Int J Pharm Sci Nanotech Vol 10; Issue 2 March April 2017...

3688 Int J Pharm Sci Nanotech Vol 10; Issue 2 March April 2017

Research Paper

Screening, Fabrication and Evaluation of Pharmaceutical Dosage Forms using P-Glycoprotein Modulators Vedamurthy Joshi1,2*, Shivakumar Inamdar3, Ankit Acharya1 and Rajesh Kowti1 1Sri Adichunchanagiri College of Pharmacy, BG Nagar, 2Research scholar, PRIST University, Thanjavur, Tamil Nadu, 3HKES’ College of Pharmacy, Gulbarga, Karnataka.

Received February 18, 2017; accepted March 6, 2017

ABSTRACT

In this project, in vitro absorption enhancement activity of P-gp substrates Fexofenadine (Fx) and Ciprofloxacin (Cp) were evaluated in everted rat gut sac model and Caco-2 cell lines. Verapamil was used as P-gp inhibitor. Piper betel, Trachyspermum ammi, Plumbago zeylanica, Trikatu, Moringaoleifera, Murraya koenigii, Ferulafoitida Zingiber officinale, Cheilocostus speciosus, Capsicum frutescens Operculina turpethum Holarrhena antidysenterica Mesuaferrea, Tinospora cordifolia, and Picrorhiza kurroa, were selected and extracted with 99% alcohol and fresh juices of Citrus limon, Punica granatum seeds were also studied. In-vitro studies depicted that Fexofenadine and Ciprofloxacin absorption was increased greater than 20% in the presence of Operculinaturpethum, Capsicum frutescens, Holarrhena Antidysenterica, Tinospora cordifolia, Trikatu, Trachyspermum ammi, Plumbago zeylanica. The flux of the ciprofloxacin transport was in the range of 9-23 mcg/min and Papp

2.6 × 10-5

cm/sec to 4.1 × 10-5

cm/sec whereas Fexofenadine flux was in the range of

2-7.7 mcg/min and Papp 4.16 × 10–6 cm/sec to 1.62 ×

10-5 cm/sec. In vitro antimicrobial activity of ciprofloxacin on selected microbes in presence of extracts also depicted synergistic activity. Histological studies revealed that there is no significant variation observed in the isolated sac in presence of the extracts. CaCo2 cell lines studies showed that, formulation enhanced the absorption of fexofenadine greater than 50%. Tablets were prepared and evaluated using the plant extracts which yielded >20% absorption enhancement of the substrates. In conclusion, tablet formulation containing the alcoholic extracts of Trachyspermum ammi, Plumbago zylanicum, Capsicum frutescens, Operculina turpethum, Holarrhena Antidysenterica, Tinospora cordifolia and Trikatu can act as an absorption enhancer for fexofenadine and ciprofloxacin. The mechanism of action of these herbs could be due to P-gp inhibition. Further clinical studies are needed to prove its efficacy in humans.

KEYWORDS: Ciprofloxacin; CaCo2 cell; Fexofenadine; P-gp inhibitor.

Introduction

Herbs and their products commonly used for treatment of a number of pathological conditions. According to a UNAIDS survey, one-third of the people in developed countries and over 80% of people from Africa depend on herbal medicines to treat common ailments such as cold, cough, inflammatory disorders, heart disease, diabetes, and central nervous system diseases (Awortwe et al., 2014). There is also much evidence that large number of people use herbal medicines along with chemical drugs. Although herbal medicine is natural, it contains a complex mixture of several active and inactive chemical constituents and involved directly and indirectly in drug metabolism and transport. To confirm patient safety and efficacy, large number of research is being conducted to determine possible harmful interactions between these herbal products and prescribed medications (Hellum et al., 2007).

During past three decades, a great deal of the work has been published on the drug absorption process, however much remained unknown. Despite many drug candidates show great pharmacological activity intravenously, fails to absorb orally. The reasons for the poor bioavailability may be due to issues with solubility and permeability. Solubility would be improved by various methods including the micronization, nanonization, complexation, pH change, co-solvent addition, solubulization with surfactants. However, permeability can be improved by the use of permeability enhancers. These agents function by modifying the a) permeant solubility, b) permeant mucus diffusivity, c) protecting the permeant from pH, enzymes present in lumen / brush border, d) preventing the attachment of the permeant from nonspecific binding site b) reducing the activity of or compete for uptake by efflux pumps in the apical intestinal epithelium and e) improving the

International Journal of Pharmaceutical Sciences and Nanotechnology

Volume 10Issue 2 March – April 2017

MS ID: IJPSN-2-18-17-JOSHI

3688

Joshi et al: Screening, Fabrication and Evaluation of Pharmaceutical Dosage Forms using P-Glycoprotein Modulators 3689

permeant permeability through gastro intestinal epithelium (LeCluyse et al., 1997)

It was identified that drug efflux pumps like P-glycoprotein (P-gp) are playing major role in altering the pharmacokinetics of various drugs and particularly associated with poor bioavailability in co-ordination with gut wall metabolism. Though much work has been pursued in the process of establishing the role of P-gp in multidrug resistance (MDR) in cancer cells, recently it is gaining importance in absorption enhancement due to its selective distribution at the site of drug absorption (Del et al., 2009).

