Toxicity of Self-nanoemulsifying Drug Delivery System Formulation of

Nigella Sativa L. Seed Oil against Adult Danio rerio

Yanti Tri Utami

and Isnatin Miladiyah

Medical student, Faculty of Medicine Universitas Islam Indonesia, Yogyakarta, Indonesia

Department of Pharmacology, Faculty of Medicine, Universitas Islam Indonesia, Yogyakarta, Indonesia

Keywords: Self-nanoemulsifying, Drug delivery system, Nigella sativa L., Toxicity, Danio rerio

Abstract: Nigella sativa L. (N. sativa L.) has been used in traditional medicine due to its numerous therapeutic effects,

but its oral form has low solubility and has a minor therapeutic effect. The development of self-

nanoemulsifying drug delivery system (SNEDDS) formulation for N. sativa L may solve this issue. The

pharmacological activity of the SNEDDS formulation of N. sativa L. seed oil extract (NSOE) has been widely

explored but its toxicity remains unknown. This study aimed to determine the lethal concentration of NSOE

SNEDDS formulation. An experimental study was conducted using adult zebrafish, aged 4–6 months,

incubated with NSOE SNEDDS formulation at 500, 250, 125, 62.5, and 31.5 part per million (ppm)

concentrations and with non-SNEDDS formulation at 125, 62.5, 31.25, 15.625, and 7.8125 ppm

concentrations. The mortality was calculated through macroscopic examination after 24, 48, 72, and 96 hours

of exposure. The half-maximal lethal concentration (LC

) of the NSOE SNEDDS and non-SNEDDS

formulations was determined using the probit analysis. The LC

of NSOE SNEDDS formulations was

154.637 + 75.609 ppm and was not significantly different from that of non-SNEDDS (72.358 + 15.253 ppm)

(p = 0.138). The toxicity of the SNEDDS formulation of NSOE was comparable to that of the non-SNEDDS.

1 INTRODUCTION

Nigella sativa L. (N. sativa L.) is a herbal plant from

the Ranunculaceae family and is widely grown in

Mediterranean countries, the Middle East, Eastern

Europe, and West Asia (Abedi et al., 2017). Several

phytochemical studies of N. sativa L. showed that its

extract contains numerous antioxidant compounds,

including thymoquinone, carvacrol, t-anethole, and 4-

terpineol, which indirectly reduce reactive oxygen

species production and inhibit lipid peroxidation

(Amina, 2016). Thymoquinone has the most powerful

antioxidant properties (Kooti et al., 2016).

Herbal medicines are usually orally administered

since this route of administration is the safest, most

convenient, and most inexpensive (Cherniakov et al.,

2015). However, the low solubility and poor oral

bioavailability of many herbal medicines lead to less

optimal effectiveness. Therefore, researchers have

begun developing oil-based drug formulations in the

form of nanoemulsions that are expected to improve

https://orcid.org/0000-0003-4381-0129

https://orcid.org/0000-0003-1630-7130

the oral bioavailability and drug solubility of herbal

plant extracts, including the development of a self-

nanoemulsifying drug delivery system (SNEDDS)

(Abdelbary et al., 2013).

The SNEDDS formulation consists of a mixture

of isotropic oils, surfactants, and cosurfactants that

are capable of spontaneously forming oil

nanoemulsions in the gastrointestinal tract by

producing nanometer-sized droplets (<300 nm in

size) when dispersed in liquid media (Christophersen

et al., 2014; Patel et al., 2011) Drug preparations in

SNEDDS formulations have several advantages,

including the ability to maximize absorption and

transportation, modulate drug biodistribution and

disposition, and allow targeted drug delivery to

reduce the side effects. SNEDDS formulations tend

to be more physically and chemically stable for long-

term storage and allow the packaging of drugs in unit

dosage forms, using hydroxypropyl methylcellulose

or both soft and hard gelatin capsules, than other

nanoemulsion systems (Date et al., 2010).

Utami, Y. and Miladiyah, I.

