ISSARANI. by binding individual components of herbal extracts. 1. Introduction. embedded or dissolved in nanoparticles and can also

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1 Aniket et al., Page 18 International Association of Advances in Research and Development International Journal of Pharmaceutical Sciences International Journal of Pharmaceutical Sciences, 2015, 1(1),18-26 Preparation and Characterization of Soy-Phytosomes using Ethanol: Water (70:30) Solvent System 1 ANIKET*, 1 S.S. SAURABH, 1 NEHA PANDEY, 2 K.S. RATHORE 1 AJAY GAUR, 1 ROSHAN ISSARANI 1 Lachoo Memorial College of Science & Technology, Pharmacy Wing, Jodhpur (Raj.), India (342001) 2 Department of Pharmacy, College of Medicine and Health Sciences, Ambo University, Ambo, Ethiopia *Address for correspondence: E- mail: aniketsingh18@yahoo.com... Abstract: The bioavailability and absorption of water soluble phytoconstituents is erratic due to poor solubility of these constituents in gastrointestinal tract. This can be overcome by a novel delivery system known as phytosome technology in which water soluble phytoconstituents are allowed to react with phospholipids. For better and improved bioavailability, natural phytoconstituents must have a good balance between hydrophilicity (helps in dissolution in gastro-intestinal fluids) and hydrophobicity (helps to cross lipid rich cell membranes). This is achieved through phytosome technology. Phospholipids have a dual solubility and acts as an emulsifier. Phytosome technology acts as a bridge between novel and conventional delivery systems. Many products are available in the market based on this phytosome technology which includes popular herbal extracts such as Ginkgo biloba, Silybum marianum, grape seed, olive oil flavonoids etc. Keywords: - Phytosomes, liposomes, phytoconstituents, bioavailability, phospholipid complex Introduction by binding individual components of herbal extracts Phytosome is a patented technology to phosphatidylcholine, resulting in a dosage form developed by a leading manufacturer of drugs and that is better absorbed and thus, produces better nutraceuticals, to incorporate standardized plant results than the conventional herbal extracts [16-18]. extracts or water soluble phytoconstituents into The results indicate that the absorption of silybin phospholipids to produce lipid compatible from silybin phytosome is approximately seven molecular complexes, called as phytosomes and so times greater compared to the absorption of silybin vastly improve their absorption and bioavailability from regular milk thistle extract [19]. Drugs can be [1-3]. embedded or dissolved in nanoparticles and can also The term phyto means plant while some be adsorbed or coupled on the surface. means cell like. Phytosomes are one of the novel Encapsulating drugs within NPs can improve the drug delivery systems containing hydrophilic solubility and pharmacokinetics of drugs, and, in bioactive phyto-constituents of herbs surrounded some cases, enable further clinical development of and bounded by phospholipids [4]. The phytosome new chemical entities that have stalled because of technology produces a little micro sphere or little poor pharmacokinetic properties [20-22]. The major cell, which protects the plant extract or its active carrier materials of nanoparticles are synthetic constituent from destruction by gastric secretion biodegradable high molecular polymer and natural and gut bacteria due to the gastro-protective polymer. The former usually includes poly-αcyanoacrylate alkyl esters, polyvinyl alcohol, property of phosphatidylcholine [5-7]. This phytophospholipid complex resembles a little cell which polylactic acid, and polylacticglycolic acid, etc. The exhibits better pharmacokinetic and latter is usually divided into two classes: proteins pharmacodynamic profile than the conventional (albumin, gelatin and vegetable protein) and herbal extract resulting in better bioavailability [8-10]. polysaccharides (cellulose, starch and its The phytosome process has also been derivatives, alginate, chitin and chitosan, etc.) [23-26]. applied to many popular herbal extracts including 2. Materials and Methods Ginkgo biloba, grape seed, hawthorn, milk thistle, Ethanol(Jai Pharma and Chemical Works, green tea, and ginseng [11]. The flavonoid and Jaipur), Methanol(Merck Specialities Private terpenoid components of these herbal extracts lend Limited, Mumbai), Soy-Lecithin(Ases Chemicals themselves quite well for the direct binding to Private Limited, Jodhpur), Di-methyl-sulphoxide phosphatidylcholine [12-15]. Phytosome is produced (DMSO) (S.D. Fine Chem. Ltd, Mumbai), n-

