Simvastatin loaded composite polyspheres of gellan gum and carrageenan: In vitro and in vivo evaluation

International Journal of Biological Macromolecules 57 (2013) 238–244

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Simvastatin loaded composite polyspheres of gellan gum and carrageenan: In vitro and in vivo evaluation Raghavendra V. Kulkarni a,∗ , Vineeta V. Nagathan a , Prakash R. Biradar b , Akram A. Naikawadi c a

Department of Pharmaceutical Technology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586103, Karnataka, India Department of Pharmacology, BLDEA’s College of Pharmacy, BLDE University Campus, Bijapur 586103, Karnataka, India c Department of Pharmacology, BLDEA’s Shri. B.M. Patil Medical College, BLDE University, Bijapur 586103, Karnataka, India b

a r t i c l e

i n f o

Article history: Received 26 July 2012 Received in revised form 3 February 2013 Accepted 9 March 2013 Available online 17 March 2013 Keywords: Simvastatin Polyspheres Gellan gum Carrageenan Controlled release Hypolipidemic activity

a b s t r a c t We investigated the lipid lowering ability of simvastatin loaded gellan gum–carrageenan composite polyspheres, which were prepared by ionotropic gelation/covalent crosslinking method. The surface morphology revealed that the polyspheres have rough and dense surface. The drug entrapment efficiency of the polyspheres prepared by ionic crosslinking was higher than those prepared by dual crosslinking. The in vitro drug release study indicated that the ionically crosslinked polyspheres discharged the drug quickly whereas, dual crosslinked polyspheres extended the drug release for longer period. The hypolipidemic activity performed on Wistar rats indicated that the polyspheres have effectively reduced the elevated total serum cholesterol and triglycerides. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Multi-particulate matrices (designated as “polyspheres” herein) are oral dosage forms containing a number of smaller matrices with diameter ranging from 0.05 to 2 mm, which exhibit desired properties for drug delivery applications [1]. For administration of the recommended dose, these polyspheres may be filled into a sachet and encapsulated or compressed into a tablet [2]. Polyspheres are the discrete particles that make up a multiple unit system and provide many advantages over single-unit systems because of their small size. Polyspheres are less dependent on gastric emptying, thus resulting in less inter and intra-subject variability in gastrointestinal transit time [3]. Recently, much importance is being laid on the development of multiparticulate dosage forms than the single unit dosage form, because of their benefits such as increased bioavailability, reduced risk of systemic toxicity, reduced risk of local irritation and predictable gastric emptying [4]. The drug safety can also be increased using multiparticulate dosage forms, particularly for modified release systems [5,6]. Several natural and synthetic polymers are being used for development of polyspheres, but use of natural polysaccharides is the focused area of research in multiparticulate drug delivery.

∗ Corresponding author. E-mail address: pharma [email protected] (R.V. Kulkarni). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.03.027

Gellan gum (GG) is an exocellular natural anionic heteropolysaccharide produced by aerobic fermentation of the bacterium Sphingomonas elodea [7]. The gelation of gellan gum can be induced by cations as well as temperature [8,9]. GG solution becomes gel in the presence of mono and divalent cations, but, its affinity for divalent cations such as Ca2+ and Mg2+ is stronger than the monovalent ions such as Na+ and K+ [10]. In the literature, GG has been reported in the development of ophthalmic drug delivery [11], oral sustained/controlled delivery systems [12–15] and floating in situ gelling system [16]. Carrageenan (CRG) is a high molecular weight anionic heteropolysaccharide obtained from marine algae, Rhodophyceae. It is a sulfate ester of galactose and 3,6-anhydrogalactose copolymers, linked by alternating ␣-1,3 and ␤-1,4 glycosidic linkages [17]. CRG has been used in the development of drug delivery systems [18–20]. Simvastatin (SMT) is a lipid-lowering drug, used in the treatment of hypercholesterolemia. It decreases cholesterol levels by inhibiting the HMG-CoA reductase, the enzyme that reduces the biosynthesis of cholesterol. After oral administration, it shows less than 5% bioavailability and plasma half life of about 1.9 h [21]. Hence, there is a need for the development of controlled release dosage form, which could avoid the repeated dosing. Therefore, the objective of the present work was to develop the composite polyspheres of GG-CRG for controlled release of SMT and to evaluate the lipid lowering ability of the prepared polyspheres in Wistar rats. The prepared polyspheres were characterized by

