Nasal drug bringing prepared from natural stuffs is deriving importance in the sphere of pharmaceutical engineering. Mucilage isolated from Linum usitatissimum L. seeds was reported to be an effectual natural mucoadhesive agent. The present survey trades with comparision of assorted features of rhinal gels prepared from mucoadhesive agent extracted from Linum usitatissimum L. seeds and man-made polymers like HPMC and Carbopol 934 in footings of texture profile analysis, mucoadhesive strength and in vivo drug soaking up profiles. It was observed that gels formulated with the natural mucilage showed better consequences in all facets than the man-made gels. The absolute bioavailability of midazolam hydrochloride from the natural gel was 97.55 % whereas that of man-made gels were 57.33 % and 76.81 % severally.
Uniterms: Linum usitatissimum L./pharmacognosy. Linum usitatissimum L./ natural mucoadhesive agent. Natural Mucoadhesive agent/bioavailability. Natural mucilage.
Mucoadhesive nasal gels provide a steadfast platform of drug bringing to the nasal pit than the other types of rhinal preparations like solutions, sprays, insufflations, since the mucoadhesive agents make a better contact with the nasal mucous membrane which helps in heightening drug bioavailability. Assorted man-made agents are available like hydroxy propyl methy cellulose ( HPMC ) , Carbopol 934, Na alginate and so on. Nowadays, natural mucilages isolated from assorted works parts and are used as mucoadhesive agents. It has been observed that natural stuffs are biocompatible and biodegradable and hence they are more preferable than the man-made polymers. In our old work, mucoadhesive nasal gels of Versed were prepared from mucilages isolated from Linum usitatissimum L. seeds and it was observed that they gave better consequences than man-made polymers in footings of viscousness and in vitro release profiles ( Basu et al, 2009 ) .
Linum usitatissimum L. mucilage is a H2O soluble heterogenous polyose composed of xylose, arabinose, glucose, galactose, galacturonic acid, rhamnose and fruit sugar ( Cui et al, 1994 ; Erskine et Al, 1957 ; Hunt et Al, 1962 ; Muralikrishna et Al, 1987 ) . It has good water-holding capacities, owing to its pronounced puffiness capacity and high viscousness in aqueous solution ( Basu et al, 2009 ) . It has been reported that presence of many oligo- and polyoses in many substances impart mucoadhesive belongingss ( Hunt et al, 1962 ) . Since, this mucilage is a rich beginning of polyoses and has singular swelling capacity and high viscousness, it was selected to fix rhinal gels of Versed.
Midazolam is a fast acting benzodiazepine with a short riddance half life ( 1.8 6.4 hour ) . It is hence a really utile drug to utilize for short minor processs such as dental extraction. However, due to really hapless bioavailability of this drug from gastro-intestinal system ( ? 36 % ) , it is administered through intramuscular injection merely where bioavailability is over 90 % ( wikipedia ) . Nasal path will be one of the best alternate path of disposal of this drug to intramuscular injections which is one of the most risky path of drug disposal every bit good as most hard to maker due to rigorous regulations and ordinances.
Hence, an effort was made to fix mucoadhesive rhinal gels of Versed which may replace the conventional Versed injection and widen the range of fresh drug bringing system.
The present survey focuses on comparision of texture profile analyses, mucoadhesive strengths and in vivo drug soaking up profiles from rhinal gels prepared with mucilage isolated from Linum usitatissimum L. seeds and man-made polymers like HPMC and Carbopol 934.
MATERIALS AND METHODS
Midazolam hydrochloride was obtained as a gift from Sun Pharmaceutical Industries Ltd. , Gujarat, India. Linum usitatissimum L. seeds were purchased from local market. HPMC, Carbopol 934 and sodium taurocholate were purchased from Loba Chemie Pvt. Ltd. , Mumbai, India. All other reagents and chemicals used were of analytical class.