Permeability-glycoprotein (P-gp) is an energy dependent transporter protein located in the apical membrane of intestinal mucosal cells which is believed to efflux the absorbed drug back into the gut. P-gp is associated with the multidrug resistance in tumor cell, but also expressed in a variety of normal human tissues including liver, brain, kidney, gastrointestinal tract, intestine, adrenal gland, blood-brain barrier, placenta, blood-testis, blood-ovarian barriers and in various tumors (Wessler et al., 2013).

P-gp is able to recognize a variety of structurally and pharmacologically unrelated neutral and positively charged hydrophobic compounds (substrates) and appears to be the efflux transporter which may perhaps have the most significant role in limiting the intestinal absorption of many drugs. Once a P-gp substrate drug crosses the apical membrane of the enterocyte, a portion will be effluxed back to the intestinal lumen by P-gp and another part of the drug could be subjected to intestinal first pass metabolism by cytochrome P-450 (CYP) enzymes, predominantly CYP3A, which is relatively abundant in the intestinal mucosa. In addition, enzymatic processes such as phase II conjugation may also occur. Consequently, the net amount of drug absorbed into the mesenteric blood circulation is the difference between the amount absorbed by the influx process and the sum of the amount extruded by efflux transport (P- gp) together with the amount metabolized by enzymes. This fraction of drug absorbed enters into the liver via the portal vein and may be subjected to hepatic first pass elimination. The molecule may be metabolized by hepatic enzymes, transported from the hepatocyte into the bile by P-gp or continue to the systemic circulation by the hepatic vein distributing in the organism. The unchanged drug (as well as the resulting metabolites) secreted into the bile will be secreted into the duodenum. In the intestine, the compound could be reabsorbed or eliminated in the faeces (Eichhorn et al., 2012).

Interestingly several studies have demonstrated that the P-transport can be inhibited by certain agents called as P-gp inhibitors. Example includes verapamil and cyclosporine which can improve the bioavailability of a number of substrates molecules. Co-administration of certain dietary substances like piperin, major constituent from pepper, showed to inhibit the p-gp effectively in human intestinal cells (Bhardwaj et al., 2002). Natural compounds like zinger, liquorice, black cumin etc are known to increase the bioavailability of drugs probably

by inhibiting P-gp (Drabu et al., 2011). Recent studies revealed that co-administration of P-gp inhibitors may possess many advantages such as a) Bioavailability improvement b) Low dose administration c) Brain targeting using p-gp inhibitors d) Cancer therapy e) Antimicrobial efficacy improvement f) Minimizing multidrug resistance g) Contributes in development of new drug molecules and pharmacokinetic research etc. Studies also showed that herbs and dietary supplements possess p-gp modulation activity and may interfere in pharmacokinetic properties of the drug (Li et al., 2011). In addition natural compounds are safer than the synthetic one with less toxicity and are more biocompatible. Hence, this research work have been designed to screen 17 alcoholic herbal extracts / juices for in-vitro absorption enhancement activity using ciprofloxacin and fexofenadine as model substrates and Verapamil as standard P-gp inhibitor using everted gut sac model. Plants were chosen based on the folklore use, Ayurvedic literature and chemical constituents of the plant.

Materials and Methods

Materials

Verapamil and Ciprofloxacin were purchased from Yarrow chemicals, Mumbai. Fexofenadine was gifted by Microlabs, Bangalore. Piper betle (Betle leaf), Trachyspermum ammi (Ajwain seeds) Plumbago zeylanica (Chitrak), Trikatu (Equal mixture of Black Pepper (Piper nigrum), Indian Long Pepper (Piper longum) and Ginger (Zingiber officinale), Moringa oleifera (Drum stick) Murraya koenigii (Curry leaves), Ferula foitida (Asafoitida), Zingiber officinale (Ginger), Cheilocostus speciosus (Crepe ginger), Capsicum frutescens (Capsicum), Operculina turpethum (Ttrivrit), Holarrhena antidysenterica (Kutaja), Mesua ferrea (Nagakesara) Tinospora cordifolia (Guduchi), Picrorhiza kurroa (Kutaki), Citrus limon (Lemon), Punica granatum (Pomagrante seeds) were purchased from local market of Mysore, India. All other reagents used were analytical grade. Milli Q water (HPLC grade) was used throughout experiment.