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Table 1 The formulation details of simvastatin loaded polyspheres. Formulation codes

Gellan gum (%, w/v)

Carrageenan (%, w/v)

Simvastatin (%, w/w of polymer)

Zinc sulfate (%, w/v)

Glutaraldehyde (%, w/w of polymer)

GC1 GC2 GC3 GC4 GC5 GC6 GC7 GC8 GC9 GC10 GC11

0.50 0.75 1.00 1.00 1.00 1.00 0.50 0.75 1.00 1.00 1.00

1.00 0.75 0.50 0.50 0.50 0.50 1.00 0.75 0.50 0.50 0.50

20 20 20 20 20 40 20 20 20 20 40

5 5 5 10 15 15 5 5 5 5 5

– – – – – – 5 5 5 10 10

Fourier transform infrared (FTIR) spectroscopy, differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), X-ray diffraction (X-RD) studies and scanning electron microscopy (SEM).

with the secondary electron image as a detector and 20 kV acceleration voltage. 2.3. Measurement of size

2. Materials and methods Simvastatin (SMT) was obtained as gift sample from Strides Arco lab Ltd. (Bangalore, India). ␬-Carrageenan (CRG) was purchased from HiMedia Laboratories Pvt. Ltd. (Mumbai, India). Gellan gum (GG) was purchased from Ozone International (Mumbai, India). Glutaraldehyde (GA; 25%, v/v) was procured from S.D Fine Chemicals (Mumbai, India). Zinc sulfate (ZnSO4 ) was procured from New Modern Chemical Corporation (Mumbai, India). Cholesterol and triglyceride kits were purchased from Transasia Bio-Medicals Ltd. (Baddi, India). Double distilled water was used throughout the study. All other chemicals were used without further purification. Male Wistar rats weighing between 150 and 250 g were used for the study. The animals were acclimatized to laboratory conditions for five days before the experiments, with adequate food and water ad libitum. Animal experimental protocols were approved by our institutional ethics committee which follows the guidelines of the committee for the purpose of control and supervision of experiments on animals (CPCSEA).

The average diameter of the polyspheres of was determined with the help of a digital micrometer (MDC-25S Mitutoyo, Tokyo, Japan) with an accuracy of 0.001 mm. A total of 100 polyspheres per batch were measured and average size was calculated. 2.4. Estimation of drug entrapment efficiency (DEE) The DEE of the polyspheres was determined by swelling method. Accurately weighed quantities of polyspheres were soaked in 100 ml of phosphate buffer (pH 7.4) for complete swelling at 37 ◦ C. Then the polyspheres were crushed and the solution was gently heated for 2 h and centrifuged to separate the polymeric debris. The supernatant solution was analyzed for the drug content using UVvis spectrophotometer (Model Pharmaspec UV-1700, Shimadzu, Japan) at 238 nm. The DEE was calculated using the following equation: Drug entrapment efficiency =

