COMPLETE SPECIFICATIONS
Back Ground of the Invention
Navel has been used as a drug application site in time tested traditional medicinal system
for the treatment of various diseases. Use of cow ghee and mustard oil in the navel as a
remedy for dry and chapped lips, use of asafoetida paste and castor oil on navel of
infants to treat the flatulence and constipation etc. are some common remedies. Navel is
composed of thinnest skin. Behind the navel, small intestine and other organs are
situated. Navel skin is devoid of fatty tissues and is the thinner most area of the
abdominal wall. The periphery of navel is abundant in blood vessels and nerve supply so
it can serve as a potential absorbing site.Superficial epigastric artery supplies blood to
the nabhi. Superior epigastric artery supplies blood the nearby area of the nabhi. Blood
is drain away from the nabhi skin and nearby area by Inferior epigastric vein. Nabhi and
its skin is supplied by 10th pair of thoracic nerve. 11th pair of thoracic nerve supplies to
Hie skin around the nabhi. Abdominal aorta is situated beneath the umbilicus and it
extends up to a point 2 cm below and left to umbilicus. About 7 to 8 cm above the
umbilicus, pancreas is situate and transvere colon is situated slightly above the umbilicus
.Seventh thoracic nerve ends at a level of xiphoid process and 10th supplies the vicinity of
umbilical and twelfth one supplies midway between umbilicus and upper part of
symphysis pubis. Navel offers an excellent site for application of drug or drug loaded
device. Due to the specific shape of navel drug or drug loaded device can be secured in
place for a longer time with minimum chances of removing the drug from the
application site. Skin folds of the navel provide larger surface area for better drug
absorption. Navel area is rich in blood capillaries and nerves ending. Due to absence of
fatty tissues these are closer to the skin hence better systemic delivery of drug is
possible. Navel as a drug delivery route offers several additional advantages like drug
delivery through nerves ending present at navel area, bypassing first pass metabolism,
improved compliance of geriatric, paediatric and bed ridden patients. This route offers
better bioavailability with decreased toxicity.
Aegle marmelos fruit pulp was obtained from fruits of Aegle marmelos of family
Rutaceae. Aegle marmelos ripened fruit pulp contains coumarins, contains alkaloids,
pectin, alpha and beta-amyrin, beta-sitosterol, and polysaccharides. Aegle marmelos
fruit pulp is commonly consumed by human being as a refreshing drink in combination
with water, sugar and lemon etc. and to treat gastrointestinal problems, piles, oedema,
jaundice, vomiting, obesity, paediatric disorders, gynaecological disorders, urinary
complaints and as a rejuvenative. A. marmelos also known to have has proven
antioxidant, anti-diarrhoeal, antipyretic, wound healing and cardio tonic activity. Based
on our literature survey it was observed that no attempt has been made to isolate
biopolymer from Aegle marmelos fruit pulp. This invention explores the method for
isolation of biopolymer from Aegle marmelos fruit pulp.
Rosiglitazone andEmtricitabine were model drug candidates for current invention.
Rosiglitazone is anti-diabetic and emtricitabine is nucleoside reverse transcriptase
inhibitor. Emtricitabine is lipophilic drug whereas emtricitabine has good water
solubility.
The invention explores the potentiality of trans-nabhi route as a drug delivery platform
because of its unique histology, blood and nerve supply. Rosiglitazone and emtricitabine,
loaded bioadhesive films were prepared using a novel bio film former obtained from
Aegle marmelos fruit pulp along with co-processing agents. The films were evaluated
for its properties.
DETAILED DESCRIPTION OF THE INVENTION
1. Isolation of biopolymer from Aegle marmelos fruits pulp
Ripened Aegle marmelos fruits were procured. Fruits were cut and pulp was collected
and seeds along with the jelly like coverings were separate. 200 grams of seed free fruit
pulp was taken, washed with distilled water and minced with 500 ml of distilled water.
