Title of Invention

METHOD FOR OBTAINING NOVEL DIHYDROXY OXYBIS COMPOUNDS

Abstract A method of obtaining anti-diabetic/anti-hyperglycemic/anti-hyperlipidemic novel compounds of generic formula I, wherein R = CH3 or H said method comprising:- a) extracting pulverized plant material (seeds of Eugenia Jambolana) by soaking in a polar organic solvent such as herein described; b) fractionating in the manner such a herein described the extract so obtained from step a) applying different solvents such as herein described, c) undertaking two stage column chromatographic separations, d) isolation of the active fraction followed by purification thereof and identification of bioactive molecules of formula la and/or formula lb, therefrom.
Full Text METHOD FOR OBTAINING NOVEL DIHYDROXY OXYBIS COMPOUNDS
FIELD OF THE INVENTION
The present invention in general relates to novel dihydroxy oxybis compounds for
treatment of diabetes and method of obtaining the same. In particular, the present
invention relates to isolation, identification and purification of such compounds from
the seeds of Eugenia Jambolana.
BACKGROUND OF THE INVENTION
Since very ancient period; the medicinal plants are being used for the treatment and
management of many diseases. The seed and pulp of Eugenia jambolana (belongs
to the family: Myrtaceae) are being widely used as crude drug; either in single or in
combination for the treatment and management of diabetes mellitus. Moreover, in
different medicinal books this plant is also described as antidiabetogenic plant having
antihyperglycemic effect.
However, no reports are hitherto available for obtaining novel compounds from seeds
of Eugenia Jambolana for treatment of diabetes, so that effective pharmaceutical
compositions can be arrived at for treatment of diabetes, hyperglycemia and
hyperlipidimia.
Accordingly, their was a long felt need for obtaining novel compounds from seeds of
Eugenia Jambolana for treatment of diabetes, so that effective pharmaceutical
compositions can be arrived at, for treatment of diabetes.
Re present invention meets the aforesaid long felt need.
OBJECTS OF THE INVENTION:
It is the principal object of the present invention to provide novel dihydroxy oxybis
compounds for treatment of diabetes, hyperglycemia, hyperlipidimia. It is a further
object of the present invention to provide dimethyl 4,4' oxybis (3,5 dihydroxy
benzoate) for treatment of diabetes, hyperglycemia, hyperlipidimia.
It is another object of the present invention to provide 4,4' oxybis (3,5 dihydroxy
benzoic acid) for treatment of diabetes, hyperglycemia, hyperlipidimia.

It is a further object of the present invention to provide effective pharmaceutical
compositions for treatment of diabetes, hyperglycemia, hyperlipidimia.
It is another object of the present invention to provide a pharmaceutical
compositions and a method therefore for treatment of diabetes, hyperglycemia and
hyperlipidimia, which reveal no side effect.
It is yet another object of the present invention to provide a chemical method for
obtaining dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) and/or 4,4' oxybis (3,5
dihydroxy benzoic acid) from seeds of Eugenia Jambolana for treatment of diabetes,
hyperglycemia, hyperlipidemia.
It is a further object of the present invention to provide a chemical methodology
including chemical isolation, identification, chemical purification, chemical treatment
followed by bio efficacy testing, chemical characterization (by IR and NMR) and
structural elucidation (by Mass Spectroscopy) of most effective solvent fraction of
seeds of Eugenia Jambolana.
How the foregoing objects are achieved and the other aspects of the present
invention, will be clear from the following description which is purely by way of
understanding and not by way of any sort of limitation.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a method of obtaining anti-diabetic/anti-
hyperglycemic/anti-hyperlipidemic novel compounds of generic formula I, wherein
R= CH3 or H


said method comprising:-
a) extracting pulverized plant material (seeds of Eugenia Jambolana) by soaking
in a polar organic solvent such as herein described;
b) fractionating in the manner such a herein described the extract so obtained
from step a) applying different solvents such as herein described,
c) undertaking two stage column chromatographic separations,
d) isolation of the active fraction followed by purification thereof and
identification of bioactive molecules of formula la and/or formula lb,
therefrom.
In accordance with preferred embodiments of the method of the invention:
-said compounds are of formula la and/or formula lb wherein R. =- C-H-2 • ar|d H
respectively,

-said solvents applied in step b) includes any one or combination of two or more of
n-hexane, chloroform, Ethylacetate and n-Butanol.
-said active fraction includes ethylacetate fraction and said step c) involves
subjecting said fraction to column chromatography using silica gel (100-200 AST
mesh) as adsorbent and chloroform, ethylacetate and methanol as eluting solvents
with gradient techniques.

-separation and purification of compounds of formula la and formula lb and carried
out on combiflash companion chromatographic system using Residep normal phase
column (40gm) filled with silica gel (230-400 ASTM) as separating column.
-said compound of formula la is dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) and
said compound of formula lb is 4,4' oxybis (3,5 dihydroxy benzoic acid)
The present invention also provides a process for manufacturing a pharmaceutical
composition comprising undertaking the method of obtaining as described
hereinbefore and combining said compounds of formula la and lb in synergistically
effective proportioned with suitable pharmaceutical carriers whereby effective anti
diabetic, antihyperglycemic and anti-hyperlipidemic traits are revealed by said
composition.
The present invention also provides a process for manufacturing a pharmaceutical
composition comprising undertaking the method of obtaining described hereinbefore,
so that any one of said compounds of formula la or lb are extracted followed by
combining said compounds with suitable pharmaceutical carriers in synergistically
effective proportions, whereby effective antidiabetic, anti-hyperglycemic and anti-
hyperlipidemic traits are revealed by said composition.
The present invention also provides Pharmacuetical compounds of generic formula I
Wherein R= CH3 or H,

said compound being chemically obtained by method as described hereinbefore.

