Olive Leaves (Olea Europaea L) Extract Loaded Lipid Nanoparticles: Optimization Of Processing Parameters By Box-Behnken Statistical Design, In-vitro Characterization, And Evaluation Of Anti-oxidant And Anti-microbial Activity

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Abstract: The present study aimed to prepare and evaluated solid lipid nanoparticles(SLNs) of olive eaves extract powder(OLP)which contained many anti-oxidant and antimicrobial agents like oleuropein, a natural polyphenol. The major issue concerning OLP was the instability due to environmental conditions and hence compromised bioactivity. To overcome this problem, SLNs were designed by hot homogenous followed by sonication technique to protect the drug and improve its antioxidant and antimicrobial activity. Lipids like compritol 888ATO and surfactants like tween 80 were used for the development and stabilization of SLNS and optimization was done by Box-Behnken statistical design(3x3).The optimized batch (F9)showed particle size,entrapment efficiency,PDI,and zeta potential 277.46 nm,80.48%,0.275. and -23.18 mV respectively. Optimized formulation (F9) exhibited a sustained release pattern up to 24 h with first-order release kinetic（R²=0.9984） and the mechanism of drug release was found to be Fickian diffusion type (n=0.441). Upon the stability study, it could be found that the SLNs formulation was stable. Anti-oxidation and anti-microbial studies were conducted on optimized formulation and findings suggested that SLNs showed an improved radical scavenging activity and anti-microbial activity against Gram-positive(Staphylococcus aureus)and Gram-negative(Pseudomonas aeruginosa)bacteria. Finally, it was concluded that developed SLNs were able to protect and be suitable for the delivery of OLP.

Keywords: Olea europaea, oleuropein, solid lipid nanoparticles, olive leaves extract,anti-oxidant activity, anti-microbial activity

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1 Introduction

The Dicotyledons Oleaceae family consists of about 30 genera of deciduous plants including olive trees and it has related around 600 species. Oleaceae members are well grown in topical and sub-temperate reasons of Asia?. Olive tree(Olea europaea) oil obtained from different parts like fruit, leaves, etc. has many health benefits upon regular consumption. Traditionally it was used as a folk medicine for remedying various types of fevers and other problems associated with the body2).

Olive leaves extract exhibited several therapeutic activities like anti-hypertensive, hypoglycemic, antimicrobial, hypouricemic, etc. Anti-viral activity against HIV-I infec-on has been demonstrated and reported. In addition, oleuropein(a typical secoiridoid)obtained from the olive plant exhibited hypocholesterolemic and hypoglycemic activity with potent anti-oxidant and as well as anti-inflammatory activity. Degradation product of oleuropein ie. hydroxytyrosol also exhibited all above mention activities along with potential free radical scavenging action5. bioflavonoids Association all these therapeutic activities concerned to olive plant makes it an important contributor in the field of health· 4).

In this project, the authors are concerned with the anti-microbial and anti-oxidant properties of extract of Olive leaves. The anti-microbial activity of the olive extract is due to p-hydroxybenzoic acid, cyclotrisiloxane hexamethyl, Cyclopentasiloxane octamethyl, and Cyclopentasiloxane desmethyl, vanillic, caffeic, protocatechuic, syringic, gallic acid, etc. Other ingredients like oleuropein, quercetin, tyrosol, and oleanolic acid are also responsible for anti-microbial activity. The antimicrobial potential of all these substances has been reported previously by several researchers against bacteria, yeast, fungi, viruses, retroviruses, and other parasites,2).

Cistanche can anti-aging

Dietary intake of natural anti-oxidant sources like fruits and leaves have been gained a lot of attention and showing a positive effect on the health of human beings. Results of many epidemiological studies explored that the intake of polyphenol-rich food reduces the incidence of coronary artery disease(CHD). The CHD like MI(myocardial infarction), IS(ischemic stroke), etc are associated with athero-sclerosis". Recent studies explored that oxidative damage is an important etiological factor for the progress of atherosclerosis, Especially, according to the oxidative stress theory, oxidative modification of low-density lipoprotein(LDL)is thought to play a key role in the development of atherosclerosis. Therefore, the inhibition of such a process is considered to be an important therapeutic approach. Various important constituents like Oleuropein (hydroxytyrosol, tyrosol), verbascoside(also known as ac-toeside or Kalinin), ligstroside,apigenin-7-glucoside,di-cosmetic-7-glucoside, luteolin, catechin, etc were obtained for the leaves of olive and exhibiting anti-oxidant activity as reported previously. Hydroxytyrosol is the principal olive leaf oleuropein that has patent anti-oxidant properties).