The mixture was subjected to stirring for 10 minutes using mechanical stirrer at 2000
rpm. Mixture was strained through muslin cloth and filtrate was stored in a separate
glass container. The residue obtained was again subjected to above process for two more
times. All portions of filtrate were combined and centrifuged (Remi R-24) at 5000 rpm
for 10 minutes. Supernatants were collected, treated with acetone in anoptimized ratio
(1:1 to 1:4) and kept in refrigerator for 5 hours. Mixture was centrifuged at 5000 rpm for
15 minutes; supernatant liquid was decanted and discarded. Bioniaterial was collected
and treated with 50 mL of acetone for 5 times. The bio-material obtained was dried in a
desiccators containing fresh anhydrous calcium chloride for 24hours. Biomaterial was
further purified by hot dialysis method using dialysis membrane (ORCHID scientific
dialysis apparatus). Biomaterial was screened through # 120 and stored in an airtight
light resistant container for the further use. The procedure was optimized by repeating
the procedure for 6 times and the percentage yield was calculated and reported.
2. Characterization of biomaterial isolated fromAegle marmelos fruits pulp
a. Physicochemical Properties of Coelogyne ovalis petiole biomaterial
Aegle marmelos seed coat pulp biomaterial was gritty, irregularly texturedmaterial
withbrownish-orange colour and characteristic odour with mucilaginous taste. It has
shown a positive chemical test for carbohydrates and absence of proteins. Isolated
biomaterial was water soluble and insoluble in methanol, ethanol, Carbon tetrachloride
Chloroform and acetone. Colour changing point of the biomaterial was in a range of
177-181 °C
b. Spectral Analysis of Aegle marmelos fruit pulp biopolymers
Aegle marmelos fruit pulp bio-polymer was subjected for spectral studies such as UV,
IR, NMR and Mass spectroscopy along with SEM and DSC analysis.UV spectroscopy
of Aegle marmelos fruit pulp bio-polymer has shown a Xmax at wave length of 291 nm.
IR spectra of Aegle marmelos fruit pulp biopolymer were recorded from' a bio-polymer
containing disks of KBr at frequency regions of 4000-400Dcm-'.Shoulders at different
frequencies has confirmed the presence of various functional groups such as amides
(3399.5 cm"1 632.12 cm-11; NH stretch of RCONH2,),alkanes (2931.31 cm"1; CH stretch
of RCH2CH3), aromatic (1415.16 cm"1; Ar C-C stretch of C-C in ring), carboxylic
(1307.21 ,1269.22 cm"1 ;C-0 stretch of RCO-OH), alkenes (891.30 cm",;R2C=CH2),
amine (891.30 cm'1,670.23 cm"1; NH-wag amine RNH2 and R2NH), alkynes (610.23
cm"1 #C-H bend of RC#CH).
H]-NMR spectra of Aegle marmelos fruit pulp biopolymer has revealed the presence
of various types of protons associated with different type of functional groups such as
Alkyl-RCH2CH3 (5- 1.163-1.9551 PPM), Ketone-RCOCH3 (8- 2.3828-2.7648 PPM),
Ether-ROCH2R (8- 3.0252-3.9592 PPM), Alcohol-HOCH2R(8- 4.0225-4.4243 PPM),
Vinylic-R2C=CH2 (8- 5.0134 PPM) and Amino-R-NH2 (8- 1-5 PPM) which has
confirmed the presence of these functional groups in the biopolymer. From the integral
values of peaks total 164 protons were calculated corresponding to various peaks present
in the spectra at different chemical shift values.
In case of Aegle marmelos fruit pulp biopolymer, LC-MS spectra has shown base
peak at m/z value of 141.1 with a relative abundance of 2406.With respect to base peak,
molecular ion peak was detected at m/z value of 413.2 with a relative abundance of 265.
SEM ofAegle marmelos fruit pulp bio-polymer (at 200 x and 600 X) magnification
revealed that bio-polymer consists of mixture of slightly rough, irregular, and cuboidal
crystal like particles with size up to200 um.