The present invention also provides a method of chemically obtaining chemical
compounds from seeds of Eugenia jambolana for treatment of
diabetes/hyperglycemia/hyperlipidemia substantially as herein described.
BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS
The nature and scope of the present invention, will be better understood from the
accompanying drawings, which are by way of illustration and not by way of any sort
of limitation.
In the accompanying drawings-
Figure la illustrates the results of combiflash companion chromatographic separation
of dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) [hereinafter referred to as P1] and
4,4' oxybis (3,5 dihydroxy benzoic acid) [hereinafter referred to as P2].
Figures lb and 1c illustrate UV spectrum of P1 and P2 respectively.
Figures 2a illustrate HPTLC finger printing of P1, P2 and P3 being mixtures of 8
flavonoids.
Figure 2b illustrates HPTLC finger printing of P1 and P2.
Figure 3a illustrates HPLC chromatogram of P1 and P2 on carbohydrate column at
220nm.
Figure 3b illustrates HPLC chromatogram of P2 on carbohydrate column at 220nm.
Figure 3c illustrates HPLC chromatogram of P1 and P2 on C18 column at 220nm.
Figure 3d illustrates PDA spectrum of P1 and P2 on waters HPLC.
Figure 4a illustrates I.R. spectrum of P1 in KBr Disc.
Figure 4b illustrates I.R. spectrum of P2 in KBr Disc
Figure 5a illustrates H NMR spectrum of P1 in CD3OD
Figure 5b illustrates H NMR spectrum of P2 in CD3OD
Figure 6a illustrates product ion spectrum and structure of P1 at m/z 368.85
Figure 6b illustrates product ion spectrum and structure of P1 at m/z 183.53 and m/z
167.28.
Figure 6c illustrates product ion spectrum and structure of P2 at m/z 340.67.
Figure 6d illustrates product ion spectrum and structure of monomers at m/z 169.44
and 153.32
DETAILED DESCRIPTION OF THE INVENTION
The following describes some preferred embodiments of the present invention, which
are purely for the sake of understanding the performance of the invention, and not
by way of any sort of limitation.


The present invention deals about two new potent diabetic (Diabetes mellitus)
therapeutic agents; identified as dimethyl 4, 4' oxybis (3, 5 dihydroxy
benzoate) and 4, 4' oxybis (3, 5 dihydroxy benzoic acid) from the seeds of
Eugenia jambolana, and to a process for obtaining such compounds chemically.
The compounds according to the present invention have the following generic
structures, as I below

R may be CH3 or H. When R is CH3, the compound has a structure la as , shown
below and it is Dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) [Pi]

When R is H, the compound has a structure lb, as shown below and it is 4,4' oxybis
(3,5 dihydroxy benzoic acid) [P2]


Here seeds of Eugenia jambolana have been used for the preparation of
hydromethanolic (MeOH: H2O:: 60:40) crude extract followed by bio activity guided
solvent fractionations using different solvents (n-Hexane, Chloroform, Ethyl acetate
and n-Butanol). Most effective fraction (Ethyl acetate) is further used for two stages
column chromatography for isolation, identification, purification and chemical
treatment followed by bio efficacy testing, chemical characterization ( by IR and
NMR) and structural elucidation ( by Mass Spectroscopy ).
The present invention provides an efficient technique for successful separation,
identification and characterization of new bio molecule(s) as nontoxic herbal
therapeutic agent(s) as well as to establish their in vivo therapeutic effects(s) on
streptozotocin induced experimental diabetic rats. This invention is the first finding of
dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) and 4,4' oxybis (3,5
dihydroxy benzoic acid) as curative natural diabetic ( Type-II) therapeutic agents
from the seeds of E. jambolana. These two new molecules not only possess anti-
hyperglycemic property but also restore normal body weight, lipid profile and the
glycogen content of hepatic and skeletal muscle. Moreover, these two therapeutic
agents do not have any acute or chronic toxic (haematotoxicity, hepatotoxicity and
renal toxicity) effect on normal experimental animals (rats and mice) when
administered orally at their respective therapeutic doses. Long duration therapeutic
study (six months) with these new therapeutic agents reveals that plasma insulin
and C-peptide levels have significantly been increased in experimental diabetic rats.
These findings indicate that these two molecules have a prospective positive effect
on the regeneration of insulin secreting pancreatic beta cells.

Extract Preparation
Dried seeds of Eugenia jambolana were procured. Maceration of the pulverized
seeds (4.8Kg) was carried out at room temperature (to avoid any degradation or
deactivation of active molecules) in 20L percolator with hydromethanolic solution
(MeOH:H2O :: 60:40). The slurry was stirred intermittently for 1 hr and then left
for overnight. The chemical extraction was carried out for consecutive three
days; each day with 5L freshly prepared hydromethanolic solution. The chemical
extract was filtered first through cotton wool and then through Whatman filter
paper (No-1) and finally subjected to evaporation on Rota vapour (BUCHI-R-124,
Switzerland) under reduced pressure (100 mbar) at 40°C for complete removal of
methanol. The plain aqueous filtrate (890 gm w/w) was lyophilized on VirTis
Bench top K Lyophilizer. The Lyophilized extract was stored at 2°- 8°C under
vacuum for subsequent fractionation and experimental studies. This scheme is
schematically provided below and hereinafter referred to as scheme I


