Formulation and Optimization of Immediate Release Cajanus Cajan Starch-Based Tablets Containing Metronidazole

Starch is found almost in all organs of plants as a carbohydrate reserve. It is considered one of the most commonly used pharmaceutical additives, mainly in tablet dosage forms; it used as a tablet binder when incorporated through the wet granulation process or as a disintegrant.Cajanus cajan has a high level of carbohydrate, which makes it another potential choice as a source for starch. This study aims to investigate and optimize the effect of Cajanus cajan starch concentrations as well as wet massing granulation time on physicochemical properties of metronidazole tablets. The hardness, friability percentage, and disintegration time of prepared tablets were determined, and the central composite design was employed in the optimization process. Then the tablets of optimized batch were compared against those tablets in which maize starch and sodium starch glycolate were used instead of Cajanus cajan starch. The results indicated that metronidazole tablets containing the upper level of starch paste (Cajanus cajan and/or maize starch paste) exhibited better percentage friability, hardness, and disintegration time than those formulated with lower levels and those without starch paste. The study showed that experimental design is a useful technique for optimizing Cajanus cajan starch-based tablets, which enabled a better understanding of how different variables could affect the responses. In addition, the study demonstrated that incorporation of Cajanus cajan starch in tablets formulation led to improvement of its physical properties compared to the formulations of maize starch and sodium starch glycolate respectively. CONTACT Ahmed Abdalla Bakheit Abdelgader ahmaedabdella@ymail.com Department of Pharmaceutics, Faculty of Pharmacy, The National Ribat University, Khartoum, Sudan. © 2020 The Author(s). Published by Oriental Scientific Publishing Company This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC-BY). Doi: http://dx.doi.org/10.13005/OJPS05.01-02.05 Oriental Journal of Physical Sciences www.orientaljphysicalsciences.org ISSN: 2456-799X, Vol.05, No.(1-2) 2020, Pg. 16-29 Article History Received: 19 June 2021 Accepted: 21 August 2021


Introduction
Pharmaceutical excipients refer to all materials other than the active drug added intentionally to the formulation of a dosage form. 1 They are essential components in pharmaceutical formulation, to assist with many essential aspects during the pharmaceutical product development process such as processability during the manufacturing, increase stability, and granting the finished products with distinct or particular taste and colour. 2 Excipients are obtained from different origins: animals such as gelatin and stearic acid, plants such as starches and cellulose, minerals such as calcium phosphate, and synthetics like polyethylene glycols(PEGs) and povidone. 3 Presently, there is a need for safe and natural excipients with various uses due to the concerns raised with the use of synthetic polymers and animal-based products. 4 Starch is a natural carbohydrate reserve found in almost all parts of plants; it is widely abundant and considered one of the most commonly used pharmaceutical additives, mainly in tablet dosage forms. Starch paste has been employed as a tablet binder in a concentration ranging from 3% to 20% w/w and incorporated into it via the granulation technique. 5 Also, starch is used as a disintegrant agent at concentrations ranging from 3 to 25% w/w. 5 At present, commercial starch is traced from a constrained range of crops, comprising maize, wheat, tapioca, & potatoes. [6][7] The physicochemical properties and pharmaceutical benefits of starch and its use vary according to its biological origin and source, and that arises from its different amylose/ amylopectin ratio; due to that, a quest for new sources of starch with different physicochemical and functional properties is a rich area of scientific research. [8][9] Sparing investigation and study have been performed on starch from Cajanus cajan regarding its extraction, physicochemical characterization, and pharmaceutical use. Also, few researchers had declared that Cajanus cajan contains high carbohydrate sources, making it a prospective origin for starch. 5 Metronidazole is a nitroimidazole compound, 10 used widely as an antibiotic against anaerobes, 11 and for treatment of infection caused by protozoa. 12 The MNA powder possesses poor compression characteristics. [13][14] MNA is formulated and used in different strengths and dosage forms such as tablets, syrups, creams, and gels. 12 After the oral administration, the MNA absorbed wholly and rapidly. 10,12 Various binders and disintegrants sourced from natural origins such as starch, gum, and mucilage from cashew, Colocassia esculenta, Albelmoschus esculentus, Cochorus olitorious, Aloe barbadensis, Spondias purpurea, Brachystegia eurycoma, Irvingia gabonensis, Cyperus esculentus L., Triticum aestivum, cassava, and Zingiber officinale in addition to sodium starch glycolate, gelatin, microcrystalline starch methylcellulose and maize starch BP have been evaluated and used in metronidazole tablet formulation. [12][13][15][16][17][18][19][20][21][22][23][24][25] Most scientific experiments measure the effect of one or more factors (independent variables) on the experiment's outcome (response or dependent variable). One tool to measure this effect is the experimental design or design of experiments (DoE). 26 Statistical DoE allows all potential factors to be evaluated simultaneously and systematically. Experimental design is an instrument used in evaluating the effects of formulation factors on different primary responses and possible interaction effects; thus, critical parameters are analyzed based on statistical analysis. Final formulation parameters are then defined using optimization, where the levels of all critical factors are determined. 27  Central composite design (CCD) is second-order design most frequently employed to study & optimize tablet formulations. Using CCD, response surfaces that permit the rating of each factor depending on its significance on the responses studied are possibly created. Using these tools, predicting the formulation composition will produce the desired response in a shorter time and less experimental effort. 29 The central composite design is composed of factorial experiment, axial points & center points. In recent years, these designs are extensively used to optimize various drug delivery technologies such as sustained-release tablets, liposomes, microspheres and nanoparticles. 28 This study aims to investigate and optimize the effect of non-conventional used Cajanus cajan starch (CCS) concentrations and wet massing granulation time on physicochemical properties of metronidazole (MNA) tablets.