Toxicity of Self-nanoemulsifying Drug Delivery System Formulation of Nigella Sativa L. Seed Oil against Adult Danio rerio.

DOI: 10.5220/0010487300350042

In Proceedings of the 1st Jenderal Soedirman International Medical Conference in conjunction with the 5th Annual Scientiﬁc Meeting (Temilnas) Consortium of Biomedical Science Indonesia

(JIMC 2020), pages 35-42

ISBN: 978-989-758-499-2

Previous studies have focused on the

pharmacological effectiveness of SNEDDS

formulations; however, their toxicity in living cells,

including the SNEDDS formulation of N. sativa L.

seed oil extract (NSOE), has not been widely

investigated. Toxic impacts are important and

inseparable from the development of new drugs

(Parasuraman, 2011). Thus, this study aimed to

determine the toxicity of NSOE in a SNEDDS

formulation in comparison with the toxicity of the

non-SNEDDS formulation on adult zebrafish.

2 MATERIALS AND METHODS

This study was approved by the Medical and Health

Research Ethics Committee of the Faculty of

Medicine of Universitas Islam Indonesia with

protocol Number 37/Ka.Kom.Et/70/KE/V/2019.

2.1 Animal Subjects

Both male and female adult zebrafish (Danio rerio)

were used in the study. The fish were 4–6 months old

and healthy, as characterized by active swimming.

The fishes that died before the research took place

were excluded. Prior to treatment, the fish were

adapted for one week. Zebrafish were identified at the

Biology Research Center, Indonesia Academy of

Science at Bogor, Indonesia.

2.2 Preparation of N. sativa L. Seed Oil

Extract and SNEDDS Formulation

NSOE was prepared following the previous study by

Akrom et al. (Akrom & Fatimah, 2015). N. sativa L.

seeds were dried and mashed to form a powder.

Overall, 1 kg N. sativa L. seeds was soaked in 1 L of

70% ethanol for 48 hours and filtered to separate the

macerated yield from the residue. The product of the

maceration process was collected, and an evaporator

was used to remove the 70% ethanol solvent and form

a thick NSOE. The viscous NSOE was kept upright

until it divided into two phases, the extract and the oil

phases. The oil phase was then used for the SNEDDS

formulation.

In this study, NSOE was produced with the

maceration method using 70% ethyl alcohol. This

method is simple, easy to perform, inexpensive, and

can protect the thermolabile compounds in N. sativa

L. seed from damage (Savitri et al., 2017). Different

concentrations of surfactants and cosurfactants were

added to the oil phase after maceration to prepare the

SNEDDS formulation. The determination of the

optimal NSOE SNEDDS was based on previous

research (Wahyuningsih & Putranti, 2015), in which

the best composition was NSOE 0.154 parts, Tween

80 0.587 parts, and polyethylene glycol 400 (PEG

400) 0.259 parts. Therefore, the formulation prepared

consisted of 0.532 mL of NSOE, 2.047 mL of Tween

80 (surfactant), and 0.258 mL of PEG 400

(cosurfactant) (Wahyuningsih & Putranti, 2015)

2.3 Formulation Stability Test

Stability of SNEDDS formuations was evaluated

using heating-stability, freeze-thaw, and

centrifugation tests (Senapati et al., 2016). The

heating-stability test was conducted by storing

SNEDDS samples in a refrigerator at 4C for 24 hours

and then by transferring them to an incubator at 40C

for 24 hours (48 hours per cycle). The freeze-thaw

test was conducted by storing SNEDDS samples at

temperatures between −21C and 25C for 48 hours.

The centrifugation test was performed by

centrifuging SNEDDS samples at 5000 rpm for 30

minutes. All tests were repeated six times (six cycles);

then, organoleptic observations and instability

parameters (phase separation and precipitation) were

recorded.