2 Aniket et al., Page 19 Hexane(S.D. Fine Chem. Ltd, Mumbai), Whatman filter paper-42(whatman International Limited Maidstone, England), De-ionized water and other chemical and solvents were of analytical grade/ip/equivalent gradeand procured from laboratory. 2.1 Formulation of Soy extract-phytosomes The of phytosomes consists of two stages: Preparation of soy extract- phospholipid complex Preparation of soy-phytosomes using a suitable method. 2.2 Formulation Design For of soy-phytosomes, it is proposed to use the following formulation composition in Table 1. Use of ingredients: Uses of the ingredients which are presented in the formulation composition table are: soy extract as active moiety having anti-cancer property; soy lecithin as emulsifier, stabilizer and lubricant agent; ethanol, methanol and de-ionized water used as solvent system in formulation. 2.3 Preparation of soy extract-phospholipid complex: Selection of the appropriate solvent system: Various types of solvent systems were used to achieve homogenous dispersion of extract and phospholipid, which were as follows: Methanol: According to literature survey methanol was selected as solvent. 5 ml of extract and 5 ml of phospholipid (soy-lecithin) were taken (i.e. 1:1 ratio) in a conical flask. To this mixture, 20 ml of methanol was added (i.e. 1:2) proportion and the mixture was stirred using magnetic stirrer at 800 rpm for 1 hr. Mixture of extract and phospholipid was not completely dissolved in methanol solvent. Therefore another solvent system was used. Methanol: Water (60:40; v/v): Due to slight solubility in methanol of phospholipid mixture, another solvent system comprising methanol and water in ratio of 60:40 was used. SE and PL (soylecithin) was taken in conical flask, each 5mL and mixed with 20 ml solvent system and mixture was stirred using magnetic stirrer at 800 rpm for 1 hr. This solvent system proved to be better solvent for SE-PL complex. However, after keeping the solution for about 24hrs, phase separation of the phospholipid and solvent not occurred. So this solvent system could be used. Methanol: Water (40:60; v/v): The proportion of methanol and water was changed to 40: ml of solvent system was prepared by adding 8mL of methanol and 12mL of de-ionized water. Mixture was stirred at 800 rpm for 1 hr. Using methanol: water (40:60; v/v), a better extract-phospholipid mixture could be prepared. No phase separation of solvent and PL occurred even after 24 hrs. So this solvent system could be used for of SE-PL complex. Ethanol (99.5%): Ethanol is the best solvent for phospholipid (soy-lecithin). The SE-PL in a ratio of 1:1 was mixed with ethanol in 1:2 ratios and mixture was stirred at 800 rpm for 45 min, to avoid phase separation. Phospholipid-extract mixture was better solubilized in ethanol and there no phase separation occurred even after 24 hrs in mixture. This indicates that ethanol is more preferable for the of SE-PL mixture. Ethanol: Water (70:30): Since ethanol is highly volatile in nature so it necessitated adding deionized water and an ethanol: water in a ratio of 70:30 was used to check the solubility of SE-PL mixture. Mixture was stirred at 800 rpm for 30 min, for obtaining homogenous mixture. 2.4 Determination of appropriate ratio of soy extract phospholipid: The formulation components were placed in a 100 ml conical flask and the contents were magnetically stirred for 30 min followed by Sonication for another 15 min. 2.5 Preparation of soy-phytosomes The spray drying method was used for of phytosomes from selected batches F1, F2 and F4 of SE-PL mixture. 