Preparation of rhinal gel incorporating Versed
Mucoadhesive nasal gels were prepared by fade outing midazolam hydrochloride in rhinal solution ( 0.65 % NaCl, 0.04 % KH2PO4, 0.09 % K2HPO4 and 0.02 % benzalkonium chloride ) ( pH 6 ) in a changeless stirring status. Needed sums of LUM and man-made polymers ( HPMC and Carbopol 934 ) were added to the solution and stirred on a magnetic scaremonger until a unvarying solution was obtained which was kept at 4 & A ; deg ; C overnight to let complete swelling so that a homogeneous gel was formed. Penetration foil like Na taurocholate, and Na thioglycollate were besides used in the preparations at a concentration of 0.50 % ( w/v ) .
In our old work it was shown that Na taurocholate produced better drug release profiles than Na thioglycollate ( Basu et al, 2009 ) and therefore, for the in vivo survey, rhinal gels incorporating 0.5 % w/v Na taurocholate were administered to the coneies. Composition of rhinal gels used in the present survey is provided in Table I.
TABLE I- Composition of rhinal gels alongwith preparation codifications
Formulation Midazolam HCl LUM HPMC Carbopol 934 Sodium taurocholate
codification ( % w/v ) ( % w/v ) ( % w/v ) ( % w/v ) ( % w/v )
F1 5.0 3.0 – – 0.5
F2 5.0 4.0 – – 0.5
F3 5.0 5.0 – – 0.5
F4 5.0 – 3.0 – 0.5
– 4.0 – 0.5
– 5.0 – 0.5
– – 3.0 0.5
– – 4.0 0.5
– – 5.0 0.5
Word picture of gels
Determination of texture profile analysis
Texture profile analyses of the gels were evaluated utilizing QTS-25 Texture Analyser ( Brookfield Engineering Labs. , USA ) to find the mechanical parametric quantities like hardness, coherence and adhesion. An analytical investigation of diameter 1.2 centimeter was down twice into each sample to a fixed deepness ( 15 millimeter ) , at a defined rate ( 30 mm/min ) , with a defined recovery period ( 15 s ) , between the terminal of the first compaction and the beginning of the 2nd. A trigger force of 4 g was applied. At least six replicate analyses of each sample were performed at 37±1 & A ; deg ; C. Data aggregation and rating were done by Texture pro package, version 2.1 ( Cevher et al, 2008 ) .
TABLE II – Valuess of texture profile analysis and mucoadhesive strength of conventional gels.
Data represent mean ± SD ( n=6 )
Formulation Hardness ( g ) Adhesiveness ( gs ) Cohesiveness Mucoadhesive
codification strength ( g )
28.00 ± 1.11
38.33 ± 0.69
55.00 ± 0.38
11.00 ± 0.50
14.00 ± 0.48
18.00 ± 0.36
15.00 ± 0. 51
21.00 ± 0.36
27.05 ± 0.32
-89.00 ± 7.12
-95.00 ± 2.65
-112.08 ± 5.89
– 30.60 ± 3.03
– 40.62 ± 4.88
– 51.26 ± 6.45
-45.03 ± 5.02
– 69.36 ± 8.56
-80.36 ± 7.98
1.01 ± 0.05 16.26 ± 0.98
0.99 ± 0.02 19.76 ± 0.85
0.97 ± 0.03 22.45 ± 1.02
1.15 ± 0.01 8.00 ± 0.85
1.02 ± 0.05 9.50 ± 0.36
1.00 ± 0.03 11.95 ± 0.56
1.10 ± 0.05 9.63 ± 1.02
1.06 ± 0.04 13.22 ± 1.05
0.96 ± 0.03 16.39 ± 0.89
Evaluation of mucoadhesive strength
Mucoadhesive strengths of the gels were determined by mensurating force required to detach nasal mucose membrane from the gel utilizing the same texture analyzer. Freshly excised caprine animal nasal membrane was attached to the upper investigation of the instrument, and fixed sum of gel was kept below that. The upper investigation was so lowered at a velocity of 10 mm/min to touch the surface of the gel. A force of 0.1 N was applied for 5 min to guarantee intimate contact between the membrane and the gel. The surface country of exposed mucose membrane was 1.13 cm2.
