INTRODUCTION:

The liver is an important organ for the maintenance of metabolic functions and detoxification of endogenous and exogenous substances like drugs and Xenobiotics. Drug induced liver injury is an unresolved problem and often limits drug therapy in clinical practice. Liver diseases like hepatitis B virus infections, liver cirrhosis and hepatocellular carcinoma are significant in health challenge worldwide due to lack of curative treatment options besides liver resection and transplantation¹

Silybum marianum, commonly known as milk thistle in the family Asteraceae, is one of the oldest and thoroughly researched plants of ancient times for the treatment of liver and gallbladder disorders, including hepatitis, cirrhosis, jaundice and other toxin poisonings. Silymarin, the active component of this plant, is a standardized extract consisting of 70 to 80 percent silymarin flavonolignans like Silybin A&B, Isosilybin A&B, Silydianin, Silychristin and flavonoids like Taxifolin, Quercetin and the remaining 20-30 percent consisting of a chemically undefined fraction comprising of polymeric and oxidized polyphenolic compounds². This plant was used to safeguard and also regenerate the liver cells in various diseases affecting liver. It also has strong antioxidant action via free radical scavenging activity and inhibits lipid peroxidation. It prevents the entry of harmful chemicals like heavy metals, pesticides, alcohols, carbon tetra chloride etc into liver, thereby protecting the liver cells from further damage. It inhibits the hepatotoxin’s binding to receptor sites, protects hepatocyte membranes, enhances liver parenchyma regeneration and increases glutathione levels^3,4

Adult dose of Silymarin is 240-800mg/day in two or three divided doses orally. It is a non-lipophilic and poor water soluble compound with water solubility of 0.04mg/ml. Only 20-30% of oral Silymarin is absorbed from gastrointestinal tract where it undergoes extensive entero-hepatic circulation. Therefore, absorption of Silymarin from the gastrointestinal tract is low, making bioavailability poor. The low bioavailability, short half-life, and high frequency of administration of Silymarin presented immense scope for the development of nanoparticulate drug delivery system.⁵

Nanoparticles are solid colloidal particles with diameters ranging from 1 -1000 nm. The colloidal carriers based on biodegradable and biocompatible polymeric systems have a major role in the controlled and targeted drug delivery.⁶ Most of drug candidates have problems like poor absorption, rapid metabolism and elimination, toxicity due to drug distribution to other tissues, poor drug solubility, unpredictable bioavailability, etc. These problems can be minimized by formulating in nanoparticle form. Its properties like small size, high surface area, ease of getting suspended in liquids, deep access to cells and organelles and variable optical and magnetic properties makes them unique. Polymeric nanoparticles consist of a polymeric matrix and an incorporated drug. When drugs are formulated in nanoparticle form, their volumes of distribution are reduced and pharmacokinetics properties are improved and drug toxicity is reduced. These polymeric nanoparticles are made from copolymers which shows increased half-life and reduced Mononuclear Phagocytic System uptake. PLA and PLGA degrade by hydrolysis to Lactic Acid and Glycolic acid via Natural pathway in body. Nanoparticles are sub-nanosize colloidal structures composed of synthetic or semi-synthetic polymers⁷

In case of Conventional Dosage form for the treatment of liver disease, it very difficult to treat liver disease because liver is the major organ for metabolism, so drug gets metabolised by liver (by Phase1 and Phase 2 Metabolism) before reaching site of action. Therefore development of a targeted Drug Delivery System is essential. Currently, the whole set of targeting protocols is under development that includes many different approaches to targeted drug delivery. Not necessarily, these approaches involve the use of specific targeting moieties. In certain cases, various physical principles and some physiological features of the target could also be utilized for a successful targeting of pharmaceuticals and pharmaceutical carriers. There are two methods for Drug targeting i.e. a) Active targeting, b) Passive Targeting

Passive targeting:

Passive targeting defines the transportation of nanoparticles through leaky tumor capillary fenestrations into the tumor interstitium and cells. It is achieved by passive diffusion or convection or also refers to the build-up of nanoparticle therapeutics at a specific body site due to certain anatomic or pathophysiological features⁸. Size properties (typically < 200nm in diameter) of nanoparticles greatly facilitates passive liver targeting. This effectively builds up a high local concentration of nanoparticles via diffusion to the various liver cells.