Solvent Fractionation
The lyophilized crude extract (600gm) was dissolved in 2L of hydromethanolic
solution (MeOH: H2O::60:40) and solvent partitioning was carried out using various
solvents (n-Hexane, CHCI3, EtOAc and n-Butanol) with increasing polarity. Progress
in fractionation was monitored by planner chromatography (TLC) using various
mobile phase systems. All fractions were filtered and dried separately under reduced
pressure (20 mbar - 200 mbar by Rotavac Vacuum Pump, Germany) on Heidolph
Laborota-4000 (Germany) keeping bath temperature at 40°C. The residual
hydromethanolic fraction was finally lyophilized after complete removal of methanol.
Finally all fractions were subjected to the bio-activity study to identify most active
fraction responsible for anti-hyperglycaemic activity. These steps are schematically
provided below and hereinafter referred to as scheme - II


Bioactivity study
In vivo anti-hyperglycaemic study of different solvent fractions was carried out at
various dose levels (50 - 300 mg/kg) on diabetic Wistar rats to identify the most
active fraction. Finally ethyl acetate fraction (46.8 gm w/w) was found to be the
most active fraction that exhibited significant anti-hyperglycaemic activity (reduction
in fasting plasma glucose level by 35.9% after 3hrs of oral administration) at a dose
of 200mg/Kg of body weight. Ethyl acetate fraction was finally subjected to column
chromatography for isolating active molecules.
Isolation and Identification of Bio-active Compounds from Active
Fraction
Isolation through Column Chromatography
20 gm of ethyl acetate fraction was subjected to column chromatography (Scheme-
Ill) using Silica gel (100-200 ASTM mesh) as adsorbent and CHCI3, EtOAc and
MeOH as eluting solvents with gradient technique. Fractions found similar on TLC
were combined together and finally dried under reduced pressure on Laborota-4000
at 40°C. Activity of different fractions was carried out at various dose levels and
finally the combined fraction No -2 (7.5gm w/w) containing both Pi and P2 as main
constituents was found to be anti-hyperglycaemic in nature. These steps are shown
below schematically and hereinafter referred to as scheme - III


Separation and Purification of compound P1 and P2
Further separation and purification of P1 and P2 were carried out on Combiflash
Companion Chromatographic system using Redisep normal phase column (40gm)
filled with silica gel (230 - 400 ASTM Mesh) as separating column. 4gm of combined
column fraction (No-2) was adsorbed on Si gel (230-400 ASTM Mesh) to obtain a
free flowing powder. The free flowing adsorbed powder was filled in sample cartridge
(size- 5gm) and then subjected to chromatographic separation (Fig.la) using CHCI3
and CH3OH as mobile phase with linear gradient elution (CHCI3 100-0%; CH3OH O-
100%) technique. The results are illustrated in the accompanying Figure la. Flow
rate of the mobile phase was 40ml/min and detection of the compounds was done at
254 nm. The column eluents were collected in different collection tubes. The
identification of pure fraction was further confirmed by TLC (Normal phase and
Reverse phase) using various solvent systems as mobile phase. Pure fractions
containing P1 were combined together and dried under reduced pressure at 40°C to
obtain pure compound P1 (810 mg w/w) as pale yellow powder. Similarly pure
fractions containing P2 were combined together to obtain pure P2 (3.1 gm w/w) as
white crystalline powder. Both the compounds; P1 and P2 were subjected to activity
study at various dose levels (2 mg, 5 mg, 10 mg and 20 mg/Kg of body weight) and
found to be anti-hyperglycaemic. Biological studies on various parameters were
carried out both in separate and combined form to identify its mode of action and
also to establish its anti-diabetic potency. Identification, characterization and others
instrumental analysis of the compounds were also carried out to establish their
chemical identities and characteristics. HPLC purity of P1 and P2 was established as
98.5% and 99.2% respectively. These steps are provided below schematically and
hereinafter referred to as scheme - IV.


Instrumental Analysis, Chemical Characterization and Structural
Elucidation of Compounds P1 and P2
Determination of A max
Both P1 and P2 were separately dissolved in and diluted with HPLC grade methanol to
prepare the solutions of 25pg/ml and the compounds were scanned over entire UV
range (400 - 199 nm) for recording of UV spectra to determine the A max of the
compounds. The UV spectrum have been illustrated in the accompanying figures IB
and 1C.
HPTLC Fingerprinting
HPTLC chromatography was performed on 10 cm x 10 cm precoated silica gel G 60F.
254 plates (E. Merck). Ethyl acetate fraction, P1 and P2 were separately dissolved in
and diluted with HPLC grade methanol to prepare the solutions of 0.5 mg/mL. 5 µL of
each sample solution was applied to the HPTLC plates by spray-on technique. The
first sample band was at 18.0 mm from the edge of the plate, length of each band
was 6.0 mm and the distance between sample bands was 15.0 mm. Plates were
developed in a Camag twin-through glass TLC chamber (10 cm x 10 cm) previously
saturated with mobile phase (EtOAc:MeOH:H2O :: 100:13.5:10 v/v) for 30 min. The
plates were dried under stream of hot air and then examined in a Camag UV cabinet
at λ = 254 nm and 366 nm. The results are shown in the accompanying figures 2a
and 2b.
High performance liquid chromatography
The analytical RP-HPLC was performed to establish the chromatographic purities of
both P1 and P2. RP-HPLC analysis was carried on Thermo Hypersil BDS C18 (4.6 x
250 mm, 5 urn) column using a premixed solvents (Water: ACN: MeOH :: 40:30:30)
as mobile phase with a flow rate of 1.0 mL/min and isocratic elution technique. The
column temperature was maintained at 30°C and detection was performed at 220
nm. HPLC analysis was also carried out using (a) carbohydrate column (300 x 7.8
mm, 5 urn ) maintained at 30°C and (b) 0.001M H2SO4 (pH; 3.0) as mobile with an
isocratic flow rate of 1ml/min. For both the HPLC analyses, P1 and P2 were separately
dissolved with the mobile phase to prepare the solutions of 50 µg/mL. The sample
solutions were sonicated for 15 min and then filtered through Millipore Millex syringe
filter unit (0.45 µm). 10µL of each sample solution was injected through auto injector
fitted with 200 µL syringe and 100 µL sample loop. The compounds were scanned