Methods Sample preparation All plants were purchased from the local market from

Mysore in dry form (except Citrus limon and Punica granatum). Dried powders were extracted with 99% ethanol using a Soxhlet extractor apparatus. The 100g power was put in soxhlet thimble and put into a soxhlet thimble tube. 1000 mL of ethanol was added to a soxhlet flask, and then extracted at 60°C until the extract was not clear or about 8 hour. The ethanol was removed under pressure using a rotator evaporator (Buchi Rotavapor R-300, Singapore). Semi solid concentrate was collected and stored in an airtight plastic container for further use.

Phytochemical Evaluation Qualitative estimation Phytoconstituents such as carbohydrates, proteins,

Alkaloids, glycosides, terpenes, steroids, flavanoids,


tannins and saponins were determined using crude extracts. Methods for phytochemical evaluation were employed as per (Kokate, 2015)

Screening of P-Glycoprotein (Pgp) Modulation Activity Composition and preparation of Krebs-Ringer’s solution (KRS) Krebs-Ringer’s solution of composition (g/L): NaCl

6.9 g, KCl 0.35 g, MgSO4.7H2O 0.29 g, KH2PO4 0.16 g, NaHCO3 2.1 g, CaCl2 0.28 g and 0.2% glucose (pH 7.3). Except CaCl2, all ingredients were dissolved in 600 mL of purified water in 1 litre volumetric flask. In a separate 250 mL beaker containing 100 mL purified water, CaCl2 was dissolved. The CaCl2 solution was added to the above solution and made up to 1000 mL using purified water.

Everted gut sac preparation Male albino rats weighing 170-220g on standard feed

and tap water were used throughout study and were maintained in standard lab conditions (22 ± 3oC) with 12 hour light and 12 hour dark exposure. Animals were fasted overnight before scarification. After being fasted overnight with free access to water, the rats were anesthetized by chloroform. Their abdomen opened with a middle incision. The underlying mesenterium and fat were removed, and the sacs were carefully everted with a glass rod (2.5mm diameter). A segment, 3-4 cm distant to the ileum excised, and it was removed of the mesenteric attachments carefully without damaging the intestinal architecture. The intestinal segment was transferred to a petri dish containing Kreb's medium. The tissue medium was continuously bubbled with a gas mixture of 95% O2

and 5% CO2. Isolated segment was tied to the assembly and studied further.

Sampling 0.2 mL - 1 mL from the serosal fluid was withdrawn

at every 5 min intervals in 10 mL volumetric flask using glass syringe. Serosal fluid loss was compensated by replacing respective volume of blank KRS solution into the serosal fluid. Volume of the sample withdrawn was made up to 10 mL using blank KRS absorption was estimated.

Permeability determination The apparent permeability (Papp) value was calculated

according to the following equation: Papp = (dQ/dt) × 1/ (A*Co)

Papp is apparent permeability co-efficient; dQ/dt the cumulative amount of drug (Q) appearing in the acceptor (serosal) compartment as a function of time, A is surface area of the intestine (cm2) Co is the initial concentration of drug in the donor compartment (μg/mL). After each experiment, intestinal segment is cut open and surface area is determined by measuring length and breadth.

Caco-2 cell line studies This model is widely accepted by research scientists.

Caco-2 cell line is a continuous line of Human epithelial colorectal cells derived and isolated from a colon adenocarcinoma. When cultivated under particular conditions, these cells have the ability to spontaneously

differentiate into intestinal cells to form a functional polarized epithelium mimicking the gastrointestinal barrier (including the presence of microvilli, tight junctions, specific transporters, metabolic enzymes, etc.). The Caco-2 assay allows the evaluation of the intestinal permeability of a chemical compound, i.e. its ability to cross the intestinal barrier in order to reach the bloodstream and circulate throughout the body.