Active targeting:

Active targeting defines the specific delivery of the therapeutic agent to the diseased cell and minimizes unwanted side effects on normal liver cells by non-specific cellular uptake. The diverse physiological functions of the human liver is achieved by the specific activities of various cell types, including the non-parenchymal sinusoidal endothelial cells (SECs), Kupffer cells (KCs), hepatic stellate cells (HSCs) and the predominant parenchymal hepatocytes.⁹

Biodegradable nanoparticles (NPs) are effective drug delivery devices. Various polymers have been used in drug delivery research as they can effectively deliver the drug to a target site and thus increase the therapeutic benefit, while minimizing side effects¹⁰. The controlled release (CR) of pharmacologically active agents to the specific site of action at the therapeutically optimal rate and dose regimen has been a major goal in designing such devices.^11,12. In this study, we are focusing on the physicochemical characterization of Silymarin like morphology, size, solubility, pH, Partition coefficient, Surface area flow property, drug content and release study. This data can be useful in developing controlled release formulation of Silymarin nanoparticles.¹³

MATERIAL AND METHODS:

Material:

The Silymarin was purchased from Sigma Aldrich, India, Poly Lactic-co-Glycolic Acid (PLGA) (75:25) was obtained as gift sample from Hindustan latex limited(HLL), Akkulam, Trivandrum and Acetone and Methanol (Analytical grade)was purchased from SD Fine, Nashik, Polyvinyl alcohol, Tween 80, Acetonitrile (HPLC grade), Manitol, Dipotassium Hydrogen Phosphate and Phosphoric acid (analytical grade)was supplied by M/S SD Fine chemicals, Mumbai, India. The HPLC grade water was prepared using Milli-Q Academic, Bangalore. All chemicals used in the study were of analytical grade and used without further purification.

Experimental Studies:

Determination of solubility:

The Silymarin was evaluated for solubility in water, Acetone, Methanol, Diethyl Ether, Chloroform and Ethanol in accordance with the British pharmacopoeia specifications.¹⁴

pH Determination:

This was done by shaking a 1%w/v dispersion of the sample in water for 5 min and the pH determined using a digital pH meter¹⁵. The data presented here is for triplicate determinations.

True density:

True density of Silymarin was determined by liquid displacement method. It is calculated from the volume of intrusion fluid (toluene) displaced in the pycnometer by a given mass of powder.

D = –––––––

Vp-Vi

Where, D is true density, Vp is the total volume of the pycnometer and Vi is the volume of intrusion fluid in the pycnometer containing the mass of powder (M). All the estimations were done in triplicate and their average is reported in table 1.

Determination of bulk density, bulkiness and compressibility index:

The bulk density of Silymarin was determined by the three tap method¹⁶. 10g of Silymarin powder was carefully introduced into a 100 ml graduated cylinder. The cylinder was dropped onto a hard wood surface 3 times from a height of 1inch at an interval of 2 seconds. The bulk density was obtained by dividing the weight of the sample by volume of the sample contained in the cylinder. Reciprocal of bulk density or the specific bulk volume gave the bulkiness. The percent compressibility index (I)¹⁷ of the Silymarin was calculated using following formula and the results are given in Table 1.

Vo - VF

Compressibility Index = ----------------- X100

Vo = Unsettled apparent Volume

VF = Final Tapped Volume

Angle of repose:

The static angle of repose Ø, a, was measured according to the fixed funnel and free standing cone method ^17,18.A funnel was clamped with its tip 2cm above a graph paper placed on a flat horizontal surface. The powders were carefully poured through the funnel until the apex of the cone thus formed just reached the tip of the funnel. The mean diameters (D) of the base of the powder cones were determined and the tangent of the angle of repose calculated using the equation:

Tan Ø= 2h/D

Where h = height of pile

D = Diameter of pile

The data presented here is obtained from triplicate determinations.