2.6 Optimization of the process variables: To prepare soy-phytosomes using the spray drying process, process variables to be optimized were: i. Stirring speed and stirring time ii. Sonication time and sonication temperature iii. Spray drying: a) Feed flow rate; b) Inlet/Outlet temperature; c) Aspirator flow rate; d) Air flow rate and pressure. Stirring speed and Stirring time: For optimization of the stirring speed and stirring time for obtaining soy-phytosomes, SE-PL mixture was prepared into various batches which were denoted in the following Table 2. Sonication time and temperature: For optimization of the Sonication time and sonication temperature for obtaining soyphytosomes, SE-PL mixture was prepared into

3 Aniket et al., Page 20 various batches and using bath Sonicator, sonication time and temperature were studied as discussed in following Table 3. Table 1: Formulation composition of Soy-Phytosomes Ingredients (ml) F1 F2 F3 F4 F5 F6 Soy-powder extract Phospholipid (Soy lecithin) Methanol: Water (60:40) Methanol: Water (40:60) Ethanol: Water (70:30) Table 2: Optimization of stirring speed and stirring time for soy-phytosomes S. No Formulation ratio Stirring speed Stirring time Product state on keeping for 24 hrs. (rpm) (min) 1. 1: Instable mixture 2. 1: Improper mixing 3. 1: Better than above two but nonhomogenous mixture 4. 1: Homogenous mixture obtain but phase separation started 5. 1: Good homogenous mixture 6. 1: Good homogenous mixture 7. 2: Instable mixture 8. 2: Improper mixing 9. 2: Better than above two but nonhomogenous mixture 10. 2: Good homogenous mixture 11. 2: Good homogenous mixture 12. 2: Acceptable homogenous mixture Table 3: Optimization of sonication time and sonication temperature for soy-phytosomes S. Formulation ratio Sonication time (min) Sonication temp. range Product state after completion of sonication 1. 1: C No proper mixing of extract and phospholipid 2. 1: C Better mixing than previous 3. 1: C Result in a good mixture 4. 1: C Good mixing of the soy extract and phospholipid occurred 5. 1: C Result in an excellent mixing of the Soy extract and Phospholipid with solvent system 6. 1: C Homogenous mixing but phase degradation occurred 7. 2: C Homogenous mixing but phase degradation occurred 8. 2: C Result in an excellent mixing of the Soy extract and Phospholipid with solvent system Good mixing of the soy extract and phospholipid occurred 9. 2: C 10. 2: C Result in a good mixture 11. 2: C Better mixing than previous 12. 2: C No proper mixing of extract and phospholipid Spray Drying Soy-phytosomeswere prepared by spray drying method. Aqueous dispersion of SE-PL mixture of 1:1 and 2:1 ratios were spray dried and various process parameters were studied. The mixtures were sonicated up to 15 min. Then the mixtures were spray dried using Lab spray dryer (LU-222 Advanced Labultima, India) with 0.7mm nozzle. Parameters of spray dryer were changed and products were collected separately in each run. Process variables that were studied during spray drying method were as follow: - a) Feed flow rate

4 Aniket et al., Page 21 (F.F.R.); b) Inlet/Outlet (I/O) temperature; c) Aspirator flow rate; d) Air flow rate and pressure. There are various process variables studied are summarized in Table 4 and 5 under 4(a), 5(a) and 4(b), 5(b) where 4(a) and 5(a) provide data on aspirator flow rate of 25 liter/minute and air flow rate of 1 kg /cm 2 and vacuum air pressure will be of approximate 6±1 psi; and 10(b) and 11(b) provide data on aspirator flow rate of 50 l/min and air flow rate of 1 kg /cm 2 and vacuum air pressure of approximate 6±1 psi. Table 4: Process variables for formulation ratio 1:1-4 (a)- At aspirator flow rate of 25 l/min F.F.R. (ml/min) I/O Temp. (in o C) Inference 65/ /65 No phytosomal powder occurred at first three temperature range but a very low yield of powder found at last temperature range