Polysaccharide-Based Controlled Release Systems for Antipsychotic Therapeutics Delivery

Researches in pharmaceutical (cid:977)ields are now focusing on biopolymer-based drug delivery systems for treatment of mild to severe diseases. Amongst all biopolymers, pectin is one of the frequently available and eco-friendly bio-polymer extracted from different fruit peels and abundantly available in nature. This study focuses to extract the pectin from two different fruits using different methods namely hot water-based extraction and cold maceration, involving two different fruit peels. Post extraction and isolation of polymer, study was performed for characterization of pectin powder by particle shape, particle size distribution, surface area angle of repose, (cid:977)lowability and tapped density. The obtained pectin was used for anti-psychotic drug delivery when incorporated into microspheres by solvent evaporation method. It’s surface morphology and release kinetics was analyzed using in vitro methods and in vivo models and kinetics of drug release (goodness of (cid:977)it) was assessed. The result revealed that the pectin extracted from mango peel using soxhlation method showed good (cid:977)low rate, minimum particle size and shape distribution, porosity etc. Moreover, it showed the better drug release data in both invitro and in-vivo analysis. The study concludes that in future the natural biopolymer like pectin stands as a potential candidate for formulation of drug release system for ef(cid:977)icient delivery of drug.


INTRODUCTION
In the past decade, systematic drug delivery plays a key role in various clinical aspects. In this connection numerous nanomaterials have been used as vehicle for drug delivery, but it cruelly hampered owing to low ef icacy of delivery to the target site and fast discharge from the body before instigating their action (Reis, 2019). Consequently, the researchers focused on bioinspired drug delivery system like biopolymers.
Polysaccharides like starch, pectin, cellulose are different natural polymers which have been used for delivering various types of drug doses. They are applicable in various ields like food industry, pharmaceutical industry and cosmetics sector (Shirwaikar et al., 2008;Patil et al., 2013). Most of the natural polymers are built by the process of condensation followed by polymerization. The advantage of use of biopolymers are bio degradable in nature, nontoxic/ non -in lammatory, they are also biocompatible as their rate of degradation is inversely pro-portional to chemical modi ication. Compare to synthetic polymer the natural polymer is highly porous, able to attached with other molecule as they have various functional groups (Kuo et al., 2013;Baggi and Kilaru, 2016). Moreover, it is cost effective renewable sources.
Pectin is one of the most abundant, plant derived and naturally available biopolymer using in various ields like drug delivery, Nutraceuticals, tissue engineering, Pharmaceutics etc. (Masheer, 2013). It is nontoxic, eco-friendly polymer used as gelling and emulsifying agent in food industries (Noreen et al., 2017). In drug delivery system the hydrophilic properties of pectin decrease the ability to control ef icient drug release in target site (Rao et al., 1995;Ando et al., 2012;Ruoslahti et al., 2010). To overawed this disadvantage physical, chemical and enzymatic modi ications of pectin is in practice. Recently the pectin uni ied with various compounds like cellulose, starch, chitosan, PEO, collage metal-oxides etc. and applying in wide areas.
This pectin composites with LDH are applied to control drug release in stomach and intestine for colonic disease and ulcerative crohn's disease (Guo et al., 2010;Leroux et al., 2012) Pectin collagen composites are used as bone substitute, salicylate-based composite has antimicrobial activity it is help to prepare for food packaging (Gorrasi and Bugatti, 2016). Pectin CMC based composite are used for scaffold production in tissue engineering (Agoda-Tandjawa et al., 2012). Pectin based powders and nanoparticles are applied in colon speci ic drug delivery medium and wound dressing (Wong et al., 2011;Rossi et al., 2010).
Apart from these, extra ordinarily the pectin is involved in brain targeting drug delivery system, numerous approaches were established to progress the brain speci ic drug delivery by engineering nanoparticles-based drug delivery systems. While remarkable growth has been made, but the drug delivery productivity is still remote (Gao, 2016). The pectin based nasal inserts can improve bioavailability of antipsychotic drugs with chlorpromazine hydrochloride due to its mucoadhesion capacity (Luppi et al., 2010) and low methylated pectin have been exposed to gel and be retained in the nasal cavity after deposition so it could expand the pro iciency of the direct nose to brain pathway particularly for drugs for treatment of central nervous system disorders (Charlton et al., 2007). In the present study focus the antipsychotic drug release system using pectin derived from different fruits and applied with Chlorpromazine for the treatment of psychiatric disorders (Ranbhise and Kamat, 2014).

MATERIALS AND METHODS
The Mango and Orange peels were obtained from local market of Lucknow, Uttar Pradesh. The drug Chlorpromazine was procured from Salvavidas T M Pharmaceuticals private limited, Surat, Gujrat. Other excipients like Poly-Lacto Glycolic Acid, Dichloromethane and Ethanol were purchased from Sigma and Himedia. The synthetic pectin was procured from Sigma Aldrich. The fruit peels were subjected to different temperature and solvent conditions for cold soak (maceration) and hot water-based extraction.

