• Nu S-Au Găsit Rezultate

View of Itraconazole Loaded Nano structured lipid Carriers based In-situ Gel: Formulation, Optimization, Ex-Vivo Permeation and In-vitro Anti-Fungal Activity

N/A
N/A
Protected

Academic year: 2022

Share "View of Itraconazole Loaded Nano structured lipid Carriers based In-situ Gel: Formulation, Optimization, Ex-Vivo Permeation and In-vitro Anti-Fungal Activity"

Copied!
8
0
0

Text complet

(1)

Itraconazole Loaded Nano structured lipid Carriers based In-situ Gel: Formulation, Optimization, Ex-Vivo Permeation and In-vitro

Anti-Fungal Activity

Priyanka Singh1, Hema Jaiswal1, Avneet Kaur Lamba2, Rakesh Pahwa3, Sheetal Devi4, Ravi Shankar5, Manish Kumar6*,

Abhishek Tiwari

7

, Varsha Tiwari

7

1

Institute of Pharmaceutical Sciences and Research, Unnao, UP, India

2

Department of Pharmaceutical Sciences, Gurugram University, Gurugram – 122003

3

Institute of Pharmaceutical Sciences, Kurukshetra University, Kurukshetra-136119, Haryana, India

4

Global Research Institute of Pharmacy, Nachraun, Radaur, Yamunanagar, Haryana, India

5

Sagar Institute of Technology and Management, Dept. of Pharmacy, Barabanki, U.P., India

6

M M College of Pharmacy, Maharishi Markandeshwar (Deemed to be University), Mullana, Ambala, Haryana, India

7

Department of Pharmacy, Devsthali Vidyapeeth College of Pharmacy, Lalpur, Rudrapur (U.S. Nagar), Uttrakhand, India

*Corresponding Author: [email protected]

Abstract

The current study aimed to design and mark Nanostructured lipid carriers (NLCs) combined with Itraconazole for topical fungal activity. Itraconazole is a broad-based antifungal agent derived from azole that has an anti-fungal, yeast, fungus, and dermatophytes-based action on cell membrane disruption. Itraconazole-NLCs were developed using the high-density homogenization method Oleic acid as a liquid lipid, Glyceryl monostearate as a solid lipid, and Poloxamer 188 as a surfactant.

Advanced Itraconazole-NLC is targeted at a wide range of pharmaceutical parameters including particle size, Entrapment efficiency, drug content, and compound drug release. A separate Calorimetry (DSC) study was performed on NLCs to detect changes in drug and lipid conversion. In vitro, drug release studies have been found to vary widely and the % CDR have values b / w 55.37 ± 2.26 - 92.54

± 1.18% depending on the type of polymer and the particle size. Exfoliation of Ex-vivo skin and Pharmacodynamic studies have shown that Itraconazole-loaded NLCs have better penetration potential than commercially available compounds and are able to reduce fungal infections to a large extent. In- vitro antifungal activity shows that itraconazole-NLCs were effective in reducing the growth of Candida Albicans. Therefore, the study concludes that Itraconazole-loaded NLCs have a persistent release profile and have the potential for the treatment of fungal topical infections.

Keywords: Itraconazole, Nanostructure lipid carriers, Topical delivery, Anti-fungal infection, High

pressure homogenization

1. Introduction

In the current situation there are a wide variety of fungal infections affecting human population all over the world.

The people are found to be affected either or systemic and more severe fungal infection. The antifungal drugs commonly utilized for this purpose are associated mainly with liver toxicities and enhanced levels of estrogen causing a wide variety of allergic reaction as adverse drug reactions.

(2)

molds, yeasts, dermatophytes and Gram-positive bacteria. Itraconazole is mainly preferred for the treatment of topical infections as like tinea pedis, candidiasis. It is practically insoluble in water having oral bioavailability of itraconazole is 55%, and is maximal when taken with a full meal2, 3.

Itraconazole inhibits the conversion of lanosterol to ergosterol by interacting with 14-α demethylase, a cytochrome P-450 enzyme. Ergosterol is an important part of cell membranes and helps in the functionalization of the structural and functional role of the membrane. The inhibition of its synthesis leads to an increase in cellular penetration leading to cell leakage. Itraconazole can also inhibit chronic respiration, interact with phospholipids, inhibit mycelial conversion, inhibit purine uptake, and inhibit triglyceride and/or phospholipid biosynthesis.

