Polyacrylamide grafted Eucalyptus camaldulensis (EC-g-PAM) gum as an efficient binding agent in drug formulations

The use of plant based gums in pharmaceutical sphere is desirable binding agents during pharmaceutical formulations. In this research, Eucalyptus camaldulensis gum is modified by microwave irradiation in order to estimate its binding characteristics for the fabrication of varied pharmaceutical formulations. Gum is analyzed in three forms; native, purified and grafted EC-g-PAM. The distinctive chemical assays for the characterization of carbohydrates indicated the existence of reducing sugars in all three types native, purified and grafted EC (EC-g-PAM) gum extracts. The relatively high phenolic contents i.e. 0.159 μg ml−1 GAE of grafted EC extract indicate considerable antioxidant potential worthy of further investigations. In case of antimicrobial assay, grafted gum proved to be highly effective and produced a wider ring of no bacterial growth with for E. coli while showed comparatively lesser change in the surrounding S. aureus concentration. Owing to its non-toxicity, it is incorporated into the paracetamol and it revealed excessive controlled drug-release profiles. Grafted gum possessed significantly controlled drug release profile, thus, the drug formulations based on the proposed gum, could be more beneficial site-specific oral drug carrier system.


Introduction
A major challenge associated with the development of robust and optimized therapeutic formulations is the advancement of drug carrier technologies that may facilitate innocuous distribution of therapeutics within the patient's body and may customize their release profiles, assimilation, dissemination and elimination as required to procure their desired therapeutic efficacy coupled with patients' compliance and convenience [1][2][3]. Advanced drug formulation strategies empower the pharmaceutical sphere with all the realistic tools required to develop and formulate up-to-date drug systems day-by-day with a view to comply with the up surging therapeutic demands [4,5]. At the moment, nearly all drug manufacturing approaches predominantly incorporate inert pharmaceutical excipients mainly as binders, in conjunction with pharmacologically active ingredients to achieve extended release of therapeutic agents in finalized dosage designs [6,7]. For the most part, binders are employed in formulations to impart adequate mechanical characteristics by enhancing the adhesion and cohesion existing between individual components of powdered mixtures, as a consequence promoting the toughness of the finalized formulations produced [8,9].
Binders are distinguished as familiar inactive additive substances to be incorporated in the formulations, along with the active pharmaceutical ingredients (APIs) to ensure appropriate tensile strength and to compensate granulate compaction by offering requisite cohesiveness to the fine powders, thus permitting the formulations to disintegrate and disseminate upon ingestion providing the APIs for assimilation [10,11]. At the moment, development of plant-derived natural polysaccharides as binders has evoked considerable attention in biochemistry and biopharmaceutics on account of their availability, biodegradability, biosafety, renewability, 2. Experimental 2.1. Chemicals and reagents All of the analytical grade chemicals and reagents utilized in this research were either purchased from E. Merck (Darmstadt. Germany) or Sigma-Aldrich Chemical Company (St. Louis, MO) unless otherwise observed. Acetone, Barfoed's reagent, Benedict's reagent, Ciprofloxacin, Crystal violet stain, DMSO solution (Dimethyl sulphoxide), Distilled Water, DPPH Solution 1,1-Diphenyl-2-Picrylhydrazyl, Ethanol, Fehling Reagent A and B, Folin-Ciocalteau reagent, Gallic acid, Glacial acetic acid, Folin-Ciocalteau reagent Hydrochloric acid, Magnesium stearate, Maize starch, Methanol, Methylene blue solution, α-Naphthol, PBS (Phosphate buffer saline), Polyacrylamide, Sodium carbonate, Sodium hydroxide, Sulfuric acid, Triton X-100. The crude Eucalyptus camaldulensis gum (kino) was procured in the form of dried exudations from the local market of district Multan situated in Punjab, Pakistan. The gum was well-pulverized to a fine powder prior to utilization for the research.