DSC ofAegle marmelos fruit pulp bio-polymer has shown a broad endothermic peak
between with a Tm at 132.20 °C) with peak area of 341.445 mJ and peak height of
14.6772 mW. The heat capacity AH and heat of fusion AHf of the endothermic bend was
found to be 34.1445 J/g and 0.0341 kJ/mole respectively:
Results outcomes of spectral studies as well as results of DSC and SEM analysis
clearly indicate that biomaterial isolated from Coelogyne ovalis petiole biomaterial was
polymeric in nature.
3. Bioadhesive study ofAegle marmelos fruit pulp biopolymers
In-vitro shear stress study results showed that depending upon the contact time (0.5,10,
15 and 20 minutes) and concentration (1, 2 and 3 % w/v) shear strength of Aegle
marmelos fruit pulp was found to be in a range of 3.0±1.5 to 247.3±9.5.
In Park and Robinson method bioadhesive strength of Aegle marmelos fruit pulp was
6.11±1.34 grams and Results of rotating cylinder method were found to be in
261.00±8.19 minutes for that ofAegle marmelos fruit pulp biopolymer
4. Drugs -Aegle marmelos fruit pulp biopolymers interaction study
The pure drugs along with isolated Aegle marmelos fruit pulp biopolymers were
subjected to interaction study. For this both dry and wet methods were^adopted. In the
procedure drug and Aegle marmelos fruit pulp biopolymers were taken in a series of
ratios of 1:1, 1:2, 1:4, 1:6 and 6:1 ratio and kept at different sets of temperature for time
period as specified in the Table 1 and 2. After the specified time period, all samples
were dissolved in methanol, filtered and scanned for Xmax of respective drugs between
200 to 800 nm using UV spectroscopy.
observed in all drug-polymer mixture ratios kept at different conditions which indicates
that drugs and biopolymers are compatible and devoid of reacting functional groups.
5. Formulation of drug loaded bioadhesive films of Aegle marmelos fruit pulp
biopolymer
a. Rosiglitazone loaded bioadhesive films
Rosiglitazone was weighed mixed with flexiciser dextrose, biopolymer, standard
polymer and double distilled water in quantities as specified inTable 3. This mixture
was sonicated in a bath sonicator at 500 hertz for two cycles of 90 seconds each. From
the mixture drug loaded films AMFRl, AMFR2, AMFR4 and AMFR6were prepared by
solvent casting.One rosiglitazone loaded film PCR4 with standard polymer Polycap
6500 was also formulated similarly. .
Table 3: Formulation of Rosiglitazone bioadhesive films using Aegle marmelos
fruit pulp biopolymers and Standard polymer Polycap-6500
b. Emtricitabine loaded bioadhesive films
Emtricitabine was weighed mixed with flexiciser dextrose, biopolymer, standard
polymer and double distilled water in quantities as specified in Table 4. This mixture
was sonicated in a bath sonicator at 500 hertz for two cycles of 90 seconds each. From
the mixture drug loaded films AMFEl, AMFE2, AMFE4 and AMFE6were prepared by
solvent casting.One rosiglitazone loaded film PCE4 with standard polymer Polycap
6500 was also formulated similarly.
Table 4: Formulation of Emtricitabine bioadhesive films using Aegle marmelos
fruit pulp biopolymers and Standard polymer Polycap-6500
6. Evaluation of Drug loaded bioadhesive films
a. Evaluation of Rosiglitazone and emtricitabine loaded bioadhesive films
Rosiglitazone and emtricitabine loaded bioadhesive films were evaluated for various
parameters such as thickness, % drug content, folding endurance, % swelling index, %
moisture absorption, % moisture loss, average surface pH and water vapour transmission
rate (WVTR) and results are reported in Table 6 (a) and 6(b).
It has been found that rosiglitazone loaded films of Aegle marmelos fruit pulp
biopolymer have shown folding endurance, thickness, drug content and surface pH in a
range of 302±7.4 to 389±7.4, 96.00±2.00 to 98.67±1.53 %, 0.31±0.06 to 0.66±0.06 mm
and 6.30±0.10 to 6.83±0.12 respectively. Values for % Swelling index, % moisture Loss
and % moisture absorption for rosiglitazone loaded bioadhesive films were 9.56±9.62
to38.89±9.62, 5.56±9.62 toll.ll±2.75 and 5.56±9.62 to22.22±11.00 respectively.