2.4 Determination of Globule Size,

Zeta Potential, Polydispersity

Index, and %Transmittance

The NSOE SNEDDS formulation was diluted with

water at a ratio of 1:25 on a magnetic stirrer until a

nanoemulsion was formed. The nanoemulsion was

then put into a cuvette and measured using a Particle

Size Analyzer (SZ 100, HORIBA) to determine

globule size, zeta potential, and polydispersity index

(PDI). The %transmittance was measured by adding

5 mL distilled water to 0.1 mL SNEDDS formulation

of NSOE and then rotating them in a vortex for 30

seconds. The transmittance was read using a UV-Vis

spectrophotometer (UV-Vis UH5300, Hitachi) at a

wavelength of 650 nm with distilled water as the

comparison (Ujilestari et al., 2018).

2.5 Determination of Half-Maximal

Lethal Concentration (LC

value)

The toxicity in zebrafish was evaluated for 96 hours

in each test group. Subjects were exposed to five

concentrations of NSOE SNEDDS (500, 250, 125,

62.5, and 31.5 part per million (ppm)) and five

concentrations of NSOE non-SNEDDS (125, 62.5,

31.25, 15.625, and 7.8125 ppm). One subject group

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

was given a combination of cosurfactants-surfactants

to ensure that the use of surfactants and cosurfactants

was not toxic to zebrafish. Each observation was

performed in three replications. Zebrafish mortality

was characterized by the absence of movement and

tail response when touched. Dead fish were removed

each day from the test aquarium, and their mortality

was calculated (The Organization for Economic Co-

operation and Development [OECD], 2018). The

half-maximal lethal concentration (lethal

concentration 50% or LC

) value was the

concentrations of NSOE SNEDDS and non-

SNEDDS (ppm) that could kill 50% of the zebrafish

in each test group. The LC

values were calculated

from the equation for the linear regression line

between the concentration versus the percent

mortality of zebrafish. After 96 hours, the mortality

in each group was calculated in percent.

2.6 Statistical Analyses

The death percentage per concentration for each

formulation was analyzed using the probit analysis

(SPSS software version 21) to determine the LC

values. The average of IC

values of NSOE

SNEDDS and non SNEDDS were compared using t-

test and p value < 0.05 was considered as significant.

3 RESULTS.

Preparation of NSOE and NSOE SNEDDS

Formulation: The extraction process produced

NSOE, which was then formulated into a SNEDDS

preparation. The procedure resulted in a water-

soluble SNEDDS preparation characterized by a

clear, transparent, slightly misty solution.

Formulation Stability Test: The three stability

tests indicated that the NSOE SNEDDS had good

physical stability, characterized by no organoleptic

changes before or after the test, no phase separation,

and no formation of crystal or sediment (Figure 1 and

Table 1).

Globule Size, Zeta Potential, PDI, and

%Transmittance: The particle size was in the

50200 nm range, and the potential zeta value was

lower than 30 mV, demonstrating the good stability

of the nanoemulsion of NSOE. The PDI value was

0.499 and %transmittance was 64.414%, indicating

that the SNEDDS preparation was not fully

monodispersed (Table 2).

Value of Formulations: The toxicity of the

test compound was calculated from the zebrafish

mortality rate for 96 hours of treatment (Table 3).

Combinations of the surfactant-cosurfactant (SCS)

showed no subject deaths in all three replications.

This indicated that using surfactants-cosurfactants in

preparing NSOE SNEDDS was safe and nontoxic to

zebrafish. The LC

values for the NSOE SNEDDS

and non-SNEDDS formulations calculated using a

probit analysis are listed in Table 4. As shown in

Table 4, although not statistically significant (p value

> 0.05), the toxicity of the NSOE SNEDDS

formulation was lower than in the non-SNEDDS.

Therefore, the toxicities of NSOE SNEDDS and non-

SNEDDS formulations were comparable.

(a) (b) (c)

Figure 1: Appearance of NSOE SNEDDS formulation (left, before the test; right, after the test); (a) before and after the

heating-stability test; (b) before and after the freeze-thaw test; and (c) before and after the centrifugation test.

Table 1: Organoleptic appearance, phase separation, and precipitation before and after stability tests.