Caco-2 cells are grown in trans well plates in DMEM-based culture medium (Dulbecco’s modified eagle medium) for 18-21 days until a constant trans epithelial electrical resistance (TEER) is achieved, indicating that tight junctions have formed in the monolayer. Before the initiation of permeation experiments, the cell membranes are equilibrated in transport buffer. Hank’s Balanced Salt Solution (HBSS) of different formulations and simulated intestinal fluids (SIFs) are added to the apical part of the trans well as the transport buffer, and in all wells, HBSS buffered with 10 mm TEER of the monolayer in each well is measured as the initial value, and permeation studies are initiated by replacing transport buffer in the apical chamber of the trans well with the drug dissolved in the same transport buffer. The drug concentration changes over time are analyzed with UV Method / HPLC method to calculate the Papp using the following equation and expressed in cm/s

Calculation of permeability The apparent permeability (Papp) value was calculated

according to the following equation: Papp = (dc × v)/ (dt × A × Co)

Where Papp Apparent permeability coefficient (cm/s), dC/dt-flux across monolayer (mM/sec), V-volume of the chamber (ml), A- surface area (sq cm), Co- initial drug concentration (mM)

Histological studies A small piece of the everted intestine was fixed with

10% formalin for 24 hour and then dehydrated with a graded alcohol series and fixed by using paraffin wax. It was stained by H&E stain and specimens were observed and photo micro-graphed under a co-focal microscope.

Antimicrobial efficiency of ciprofloxacin Antimicrobial efficacy of the ciprofloxacin was

determined in presence and absence of selected herbal extracts against ATCC strains of Staphylococcus aureus, Klebsiella pneumonia and clinical isolates of Pseudomonas aeruginosa and E.Coli by cup plate diffusion method.

Tablet formulation The selected ingredients were passed through sieve

no 40 separately and weighed. Weighed ingredients were transferred into mortar and mixed for 15 minutes using pestle. Binder solution, 5 % starch paste was added to make damp mass and passed through sieve number 10 to produce granules. The granules were dried in the hot air oven for 1 hour and passed through sieve number 10. Lubricant, Magnesium stearate and talc were added. The granules were evaluated for its flow properties such as, Bulk density, tapped density, percentage compressibility, and angle of repose. The tablets were compressed into


tablets and were evaluated for general appearance, hardness, friability, drug content, weight uniformity, disintegration and in vitro dissolution testing.

Stability studies Tablets were packed in a bulk packing and kept at

40oC/ 75% RH, and 250C/ 60% RH for two month. Tablets were analyzed for its parameters at 15 days interval.

FTIR Studies FTIR studies were carried out to determine the drug

excipient interaction. In this study, IR spectra of the samples were taken using the FTIR instrument and percentage transmittance was recorded.

Results and Discussion

It is challenging to understand the mechanism of absorption of food as well as the drug as it keeps varing from subject to subject’s condition. Certain challenges of the current situation such as multi drug resistance, antibiotic resistance, increasing drug tolerance, poor drug absorption in spite of being very soluble, triggers a scientist to think towards certain mechanism adopted by the cell in the course of its evolution. One of such interesting mechanism includes the presence of P-glycoprotein (Del et al., 2009). Studies revealed that the P-gp is responsible for the lowering the bioavailability of many drugs by effluxing the drug back to the lumen. Certain agents like verapamil inhibits the P-gp thereby ceasing the drug efflux consequently improving the bioavailability of the drug. Hence, P-gp inhibitors are much focused to improve the bioavailability of the P-gp substrates. Studies also revealed that natural herbs such as pepper, curcumin etc, also possess such P-gp inhibition activity. Hence, this project was undertaken to screen 17 herbs / herbal extracts for its absorption enhancement activity and formulate the suitable candidates into tablet dosage form, which, not only reduce to dose but also improves bioavailability.

In this work, Fexofenadine and Ciprofloxacin drugs were chosen as the P-gp substrates and Verapamil as P-gp inhibitor. Few studies in MDCKI and MDCKI-MDR1 cells indicate that ciprofloxacin is a substrate of P-gp but data from MDCKII, MDCKII-MDR1, LLC-PK1 and L-MDR1 cells indicate that ciprofloxacin is not a substrate of P-gp (Park MS et al., 2014). Hence, ciprofloxacin was selected to seek more information about its absorption. Calibration curves of Ciprofloxacin, Fexofenadine and Verapamil were prepared in Milli Q water (HPLC grade water) as well as in Krebs’ Ringer solution in 278nm, 220nm and 276nm using UV Visible spectrometry. Linearity was observed in all graphs indicating the samples obey Beer Lambert range. As the absorption maxima of Ciprofloxacin and Verapamil were similar, titrimetric method was followed to estimate Verapamil when used in combination with substrates (Joshi et al., 2016).

Photochemical evaluation of herbal extracts was summarized in Table 1. All ethanolic extracts possess carbohydrate except Ferula foitida, Capsicum frutescens, and Operculina turpethum. Alkaloid was present in all plant extracts except Ferula foitida and Punica granatum. Terpenoids and steroids are present in Piper betle, Trachyspermum ammi, Plumbago zeylanica, Trikatu, M. oleifera, Murraya koenigii, Capsicum frutescens, Operculina turpethum, Holarrhena Antidysenterica, Tinospora cordifolia and Picrorhiza kurroa. Tannins are present in all except Plumbago zeylanica, Moringa oleifera and Punica granatum. All the phytochemical investigations were carried out in triplicate.