Besides this activity, olive leaves extract exhibited other pharmacological activities like anti-inflammatory",12),anti-cancer8.14), anti-viral5),hypoglycemicl4), hypolipidemic17, anti-platelet aggregation. Agency like EFSA(Euro-pean Food Safety Agency)and EMA(European Medicine Agency)gave their assessment of the therapeutic applicability of olive leaf extractants. Along with the major applicability, the extraction products of olive leaves are suffered from instability problems in atmospheric conditions(temperature, light, and oxygen)as well as biological conditions and hence exhibited poor bioactivity20,21). buy cistanche To improve the stability, various nano-based formulations have been developed to explore the various activity of olive leaf extract. These formulations include nano-emulsion (W/O as well as multiple types)2), inclusion complexes, spray dry products?4),electrostatic extrusion25, bipolar complexes), nano-liposomes20, Nano-emulsion, micro-encapsulation,PLA nanoparticles2,and NLCs0.Various problems are associated with these formulations like low yield value, time-consuming procedure, compromised entrapment efficiency, residual solvent contamination, and high particle size. So, to regulate these disadvantages, lipid nanoparticles came into existence. Lipid nanoparticles es-specially solid lipid nanoparticles(SLNs)offer many advantages like better entrapment efficiency, controlled particle size, biocompatibility, biodegradability, and ease of preparation. Moreover, lipids used for the preparation of SLNs have some synergistic action on anti-inflammatory activity along with the entrapped drug. To date, no formulation based on solid lipid nanoparticles(SLNs)for exploring its anti-microbial and anti-oxidant properties as well to improve the stability of olive leave extract product has been developed.

Therefore, the research work was aimed at the"development of solid lipid nanoparticles(SLNs)for Olive leaves (Olea europaea L.)extract powder(OLP). The active principles were separated by the extraction of olive leaves using ethanol. The solvent was removed by drying to get the dry powder product. Olive leaves extract power(OLP)was incorporated into SLNs(OLP-SLNs)and optimization was done by expert design software(Model 8.0.7.1)using Box-Behnken design. Three factors like a drug(OLP)to lipid ratio, surfactant concentration(%, Tween 80), and homogenization speed(rpm)were taken as independent parameters, and factors like particle size, entrapment efficiency, and polydispersity index(PDI)were selected as de-pendent parameters. Optimized formulation was evaluated for morphological study, DSC study, in-vitro release study, and stability study. Finally, antimicrobial and anti-oxidation activities were evaluated to check the anti-microbial and anti-oxidant potential of developed SLNs formulation.

2 Materials and Methods

Olive leaves were obtained from the local garden. Fresh leaves were dried and powdered for extraction. Various lipids like precious ATO5, compritol 888 ATO were procured from Gattefosse(Germany). Glyceryl monostearate (GMS), palmitic acid, stearic acid tween 80, etc were purchased from central drug house ltd(New Delhi, India).2, 2-Diphenyl-1-Picryl hydroxyl-hydrate free radicals(DPPH)were procured from Merck(Darmstadt, Germany). Ascorbic acid, potassium dihydrogen phosphate, ammonium acetate, methanol, sodium hydroxide, poloxamer 188, ethanol, nutrient agar, and dialysis bag(M.Wt cut off 12,000 kD)were procured from Sigma Aldrich(St Louis, USA). All other chemicals used for the study are analytical grade.

2.1 Extraction of phenolic components

Fresh leaves of olive were dried and powdered for ex-traction. The active principles were extracted from the power with the help of solvent(ethanol: water, 4:1 v/v). A sufficient quantity of powder(250 mg) was taken in the extraction flask and the solvent was added(1000 mL)ethanol: water, 4:1 v/)and the mixture was continuously agitated for the duration of 24 h. After this step, the physical mixture was filtered and the solvent was evaporated to dryness at 40℃ to get the powder).

2.2 Preparation and optimization of SLNs: Preliminary

screening study and pre-optimization of formulation variables For the selection of suitable lipid, surfactant, homogenization speed & time, sonication time, etc., preliminary studies were conducted. The selection of suitable lipid depends upon the drug solubility in lipid and the selection of surfactant is depending upon the solubility of lipid in surfactant.

The concerned lipid(100 mg)was melted above its melting points(around 10℃ above)in the vial followed by the incorporation of the drug(OLP)with continuous shaking. The appearance of light pale color indicates the endpoint. Similar experiments were performed for the selection of suitable surfactants.