2.1. Preparation of polyspheres Drug–polymer dispersion was prepared by mixing the accurately weighed quantities of GG, CRG and SMT with double distilled water using a magnetic stirrer. Twenty milliliters of the dispersion was taken into 25 cc disposable syringe fixed with # 23 needle and added dropwise into an aqueous solution of ZnSO4 under constant stirring. For hardening, the polyspheres were left in the in the ZnSO4 solution for additional 15 min and then separated from ZnSO4 solution; dried at 40 ◦ C for 12 h. Further, the polyspheres were dual crosslinked by placing in a solution containing different concentrations of glutaraldehyde (GA) and 1 N HCl for 30 min at 50 ◦ C. After which they were washed with distilled water repeatedly to remove the unreacted GA. The polyspheres were dried at 50 ◦ C for 2 h and stored in a closed container fro further evaluation. The ingredients used for the preparation of polyspheres are summarized in Table 1. 2.2. Scanning electron microscopic (SEM) analysis The shape and surface morphology of the polyspheres was examined by using SEM analysis. The photographs were taken by placing the polyspheres on stub of the instrument using double sided adhesive tape; further polyspheres were sputter coated with platinum with the help of sputter coater (Edward S 150, UK). The coated polyspheres were observed under SEM (JEOL, JSM-6360, Kyoto, Japan) at the required magnification at room temperature

experimental drug content × 100 theoretical drug content (1)

2.5. Fourier transform infrared (FTIR) spectroscopy FTIR spectra of the samples were recorded using FTIR spectrophotometer (8400S, Shimadzu, Japan). The samples were crushed with potassium bromide to get thin pellets under a pressure of 600 kg and were scanned between 450 and 4000 cm−1 . 2.6. Differential scanning calorimetric (DSC) analysis The DSC analysis of SMT, drug-free GC6 polyspheres and drugloaded GC6 polyspheres was carried out using a microcalorimeter (DSC Q20 V24.4 Build 116, TA Instruments, USA) and thermograms were obtained. The samples were heated in the temperature of 0–300 ◦ C at a heating rate of 10 ◦ C/min under argon atmosphere. 2.7. Thermogravimetric analysis (TGA) TGA was performed on GC3 and GC9 polyspheres using a microcalorimeter (DSC Q20 V24.4 Build 116, TA Instruments, USA) under a dynamic argon atmosphere flowing at a rate of 50 ml/min and at a heating rate of 10 ◦ C/min. in the temperature range 0–600 ◦ C.

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2.8. X-ray diffraction (XRD) studies The XRD analysis of SMT, drug-free GC6 polyspheres and drugloaded GC6 polyspheres was carried using a Philips, PW-171, X-ray diffractometer with Cu-NF filtered CuK␣ radiation. Quartz was used as an internal standard for calibration. The powder X-ray diffractometer was attached to a digital graphical assembly and computer with Cu-NF 25 KV/20 mA tube as a CuK␣ radiation source in the 2 range 0–50◦ . 2.9. Dynamic swelling study The dynamic swelling behavior of the polyspheres was studied by mass measurement. The 50 mg of polyspheres were incubated with 25 ml phosphate buffer solution pH 7.4 at 37 ◦ C. The polyspheres were taken out at different time intervals and blotted carefully without pressing hard to remove the excess surface liquid. The swollen polyspheres were weighed using the electronic microbalance. The percent water uptake (Q) at different time intervals was calculated using the following equation: Q =

W2 − W1 × 100 W1

(2)

where W1 is mass of the dry polyspheres and W2 is the mass of swollen polyspheres. 2.10. In vitro drug release study The drug release study was performed using a USP-23 dissolution tester (Electrolab TDT-06P, (USP), Mumbai, India), at 37.0 ± 0.5 ◦ C and 50 rpm. The study was performed in 900 ml acidic medium (pH 1.2) for 2 h and in alkaline medium (pH 7.4 phosphate buffer) till end of the study. At pre-determined time intervals, samples (5 ml) were withdrawn and replaced with the same volume of fresh medium. Further, samples were filtered through a 0.45 mm membrane filter and the drug concentration was determined using a UV-vis spectrophotometer at a max of 238 nm following suitable dilutions. 2.11. In vivo hypolipidemic study in Wistar rats

Fig. 1. SEM photographs of GC4 polyspheres (A), and surface morphology (B).