Procedure for the preparation of gum
The estimated amount of gum powder was soaked over-night in sufficient distilled water which swelled up to produce a gel-like mass. That concentrated viscous solution was further treated for purification by means of washing with excess of absolute ethanol along with continuous stirring. The precipitated gum formed as a reddish mass was obtained and dried out within hot-air oven at 40 to 50°C. The obtained dried gum product was pulverized and powdered, passed through sieve and eventually stored in an air-tight container for further study.
As an alternative to the free radical initiator approach, microwave irradiation mechanism was employed exclusively to create free-radical regions on the polymer backbone. The general scheme of the methodologies employed for grafting of gum was as described elsewhere [28]. 1 g of E. camaldulensis gum powder was dissolved in 40 ml of distilled water. In 10 ml of distilled water, estimated quantity of acrylamide was dissolved and then mixed with the E. camaldulensis gum solution. The two solutions were transferred to a 1000 ml reaction vessel that was eventually kept onto the turn-table microwave oven (Dawlance DW-20M) for irradiation at power of 900 W for requisite duration of time varying from 2 to 4 min. The leftover gel-like mass was allowed to cool and washed with acetone. The precipitated polyacrylamide grafted polymer (EC-g-PAM) was obtained and dried out within hot-air oven. Subsequent to that, it was well-pulverized and sieved.
2.4. Physicochemical characterization of E.camaldulensis gum FTIR analysis of native, purified and modified E.camaldulensis gum was carried out in the range of 4000 to 500 cm −1 for functional group analysis during chemical and biochemical graft-modification processes. The elemental investigation was done by Elemental Analyzer. The estimation of eight discrete elements Carbon, Oxygen, Magnesium, Silicon, Sulphur, Potassium, Aluminium and Calcium was undertaken. Scanning electron microscope (SEM) (Model: JSM 5910) was used to analyze the external morphology of native, purified and grafted specimens of gum. Thermo-gravimetric (TGA) instrument (Perkin Elmer, USA) was employed for the evaluation of time-based temperature dependence with reference to modified and unmodified gum specimens. The temperature range of 30°C to 1200°C was set with the constant heating rate of 10°C per minute in nitrogen atmosphere.
2.5. Quantitative analysis of carbohydrates, antimicrobial assay, bacterial assay and biofilm inhibition analysis Experimental detail of these tests in given in supporting information.

In vitro toxicity analysis by hemolytic activity
In vitro hemolytic capabilities of crude, purified and processed gum extracts were estimated. The extracts were prepared with varied concentrations i.e. 2%, 4%, 6%, 8% and 10% of gum. Hemolytic activity of the samples was conducted by following the procedure mentioned as follows.
2.6.1. Collection and count of erythrocytes About 3 ml of fresh human blood was obtained in heparinized tubes, mixed gently and then poured into 15 ml sterile falcon tubes followed by centrifugation at 850 × g for around 5 min. The supernatant was decanted off and three times rinsing of RBCs was done by using 5 ml of isotonic sterile phosphate buffer saline (PBS) solution chilled at 4°C and pH adjusted at 7.4. The washed red blood cells were suspended within 20 ml volume of chilled PBS. Counting of erythrocytes was performed on Hemocytometer.
2.6.2. Collection of supernatant for hemolytic activity 20 μl of gum extract sample was taken in Eppendorf tube and 180 μl diluted blood cell suspension was added and further incubated for half an hour at 37°C along with agitation. Then, these Eppendorf were positioned on ice for 5 min and centrifuged for other 5 min at 1310 × g. 100 μl of supernatant was withdrawn from the tubes and diluted by adding 900 μl of chilled PBS. Then, all the Eppendorf tubes were sustained on ice. The supernatant was used to measure the absorbance of extracted hemoglobin from erythrocytes at 576 nm.
2.6.3. Analysis of hemolytic activity (estimation of released hemoglobin) 200 μl of the prepared mixture was placed into 96 well-plates. 0.1% triton X-100 was referred to as positive control while phosphate buffer saline solution (PBS) was referred in every analysis as a negative control. At 576 nm, absorbance was recorded that showed the amount of hemoglobin within the extracellular environment. Every assay was executed in triplicate. The %age hemolysis was calculated by using the following formula:

