Formulation development & Evaluation of Phospholipids Complex of Euphorbia neriifolia Linn Extract

Phytoconstituents have been utilized as medicines for thousands of years, yet their application is limited owing to major hurdles like deficit lipid solubility, large molecular size and degradation in the gastric environment of gut. To overcome this hurdle, Novel Drug Delivery System (NDDS) proves to be a promising method for formulations containing phytoconstituents. In present work, phyto-phospholipids complex (phytosomes) of Euphorbia neriifolia L. has been prepared to improve the absorption and bioavailability of phytoconstituents. Euphorbia neriifolia L. consists of flavonoids, saponins and polyphenols which possess various medicinal properties. These constituents are hydrophilic in nature and have large molecular size, thus causing poor absorption. Phyto-phospholipids complex of Euphorbia neriifoliahas been prepared by taking Phosphatidylcholine (PC), Cholesterol and Euphorbia neriifolia extract in different ratio and the formulation been optimized to achieve maximum entrapment efficiency and smaller molecular size. The prepared phytosomes has been evaluated by Optical Microscopy, Transmission Electron Microscopy, Dynamic Light Scattering and UV/Vis Spectroscopy for physical appearance, entrapment efficiency, particle size, zeta potential and dissolution rate. The result so obtained indicates the improvement in the absorption rate and bioavailability of the phytoconstituents. Thus, Novel Drug Delivery System (NDDS) possess a great potential in overcoming the challenges of plant based formulations. 
Keywords: Euphorbia neriifolia, Phyto-phospholipids Complex, Novel Drug Delivery System, Phytosomes


Herbal Medicines
From last thousands of years, the traditional knowledge of herb drugs has been spread from the old generation to the new generation. The newer generation across the world studies plants as a future source of drugs because herbal plant medicines have a strong traditional or conceptual base. It is a possible lead to treat different diseases with least adverse effects. Natural products from medicinal plants have been the source of the management of human disease [1][2] . Plants fulfills the needs of not only human being but also the entire animal kingdom, particularly because of the presence of different bioactive compounds. Ethnopharmacology is the culturally diverse study of how individuals get medicines from fungi, plants, animals or remaining different naturally occurring resources. At present, the field mainly has persistent on developing new drugs on the basis of the medicinal plants use by native peoples. The revelation of the indigenous information about remedial plants may hold clues for relieving diseases, has ended up being a good among the most extensively used conflicts for preserving society and an organic decent variety 3 . The conventional utilization of restorative plants by indigenous networks that reflects the social perspectives just as biodynamic components that have the enormous pharmacological potential to fix numerous illnesses 4-5 .

Polyphenols
Plants are blend from a huge scope of natural aggravates that are customarily delegated essential and auxiliary metabolites. Essential metabolites are aggravates that have basic jobs related with photosynthesis, breathing, development, and improvement. These incorporate nucleotides, amino acids, acyl lipids, phytosterols, and natural acids. Different phytochemicals, huge numbers of which gather in shockingly high focuses in certain species, are alluded to as auxiliary metabolites. There are three noteworthy gatherings on their biosynthetic inceptions can be isolated by the plant auxiliary metabolites [6][7] . (i) flavonoids and unified phenolic and polyphenolic mixes, (ii) terpenoids (iii) nitrogen-containing alkaloids and sulfurcontaining mixes.

Nanopharmaceuticals
Nanoformulations have attracted the interest of many researchers for drug delivery applications. These nano-formulations enhance the properties of conventional drugs and are specific to the targeted delivery site. Dendrimers, liposomes, polymeric nanoparticles, nanoemulsions and micelles are some of the nanoformulations that are gaining importance in the Pharmaceutical industry for enhanced drug formulation 8-12 .

