dose in
rals which on extrapolation comes to be 300-4QO mg daily human dose (70 kg) in
single or divided doses.
Characterisation of agnuside 1
1 obtained as crystalline compound, mp 148-50°C IvT : -466, UV-232 nm, IR (KBr)
spectrum showed absorptions at 3400, 1700, 1642 and 1618 cm"1. !H NMR (200
MHz, CD3OD) 5 6.35 (dd, J2,6, H-3) 5.12 (dd, J, 4,6, H-4) 2.70 (m-H-5) 4.48 (m, H-
6) 5.82 (s, H-7) 2.94 (m, H-9) 5.05 (s, H-10) 4. 69 (d, J 8, H-l') 3.65 (m, H-2') 7.92
fdd,/2,7, H-2 "6") 6.84 (dd, 7,2,7, H-3 ") 13CNMR 5 97.92(C-1), 141.50 (C-3),
105.35 (C-4), 46.04 (C-5) 82.58 (C-6) 132.09 (C-7), 142.55 (C-8), 46.04 (C-9) 63.46
(C-10), 100.07 (C-l1) 74.56 (C-21), 77.61 (C-31) 71.01 (C-41), 77.82 (C-51) 62.49 (C-
6') 121.81 (C-l") 132.73 (C-2",C-6"), 16.08 (C-3", 5"), 163.50 (C-4 "), 167.70 (CO)
Detailed description of the invention
Accordingly, the present invention provides a method for treating and/or preventing
hepatic disease conditions in mammals including human beings, said method
comprising the steps of administering to the mammal an effective amount of 10-O-phydroxybenzoylaucubin
compound of Formula 1, also called Agnuside, optionally
individual or in combination with one or more pharmaceutically acceptable additives.
Another embodiment of the present invention wherein the said composition reduces
the elevated levels of serum glutamin-pyruvic transaminase (GPT) about 70%.
In yet another embodiment of the present invention wherein the said composition
reduces the elevated levels of serum glutamin-oxalo acetic transaminase (GOT) about
60%.
In still another embodiment of the present invention wherein the said composition
reduces the elevated levels of serum alkaline phosphatase (ALP) about 62%.
In yet another embodiment of the present invention wherein the said composition
reduces the elevated levels of serum tryglycerides about 70%.
In still another embodiment of the present invention wherein said composition against
the elevated level of bilirubin about 72%.
In yet another embodiment of the present invention wherein compound agnuside is
obtained from the whole plant.
In yet another embodiment of the present invention wherein 10-O-phydroxybenzoylaucubin
is of concentration ranging between about 20 to 200 mg /kgbody
weight.
In still another embodiment of the present invention wherein 10-O-phydroxybenzoylaucubin
is of concentration is about 50 mg/kg-body weight.
In yet another embodiment of the present invention wherein the pathological
condition is selected from liver disorder
In still another embodiment of the present invention wherein the subject is selected
from mammals and animals preferably humans.
In yet another embodiment of the present invention wherein said composition is used
singly or in combination with pharmaceutically acceptable carriers.
In still another embodiment of the present invention wherein said composition is
administered to subject in combination with pharmaceutically acceptable additives,
carriers, diluents, solvents, filters. Lubricants, excipients, binder or stabilizers.
In yet another embodiment of the present invention wherein the desired dosage is
administered for both preventive and curative properties.
In still another embodiment of the present invention wherein said composition is
administered, orally or by any clinically/medically accepted methods.
In yet another embodiment of the present invention wherein the preferred dosage for
human beings is about 5 mg/Kg of body weight.
In still another embodiment of the present invention wherein the various physical
forms in which the composition is available, e.g. powder, tablet, capsule, syrup,
granules, emulsion, aerosoal, or beads.
In yet another embodiment of the present invention is useful for Liver cirrhosis,
Galactosemia, Hemoanigoma, Hemochromatosis, Hepatitis A, Hepatitis B, Hepatitis
C, Hepatitis D, Hepatitis E, Hepatitis G, Alcholic Liver disease, Autoimmune
hepatitis, Cancer of Liver, Biliary Atresia, Glycogen Storage Disease 1, Alpha-1-
antitrypsin deficiency, Alagille syndrome, Byler Disease, Caroli disease, Fatty liver,
Itching in Liver, Primar Biliary Cirrhosis, Sclerosing Cholangitis or Protoporphyria
Erythroepatic.
