AHasrawati1*imageIrsan Rizaldi2, Neneng Amelia Bakri2Deisy Febrianti2, A Mumtihanah Mursyid1

1Pharmaceutical Department, Faculty of Pharmacy, Universitas Muslim, Indonesia.

2Faculty of Pharmacy, Universitas Muslim, Indonesia.


Objective: Thymoquinone is a main component of Black Cumin (Nigella  sativa Linn.) with various pharmacological activities, but has poor stability and bioavaibility. The purpose of this study was to carry out the preparation and characterization of timoquinone nanoparticles PEGylation. 

Methods: The Thymoquinone nanoparticles  (TQ-NP) were made with PEGylation using PEG 6000 with the concentrations on each preparation of 3 mM (A), 4 mM (B), and 5 mM (C) then were evaluated by the parameter of yield percentage Entrapment Efficiency (EE) and Drug Loading (DL), drug release, size and distribution particle, morphological analysis and Fourier Transform-Infrared spectrophotometer (FTIR). 

Results: Thymoquinone nanoparticle was PEGylated with PEG 6000  has the highest efficiency entrapment of 99.9718±0.029% in formula A, with the capacity of drug loading 0,66%. Formulation A release 99.9718±0.029% of Thymoquinone at 50 minutes. The morphological observations with Scanning Electron Microscope (SEM) showed spherical nanoparticles morphology. 

Keywords: Black Cumin, Nanoparticles, PEG 6000, Pegylation, Thymoquinone.




Black cumin mostly used as a food additive and has properties to treat several diseases due to its various chemical properties1. Black cumin oil has various therapeutic benefits such as dysentery, headaches1, gastrointestinal problems2,3, eczema, hypertension4, and obesity, etc. Thymoquinone (TQ) is one of the major active chemical component of black cumin oil (Nigella  sativa Linn)5. TQ can increase the immune system of patients with bronchial asthma due to allergies, and the main properties as an anti-inflammatory1,6 and antioxidants. In  a study conducted by Salim et al., showed that TQ has an IC value of 1.5 ±0.04 μg/mL7. Thymoquinone as an anti-inflammatory works by inhibiting the cyclooxygenase (COX) and lipoxygenase (LO) pathways6. In addition, black cumin oil also contains chemical components such as, nigellienine, nigellamine-n-oxide, essential oils, fatty oils, alkaloid group compounds, saponins, steroids, isokuinolin alkaloids, oleats, and linolenate8.

Thymoquinone is an essential oil that has volatile properties, low melting point and is easily oxidized, so it will be difficult to formulate into a pharmaceutical dosage form9. In addition, thymoquinone is also difficult to dissolve in water, and has a poor bioavailability10. To overcome this problem a nanoparticle preparation was developed as a thymoquinone carrier11

Nanoparticles are colloidal particles that range in size from 1-100 nm. The drug is dissolved, adsorbed, encapsulated or attached to the nanoparticle matrix12. The advantage of nanoparticles is that it increases the bioavailability of the drug, small doses, reduces side effects, increases the surface area to produce rapid solubility of active ingredients in the fluid environment, such as in the digestive tract13. If the dissolution rate is high, the absorption and bioavailability of the drug will also be better14. Within a few years, many methods have been designed in the development of formulations to improve the characteristic and pharmacokinetics properties of a drug compound to produce the maximum benefit of therapeutic action12. One of them is pegylated nanoparticles. Pegylation is a modification of protein, peptide and non peptide molecules by forming bonds or links with one or more chains of polyethylene glycol (PEG). Polyethylene glycol (PEG) is a polymer that has been approved by the FDA in the application of nanomedicine and biomedicine. A good nanoparticle formulation should have a high drug loading capacity15. The molecular weight of polyethylene glycol (PEG) used can affect the loading capacity of the drug so that it is necessary to optimize the type and concentration of PEG used based on its molecular weight16. Based on this, it is necessary to do research on the preparation and characterization of thymoquinone nanoparticles so that they can be used for drug delivery.



Thymoquinone (TQ) was obtained from Sigma Aldrich USA. The TQ was prepared TQ was prepare into the form nanoparticles PeGylated. In this study, the TQ was quantified using spectrophotometer UV-Vis. 

Standard Solution preparation 

A stock solution of 100 ppm of Thymoquinone concentration was made by weighing 10 mg of TQ and dissolving it in 100 mL of phosphate buffer pH 7.4. The solution is used to determine the maximum wavelength of TQ. The λmax was observed with a spectrophotometer UV-VIS at 200 – 400 nm. 

Preparation of Thymoquinone Nanoparticle (TQ-NP)

Nanoparticles are made by the pegylation method. A series of concentrations of PEG 400, PEG 4000 and PEG 6000 were made as polymers, Tween 80 as a surfactant in the optimization of nanoparticles can be seen in Table 1. Each formula was made as much as 5 mL with 1 mg of TQ. Dissolved 1 mg of TQ in 70% ethanol. Tween 80 added 0.03 mL into the mixture until it was homogeneous, then the mixture was slowly dripped into polyethylene glycol (PEG) while being stirrer at a speed of 700 rpm for 6 hours. The TQ-PEG dispersion was then rotavapory to remove solvents and made into powder with freeze drying technique to obtain TQ-NP PEGylated5.

Characterization of TQ-NP

TQ-NP Morphology

Morphological observations of TQ-NP were carried out using Scanning Electron Microscope (SEM). The TQ-NP were dropped over cooper gird then coated with an auto carbon coated (JOEL JEM, Japan) tool for 5 seconds, dried at room temperature for 24 hours. TQ-NP was analyzed by accelerating the voltage at 120 kV and magnification of 60,000.