Materials and Methods Materials
Cajanus cajan seeds were used to extract CCS in our pharmaceutics laboratory, The National Ribat University (Khartoum, Sudan

Drug-Starch Compatibility Study
The method described by Khalid et al. 14 was used to perform the compatibility studies using FTIR (IR Affinity-1, Shimadzu, Japan).

Tablets Preparation
MNA tablets of 200 mg each were prepared using the wet granulation technique.  (2) were blended for 10 min in polyethylene pouch. The slurry was prepared in stainless steel pot by mixing equal weights of CCS (1) and cold water.
Hot water heated at 85 ᴼ C for 30 min was added to slurry until the quantity of water added amounted to 25% w/w of the total formula weight, continuously stirred until a smooth paste was obtained. The paste was cooled and then added to the prepared MNA, PVP, and CCS (2) mixture. An amount of water equal to that lost during evaporation was used to rinse the pot and added to mixture. The granulation process was carried out in a planetary mixer at 34 rpm for determined time with a pause at one and two thirds of the time to abrade the blades and sides using a spatula. The obtained wet mass was then screened through BSS# 12 sieve and dried to loss on drying (LOD) between 2.9 and 4.5% w/w. After that, the resizing of dried granules was performed through the BSS # 18 sieve. The remaining half of the starch, i.e. CCS (2), was used to serve as extra granular disintegrant. So it was mixed with the dried granules for 3 min, then the mixture was lubricated with 0.5 % w/w Mg. St. for 2 min before being compressed into round flat-surfaced tablets using a single punch tabletting machine (Erweka Type EP-1, Heusenstamm, Germany) with 9 mm tooling. The compressional force was 4-8 KP. To minimize interference between CCS (2) and CCS (1), CCS (2) was added as a dry powder to all formulae and at the same quantity, while CCS (1) was added as a paste.
The center point was studied in quintuplicate. All other formulations and process variables were kept constant.
Thirteen formulations (F1 to F13) were prepared as per the experimental design, as seen in Table  (2). A second-order model was established for the responses, and a polynomial equation (1) was generated. The validity of the mathematical model was checked through analysis of variance (ANOVA).
A, B represent the main effects, A 2 and B 2 the quadratic effects, and AB is the interaction effect. Y represents responses (disintegration time, friability, and hardness of tablet); b 0 is an intercept representing the arithmetic average of all quantitative outcomes of 13 runs, and b 1 , b 2 , b 12 , b 11 , b 22 represent the regression coefficients.