over entire UV range on 3D spectral mode to record the PDA spectra. This is shown
in the accompanying figures 3d. HPLC chromatograms of both the compounds were
recorded at 220 nm as shown in the accompanying figure 3a, 3b and 3c. All the data
of Waters alliance HPLC system were acquired and processed using Waters
Millennium32 software and the data of Agilent Technologies 1200 series HPLC
system were acquired and processed using HP Chemstation B.02.01 (244)
software.
Infrared Absorption Spectroscopy (I.R. Spectra)
2 mg of each of P1 and P2 were separately mixed with 100 mg of KBr (previously
dried at 120°C for 2 hours) and the transparent film was prepared using hydraulic
pellet press (pressure applied 100 Kg/cm2 for 1 min). I.R. spectrum was recorded
over the range of 4000 cm-1 to 400 cm-1 as shown in the accompanying figures 4a
and 4b.
Nuclear Magnetic Resonance Spectroscopy (NMR Spectra)
2.5 mg of each of P1 and P2 was dissolved separately in 0.7 ml of CD3OD and 1H
spectra were recorded at 300 MHz. The results are shown in the accompanying
figures 5a and 5b. 25 mg of each of P1 and P2 was dissolved separately in 0.7 ml of
CD3OD and 13C NMR spectra of P1 and P2 were recorded at 75 MHz using broad band
decoupling mode. The results are shown in the accompanying figures 5c and 5d.
LCMS / MS Study
HPLC-MS experiments were carried out employing a nova-pak C18 (3.9 X 150 mm, 5
urn) column with a flow rate of 1 mL/min. An isocratic flow of premixed solvents
(water: ACN: MeOH :: 40:30:30) was used for 15 min. Both P1 and P2 were
separately dissolved in and diluted with HPLC grade methanol to prepare the
solutions of 500ug/mL. The prepared solutions were filtered through Millipore Millex
syringe filter unit (0.22 urn) and then 20 uL of each test solution was injected
separately via auto injector for LCMS analysis. For direct MS study, 10 ug/mL
solutions of both Pi and P2 were prepared separately and then filtered through
Millipore Millex syringe filter unit (0.22 urn). 40 uL of each of filtered test solution
was injected directly to the Mass instrument through Hamilton syringe. Four
channels (two for positive mode and two for negative mode) were selected at cone
voltages of 20V and 40V. Capillary voltage, desolvation temperature and source
temperature were maintained at 3.0 kV, 250° C and 120° C respectively. Nitrogen
gas was used as desolvation gas and cone gas at a flow rate 500 L/hr and 50 L/hr

respectively. All the data were acquired and processed using Waters Mass Lynx 4.0
software.
Results and Discussion
Compound P1: Obtained as pale yellow amorphous powder, m.p. 183-184.9°C. Its
molecular formula C16H14O9 (I) was deduced from its LCMS spectrum at m/z 368 (M+
+ H2O; 350 + 18) in association with its 13CNMR spectrum accompanying figure 6a.
Positive reaction with FeCI3 to give deep coloration implied the presence of phenolic
groups. IR spectrum showing absorptions at 3507 (br), 1697 and 1618 cm-1
suggested the presence of hydroxyl, carbonyl and aromatic functionalities in the
molecule. 1HNMR accompanying (Fig.5a) and 13CNMR spectra showed signals typical
of galloyl pattern. 1HNMR exhibited equivalent signal for four aromatic protons at 5
7.03 indicating the presence of two galloyl moieties in the molecule at para position
successively due to equivalent signals for four aromatic protons. Mass spectrum
accompanying (Fig.6b) exhibiting fragment ion peaks at m/z 184 (C8H8O5), m/z 168
(C8H8O4) further supported the linkage as shown in IIa below. Singlet at 5 3.8 for six
protons indicated methoxylation of carboxylic hydroxyl group. Compound P1 was
therefore characterized as Dimethyl 4, 4'- Oxybis (3, 5-Dihydroxybenzoate). Its
13CNMR is summarized in Table 1.

Compound P2: Obtained as off white amorphous powder, m. p. 243.6-244.8°C
Molecular formula C14H10O9 (I) was deduced from LCMS spectrum at m/z 340 (M+ +
H2O; 322 + 18) in association with its 13CNMR spectrum accompanying figures 6c. Its
positive reaction to give a dark blue colour with FeCI3 indicated the presence of
phenolic group. IR absorptions at 3557(br), 1698, 1615 cm-1 further supported the
presence of hydroxyl, carbonyl and aromatic functionalities in the molecule. 1HNMR

accompanying (Fig.5b) and 13CNMR spectra showed signals typical of galloyl pattern.
1HNMR exhibited signals due to the presence of equivalent protons at d 7.03 as
sharp singlet integrating for four protons of two gallic acids. The linkage was
established to be attached at C-4 and C-4' successively due to the presence of
double intensity signals in NMR for each aromatic protons which was further
supported by mass spectrum accompanying (Fig.6d) exhibiting the presence of
fragment ions at m/z 170, m/z 154 as shown hereinafter.