The formulation GC11, which has shown satisfactory in vitro drug release, was selected for in vivo hypolipidemic activity. The plasma lipid profiles were determined in healthy Wistar rats weighing between 200 and 250 g. Animals had free access to food and water during the study. The animals were divided into three groups of six animals each, viz., control group, standard group and test group. The treatment was given for 14 days. All groups received daily 2 ml of coconut oil orally throughout 14 days. Standard groups and test groups additionally received pure simvastatin (10 mg/kg body weight) and polyspheres GC11 (equivalent to 10 mg/kg body weight) respectively as 2% acacia aqueous suspension. Blood samples were collected under light ether anesthesia by retro-orbital puncture at fixed time intervals, viz., 0th day, after 7th day and after 14th days. Plasma was separated by centrifugation at 3000 rpm for 20 min and used to measure lipid profiles. The plasma samples were analyzed for total cholesterol (CH) and triglycerides (TG) using semi auto analyzer and in vitro diagnostic kits (Erba Ltd., India). Briefly, fixed volumes of sample and standard were mixed with the working reagent separately, followed by incubation at 37 ◦ C for 10 min. Absorbance was read at 505 nm for CH and at 546 nm for TG determinations. Statistical analysis for the determination of differences between the lipid profiles of experimental animals was done by the unpaired t-test and ANOVA. The ‘P’ values less than 0.05 were considered as statistically significant.

3. Results and discussion During the preparation of polyspheres, as soon as a drop of the GG-CRG solution was added to zinc sulfate solution, ionic cross-linking occurred between two GG polymer chains and different parts of the same polymer chain including CRG to form a ionically crosslinked polyspheres. Further, on treatment of these polyspheres with GA, a bi-functional covalent crosslinking agent, an acetal structures were formed between the CHO groups of GA and OH groups of GG and CRG, thus forming a dual crosslinked composite polyspheres network [22]. The prepared polyspheres were having rough and dense surface as shown in Fig. 1 and they were in the size range of 1044–1292 ␮m (Table 2). Size of the dual crosslinked polyspheres (GC7–GC11) was smaller, as compared to ionically crosslinked polyspheres (GC1–GC6). This may be attributed to the rapid de-swelling of polymer matrix, due to the formation of covalent crosslinks between the polymer chains. With an increase in concentration of crosslinking agents, a decrease in size of the polyspheres was observed. This may be due to the formation of more rigid polymer network at higher crosslink densities. This is in agreement with the earlier reports [23]. Also by increasing the concentration of CRG, an increase in size of the polyspheres was observed. This could be attributed to the formation of larger droplets due to increase in the viscosity of the solution with increasing concentration of CRG

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Table 2 Average size, drug entrapment efficiency (DEE), diffusion coefficients (D) and release exponent (n) of polyspheres. Polyspheres GC1 GC2 GC3 GC4 GC5 GC6 GC7 GC8 GC9 GC10 GC11

Average size (␮m) ±SD 1292 1282 1232 1130 1100 1143 1099 1276 1151 1044 1048

± ± ± ± ± ± ± ± ± ± ±

3.14 2.31 3.34 1.58 1.98 2.87 4.65 5.62 1.93 1.91 5.68

D (cm2 /s)

DEE (%) 78.83 79.20 80.29 81.02 83.21 84.31 77.37 78.91 79.56 79.93 80.29

± ± ± ± ± ± ± ± ± ± ±

0.047 0.014 0.054 0.074 0.063 0.045 0.078 0.098 0.054 0.074 0.024

−5

7.31 × 10 7.02 × 10−5 6.85 × 10−5 6.34 × 10−5 6.11 × 10−5 6.18 × 10−5 5.54 × 10−5 5.31 × 10−5 5.01 × 10−5 4.53 × 10−5 4.74 × 10−5

n

ra

0.51 0.55 0.58 0.61 0.67 0.64 0.68 0.71 0.74 0.79 0.75

0.91 0.88 0.93 0.97 0.98 0.89 0.91 0.93 0.97 0.98 0.96

All the values are average of three determinations. ± indicates SD values. a Correlation coefficient.