Calibration curve of Versed in coney plasma
Stock solutions of Versed were prepared in a mixture of 10 millimeters phosphate buffer ( pH 6.0 ) : acetonitrile ( 80:20 ) to give concluding concentrations of 1 mg/ml. Aliquots of 0.1, 0.5, 1.0, 2.0, 6.0, 8.0 and 10 µg/ml of Versed by appropriate dilutions of the mention solution with the solvent mixture. Plasma criterions for standardization curves were prepared by spiking 1.0 ml aliquots of pooled drug free plasma with 100 µl of the above mentioned working solutions, to do midazolam plasma criterions runing from 10 to 1000 ng/ml.
TABLE III- Interday and intraday truth and preciseness informations for quantitation of Versed in coney plasma
Amount of drug added ( ng/ml )
Concentration in plasma Accuracy Precision ( % CV )
( ng/ml )
Intraday Interday Intraday Interday Intraday Interday
9.91±0.24 9.55±0.38 99.10 95.50 2.42 3.97
199.05±2.15 195.99±4.51 99.53 98.46 1.08 2.30
599.30±2.65 596.08±6.58 99.88 99.34 0.44 1.10
998.05±3.95 994.09±5.01 99.80 99.41 0.39 0.50
In vivo drug soaking up survey
Choice of animate beings
In vivo drug soaking up survey was conducted with anterior blessing of the Institutional Animal Ethics Committee and it was conducted harmonizing to the institutional guidelines of Animal Ethics Committee of Dr. B. C. Roy College of Pharmacy and Allied Health Sciences, West Bengal University of Technology, as recognized by the Committee for the Purpose of Control and Supervision on Experiments on Animals, India.
In vivo surveies were conducted on 12 New Zealand albino male rabbits weighing between 2 and 2.5 kilogram. Based on consequences of texture profile analyses, mucoadhesive strengths and in vitro drug release form ( Basu et al, 2009 ) , rhinal gels incorporating 3 % w/v LUM, 5 % w/v Carbopol 934 and 5 % w/v HPMC along with 0.5 % Na taurocholate were selected for in vivo survey. The animate beings were kept in single metal coops and maintained at 25 & A ; deg ; C for 10 yearss prior to the experiment. They were provided with standard diet and H2O ad libitum. The coneies were kept in fasting status for 24 H before the experiment commenced. The coneies were grouped into four ( group I, II, III and IV ) , each group incorporating three coneies. Group I was administered endovenous bolus injection of Versed. Groups II, III and IV were administered rhinal gels of Versed prepared with LUM, Carbopol 934 and HPMC. Single dosage of Versed ( 2 mg/kg organic structure weight of coney ) was administrated intravenously to compare the pharmacokinetic parametric quantities. No anesthesia was used for the endovenous survey. Midazolam was injected through cannulated fringy ear vena. After every 20 min, each coney was administered tierce of the initial dosage of xylazine and ketamine intramuscularly to keep a light plane of anesthesia. In instance of nasal gels, the dosage of Versed that was administered was besides 2 mg/kg organic structure weight of coney.
Collection of blood samples
Two milliliters of blood samples were collected earlier endovenous injection and so at 5- , 10- , 15- , 20- , 30- , 45- , 60- , 90- , 120- , 180- , 240- and 300-min intervals in eppendorffs incorporating Lipo-Hepin Na ( 100 U/ml ) . Immediately after each blood sample aggregation, the catheter was flushed with 0.2 milliliters of a 10 % ( v/v ) heparin/normal saline solution to forestall blood coagulating inside the catheter. The blood samples were kept on ice and centrifuged at 3,000 revolutions per minute for 10 min instantly after aggregation to divide the plasma and stored at ?20 & A ; deg ; C until the clip of analysis.
