1.1 INTRODUCTION:

Over the past century, phyto-chemical and phytopharmacological sciences established the composition, biological activities and health promoting benefits of numerous plant products (Bhattacharya et al, 2011.). But most of the biologically active constituents of plants are poorly soluble in water and organic solvents make the bioavailability erratic and poor. Several plant polyphenolics and terpenes are ubiquitous in plants, but there poor solubility in different phase is the major concern limiting their utilization. The limited solubility both in water and in lipophilic phases, as well as hydrolytical instability at physiological pH values make several botanical extracts containing polyphenolics and terpenes and its individual constituents very poorly absorbed both orally and topically.

Bioavailability can be improved by using novel delivery systems which can enhance the rate and the extent of solubilization into aqueous intestinal fluids and the capacity to cross biomembranes (www.phytosome. info, 2013). Recently novel drug delivery system got attention by many researchers in phytoformulation fields. The variety of novel herbal formulations like polymeric nanoparticles, nanocapsules, liposome, phytosomes, nanoemulsions, microsphere, herbosome and ethosomes has been reported using bioactive and plant extracts (Ghosh et al, 2009, Awasthi et al, 2011 and Parveen et al, 2011.). The novel formulations are reported to have remarkable advantages over conventional formulations of plant actives and extracts which include enhancement of solubility, stability, bioavailability, protection from toxicity, physical and chemical degradation, improved tissue macrophages distribution and sustained delivery. Thus novel drug delivery systems of herbal drugs have a potential future for enhancing the activity and overcoming problems associated with plant medicines (Ajazuddin et al, 2010). There are several polyphenolics such as curcumin, silybin, green tea flavan-3-ol, grape seed proanthocyanidin have several activity but having poor bioavailability (Kidd et al, 2009).

2.1. MATERIAL AND METHODS:

2.1.1Method of preparation of phytovesicles:

On the basis of various literature serve total four method of preparation of phytosome

were found i.e.

(1) Solvent evaporation method (Minakshi et al, 2012 and Freag et al, 2013)

(2) Salting out Techniques

(3) Lyophilization Techniques

(4) Mechanical Dispersion method

METHODOLOGY:

2.2.1 Preformulation studies (Gupta et al, 2011)

2.2.1.1 Melting Point:

Melting point of Rosmarinic acid was determined by digital melting point apparatus.

2.2.1.2 Solubility study:

Solubility of rosmarinic acid, rosmarinic acid–phospholipid complex, physical mixture and phytovesicle of rosmarinic acid and phospholipid were obtained by adding a weighed amount of the each samples to 5 ml of different solvent system viz. water, methanol, chloroform, dichloromethane and hexane in sealed glass vial at room temperature. Each glass vials were then vortexed for 5 minutes and then observes visually.

2.2.1.3 Determination of λmax of rosmarinic acid:

The λmax of rosmarinic acid in ethanol: PBS (1:9) was determined using UV spectrophotometer.

The λmax was determined by scanning different concentration of RA in ethanol: PBS in UV range of 200 – 400 nm.

2.2.1.4 Preparation of calibration curve of rosmarinic acid:

Calibration curve of RA was prepared in ethanol: phosphate buffer (1:9) at λmax 324 nm. The stock solution of rosmarinic acid (100μg/ml) was prepared. From this stock solution a range (2-10μg/ml) of dilutions were prepared and scanned for their ultraviolet absorbance at 324 nm using UV-spectrophotometer.

Preparation of phytovesicles of rosmarinic acid:

2.2.2.1 Solvent evaporation technique:

Solvent evaporation technique followed by precipitation is a most suitable method for phytovesicle formation (Gupta et al, 2011). Phytovesicles were fabricated in two steps;

Step 1: Formation of phospholipid-drug complex

Step 2: Preparation of phytovesicle:

2.2.3 Optimization via response surface methodology:

The prepared phytovesicle was further optimized on the basis of entrapment efficiency and drug release using Stat Ease Design of Experiment trial version 9.0.3.1.