during passing through a needle. Please note that, as the amount of SMT increases, the size of polyspheres also increases, because SMT might have occupied the internal spaces between polymer segments. This is also in agreement with the previously published results [24]. The polyspheres have shown the DEE in the range of 77.37–84.31%. Results show that the DEE of the polyspheres prepared by ionic crosslinking was higher than those prepared by dual crosslinking (Table 2). In case of ionically crosslinked polyspheres, DEE of the polyspheres prepared with lower concentration of ZnSO4 was lowest when compared to those prepared with higher concentration of ZnSO4 . This may be due to the fact that, at lower concentration of ZnSO4 , the polymer matrix is flexible and porous due to insufficient crosslinking. This results in higher leakage of drug into crosslinking solution from polymer matrix, which results in lower DEE. Whereas, at higher concentration of ZnSO4 , the polymer matrix is stiff and leakage of drug from matrix is low resulting in high DEE. This also concur earlier reports [25].

3.1. FTIR spectroscopy The chemical stability of drug in the polyspheres was studied by FTIR analysis and is presented in Fig. 2. The spectra of SMT (A) showed the characteristic peaks at 3550 cm−1 due to stretching vibrations of OH groups, the peaks at 3011 cm−1 , 2963 cm−1 , and 2876 cm−1 are due to CH stretching vibrations, and peak at 1710 cm−1 is assigned to stretching vibrations of ester and lactone carbonyl functional groups. Whereas in the spectra of drug loaded polyspheres (B), the same characteristics peaks related to SMT were noticed with slight shift. This confirms the chemical stability of SMT in polyspheres matrix. 3.2. DSC analysis The DSC analysis of plain SMT (A), drug-free GC6 polyspheres (B) and drug-loaded GC6 polyspheres (C) was carried out and the results are shown in Fig. 3. The drug-free polyspheres have displayed an endothermic peak at 272.05 ◦ C, while drug-loaded

Fig. 2. FTIR spectra of simvastatin (A), and GC6 polyspheres (B).

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2 0

A

Heat flow (mW)

-2

B B

-4

C 272 oC

74 oC

262 oC

-6 -8

A

C

-10

140 oC

64 oC

-12 88

38

138

188

238

288

Temperature Fig. 3. DSC thermograms of simvastatin (A), drug-free GC6 polyspheres (B) and drug-loaded GC6 polyspheres (C).

polyspheres demonstrated an endothermic peak at 262.38 ◦ C. This small decrease in melting temperature may be ascribed to the formation of loose polymer network as a result of creation of extra free space after drug entrapment. While, the endothermic peaks at 74.34 ◦ C and 64.05 ◦ C in the drug-free GC6 polyspheres and drugloaded GC6 polyspheres may be attributed to loss of water. The plain SMT has shown a sharp endothermic peak at 140 ◦ C due to melting of the drug, but this peak is not seen in the drug-loaded polyspheres. This confirms the amorphous dispersion of drug in the polyspheres matrix. 3.3. TGA The TGA thermograms of the polyspheres GC3 (A) and GC9 (B) are presented in Fig. 4. The decomposition of GC3 matrix started at 92 ◦ C and 28.99% mass loss was observed up to 92 ◦ C. This may be due to the evaporation of water from the polymer matrix. A consequent mass loss of 20.37% was seen between 92 and 267 ◦ C. Further, a 16.79% of mass loss was observed between 267 and 507 ◦ C and it arrived at a value of 71.15% at 600 ◦ C. This could be due to the decomposition of the polyspheres matrix. While, the decomposition of GC9 started after 66 ◦ C and 9.96% mass loss was observed up to 66 ◦ C, which may be due to the loss of water from the polymer matrix. A mass loss of 18.51% was observed between 66 and 258 ◦ C. Further, 11.31% mass loss was observed between 258 and 502 ◦ C and finally it arrived at a mass loss of 62.21% at 600 ◦ C. The mass loss in case of GC9 matrix was constant and residual mass 110

Thermogram A: 71.15% mass loss Thermogram B: 62.21% mass loss

100

(B)

90

Weight (%)

80

(A)