Phyto-phospholipids
Phyto-phospholipid complexes are formed by interactions between active constituents and the polar head of phospholipids 13 . Interactions between active constituents, and phospholipids enable phospholipid complexes to be an integral part in which the phospholipids head group is anchored, but the two long fatty acid chains do not participate in complex formation. The two long fatty acid chains can move and encapsulate the polar part of complexes to form a lipophilic surface. The active ingredient is an integral part of the membrane, being the molecules stabled through hydrogen bonds to the polar head of the phospholipids.

Plant Profile (Euphorbia neriifolia L.)
Euphorbia neriifolia Linn (Indian Spurge tree, Hedge Euphorbia commonly known as Snuhi) belong to the family Euphorbiaceae, is one of the different species of Euphorbia genus plants, with wide range of local medicinal uses throughout the areas in which it is grown. This is one of the herbs extensively used in the Indian system of medicine. It is used as analgesic, hepatoprotective, immunostimulant, antiinflammatory, mild CNS depressant, wound healing, radioprotective agent. A significant percentage is succulent, but they are mostly originating from Africa and Madagascar 8 .

Methodology 3.1 Collection of Plant material
Leaves of Euphorbia neriifolia Linn was collected from Ashoka garden Bhopal (M.P), India in month of February, 2021.

Identification of Plant material
The plant has been identified and authenticated by botanist at Department of Botany, Shaheed Bhagat Singh Govt. College, Jaora (M.P.)

Extraction procedure
Following procedure was adopted for the preparation of extract from the shade dried and powdered herbs [15][16]  extraction was continued till the defatting of the material had taken place.

Extraction by maceration process
Defatted Euphorbia neriifolia Linn dried plant material were exhaustively extracted with hydroalcoholic solvent (Ethanol: Water: 70:30). The extract was evaporated above their boiling points. Finally the percentage yields were calculated of the dried extracts.

Determination of Percentage yield
The percentage yield of yield of each extract was calculated by using formula: The crude extracts so obtained after the maceration process were further concentrated on water bath till the evaporation of solvents to obtain the actual yield of extraction. The percentage yield calculation of extract is a very important concept in phytochemical extraction.
To evaluate the standard extraction efficiency for a particular plant, different parts of same plant or different solvents shall be used. The yield of extracts obtained from sample using hydroalcoholic solvent is depicted in the table 3.1.

Qualitative phytochemical tests
The extracts were subjected to various qualitative tests to detect the presence of plant constituents [17][18][19] .  20 . Preparation of Standard: 10 mg Gallic acid was dissolved in 10 ml methanol, various aliquots of 10-50µg/ml was prepared in methanol Preparation of Extract: 10 mg of dried extract was dissolved in 10 ml methanol and filter. Two ml (1mg/ml) of this extract was used for the estimation of phenol. Procedure: 2 ml of methanolic extract and each standard was mixed with 1 ml of Folin-Ciocalteu reagent (previously diluted with GA: distilled water 1:10 v/v) and 1 ml (7.5g/l) of sodium carbonate. The mixture was vortexed for 15s and allowed to stand for 10min for colour development. The absorbance was measured at 765 nm using a spectrophotometer.  Preparation of extract: 10 mg of dried extracted dissolve in 10 ml methanol and filter. Three ml (1mg/ml) of this extract was used for the estimation of flavonoid. Procedure: 1 ml of 2% AlCl3 methanolic solution was added to 3 ml of extract or standard and allowed to stand for 15 min at room temperature; absorbance was measured at 420 nm.

Formulation development of phospholipids complex
The complex was prepared with phospholipids, Cholesterol and Euphorbia neriifolia Linn extract in the ratio of 1:1:1, 1:2:1, 2:1:1, 2:3:1 respectively 21 . Weighed amount of extract, phospholipids and cholesterol were placed in a 100ml round-bottom flask and 25ml of dichloromethane was added as reaction medium. The mixture was refluxed and the reaction temperature of the complex was controlled to 50°C for 3 h. The resultant clear mixture was evaporated and 20 ml of n-hexane was added to it with stirring. The precipitate was filtered and dried under vacuum to remove the traces amount of solvents. The dried residues were gathered and placed in desiccators overnight and stored at room temperature in an amber colored glass bottle.