Mycelium fungi can deeply penetrate the skin layers and causes the fungal infection. To overcome this problem, improvement in the activity of the active agent for the antifungal treatment is required 4. Hence, the current study is done with the aim of enhanced penetration of itraconazole through the deeper layers of the skin by encapsulating the drug in NLCs containing hydrogel for better therapeutic achievements.

So, the overall aim of the present study was to formulate Itraconazole loaded NLC gel formulation for topical administration and characterize it on different physical, pharmaceutical and biological parameters.

2. Materials and Methods 2.1 Materials

Itraconazole was received as a gift sample from Vital Laboratories Private Limited (Gujarat, India). Glyceryl monosterate and Oleic acid was obtained from Central Drug House Ltd. Vardaan House, Daryaganj, New Delhi (India). Tween80 was obtained from Central Drug House Ltd. Vardaan House, Daryagan (New Delhi, India).

Sodium hydroxide pellets, HCL (Concentrated), Methanol AR were obtained from Qualikems Fine Chemicals (Mumbai, India). All reagents and solvents used were of systematic rank.

2.2 Experimental design of Itraconazole loaded NLCs

A complete 32 factorial was used to design the tests. These two independent variables have been used in three different levels to obtain the best composition. Independent variables are lipid concentrations and filactants of surfactants while the dependent variables are particle size, efficiency of percentage inclusion and cumulative drug release. Nine coded forms such as F1 to F9 were prepared using three different levels of lipid ratio and the surfactant and characterization of parameters was done (Table 1).

2.3 Selection of binary lipid phase

Itraconazole was dissolved in a mixture of solid and liquid lipids of various compounds and was tested by a binary lipid system with excellent melting potential. The most pronounced formulation: liquid lipid was ratios viz., 95: 5, 90:10, 85:15, 80:20, 70:30, and 60:40. The binary mixture was then stirred at 85⁰ C for an hour at a speed of 200 rpm using a magnetic stirrer. The miscibility of the two lipids was evaluated using a smeared coated sample of a solid mixture. Visual aids are used to determine the presence of any droplets of liquid oil in the filter paper. For the development of itraconazole NLC, a binary mix showing a melting point above 40⁰ C was selected if it did not reveal the presence of oil droplets on the filter paper6.

2.4 Preparation of NLCs

The lipid phase was accurately weighed and heated at approximately 85 C well above the solid phase melting point and an approximate amount of the drug was added. The step was further followed by the addition of aqueous surfactant simultaneously at same temperature. The lipid mixture was gradually and continuously administered into a hot surfactant solution using a mechanical stirrer for the preparation of the main emulsion7,8. The NLC system was developed using the primary emulsion with the help of a high-pressure homogenizer (Sunrise Pvt. Ltd., Mumbai, India) at 15000 PSI. The distributed NLC distribution was allowed to cool and attain room temperature at a very slow rate and then lyophilization was done to attain long-term stability. Samples were frozen at -78ºC for 10 h followed by lyophilization of 36 h using mannitol as a cryoprotectant.

Evaluation and Characterization of Itraconazole loaded NLC Particle size analysis

Photon correlation spectroscopy (PCS) with a Zetasizer (Malvern Instruments, Worcestershire, UK) was utilized for evaluating and characterizing the particle size. The PCS provides the mean particle size (z-average).

Entrapment Efficiency (EE) and Drug-loading capacity (DL)

(3)

The drug-loaded NLC dispersion (1ml) was added to 9.0 ml methanol and the dispersion was centrifuged using High-Speed Refrigerated Centrifuge (Labtop Instruments Pvt. Ltd, palaghar, Maharashtra) for 45 min at 15,000 rpm. The solution was filtered using Millipore membrane (0.2 µm) to separate the dissolved portion from the undissolved part. The filtrate was obtained and was further diluted to optimum concentrations with methanol and the absorbance was measured utilizing U.V. spectrophotometer (Perkin Elmire, Lamda 1050+, Ohio, US) at ʎmax

of 262 nm. The percent entrapment efficiency (EE %) and Drug loading was determined utilizing the equations mentioned below9.

