INTRODUCTION:

Many body functions has shown circadian rythms like stomach pH, Heart beat, perfusion, blood pressure and basal body temperature. Certain diseases like Rheumatoid arthritis, bronchial asthma, hypertension, angina pectoris and myocardial infraction show symptoms at regular intervals.¹

In the early morning Patients with RA experience stiffness, pain, and swelling in afflicted joints, which tends to become decreased toward the afternoon. This has been primarily explained by the increased blood levels of pro-inflammatory hormones and cytokines, such as melatonin, TNFα, IL-1, and IL-6, in the early morning than in the afternoon as well as insufficient levels of anti-inflammatory cortisol, which rises later in the morning. As the symptoms in Rheumatoid artritis are shown circadian rythms require pulsatile drug delivery.²PDDS are based on the principle of burst release of a maximum amount of the drug within a short time period after a predetermined off release period, lag time³.

In the pulsatile drug delivery system, the drug delivered at the required site in required amount according to the increased levels of certain inflammatory agents or hormones within the body. As symptoms in the diseases like arthritis, asthma, hypertension shown at regular time intervals these pulsatile drug delivery system holds good promises of releiving the symptoms.⁴

The main object of the currentt work was to formulate floating pulsatile Ketoprofen tablets with a predetermined lag time of 4hrs.In Arthritis the symptoms worsen during early hours. hence Ketoprofen widely used to treat arthritis was selected and delonix regia gum obtained from seeds was used as natural pulsatile polymer. To decrease the disintegration time Dehydrated banana pwder was used as natural super disintegrant. The aim of the currnt research work is to attain the lag time by incorporating floating polymers and pulsatile polymers in a single formulation.

MATERIALS AND METHODS:

The drugs and excepients were obtained from yarrow chemicals, Mumbai. Raw bananas and ground nuts were purchased from local market, Tagarapuvalasa, Visakhapatnam, Andhrapradesh.

Flow chart of work:

· Collection and extraction of gum from the seeds of delonix regia

· Preformulation studies for delonix seed powder

· Preformulation studies for drug

· Construction of standard graph for Ketoprofen in 0.1N HCl and pH 7.4 Buffer solutions

· Preparation of Core tablets

· Coating pulsatile layer for core tablet

· Preparation of Press coated tablet by coating floating layer for core compressed tablet

· Evaluation of Press coated tablets

· Optimizing the formula

Isolation of the gum from the seeds of Delonix Regia⁵

The pods of Delonix regia, were collected and these pods were soaked in the water for an overnight to separate the seeds from the pods. The seeds contain the three parts seed kernel, endosperm, and dicotyledon. The seeds (150g) were boiled in the distilled water for 3 h until the seed kernels were swelled which was then removed by the hands. The gum part was separated from the yellow dicotyledons as shown in figure 4 The gum portion was dried in an oven at 45°C for 12h and then was grounded in the mixer grinder. The resulting powder was passed through 60 # sieve. The obtained powder is store in desiccator for further use.

Fig 1: Mucilage from Delonix pods

Preparation of peanut husk powder⁶:

The peanut seeds were collected from the market. These seeds were dried in the hot air oven at 40^oC for three hours and the surface layer was removed from the seed crushing them with hands. The husk was sifted into fine powder by using a mixer grinder. Obtained peanut husk powder was passed through sieves no.100 and the fine powder was stored in a desiccator for further use. A fine and characteristic flavoured powder with light brown colour was obtained after milling the husk of peanut of plant Peanut.

Construction of standard graph for Domperidone in various solvents⁸:

To construct calibration curve of pure Domperidone 50mg drug was dissolved in 50ml solvent (0.1NHCl, pH7.4 phosphate buffer). Using this stock solution 10µg/ml, 20µg/ml, 40µg/ml, 60µg/ml, 80µg/ml and 100 µg/ml were prepared. The concentration of the drug for the above solutions were measured by UV spectrophotometer at λ_max 257nm.

Preparation of core tablets by direct compression⁹:

The inner core tablets were prepared by direct compression method. Formulattions F1 toF4 were taken to comapare the superdisintegrant activity of natural super disintegrants with synthetic super disintegrants. For F1, F2 formulations sodium starch glycolate was used as superdisintegrant and for F3, F4 dehydrated banana powder was used as superdisintegrant, As shown in Table 2 the core tablets were prepared.