Determination of Partition Coefficient:

10mg drug was added to 50ml of n-Octanol (pre saturated with water), it was shaked and then 50ml of distilled water (pre saturated with n- Octanol) was added to it and the mixture was shaken using mechanical shaker for 24 hours. After 24 hours, both phases got separated. Absorbance was taken for both the phases and concentration was calculated in each phase^19,20

Drug concentration in n-Octanol

Partition Coefficient = ––––––––––––––––––––––––––––––

Drug concentration in water

Percentage of moisture loss:

The Silymarin loaded nanoparticles were evaluated for percentage of moisture loss which gives an idea about its hydrophilic nature. The nanoparticles were weighed initially and kept in desiccators containing calcium chloride at 37 °C for 24 hours. When no further change in weight of sample was observed, the final weight was noted down^21,22

Initial weight- final weight

% moisture content = ––––––––––––––––––––––––– X 100

Initial weight

Drug polymer interaction studies by FT-IR:

Drug-polymer interaction was studied by FT-IR spectroscopy using the instrument Shimadzu FT-IR-8400S. The spectra was recorded for Silymarin, PLGA, and physical mixture of Silymarin: PLGA (1:1). Samples were prepared in KBr disks (2 mg sample in 200 mg KBr) with a hydrostatic press and pressed for 2 for 3 minutes. The scanning range was 400 - 4000 cm^-1and the resolution was 4 cm^-1.^23,24

Preparation of Nanoparticles:

Nanoparticles were prepared using solvent evaporation technique. The method involves preparation of an organic phase (Acetone) containing drug and polymer PLGA and aqueous phase containing PVA (Polyvinyl alcohol) dissolved in water. The organic phase was added drop by drop into the aqueous phase during homogenization. The emulsion was broken down in to Nano droplets by homogenizer (Ultra turrex-T25, IKA laboratories, India), which formed O/W emulsion. Continuous homogenization was carried out for specific time periods. Solvent is evaporated by heating the suspension on magnetic stirrer for 2 hours. Filtrate was 2- 3 times centrifuged in cooling centrifuge (Remi equipment’s, India) for 15 mins at 15000 rpm. After washing 2 -3 times using distilled water, collect the supernant, add manitol and dry the formed nanoparticles by spray dryer^25,26

Particle Size Analysis of Nanoparticles:

Average particle diameter and size distribution of nanoparticles were determined by laser diffractometry using a Mastersizer 2000 (Malvern Instruments, Malvern, UK). Approximately 10mg of nanoparticles were dispersed in 2 to 3 ml distilled water containing 0.1% Nonidet P40 for several minutes using an ultrasonic bath. Then, an aliquot of the nanoparticle suspension was added into the small volume recirculation unit²⁷, which was subsequently circulated 3500 times per minute. Each sample was measured in triplicate for the analysis and the results are given in table 2.

Encapsulation efficiency (EE):

Nanoparticles equivalent to 3mg of Silymarin were dissolved in 10ml of methanol. Then sonicate it for 15 min. The solution was filtered and absorbance was measured by a UV spectrophotometer (Shimadzu 1800, Japan) at 288nm.^28,29. The % entrapment efficiency was calculated from following formula

Actual weight of drug in sample

Encapsulation efficiency = ––––––––––––––––––––––––––– X100

Nanoparticle sample weight

In-Vitro Drug Release Study:

The in vitro release of drug from the nanoparticulate formulations was determined using membrane diffusion technique by using 100μm cellophane membranes. Silymarin loaded PLGA NP (dried product) equivalent to 10mg of drug from each batch were taken and suspended in 100ml of buffer adjusted to pH 4.6 using acetic acid. Suspension was placed in donor compartment which was immersed in a beaker containing 250ml of buffer as diffusion medium (receiver compartment).The whole assembly was stirred on magnetic stirrer maintaining the temperature at 37°C .10 ml of diffusive liquid was withdrawn at various time intervals of 1, 2, 4, 8, 24, 28, 32 and 36hrs. Sink condition was maintained. Solutions were analyzed in UV spectrophotometer (Shimadzu 1800) at 288nm³⁰