Compound was therefore characterized as 4,4'- Oxybis (3, 5-Dihydroxybenzoic
acid). Its 13CNMR is summarized in Table 1.


Compound P1: Characterized as Dimethyl 4,4'-Oxybis (3,5-
Dihydroxybenzoate), Pale yellow amorphous powder, m. p. 183-184.9°C,
molecular formula C16H14O9, dark blue colour reaction with FeCI3, Rf 0.83 (EtOAc :
MeOH: H2O :: 100:13.5:10), A max (MeOH, c = 0.025mg/ml) nm: 273.3, 217.8, IR
(KBr) v max cm-1 : 3507, 3469, 3307, 1697, 1618, 1540, 1470, 1438, 1384, 1314,
1252, 1260, 1196, 1037, 1004, 859, 768, 746,641,554. 1H NMR (CD3OD) δ ppm:
7.03, 4H; H-2, H-6, H-2', H-6', 3.8(s, 6H; O-CH3). 13C NMR (CD3OD): Table 1. LCMS
m/z: 368 (M + H2O; 350 + 18), 184 (C8H8O5, Methyl gallate), 168 (C8H8O4).
Compound P2: Characterized as 4,4'-Oxybis (3,5 - Dihydroxybenzoic Acid), Off
white amorphous powder, m. p. 243.6-244.8°C, Molecular formula C14H10O9, dark
blue colour reaction with FeCI3, Rf 0.54 (EtOAc : MeOH : H2O :: 100:13.5:10), A max
(MeOH, c = 0.025mg/ml) nm: 260.3, 213.1, IR (KBr) v max cm-1 : 3557, 3084(br),
1698, 1615, 1583, 1542, 1511, 1449, 1397, 1338, 1260, 1195, 1111, 1056, 882,
812, 757. 1H NMR (CD3OD) δ ppm: 7.03, 4H; H-2, H-6, H-2', H-6 y 13C NMR
(CD3OD): Table 1. LCMS m/z: 340 (M + H2O; 322 + 18), 170 (C7H6O5, gallic acid),
154 (C7H6O4).
1. Acute therapeutic effects of P1 and P2 (Effects after single oral
administration)
(a) On fasting serum glucose levels in hyperglycemic Wistar rats
(i) After 3hrs of single oral administration of Pi{Dimethyl 4,4'oxybis (3,5 dihydroxy
benzoate)} and P2{4,4' oxybis (3,5 dihydroxy benzoic acid)} at a dose of 10 mg/kg
and 20 mg /kg of body weight respectively, the fasting blood glucose level in
moderate diabetic (FSO200 mg/dL) Wistar rats were reduced by 37.5 % and 32.5
% respectively.
(ii) The fasting serum glucose level in severe diabetic (FSG>300 mg/dL) Wistar rats
were reduce by 26.48 % and 24.74 % respectively after 3 hrs of oral administration
of P1{Dimethyl 4,4'oxybis (3,5 dihydroxy benzoate)} and P2 {4,4' oxybis (3,5
dihydroxy benzoic acid)}at a dose of 10 mg/kg and 20 mg /kg of body weight
respectively.


(b) On fasting serum glucose levels in normoglycemic Wistar rats
No acute hypoglycemic effect was observed in normoglycemic Wistar rats after
single oral administration of P1 (10 mg /kg) and P2 (20 mg /kg) at their respective
therapeutic doses.
2. Chronic therapeutic effects of P1 and P2 on experimental animals (Effects
after daily oral administration)
(i) Short duration (21, 30 and 60 days) therapeutic effects
(a) 30 days oral administration (twice a day in separate and combined form) of P1
(10 mg/Kg), P2 (20 mg/Kg) and P1P2 (10 +20mg/Kg); resulted significant recovery in
serum glucose levels in streptozotocin induced experimental diabetic rats.
(b) The gradual loss in body weight which was evident in all diabetic rats, was
reversed and a significant improvement in body weight was observed only after three
weeks daily (twice a day) oral administration of P1 and P2 at their respective
therapeutic doses.

(c) The glycogen content of skeletal muscle and liver which was decreased
significantly in streptozotocin induced severe diabetic rats; was found to be
recovered significantly towards the normal control level after oral administration
(twice a day) of P1 and P2 at their respective therapeutic doses for 30 days
(d) The serum cholesterol, triglycerides and free fatty acids which were increased
significantly in streptozotocin induced diabetic animals; were found to be recovered
towards the normal levels after regular oral administration (twice a day) of
P1(10mg/kg) and P2(20mg/kg) for 30 days.
(f) After daily oral administration (twice a day for 60 days) of P1(10mg/kg) and
P2(20mg/kg), the fasting serum glucose levels in moderate diabetic rats were
reduced significantly (32-35%).
(ii) Long duration (90 days and 180 days) therapeutic effects
After monotherapy (twice a day for 90 days) with the isolated active bio-molecules
P1(10mg/kg) and P2(20mg/kg), the fasting serum glucose levels, body weights and
glycogen content of hepatic and skeletal muscle in experimental rats were found be
heading towards normal values.