Emtricitabine loaded films of Aegle marmelos fruit pulp biopolymer have shown folding
endurance, thickness, drug content and surface pH in a range of 287±3.5 to 370±3.5,
97.00±2.65 to 98.00±1.00 %, 0.28±0.05 to 0.63±0.05 mm and 6.37±0.50 to6.50±0.53
respectively. Values for % Swelling index, % moisture Loss and % moisture absorption
for emtricitabine loaded bioadhesive films were 5.56±9.62 to25.40±2.75,5.56±9.62
tol 1.11±0.00 and 11.11±9.62 to23.81±12.60 respectively. WVTR was found to be in the
range of 60±17.32 to 163±11.55 and 287±3.5 to 370±3.5 for rosmlitazone and
emtricitabine loaded bioadhesive films respectively. /'
b. Ex-vivo Permeation study
Results of permeation study reveals permeation of 96.06±0.6020% of rosiglitazone
within 72 hours. Flux at steady state (Jss) was calculated as 18.2xl0"3 mg/cm2 /hour.
Permeability coefficient Kp for rosiglitazone was found to be 9.099x 10"3 cm/hr.
Permeation of emtricitabine was found to be 54.84±7.01 % within 72 hours. Value of
flux at steady state and permeability coefficient Kp for emtricitabine was recorded as
8.709xlO"3 mg/cm2/hour and 4.355x 10'3 cm/hr respectively.
c. In vitro diffusion Study
Diffusion study of all rosiglitazone and emtricitabine loaded bioadhesive films were
carried out by modified MS diffusion cell apparatus using Dialysis membrane (High
media, Av. flat width 32.34mm, Av. diameter 21.5 mm, and capacity approx. 3.63
ml/cm).
Consideringt5o% and tgo% values for drug release and concentration of biopolymer used as
screening parameters best formulation was selected. Rosiglitazpne loaded bio-films of
biopolymer Aegle marmelos fruit pulp have shown overall performance as
AMFR1>AMFR2>AMFR4>AMFR6. AMFRl was further compared with
Rosiglitazone loaded standard film PCR4 and performance of AMFRl was better than
Rosiglitazone loaded film PCR4. Best fit kinetic model for AMFRl was found to be
Peppas Korsmeyer with R2 value of 0.9531. Value of diffusion coefficient for this
Rosiglitazone loaded bio-film was 0.6698 which indicates that mechanism of drug
release was anomalous transport.
In case of Emtricitabine loaded bio-films of biopolymer Aegle marmelos fruit pulp order
of overall performance was found to be as AMFE4>AMFE1>AMFE6>AMFE2.
AMFE4 was further compared with Emtricitabine loaded standard film PCE4 and
performance of AMFE4 was better than Emtricitabine loaded standard film PCE4. Best
fit kinetic model for AMFE4 was found to be Higuchi Matrix with Rvalue of 0.9372.
Value of diffusion coefficient for this Emtricitabine loaded bio-film was 0.6254 which
indicates that mechanism of drug release was anomalous transport.
In vitro drug release profile of rosiglitazone loaded bioadhesive films of Aegle
marmelos fruit pulp biopolymer showed t5o% and t8oo/o values in a range of 3.8 to 22 hours
and 8.7 to more than 24 hours respectively. Emtricitabine loaded Aegle marmelos fruit
pulp biopolymer showed tso% and tso% values in a range of 2.7 to 3.6 hours and 3.5 to
more than 24 hours respectively.
d. In-Vivo Study
In vivo study was conducted with best bioadhesive film of rosiglitazone AMFRl.The
Trans -nabhi patch of AMFR1 formulation was formulated and applied in the navel area
of rabbits. From the drug plasma concentration vs. time profile the; value of Cmax and
Tmax was found to be 26.37^ gms/mL and 3 hours respectively. From the drug plasma
concentration vs. time profile area under the curve was calculated and value for AUG
(from t=0 to t=29) was found to be 442.407 p. gms mL"1 hour.
e. Stability Studies of Rosiglitazone loaded bio-film AMFR1
Samples from formulation AMFR1 were placed in different storage conditions such asr
at40±2°C and 75±5% RH, 4°C, 30 °C and 50°C for 3 months. The samples were taken at
30 days interval up to 3 months and analysed for change in drug release and physical
properties.