ysis of RBCs %age
Absorbance of sample Absorbance of triton X 100 100

Preparation of tablets
Direct compression methodology was employed for the formation of tablets. The active pharmaceutical ingredient was paracetamol. Magnesium stearate (2.5% w/w) was used as lubricant and maize starch (5% w/w) was employed as the dis-integrant. All material was processed in a climate-controlled room at 21°C temperature.

Results and discussion
3.1. Polyacrylamide grafted Eucalyptus camaldulensis (EC-g-PAM) gum synthesis The synthesis of polyacrylamide grafted Eucalyptus camaldulensis (EC-g-PAM) gum is carried out through microwave irradiation that assisted the creation of radicals on the gums' polymeric backbone. Therefore, for the most part, synthesis is based on free radical generation [35]. The synthesized polymer are obtained in the uttermost proportion at an irradiation time-span of 4 min. However, by means of incremental time duration, additional modification percentage can be attained as well. The contemporary investigations revealed the alteration of native gum utilizing polyacrylamide under time-specific irradiation. Graft copolymerization serves as a specified modification technique by which grafting mechanism is propagated through radical sites generation, and as a consequence, homo-polymerization is reduced to minimal. Besides, microwave irradiation for extended time-durations induces degeneration of the polysaccharide backbone. This serves as an alternative to graft copolymerization and stimulates the generation of additional homo-polymers. On exposure to microwave radiations, hydroxyl (-OH) groups possessing polar-nature assimilate microwaves, as a result, distinctive immobilized and localized rotations cleave the polar bonds and generate free radical sites within the E. camaldulensis molecules. Meanwhile, absorption of microwaves occurs at certain infinitesimal polar molecules such as water (H 2 O) as well, yet, free radicals are never generated and only thermogenesis along with rotations be the ultimate outcomes of that absorbed heat. As polyacrylamide has been a well-known responsive towards grafting reactions, so appropriate substitutions carried out on the exteriors of polysaccharide can intensify hydrophobic character. Owing to these substitutions, modified EC-g-PAM gum demonstrate enhanced hydrophobicity and diminished aqueous viscosity. Moreover, bio-compatibility and water consistency are the two additional indispensable characteristics possessed by the Eucalyptus gums.