% EE =

(Total) W

(Free) W

× (Total)

W

x 100

% DL =

(Lipid) W

(Free) W

× (Total)

W

x 100

Wtotal = the weight of drug

Wfree = weight of drug in supernatant Wlipids = weight of lipid

Evaluation of Itraconazole loaded NLCs In vitro drug release study

Dialysis bag technique was utilized to carry out in-vitro drug release studies of NLCs. The activation of dialysis membrane was done and further the experiments were performed under sink conditions. The process involved loading of dialysis bag with 10 mg of each formulation separately and immersing it into 200 ml phosphate buffer solution pH 6.8 with added 0.8 % tween solution. The system was magnetically stirred at 32°C at pH 6.8. The aliquots were taken at predetermined intervals from the receiver solution and were replaced with equal volumes of buffer solution. The concentration of the drug was determined spectrophotometrically at λmax 262 nm. The release studies were performed in triplicate10, 11.

Transmission electron microscopy (TEM)

TEM studies were performed to characterize the surface characteristics and other physical characteristics of the NLCs using TEM (HITACHI High Tech Global, china). A drop of the dispersion was added on a paraffin sheet followed by the coating of carbon grid over the sample. The system was allowed to stand for 1 min for proper adherence on the substrate. Removal of the un-trapped NLC was done by adsorbing the drop on filter paper. Then the grid was placed on the drop of phosphotungstate (1%) for 10 s. further the sample was air dried and utilized.

Preparation of Itraconazole loaded NLCs Based Gels

The hydrogels were formulated using the polymer Carbopol_ 940P. The gel was prepared by dispersing the Carbopol polymer in water and subsequently adding the optimized formulation followed by subsequent addition of triethanolamine (TEA) to increase the pH to the desired value for formation of gel. Three formulation codes were prepared for hydrogels in which the final concentrations of the Carbopol were varied as detailed in table.

Evaluation of NLCs based gel:

Viscosity

The viscosity of the optimized NLCs gel formulation was determined utilizing Brookfield viscometer (AMETEK Instruments India Pvt. Ltd., Bangalore) with spindle No. 64 at 50 rpm at temperature of 25 ± 0.5⁰C.

Determination of pH

Weighed quantity (1gm) of the NLC gel preparation was utilized to determine the pH. The gel was added to a volumetric flask and a specified amount of distilled water (0.2% strength 50 ml) was added and homogenous dispersion was obtained. The pH was determined using digital pH meter (Royal Scientific, Mumbai, India).

Spreadability

The spreadability study was performed utilizing a glass plate and marking a circle of 1 cm diameter. The formulated gel (0.5g) was placed over glass A within the specified area, and above it another glass plate is placed. Finally half kg weight was placed over the glass plate for 5-10 min. gel spreading diameter was noted and compare with the

(4)

Ex vivo permeation studies

Abdominal skin of Albino rat was utilized for carrying out ex-vivo skin permeation studies due to easy availability and ease of study. Wistar Albino Rats of 20-25 weeks and weight of 200-250 g having any sex were utilized. The hairs were first removed using hair removal cream followed by removal of skin. The subcutaneous fat was removed with a scalpel and the skin was washed many times with water and finally clear skin was placed in phosphate buffer saline (pH 6.8). The skin was then mounted on the Franz diffusion cell between the receiver and the donor compartment with SC facing upwards towards the donor compartment and the receiver chamber was filled with 20 ml diffusion medium (PBS pH 6.8 with 0.8 % v/v tween 80. The entire system was placed over a magnetic stirrer for continuous agitation. The formulation to be tested (equivalent to drug of 10 mg) were applied drug to the skin in the donor compartment including the plain drug solution, marketed preparation, optimized NLC formulation and optimized NLC gel. Aliquots were taken out of specific amount through the receptor compartment at fixed time intervals, and fresh diffusion medium was added to maintain sink condition. The studies were performed for 24 h as per the clinical settings desired and the concentration of drug was determined using UV spectrophotometer at λmax 262 nm. The amount of drug permeated, Flux (µg/cm2/h) and the values of permeability coefficient (Pb) [cm-2h-1] were calculated using the formulas mentioned12, 13.

In vitro antifungal activity

In vitro antifungal activity was determined using cup-plate method for calculating zone of inhibition. In this method, firstly we prepared the dextrose agar media. Then sterilization was done by autoclaving and the sterilized media was transferred into the Petri plate for solidify. After that inoculation of the fungal suspension was done in solidified media. The fungal growth was observed. Pre-sterilized steel borer was utilized to create bores of specific size (equivalent to McFarland standard no. 0.5). Itraconazole standard solution, marketed formulation, NLCs based gel were placed in the specified bores in the solidified agar media14,15. The plates were allowed for incubation for a period of 48 hours and zone of inhibition was measured after incubation period.