Particle Size

TQ-NP particle size was determined using a NicompTM 380 ZLS Submicron particle size analyzer. A total of 2 drops of pH 4.0 TQ-NP added 5.0 mL aquadestillata, then mixed by flipping through a conical tube. After that, 3.0 mL was taken and put into cuvettes to analyze distribution and particle size. 


 The Entrapment Efficiency  (EE)and The Loading Drug

5 mg of TQ-NP were centrifuged at room temperature with 15.000 rpm a, for 30 minutes and then  absorbance was measured with UV-Vis spectrophotometry. Entrapment Efficiency of TQ-NP was calculated by the equation:


Drug Release Study

The release of TQ-NP carried out in vitro using franz diffusion cells, the membrane used was removable python morolus skin. The medium fluid in the receptor compartment used was a phosphate buffer pH of 7.4 and maintained at a temperature of 35±0.5°C as much as 50 mL. The snake skin is then placed between donor compartments with receptor compartments, samples weighed as much as 5 mg are applied to the surface of the skin. Medium liquid is flowed through the bottom of the skin membrane with the help of a magnetic stirrer at a speed of 100 rpm. Sampling was carried out at the 10, 20, 30, 40, 50, 60, 70, 80, 90, 110, and 120 minute where 5 mL samples were taken from the receptor compartment using a syringe and immediately replaced with a 5 mL medium solution. The samples were then measured for absorbance using UV-Vis spectrophotometry at a maximum wavelength of 260 nm.




The development of nanoparticles using thymoquinone as an active ingredient is based on volatility, low melting point and easily oxidized10.Thymoquinone is also difficult to dissolve in water, so it has a small bioavailability9

Therefore, thymoquinone is made in the form of nanoparticles to improve the bioavailability of drugs, improve the physical properties of chemicals10 and protect medicinal ingredients so as to provide an effective therapeutic effect17. 

In this study, the resulting nanoparticle TQ-NP was characterized by evaluating nanoparticle morphology, size and distribution of particles, measurement of entrapment efficiency (EE) and drug loading (DL), and nanoparticle release. In this study thymoquinone was prepared to form nanoparticles with PEGylated method using PEG 6000 as a polymer. PEG was chosen as a polymer used because it was 'safe' in the body and approved for use as excipients in many pharmaceutical formulations16. PEG has been widely used in various nanoparticle systems to increase surface hydrophilicity and half-life by interacting with blood and mononuclear phagocyte cell systems. Pegylation is formed because physical adsorption or covalent grafting results in a layer of PEG on the surface of the particles so that it can increase the stability of the medicinal material17.

In making the pegylation method thymoquinone and PEG were dissolved in 70% ethanol, then tween 80 was added as a surfactant as much as 0.03 mL, the distrer was at 700 rpm, dropped thymoquinone and distrerer dispersion for 6 hours, using a rotary evaporator at 40 0C at a speed 50 rpm and mixed with freeze drying technique to form thymoquione nanoparticles.

The optimization parameter is to see the formation of particles after being pollinated by the technique of freeze drying. The optimization results showed that only PEG 6000 produced powder (solid particles), can be seen in Table 1. 

The Efficiency of Entrapment (EE) and The Loading of Drug (LD)

The Efficiency of Entrapment (EE) shows the ability of polymers to entrapment drugs, while DL shows the ability of drugs to be absorbed into the polymer matrix. EE can be influenced by the concentration of the polymer used in making nanoparticles19. The EE test results show that all preparations have% EE, which are 99.9718±0.029%, 99.9628±0.026%, and 99.9363±0.049%, respectively. From the results above it can be seen that the higher the polymer concentration will cause the EE value to be low. This can be caused by the high concentration of polymers that will produce a solution with high viscosity so that the drug ingredients will be difficult to diffuse into the polymer matrix18


Research conducted by Odeh et al., 20 who conducted a study of the efficiency of loading timoquinone in liposomes showed a 90% absorption efficiency of timoquinone and the use of Triton X-100 showed that only 49.6% of the drug was absorbed, the value was smaller than the results of research that has been done.

The results of the DL test showed that the preparations A (3 mM), B (4 mM), and (5 mM) were 1.54%, 1.02%, and 0.66% respectively. From these results it is known that preparation A has a high DL value compared to preparations B and C. An ideal nanoparticulate system has a high drug loading capacity thereby reducing the material used for drug delivery18. Drug and drug loading is very dependent on drug solubility in materials or polymers22, polymer composition, polymer molecular weight, and drug and polymer interactions23.

Particles Size 

Particle size are very important characteristics in nanoparticle systems. The particle size will determine the drug distribution in vivo, the fate of the drug in the biological system and its toxicity and the ability to target drugs in the nanoparticle system24. The size and distribution of particle also affects drug absorption, drug release and stability of the nanoparticles16.The main application of nanoparticles is drug release and drug targeting. It has been found that particle size affects drug release. Small particles produce a larger surface area. As a result, most drugs that are inserted will spread to the surface of the particles to facilitate drug release faster. Instead, the drug will experience slower diffusion in larger24. Measurement of size and distribution of nanoparticles using preparations that have good percent EE and in vitro release values. The results showed that preparation A had an average particle size of 10 nm, which is in the range of size of the nanoparticles (10-100 nm).

The Polydispersity Index is a parameter to determine the particle size distribution of nanoparticles with a range that can be absorbed by the PEG 6000 polymer matrix. Preparations A (3 mM) have IP> 0.5 which is 0.350. According to Avadi in 2010, particles with IP> 0.7 have a very wide size distribution. The smaller IP, shows the particle size is uniform. So that it can be concluded that thymoquinone nanoparticles formed monodispersion or relatively homogeneous.