2.2.3.1 Experimental design:

The batches were prepared as indicated by the software generated runs using Central Composite Design.

2.2.3.2 Response surface methodology:

Central composite design (CCD) of response surface methodology (RSM) was employed to produce phytovesicles of RA using a polynomial equation with the help of Design-expert 8.0.7.1 software (Trial version; Stat- Ease Inc., USA). In this design, the individual effect of two independent variables namely phospholipid-to-RA ratio (X1, mol/mol) and refluxing time (X2, in hrs) were analyzed on the entrapment efficiency (Y1) and cumulative percent drug release (Y2) (dependent variable). The lower and higher value of drug-polymer ratio and refluxing time were selected on the basis of preliminary experimental results.

Briefly, these two factors with three levels each were evaluated and experimental trials were performed at all 13 possible combinations (Table 1.1 & Table 1.2). Entrapment efficiency and cumulative percentage drug release were taken as the response variable (Malakar et al, 2014).

The level values of two independent variables and the composition of the central composite design batches were presented in Tables 1.1 and1.2

Table 1.1 Coded Levels and “Real” Values for each factor under study

Variable	Code		-x	-1	0	+1	+x	∆x1
DPR (drug: polymer)	X1	29.5:100		1:2	1:1	3:2	170.5: 100	0.5
RT (refluxing time)	X2	1.18		2	4	6	6.82	2

Table 1.2 Composition of batches

Run	Standard	Factor1(DPR)	Factor2(RT)
1	2	1	(-1)
2	9	0	0
3	7	0	(-1.41)
4	10	0	0
5	5	(-1.41)	0
6	6	(1.41)	0
7	8	0	1.41
8	1	(-1)	(-1)
9	13	0	0
10	12	0	0
11	4	1	1
12	11	0	0
13	3	(-1)	1

2.2.4 Characterization of phytovesicles:

The prepared batches were characterized to obtain the dependent variables (responses).

2.2.4.1 Determination of drug encapsulation efficiency [DEE]:

Accurately weighed phytovesicle which is equivalent to 10mg of rosmarinic acid were taken in a centrifuge tube and added 1ml of alcohol and vortex it for 5min. After vortexing the sample was sonicated using small probe sonicator for 5min. The energy was provided in pulsative mode as continuous energy produced heat that may cause drug and phospholipids degradation. Both these operation i.e. vortex and sonication lead complete leach out of drug from phytovesicles into the phosphate buffer. 39 Finally volume was make up to 10ml by using phosphate buffer saline (PH-7.4). Finally, the encapsulation efficiency of the sample was determined using a UV–visible spectrophotometer by measuring absorbance at 324nm λmax. The % DEE of phytovesicles was calculated using this following formula (Brewster et al, 2007)

% drug entrapment efficiency = Actual Drug Content in Phytovesicle/ theoretical content X 100

2.2.4.2 In vitro drug release profile:

In vitro release studies of Rosmarinic acid from prepared phytovesicle and pure Phytomolecules were performed at room temperature by using USP dissolution apparatus. A 200mL closed conical flask containing PBS: Ethanol (9: 1) solutions were used as a dissolution media. Weighed amount of rosmarinic acid and phytovesicles equivalent to 10mg of RA were loaded into gelatin capsules separately. The capsules were then placed into basket followed by dipping into dissolution media. The temperature of media is maintained as 35±0.5ºC. 2ml of sample was withdrawn at regular time intervals, and soon after same amount of fresh buffer was placed into dissolution vessel keeping the sink condition throughout the experiment. The collected samples were analyzed to find out the amount of drug release from the vesicle by using UV–visible spectrophotometer, measuring absorbance at λmax of 324 nm (Brewster et al, 2007)

2.2.4.3 Phase solubility study:

Phase solubility study of complexing agents on the compound being solubilized is a traditional approach to determine not only the value of the stability constant but also to give insight into the stoichiometry of the equilibrium (Campos et al, 2014).