Table 2: Formulation of core tablets

Ingredients	F1	F2	F3	F4
Ketoprofen	80mg	80mg	80mg	80mg
Sodium Starch Glycolate	2mg	5mg	-	-
Dehydrated Banana Powder	-	-	5mg	10mg
Microcrystalline Cellulose	20mg	20mg	20mg	20mg
PVP K 30	5mg	5mg	5mg	5mg
Magnesium Stearate	2mg	2mg	2mg	2mg

Formulations of the Pulsatile Release Tablet (PRT)⁹

The core tablets were used for the coating with pulsatile polymers by compression. The powder mixture used as an erodible outer shell contained HPMC K15 M, Ethyl cellulose, and Delonix regia as shown in tables 2 and 3. Half of the barrier layer material was weighed and transferred into an 11-mm die, then the core tablet was placed at the center, and the remaining half of the barrier layer materiel was added into the die. The tablets were compressed at a pressure of around 300 kg/cm². The same procedure was applied to all of the powders. In the F1, F2 Formulations Ethyl cellulose and HPMC K 15 M used as pulsatile polymers and in F3, F4, formulations Delonix regia seed powder is used for pulsatile drug release with increased concentrations.

Table 3: Formulation of Pulsatile relase tablets:

Ingredients	F1	F2	F3	F4
Ethyl Cellulose	240mg	-
HPMC K 15	-	240mg	-	-
Delonix Regia	-	-	120mg	240mg
PVP K 30	5mg	5mg	5mg	5mg
Magnesium Stearate	2mg	2mg	2mg	2mg
Talc	2mg	2mg	2mg	2mg

After preparing these tablets were subjected to drug relese and lag time of 1hr was obtained, to increase lag time the compressed tablets were coated with floating polymers for F1 and F2 sodium bicarbonate and citric acid were used and for F3 and F4 peanut husk powder was used.

Table 4: Formulations of the Floating-Pulsatile Release Tablet (FPRT)¹⁰

Ingredients	F1	F2	F3	F4
HPMC K 100 M	50mg	50mg	50mg	50mg
MCC	40mg	50mg	50mg	50mg
Mg Stearate	10mg	10mg	5mg	5mg
Lactose	20mg	30mg	30mg	30mg
Sodium Bicarbonate	25mg	50mg	-	-
Citric Acid	5mg	5mg	-	-
Peanut husk powder	-	-	30mg	50mg

The floating-pulsatile release tablet was designed to comprise a pulsatile release tablet with a top cover containing a floating buoyant layer. The buoyant layer in F3 and F4 included the peanut husk powder, HPMC K 100 M. The tablet flows in the gastric fluid because of light weight of peanut powder. The buoyant layer in F1 and F2 included sodium bicarbonate and citric acid, which were fabricated so that upon arrival in the stomach, carbon dioxide is liberated by the acid present in gastric fluid and entrapped in the swelled polymer. This produced an upward motion of the dosage form and maintained its floating nature. In this case floating of tablets was necessary to increase the lag time such that the tablets after 4hrs would sink to intestinne where there was a burst release of drug occurs.

Fig 2: Press coated tablets of Ketoprofen

Evaluation of Press coated tablets¹¹:

Physical Evaluation:

Thickness:

The thickness of all tablets was measured using a vernier calliper.

Hardness:

The Monsanto hardness tester was used for the determination of hardness of the tablets. The tablet was placed in contact between the plungers and the handle was pressed. The force of fracture was recorded.

weight variation:

The weight of all the tablets were taken individually on an electronic balance and the weight variation was calculated.

Friability:

For each formulation, the friability of the tablets was determined using the Roche friabilator. In this test, 20 core tablets were weighed and the initial weight of these tablets was recorded and placed in Roche friabilator tablets were subjected to the combined effect of shock abrasion by utilizing a plastic chamber which revolves at a speed of 25rpm, The tablets were removed from friabilator, dusted off the fines and reweighed and the weight was recorded. Percent friability (%F) was calculated as follows:

% Friability = (loss in weight/initial weight) x 100

The test was carried out as per Indian Pharmacopoeia (IP) 2010 guidelines.