Characterization of EC-g-PAM gum
A comparison of FTIR analyses is done for crude, purified and synthesized E. camaldulensis (EG-g-PAM) gum that presented diverse changes in their chemical organizations. Figure S1 is available online at stacks.iop.org/ MRX/7/045307/mmedia shows the FT-IR spectrum of native E. camaldulensis gum. In FTIR spectrum, the fundamental vibrations observed within the 4000-2500 cm −1 region are attributed to -OH, CH and -NH stretching, which are abundantly present in gum. Moreover, C-H stretching vibrations of methylene (-CH 2 ) groups on the gum's polymeric backbone, less intense peak appeared at 1200 cm −1 . Similarly, peaks appearing in the 2000-1500 cm −1 region correspond to carbonyl and C=C stretching, the most intense band in the 1830-1650 cm −1 region of the spectrum is due carbonyl groups. FT-IR spectral observations of purified E. camaldulensis gum is also shown in figure S2. A broader peak at 3350 cm −1 is due to hydroxyl group stretching vibrations which also exhibited hydrogen bonding with water molecules. One other at 2916 cm −1 corresponds C-H stretching vibrations of methylene (-CH 2 ) group. IR band appearing at 1740 cm −1 and 1200 cm −1 correspond to carbonyl group and C-C stretching vibrations. Distinct peaks between 1200 cm −1 and 800 cm −1 for glycoside linkages represent highly coupled C-C-O, C-O-C and C-O-H stretching modes. In case of EG-g-PAM, IR spectrum is different from those of crude and purified gums. FTIR spectrum of EC-g-PAM ( figure S3) the peak at 3409 cm −1 show overlapping of -OH stretching band of EC and N-H stretching band of amide (-NH 2 ) group. A clear peak at 3000 cm −1 in EG-g-PAM is observed due to C-H stretching vibrations, while the detection of other bands at 1740 cm −1 , 1676 cm −1 and 1410 cm −1 are predicted as C-O and C-N stretching vibration bands. During graft-copolymer synthesis of E. camaldulensis gum along with polyacrylamide, a distinct peak at 1289 cm −1 is characteristic for C-N stretching of secondary amide groups and hence authorized grafting. These additional bands within grafted EC as compared with crude EC confirmed the grafting of polyacrylamide chains onto the polymeric backbone of EC. Scanning electron microscope of native, purified as well as modified EC-g-PAM gum provides practical comprehension with reference to graft copolymerization. A comparative analysis of the SEM micrographs of crude EC gum and EC-g-PAM gum is shown in figure 1. SEM image show the profound morphological modifications, evidenced in the form of transition from granular to fibrillar arrangement, which is due to the grafting of polyacrylamide (PAM) chains onto EC gum's polymeric backbone. Prior to modification, particulates of the native gum exhibited rough and irregular external morphologies ( figure 1(A)). On the other hand, SEM micrographs of purified gum revealed roughness, yet to a lower extent, as compared to the crude gum ( figure 1(B)) and lesser topographical irregularities are observed in case of purified gum. It is, therefore, evident that granular morphology of EC is lost as a consequence of grafting and got transformed into fibrillar form and an even, smooth and non-porous texture is visible for grafted product (figure 1(C)). From these results, two specifications could be conferred; at first, when dissolved in water, the native E. camaldulensis gum converted into viscous solution, primarily due to the hydrogen bonding. Secondly, the synthesis of E. camaldulensis gum utilizing polyacrylamide imparted softness to the micelles' particulates that organized themselves homogeneous in all respects, along with even and non-porous textures and reduced viscosities for aqueous solutions. So, the grafting mechanism can be authenticated by SEM showing difference is surface of E. camaldulensis gum which changed into fibrillar form, from irregular surface morphology.