2.2.4.4 Fourier transform infra-red (FT-IR) analysis:

The spectroscopic evaluation of the formed complex can be confirmed by FTIR simply by comparing the spectrum of the complex and the individual components and that of the mechanical mixtures (Shukla et al, 2012). The spectrum was recorded between 400 and 4000 cm-1.

2.2.4.5 Vesicle shape/surface morphology/internal structure:

(a) Transmission electron microscopy:

Vesicular systems were visualized by Transmission Electron Microscope. A drop of formulation i.e. phyto-vesicles was placed on different carbon coated copper grids to leave a thin film on the grids. Then, the film was negatively stained with 1% phosphotungstic acid by placing a drop of the staining solution on to the film and the excess of the solution was drained off with a filter paper. The grid was allowed to dry and formulations were viewed under the transmission electron microscope and photographs were taken at suitable magnification (Gupta et al, 2011).

(b) Scanning Electron Microscope:

The particle size and surface morphology of the optimized phytovesicle loaded with rosmarinic acid was examined by means of a JEOL, Japan (Model-JSM-6490 LV) scanning electron microscope (SEM). (Pouchers 10th edition).

2.2.5 Preparations of cream base for incorporation of rosmarinic acid and phytovesicles:

Choices of excipients for o/w cream semisolid vehicles were developed with the reference to earlier published research data with slight modification.

RESULTS AND DISCUSSIONS:

3.1 Preformulation study:

3.1.1 Melting point:

The melting point of rosmarinic acid was found to be 171-176ºC.

3.1.2 Solubility:

The solubility of rosmarinic acid, lecithin, RA-PC complex and phytovesicle of rosmarinic acid is given in Table 2.1. The data indicate the RA-PC complex and their phytovesicles both are soluble in aqueous and organic phase. Whereas rosmarinic acid and lecithin have not significant solubility for both the phases. So on the basis of this parameter it could be anticipated that phytovesicle and their complex may maintain the balance hydrophilicity and lipophilic balance, which is required for better bioavailability.

3.1.3 Scanning of rosmarinic acid for λmax:

Spectrophotometric studies were carried out in order to determine the λmax of rosmarinic acid in ethanol: PBS (1:9). λmax was determined by scanning RA in test medium in the range of 200-400 nm. The observed absorbance maxima was found to be 324 nm.

3.1.4 Preparation of calibration curve:

The calibration curve of drug was prepared in the concentration range of 2-8 μg/ml in ethanol: PBS (1:9) at λmax 324 nm. Absorbance of each dilution was taken and a grap was plotted between absorbance and concentration and standard curve of rosmarinic acid was obtained.

Figure 3.2 Calibration curve of Rosmarinic acid in ethanol: PBS (1:9) at λmax 324 nm

3.1.4.1 Statistical Test:

Calibration curve data was subjected to linear regression analysis and various parameters were calculated. Rosmarinic acid was found to obey beer –Lambert law in the concentration range of 2-10 μg/ml with regression coefficient (r2) value 0.9993 in ethanol: phosphate buffer (1: 9). The regression equation was calculated as Y=0.0578x-0.0022.

3.2 Optimization of phytovesicles preparation:

Response surface methodology was used to analyze the influences of main effects (factors) on responses (DEE and CDR). It is a widely applied approach in the development and formulation optimization of drug delivery devices in pharmaceutical technology (Malakar et al, 2014). The 3D response surface graph is very useful in learning about the main and interaction effects of the independent variables (factors). In the present investigation, a total 13 trial formulations were proposed by the central composite design for two independent variables i.e. drug polymer ratio and refluxing time which were varied at three levels (low, medium and high). The effect of these independent variables on DEE and CDR were investigated as response parameters. According to central composite design, various trial formulation of RA, phytovesicles were prepared (Table 2.2). The result of ANOVA indicated that the investigated models were significant for all response parameters (Table 2.3). Out of all 13 batches, the optimized batch was found to have the percent entrapment efficiency of 71.798 and cumulative percentage release of 75.708 in 10h with desirability factor of 0.972.