F.F.R. (ml/min) 1 70/50 100/70 110/80 115/85 110/75 120/80 95/75 115/80 125/85 100/80 120/85 130/90 4(b) - At aspirator flow rate of 50 l/min I/O Temp. (in o C) 65/45 95/65 In first two temperatures range, there was no phytosomal powder but in last two ranges a feasible quantity of powder seen in collector. Same condition as previous parameters but better yield found on 115/85 temperature range was noticed in this section. Good yield of phytosomal powder occurred at 120/80 temperature but remaining parameters were failed to produce phytosomes. A feasible quantity of powder at 115/80 and 125/85 temperatures occurred but remaining ranges were again failed. Not much quantity of powder had seen but at 120 C small amount of phytosomes yield in this section. Inference No phytosomal powder occurred at first three temperature range but very minute quantity of powder found at last temperature range. 70/ /70 110/80 Good yield of phytosomal powder occurred at 110/80 temperature but remaining parameters were failed to produce powder /85 Better yield of powder occurred at 115/85 temperature but remaining parameters were failed to produce powder in a feasible amount /75 120/80 In first two temperatures range, there was no phytosomal powder but after that, in last two ranges a feasible amount of powder had seen. 95/ /80 A very low yield of phytosomal powder occurred at last two temperature ranges. 125/ /80 120/85 130/90 Not much quantity of powder had seen but at 120 C and 130 C small amount of phytosomes yield in this section. Table 5: Process variables for Formulation ratio 2:1-5(a)-At aspirator flow rate of 25 l/min F.F.R. (ml/min) I/O Temp. (in o C) Inference 65/45

5 Aniket et al., Page No phytosomal powder occurred at all temperature range but a very viscous 95/65 liquid found at last temperature range. 70/50 100/70 110/80 115/85 110/75 120/80 95/75 115/80 125/85 100/80 120/85 130/90 5 (b)- At aspirator flow rate of 50 l/min F.F.R. (ml/min) I/O Temp. (in o C) 65/ /65 70/ /70 110/ / /75 120/80 95/ /80 125/85 100/ /85 130/90 3. Characterization of the prepared soyphytosomes : Behavior of phytosomes in both physical and biological systems is governed by factors such as physical size, membrane permeability, percentage of entrapped solutes and chemical In last temperature range a feasible quantity of phytosome powder had seen in collector. Same as previous section but good yield of phytosomes at 115/85 temperature range was noticed. Good yield of phytosomal powder occurred at 120/80 temperature but remaining parameters were failed to produce powder. A very less feasible quantity of powder at 115/80 and 125/85 temperatures occurred but remaining ranges were again failed. Not much quantity of powder had seen but at 120 C small amount of phytosomes yield in this section. Inference No phytosomal powder occurred at first three temperature range but feasible quantity of powder found at temperature. Good yield of phytosomal powder occurred only at 110/80 temperature. Very high amount of phytosomal powder occurred at and 115/85 temperature ranges. A good yield of phytosomes seen at 120/80 temperature. A very low yield of phytosomal powder occurred at last two temperature ranges. Not much quantity of powder had seen but at 120 C and 130 C small amount of phytosomes yield in this section. compositions as well as quantity and purity of starting material. Phytosomes can be characterized in terms of their physical attributes i.e. shape, size, distribution, percentage drug captured, entrapped volume, percentage drug released and chemical