After oral treatment (twice a day for 180 days) separately with P1 (10mg/kg) and P2
(20mg/kg); the fasting serum glucose levels, lipid profiles and glycogen content of
skeletal muscle and hepatic tissue in experimental rats were found to be restored
towards normal control values.
Long duration therapeutic study (three and six months) with these new therapeutic
agents revealed that plasma insulin and C-peptide levels were significantly increased
in experimental diabetic rats. These findings indicate that these two molecules have
prospective positive effect on the regeneration of insulin secreting pancreatic beta
cells.
Oral Glucose Tolerance Test (OGTT)
Test Procedure:
After daily oral dose (twice a day) of P1 (10 mg/kg) and P2 (20 mg/kg) for 120 days,
all the treated diabetic (FSG>300 mg/dL) rats were subjected to oral glucose
tolerance test to assess post therapeutic effects of these isolated molecules on
glucose tolerance level in the diabetic animals. Prior to the test; all the rats were
divided into five groups and were kept under overnight fasting. Prior to glucose load;
fasting serum glucose level of all the rats were measured by GOD/POD method. The
rats of all groups were then loaded with glucose (2g/kg, p.o.) solution. After glucose
load; blood samples of all the treated rats were collected at a specified interval by
puncturing of retro-orbital plexus using Micro Hematocrit capillary tubes under light
anesthesia.
Observations:
Glucose tolerance levels of all treated rats were significantly improved in compare to
all untreated diabetic rats and this may be due to either decrease in the resistivity of
the insulin receptor sites or regeneration of insulin secreting p cells or both of them .
Animals treated with P2 showed better glucose tolerance in compare with P1.
The fasting serum glucose levels in both the treated groups were found to return to
their respective basal values within 2.0 hrs of glucose load where as in diabetic
control animals; serum glucose levels were found to return to the basal values after
3.0 hrs of oral glucose load.
Effects after withdrawals of therapeutic application of P1 and P2
This study was carried out to evaluate the post therapeutic effect on fasting serum
glucose levels in experimental diabetic rats after regular oral dose (twice a day for


120 days) of P1 (10 mg/kg) and P2 (20 mg/kg) followed by one month withdrawal of
the said therapy.
Observations:
It was found that during oral therapy with P1 and P2 at their respective therapeutic
doses; the recovery from the patho-physiological condition of diabetes was
permanent and irreversible.
It was also observed that the therapeutic potency of these newly discovered
molecules towards the improvement in diabetic condition was much better than the
established oral hypoglycemic agents like glipizide.
Biological safety test of isolated molecules
1. Acute Toxicity Test (As per Indian Pharmacopoeia 1996, Vol II)
(i) Acute Toxicity Test in Mice (14 days observation)
Test procedures
Acute toxicity levels of active compound P1 and P2 were evaluated orally in Swiss
albino mice of either sex weighing between 17-22 gm .The mice were divided in
three groups comprising 10 mice in each group (5 male and 5 female). The first and
second group was fed with the active compound P1 and P2 respectively at a dose of
0.4mg/20gm/ml i.e. 20 mg/kg of body weight. The third group was fed with sterile
drinking water (lml/20gm of body weight) and was marked as control. Before and
during experiment all mice were housed in an air conditioned room (at 23°- 25°C
and relative humidity 44-56%) with 12hr/12hr light/dark cycle and maintained with
free access to drinking water (Aqua guard H2O) and standard pallet diet (supplied by
Golden Feeds; Mehrauli; New Delhi). All the treated mice were monitored closely;
initially after every 30 mins for 2hrs; then after every lhr of interval for 4hrs and
finally after every 8hrs for remaining period of time. All the treated animals were
observed for any general change in the behaviour, retardation in body weight or any
eventual drug related mortality during the period of study (2 weeks).
Observations:
(a) No mortality of experimental mice was found during period of observation.
(b) No abnormal sign of toxicity was observed in experimental animals (mice) of
both sexes during 14 days of observation period.


(c) The experimental result reveals that the isolated molecules (Pi and P2) do not
have any acute toxic effect in experimental animal (mice).
(ii) Acute Toxicity Test in Wistar Rats (14 days observation)
Test procedures
Acute toxicity of active compound P1 and P2 was also evaluated in rats of either Sex
weighing between 100-150 gm. The rats were divided in three groups comprising 10
rats in each group (5 male and 5 female). The first and second group was fed with
active compound P1 and P2 respectively at a dose of 2000 mg/kg/5ml which was
100-200 times more than the respective therapeutic dose of these molecules. The
third group was fed with sterile drinking water (5ml/kg) and was marked as control.
Before and during the experiment all rats were housed in an air conditioned room
(at 23°- 25°C and relative humidity 44-56%) with 12hr/12hr light/dark cycle
and maintained with free access to drinking water (Aqua guard H2O) and standard
pallet diet (supplied by Golden Feeds; Mehrauli; New Delhi). All the treated rats
were monitored closely; initially after every 30 mins for 2hrs; then after every lhr of
interval for another 4hrs and finally after every 8hrs for remaining period of time. All
the treated animals were observed for any general change in the behaviour,
retardation in body weight or any eventual drug related mortality during the period
of study (2 weeks). Following signs were monitored with the help of experienced
veterinarian and pathologist.