The model equation relating DEE as response:

DEE (%) = 71.80 + 4.11*A + 0.42*B -0.15*AB – 4.51*A2-1.00*B2………………………………...Eq. (1)

[F-value 127.05 r2= 0.9906]

The model equation relating CDR as response:

CDR (%) = 75.71+2.33*A-0.73*B-0.57*AB-7.44*A2-4.94*B2…………………………………………...Eq. (2)

[r2 = 0.9850 F-value= 78.75]

Table 2.2: Levels of independent variable along with their response variable result:

Batch	DPR	RT	EE*	CDR**
1	-1	-1	62	61.78
2	1	-1	70.01	68.37
3	-1	1	63.27	60.55
4	1	1	71.22	64.87
5	-1.41	0
6	1.41	0	68.11	62.98
7	0	-1.41	69.12	65.67
8	0	1.41	71.02	64.87
9	0	0	73.45	76.39
10	0	0	72.65	76.11
11	0	0	71.43	75.3
12	0	0	71.23	75.32
13	0	0	72.21	75.38

*Entrapment efficiency

**Cumulative drug release

Table 2.3 Summary of ANOVA for response parameters

Source	Sum of square	d.f.	Mean square	F value	p-value prob>F
For DEE%
Model	137.86	5	27.57	127.05	<0.0001
A	81.27	1	81.27	374.49	<0.0001
B	1.42	1	1.42	6.56	0.0428
AB	0.090	1	0.090	0.41	0.5434
A²	85.78	1	85.78	395.29	<0.0001
B²	6.54	1	6.54	30.13	0.0015
For CDR
Model	424.89	5	84.98	78.75	<0.0001
A	26.01	1	26.01	24.10	0.0027
B	4.29	1	4.29	3.98	0.0931
AB	1.29	1	1.29	1.19	0.3165
A²	233.35	1	233.35	216.24	<0.0001
B²	158.06	1	158.06	146.47	<0.0001

Table 2.5 In vitro percent cumulative drug released data of RA and different batches of phytovesicles

Time (hr)	RA	B1	B2	B3	B4	B6	B7	B8	B9	B10	B11	B12	B13
0	0	0		0	0	0	0	0	0	0	0	0	0
0.25	20.59	12.38	13.29	11.58	13.01	12.54	13.01	12.99	13.99	14.32	12.92	13.64	14
0.5	42.65	16.98	17.02	15.62	17.34	15.43	15.52	16.62	21.64	19.51	18.61	19.32	19.45
1	65.38	17.61	19.61	15.59	18.61	17.43	18.56	18	30.09	27.83	29.31	26.2	23.65
2	83.45	24.88	29.88	22.37	24.54	25.61	20.32	22.67	37.2	33.8	38.93	32.91	29.01
3	100	35.12	35.12	31.89	37.45	36.07	28.67	32.7	45	47.34	50.19	48.1	37.1
4	100	41.32	43.32	39.45	44.08	42.9	42.76	43.09	58.19	59.15	61	53.66	42.45
5	100	48.75	50.75	46.56	52.43	49.38	48.99	50.55	62.15	65.99	65.33	61.91	63.12
6	100	53.7	59.7	52.39	57.61	54.68	52.67	56.04	67.43	70.61	69.21	67.45	69.22
8	100	56.34	64.34	55.62	60.89	57.39	58.11	61.84	70.03	72.45	70.11	71.53	72.59
10	100	61.78	68.78	60.55	64.87	62.98	65.67	64.87	76.39	76.11	75.3	75.38	75.38

Table 3.1 Stability Study Parameter of optimized batch

Storage condition	Room Temperature		40ºC/75%RH				Specifications
Period	Initial		1 Month		2 Months
Formulations	Reference	Optimized batch	Refer Ence	Optimized batch	Reference	Optimized batch
Parameters	Observations
Physical Appearance							No change should observe
Hardness (N)	14 g	14.2 g	14.2 g	14.1 g	14.2 g	14 g	NMT 20 gm
consistency	2.2 mJ	2.2 mJ	2.1 mJ	2.2mJ	2.3mJ	2.2mJ	NMT 5 mJ
spreadablity	3.4 mJ	3.3 mJ	3.2mJ	3.3mJ	3.4mJ	3.3mJ	NMT 4 mJ
IR SPECTRO SCOPY	IR SPECTRO SCOPY	Same as reference	Same	Same	Same	Same	NO new peak or broading of peak is shown during whole stability study
Entrapmment Efficiency
Cumulative Drug Release (10 hours)	99.6	99	98.9	97.7	98.6	97.25	NLT 85% in 30 min.