One more embodiment of the present invention a process for the isolation of
compound 10-O-p-hydroxybenzoylaucubin compound of Formula 1, also called
Agnuside, said compound isolated from aerial part/whole body comprising of steps:
(a) powdering the plant material by known methods
(b) preparing the aqueous alcoholic extract by percolation
(c) concentrating the alcoholic extract by conventional method
(d) removing fatty non-polar constituents by triturating the extract with
solvents such as ethylene chloride, methylene chloride, chloroform or
ethyl acetate
(e) adsorbing the residue extract over silica gel
(f) isolating of 2'-p-Hydroxy benzoyl mussaenosidic acid from the
adsorbed extract by column chromatography
Brief Description of the accompanying drawings
Figure 1 represents the compound 10-0-p-hydroxybenzoylaucubin (Agnuside) of formula 1.
Figure 2 represents flow-sheet for isolation of 10-O-p-hydroxybenzoyIaucubin)
(Agnuside).
The invention is described in detail by the examples given below which should not be
construed to the limit of scope of the present invention
Example 1
The shade dried and powdered leaves of (1 kg) V. negundo were extracted with 80%
ethanol (4 x 3L) by percolation. The pooled extract was concentrated under
reduced pressure at below 50°C to 1 litre of aqueous concentrate. The aqueous
concentrate was washed with ethyl acetate (3 x 500 ml) and further concentrated to
400 ml. This syrupy residue was adsorbed over silica gel (400 g) and allowed to dry at
room temperature. The slurry was put on silica gel column and eluted with mixture of
chloroform-rnethanol (19:1), furnished 1 (2.1g) (coded as 033 (1)
Example 2
The finely ground leaves (200 g) of Vitex negundo were extracted thrice with
petroleum ether (60-80°) ( 1 litre for the first extraction and 600 ml each for
two subsequent x extractions) The drug was freed of solvent at room temperature. It
was then extracted with ethanol (1 litre for the first extraction and then thrice with the
same solvent 600 ml each time) Evaporation of ethanol in a rotary film evaporator
yielded 30 g of extract (coded as
Example 3
Treatment of experimental animals with the 033 and 033(1) against CCU induced
liver damage reduced the elevated levels of serum OPT, GOT, ALP, bilirubin, TG and
hepatic lipid peroxidation and increased the GSH level. A comparison with the known
hepatoprotective agent silymarin revealed that 033 and 033(1) exhibited higher
protective potential in most of the parameters with respect to their effect on elevated
levels of above parameters by CCU The hepatoprotective activity observed with 033
and 033(1} was: serum GPT- 67.52 & 59.52, GOT- 58.91 & 57.42, ALP- 58.26 &
60.17, Bilirubin- 60.00 & 71.79, TG- 46.47 & 38.53 and in liver homogenate LP-
55.71 & 41.48 and GSH- 61.42 & 38.12% respectively. The same with silymarin was:
58.44, 51.63, 52.26, 57.50, 41.30, 62.05 & 60.15 respectively (Table-1). Example 4
Treatment of animals against the galactosamine (GaIN) induced hepatic damage also
reduced the elevated levels of serum GPT, GOT, ALP, bilirubin, TG and hepatic lipid
peroxidation and increased the GSH level . The hepatoprotective activity observed
with 033 and 033(1) was 59.58 & 44.97, 56.62 & 44.05, 54.28 & 47.66, 57.71 &
66.66, 57.89 & 49.33 percent in serum GPT, GOT, Bilirubin, ALP, and TG
respectively and 67.51 & 48.34, 66.56 & 55.13 percent in hepatic lipid peroxidation
(LP) and GSH respectively. The same with silymarin was 57.38, 55.40, 60.00,
53.54,46.17, 69.59, 67.18 percent respectively (Table-2).
Advantages of the present invention over currently used plant based
hepatoprotectives:
1. Agnuside is more potent than the commercially available herbal hepatoprotective
agent silymarin.
2. Silymarin is a mixture of three constituents whose relative proportion varies from
batch to batch while agnuside is a pure compound.
Values represent the mean percent hepatoprotective activity of six animals in each
group.
H: Hepatoprotective activity was calculated as (1-(T-V/C • V)} x 100
whereT is mean value of drug and CCU,
"C" is mean value of CCU alone and "V" is the mean value of vehicle treated animals.
Unit: each unit is (mole pyruvate/min/L.
b: is 11 mole of p-mtropheno\ formed/mini U
c: is n moles MDA/g liver.,
d: is n mole GSH/g liver
Values represent the mean percent hepatoproteclive activity of six animals in each
group.