Energy dispersive x-ray spectroscopy (EDS) analysis support our observation ( figure 2). The percentage of each element in crude, purified and EC-g-PAM gum are mentioned in tables S2-S4 (supporting information) respectively. The elemental analysis (atomic percent as well as mass percent) of native gum samples indicate the highest concentrations for element carbon i.e. 51.81% and 59.30% respectively where oxygen exhibited second highest fractions by weight (46.71%) followed by potassium (0.72%) and calcium (0.41%). Four elements including potassium, calcium, magnesium and sulphur are also present in trace concentrations, not exceeding 1%. In case of purified EC gum, the sequential order of percentage composition by weight of the elements as carbon, oxygen, potassium, calcium, magnesium, silicon, sulphur and aluminium with 52.81%, 45.09%, 0.63%, 0.44%, 0.38%, 0.32%, 0.23% and 0.12% respectively. The atomic fractions provide a similar sequence for these elements except for magnesium (0.21%) that show slightly higher concentration in comparison with potassium (0.15%) However, the enhanced percentages of carbon and some other elements in correspondence to the native EC gum can be attributed to removal of impurities from the gum. For polyacrylamide grafted Eucalyptus camaldulensis gum (EC-g-PAM), carbon maintain its utmost bulk proportions i.e. 53.24% followed by oxygen with 45.37% weight composition. Other elements such as potassium, calcium, silicon, sulphur and magnesium are present in minute mass concentrations viz. 0.59%, 0.34%, 0.19%, 0.15% and 0.12% respectively (table S4). Similarly, the atomic fractions are observed to be 60.65%, 38.80%, 0.21%, 0.12%, 0.09% for carbon, oxygen, potassium, calcium, silicon respectively, however, sulphur and magnesium show the least and comparable atomic proportions of 0.07%.
Thermo-gravimetric analysis (TGA) is carried out to access the changes in masses of the unmodified, purified and modified samples of E. camaldulensis gum with respect to temperature. Four distinctive zones are observed in the TGA curve of native EC gum, characteristic of the percentage reduction in weight. Figure S4 (supporting information) is the TGA for native E. camaldulensis gum and which reveal that initial decomposition set about at 49.66°C after a time-span of 2.50 min with a correspondent 8.42% loss in mass that could be assigned to the removal of moistness in the EC gum sample. The succeeding zone of weight loss within the range of 87.87°C to 241.61°C showing 5.70% additional reduction in weight which occurs due to thermal decomposition of lower molecular compounds within the polysaccharide chains. The significant change is observed in the range of 241.61°C to 395.64°C and percentage reduction in weight is approximately 20.74% which may be due to partial decomposition of higher molecular weight compounds. A weight loss of 12.85% is recorded within the temperature range of 395.64°C to 618.40°C that characterize the decomposition of the polymeric backbone incorporating primary alcoholic functionalities. A time-lapse of about half an hour, resulted in an approximate reduction of 50% in mass at 618.40°C. Similarly, TGA curve of purified gum also exhibit similar four distinctive thermal peaks, comparable to that of the crude gum on the whole, along with the similar % age mass reduction (figure S5). After initial 9% loss, all the subsequent degradation peaks are sharper, in comparison to crude EC gum, particularly attributed to high purity of the gum. Figure S6 show the TGA curves of EC-g-PAM where weight loss is distinctive and steeper, in comparison to native and purified gums. Thermal decomposition at 87.58°C show the thermal stability of the graft copolymer, in comparison to crude gum that formerly exhibit the initial decomposition at 49.65°C. Thus, the earliest weight loss from 87.58°C to 245.16°C is observed to be 7.05%. Then, weight loss from 87.58°C to 245.16°C indicate the degradation of the simpler, low molecular weight compounds existing within the polysaccharide chains. Third zone start from 245.16°C to 313.89°C is due thermal decomposition of somewhat more complex components such as secondary alcoholic groups (-CHOH). Fourth region of degradation from 313.89°C to 485.69°C represent the degradation of remaining polymeric backbone i.e. -CH 2 OH functionalities. One more distinctive zone is evident between 485.69°C and 614.84°C, which is due to the grafted amide group (-CONH 2 ) decomposition on the polysaccharide chain of EC gum. This distinct curve is due to amide group which confirms the graft modification process.