All the extracts were tacky, dark and possessed characteristic intense odour. Initially, absorption studies with ciprofloxacin were carried out in two concentrations of 1.5mM and 3.0 mM in mucosal part in presence and absence of herbal extracts/Verapamil (Joshi et al., 2016).

TABLE 1

Observation of phytochemical constituents present in various plant extracts.

S. No. Scientific Name Carbohydrates Proteins and Amino-acids Alkaloids Glycosides

Terpenoids and Steroids Flavonoids Tannins Saponins

1. P. betle + - + + + - + - 2. T. ammi + + + + + + + + 3. P. zeylanica + + + + + - - - 4. Trikatu + + + + + + + + 5. M. oleifera + - + + + + - - 6. M. koenigii + + + + + - + - 7. F. foitida - - - - - - + - 8. Z. officinale + + + + - + + + 9. C. speciosus + + + + - - + -

10. C. frutescens - - + + + - + 11. O. turpethum - - + + + + + - 12. H. Antidysenterica + + + + + + + + 13. M. ferrea + + + + - + + + 14. T. cordifolia + + + + + + + - 15. P. kurroa + + + + + + + - 16. C. limon + + + - - - + + 17. P. granatum + + - + - + - -

(+) indicates presence of Phytochemical, (-) indicates absence of Phytochemical constituents


The results indicated that the absorption was concentration dependent (10.124 to 15.245 × 10-5 cm/sec). Later the studies were carried out in 2000mcg/mL concentrations in mucosal side. Results showed in the Table 2, flux (dQ/dt) was in the range of 9-23 mcg/min and Papp 2.6 × 10-5 cm/sec to 4.1 × 10-5 cm/sec. Significant increased absorption of Ciprofloxacin was noticed in the following order Operculina turpethum> Capsicum frutescens >Holarrhena Antidysenterica >Verapamil >Tinospora cordifolia> Trikatu>Trachyspermum ammi>Plumbago zeylanica> Citrus limon>Murraya koenigii>Punica granatum>Zingiber officinale>Mesua ferrea> Cheilocostus speciosus> Piper betle. The in-vitro absorption was found unaltered in case of Moringa oleifera and Ferula foitida. As data showed in Table 2, extracts showing > 20 % increase in the absorption was considered to formulate it into tablets. Antimicrobial study was also carried out with Ciprofloxacin efficiency. This study, however, may not directly relate to the P-gp absorption, but explains the added advantage of combining the extracts into the formulation for better antimicrobial activity of ciprofloxacin respectively. The extracts didnot show any antimicrobial activity of their own, but showed synergistic activity in presence of ciprofloxacin. From the Figure 7, following extracts depicted greater activity against microbes. Trikatu> Operculina turpethum>Piper betle> Murrya Koenigii. It’s our intuition that, herbal extracts, though hardly published, possess the similar activity against microbes

as it shows on eukaryotic cells and may vary in their intensities. Hence, this antimicrobial activity can adds value and signifies the P-gp activity. In case of Fexofenadine substrate, the activity was much clear and depicted in the Table 3. Increase in the absorption of the Fexofenadine in descending order as shown in the Table 3. Trikatu > Operculina turpethum > Verapamil > Capsicum frutescens > Piper betle> Trachyspermum ammi > Zingiber officinale > Tinospora cordifolia > Plumbago zeylanica >Picrorhiza kurroa > Citrus limon > Holarrhena antidysenterica > Mesua ferrea > Ferula foitida > Murraya koenigii > Moringa oleifera > Punica granatum > Cheilocostus speciosus. As per the data depicted in Table 3, flux (dQ/dt) was in the range of 2-7.7 mcg/min and Papp 4.16 × 10–6 cm/sec to 1.62 × 10–5 cm/sec. High Papp values were observed in Ciprofloxacin than Fexofenadine. The flux (dQ/dt) values were also greater than in case of Ciprofloxacin than Fexofenadine. The reason would be due to the more channels available for Ciprofloxacin absorption than Fexofenadine (P-gp alone). Further specific experiments need to confirm the same. When Papp values of both the substrates were compared the following herbal extracts remained constant in the first line where greater than 20 % activity was observed. The extracts include Operculina turpethum, Trikatu, Capsicum frutescens, Holarrhena Antidysenterica, Plumbago zeylanica. Hence, these were selected for the tablet preparation with 25 mg as dose.