6 Aniket et al., Page 23 composition. Some of characterization of prepared soy-phytosomes was performed as follow: 3.1 Physical appearance: Various parameters like color, odor, taste and physical state of were characterized and summarized in Table Visualization of Shape: Shape of prepared batches of phytosomes and SE-PL mixture was optimized by using optical microscopy. Slides of phytosomes and SE-PL mixtures were prepared and studied under microscope. 3.3 Vesicle size determination: The vesicle size of phytosome mixture can be determined by optical microscopy. The size distribution of soy-phytosome is presented in Table Drug content determination: Accurately weighed one gm of phytosome was dissolved in q.s. 100 ml solvent system. The volumetric flask was kept for 2 hrs and shaken well to mix properly. The solution was filtered through Whatmann filter paper and suitably diluted. The absorbance of the solution was measured spectrophotometrically at 281.4nm. The drug content of both ratios of phytosome formulations was determined which is shown in Table Results and Discussion The ethanol: water in ratio of 70:30 proved to be best solvent system amongst all solvent Table 6: Optimized process variables for formulation ratios 1:1 and 2:1 Formulation Feed Inlet/outlet Aspirator Air flow rate temp. flow rate flow Ratio (ml/min) range ( C) (l/min) rate and pressure 1:1 3 and and 3 120/ kg/cm 2 2:1 2,3 and 4 120/ and 6±1 psi 120/ and / Table 7: Physical appearance of prepared phytosome-batches Sr. Parameters Observation 1:1 2:1 1. Color Yellow in color Light yellow in color 2. Odor Odorless Odorless 3. Taste Tasteless Tasteless 4. Physical state Powder Powder Table 8: Size distribution of prepared soy-phytosomes Sr. Vesicle Size Average Vesicle 1:1 ratio 2:1 ratio Size I II I II ± S.D. (µm) 1:1 ratio 2:1 ratio ±7 85± ±21 50±28 systems with no phase separation even after 24 hrs. So this solvent system was selected for of the phytosomes. Formulation composition F1, F2, F4 resulted in formulation of homogenous SE-PL mixture without any evidence of phase separation, even after 24 hrs. In formulation composition F3 and F5 a homogenous phase was obtained but it tended to phase separation on standing for 24 hrs. F6 formulation composition was viscous not suitable for spray-drying. So formulation of SE- PL mixture having 1:1 ratio of SE: PL i.e. F1, F2 and F4 formulas shall be used for of phytosomes. On stirring speed of 700 rpm, there were no was showing homogenous mixture on stirring time range from min for both ratios 1:1 and 2:1 whereas on stirring speed of 800 rpm for 1:1 stirring time range was min and for 2:1 time range of stirring was min. Best stirring speed for both ratios 1:1 and 2:1 was 800 rpm. Similarly suitable stirring time range for phytosome for 1:1 was min whereas for 2:1 was min ±0 75± ±7 30± ±14 70± ±7 90± ±0 105± ±7 60± ±21 70± ±21 45±7 Average vesicle size: (I) For 1:1 ratio = 1095/10= µm (II) For 2:1 ratio = 680/10= 68 µm Table 9: Drug content of prepared phytosome formulation Sr. Formulation Drug content (%)* 1. F1 (1:1) 96.76± F2(2:1) 92.51±0.21 * Data indicates mean ± S.D. of triplicate determinations Table 10: Optimized process variables for formulation ratio 1:1 Formulat ion Ratio Feed flow rate (ml/min ) Inlet/outle t temp. range ( C) Aspira tor flow rate (l/min) Air flow rate and pressure 1:1 3 and / kg/cm 2 and 6±1 psi Table 11: Physicochemical characterization of prepared phytosomes Sr. Parameters 1:1 Observation 2:1

7 Aniket et al., Page Physical appearance Color Odor Taste Physical state Yellow in color Odorless Tasteless Powder Light yellow in color Odorless Tasteless Powder At sonication temperature range C, better homogenous mixture of soy-extract and phospholipid was obtained at sonication time of 45 min for 1:1 ratio and for 2:1 ratio, sonication time was 30 min. Whereas at sonication temperature range C, best homogenous mixing occur at sonication time of 15 min and 30 min for 2:1 ratio and 1:1 ratio, respectively. At temperature range of C and C other sonication time intervals was not sufficient to produce a best stable homogenous mixture after keeping them on 24 hrs, since either they lead to phase separation or no properly form a complex mixture. Phytosomes were prepared by spray drying technique using Lab spray A 2. Vesicle size determination 3. Drug content determination µm 68 µm (96.76±0.47) % (92.51±0.21) % dryer (LU-222 Advanced Labultima, India) with 0.7mm nozzle and various process parameters were studied during of phytosomes. Phytosomes were prepared at parameters which were presented in TABLE 4 and 5. Prepared phytosome batches of 1:1 and 2:1 ratios were odorless and tasteless but they varied in color and physical state, 1:1 ratio batch was yellow in color and fine powder whereas 2:1 ratio batch was light in color and crystalline powder. This variation can be due to variation in the concentration of the soy extract phospholipid and solvent medium and spray dryer parameters. Optical micrographs were captured as shown in Figure 4. B Figure. 