Extraction and isolation of polymer using cold soak/ maceration
Fresh fruit peels were comminuted for easy solvent capturing. 100g of fruit peels were soaked individually in Acidi ied water and cyclohexane diamine tetra acetic acid (Trans-CDTA) (4% v/v) in 1:10 ratio with pH 2.0 (0.1N citric acid as pH modi ier) for 30 days with occasional stirring. Throughout the experiment temperature was maintained at 20 • C to 30 • C. Then the pectin was precipitated using 95% ethyl alcohol and incubate for 6 hours at RT.
After incubation, the entire content was iltered using four-fold muslin cloth and the iltrate was treated with 95% ethyl alcohol again and centrifuged at 10000 rpm for 30 min. collect the pellet and washed with acetone of LR grade and dried in hot air oven below 40 • C for 5-6 hrs. The Powder was obtained and passed through sieve number #80 and stored in refrigerator.

Extraction and isolation of polymer using hot water-based extraction
The process of Soxhlation was used for carried out the extraction process. 100g of dried peel powder was taken in round bottom lask with 1:8 ratio of acidi ied water (pH 2) and heated up to 75 • C for 6h to 8h after irst siphon cycle. During the procedure, the temperature was continuously monitored, and pH was sustained. After the soxhlation procedure, the ethyl alcohol was mixed in 2:1 to the lask and continuously stirred at 4 • C for 2 hrs for precipitation. Precipitated pectin was iltered through four-fold muslin cloth and washed three times with iso-propyl alcohol. The precipitate was dried below 40 • C in hot air oven, sieved through sieve size #20 and stored in refrigerator (Srivastava et al., 2017).
The extracted pectin from fruit peels were characterized for various micromeritic properties, low behavior, swelling properties etc. (Srivastava et al., 2017). The surface morphology of pectin was studied using Scanning Electron Microscopy (Sophisticated Analytical Instrumentation Facility (DST)"

Formulation of Microspheres Using Extracted Polymers
1gm of Pectin from various peels were separately dissolved in 400 ml of water followed by 1% Ammonium cerium (IV) sulphate dihydrate (0.005 M) was added and continuously stirred at RT for 2 hrs. Then Methylated PEG solution (1% w/v) was mixed. The reaction mixture was continuously stirred at RT for 48 hrs (Kim et al., 2006;Kumbhar et al., 2016;Vanitha and Desu, 2019).
Next to that 4% of PEG 6000 (dissolved in ethanol) was added into this complexed solution inally the 100mg of Chlorpromazine (dissolved in ethanol) was also added in a streamlined low under continuous stirring.
The above solution was then added in a thin stream to a beaker containing a mixture of light liquid parafin (90 ml), n-heptane (10 mL) and Polysorbate 80 (2 % V/V) in liquid paraf in and continuously stirred at 1000 rpm using mechanical stirrer at RT until the ethanol was evaporated completely for microsphere preparation. The formed microspheres were iltered through Whatman no. 1 ilter paper and washed 4-5 times with petroleum ether allowed to dried at RT for 24 hrs (Patil et al., 2013).
The obtained microparticles were characterized by observing through optical microscopy, particle size and distribution, product yield, encapsulation ef iciency and loading ef iciency, micromeritic properties of particles, scanning electron microscopy and invitro release characteristics (Jelvehgari et al., 2011). Table 1 For the drug entrapment study the microparticles equivalent to 10 mg Chlorpromazine were as dissolved in 10 ml 0.

Drug Release Study
The invitro release of microparticles were taken in SGF (Simulated Gastric Fluid) of pH 1.2 prepared using 0.1 N HCl in 600 ml of luid. The irst two hour of release was taken in SGF. Further release data was collected in phosphate buffer of pH 6.8 for one hour, followed by release study in PBS of pH 7.4 for 9 hours. 5ml aliqoutes were collected at predetermined interval and subsequently were replaced by 5ml of fresh buffer. The samples were analyzed by UV Spec. at 250 nm. The drug release was calculated and kinetic pro ile was determined.
The kinetic models like Zero-order equation, Firstorder equation, Higuchi kinetics and Korsemeyer-Peppas models were applied to all the formulations, suitable model was studied using regression coef icient (Schneider et al., 2010).
The in-vivo analysis of drug release from microparticles were tested using Pole Climb Avoidance Test, which is a behavioral model for studying effect of antipsychotics on rats. The analysis with Cooks Pole Climbing Apparatus in Male Wistar rats (6 in each group). The dose calculated was 10mg/kg bodyweight of the rats and the end point analysis sated the fact that neuroleptics enhance the time taken to climb the pole (expressed as latency) in Electric shocks through grid loor (Radahmadi et al., 2013). The latency in seconds were plotted for all the four formulations and was analyzed using a Kruskal-Wallis nonparametric one-way analysis of variance corrected for formulations and ties. All data were reported as the mean ± standard error in spite of the probable no normality of the distribution of scores, because it seems these parameters provide a clearer indication. The P value less than 0.05 (P < 0.05) was considered as signi icant.