TABLE 2

Table indicating the effect of herbal extract on the in-vitro absorption of ciprofloxacin.

S. No. Scientific Name Common name Effect on ciprofloxacin dQ/dt Papp % Change

1 Ciprofloxacin - - 12.995 2.61996 E-05 0 2 Ciprofloxacin + Verapamil - Increased 18.495 3.66964 E-05 28.60450514 3 Piper betle Betle leaf Increased 14.537 2.69204 E-05 2.677428231 4 Trachyspermum ammi Ajwain Increased 18.302 3.45582 E-05 24.18693885 5 Plumbago zeylanica Chitrak Increased 20.078 3.38013 E-05 22.48948858 6 Trikatu Trikatu Increased 18.495 3.58291 E-05 26.87628086 7 Moringa oleifera Drum stick Not altered 10.84 2.41071E-05 -8.679808841 8 Murraya koenigii Curry leaves Increased 16.93 3.30664 E-05 20.76672446 9 Ferula foitida Asafoitida Not altered 9.74 2.24424 E-05 -16.74152977

10 Zingiber officinale Ginger Increased 16.579 3.17605 E-05 17.50896003 11 Cheilocostus speciosus Crepe ginger Increased 18.302 2.85969 E-05 8.383007674 12 Capsicum frutescens Capsicum Increased 21.034 3.76953 E-05 30.49645812 13 Operculina turpethum Ttrivrit Increased 21.304 4.16094 E-05 37.03439003 14 Holarrhena Antidysenterica Kutaja Increased 22.594 3.69183 E-05 29.03357871 15 Mesua ferrea Nagakesara Not altered 19.774 3.08969 E-05 15.20308519 16 Tinospora cordifolia Guduchi Not altered 17.828 3.59435 E-05 27.10904196 17 Picrorhiza kurroa Kutaki Increased 17.34 3.17815 E-05 17.56343714 18 Citrus limon Lemon Not altered 15.398 3.3329 E-05 21.39100072 19 Punica granatum Pomagrante seeds Not altered 18.785 3.25112 E-05 19.41375025

TABLE 3

The effect of herbal extract on the in-vitro absorption of fexofenadine.

S. No. Scientific Name Common name Effect on

Fexofenadine dQ/dt Papp % Change

1 Fexofenadine - - 2.0645 4.16233E-06 - 2 Fexofenadine + Verapamil - Increased 6.6667 1.32275E-05 68.53277836 3 Piper betle Betle leaf Increased 4.5 7.26E-06 42.65232975 4 Trachyspermum ammi Ajwain Increased 3.2366 5.94956E-06 30.03965277 5 Plumbago zeylanica Chitrak Increased 3.3871 5.23994E-06 20.56528418

TABLE 3 Contd…


S. No. Scientific Name Common name Effect on

Fexofenadine dQ/dt Papp % Change

6 Trikatu Trikatu Increased 7.7742 1.61962E-05 74.3006291 7 Moringa oleifera Drum stick Not altered 2.3656 4.23941E-06 1.818181818 8 Murraya koenigii Curry leaves Not altered 2.2043 4.30528E-06 3.320220299 9 Ferula foitida Asafoitida Not altered 2.6452 4.45313E-06 6.530291109

10 Zingiber officinale Ginger Increased 3.0968 5.93252E-06 29.83870968 11 Cheilocostus speciosus Crepe ginger Not altered 2.6344 3.74206E-06 -11.23107307 12 Capsicum frutescens Capsicum Increased 5.6882 1.01939E-05 59.16824197 13 Operculina turpethum Ttrivrit Increased 7.086 1.38399E-05 69.92510647 14 Holarrhena Antidysenterica Kutaja Not altered 2.828 4.62084E-06 9.92272783 15 Mesua ferrea Nagakesara Not altered 2.914 4.55309E-06 8.58231163 16 Tinospora cordifolia Guduchi Increased 2.6129 5.26795E-06 20.98765432 17 Picrorhiza kurroa Kutaki Increased 2.8065 5.14379E-06 19.08045977 18 Citrus limon Lemon Not altered 2.9032 4.88759E-06 14.83870968 19 Punica granatum Pomagrante seeds Not altered 1.7634 3.80052E-06 -9.520062943

Fig.1. Cumulative amount of drug release during the in-vitro absorption studies with ciprofloxacin.

Fig. 2. Bar graph indicating the Papp of ciprofloxacin absorption in presence of herbal extracts.


Fig. 3. Cumulative amount of drug release during the in-vitro absorption studies with fexofenadine.

Fig. 4. Bar graph indicating the Papp of Fexofenadine absorption in presence of herbal extracts.