Determination of % Drug Content:

The tablets were crushed and powder equivalent to 20 mg of ketoprofen was weighed accurately and dissolved in pH 7.4 phosphate buffer. The solutions were filtered through a membrane filter (0.45mm). The drug content was analyzed at 257nm by a UV spectrophotometer.

Disintegration Test:

To test the disintegration time of core tablets, one tablet was placed in each tube and the basket rack was positioned in a 1-liter beaker containing 7.4 phosphate buffer solution at 37°C±1°C such that the tablet remains 2.5cm below the surface of the liquid.

In Vitro Buoyancy Determination:

The floating behavior of the tablets was determined using the USP dissolution apparatus-II in 900ml of 0.1 N HCl, which was maintained at 37±0.5°C and rotated at 50rpm.

In Vitro Drug Release:

To study the the drug release from the tablet’s dissolution study was conducted by using USPII dissolution. The dissolution test was performed totally 8 hrs, the first 4hrs 0.1N hcl is used as dissolution medium. The pH 7.4 phosphate buffer is used as dissolution media for rest of 4 hrs. The dissolution test was performed using 900ml of 0.1 N HCl at 37±0.5°C and 50rpm. A sample (5ml) of the solution was withdrawn from the dissolution apparatus hourly for 8 hrs, and the samples were replaced with fresh dissolution medium. The samples were filtered through a 0.45-μm membrane filter and diluted to a suitable concentration with respective dissolution media. The absorbance of these solutions was measured at 257nm using UV spectroscopy.

RESULTS AND DISCUSSION:

Aqueous extract of Gum obtained from the seeds of delonix was shown in the figure .The pre formulation studies were done for delonix regia powder and the results were given in the following table .The FTIR graph of delonix has shown peaks at 3284.94cm^-1 represents O-H streching vibration.The band at 2923.11cm^-1 due to C-H streching of the –CH2 group.The bands due to ring streching of galactose and mannose appears at 1657 and 1636.54cm^-1.The bands in the region 1350– 1450 cm−1due to symmetrical deformation of CH2 and C-OH groups. The bands due to primary alcoholic –CH2OH stretching and -CH2 twisting vibrations appear at 1050 to 1021 cm−1. The weak bands around 870cm−1 are due to ring stretching and ring deformation of α-D-(1-4) and α-D-(1-6) linkages.

Table 5: Charecterisation of Delonix mucilage:

Parameter	Value
pH of 1%w/v^*	6.2
Viscosity of 1%w/v^*	97cps
water	Fairly soluble
Angle of repose (⁰)	28
Hausner ratio	1.26
Carrs index	15

*1%w/v mucilage was prepared by dissolving 1g mucilage in 100ml water.

Fig 3: FTIR graph of delonix mucilage

The FT-IR spectra of pure ketoprofen showed characteristic symmetric carbonyl peaks at 1693.77cm^-1 and 1655.0 cm^-1 due to dimeric carboxylic and ketonic group stretching vibra-tions, respectively.

The characteristic acid carbonyl stretching band of keto-profen was unchanged in formulations with ethylcellulosepolymer and different excipients hydroxypropylmethylcellulose (HPMC), Delonix regia as shown. These studies conﬁrm the lack of a possible interaction between the drug, the polymer and drug and delonix regia mucilage.

Fig 5: FTIR graph for mixture of HPMC and Ketoprofen

Fig 6: FTIR of Ketoprofen and delonix regia mucilage

Fig7: Calibration curve of Ketoprofen in 0.1N HCl

Fig 8: Calibration curve of Ketoprofen in pH

7.4 phosphate buffer

The standard graphs were constructed in 0.1NHCl and pH 7.4 phosphate buffer and the graphs has shown increased absorbance values with increasw concentration with r² values of 0.99.

The tablets evaluated for thickness, hardness, friability and all the values were given in the table 2

Before coating with floating polymers dissolution studies were performed and the lagtime obtained is more than 8hrs. So to improve lag time the core tablets were coated with floating polymers.Afetr coating with floating polymers the required lag time was obtained.the dissolution data was fitted into various kinetic models and all the graphs were given in figure no .Based on r² values the drug release follows order and exhibit lag time followed by burst release.The drug release from compression coated tablets occured in following steps 1)Floating of the drug in stomach 2)entry of the drug into intestine 3) Entrry of dissolution solvent into the compressedcoated tablet 4)Swelling or erosion of hydrophilic polymers 5) Breakdown of outer coating into two halves due to swelling or erosion of hydrophilic polymers used in coating.6)Burst release due to super disintegrants used in inner core tablets.