70 60 50 40 30 20 10

Fig. 5. X-ray diffractograms of simvastatin (A), drug-free GC6 polyspheres (B) and drug-loaded GC6 polyspheres (C).

was higher than GC3 polyspheres. In case of GC9 polyspheres, the polymer matrix is stiff due to dual crosslinking as compared to GC3, which were prepared only by ionically crosslinking. This denotes that the thermal stability of GC9 polyspheres is higher than the GC3 polyspheres. 3.4. XRD studies The X-ray diffractograms of SMT, drug-free polyspheres (GC6) and drug loaded polyspheres (GC6) are presented in Fig. 5. We noticed the intense peaks between the 2 of 13◦ and 35◦ for SMT, due to its crystalline nature. While both drug-free and drug loaded polyspheres, did not show the intense peaks between the 2 of 13◦ and 35◦ . We also noticed almost identical diffractograms of both drug-free and drug loaded polyspheres. This indicates the amorphous dispersion of the drug after loading into polyspheres matrix. 3.5. Dynamic swelling studies The dynamic swelling pattern of polyspheres is shown in Fig. 6. The swelling of polyspheres depends upon the concentration of CRG and extent of crosslinking of the polymer matrix. The swelling of polyspheres increased with an increasing amount of CRG in the polyspheres and it decreased with increasing amounts of ZnSO4 and GA. This may be due to the formation of stiff polyspheres matrix. At lower crosslink density, the polymer matrix is flexible with more hydrodynamic free volume and can suck up more solvent, leading to higher swelling, on the other hand the increased swelling of polyspheres with an increasing amount of CRG may be due to formation loose polyspheres network [26]. On the whole, swelling of the ionically crosslinked polyspheres (GC1–GC6) was greater than the dual crosslinked polyspheres (GC7–GC11). 3.6. In vitro drug release

0 0

100

200

300 400 Temperature (oC)

500

Fig. 4. TGA thermograms of GC3 (A) and GC9 (B) polyspheres.

600

The in vitro drug release behavior of the polyspheres is illustrated in Fig. 7. The ionically cross-linked polyspheres (GC1–GC6) liberated the drug quickly as compared to dual cross-linked

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5 4.5 4 3.5

Wt/Wo

3 2.5 2 1.5 1 0.5

GC1

GC2

GC3

GC4

GC5

GC6

GC7

GC8

GC9

GC10

GC11

0 1

0

2

3

4

5

Time (hr) Fig. 6. Swelling behavior of polyspheres in phosphate buffer pH 7.4.

polyspheres (GC7–GC11). The release of drug was continued for 8 h through dual cross-linked polyspheres depending upon the formulation variables. The polyspheres that were prepared with higher amount of ZnSO4 and GA released the drug slowly. This could be due to the fact that at higher crosslinking, free volume of the matrix decreases, thus hindering the movement of solutes through the polyspheres matrix. This could decrease the swelling as well as drug release rate from the polyspheres. An increase in amount of the CRG and initial drug loading showed increase in drug release rate. The diffusion coefficient values (D), for the drug transport through the polyspheres were calculated using the equation [27]:

 D=

r 6M∞

2 

(3)

where  relates to slope of the linear portion of the plot of Mt /M∞ versus t1/2 , r relates to radius of the polyspheres and M∞ relates to the total amount of drug loaded. The diffusion coefficients were calculated based on the Fickian diffusion model and the D values are summarized in Table 2. These D values suggest that the extent of crosslinking had an influence on the drug release behavior of polyspheres. The values of D were decreased with increasing amount of crosslinking agents. This may be ascribed to the reality that with increasing the concentration of crosslinking agent, a stiffer matrix is likely to be formed, which would forbid the diffusion of drug 100 90 80 70

% Drug released

Fig. 8. Serum total cholesterol (A) and triglycerides (B) of experimental rats obtained from pharmacodynamic activity.

molecules. Also, the D values were increased with an increasing the amount of CRG and initial drug loading in the polyspheres. This may be due to the formation of loose matrix. To determine the drug release mechanism from the polyspheres, release data was treated with an empirical equation [28]: Mt = Kt n M∞