1: SE-PL mixture with Ethanol: Water (70:30) solvent system (A) During formulation; (B) After kept 24 hrs-no phase separation 1:1 2:1 Figure. 2: SE-PL mixture with Ethanol: Water (70:30) solvent system after Stirring and Sonication (A) (B) (C) (D) Figure. 3: Spray drying-(a) and (B) before spray drying (C) and (D) are spray dried powder formulation of 1:1 and 2:1 ratio respectively

8 Aniket et al., Page 25 (A) (B) Figure. 4: Visualization of shape - (A) SE-PL mixture and (B) Spray dried Phytosomes A B Figure.5: Vesicle size determination (A) 1:1 ratio and (B) 2:1 ratio 100µm 100µm For 1:1 ratio of SE-PL mixture average vesicle size was µm whereas vesicle size for 2:1 ratio was 68 µm obtained and shown in Figure3. This difference in size is due to various spray drying parameters and concentration of soy-extract used for formulation. For 1:1 ratio of SE-PL mixture average vesicle size was µm whereas vesicle size for 2:1 ratio was 68 µm obtained and shown in Figure 5. This difference in size is due to various spray drying parameters and concentration of soy-extract used for formulation. The drug content of the formulations ranged between (92.51±0.21) percent to (96.76±0.47) percent. Conclusion In the present dissertation work, an attempt was made to prepare soy-phytosomes. For the purpose, extract of soybean was prepared and subjected to phyto-chemical screening. The chief constituent in extract was found to be flavonoid which is reported to be responsible for anticancer activity; % yield of extract was fond to be 14% w/w. Solvent system for of SE-PL complex was optimized. Amongst the various solvent systems investigated: methanol, ethanol, methanol: water (40:60), methanol: water (60:40), ethanol: water (70:30), three solvent systems were found to be promising, namely- methanol: water (40:60) and (60:40) and ethanol: water (70:30). The ratio of SE: PL for the of complex was optimized. Amongst the various ratios (1:1 and 2:1) ratio of 1:1 was best suited for the formation of phytosomes. Two formulations were prepared successfully using soy-extract and phospholipid in ratio of 1:1 and 2:1; with solvent system ethanol: water (70:30) using the optimized spray drying condition. Acknowledgements Authors are profusely thankful to Lachho Memorial College of Science and Technology, Jodhpur and BN Institute of Pharmaceutical Sciences, Udaipur staff for their constant and perennial support and friends Vikas Mehta, Rekha Singh Saurabh and Sandeep Arora. References 1. Ajazuddin SS. Elsevier- Fitoterapia, 81, Kareparamban JA. International Journal of Research in Pharmacy and Chemistry; 2(2), Suryawanshi JAS., Journal of Medical Genetics and Genomics; 3(6), Mohd AA. Elsevier- Drug Discovery Today Bombardelli E. BollChim Farm.; ,130, Bombardelli E, Spelta M. Cosm. & Toil , 106, Murray D. (Online available atwww.doctormurray.com/articles/silybin.htm).retrie ved on December20, 2015) Choubey A. International Journal of Pharmaceutical Sciences and Research; , 2(4), Chowdhury RS. International Research Journal of Pharmacy; 60-64, 3(10), Jain N. International Journal of Pharmaceutical Sciences and Drug Research; , 2(4), Vangapelli S. International Journal of Pharmaceutical Research and Development; , 3(6), Tripathy S. Journal of Drug Delivery and Therapeutics; , 3(3), Retrieved on December21, Retrieved on December21, Rathore P, Swami G. International Journal of Pharmaceutical Sciences and Research, (IJPSR); 3: 3, Retrieved on December22,

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