Evaluation of Prepared phospholipids Complex
The prepared phytosomes has been evaluated by Optical Microscopy, Transmission Electron Microscopy, Dynamic Light Scattering and UV/Vis Spectroscopy for physical appearance, entrapment efficiency, particle size, zeta potential and dissolution rate.

Microscopic observation of prepared phospholipids complex
An optical microscope with a camera attachment was used to observe the shape of the optimized phospholipids complex formulation. The spectrum of extract and phospholipids complex was authenticated by FTIR spectroscopy. The presences of characteristic peaks associated with specific structural characteristics of the drug molecule were noted. Various peaks of the drug are shown in Figure 3.4 and the wave numbers are listed in Table  7.6.  shows stretching vibrations at 1600.9880 cm-1 attributed predominantly to the overlapping stretching vibrations of alkenes (C=C) and carbonyl (C=O) character. Infrared of extract show stretching vibration at 2941.0572cm -1 due to O-H groups, C=C aromatic stretching vibration at 1436.7392cm -1 . When the data obtained from FTIR spectra is compared with the spectra studied it was observed that there are similar peaks for functional groups in phospholipids complex. From the FTIR data of the physical mixture it is clear that functionalities of drug have remained unchanged including intensities of the peak. This suggests that during the process drug, Phospholipids and Cholesterol has not reacted with the drug to give rise to reactant products. So there is no interaction between them which is in favor to proceed for formulation of phospholipids complex as drug delivery system.

Entrapment efficiency and particle size analysis
Entrapment efficiency is an important parameter for characterizing phospholipids complex. In order to attain optimal encapsulation efficiency, several factors were varied, including the concentration of the lipid, concentration of drug and concentration of alcohol. The entrapment efficiency of all the prepared formulations is shown in Table 3.8. The entrapment efficiency of the phospholipids complex was found in the range of 45.65±0.25 to 71.12±0.42%. Particle size of all formulations found within range 312.25±0.58-465.25±0.32nm. Concentration of lipid has shows significant impact on size of phospholipids complex. Formulation F10 was found best one which is further evaluated for drug release study, transmission electron microscopy (TEM), and stability studies.

Transmission Electron Microscopy (TEM)
TEM characterization revealed that the phospholipids complex is spherical in shape (Figure 3.6). However, some variation in size distribution was observed in the TEM image, which might be attributed to an uncontrolled charge neutralization process involved between oppositely charged chains occurring during the formation of phospholipids complex.      This suggests that during the process, Phospholipids and Cholesterol has not reacted with the drug to give rise to reactant products. So there is no interaction between them which is in favor to proceed for formulation of phospholipids complex as drug delivery system. Entrapment efficiency is an important parameter for characterizing phospholipids complex. In order to attain optimal encapsulation efficiency, several factors were varied, including the concentration of the lipid, concentration of drug and concentration of alcohol. The entrapment efficiency of all the prepared formulations were checked. The entrapment efficiency of the phospholipids complex was found in the range of 45.65±0.25 to 71.12±0.42%. Particle size of all formulations found within range 312.25±0.58-465.25±0.32nm. Concentration of lipid has shows significant impact on size of phospholipids complex. Formulation F10 was found best one which is further evaluated for drug release study, transmission electron microscopy (TEM), and stability studies. When the regression coefficient values of were compared, it was observed that 'r 2 ' values of Higuchi was maximum i.e. 0.993 hence indicating drug release from formulations was found to follow Higuchi release kinetics. Results of stability studies clearly indicates that optimized batches of phospholipids complex were stable over the chosen temperature and humidity conditions up to 3 months as were found no significant variation in physical appearance and % drug content. It can be concluded that the phospholipids complex containing Euphorbia neriifolia can provide a convenient and safe alternative to dosage form.