CONCLUSION:

From the present studies it could be concluded that RA successfully complexed with phospholipids to form phytovesicles. The results of the central composite design study confirmed that the values of phospholipids to RA ratio and refluxing time significantly influenced the dependent variable (Entrapment efficiency and cumulative drug release). SEM, TEM and FTIR spectroscopy confirmed the formation of phytovesicles and non-covalent bonding (such as hydrogen bond) between RA and phospholipids thereby forming supra molecular structures. Stability study of final trial for 2 month were done and it indicates that optimized formulation were stable.

REFERENCE:

1. Mahalingam, R.; Li, X.; Jasti, B. semisolid Dosages: ointment, Cream, and gels. pharmaceutical science encyclopedia 2010 , 1-46.

2. Lackmann, L.; Lieberman, H. A..; Kanig, J. L.; A theory and practice of Industrial pharmacy, 3RD edition 534.

3. Mohan H (2005).Textbook of pathology 5th edition published by jaypee brothers new delhi

4. Tripathi, K. T.; Essential of medical pharmacology, 6th edition, japee brothers publisher.

5. Pouchers perfume cosmetic and soap 10th edition edited by Hilda butler Springer international edition page 430

6. Bhattacharya, S. Phytosomes: The New Technology for Enhancement of Bioavailability of Botanicals and Nutraceuticals. International Journal of Health Research. 2009, 2(3), 225-232.

7. Ghosh, A.; Mandal, A. K..; Sarkar, Sibani.; Panda, Subhamay.; Das. N. Nanoencapsulation of quercetin enhances its dietary efficacy in combating arsenicinduced oxidative damage in liver and brain of rats. Life Sciences. 2009, 84, 75–80.

8. Tsai, Wen-che.; Li, Wei-Chu.; Yin, Hsin-Yi, Yu, Ming-Chiang.; Wen, Hsiao-Wei.; Constructing liposomal nanovesicles of ginseng extract against hydrogen peroxideinduced oxidative damage to L929 cells. Food Chemistry 2012, 132, 744–751.

9. Awasthi, R.; kulkarni, G. T.; pawar , vivek. k. phytosomes: an approach to increase the bioavailability of plantextracts. International Journal of Pharmacy and Pharmaceutical Sciences. 2011, 3(2). 1-3.

10. Parveen, R.; Baboota, S.; Ali, J.; Ahuja, A.; Vasudev, Suruchi. S.; Ahmad, S. Oil based nanocarrier for improved oral delivery of silymarin: In vitro and in vivo studies. International Journal of Pharmaceutics. 2011, 413, 245– 253.

11. Shukla, A.;. Pandey, V.; Shukla, R.; Bhatnagar, P.; Jain, S. Herbosomes: A Current Concept of Herbal Drug Technology an Overview. Journal of Medical Pharmaceutical And Allied Sciences. 2012, 01, 39-56.

12. Mishra, N.; Yadav, N. P.; Meher, J. G.; Sinha, P. Phyto-vesicles: conduit between conventional and novel drug delivery System. Asian Pacific Journal of Tropical Biomedicine. 2012, 1728-1734.

13. Kim, H.-J.; Kim, T.-H.; Kang, K.-C.; Pyo, H.-B.; Jeong, H.-H. Microencapsulation of rosmarinic acid using polycaprolactone and various surfactants. International Journal of Cosmetic Science, 2010, 32, 185–191.