RESULTS AND DISCUSSION
The properties of pectin power were studied based on density and porosity and powder lowability these are important pharmaceutical properties that can be derived from particle shape, particle size distribution, surface area angle of repose, low through an ori ice, and tapped density. These are the Traditional tests through a long history of extensive use, and reference in USP/EP guidelines (Ramankannan et al., 2013). In this study the obtained pectin powders from various methods Polymer Characterization & Flow Property Study was mentioned in Table 1 & Table 2.
One of the challenges of increasing the lowability by using low additives in pharmaceutical aspects. (Rao and Sahoo, 2017;Sahoo et al., 2017) Various parameters such as angle of repose, Carr's index, Hausner ratio are used to express lowability of powders (Hadjitto is et al., 2017). A good low is indicated by a Hausner ratio greater than 1.25, and a poor low may have a value of 1.5 (Clayton, 2019). Nearly all the pectin powders showed same ratio from 1.2 to 1.3 so compare to others the mango peel pectin prepared from soxhalation method (F2) showed fair low rate compare to others. Carr's index calculation 5 -21% is good low rate (Patil et al., 2018) all the powders are showed less than 20% except F1 and the Angle of repose < 30 • is mentioned as good low rate , here all the powders showed less than 21 • So based on this equations all the four (F1-F4) powders showed good lowability but compared to other commercial pectin the pectin prepared from Mango peel (Soxhlation) F2 showed excellent lowability.
Study of particle shape, particle size distribution using microscopic analysis showed in Figure 1 & Figure 2 and Table 3.
The SEM micrographs in Figure 2 revealed that the resulting formulation were in a range of spherical to less spherical in nature with rough surfaces containing cracks and holes over its surface. The reason    behind this morphology change can be attributed to the faster evaporation of ethanol forming a pore like structure. From various SEM photographs, the size, mean diameter and its distribution of the microsphere the size of the prepared microspheres was uniform and appropriate for the study, but the shape uniformity more in F2 pectin compare to others.
The mean particle size of the microsphere showed signi icant increase with the increase in polymer concentration and Particle sizes of the microspheres were found to be mainly affected by the concentration of polymer and stirring time used in the formulations. The size ranges vary from 295 µm to 469 µm, smaller in size and shape uniformity of particle is better for various application in this connection the F1 and F2 pectin are exhibited smaller in size.
The percentage encapsulation ef iciency of microparticles varied from 17.8% to 72.7%. The percentage loading of drug was increased by increasing the ratio of polymer as found high in all the formulation. Encapsulation ef iciency is the amount of drug entrapped within a colloidal system after a formulation process (Wake and Kshirsagar, 2017). Better drug loading was achieved by increasing the ratio of polymer. The percentage yield for all the microparticles was found in the range as 25-81%. All the characteristic study result revealed that the F2 and some what the F1 pectin is better can applicable for various drug delivery systems.
In the invitro release pattern the obtained pectin entrapped drug is compared with comm0ercial pectin entrapped drug and released kinetics was studied ( Figure 3 & Table 4 ). he According to the release pattern, it was observed that commercially obtained pectin released around 99% of drug at a time span of 330 minutes. The release could not be extended over 5 hours 30 minutes in comparison to 460 minutes release obtained from extracted pectin F2 Figure 3. This can be attributed to the reason that acid-based extraction of pectin involves acids, which are the strongest extracting agents as they facilitate extraction of insoluble pectin that is tightly bound to the cell matrix of the plant material and result in higher yields. On the other hand, commercially available pectin is obtained by either enzymatic extraction correlating with quicker and higher yield.
The In-vivo release pattern in rat model, compare to control all commercial and obtained pectin showed highest latency time. The latency time was had signi icantly high in F2 and F3 compare to control (P < 0.001) and F1 and F4 is also signi icantly increased (P < 0.05) Figure 4. Finally, this study concludes that the natural pectin microencapsulation is used for good drug release in delivery system for psychiatric treatment.

CONCLUSION
The above studies identi ies the fact that waste of fruits that is peels of Orange & Mango serve as potential candidate for pectin collection. More so ever, the pectin extracted by cold maceration technique is competitive enough to hot water based extraction in the view point that loss due to temperature rise and denaturation due to temperature rise do not occur. The study is supported by invito as well as invivo release data which envisage the path to cold maceration as prominent technique for extraction. Formulation of even surfaced microspheres by solvent evaporation method rules the fact that pectin is potential candidate to encapture the drug in ef icient way and deliver at the required site as desired.

ACKNOWLEDGEMENT
The researcher is thankful to Rameshwaram Instititute of Engineering & Technology for providing platform for research.