H: Hepatoprotective activity was calculated as {I- (T - V / C - V)} x 100 where "T" is
mean value of drug and GalN, 'C' is mean value of GalN alone and "V" is the mean
value of vehicle treated animals.
Unit: each unit is pinole pyruvate/min/L.
b: is M, mole of /j-nitrophenol formed/min/ L,
c: is n moles MDA/g liver.,
d: is p. mole GSH/g liver.,



We Claim
1. A method for the isolation of compound 10-O-p-hydroxybenzoylaucubin
compound of Formula 1, also called Agnuside, said compound isolated from
aerial part/whole body comprising of steps: 1. powdering the plant material by
known methods 2. preparing the aqueous alcoholic extract by percolation 3.
concentrating the alcoholic extract by conventional method 4. removing fatty non-
polar constituents by triturating the extract with solvents such as ethylene
chloride, methylene chloride, chloroform or ethyl acetate 5. adsorbing the residue
extract over silica gel 6. isolating of 2'-p-Hydroxy benzoyl mussaenosidic acid
from the adsorbed extract by column chromatography.
2. The method as claimed in claim 1, wherein compound agnuside is obtained from
the whole plant.
3. The method as claimed in claim 1, wherein compound 10-O-p-
hydroxybenzoylaucubin is of concentration ranging between about 20 to 200
mg/kg-body weight.
4. The method as claimed in claim 1, wherein 10-O-p-hydroxybenzoylaucubin is of
concentration is about 50 mg/kg-body weight.
5. The method as claimed in claim 1, wherein said composition is used singly or in
combination with pharmaceutically acceptable carriers.
6. The method as claimed in claim 1, wherein said composition is administered to
subject in combination with pharmaceutically acceptable additives, carriers,
diluents, solvents, filters. Lubricants, excipients, binder or stabilizers.
7. The method as claimed in claim 1, wherein said composition is administered,
orally or by any clinically/medically accepted methods.
8. A use of Agnuside for treating and/or preventing hepatic disease conditions in
mammals including human beings, said method comprising the steps of
administering to the mammal an effective amount of 10-O-p-
hydroxybenzoylaucubin compound of Formula 1, also called Agnuside, optionally
individual or in combination .wherein the said composition reduces the elevated levels of serum glutamin-pyruvie transaminase (GPT) about 70%.
9. A use as claimed in claim 8, wherein the said composition reduces the elevated
levels of serum glutamin-oxalo acetic transaminase (GOT) about 60%.
10. A use as claimed in claim 8, wherein the said composition reduces the elevated
levels of serum alkaline phosphatase (ALP) about 62%.
11. A use as claimed in claim 8, wherein the said composition reduces the elevated
levels of serum tryglycerides about 70%.
12. A use as claimed in claim 8, wherein said composition against the elevated level
of bilirubia about 72%.
13. A use as claimed in claim 8, wherein compound agnuside is obtained from the
whole plant.
14. A use as claimed in claim 8, wherein 10-O-p-hydroxybenzoylaucubin is of
concentration rangiy beeween : about 20 to 200 mg/kg-body weight.
15. A use as claimed in claim 8,wherein 10-O-p-hydroxybenzoylaucubin is of .
concentration is about 50 mg/kg-body weight.
16. A use as claimed in claim 8, wherein the pathological condition is selected from
liver disorder.
17. A use as claimed in claim: 8, wherein said composition is used singly or in
combination with pharmaceutically acceptable carriers.

Documents:


Patent Number 235713
Indian Patent Application Number 2498/DELNP/2004
PG Journal Number 34/2009
Publication Date 21-Aug-2009
Grant Date 12-Aug-2009
Date of Filing 26-Aug-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 ANIL PRABHAKAR REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
2 BISHAN DATT GUPTA REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
3 NARESH KUMAR SATTI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
4 SWADESH MALHOTRA REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
5 VIJAY KUMAR SHARMA REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
6 BAL KRISHAN CHANDAN REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
7 SHANKAR LAL REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
8 KRISHAN AVTAR SURI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
9 KULDEEP KUMAR GUPTA REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
10 RAKESH KAMAL JOHRI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
11 BUPINDER SINGH JAGGI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
12 KASTURI LAL BEDI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
13 OM PARKASH SURI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
14 GHULAM NABI QAZI REGIONAL RESEARCH LABORATORY (CSIR) JAMMU.
PCT International Classification Number A61K 31/352
PCT International Application Number PCT/IB03/01810
PCT International Filing date 2003-05-09
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/397,359 2002-05-10 U.S.A.