Preparation of fungal inoculums

Candida albicans culture was obtained from Microbiology Department, MMIMS, Mullana, Ambala, India. The Candida albicans was subcultured and allowed for 24 hr to grow at 25 °C.

The conidial suspension density was adjusted to be approx. to 1 × 106 CFU/ml by hemocytometer and it was used for the inoculum16.

Results and discussion Particle size

The particle size of the optimized NLC formulation was found to be in nanosized range (161.5 nm) with low polydispersity index (0.338 ± 0.16) as seen in figure 1. The size of the NLC particle was inversely dependent on the concentration of liquid lipid and also on concentration of surfactant.

Figure 1.Particle size of optimized ITRACONAZOLE loaded NLCs formulation

(5)

Entrapment efficiency and drug loading

The Entrapment efficiency and drug loading was found to be 98.26 ± .68 and 19.8 ± 1.12. The NLCs have small sections of liquid lipid in solid lipid matrix in which drug solubility is higher which increases and resulting into increased total drug loading capacity. And liquid lipid also affects the entrapment efficiency and helps in the loading of larger amount of drug (table 2).

Optimization of Itraconazole loaded NLCs

The selection of optimized formulation was done on the basis of 32 full factorial design. The software was used to study the effects of the variables in the formulation of NLCs over different parameters. The independent variables were the ratio of glyceryl monostearate and oleic acid and the concentration of surfactant while Particle size (Y1), Entrapment Efficiency (Y2) and Drug Loading (Y3) were dependent variables to be studied. Table 2 shows the experimental design of Glyceryl monosterate and oleic acid nanoparticles and the results of measured responses. It was found that with enhancement in concentration of lipid the particle size increases and also the reduction of the volume of organic phase leads to increased particle size. The results also indicated that increase in polymer concentration and less solvent concentration resulted in better drug entrapment of drug into the matrix.

Response surface plots

The response surface plots were formulated using the Design Expert software and the resulting plots are presented in Fig 2(a) for Itraconazole NLCs. Fig 2(a) clearly revealed that the particle size of Itraconazole NLCs were dependent on the lipid concentration and there was increase in particle size with subsequent increase in lipid concentration and decrease in the volume of the aqueous phase9,11. Fig 2(b) shows the effect of liquid concentration on Drug entrapment efficiency and the figure indicates that there is an increase in drug entrapment due to an increase in liquid lipid concentration. Fig 2(c) shows that with increase in concentration of lipid and surfactant there is sharp increase in the amount of drug able to be loaded in the NLCs.

Figure 2 Response surface plots of factors (particle size, entrapment efficiency, drug loading)

Evaluation of NLCs In vitro drug release study

The in vitro drug release profiles (figure.3) were studies and it was found that the % CDR varies widely b/w 55.37 ± 2.26 – 92.54 ± 1.18%. The variability was observed due to the variation in particle size among all the ten formulations resulting as a result of different lipid ratios utilized.

Formulation F5, F6 & F7 exhibited more than 80% CDR and among these F5 displayed maximum CDR of 92.54 ± 1.18% due to small sized particles and optimum entrapment efficiency NLCs developed from Span 80 (F1-F4) showed show & incomplete CDR OF (55.37± 2.26 – 78.34 ± 1.32) and when compared to F5 – F7 made with Poloxamer 188 (80.92 ± 2.1 – 92.54 ± 1.18). The determinants of this variability may be attributed to particle size and PDI that was in turn affected by type of copolymer used (Harada et al., 2011). Thus F8 – F11 prepared with PF127 produced large sized Nanostructured lipid carriers. This led to incomplete drug release which was varied from (61.01 ± 2.12 – 71.06 ± 1.82 %).

(6)

Figure 3 In vitro release of ITRACONAZOLE loaded NLCs formulations (F1-F11)

Transmission electron microscopy (TEM)

The TEM studies were performed to gather detailed information about the morphology of the NLCs systems. It was observed that drug loading resulted in increased particle size. This was attributed to the accommodation of the drug in sufficient space in the lipid matrix. The TEM images (Figure 4) clearly revealed that the drug is dispersed homogenously in the lipid matrix. The NLC particle size was found to be in range of 160-500 nm and it was found that the follow a normal distribution curve around mean value. The small value of PDI (0.38) obtained through photon correlation spectroscopy demonstrates the uniformity of particle size distribution.