Rearward stage HPLC was used to quantitate Versed in plasma samples. Midazolam was extracted with 3 milliliters of cyclohexane/diethyl quintessence ( 3:7 ) after the add-on of 10 ?l of 2 % Na hydrated oxide ( Shih et al, 2002 ) . The organic stage was removed and evaporated to dryness under N, and the residue was reconstituted in 200 ?l of the nomadic stage ( 10 millimeter phosphate buffer ( pH 6.0 ) /acetonitrile, 80:20 ) . From the mixture, 100 ?l was injected for chromatographic analysis. The nomadic stage consistedof phosphate buffer/acetonitrile ( 80:20 ) v/v. The nomadic stage was delivered into the HPLC setup at a flow rate of 1 ml/min ( isocratic pump, Model LC-10AS, Jasco, Japan ) . The sensing wavelength was 218 nanometer ( ultraviolet variable wavelength sensor, Model SPD-10A ) , and a C18 column was used. All checks were performed at ambient temperature.
Pharmacokinetic parametric quantities like extremum plasma concentration ( Cmax ) , clip to make peak plasma concentration ( tmax ) , country under the ( concentration-time ) curve ( AUC ) , average abode clip ( MRT ) , elimination half life ( t1/2 ) and entire organic structure clearance ( CL ) were calculated following non-compartment theoretical account by Kinetica 4.4, PK/PD Analysis, Thermoelectron Corporation. All the parametric quantities were calculated for i.v. bolus injection of Versed and in situ rhinal gels. Fraction of dose absorbed ( F ) was calculated by the undermentioned equation:
F = AUC ( nasal ) Ten Dose ( four )
AUC ( i.v. ) Ten Dose ( nasal )
where Dose ( four ) =dose of Versed given as i.v. solution, Dose ( nasal ) =dose of Versed in rhinal gels, AUC ( i.v. ) =AUC after i.v. disposal of Versed and AUC ( nasal ) =AUC after rhinal disposal of Versed.
TABLE IV – Comparative pharmacokinetic parametric quantities of midazolam hydrochloride following disposal of endovenous and rhinal gels in coneies ( dose=2 mg/kg )
Pharmacokinetic Intravenous HPMC CP LUM
parametric quantities solution
Cmax ( ng/ml ) 573.64 ± 5.23 151.47 ± 7.22 164.07 ± 6.85 181.12 ± 6.21
Tmax ( min ) – 60.00 ± 5.68 45.00 ± 3.66 30.00 ± 4.75
AUClast 32671.50 ± 90.56 18034.30 ± 101.45 22074.10 ± 96.72 6168.80 ± 123.67
AUC excess 865.87 ± 37.25 1191.83 ± 45.26 3684.71± 60.58 5546.92 ± 48.76
AUCtotal 33537.40 ± 101.25 9226.10 ± 97.25 25758.90 ± 95.46 32715.72 ± 98.77
MRT ( min ) 71.61 ± 5.05 130.16 ± 6.58 162.50 ± 7.25 190.07 ± 8.05
T1/2 ( min ) 58.78 ± 6.32 68.44 ± 5.28 99.68 ± 4.89 122.55 ± 5.87
Clearence X 10-5 5.79 ± 6.95 10.11 ± 3.66 7.53 ± 2.89 6.67 ± 4.22
( mg/ Kg*min/ ( ng/ml ) )
The present survey determines the assorted mechanical belongingss every bit good as mucoadhesive strengths of the gels with the aid of QTS – 25 Texture Analyser. Results of the texture profile analysis and mucoadhesive strengths are tabulated in Table I. Hardness of LUM gels ranges from 28.00 ± 1.11g to 55.00 ± 0.38g. In instance of HPMC gels, it ranges from 11.00 ± 0.50g to 18.00 ± 0.36g and for Carbopol 934 it varies from 15.00 ± 0.51g to 27.05 ± 0.32g. Valuess of adhesion of LUM, HPMC and Carbopol 934 gels varies from -89.00 ± 7.12gs to -112.08 ± 5.89gs, -30.60 ± 3.03gs to -51.26 ± 6.45 gs and -45.03 ± 5.02gs to -80.36 ± 7.98 gs, severally.
Coherence of LUM gels ranges from 1.01 ± 0.05 to 0.97± 0.03. For HPMC gels, it varies from 1.15 ± 0.01 to 1.00 ± 0.03 and for Carbopol 934 gels, it varies from 1.10 ± 0.05 to 0.96 ± 0.03.