(4)

Here, Mt is the quantity of drug released at time t, and M∞ is the total amount of drug in the polyspheres, ‘n’ values are the release exponents, which indicate the type of release mechanism. The ‘n’ values along with the correlation coefficients have been computed and summarized in Table 2. The values of ‘n’ depend upon the concentrations of cross-linking agents and amount of CRG; we observed increase in ‘n’ values with increase in amounts of cross-linking agents, while ‘n’ values decrease as concentration of CRG increases. The calculated ‘n’ values suggest that the mechanism of drug release follows non-Fickian transport. 3.7. In vivo hypolipidemic study

60 50 40 30 20 10

GC1

GC2

GC3

GC4

GC5

GC6

GC7

GC8

GC9

GC10

GC11

0 0

1

2

3

4

5

6

7

Time (hr) Fig. 7. In vitro release profiles of simvastatin from polyspheres.

8

The in vivo performance of prepared polyspheres has been evaluated using pharmacodynamic activity. The serum lipid profiles of all the three groups of animals at different time intervals are presented in Table 3 and Fig. 8. No significant difference was observed between three groups of animals on day zero (initial day) due to random sampling of animals. After 7th day of treatment, the animals of control group demonstrated significant increase in CH and TG. This indicated the inducement of hypercholesterolemia as a result of feeding of coconut oil. While, animals of standard and test groups showed very slight increase in CH and TG levels in the plasma (P < 0.05). After 14th day of treatment, animals of control group once again showed a noticeable increase in total CH and TG (P < 0.05). But animals of standard and test group showed a

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Table 3 Serum total cholesterol and triglycerides of experimental rats at 0th day, 7th day and 14th day time intervals. Groups

Time intervals

Total cholesterol (mg/dl)

Triglycerides (mg/dl)

Control

0th day 7th day 14th day

56.42 ± 102 91.89 ± 1.36 148.65 ± 2.36

105.11 ± 3.12 160.71 ± 2.13 208.15 ± 1.57

Standard

0th day 7th day 14th day

61.80 ± 2.10 85.08 ± 4.30 106.48 ± 1.32

113.28 ± 1.56 127.65 ± 1.84 135.20 ± 2.10

Test

0th day 7th day 14th day

59.65 ± 0.987 68.37 ± 2.36 77.31 ± 3.21

99.43 ± 2.12 103.51 ± 3.12 108.39 ± 2.96

Results are mean ± SD, n = 6.

slight increase in CH and TG levels. Among the animals of test and standard groups, the test group animals (treated with polyspheres) showed less elevation in CH and TG levels as compared to standard group animals (treated with pure SMT). This indicates that the prepared polyspheres were capable of releasing the SMT in a controlled manner to maintain the constant plasma concentration. 4. Conclusions The GG and CRG based composite polyspheres were prepared by ionotropic gelation and covalent crosslinking method for the controlled release of lipid lowering drug, SMT. The polyspheres were produced with entrapment efficiency as high as 84.31%. The FTIR spectra confirmed the stability of drug in the polyspheres matrix. The DSC and XRD studies confirmed the amorphous dispersion of the drug in polyspheres. The swelling and drug release ability of the polyspheres depend upon the amounts of crosslinking agents and CRG used in the preparation of polyspheres. The ionically crosslinked polyspheres liberated the drug rapidly while, dual crosslinked polyspheres extended the release for longer period. The hypolipidemic activity performed on Wistar rats indicated that the polyspheres reduced the total cholesterol and triglycerides in the blood plasma effectively as compared to pure SMT. It can be concluded from the findings of this study that, the prepared polyspheres are versatile delivery systems for SMT, which could effectively lower the plasma levels of cholesterol and triglycerides. Acknowledgements One of the authors (Prof. R.V. Kulkarni) is grateful to Vision Group on Science and Technology, Government of Karnataka, India