Differential Scanning Calorimetry (DSC):

The thermal properties of Silymarin, PLGA, and Silymarin-loaded PLGA nanoparticles were investigated by Differential Scanning Calorimetry (DSC). Samples (3-5mg) was sealed in Aluminum pans with lids and heated at a rate of 10ºC/min using dry Nitrogen as carrier gas with a flow rate of 25ml/min. The heat flow being recorded from 30 to 400°C. Indium was used as the standard reference material to calibrate the temperature and energy scales of the DSC instrument.

Zeta potential:

The electrophoretic mobility and zeta potential were measured using a zeta potentiometer. To determine the zeta potential, nanoparticle sample was diluted with Potassium Chloride (0.1mM) and placed in the electrophoretic cell where an electric field of 15.2 V/ cm was applied. Each sample was analyzed in triplicate.

Effect of temperature and humidity:

Effect of temperature and humidity of the prepared nanoparticles were studied by storing optimized formulation at 4±2˚C and at room temperature maintaining a 45% Relative Humidity for 30 days in stability chamber³¹. Two parameters namely entrapment efficiency and in-vitro release studies were carried out.

RESULTS AND DISCUSSION:

The results of Solubility, True density, Bulk density, and Compressibility index, Angle of repose, Moisture content, pH, Partition Coefficient and Melting point determination are given in Table 1.

Table 1: Physiochemical properties of Silymarin

Sl No.	Parameters	Results
1	Description	Silymarin occurs as yellowish powder
2	Solubility	Low water solubility (0.04mg/mL) of Silymarin is reported. Solubility of Silymarin in various other solvents like Transcutol is 350.1 mg/mL, Ethanol is 225.2 mg/mL, Polysorbate 20 is 131.3 mg/mL Glyceryl monooleate is 33.2 mg/mL
3	Tap density(gm./cc)	0.57±0.64
4	Bulk density(gm./cc)	0.73±0.06
5	Compressibility index (%)	16.57±0.3
6	Angle of repose (⁰)	29.5±0.5
7	Moisture content (%)	9.32±0.45
8	Partition coefficient	1.82±0.53
9	Melting point(⁰C)	132

Particle size distribution of Silymarin nanoparticles:

Particle size distribution of drug has influence on bulk properties of pharmaceutical interest such as flow properties, packing densities, compressibility, segregation characteristics etc. Hence, it must be the aim of pharmaceutical technologist to study the particle size distribution. The particle size distribution of Silymarin nanoparticles is shown in Table 2

Table 2: Particle size distribution of Silymarin nanoparticles

S. No.	Size Range(µm)	Number of particles
1	0-30	30
2	30-60	120
3	60-90	220
4	90-120	142
5	>120	20

Figure 1: Particle size distribution of silymarin nanoparticles

Drug –Excipients accelerated compatibility study:

Accelerated compatibility study of Drug –Excipients was based on physical observation and in the assay no colour change was observed. Based on the assay, it was found that there was no significant change in the estimated amount of Silymarin indicating that the drug is compatible with the added ingredients. The results of the study are given from Tables 3 to 5.

Table 3: Physical characteristics of individual drug and excipients

S.No.	Sample	Initial colour	Final Colour
1	Silymarin	Yellowish powder	Yellowish powder
2	PLGA	Fine White crystalline powder	FineWhite crystalline powder

Table 4: Physical characteristics of drug –excipients mixture

S.No.	Sample	Initial colour	Final colour
1	Silymarin	Yellowish powder	Yellowish powder
2	Silymarin+PLGA	Yellowish White	Yellowish White

Table 5: Chemical characteristics of drug –excipient mixture

S. No.	Sample	Initial Assay	Final Assay
1	Silymarin	99.79	99.75
2	Silymarin +PLGA	99.87	99.82

Drug-Excipient Compatibility Studies by FTIR:

The drug polymer compatibility study is done by FTIR. Here IR spectra of physical mixture when compared with individual spectra of drug and polymer showed no significant change in position of IR absorption peaks which conforms that the drug and polymer have no compatibility problems.

