COMPLETE SPECIFICATIONS
Back Ground of Invention
Biopolymers are polymeric biomolecules obtained from the living sources. Biopolymers
may be poly nucleotides, polypeptides or polysaccharides. Biopolymers in contrast to
synthetic polymers are known to have well defined structure and are biodegradable or
compostable. Biopolymers are a potential excipient in drug delivery system due to their
inertness and biodegradable nature. Due to their biological origin they have good
compatibility and reduced toxicological properties.
Biofilm former was isolated from petioles of Coelogyne ovalis. Coelogyneovalis
belongs to familyorchidaceae and commonly known as Oval Coelogyne, Coelogyne
decora, Coelogyne longiciliata, Pleione fuliginosa etc. Coelogyne ovalis is a wildly
grown orchid found in Himalayas, from Uttarakhand, at altitudes of 1300-1700
m.Coelogyne ovalis has 2, 7-Dihydroxy-3, 4, 6-trimethoxy-9, 10-Dihydrophenanthrene
as chemical constituents, some phenolic constituents, bibenzyle 3'-0-methylbatatasin III
and sterols. The pseudobulbs of the plant contain a translucent and viscous liquid which
has been used as a natural occurring adhesive by the natives of Uttarakhand villages on
different occasions. Traditionally the liquid obtained from these pseudobulbs is also
used to treat cattle for fever, infection and other-diseases. Coelogyne ovalis is known to
have antifungal,aphrodisiacand spasmolytic activity.Based on our literature survey it
was observed that no attempt has been made to isolate biopolymer from Coelogyne
ovalis petioles.
In present research work emtricitabine has been chosen as a model drug to formulate
emtricitabine loaded bioadhesive films and biogels. Emtricitabine is a nucleoside reverse
transcriptase inhibitor (NRTI) and it is used for the treatment of HIV infection in adults
and chronictypeBhepatitis. The drug is an analogue of cytidine. Emtricitabine inhibits
reverse transcriptase, thus prevents copying of HIVRNA into new viral DNA. Treatment
of HIV-1 infection in adults. Emtricitabine is fairly water soluble drug (112 mg/ml).
After oral administration it shows rapid absorption (up to 93%) and elimination half-life
of 10 hrs. Its systemic bioavailability is 93%. Overdose could lead to serious liver
I
problems like hepatotoxicity, with liver enlargement and steatosis or a lactic acidosis.
Autoimmune disorder is also some times have been observed.
Tans-nabhi drug delivery is a novel concept to deliver the drug locally or systemically.
The unique size of navel, special histology of navel skin offers excellent site for drug
delivery. Navel is rich in blood vessels, nerve endings and composed of skin devoid of
fatty tissues and due to this unique properties drug can be delivered through the nerves
or through blood vessels. This route of drug administration also carries all additional
advantages of conventional transdermal drug delivery. The invention explores the
potentiality of Coelogyne ovalis petiole biopolymer as a versatile excipient due to its
inbuilt suspendability, filmability and gelability.
DETAILED DESCRIPTION OF THE INVENTION
1. Isolation of Biomaterial from Coelogyne ovalis Petioles
Coelogyne ovalis petioleswere collected. 200 grams of petioles were taken, washed with
distilled water and cut in small pieces. Pieces of petioles were crushed with 500 ml of
distilled water. The mixture was subjected to stirring for 30 minutes using mechanical
stirrer at 2000 rpm. Mixture was strained through muslin cloth and filtrate was stored in
a separate glass container. The procedure was repeated for two more times with the
residue. All portions of filtrate (1500 mL) were combined and centrifuged (Remi R-24)
at 5000 rpm for 30 minutes. Supernatants were collected, treated with acetone in a ratio
of 1:5 and kept in refrigerator for 12 hours. Mixture was centrifuged at 5000 rpm for 15
minutes; supernatant liquid was decanted and discarded. Residue was collected and
treated with 50 mL of acetone for 5 times. The bio-material obtained was kept 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 from Coelogyne ovalis petiole
a. Physicochemical Properties of Coelogyne ovalis petiole biomaterial
Coelogyne ovalis biomaterial was smooth fine textured, brown coloured with
characteristic odour and mucilaginous taste. Biomaterial showed positive chemical test
for protein and absence of carbohydrates. Isolated biomaterial was water soluble and
insoluble in methanol, ethanol, Carbon tetrachloride Chloroform and acetone. Colour
changing point of the biomaterial was in the range of 222-224 °C
b. Spectral study of Coelogyne ovalis petiole biomaterial
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
ofCoelogyne ovalis petiole biopolymer has shown a A^ax at wave length of 296 nm. IR
spectra of Coelogyne ovalis petiole were recorded from a bio-polymer containing disks
of KBr at frequency regions of 4000-400ncnT1.Shoulders at different frequencies has
confirmed the presence of various functional groups such as Carboxylic (3391.14 cm"1
;C=C-CO-OH), alkanes (2931.22 cm"1 ; CH stretch of RCH2CH3), ketones (1740; CO
stretch doublet of R2CO), amine (1314.40 cm"1, 1256.22 cm"1; Ar-N stretch of Ar2NH),
ether (1154.20 cm"1 , 1078.18 cm"1; C-0 stretch of R-O-R), alcohol (1026.16 cm^C-O
stretch of R2CHOH). H'-NMR spectra ofCoelogyne ovalis petiole has shown signals at
different chemical shift values which has confirmed the presence of different type of
functional groups such as Alkyl- RCH3 (8- 0.7884-.8575 PPM), Alkyl-RCH2CH3 (8-
1.2474-1.8367 PPM), Ketone-RCOCH3 (8- 2.0298-2.3385 PPM), Alcohol-HOCH2R, (8-
3.1706-3.9728 PPM and 8- 4.0451-4.1204 PPM), Vinylic-R2C=CH2(8- 4.4-5.5 PPM and
8- 5.1427-5.4314 PPM), Phenolic ArOH (8- 7 PPM) and aromatic Ar-H (8- 8.3 PPM).