Before the test Afte

the test

Organoleptic appearance Organoleptic appearance Phase separation Precipitation

Heating-stability test

Brownish-yellow,

clear, smelling typical of

N.sativa L. oil

Brownish-yellow,

clear, smelling typical of

N.sativa L. oil

None None

Freeze-thaw test

Centrifu

ation test

Toxicity of Self-nanoemulsifying Drug Delivery System Formulation of Nigella Sativa L. Seed Oil against Adult Danio rerio

Table 2: Particle size, zeta potential, PDI, and %transmittance of NSOE SNEDDS formulation.

Particle size (nm) Zeta potential (mV)

PDI %Transmittance (%)

139.2

59.5

0.499 64.414

Table 3: Deaths of zebrafish in a 96-hour observation of each test group.

Compound Concentration

Number

subjects

Number of deaths Mortality (%)

Mortality

average

(

)

R1 R2 R3 R1 R2 R3

NSOE

SNEDDS

S1 (500 ppm) 7 7 7 7 100 100 100 100

S2 (250 ppm) 7 4 7 7 57 100 100 86

S3 (125 ppm) 7 0 3 7 0 42 100 47

S4 (62.5 ppm) 7 1 1 0 14 14 0 9

S5 (31.5 ppm) 7 0 1 0 0 14 0 5

NSOE non-

SNEDDS

NS1 (125 ppm) 7 7 7 7 100 100 100 100

NS 2 (62.5 ppm) 7 0 5 0 0 71 71 47

NS 3 (31.25 ppm) 7 0 0 0 0 0 0 0

NS 4 (15.625 ppm) 7 2 0 3 29 0 43 24

NS 5 (7.8125 ppm) 7 0 0 0 0 0 0 0

SCS (control)

125 ppm

7 0 0 0 0 0 0 0

Abbreviations:

S1–S5, SNEDDS concentration 1–5

NS1–NS5, non-SNEDDS concentration 1–5

SCS, surfactant-cosurfactant

R1, replication 1; R2, replication 2; R3, replication 3

Table 4: LC

values of NSOE SNEDDS and non-SNEDDS formulations.

Compound Replication LC

value* (ppm) Average LC

+ SD (ppm) p value **

NSOE SNEDDS

1 237.227

154.637 + 75.609

0.138

2 137.860

3 88.826

NSOE non-

SNEDDS

1 84.037

72.358 + 15.253

2 55.101

3 77.936

Note:

* LC

values were obtained from the mortality percentage data at five concentrations of NSOE SNEDDS and non-

SNEDDS formulations and analyzed using a probit analysis to obtain concentrations that caused the death of 50% of

subjects.

** p value was significant at 0.05

4 DISCUSSIONS

This study investigated the toxicity of NSOE

SNEDDS against adult zebra fish compared to the

non SNEDDS. The results showed that the toxicity of

NSOE SNEDDS lower than non SNEDDS, however,

this difference was non statistically significant.

Surfactant selection is a crucial part in the

preparation of SNEDDS formulations. Surfactants

play an important role in forming nanoemulsions and

reducing the surface tension between the two phases

JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

(Temilnas) Consortium of Biomedical Science Indonesia (KIBI )

(oil and water) for good emulsion dispersal (Patel et

al., 2011). Surfactants also stabilize nanoemulsion

preparations by maintaining the physical properties of

the preparation and preventing damage to the

bioactive compounds during processing and storage

(Chuacharoen et al., 2019). Tween 80 is an n-

hexane/water emulsion with non-ionic, nontoxic, and

biocompatible properties that result in a low level of

toxicity, making it safe to use. Tween 80 surfactant is

widely used in the processing of nanoemulsion

preparations in the pharmaceutical industry (Prieto &

Calvo, 2013).