In order to confirm the same, selected samples were out sourced for everted sac technique and CaCo2 cell line studies and similar results were obtained (data not produced). CaCo2 cell line studies were conducted for three samples, plane fexofenadine, fexofenadine+ verapamil, Fexofenadine + selected extracts. Results indicated that the permeability increased greater than the positive control verapamil (data not produced). This confirms that, the selected herbal extracts could be permeation enhancers for most of the drugs irrespective of the P-gp.

Histological studies (Figure 5) depicted that, there is no significant change observed in both mucosal side as well as serosal side. However, at higher concentration of herbal extracts, it was observed that the epithelium was elongated with central nucleus. Reason for the change was remained un-understood. FTIR studies showed that there was no interaction among the excipients (Figure 8).

Granules prepared with the above extracts, initially were tacky, and hence mixed with lactose monohydrate, filler, along with flow property enhancers such as magnesium stearate and talc. Small concentration of starch (5% w/v) solution was used as binder. Flow of the granules was good (Table 5) showing Carr’s index 14-16, angle repose was 23o. Granules were compressed into tablets, which also show disintegration and dissolution as per IP (Figure 9). Stability studies also showed no significant change in the formulation (table 7).

TABLE 4

Tablet formulation formula.

S. No. Ingredient Qty per tablet 1. Ciprofloxacin 250 mg2. Trachyspermum ammi 25 mg3. Trikatu 25 mg4. Plumbago zylanicum 25 mg5. Capsicum frutescens 25 mg6. Operculina turpethum 25 mg

TABLE 4 Contd…

Joshi et al

S. No. 7.

8. 9.

10. 11. 12.

TABLE 5

Results of t

Bulk densTapped dCarr’s indHaunser’sAngle of r

(d)

l: Screening, Fa

IngredienHolarrhena AntidysentericaTinospora cordiLactose monohyStarch 5 % Magnesium steTalc

the flow properti

sity gm/ml (meaensity gm/ml (mdex (mean ± sd) s ratio (mean ± repose (°)

(a) Normal Int

) Verapamil + Ci

(g) T. amm

(j) Moringa ol

(m) Zingiber of

abrication and

nt Qty p

ca 2

difolia 2ydrate 10

earate

ies of the granul

n ± sd) 0mean ± sd) 0

1sd) 1

2

testine

iprofloxacin

mi

leifera

fficinale

Evaluation of P

per tablet 25 mg

25 mg 00 mg QS 1 % 1%

les.

.526 ± 0.009

.696 ± 0.019 14.81 ± 1.38 1.46 ± 0.025 23.6 ± 0.93

(e)

Pharmaceutica

T

T

T

S

(b) Ciproflox

) Verapamil + Fe

(h) Plumbago ze

(k) Murraya k

(n) C. specio

l Dosage Form

TABLE 6

Tablet evaluation

Weight VariatioHardness meanFriability meanDrug contain mThickness meanDisintegration t

TABLE 7

hort-term stabil

S. No. Days

1 152 303 454 60

xacin

exofenadine

eylanica

koenigii

osus

ms using P-Glyc

n results.

on mean ± sd (g) n ± sd (kg/cm2)

n ± sd (%) ean ± sd (%)

n ± sd (mm) time mean ± sd (

lity studies resul

Drug content 40oC/ 75% RH

98.5697.5597.2595.6

coprotein Modu

0.721±4.13±10.19±098.58±3.01± 0

(min) 15 ± 0.

lts.

Drug content 25oC/ 60% RH

98.88 98.25 97.69 96.52

(c) Fexofena

(f) Piper be

(i) Trikat

(l) Ferula fo

(o) C. frutes

ulators 3

±0.003 16 0.010 ±0.70 0.02 .97

adine

etel

tu

oitida

scens

695


(p) Operculina turpethum (q) H. Antidysenterica (r) Mesua ferrea

(s) Tinospora cordifolia (t) Picrorhiza kurroa

(u) Citrus limon

(v) Punica granatum

Fig. 5. Histological studies of the (a) Normal Intestine, (b) Ciprofloxacin (c) Fexofenadine (d) Verapamil + Ciprofloxacin (e) Verapamil + Fexofenadine (f) Piper betel, (g) Trachyspermum ammi, (h) Plumbago zeylanica, (i) Trikatu, (j) Moringaoleifera, (k) Murraya koenigii, (l) Ferula foitida (m) Zingiber officinale, (n) Cheilocostusspeciosus, (o) Capsicum frutescens (p) Operculina turpethum (q) Holarrhena Antidysenterica (r) Mesua ferrea, (s) Tinospora cordifolia, (t) Picrorhiza kurroa, (u) Citrus limon, (v) Punica granatum.