From the integral values of peak it has been found that in the biopolymer 235 protons of
different chemical environments were present. Base peak for Gefelogyne ovalis petiole
biopolymer in LC-MS spectra was observed at m/z value of 351.1 with a relative
abundance of 336 whereas molecular ion peak was detected at m/z value of 977.3 with a
relative abundance of 36. SEM of Coelogyne ovalis petiole biopolymer (at 600 X and
1.50 K X magnification) has shown that polymer particles were irregular cuboidal in
shape and with flaky and rough surface topology. DSC of Coelogyne ovalis petiole
biopolymer has shown a broad endothermic bend ranging from 81-135 °C (peak 111.10
°C) with peak area 2032.181 mJ , peak height 12.0445 mW, heat capacity AH
203.2181 J/g and heat of fusion AHf 0.2032 kJ/mole.
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. Screening for various inbuilt properties.
Examplel: Screening of Coelogyne ovalis petiole biopolymer for Suspendability
A 2% suspension of calcium carbonate was prepared using varying concentrations of
Coelogyne ovalis petiole biopolymer (CO) and standard polymer tragacanth and
bentonite as shown in the Table 1.
Table 1: Formulation of 2% Calcium carbonate suspension with varying
concentrations of Coelogyne ovalis petiole biopolymer and standard polymers
Sedimentation volume F was calculated by dividing initial volume of suspension (at t=0)
by volume of sediment at different time. Results revealed that Coelogyne ovalis petiole
biopolymer has good suspendability. F value for suspension prepared with 400 and
600mg Coelogyne ovalis petiole biopolymer was found to be in the range of 0.47 to 0.62
and 0.61 to 0.85 which was better than the F value of suspension prepared with standard
polymer tragacanth (0.14 to 0.20) and bentonite (0.19 to 0.30).Based on the result it was
concluded that Coelogyne ovalis petiole biopolymer have a promising property of
suspendability.
Example2: Formulation of emtricitabine loaded bioadhesive films using Coelogyne
ovalis petiole biopolymer
Emtricitabine was weighed mixed with flexiciser dextrose, Coelogyne ovalis petiole
biopolymer and double distilled water in quantities as specified inTable 2. This mixture
was sonicated in a bath sonicator at 500 hertz for two cycles of 90 seconds each. From
the mixture drug loaded films COEl, COE2, COE4 and COE6 were prepared by solvent
casting. An emtricitabine loaded film PCE4 with standard polymer Polycap 6500 was
also formulated similarly.
Table 2: Formulation of Emtricitabine bioadhesive films using Coelogyne ovalis
petiole biopolymers and Standard polymer Polycap-6500
Diffusion study of all emtricitabine loaded films COEl, COE2, COE4 and COE6 and
PCE4 was carried out by modified MS diffusion cell apparatus/using dialysis atus/ius membrane
(High media, Av. flat width 32.34mm, Av. diameter 21.5 mm, and capacity approx. 3.63
ml/cm).
Best formulation was selected based on tso% and tgo% values for drug release.The release
performance of emtricitabine loaded bioadhesive films of Coelogyne ovalis petiole
biopolymer was asCOE6>COEl>COE4>COE2. COE6 was further compared with
Emtricitabine loaded standard film PCE4 and performance of COE6 was better than
Emtricitabine loaded standard film PCE4. Best fit kinetic model for COE6 was found to
be Higuchi Matrix with R value of 0.9294. Value of diffusion coefficient for this
Emtricitabine loaded bio-film was 0.6067 which indicates that mechanism of drug
release was anomalous transport i.e. release was due to combination of many
mechanisms.In vitro drug release profile of emtricitabine loaded bioadhesive films of
Coelogyne ovalis petiole biopolymershowed tso% and tso% values in a range of 1.4 to 12
hours and 2.6 to more than 24 hours respectively. Emtricitabine loaded bioadhesive
films were also evaluated for different parameters. The weight uniformity, % swelling
index, thickness and folding endurance of the emtricitabine loaded bioadhesive films
were found to be in the range of Weight (uniformity) 12±0.58 to 42±0.58 mg,
25.33±0.00 to 35.33±0.00 %, 0.20±0.05 to 0.47±0.05 mm and 326±3.5 to 370±3.5
folding cycles respectively.