14. Ajazuddin, & Saraf, s. Applications of novel drug delivery system for herbal formulation., Fitoterapia 2010,81, 680–689.70

15. Kidd, P. M. Bioavailability and Activity of Phytosome Complexes from Botanical Polyphenols: The Silymarin, Curcumin, Green Tea, and Grape Seed Extracts. Alternative Medicine Review. 2009, 14, 226-246.

16. Patel, J.; Patel, R.; Khambholia, K.; Patel, N. An overview of phytosomes as an advanced herbal drug delivery system. Asian Journal of Pharmaceutical Sciences. 2009, 4 (6), 363-371.

17. Semalty, A.; Semalty, M.; Rawat, M. S. M.; Franceschi, F. Supramolecular phospholipids– polyphenolics interactions: The PHYTOSOME® strategy to improve the bioavailability of phytochemicals. Fitoterepia , 2010, 81, 306–314.

18. Khan, J.; Alexander, A.; Ajazuddin.; Saraf, S.; Saraf, S. Recent advances and future prospects of phyto-phospholipid complexation technique for improving pharmacokinetic profile of plant actives. Journal of Controlled Release. 2013,168, 50–60.

19. S, minakshi.; A, S.; M.; B, Kolhe.; Deul.; M, J.; N. Herbosomes: Herbo-Phospholipid Complex An Approach For Absorption Enhancement. International journal of biological & pharmaceutical research. 2012 3(8) 946-955

20. Freag, M. S.; Elnaggar, Y. S.; Abdallah, O. Y. Lyophilized Phytosomal nanocarriers as platforms for enhanced diosmin delivery: optimization and ex vivo permeation. International journal of nanomedicine. 2013,8,2385–2397.

21. Song, Y.; Zuang, J.; Gua, J.; Xiao, Y.; Ping, Q. Preparation and properties of a silybin- phospholipid complex. Pharmazie, 2008,63, 35-42.

22. Jain, N.; Gupta, B. P.; Thakur, N.; Jain, R.; Banweer, J.; Jain, D. K.; Jain, S. Phytosome: A novel drug delivery system for herbal medicine. International Journal of Pharmaceutical Sciences and Drug Research 2010; 2(4), 224-228.

23. Manthena, S.; Srinivas, P.; Sadanandam. Phytosomes in herbal drug delivery International Journal of Pharmaceutical Sciences and Drug Research 2010; 2(4), 224-228.

24. Singh, A.; Saharan, V. A.; Singh, M.; Bhandari, A. Phytosome: Drug Delivery System for Polyphenolic Phytoconstituents. Iranian journal of Pharmaceutical Sciences. 2011, 7(4), 209 219. 71

25. Wirtz-peitz et al,. nov 9 1982, united state patent, Rosmarinic acid phospholipid complex

26. Mert, T.; Tugtag, B.; kilin, M.; Sahin, E.; Oksuz.; H.; Gunes, Y. Preventive and therapeutic effects of a beta Adrenoreceptar agonist, Dobutamine, in carrageenan induced inflammatory nociception in Rats. Inflammation ,2014, DOI 10.1007/s10753- 014-9912-3,

27. Adedapo, AA.; Ofuegbe, SO.; Anti-inflammatory and antinociceptive activities of the aqueous leaf extract of phyllanthus amarus schum (Euphorbiaceae) in some laboratory animals. J basic clin physiol pharmacol .2014, DOI: 10.1515/jbcpp-2013-0126.

28. Wen, M. M.; Farid, R. M.; Kassem, A. A. Nano-proniosomes enhancing the transdermal delivery of mefenamic acid. Journal of liposome research, doi:10.3109/08982104.2014.911313.

29. Zheng, c. j.; Deng, X. H.; Wu, Y.; Jiange, Y. P.; Zhu, J. Y.; Qin, L. P. Antiinflammatory effects and chemical constituents of veronicastrum axillare. Phytotherapy Research ,2014, DOI: 10.1002/ptr.5168.