Observations
There were no abnormal clinical signs of acute toxicity or any eventual drug related
mortality during the period of observation. The experimental result reveals that the
isolated molecules (P1 and P2) are practically safe for therapeutic application.
2. Sub Chronic Oral Toxicity Test (Repeated dose for 90 days in rodents as
per 408 OECD guideline; 1995)
(i) Toxicity test in mice
Test procedures
Sixty Swiss albino mice of either sex weighing between 17-22 gm were taken for this
experimental study and after adequate randomization these mice were divided into
three groups having 10 male and 10 females in each group. The ears of each mouse
were punched by suitable device to assign a unique identification. Male and female
mice of each group were kept in separate cages. First group of mice was fed with
active compound P1 (0.4 mg/20 gm of body weight/mL i.e. 20 mg/kg of body
weight); second group of mice was fed with active compound P2 (0.4 mg/20 gm of
body weight/mL i.e. 20 mg/kg of body weight) and third group of mice marked as
control group, was fed with sterile drinking water (1 mL/20gm of body weight). All
doses were given every day for 90 days and during this period; treated animals were
observed after every 8 hrs (at 2PM, 10PM and 6AM) for any drug related
abnormalities or mortality. Before and during experiment all rats were housed in an
air conditioned room (at 23°- 25°C and relative humidity 44-56%) with 12hr/12hr
light/dark cycle and maintained with free access to drinking water (Aqua guard H2O)
and standard pallet diet. At the end of experiment; the blood samples of treated
mice were collected retro-orbitally from the inner canthus of the eye using Micro
Hematocrit capillary tubes for toxicological evaluation through haematological study
(WBC and RBC counts, estimation of Hb% , MCV, HCT and MCH ).
(ii) Toxicity test in rats
Test procedures
Thirty healthy male Wistar rats (normoglycaemic) weighing between 150-200 gm;
were taken for this experimental study and after adequate randomization they were
divided into three groups having 10 animals in each group. The ears of each rat were
punched by suitable device to assign a unique identification. First group of rat was
fed with active compound P1 (20 mg/kg/2mL); second group of rat was fed with
active compound P2 (20 mg/kg/2mL) and third group of rat; marked as control
group, was fed with sterile drinking water (2mL/kg). All doses were given every day


for 90 days and during this period after every 8hrs of interval; all the treated rats
were observed regularly (at 2PM, 10PM and 6AM) for any abnormal symptoms or
mortality. Before and during experiment all rats were housed in an air conditioned
room (at 23°- 25°C and relative humidity 44-56%) with 12hr/12hr light/dark cycle
and maintained with free access to drinking water (Aqua guard H2O) and standard
pallet diet (supplied by Golden Feeds; Mehrauli; New Delhi). On 91st day of
experiment; blood samples of overnight fasted animals were collected retro-orbitally
from the inner canthus of the eye using Micro Hematocrit capillary tubes (Top-Teck
Bio-medicals; New Delhi) for evaluation of haematoxicity [WBC and RBC counts,
estimation of Hb%, MCV, MCH and PCV (HCT)], renal toxicity (Serum Urea, Uric acid,
Creatinine and Blood Urea Nitrogen) and hepatotoxicity (Serum GOT, GPT, ACP, ALP,
Albumin, Globulin and Albumin/Globulin ratio )
At the same time blood samples of diabetic treated [treated individually with P1
(n=10) and P2 (n=10) for 90 days at a dose of 20 mg/kg)] and diabetic control (90
days untreated; n=10) were also collected for comparative haematology and clinical
biochemistry. After the collection of blood samples; all the experimental animals
were scarified ( by cutting of jugular vein under light anesthesia ) and important
organs like kidney, liver, heart, spleen and pancreas were dissected out, weighed
and then carefully examined visually for any abnormalities or undesirable changes.
For histopathological study all the collected organs were fixed appropriately in 10%
formalin and histological section were prepared as per standard protocol. Finally all
the prepared sections were stained with haematoxilin - eosin and studied under light
microscope for any pathological and morphological changes.
Observations: Experimental findings revealed that
(i) Both of these new therapeutic agents did not produce any chronic toxic effect as
well as drug related mortality in experimental mice and rats.
(ii) These therapeutic agents (P1 and P2) did not produce any hematotoxicity,
hepatotoxicity and renal toxicity in experimental animals.
(iii) These therapeutic agents did not produce any significant pathological as well as
morphological changes in the vital organs of the experimental animals and thus both
of these naturally occurring therapeutic agents may safely be used in prolonged
therapeutic application for the treatment of diabetes mellitus, for the prevention of


diabetic complications and to counter abnormal patho-physiological conditions in
diabetic patients.
Both P1 and P2 as diabetic therapeutic agents not only regulate the glycemic indices
but also assist in normalizing the parameters of lipid profile which are deviated from
the normal values in streptozotocin induced diabetic rats. Thus these molecules are
found to be anti-hyperglycemic as well as anti-hyperlipidemic in nature.
Both of these therapeutic agents help in restoration of normal body weight as well as
liver and muscle glycogen content which are decreased significantly in diabetic
animals.
Both of these therapeutic agents help in the synthesis of insulin and C-peptide
probably by regenerating the pancreatic beta cells which have been reflected here by
six months duration of experiment.
Active therapeutic agents P1 and P2 do not produce any acute hypoglycemic effect in
normoglycaemic animals (rats).
Acute and Chronic toxicity study on experimental animals reveal that both these
newly discovered natural therapeutic agents are free from any adverse drug reaction
as well as undesirable side effects and thus these agents are safe for long duration
therapeutic application. Effective proportions of P1 and/or P2, when combined with
suitable pharmaceutical carriers reveal antdiabetic, anti-hyperglycemic as well as
anti- hyperdipidemic traits.
The present invention has been described with reference to some examples,
drawings and preferred embodiments and the present invention includes all
legitimate developments within the scope of what has been described hereinafter
and claimed in the appended claims.