CONCLUSION:

The lag time was obtained due to outer coating of compression coated tablets with floating polymers. In pH 7.4 phosphate buffer the burst release of the drug occurred. F4 was optimised formula based on lag time (4hr) and disintegration time(30sec) of core tablets. Based on the results of current work it has been concluded that the mucilage from the delonix pods has potent application as pulsatile polymer.

Fig 9: Dissolution profile for F1 to F4

Fig 10: First order curve for F1 to F4

Table 7: r2 values of various pharmaco kinetic models

Kinetic model	R²Values
Kinetic model	F1	F2	F3	F4
Zero Order	0.8852	0.8023	0.9051	0.855
First Order	0.905	0.840	0.895	0.0.864
Higuchi	0.7752	0.7114	0.8184	0.7675
Korsemeyer Peppas	0.9407	0.878	0.8619	0.9365
n values (Korsemeyer Peppas	0.44	0.45	0.52	0.38

LIST OF ABBREVIATIONS:

RA-Rheumatoid arthritis

TNF-Tumor necrosis factor

IL-Interleukins

PDDS-Pulsatile drug delivery system

HCl-Hydrochloric acid

FTIR: Fourier infrared spectroscopy

HPMC-Hydroxypropyl methyl cellulose

UV-Ultra visible

ACKNOWLEDGEMENT:

The authors are thankful to Dr. Jagadeesh Panda Principal, Raghu College of Pharmacy for providing necessary support for the present reserch work.

REFERENCES:

1. Rizwana Rashid, Muhammad Zaman, Mahmood Ahmad, Mahtab Ahmad Khan, Muhammad Hammad Butt, Ahmad Salawi and Rai Muhammad Sarfraz, Press-Coated Aceclofenac Tablets for Pulsatile Drug Delivery: Formulation and In Vitro Evaluations. Pharmaceuticals. 2022, 15, 326. https://doi.org/10.3390/ph15030326

2. Nobuaki kikyo, Circadian Regulation of Macrophages and Osteoclasts in Rheumatoid Arthritis. Int. J. Mol. Sci. 2023, 24(15), 12307; https://doi.org/10.3390/ijms241512307

3. Patel et al., 2010, Rajput et al., 2012, Jain et al., 2012.

4. V.N.L. Sirisha, M. Namrata, B. Sruthi, I. Harika, K. Kirankumar, Y. Kiran Kumar Rao, K. Pranavi. Pulsatile Drug Delivery System-A Review. Int. J. of Pharm. Res. and All. Sci. 2012; 1(3): 13- 23.

5. Sarojini Sarangapani, And Manavalan Rajappan, Pharmacognostical and Pharmaceutical Characterisation of Delonix Regia - A Novel Matrix Forming Natural Poymer. International Journal of Pharmacy. 2012; 2(3): 564-573

6. R. Sailaja, K.L. Deepthi, Jagdeesh Panda, V.V.S. Niharika, P.V. Navya, Ch. Himabindhu, V. Sri Vihari. Formulation and Evaluation of Non-Effervescent Floating Tablets of Verapamil Hydrochloride Using Peanut Husk Powder. International Journal of Pharmaceutical Science Invention. 2021; 10(9): 1-6.

7. Characterisation of mucilage from Abelmoschus manihot Linn, Amaranthus spinosus linn and Tqlinum tiangulare(jacq) willd. Leaves for pharmaceutical excepient application. Asian Journal of Biological and Life Sciences. 2019; 8(1).

8. K. Patel, Raj K. Prasad and M. Bajpai. Enhancement of Dissolution Rate of Domperidone Using Melt Granulation Technique. Der Pharmacia Lettre. 2011, 3(2): 25-33

9. M. Bajpai, D.C.P. Singh, A. Battacharya and A. Singh. Design and In Vitro Evaluation of Compression-coated Pulsatile Release Tablets of Losartan Potassium. Indian Journal of Pharmaceutical Sciences. 2012; 74(2): 101–106.

10. Nagi Reddy Dumpa1, Suresh Bandari 1 and Michael A. Repka, Novel Gastroretentive Floating Pulsatile Drug Delivery System Produced via Hot-Melt Extrusion and Fused Deposition Modeling 3D Printing, Pharmaceutics 2020, 12, 52; doi:10.3390/pharmaceutics12010052.