30. Cai, C.; Chen, Y.; Zhong, S.; Ji, B.; Wang, J.; Bai, X.; Shi, G. Antiinflammatory activity of N-butanol extract from Ipomoea stolonifera in vivo and in vitro. 2013, 9, doi:10.1371/journal.pone.0095931.

31. Yang, C. S.; Sang, S. S.; Lambert, J. D.; Lee, M.; j. Boiavailability issues in studying the health effects of plant polyphenolics compound. Molecular. Nutrtion. Food Research. 2008, 52, 139 –151.

32. Osakabe, N.; Yasuda, A.; Natsume, M.; Yoshikawa, T. Rosmarinic acid inhibits epidermal inflammatory responses: anticarcinogenic effect of Perilla frutescens extract in the murine two stage skin model. Carcinogenesis.2004, 25(4), 549—557.

33. Kidd, p.; Head, K. A Review of the Bioavailability and Clinical Efficacy of Milk Thistle Phytosome: A Silybin-Phosphatidylcholine Complex (Siliphos). Alternative Medicine Review.2005,10, 193-203.72

34. Upadhyay, K.; Gupta, N. K.; Dixit, V. K. Development and characterization of phytovesicles of wedelolactone for hepatoprotective activity. Drug Development and Industrial Pharmacy, 2011; 00(00), 000–000.

35. Gupta, N. K.;. & Dixit, V. K. Development and evaluation of vesicular system for curcumin delivery. Arch Dermatol Res. 2011, 303, 89–101.

36. Semlty,A.; Semalty, M.; Phytosome: A New HerbalDrug Delivery System RPMP Vol. 19 – Phytopharmacology & Therapeutic Values I

37. Petersen, M.; Simmons, M. S. J. Molecule of interest Rosmarinic acid. Phytochemistry. 62 (2013) 121-125.

38. Tang, Li.; Cheng, J.; Nonporous silica nanoparticles for nanomedicine application Nano Today, 2013, 8, 290—312.

39. Tan, Q.; Liu, S.; Chen, X. Wu, M.; Wang, H.; YIN, H.; He, D.; Xiang, H.; and Zhang, J.; Design and Evaluation of a novel evodiamine phospholipid complex for improve oral bioavailability. AAPS PharmSciTech, 201,13, 534-546

40. Malakar, J.; Datta, P. K.; Purakayastha, S. D.; Dey, S.; Nayak A. K.. International Journal of Biological Macromolecules 2014, 64, 181– 189.

41. Brewster, M. E.; Loftsson, T .; Cyclodextrins as pharmaceutical solubilizer Advanced Drug Delivery Reviews, 2007,59, 645–666.

42. Campos, D. A.; Madureira, A. R.; Gomes, A. M.; Sarmento, B.; Pintado, M. M. Optimization of the production of solid Witepsol nanoparticles loaded with rosmarinic acid. Colloids and Surface B: Biointerfaces, 2014,115, 109– 117.

43. Meher, J. P.; Yadav, N. P.; Sahu, J. J.; Sinha, P. Determination of required rHLB of citronella oil and development of stable cream formulation. Drug Development and Industrial Pharmacy. 2012

44. Yadav, K.. S.; Yadav, N. P.; Kumar, N.; Luqman, S. Anti-inflammatory effect of Leucas cephalotes Spreng in 12-O-tetradecanoylphorbol-13-acetate-induced inflammation in mice. Annals of Phytomedicine. 2012, 1,62-66.

Received on 02.04.2019 Modified on 21.05.2019

Research J. Pharm. and Tech. 2019; 12(11):5231-5239.

DOI: 10.5958/0974-360X.2019.00905.3

Solvent	Rosmarinic acid	Soya-lecithin	RA-lecithin Complex	Phytovesicle
Water	Turbid	Turbid	soluble	soluble
Chloroform	Turbid	Well soluble	Soluble (little bit turbid)	Soluble
Methanol	Completely soluble	Less soluble	insoluble	Soluble (residue present)
Dichloromethane	Turbid	Well soluble	soluble	Soluble (residue present)
Hexane	Insoluble	Well soluble	soluble	Soluble withresidue