Evaluation of Nanoparticles:

The formulated nanoparticles are evaluated for various properties like particle size, yield, entrapment efficiency and in vitro drug release from the nanoparticles. The formulated nanoparticles had good results of percentage yield, particle size, entrapment efficiency and in-vitro drug release at various time intervals the results are tabulated in table 6.

Table 6: Evaluation of Silymarin-PLGA Nanoparticles

Formulation	Particle size	% Yield	Encapsulation Efficiency	% Drug release
Silymarin-PLGA Nanoparticles	225.8nm	91.20	90.86%	89.12

Scanning Electron Microscopy (SEM):

Morphology of the pure drug and the nanoparticles was studied by SEM. The Fig.2 shows image of the pure drug. The Fig.3 shows image of the optimized batch. It shows the smooth surface with spherical shape indicating that the concentration of polymer and PLGA are in optimum ratio.

Figure 2: SEM image of pure drug-Silymarin

Figure 3: SEM image of optimized formulation

Effect of temperature and humidity:

Effect of temperature and humidity on the prepared nanoparticles were carried outby storing optimized formulation at 4 ± 2 ˚C and at room temperature with 45 % RH for 30 days in stability chamber. Two parameters namely entrapment efficiency and in-vitro release studies were carried out. It is found that nanoparticles stored at room temperature are not stable whereas the ones stored at 4˚C is stable. The results are given in Table 7. NPs at room temperature showed decrease in the entrapment efficiency and different release pattern. After 28 days of storing the NPs, stability testing showed the entrapment efficiency and drug release to be within standard limits.

Table 7: Effect of temperature and humidity on prepared formulations

Temperature	Parameters	0 days	7 days	14 days	28 days
4 ±2⁰ C	Entrapment efficiency	90.86	90.75	90.64	90.56
4 ±2⁰ C	%drug release (36^th hour)	89.12	89.05	88.90	88.55
At room temperature	Entrapment efficiency	90.86	90.69	90.58	90.40
At room temperature	%Drug release (36^th hour)	89.12	88.75	87.54	87.10

CONCLUSION:

The preformulation studies plays a very important role in drug product development. By the present study, we got an insight into the drug properties, changes in polymer nature after drug loading, its interaction and thereby we could analyse the risk assessment in product development. Data obtained in this studies can have a constructive effect on the subsequent development of this particular drug delivery system. In this study we completed the physicochemical characterization of Silymarin like determination of particle size, morphology of drug, Partition coefficient, solubility, Surface area, flow property, drug content and drug release. This data can be useful in developing controlled release formulations, especially nanoparticle formulation of Silymarin. From the experimental results, it can be concluded that PLGA is an ideal carrier for preparing nanoparticles of Silymarin. PLGA nanoparticles have the stability problem. Hence PVA is used both as Surfactant as well as Stabilizing agent for the production of stable nanoparticles. PVA also affect the entrapment efficiency, release rate, as well as particle size. From the above studies, it is revealed that present work represents a satisfactory preliminary study to design a control release formulation of Silymarin for prolonged period of time. Thus this data can be used for further analysis such as Laser diffraction, Thermal analysis of nanoformulation, optimisation of Silymarin loaded PLGA nanoparticles with maximum entrapment capacity, better bioavailability and better target specificity.

ACKNOWLEDGMENT:

The authors are thankful to the R&D, Hindustan Latex Limited (HLL), Akkulam, Trivandrum for providing the polymer for the study. The authors sincerely thank the Principal and Management of The Dale View College of Pharmacy and Research Centre, Trivandrum, for providing an opportunity to carry out this research work.

CONFLICTS OF INTEREST:

The authors have declared that they have no conflicts of interest with respect to current research.

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Received on 25.07.2020 Modified on 28.09.2020

Research J. Pharm. and Tech 2021; 14(10):5508-5514.

DOI: 10.52711/0974-360X.2021.00961