The addition of cosurfactants helps to reduce the

size of nanoemulsion globules compared to the use of

surfactants alone. PEG 400 is a widely used polymer

cosurfactant in drug formulations. Its strong

hydration property allows it to form a stronger

interaction between the polymer and water compared

to the polymer-polymer interaction, thereby

increasing the emulsion mucoadhesion (Chen et al.,

2019). The low toxicity of the Tween 80 and PEG 400

combination has been demonstrated in this study,

characterized by the absence of death (mortality) in

this group.

Successful nanoemulsion preparations are

marked by a clear, transparent or slightly foggy

appearance (Handayani et al., 2019), and good

physical stability. Physical stability tests of the

SNEDDS formulation of NSOE demonstrated good

stability and fulfilled the requirements of the

nanoemulsion (Senapati et al., 2016). The presence of

a surfactant and cosurfactant reduced the surface

tension between the oil and water phases of the

emulsion. The greater the reduction of surface

tension, the more stable the nanoemulsion

formulation (Villar et al., 2012).

The mean globule size of the NSOE SNEDDS

formulation was 139.2 nm, which meets the

requirements for nanoemulsion particle size in the

drug delivery system (50–200 nm), making it suitable

for use in both food and drug industries (McClements

& Rao, 2011). The smaller the active substance

particle size in a nanoemulsion formulation, the better

the stability and distribution in dissolution media. The

zeta potential is a parameter for estimating surface

loads to understand the physical stability of

preparations and is important in characterizing

nanoemulsion preparations. The zeta potential value

of the NSOE SNEDDS formulation was 59.5 mV,

which met the criterion of a stable nanoparticle of

more than +30 mV or smaller than30 mV (Kumar

& Dixit, 2017).

The PDI value was 0.499, indicating that the

particles formed in SNEDDS formulations were not

fully monodispersed. The PDI parameter indicates the

globule size distribution; the lower the PDI value, the

b etter the level of monodispersity. In essence, PDI is

a dimensionless particle heterogeneity index. A PDI

of <0.3 meets the monodispersion criterion (Danaei et

al., 2018). Efforts to reduce PDI include prolonging

the ultrasonication process up to 30 minutes

(Mahbubul, 2019). Although the PDI value in this

study was more than 0.3, this was fairly good because

the particle size distribution of a SNEDDS

formulation is deemed heterogeneous (polydispersed

particles) if PDI exceeds 0.7 (Danaei et al., 2018).

The value of %transmittance indicates the level of

clarity of SNEDDS preparations; the closer the

%transmittance to 100%, the smaller the particle size

in nanometers, and the closer the optical clarity is to

water (Khan et al., 2015). SNEDDS preparations with

nearly 100% transmittance appear clearer and more

transparent with a greater possibility of absorption in

the digestive tract (Yen et al., 2017). In this study, the

%transmittance was only 64%, indicating that the

SNEDDS formulation of NSOE was of poor quality.

The %transmittance may be improved by increasing

the surfactant concentrations. A previous study

showed that raising surfactant concentrations (Tween

80) in a SNEDDS preparation containing vitamin D

led to a greatly reduced globular size and an increased

oil-water interface. However, as the surfactant

concentration continues to increase, the globule size

also becomes larger. Thus, to produce the optimal

globule size, the ideal ratio of surfactant/oil is 1:1

(Guttoff et al., 2015). In a different study, an

increased surfactant concentration in a SNEDDS

formulation containing curcumin increased

%transmittance to 92.86%99.51% (Chuacharoen et

al., 2019). These findings suggest that higher

surfactant concentrations improve the trapping of

active compounds in the particles, leading to an

increase in %transmittance to almost 100%.

The acute toxicity test is designed to determine

the toxic effects of a particular dose in a short time.