Valuess of mucoadhesive strengths of the gels are besides displayed in Table I which shows that with addition in concentration of the gels from 3 % to 5 % , mucoadhesive strengths of LUM gels varies from 16.26 ± 0.98g to 22.45 ± 1.02g. For HPMC gels and Carbopol 934 gels, it ranges from 8.00 ± 0.85g to 11.95 ± 0.56 g and from 9.63± 1.02g to 16.39 ± 0.89grespectively.
Calibration curve of midazolam hydrochloride prepared in coney plasma was found to be additive over the concentration scope of 10-1,000 ng/ml ( r2=0.9999 ) . The experiment was repeated six times a twenty-four hours and for six back-to-back yearss. Interday and intraday truth and preciseness values are displayed in Table II.
Plasma concentration-time profiles of Versed after disposal of i.v. solution and the nasal gels are shown in Figure 1. Pharmacokinetic parametric quantities were displayed in Table III. Cmax values of i.v. injection, LUM, HPMC and Carbopol 934 gels were 573.64±5.23, 135.03 ± 10.25, 101.99 ± 4.59 and 110.52 ± 5.02 ng/ml, severally. tmax values of i.v. injection, FCM, HPMC and CP gels were 0.00, 30.00 ± 9.76, 60.00 ± 6.25 and 45.00 ± 7.88 min, severally. t1/2 values of i.v. injection, FCM, HPMC and CP gels were 58.78±6.32, 163.76 ± 7.05, 79.04 ± 3.96 and 138.78 ± 8.36 min, severally.
FIGURE 1 – Plasma concentration-time profiles of Versed hydrochloride after disposal of endovenous solution and the rhinal gels in coneies. Data represent mean ± SD ( n=3 ) .
Consequences of texture profile analysis reveal that with addition in concentration of the mucoadhesive agent, values of hardness and adhesion besides increased whereas coherence was found to diminish. Hardness of a gel determines the drug release form from the gel. From the consequences it can be said that gel incorporating 3 % LUM showed optimal hardness. Adhesiveness determines proper gel contact and keeping at the site of application, thereby taking to enhanced bioavailability of the drug. In the present survey, adhesion of gels was enhanced significantly with addition in sum of mucoadhesive agent. Cohesiveness was observed to be reduced with addition in sum of mucoadhesive agents. This happens because with addition in sum of spread solids, that is the mucoadhesive agents, semisolid nature of the gels increased, which caused the gel to go less consistent ( Cevher et al, 2008 )
Mucoadhesive strength of LUM gels was found to be higher than those prepared with man-made mucoadhesive polymers and it increased with corresponding addition in concentration of mucoadhesive agent used. This may be due to presence of certain functional groups in the mucilage were able to set up a more intimate contact with mucin of the mucous membrane ( Basu et al, 2009 ) .
Higher plasma concentration of midazolam hydrochloride is observed in instance of LUM gels in comparing to HPMC and Carbopol 934 gels ( Figure 1 ) , and consequently, absolute bioavailabilities of Versed from LUM, HPMC and Carbopol gels were reported to be 97.55 % , 57.33 % and 76.81 % , severally. From the in vivo survey we can reason that bioavailability of Versed from rhinal gels prepared from LUM was better than those prepared from man-made polymers. This confirms that LUM can be efficaciously used as a mucoadhesive agent alternatively of man-made polymers for bringing of Versed via rhinal path.
From the above survey it is confirmed that mucoadhesive rhinal gels of Versed hydrochloride prepared with mucilage isolated from Linum usitatissimum L. seeds exhibited better mechanical belongingss every bit good as in vivo drug soaking up form. Besides, bioavailability of Versed was higher from the gels prepared with this natural mucilage than from man-made gels. Furthermore, in our old work, histopathological survey confirmed that LUM can be used safely as a mucoadhesive agent. Hence, this new dose signifier of Versed with a natural mucilage is a safe and cost effectual signifier of rhinal drug bringing system.