11. Ashish babula rane, Surendra ganeshlal ghattani, Vinayak dinkar kadam, Avinash ramarao Tekkade, Formulation and evaluation of press coated pulsatile drug deliveryusing hydrophilic and hydrophobic polymers, Chem. Pharm. Bull. 57(11) 1213—1217 (2009).

12. Prasanthi D, Prashanti S, Meghana G, Formulation And Evaluation Of Press Coated Tablets of Lansoprazole, Int J App Pharm, Vol 11, Issue 4, 2019, 49-56.

13. Nazir SU, Khan RU, Amer M, Nadeem M, Sultana M, Naeem Qaisar M, et al. Formulation development, evaluation and anti-inflammatory effects of ketoprofen cream on rheumatoid arthritis patients. Int J Curr Life Sci 2013; 3:132-6.

14. Khadabadi S, Chishti N, Khan M, Tadvee A. Formulation and evaluation of press coated tablet of Ketoprofen a chrono-therapeutic approach. Int J Pharm Pharm Sci 2013; 5:733-40.

15. Mohammed S, Bhargavi C, Harish G, Riyajune A, Yasmeen M, Muralikrishna C, et al. Effect of different disintegrants on ciprofloxacin conventional tablets. Indian J Res Pharm Biotechnol 2013; 1:281-7.

16. Santanu G and Barik B. A comparative study of the pharmacokinetics of conventional and sustained-release tablet formulations of aceclofenac in healthy male subjects. Tropical Journal of Pharmaceutical Research, 9(4), 2010, 395-399.

17. Maha A, et al. In-vitro release, Thermodynamics and Pharmacodynamic Studies of Aceclofenac Transdermal Eudragit Patches. Drug Invention Today, 1(1), 2009, 16-22.

18. Kale VV, Kasliwal RH, Avari JG. Attempt to design continuous dissolution-absorption system using everted intestine segment for in vitro absorption studies of slow drug release formulations. Dissolution Technologies, 14, 2007, 31–36.

19. Mariappan TT, Singh S. Evidence of effluxmediated and saturable absorption of rifampicin in rat intestine using the ligated loop and everted gut sac techniques. Mol Pharm, 1, 2004, 363–367.

20. Bhargava A, et al. Oral sustained release dosage form: an opportunity to prolong the release of drug. IntJ ARPB, 3, 2013, 7-14.

21. Fukui E, Miyamura N, Uemura K, Kobayashi M, Preparation of enteric coated timed-release press-coated tablets and evaluation of their function by in-vitro/in-vivo test for colon targeting, Int. J. Pharm, 20, 2000, 7–15.

22. Lin SY, Lin KH, Li M, Micronized ethyl cellulose used for designing a directly compressed time-controlled disintegration tablets, J. Control. Release, 703, 2001, 321328.

23. Leaokittiul D, Lindsay B, O’Mahony B, Stanley A, Stevens HNE, Gamma scintigraphic study of time-delayed formulations in human volunteers, AAPS J, 7(S2), 2005, W5096.

24. Leaokittikul D, Eccleston GM, Stevens HNE, Effect of coating barrier formula on drug release from a pulsatile press-coated tablet, J. Pharm. Pharmacol, 53, 2001, S77.

25. Lin SY, Li MJ, Lin KH, Hydrophilic excipients modulate the time-controlled disintegrating press coated tablets, AAPS PharmSciTech, 5(4), 2004.

26. Indrajeet S. Patil, Omkar A. Patil, Girish Chandra R. Mandake, Manoj M. Nitalikar. Development and Evaluation of Telmisartan Pulsatile Drug Delivery by using Response Surface Methodology. Asian J. Pharm. Res. 2018; 8(4): 205-214.

27. Sujan Neupane, Pramod Kharel. Pulsatile Drug Delivery System: A Review. Asian Journal of Pharmaceutical Research. 2024; 14(3):309-4.

28. Rupali V. Khankari, Sneha M. Umale. A Review on Pulsatile Drug Delivery System. Asian J. Pharm. Tech. 2020; 10(2):121-124.