Results of Rosiglitazone stability samples of the first month revealed that samples at 4
°C and 40 °C+75% RH has a slightly increased drug release than the sample kept at 30
°C which may be due to the possible hydration or due to effect of temperature on the
samples, but sample kept at 50 °C has shown a retarded drug release because of the
dehydration of the sample and due to the loss of flexibility of the sample. Drug release
profile from the stability samples off J "' '" has/Shown almost similar
profile to that of samples of first month except samples kept at 50 °C where much
retarded release has been observed which may be attributed to the loss of moisture from
the film and due to the partial drug degradation within the sample. Stability samples at 4
°C, 30 °C of third month did not show any significant change in drug release than the
samples of first and second months but samples at 40 °C+75% has shown a decreased
drug release which may be due to the degradation of drug. Further samples kept at 50 °C
has shown a much retarded drug release which may be due to degradation of drug with
in the sample or sample has failed to release the drug due to change in physical
properties of the film. Samples kept at 4 °C and 30 °C have not shown any physical
change in the sample whereas samples kept at 40 °C+75% RH were slightly swelled and
more flexible. Samples at 50 °C were hard and brittle. Hence the biofilms should be
stored in airtight protected containers.
SUMMARY OF INVENTION
In present research work a novel biopolymer was isolated from the Aegle marmelos fruit
pulp by simplified method; The isolated biomaterial was evaluated for various
physicochemical properties and spectral studies. The biomaterial was used to formulate
drug loaded bioadliesive films loaded with emtrlcltabine and rosiglitazone. The
formulated drug loaded films were evaluated for various parameters and the results of
these study suggest that isolated biopolymer was having inbuilt film forming properties.
In-vitro diffusion study of the drug loaded bioadhesive films reveal that isolated
biopolymer was having good drug release retardant property. In vivo study results
showed a good bioavailability of rosiglitazone through trans-nabhi route. The proposed
mechanism for drug bioavailability may be due to permeation of drug through the transcellular
and para-cellular passive diffusion pathway and intra and inter-neural pathways
from the nerve endings present in navel area.

CLAIMS
1. A formulation process for formulating drug loaded bioadhesive films for trans-nabhi
delivery comprises of biofilm former, co-processing agent, and API.
2. The film former of claim 1 wherein isolated from the Aegle marmelos fruit pulp of
Aegle marmelos by adding optimized quantity of propanone and refrigerator to
< recover the biopolymer.
3. The API and co-processing agent of claim 1 wherein comprises of rosiglitazone and
emtricitabine as an API molecule and D-(+)-Glucose monohydrate as a flexiciser for
preparing drug loaded bioadhesive films. The process involved in the preparation of
emtricitabine and rosiglitazone loaded bioadhesive films were solvent casting
method. Rosiglitazone and emtricitabine showed a significant permeability through
the umbilical cord skin substrate with flux of 9.099x 10'3 cm/hr and 4.355x 10"3
cm/hr respectively.
4. The nature of the biopolymer of claim 2 wherein the slightly rough," irregular, and
cuboidal crystal like topology, Tm at 132.20 °C, m/z value of 413.2, 8 of 1-5 PPM
and presence of amide and amino groups confirms its polymeric nature.
5. The Aegle marmelos fruit pulp biopolymer of claim 4 showed its inbuilt
bioadhesivity in a range of 3.0± 1.5 to 247.3±9.5 grams at 1-5% concentration and
filmability at 1 -6% ratio.
6. The bioadhesive films loaded with rosiglitazone and emtricitabine of claim 3wherein
revealed In-vivo performance of rosiglitazone loaded bioadhesive film through transnavel
route in experimental animals for extended period 29 hourswithCmax of26.37n
gms/mL, Tmaxof3 hours and AUC of 442.407 ugms ml/1 hour.