Figure 4 TEM image of optimized formulation Preparation of Itraconazole–NLCs based gel

Carbopol 940 in various concentrations 1%, 1.5%, and 2% was used to formulate the Itraconazole-NLCs into the

(7)

gel. It was found that the Gel (having 1.5% Carbopol 940) was suitable for the incorporation of the NLCs because of optimum consistency.

Viscosity

Brookfield viscometer was used to determine the viscosity of the NLC enthused gel. The viscosity obtained was reported to be 581 ± 0.98 cps. It was optimum for the purpose for which it was designed to have initial resistance to flow at lower torque values but at higher shear stress it can easily spread and flow.

Determination of pH

The pH of the optimized NLCs Gel formulation was determined by Digital pH meter in triplicate at 26⁰ C was reported to be 5.7 ± 0.07. The pH of the optimized gel formulation was found to be well within the range of pH of skin thus preventing any rational problems regarding application.

Spreadability

The ideal gelling formulation is readily spread on the site of application. The conformity of the good spreading ability was proven by the sharp increase in the area of the gel spreading reported to be 6.4 ± 0.05 cm. The obtained value critically proves that the test formulation was having good Spreadability which is very essential criteria for topical application.

Ex vivo permeation studies

The study was conducted to determine the amount of drug permeated through the skin followed by a controlled or sustained release. Drug loaded Nanostructured lipid carriers can easily penetrate the skin layers. Ex vivo permeation studies was performed for drug dispersion, marketed formulation, optimized formulation (F5) and gel formulation (G5) (figure:5).

The Carbopol polymer present in gel has the tendency of bio-adhesion, that is the reason we have better adhesion to the skin and resulting into increased contact time. NLCs based gel and optimized formulation have proved to be having better skin penetration ability. This is desirable for the topical application10. As shown by fig. 5 it was proved that the developed formulation has served the basic purpose of formulation approaches.

Figure 5 Comparative ex-vivo permeation of ITRACONAZOLE loaded optimized NLC (F5), Dispersion, M.F and G5

In vitro antifungal activity

The zone inhibition value of the optimized gel formulation was found to be higher as compared to the marketed formulation, while the zone inhibition value was maximum for Itraconazole standard solution.

NLCs based gel was having higher antifungal activity as compared to the marketed formulation which can be attributed to the higher solubility of drug and subsequent better permeation ability through the skin layer and inhibit the ergosterol synthesis17,18 (Table 4).

(8)

3. Conclusion

Itraconazole loaded NLCs containing Gel formulation was successfully developed utilizing high pressure homogenization technique. The developed formulations revealed optimum particle size, entrapment efficiency and in-vitro release kinetics. The final NLC encapsulated Gel formulation was having sufficient spreadability, viscosity and permeation characteristics confirming the capability of NLCs containing gel to treat deep skin fungal infections as represented and proved by ex-vivo permeation studies.. And it can be concluded that the use of NLCs was far better than the conventional creams/gels.

References

1. Warnock, D.W., Trends in the epidemiology of invasive fungal infections. Japanese Journal of Medical Mycology, vol.48, pp.1–12, 2007.

2. Lackner, T.E., Clissold, S.P., Itraconazole: A review of its antimicrobial activity and therapeutic use in superficial mycoses. Drugs, vol.38, pp,204-225, 1989.

3. Drug Bank–Open data drug and drug target database version 3. http://www.drugbank.ca/drugs/DB04794 (Accessed on November 2012).

4. Müller-Goymann, C.C., Physiochemical characterization of colloidal drug delivery systems such as reverse micelles, vesicles, liquid crystals and nanoparticles for topical administration. European Journal of Pharmaceutics and Biopharmaceutics, vol.58, pp. 343-356, 2004.

5. O’Driscoll, C.M., Griffin, B.T., Biopharmaceutical challenges associated with drugs with low aqueous solubility – The potential impact of lipid-based formulations. Advanced Drug Delivery Reviews, vol.60, pp.617–624, 2008.

6. Gaba, B., Fazil, M., Nanostructured lipid carrier system for topical delivery of terbinafine hydrochloride, Bulletin of Pharmacy, Cairo university, vol.53, pp.147-159, 2015.

7. Muller, R., Mader, K., Gohla, S., Solid lipid nanoparticles (SLN) for controlled drug delivery – A review of the state of the art. European Journal of Pharmaceutics and Biopharmaceutics, vol.50, pp.161–77, 2000.