2.3 Experimental design

In the present study, BBD of expert design software with three factors and three levels was applied for optimization purposes. In the study, three factors like a drug to lipid ratio(A,1:3-1:6),surfactant concentration(B,%,1.5-4.5%),and homogenization speed(C,rpm,3000-6000 rpm, for two h)were taken as independent parameters and par-title size(nm, Y1), entrapment efficiency(%,Y2)and PDI (Y3)were taken as dependent parameters. The aim of applying this design was to optimize the above said three independent parameters and to achieve the optimum particle size, maximum entrapment efficiency, and least possible PDI. The data were fitted in the expert design software for BBD15). A total of 17 batches were generated with 5 batches having a similar composition(five center points)(Table 1). The second-order polynomial equation was used to demonstrate the influence of various independent parameters on particle size, entrapment efficiency as well as PDI.

2.4 Development of SLNs

OLP-loaded SLNs were prepared by hot homogenization followed by the sonication method. The composition of different OLP-loaded SLNs batches is given in Table 1. First of all, the lipid(compritol 888 ATO)was melted around 10℃ of its melting point and the desired quantity of OLP was added(1:3-1:6 drug to lipid ratio). Meanwhile, the required quantity(1.5-4.5%)of surfactant was dissolved in deionized water and the temperature of this phase was maintained the same as that of the lipid phase. The aqueous phase was then incorporated into the lipid phase gradually and subjected to hot homogenization at a variable speed ring(rpm, 3000-6000 rpm, two h)to get the course SLNs dispersion. The obtained emulsion was allowed to

cool and then sonicated for 10 min at 100% amplitude with the aid of a sonicator(Probe type, Vibra-Cell'VCX 130; Sonics, CT, USA)to obtain the final SLNs dispersion. The dispersion was collected in a glass vial and stored in a refrigerator for further study. Similarly, blank SLNs were developed without API(drug).

2.5 Characterization of OLP-SLNs

2.5.1 Evaluation of particle size, PDI, and zeta potential

Photon Correlation Spectroscopy(PCS)using a zeta sizer machine(Malvern, nano ZS 90, Malvern Instruments, UK)was used for the measurement of the average particle size and polydispersity index(PDI)of various batches. The temperature was kept at 25℃ and the scattering angle was set at 90°. cistanch Deionized water was used for the dilution of original OLP-SLNs dispersion. Zeta potential which indicates the surface charge was determined with the help of the same instrument with an electric field strength of around 20 V/cm27

2.5.2 Morphological study

A transmission electron microscope(TEM, Fei electron optics, Japan)using copper grid coated carbon was used to examine the morphology and shape of prepared OLP-loaded SLNs(optimized batch F9). Negative staining with phosphotungstic acid stain(2% w/w, duration 20-30 seconds)was performed to stain the OLP-SLNs sample followed by drying at room temperature. Finally, the sample was detected by TEM.

2.5.3 Evaluation of entrapment efficiency

Entrapment efficiency(EE)was determined by examining the un-entrapped amount of drug present in the OLP-SLNs dispersion. The centrifugation technique was used for this purpose. A measured quantity(10 mL)of OLP-SLNs was taken in a centrifuge tube and allowed to sediment with help of a cooling centrifuge(Remi, India)at 12000rpm for 15 minutes. The presence of an un-entrapped drug was analyzed by a UV-visible spectrophotometer(Model 1800, Shimadzu . Japan)at 230 nm. The percent of drug entrapment was determined by the following formula(equation 1):

2.5.4 Differential scanning calorimetry(DSC)study

The purpose of this study was to evaluate the thermal behavior of various formulations viz pure OLP, compritol 888 ATO, and optimized OLP-SLNs formulation(F9)using differential scanning calorimetry(Mettler, Toledo, USA). Each sample was packed in an aluminum pan and scanned at the temperature range of 20-350℃(rate 10℃/min)using an empty sealed pan as a reference in an inert atmosphere (nitrogen).DSC curves were obtained and interpreted. 2.5.5 Drug release study(%)

The drug release study of optimized batch(F9)was done by Franz diffusion cell using dialysis membrane. Before the application of the dialysis membrane, it was developed by treating 0.35% w/v sodium sulfite solution at 80℃ for 1-2 min followed by acidification with HSO(0.2%,v/v)and then stored for 12 h in distilled water. Saline phosphate buffer (pH 7.4)was filled in the receptor compartment and SLN dispersion(F9,1 mL)was taken in the donor compartment. The experiment was performed at 37±0.5℃ with continuous stirring(50 rpm).Aliquots(1 mL)were taken from the receptor compartment at predetermined time intervals and the amount of drug present in each sample was detected by the UV-visible spectrophotometer at 230 nm. The suitable release kinetic from optimized SLNs was detected by fitting the release data into zero, first, the Higuchi model and Korsmeyer-Peppas model as given the Table 2, and the value of R(Correlation coefficient)was determined. The model with the highest R-value was considered an optimized model8).

This article is extracted from J. Oleo Sci. 70, (10) 1403-1416 (2021)