29. M. Sukanya, V. Saikishore, P.Y. Shanmukha, K. Srikanth. Novel Approaches for Pulsatile Drug Delivery System. Research J. Pharma. Dosage Forms and Tech. 2012; 4(4): 197-201.

30. Pragya Baghel, Amit Roy, Shashikant Chandrakar, Sanjib Bahadur. Pulsatile Drug Delivery System: A Promising Delivery System. Research J. Pharma. Dosage Forms and Tech. 2013; 5(3): 111-114.

31. Jinshad K, Krishna Pillai M. Enteric coated Effervescent micro bead Drug Delivery System: A better approach for pulsatile drug delivery to Intestine. Research J. Pharm. and Tech 2019; 12(8): 4007-4012.

32. Amol V. Sawale, Vidhi Sunil Jajoo, Anuj A. Deshmukh. Formulation and Evaluation of Pulsatile Drug Delivery System for the Treatment of Arthritic Pain. Research Journal of Science and Technology. 2024; 16(3):193-2.

33. Amritha R., Sivakumar R., Y. Haribabu. A Review on Pulsatile Drug Delivery System: Drug Scheduling based on Biological Rhythm. Research Journal of Pharmacy and Technology. 2022; 15(3):1359-4.

34. Rohini Kharwade, Haripriya Nair, Dipali Masurkar, Ajay Pise, Sachin More, Shilpa Pise. Formulation and Evaluation of Chronomodulated Pulsatile Drug Delivery System for Nocturnal Hyperacidity. Research Journal of Pharmacy and Technology. 2022; 15(4):1449-4.

35. Prasanta Kumar Mohapatra, Janki Manohar, Pankaj Singh Patel, Mandeep Kumar Gupta, Bibhuti Prasad Rath. Physicochemical Characterization of Bi-Layered Terbutaline Sulfate Tablets for Chronotherapeutic Pulsatile Drug Delivery Design Based on Natural and Synthetic Polymer using Direct Compression Technique. Research Journal of Pharmacy and Technology. 2021; 14(4):1867-4.

36. Pasam Jyothirmayi, G. Devalarao, Mandava Venkata Basaveswara Rao. Optimization of Pulsatile Compression Coated Floated tablets of Tramadol HCL for Chronopharmacotherapy of Rheumatoid Arthritic pain using 23 Factorial Design. Research J. Pharm. and Tech. 2020; 13(12):5823-5830.

Property	Method
Solubility	Excess amount of drug added to the solvent and solubility observed
pH	1g powder dissolved in 100ml distilled water and then pH measured by Digital pH meter
Flow properties	Angle of repose was determined by funnel method, Bulk density and tapped density were measured by using measuring cylinder
Viscosity	The viscosity of 1% (w/v) drug solution was measured using Brookfield DV-E Viscometer.
FTIR	FTIR graphs were done for pure Domperidone, Delonix regia and in cobination with HPMC K15M
Phytochemical constituents
Carbohydrates	Molisch test (to the 100 mg dried mucilage powder add molisch's reagent and add conc. H2SO4 on the side of a test tube)
Proteins	Add Ninhydrin reagent to the aqueous extract and observe the colour of ppt
Ruthenium test	Take a small quantity of dried mucilage powder, mount it on a slide with ruthenium red solution, and observe it under microscope.
Glycosides	Keller kilani test (A solution of 0.5 ml with glacial acetic acid and 2-3 drops of ferric chloride was mixed with 2ml of extract. then 1 ml of concentrated H₂SO4, was added along the walls of the test tube. The appearance of deep blue colour at the junction of two liquids indicated the presence of cardiac glycosides.)
Alkaloids	Wagners test (to 100mg of powder 2ml of wagners reagent was added, appearance of reddish-brown precipitate in test tube indicated the presence of alkaloids)

Formulation	Thickness	Hardness Kg/cm²	Friability (%)	Lag time before addition of floating layer	Lag time after addition of floating layer	Disintegration time in pH 7.4Phosphate buffer (core tablets)
F1	1.82mm	3.2±0.11	0.56±0.12	1hr	3hrs	2min
F2	2.82mm	3.1±0.13	0.57±0.11	1.5hr	2hrs	1min
F3	3.02mm	3.5±0.17	0.60±0.19	2hr	4hr	1.5min
F4	2.5mm	3.4±0.15	0.61±0.15	3hr	6hr	30sec