Usually, acute toxicity is observed from 24 hours up

to 7 days. Such tests aim to evaluate adverse effects

on a test organism after substance exposure within 24

hours by oral, skin, or inhalation routes (Saganuwan,

2017). The test uses the LC

to indicate toxicity,

which is determined based on the mortality ratio of

experimental animals (Parasuraman, 2011). The use

of adult zebrafish to test the toxicity of a drug

candidate has currently replaced the use of mammals

to implement the principles of reduction,

replacement, and refinement in the use of animals for

research. Several studies have shown that zebrafish in

both the embryonic and adult forms have an equal

Toxicity of Self-nanoemulsifying Drug Delivery System Formulation of Nigella Sativa L. Seed Oil against Adult Danio rerio

sensitiity to chemicals, which is indicated by the

slight difference in their LC

values (Kovrižnych et

al., 2013). Embryonic and adult zebrafish also have

comparable sensitivity to cationic and nonionic

surfactants (Vaughan & Van Egmond, 2010).

The toxicity tests of the SNEDDS and non-

SNEDDS formulations of NSOE on zebrafish were

conducted for 96 hours in each test group. For the

SNEDDS formulation, 100% zebrafish mortality

occurred at 500 ppm concentration; at 31.5 ppm, the

zebrafish mortality was zero. As for the non-

SNEDDS group, 100% zebrafish mortality occurred

at 125 ppm concentration and no deaths occurred at

7.8125 ppm. The probit analysis revealed that the

value of the SNEDDS formulation of NSOE was

154.637 + 75.609 ppm, whereas the LC

of the non-

SNEDDS formulation was 72.358 + 15.253 ppm.

These LC

values showed that the SNEDDS

preparation of NSOE was less toxic than the non-

SNEDDS preparation; however, this difference was

not significant (p = 0.138).

This result is in agreement with previous studies

that showed that SNEDDS formulations were safer

than their non-SNEDDS preparations. The toxicity of

a SNEDDS formulation of Ipomoea reptans against

Vero cells using a 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl-tetrazolium bromide (MTT) assay did not

cause Vero cell death; instead, the cell culture growth

improved

(Chabib et al., 2019). Another study

showed that the preparation of arteether SNEDDS as

an antimalarial in Plasmodium yoelii nigeriensis-

infected mice showed a better bioavailability with

minimal toxicity (Dwivedi et al., 2014). In another

acute toxicity test, the SNEDDS formulation of bay

leaf chloroform extract also gave a very high LC

value; in the subchronic toxicity test, the preparation

had minimal effects on the pancreas, kidneys, and

liver at low to moderate doses. However, organ

damage was directly proportional to the increasing

dosage

(Prihapsara et al., 2018).

This study results might have implications for

nanoparticle research and might recommend against

the use of best combinations of surfactant and co-

surfactant in preparation of SNEDDS formulations.

However, further ongoing research is required to

ensure the safety of NSOE SNEDDS formulation for

oral drug delivery in animals and modification of this

formulation to guarantee their future application.

One limitation of this study was the composition

of the SNEDDS formulation of NSOE, which was

based on previous research. SNEDDS formulation

preparation using this composition resulted in good

stability as evidenced by the stability tests and

measurements of globules and zeta potential, but its

PDI and %transmittance was not optimal. Another

limitation is that only the toxicity was tested, not the

efficacy or pharmacological activity. The SNEDDS

formulation of NSOE is nontoxic, but the efficacy

remains to be determined. Thus, an investigation into

the efficacy and toxicity in one study is

recommended.

5 CONCLUSIONS

The LC

value of the SNEDDS formulation of NSOE

(154.637 ± 75.609) ppm was better than its non-

SNEDDS form (72.358 ± 15.253 ppm), but this

difference was not statistically different. Thus, the

toxicities of SNEDDS and non-SNEDDS

formulations of NSOE were comparable. The

SNEDDS preparation did not reach optimal

conditions, as indicated by good globule size and zeta

potential values but non-optimal PDI and

%transmittance values. Thus, the toxicity of the

SNEDDS formulation may improve with

optimization.

ACKNOWLEDGEMENTS

The authors would like to thank Dr. Sufi Desrini for

suggestions during the preparation of the study, and

Mr. Haryanto, Mr. Angga, and Mr. Marno for their

help during the data collection

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JIMC 2020 - 1’s t Jenderal Soedirman International Medical Conference (JIMC) in conjunction with the Annual Scientiﬁc Meeting

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