WE CLAIMS
1. A method of obtaining anti-diabetic/anti-hyperglycemic/anti-hyperlipidemic novel
compounds of generic formula I, wherein R = CH3 or H

said method comprising:-
a) extracting pulverized plant material (seeds of Eugenia Jambolana) by soaking
in a polar organic solvent such as herein described;
b) fractionating in the manner such a herein described the extract so obtained
from step a) applying different solvents such as herein described,
c) undertaking two stage column chromatographic separations,
d) isolation of the active fraction followed by purification thereof and
identification of bioactive molecules of formula la and/or formula lb,
therefrom.
a. The method as claimed in claim 1 wherein said compounds are of formula la
and/or formula lb wherein , R= CH3 and H respectively


3. The method as claimed in claims 1 and 2 wherein said solvents applied in step b)
includes any one or combination of two or more of n-hexane, chloroform,
Ethylacetate and n-Butanol.
4. The method as claimed in claim 1 to 3 wherein, said active fraction includes
ethylacetate fraction and said step c) involves subjecting said fraction to column
chromatography using silica gel (100-200 AST mesh) as adsorbent and chloroform,
ethylacetate and methanol as eluting solvents with gradient techniques.
5. The method as claimed in claim 1 wherein separation and purification of
compounds of formula la and formula lb and carried out on combiflash companion
chromatographic system using Residep normal phase column (40gm) filled with silica
gel (230-400 ASTM) as separating column.
6. The method as claimed in claims 1 to 4 Wherein said compound of formula la is
dimethyl 4,4' oxybis (3,5 dihydroxy benzoate) and said compound of formula lb is
4,4' oxybis (3,5 dihydroxy benzoic acid)
7. A process for manufacturing a pharmaceutical composition comprising
undertaking the method of obtaining as claimed in claims 1 to 6, and combining said
compounds la and lb in synergistically effective proportioned with suitable
pharmaceutical carriers whereby effective anti diabetic, antihyperglycemic and anti-
hyperlipidemic traits are revealed by said composition.
8. A process for manufacturing a pharmaceutical composition obtaining comprising
undertaking the method of as claimed in claim 1 to 6, so that any one of said
compounds of formula la or lb are extracted followed by combining said compound
with suitable pharmaceutical carriers, whereby effective antidiabetic, anti-
hyperglycemic and anti-hyperlipidemic traits are revealed by said composition.

9. Pharmacuetical compounds of generic formula I Wherein R= CH3 or H

said compound being chemically obtained by method as claimed in claim 1 to 6.
10. A method of chemically obtaining chemical compounds from seeds of Eugenia
jambolana for treatment of diabetes/hyperglycemia/hyperlipidemia substantially as
herein described.


A method of obtaining anti-diabetic/anti-hyperglycemic/anti-hyperlipidemic novel
compounds of generic formula I, wherein R = CH3 or H

said method comprising:-
a) extracting pulverized plant material (seeds of Eugenia Jambolana) by soaking in a
polar organic solvent such as herein described;
b) fractionating in the manner such a herein described the extract so obtained from
step a) applying different solvents such as herein described,
c) undertaking two stage column chromatographic separations,
d) isolation of the active fraction followed by purification thereof and identification of
bioactive molecules of formula la and/or formula lb, therefrom.

Documents:

00436-kol-2008-abstract.pdf

00436-kol-2008-claims.pdf

00436-kol-2008-description complete.pdf

00436-kol-2008-drawings.pdf

00436-kol-2008-form 1.pdf

00436-kol-2008-form 2.pdf

00436-kol-2008-form 3.pdf

00436-kol-2008-form 5.pdf

00436-kol-2008-form 9.pdf

436-KOL-2008-ABSTRACT 1.1.pdf

436-KOL-2008-AMANDED CLAIMS.pdf

436-kol-2008-assignment.pdf

436-KOL-2008-CANCELLED PAGES.pdf

436-KOL-2008-DESCRIPTION (COMPLETE) 1.1.pdf

436-KOL-2008-DRAWINGS 1.1.pdf

436-kol-2008-examination report.pdf

436-KOL-2008-FORM 1 1.1.pdf

436-kol-2008-form 13.1.pdf

436-KOL-2008-FORM 13.pdf

436-kol-2008-form 18.pdf

436-KOL-2008-FORM 2 1.1.pdf

436-KOL-2008-FORM 3 1.1.pdf

436-kol-2008-form 3.pdf

436-kol-2008-form 6.1.pdf

436-KOL-2008-FORM 6.pdf

436-kol-2008-form 9.pdf

436-kol-2008-granted-abstract.pdf

436-kol-2008-granted-claims.pdf

436-kol-2008-granted-description (complete).pdf

436-kol-2008-granted-drawings.pdf

436-kol-2008-granted-form 1.pdf

436-kol-2008-granted-form 2.pdf

436-kol-2008-granted-specification.pdf

436-kol-2008-others.pdf

436-KOL-2008-PA.pdf

436-kol-2008-pa1.1.pdf

436-KOL-2008-REPLY TO EXAMINATION REPORT.pdf

436-kol-2008-reply to examination report1.1.pdf


Patent Number 248596
Indian Patent Application Number 436/KOL/2008
PG Journal Number 30/2011
Publication Date 29-Jul-2011
Grant Date 27-Jul-2011
Date of Filing 05-Mar-2008
Name of Patentee DEBIDAS GHOSH
Applicant Address Y-8 LENIN PARK, BIDHAN NAGAR, PIN: 721101, MIDNAPORE, PASCHIM MIDNAPORE, WEST BENGAL, INDIA
Inventors:
# Inventor's Name Inventor's Address
1 GHOSH DEBIDAS Y-8 LENIN PARK, BIDHAN NAGAR, PIN:721101, MIDNAPORE, PASCHIM MIDNAPORE
2 BHAT BEENA B-906, ROYAL TOWERS SECTOR-61, NOIDA
3 PANDA DILIP KUMAR TOWN COLONY, HOUSE NO-20, MIDNAPORE TOWN, P.O.: VIDYASAGAR UNIVERSITY, PIN: 721102
PCT International Classification Number A61K31/00
PCT International Application Number N/A
PCT International Filing date
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 NA