Preparation of Tablets for Comparative Purposes
Extra batches of metronidazole tablets were formulated using the optimized formula according to Table (3). The tablets prepared using the same process as provided above except for formulations F14 and F15, in which the specified amount of granulating water was added to the powder mix composed of MNA, PVP and half the amount of disintegrant, i.e. no starch paste was used.

Evaluation of Tablet Characteristics
Stress relaxation for 24 hours was allowed for all compressed tablets before subjecting to quality control tests. The weight uniformity, friability percentage, crushing strength, and disintegration time were evaluated and determined following the method of Shailendra. 30 Also, the calibration curve for metronidazole was carried out, and the drug release profile was evaluated based on the method of Okpanachi et al. 31

Statistical Analysis
The Design Expert®8 was used to study the effect of CCS paste amount and WMT on prepared tablets' characteristics and optimize them. Also, the results obtained were expressed as a mean ± standard deviation calculated using Microsoft excel 2010 software. SPSS version 16.0 for windows (SPSS Inc. Sep 2007) was used for statistical analysis. Analysis of variance (ANOVA) followed by the Tukey HSD multiple comparisons, Kruskal-Wallis H followed by Mann-Whitney U test and t-test were used to compare the results at 95% confidence interval (p < 0.05).

Crushing Strength
The effects of % CCS paste and WMT on the hardness of tablets of formulations (F1 to F13) are depicted in Figure (3   These results indicate that the % friability value increases with the increase in WMT and decreases with the increased levels of CCS paste. These results can be visualized in Figure (4), demonstrating the effects of concentration of CCS paste and WMT on the % friability of prepared tablets. Figure (5 Where A and B representing the coded levels of the independent variables and the terms AB and A 2 / or B 2 represent the interaction and quadratic terms, respectively.

Disintegration Time (DT)
The polynomial equations were used to draw conclusions considering the magnitude of the coefficient and its mathematical sign. From equation (2), both the main effect terms A (% CCS paste) and the quadratic terms A 2 and B 2 have a dominantnegative effect on the Ln value of DT. In contrast, the interaction term A 2 B has a significant positive effect on the Ln DT. From equation (3) and as stated before, the A and A 2 B are significant model terms for response Y 2 (friability). It seems that friability has a negative relationship with the % CCS paste as the main effect and a positive relationship with the term A 2 B as an interaction term. Also, as observed from equation (4), the A, B 2 , A 2 B 2 are significant model terms for the hardness (Y3). It is concluded that hardness has a positive relationship with both A and B 2 as main and quadratic effects. In comparison, the A 2 B 2 has a negative quadratic interaction effect on this response. The three responses, hardness, friability, and DT, were chosen for both graphical and numerical optimization in this design. In order to obtain an optimized formulation, DT and friability were set to be minimized, while the crushing strength was set to be maximized. As in (Figure 6), the contour plots are used to visually search for the best compromise, which stands for the formulation with desirable values for all responses, simultaneously. Also, the defined desirable areas of all responses were overlapped to generate the region of interest.
Using the optimization module from design expert®8 software and the superior value for the responses, and based on the desirability approach for optimization, the solution indicated that the % CCS paste at level +1 (12%) and WMT at level -1 (6 min), yielding 346.5 second DT, 0.75 % friability and 7.23 kg/ cm 2 hardness with the desirability of 0.887. Table (4) presents the numerical optimization,  and Table (5) shows the selected numerical optimization solution.
The practical results obtained from the optimum formula and those predicted by the experimental design were close. Then the optimized formula was selected and used later on for comparative purposes.   (1978) suggested that the amylose was responsible for the disintegrating property. 33 An excipient's hydration and swelling capacity determine the water penetration and absorption, which precedes the tablet disintegration. 34 The amylose content of CCS and maize starch was 32.9 and 24.2. In addition, their hydration capacity was 1.99 and 1.84, and their swelling capacity was 1.46 and 1.23, respectively. The higher amylose content, hydration capacity and swelling capacity of CCS impart them good disintegration properties compared to maize starch.