8. Muller, R., Mader, K., Gohla, S., Solid lipid nanoparticles (SLN) for controlled drug delivery – A review of the state of the art. European Journal of Pharmaceutics and Biopharmaceutics. vol.50, pp.161–77, 2000.

9. Baboota, S., Al-Azaki, A., Kohli, K., Ali, J., Dixit, N., Shakeel, F., Development and evaluation of a microemulsion formulation for transdermal delivery of terbinafine. PDA Journal of Pharmaceutical Science and Technology, vol.61, chap.4, pp.276–85, 2007.

10. Sanad, R.A., AbdelMalak, N.S., elBayoomy, T.S., Badawi, A.A., Formulation of novel oxybenzone-loaded nanostructured lipid carriers (NLCs). journal of The American Association of Pharmaceutical Scientists, vol.11, chap.4, pp.1684–94, 2010.

11. Fang, J.Y., Fang, C.L., Liu, C.H., Su, Y.H., Lipid nanoparticles as vehicles for topical psoralen delivery:

solid lipid nanoparticles (SLN) versus nanostructured lipid carriers (NLC). European Journal of Pharmaceutics and Biopharmaceutics, vol.70, pp.633–40, 2008.

12. Cirri, M., Bragagni, M., Menni, N., Mura, P., Development of a new delivery system consisting drug- in cyclodextrin- in nanostructured lipid carriers; for ketoprofen topical delivery. European Journal of Pharmaceutics and Biopharmaceutics, vol.32, chap.4, pp.21–32, 2011.

13. Ananthanarayan, R., Panicker CKJ: Laboratory Control of Antimicrobial Therapy. Textbook of Microbiology, India, Universities Press., pp.246-247, 2009.

14. Sabale, V., Vora, S., Formulation and evaluation of microem ulsion-based hydrogel for topical delivery.

International Journal of Pharmaceutical Investigation. Vol.2, pp.140-149, 2012.

15. Zur Muhlen, A., Zur Muhlen, E., Niehus, H., Mehnert, W., Atomic force microscopy studies of solid lipid nanoparticles. Pharmaceutical Research, vol.13, pp.1411–1416, 1996.

16. Bachhav, Y.G., Patravale, V.B., Microemulsions-based vaginal gel of clotrimazole: formulation, in vitro evaluation and stability studies. journal of The American Association of Pharmaceutical Scientists, vol.10, chap.2, pp.476-481, 2009.

17. Patel, M.R., Patel, R.B., Parikh, J.R., Bhatt, K.K., Solanki, A.B., Investigating the effect of vehicle on in vitro skin permeation of ketoconazole applied in O/W microemulsions. Acta Pharmaceutica Sciencia, vol.52, pp.65-77, 2010.

18. Uchida, K., Tanaka, T., Yamaguchi, H., Achievement of complete mycological cure by topical antifungal agent NND-502 in guinea pig model of tinea pedis. Microbiology and Immunology, vol.47, pp.143–6, 2003.

Referințe

DOCUMENTE SIMILARE

Permeation studies through rat skin revealed that 0.5%w/w carpobol 934 showed the highest flux from all studies gel bases and high complex permeation compared to acetyl salicylic

To validate this hypothesis as well as exploring the anti-tumor activity of LPC against the breast cancer cells, an in vitro study was initiated and each of these five human

In this study, the antimicrobial activity of colloidal silver nanoparticles prepared by the sol-gel method was investigated, and the turbidity, viscosity and pH of the colloidal

The proposed method is accurate, selective and precise hence can be used for the routine quality-control analysis and quantitative simultaneous determination of Lopinavir and

1 In vitro dissolution profile of sustained release tramadol hydrochloride liquisolid compacts (F1-F9) compared with marketed formulation (MKT).. 3.5.1 Model

As it can be seen in table 3, subjects’ serum Iron mean concentration in the first stage and before the Bruce Protocol physical activity was found to be 1.007 nano grams per

Spreadability and consistency of polyacrylamide gel containing diclofenac sodium (F9) were 6.5g.cm/sec and 5mm as compared to 5.5g.cm/sec and 10mm respectively of marketed

In vitro anti-herpes simplex type-1 activity, antioxidant potential and total phenolic compounds of pomegranate (Punica granatum L.) peel extract. Anti-influenza virus activity and