for the financial support through a major grant under K-FIST (Level 2) programme. Authors are also thankful to Dr. B.G. Mulimani, Vice Chancellor, BLDE University, Bijapur for providing necessary facilities to carry out this work. References [1] N.S. Dey, S. Majumdar, M.E.B. Rao, Journal of Pharmaceutical Research 7 (2008) 1067–1075. [2] E.S.K. Tang, L.W. Chan, P.W.S. Heng, American Journal of Drug Delivery 3 (2005) 17–28. [3] F.A.A. Laila, S. Chandran, Journal of Pharmacy & Pharmaceutical Sciences 9 (2006) 327–338. [4] P. Roy, A. Shahiwala, Journal of Controlled Release 134 (2009) 74–80. [5] E. Bulgarelli, F. Forni, M.T. Bernabei, Journal of Microencapsulation 17 (2000) 701–710. [6] S.S. Bhalerao, J.K. Lalla, M.S. Rane, Journal of Microencapsulation 18 (2001) 299–307. [7] A. Rozier, C. Mazuel, J. Grove, B. Plazonnetet, International Journal of Pharmaceutics 57 (1989) 163–168. [8] S. Kanesaka, T. Watanabe, S. Matsukawa, Biomacromolecules 5 (2004) 863–868. [9] J. Horinaka, K. Kani, Y. Hori, S. Maeda, Biophysical Chemistry 111 (2004) 223–227. [10] G.R. Sanderson, R.C. Clark, Food Technology 37 (1983) 63–70. [11] P. Matricardi, C. Cencetti, R. Ria, F. Alhaique, T. Coviello, Molecules 14 (2009) 3376–3391. [12] S. Miyazaki, H. Aoyama, N. Kawasaki, W. Kubo, D. Attwoodet, Journal of Controlled Release 60 (1999) 287–295. [13] S.A. Agnihotri, S.S. Jawalkar, T.M. Aminabhavi, European Journal of Pharmaceutics and Biopharmaceutics 63 (2006) 249–261. [14] F. Kedzierewicz, C. Lombry, R. Rios, M. Hoffman, P. Maincentet, International Journal of Pharmaceutics 178 (1999) 129–136. [15] T. Coviello, A. Palleschi, M. Grassi, Journal of Controlled Release 55 (1998) 57–66. [16] P.S. Rajnikanth, J. Balasubramaniam, B. Mishra, International Journal of Pharmaceutics 335 (2007) 114–122. [17] S. Naim, B. Samuel, B. Chauhan, A. Paradkar, AAPS PharmSciTech 5 (2004) (article 25). [18] M. Hariharan, T.A. Wheatley, J.C. Price, Pharmaceutical Development and Technology 2 (1997) 383–388. [19] O. Sipahigil, B. Dortune, International Journal of Pharmaceutics 228 (2001) 119–123. [20] V.K. Gupta, M. Hariharan, T.A. Wheately, J.C. Price, European Journal of Pharmaceutics and Biopharmaceutics 51 (2001) 241–246. [21] F.R. Victoria, American Journal of Pharmaceutical Education 69 (2005) 546–560. [22] M.N. Collins, C. Birkinshaw, Carbohydrate Polymers 92 (2013) 1262–1279. [23] S.A. Agnihotri, T.M. Aminabhavi, Drug Development and Industrial Pharmacy 31 (2005) 491–503. [24] A.P. Rokhade, N.B. Shelke, S.A. Patil, T.M. Aminabhavi, Carbohydrate Polymers 69 (2007) 678–687. [25] R.V. Kulkarni, B. Sa, Current Drug Delivery 5 (2008) 256–264. [26] K.S. Soppimath, A.R. Kulkarni, T.M. Aminabhavi, Journal of Controlled Release 75 (2001) 331–345. [27] A.R. Kulkarni, K.S. Soppimath, T.M. Aminabhavi, A.M. Dave, M.H. Mehta, Journal of Controlled Release 63 (2000) 97–105. [28] P.L. Ritger, N.A. Peppas, Journal of Controlled Release 5 (1987) 37–42.