F15 with F14 and F16
The disintegration time of F14, F15 and F16 was 14.69, 9.15 and 4.99 min, respectively ( Figure 7). There is a significant difference between the DT of F15 and F14 and F16 (p < 0.05). The ranking order of DT was F16 < F15 < F14. F16 tablet, which contained 12% CCS paste as binder and 6% sodium starch glycolate as a disintegrant, exhibited faster DT than F15 (formulated with 6% sodium starch glycolate) and F14 (formulated with 6% CCS) as disintegrants. F14 tablets, which formulated with 6% CCS, exhibited higher disintegration time than those of F15, which formulated with 6% sodium starch glycolate as a disintegrant, and this indicates that the CCS at 6% concentration is ineffective as a disintegrant and has less disintegrating activity as compared with the sodium starch glycolate.

Hardness
The results of hardness are depicted in Figure (7

Dissolution Test
Dissolution testing is an indirect method for measuring drug availability, especially when assessing formulation factors and manufacturing methods that may affect bioavailability. 35 According to the US-FDA guideline, immediate-release drug products should release 85% (T 85%) of the labelled amount of drug within 30 min of study. 32 The MNA standard curve was constructed, and the drug release profile of the Fopt, F14, F15, F16 and F17 formulations is depicted in Figure (9). Tablets of Fopt, F16 and F17, which were formulated with 12% starch paste, released 98.2%, 88% and 82% of drugs within 30 min, respectively, exhibiting good drug release properties, While tablets of F14 and F15, which formulated without 12% starch paste, released 71% and 77.5% after 30 min, respectively. Also, at 15 min the tablets of Fopt, F16 (formulated with 12% CCS paste) and F17 (formulated with 12% maize paste) released 87%, 73.5% and 49%, respectively. These results indicate that the dissolution profile is improved with an increased amount of the CCS in the formulation.
The findings of this study have to be seen in the light of some limitations, which warrant additional investigation. The first is the effect of the rheological behaviour of the starch solution or paste on the release/binding ability of the starch. Also, thermal analysis of the CCS is needed using the TGA study.
Both rheological and thermal behaviours may have a significant effect on the physical properties of the prepared tablets. Future studies of the thermal effect on CCS like TGA can be conducted.

Conclusion
The main aim of this study was to optimize and investigate the effect of the concentration of CCS and the WMT on the physical characteristics of CCSbased tablets containing metronidazole. The tested parameters hardness, friability percentage and DT were chosen as responses. The study demonstrated that the experimental design technique is a valuable tool for optimizing CCS-based formulation, which enabled a better understanding of how variant, crucial variables could influence the selected responses.
An experimental design-derived optimized formula (12% CCS paste and with 6 WMT) was used for comparative study against maize starch and sodium starch glycolate, and the following can be concluded: • The presence of 12% starch paste in the formula led to decreased DT. Also, the tablets formulated with 12% CCS paste disintegrated faster than the tablets formulated with 12 % maize starch paste; this could be attributed to the higher amylose content, hydration capacity and swelling capacity of CCS compared to maize starch.

•
The results also showed that the super disintegrant sodium starch glycolate negatively affect percentage friability. • CCS paste positively affect the hardness of tablets and as effective as maize starch paste.

•
As the results indicate, the dissolution profile is improved as CCS increases in the formulation.