Qualitative analysis of carbohydrates
Carbohydrates present in native, purified and grafted EC gum extracts is done by distinctive chemical assays including Molisch's test, Benedict's test, Fehling's test, Methylene blue test and Barfoed's test, respectively. The violet-colored ring that appear at the juncture signifies the presence of carbohydrates via Molish's test. The formation of orange red colored precipitates indicate the presence of reducing sugars in Benedict's test. In Fehling's test, red-colored precipitates also confirmed reducing sugars. While methylene blue solution turned colorless which indicate exposure to the reducing sugars. The generation of brick-red colored precipitates indicate positive result for reducing sugars in Barfoed's test. Detail of these tests is given in table S5.

Antioxidant analysis
Phenolic and poly-phenolic compounds are the main class of natural antioxidants that strengthen the oxidative stability of biological systems due to their redox properties. These compounds play significant roles in neutralizing free radicals, quenching singlet oxygen, decomposing hydro-peroxides generated during normal metabolism and energy production within the body. The study allowed the evaluation of the antioxidant potentials of three extracts with different phenolic contents and as up to now no literature is reported about phenolic contents of the gum in this plant. All three extracts have comparable phenolic contents of 0.106, 0.129 and 0.159 μg ml −1 gallic acid equivalents (GAE) for crude, purified and grafted gum respectively (table S6). However, the results show strong graft-dependent increase in total phenolic contents. High phenolic contents of grafted extract indicate high antioxidant potential, attributed to varying hydrogen and electron-donating capacities of those phenolic compounds and suggested considerable antioxidant compounds worthy of further investigations. The standardized graph curve of gallic acid for the determination of whole phenolic contents is shown in the figure 3.
DPPH assay is among the most popular spectrophotometric methods for the determination of antioxidants in plant extracts and estimate the antioxidant activity of compounds. Detail of DPPH°radical scavenging of crude, purified and grafted EC gum extracts is presented in table S7 (supporting information). The obtained results suggest that all three extracts are apparently good free radical scavengers and probably have the ability to inhibit autoxidation of lipids. Thus, these extracts can be beneficial for the treatment of various diseases where lipid peroxidation serves as an important mechanism for pathogenesis. The results indicate that crude gum samples exhibit maximum activity (79%) while grafted EC gum show minimum (64%) and the purified samples have an intermediate antioxidant scavenging activity (70%) which further prove the potential antioxidant effectiveness of E. camaldulensis gum.

Antimicrobial assay
Antimicrobial test is carried out to determine the effectiveness of the E.camaldulensis gum against two resistant pathogens -E. coli and S. aureus. For that purpose, inhibition produced by crude, purified and amended gum samples are compared to establish their appropriate antimicrobial roles (table S8). Since the amount of space around each plate -zone of inhibition -indicate the lethality of that gum extract against the E. coli and S. aureus concentration, highly effective grafted gum samples produce a wider ring of no bacterial growth i.e. 16 mm wide as compared to crude and purified gum. These results are recorded with reference to E. coli which demonstrate comparatively lesser change in the surrounding E. coli concentration. Minimum zone of inhibition is observed in case of crude gum samples i.e. 12 mm wide ring whereas purified gum samples exhibit a moderate zone of inhibition i.e. 14 mm wide ring. In case of S. aureus pathogen, the effectiveness of gum decrease with grafting, exhibiting lesser bacterial sensitivity i.e. 13 mm wide region of growth inhibition as compared to crude and purified gum extracts that show maximum-14 mm wide and intermediate-12 mm wide zones of inhibition, respectively. Overall results show slightly more antimicrobial effect of gum extracts against E. coli than S. aureus. The obtained results are in agreement with the antimicrobial capability of Eucalyptus camaldulensis leaf extracts against Bacillus subtilis (Gram-positive), Klebsiella spp. (Gram-negative), Pseudomonas aeruginosa (Gramnegative), Salmonella typhi (Gram-negative), Staphylococcus aureus (Gram-positive) and Yersinia enterocolitica (Gram-negative) strains. The results further demonstrate broad spectrum action of dichloromethane fraction, methanol extracts and residues against these test organisms altogether except petroleum ether fraction that do not possess activity at all. Furthermore, the phytochemical analysis of this plant show the existence of cardiac glycosides, saponins and tannins. Thus our findings predict the use of Eucalyptus camaldulensis gum as an antimicrobial agent.

Biofilm inhibition
Bacteria are more resistant to most of antimicrobial agents within a biofilm and can withstand critical environments and escape the hosts' immune systems. In vivo studies have shown the potential of EC gum extracts to reduce the virulence of Bacillus subtilis and Escherichia coli and predicted that they can constructively influence the forthcoming human medicine either by facilitating the dispersion of pre-fashioned biofilms or inhibiting the generation of innovative biofilms. Our biofilm inhibition assay results show that biofilm formation can be prevented up to 50% by using crude gum (table S9). However, grafting reduce the biofilm dispersion to some extent but it is still very effective. Previously, no reports concerning the biofilms' inhibition capability of EC gum are available in literature. The evaluation of crude, purified and grafted EC gum samples is carried out by a rapid assay against two bacterial strains -B. subtilis and E. coli. All three gum extracts possess significant anti-biofilm potentials. Crude gum extracts demonstrate the highest proportions of biofilms dispersion i.e. 49.70% against B. subtilis, followed by 45.50% inhibition against E. coli. On the other hand, purified gum extracts also show remarkable activity against both bacterial strains. In case of E. coli, an effective anti-biofilm activity of 40.41% is evaluated that is lesser in comparison to B. subtilis (43.78%). The grafted gum extracts exhibit the best anti-biofilm activity against B. subtilis and limit the production of biofilms up to 39.12%. However, a lesser dispersion percentage of 37.23% is observed with E. coli ( figure 4).