Fig. 6. Bar graph depicting the antimicrobial activity of the ciprofloxacin in presence of various extracts.


Fig. 7. Bar Graph depicting the antimicrobial activity of the ciprofloxacin in presence of various extracts.

‐15

‐10

‐5

0

5

10

15

20

DM

SO

Cip

rofl

oxac

in

Pip

er b

etle

Pip

er b

etle

+ C

Tra

chys

perm

um a

mm

i

Tra

chys

perm

um a

mm

i +

C

Plu

mba

goze

ylan

ica

Plu

mba

goze

ylan

ica

+ C

Tri

katu

Tri

katu

+C

Mor

inga

olei

fera

Mor

inga

olei

fera

+C

Mur

raya

koen

igii

Mur

raya

koen

igii

+C

Fer

ula

Asa

foit

ida

Fer

ula

Asa

foit

ida

+C

Zin

gibe

roff

icin

ale

Zin

gibe

roff

icin

ale

+C

Che

iloc

ostu

sspe

cios

us

Che

iloc

ostu

sspe

cios

us +

C

Cap

sicu

m f

rute

scen

s

Cap

sicu

m f

rute

scen

s +

C

Ope

rcul

inat

urpe

thum

Ope

rcul

inat

urpe

thum

+C

Hol

arrh

enaA

ntid

ysen

teri

ca

Hol

arrh

enaA

ntid

ysen

teri

ca +

C

Mes

uafe

rrea

Mes

uafe

rrea

+C

Tin

ospo

raco

rdif

olia

Tin

ospo

raco

rdif

olia

+C

Pic

rorh

izak

urro

a

Pic

rorh

izak

urro

a +

C

Cit

rus

lim

on

Cit

rus

lim

on +

C

Pun

icag

rana

tum

Pun

icag

rana

tum

+C

staphyl. aureus

E-coli

P. auregenosa

K. Pneumonae


Fig. 8. FTIR studies of selected samples.


Time in minutes Cumulative % Release

0 0

15 39.68

30 63.32

45 75.59

60 87.14

75 92.88

90 97.15

Fig. 9. Drug release profile of the tablet.

Conclusions

In conclusion, tablet formulation containing the alcoholic extracts of Trachyspermum ammi, Plumbago zylanicum, Capsicum frutescens, Operculina turpethum, Holarrhena Antidysenterica, Tinospora cordifolia and Trikatu can act as an absorption enhancer for Fexofenadine and ciprofloxacin. The mechanism action of these herbs could be by P-gp inhibition. Further clinical studies on humans are desired to prove its efficacy in humans.

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Del Amo EM, Heikkinen AT and Mönkkönen J (2009). In-vitro- in-vivo correlation in P-glycoprotein mediated transport in intestinal absorption. Eur J Pharm Sci 36: 200-211.

Drabu S, Khatri S, Babu S and Lohani P (2011). Use of Herbal Bioenhancers to increase the bioavailability of drugs. Res J Pharm Biol Chem Sci Rev Artic 2: 107-119.

Eichhorn T and Efferth T (2012). P-glycoprotein and its inhibition in tumors by phytochemicals derived from Chinese herbs. J Ethnopharmacol 8: 53-59.

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Joshi V, Inamdar S, Kowti R, Sachin AB and Acharya A (2016). Absorption enhancing activity of seven herbal extracts on ciprofloxacin using everted sac method. Pharma Biolo Eva 3: 400-412.

Kokate CK (2015). Practical Pharmacognosy. 4th Ed., New Delhi; Vallabh Prakashan; 1-23.

LeCluyse EL and Sutton SC (1997). In-vitro models for selection of development candidates. Permeability studies to define mechanisms of absorption enhancement. Adv Drug Deliv Rev 23: 163-183.

Li S, Lei Y, Jia Y, Li N, Wink M and Ma Y (2011). Piperine, a piperidine alkaloid from Piper nigrum re-sensitizes P-gp, MRP1 and BCRP dependent multidrug resistant cancer cells. Phytomedicine 19: 83-107.

Park MS, Okochi H and Benet LZ (2011). Ciprofloxacin a Substrate of P-glycoprotein? Arch Drug Inf 4: 1-9.

Wessler JD, Grip LT, Mendell J and Giugliano RP (2013). The P-glycoprotein transport system and cardiovascular drugs. J Am Coll Cardiol 61: 2495-2502.

Address correspondence to: Vedamurthy Joshi, Asst. Professor, Department of Pharmaceutics, S.A.C. College of Pharmacy, B.G. Nagar, Karnataka, India. E-mail: [email protected]

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Int J Pharm Sci Nanotech Vol 10; Issue 2 March April 2017...

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