Results outcomes suggest that the Coelogyne ovalis biopolymer has promising
filmability. The formulated films have excellent drug loading efficiency. tso% and tso%
values of drug release from drug loaded bioadhesive films clearly indicate that
biopolymer is having a good release retardant property.
Example 3: Formulation of biogels from Coelogyne ovalis petiole biopolymer
Emtricitabine loaded biogels were prepared with Coelogyne ovalis petiole biopolymer.
Coelogyne ovalis petiole biopolymer, emtricitabine and other ingredients were taken in
quantities as specified in Table3and triturated until COE2- G, COE4-G and COE6-G
gels were formed. A standard gel CPE-0.5G was also formulated with Carbopol 940 by
adopting similar procedure.
Bio-gels were formulated with Coelogyne ovalis biopolymer were slightly yellow
coloured, smooth textured and odourless. pH of drug loaded bio-gels was found to be in
a range of a range of 6.8 to 7. Drug loaded bio-gels showed promising spreading ability
with no grittiness. Bio-gels were consistent and physically stable. Biogels were easily
washable.
All three emtricitabine loaded bio-gels COE2-G, COE4-G and COE6-G along
withemtricitabine loaded standard gel CPE-0.5-G were subjected for in vitro drug
release study. From the cumulative % drug release vs. time plot of the release data were
calculated which was found be more than 10 hours and 24 hours respectively. Based on
t50% and t80% values of drug release, the best biogel was selected asCOE4-G>COE2-
G>COE6-G. Upon comparing our best bio-gel COE4-G with emtricitabine loaded
standard gel CPE-0.5-G, biogel COE4-G was selected as the best formulation. For
Peppas Korsmeyer with R2 value of 0.9564 was found to be the best fit model for drug
release. Diffusion coefficient for biogel COE4-G was found to be 0.6658 which
indicates that mechanism of drug release was anomalous transport.
SUMMARY OF THE INVENTION
In present research work a novel biopolymer was isolated from the Coelogyne ovalis
petiole by simplified method. The isolated biomaterial was evaluated for various
physicochemical properties and spectral studies. The biomaterial was investigated for its
diverse pharmaceutical applications. It was used to formulate physically stable
suspension (sedimentation volume 0.09 to 0.85). Emtricitabine loaded bioadhesive films
were prepared with Coelogyne ovalis petiole biopolymer with excellent physical
properties and promising drug release retarding ability in in-vitro drug release study.
Coelogyne ovalis petiole biopolymer was also used to prepare emtricitabine loaded
biogels with good consistency and spreadability along with good drug loading efficiency
and drug release retardant properties.

CLAIMS
What is claimed is
1. The process for isolating a biopolymer from petioles of Coelogyne ovalis comprises
of addition of nonsolvent propanone to the aqueous extract and subjected for
refrigeration for the recovery of biomaterial.
2. The biomaterial of claim 1 wherein showed its polymeric nature which was
confirmed by spectral data of IR(1314.40 cm"1, 1256.22 cm"1; amine), NMR (8-
2.0298-2.3385 PPM; Ketone-RCOCH3 and 8- 3.1706-3.9728 PPM and 8- 4.0451-
4.1204 PPM; Alcohol-HOCH2R), Mass spectroscopy(m/z value of 351.1).
3. Coelogyne ovalis petiole biopolymer of claim 2 wherein showed broad endothermic
peak at 81-135 °C and flaky and rough surface topology which was confirmed in
DSC SEM analysis.
4. Biopolymer of claim 2 wherein showed its suspendability property which was
confirmed by preparing physically stable suspensions with a F value ranging from
0.09 to 0.85.
5. Biopolymer of claim 2 wherein showed its inbuilt film forming property which was
confirmed by formulating emtricitabine loaded bioadhesive films by solvent casting
method with D-(+)-Glucose monohydrate as co-processing agent.
6. Bioadhesive films of claim 5 wherein showed prolonged tso% (12 hours) and tgo%
(more than 24 hours) which confirms its excellent drug release retardant properties.
7. Biopolymer of claim 2 wherein showed its inbuilt gel forming properties by
formulating stable biogels with good spreadability and promising drug release
retardability in in-vitro drug release over an extended period of 24 hours.