3.7.
In vitro toxicity by hemolytic activity Erythrocytes membranes are affected by the consumption of natural bioactive compounds originating from ethnobotanical plants and their products (including gums). There exists negligible previous toxicological evidences with regards to E. camaldulensis gum within the established literature. The native, purified and synthesized E. camaldulensis gum samples underwent screening analyses utilizing a fast assay on human erythrocytes. The percentage lysis increased with increasing concentrations of the gum samples (table 1). Very low toxicity is observed by purified gum but somewhat comparable hemolytic capability is recorded for crude and grafted samples at elevated concentrations. The results indicate that there exists prohibitive correlation between the hemolytic activity and concentration of gum. At the same concentrations, crude extracts exhibit considerably higher hemolytic activity in comparison with other gum extracts and resulted in elevated total percentage lysis of 3.23%, 3.85%, 3.97%, 4.27% and 4.5% at 2%, 4%, 6%, 8% and 10% gum concentrations, respectively. In case of grafted gum extracts, the results demonstrate comparatively lower lysis percentage than crude extracts yet higher than that of purified gum extracts. Similarly, at gum concentrations of 2%, 4%, 6%, 8% and 10% the observed lytic percentages is 2.56%, 2.97%, 3.18%, 3.72% and 4.59%. However, at higher concentration i.e. 10%, no significant change in activity between the crude and grafted extracts is observed. Purified gum extracts on the whole exhibit lower toxicological activity i.e. 2.11%, 2.45%, 2.93%, 3.69% and 3.88% respectively at 2%, 4%, 6%, 8% and 10% gum concentrations.   . The sustained release of formulations can be attributed to their chemical binding with EC g-PAM. That tendency could be explicated by the fact that with correspondent increase in percentage grafting, the magnitude and number of grafted chains increase, hence, more effective linkage is formed among them which lowers the solubility of the polymeric matrix, resulting in lesser breakdown of the formulations and disintegration of the enclosed drug formulations [36,37].

Conclusion
The physicochemical characterization of native, purified and modified Eucalyptus camaldulensis gum are evaluated in this study. Prior to modification, native gum exhibited rough and irregular external morphologies that transformed into fibrillar structure. Grafting of purified gum lead to even and non-porous textures. In case of EC-g-PAM, thermo-gravimetric analysis (TGA) curves justified the thermal stabilization where initial decomposition curve appeared at 90°C rather than at 50°C and one more distinctive zone in EC-g-PAM correspondence to the native EC gum. The characteristics of synthesized E. camaldulensis gum are analyzed at distinctive concentrations i.e. 1%, 2%, 3%, 4% and 5% and Eucalyptus camaldulensis gum demonstrated sustained-release drug dosage formulations. This show that Eucalyptus camaldulensis gum could be advantageous as a binding agent due to remarkable mechanical strength. So, it is incorporated into the paracetamol, in vitro formulations. Drug release profile of modified EC gum revealed excessive controlled drugrelease profiles within distinctive media, comparable to that exhibited by various plants' gum binding agents. Grafted EC-g-PAM gum engrossed into the particulates, thus, minimized their enlargement within gastroenteric systems. These modifications suggest a more advantageous oral administered and site-specified drug transference system. The current investigations on Eucalyptus camaldulensis gum reveal that the gum can be employed as an innocuous potential binder within time release dosage formulations, especially for drug carrier systems designed to achieve prolonged therapeutic effectiveness through continuously releasing medication over an extended period of time, subsequent to single dose administrations.