World Journal of Pharmaceutical Research

Sunil et al. World Journal of Pharmaceutical

SJIF Research
Impact Factor 5.990

Volume 4, Issue 9, 1811-1828. Research Article ISSN 2277– 7105

DEVELOPMENT OF NANOSTRUCTURED LIQUID CRYSTALLINE

FORMULATION OF ANTIMALARIAL AGENTS ARTEMETHER AND
LUMEFANTRINE

Sunil Saini*, Gurpreet Singh1, Naresh kalara1, Charan Singh2, Geeta2, Tarun Virmani3
*1
Alwar Pharmacy College, Rajasthan, India.
2
CBS College of Pharmacy & Technology, Faridabad, Haryana, India.
3
School of Pharmaceutical Sciences, MVN University, Palwal, Haryana, India

Article Received on
ABSTRACT
04 July 2015, The main objective of the research work was to formulate a lipid based
Revised on 28 July 2015, delivery system that may be useful for increasing the bioavailability of
Accepted on 23 Aug 2015
BCS class II drugs. Such type of lipid based drug carriers may keep the
drug in dissolved state until the drug is completely absorbed. The
*Correspondence For
present work mainly focused to formulate liquid crystalline
Author
nanoparticles in combination to improve the solubility of both ARTM
Sunil Saini
Alwar Pharmacy College, and LMF which could probably improve absorption of ARTM and
Rajasthan, India. LMF drugs and may circumvent the drawback of poor bioavailability.
In-vitro dissolution study for OF1 (F9) was carried out and it had been
found that 98.00% Artemether and 89.00% Lumefantrine released within 72 hrs. OF1 was
further evaluated for various parameter including organoleptic evaluation and physical
stability and all are found within the acceptable ranges over a period of 3 months.

KEYWORD: Bioavailability, Artemether, Lumefantrine, LiquidCrystals, Nanoparticles,

Organoleptic etc.

INDRODUCTION
Malaria is most prevalent health problem in various countries, where transmission occurs
frequently and even areas, where transmission has been controlled or eliminated. Near of 300
to 500 million cases of malaria are reported annually, therefore it becomes one of the most
common infectious disease in world. According to WHO more than 90% of the 1.5 to 2
million deaths accounted for malaria each year in African children, about 1.5 million cases of
malaria are reported annually in India out of which 40-50% are associated with Plasmodium

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falciparum infestation.[1]

1.1.1Causative agents
There are 5 types of Plasmodium species which cause acute infectious disease
Malaria.Plasmodium falciparum,Plasmodium vivax,Plasmodium ovale,Plasmodium
malariae,Plasmodium knowlesi. The vast majority of deaths are caused by P. falciparum and
P.vivax, whereas P.malariae cause a milder form of malaria that is rarely fatal.[2] The
zoonotic species P.knowlesi prevalent in Southeast Asia causes malaria in macaques but can
also cause severe infections in humans.[3] Cerebral malaria is defined as a severe P.

falciparum-malaria presenting with neurological symptoms, including coma (with a Glass

Gow coma scale less than 11, or a Blantyre coma scale greater than 3), or with a coma that
lasts longer than 30 minutes after a seizure.[4]

Fig 1.1: A diagram showing the life cycle of the malaria parasite, detailing the A. Exo-
erythrocytic cycle, B. Erythrocytic cycle and C. Sporogonic cycle.
(Source:http://dpd.cdc.gov/dpdx/html/Malaria.htm)

MATERIAL AND METHODS

1.1.2 Preformulation study
1.1.3 Melting point determination
Melting point of Artemether and Lumefantrine was determined by capillary rise method using
digital melting point apparatus. Practically determined melting points were compared with
the literature values.[12]

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1.1.4 Solubility studies

1.1.5 Solubility study in water
Solubility of Artemether and Lumefantrine was determined in distilled water. The practical
values of the parameters were compared with values given in literature.

1.2.1 Solubility study in different solvents

Solubility of Artemether and Lumefantrine was determined in different solvents like
methanol, ethanol, ethyl acetate, chloroform and distilled water. The initial solubility of ART
and LMF was determined by weighing out 10 mg (or other suitable amount) of ART and
LMF. To this add 10 µl of solvent of interest. If the compound doesn’t dissolved, a further 40
µl of solvent was added and its effect noted. Successive amount of the solvent was then added
until the compounds were dissolved. This method give an approximate value of solubility.[5]

1.2.2 Partition coefficient[6]

Partition coefficient of Artemether and Lumefantrine was determined by shake flask method.
Two separate conical flasks of 50 ml were taken, into which 10 ml of water and 10 ml of n-
octanol taken separately and allowed to shake over wrist shaker for 24 hrs at 37oC to achieve
pre-saturation of both phases, allow the phases to separate. In the each flask add 10 mg of
Artemether and Lumefantrine and allowed to shake the flask over the wrist shaker for 24 hrs.
The solutions were allowed to stand at room temperature for 30 min. Two phases of octanol
and water samples were separated by separating funnel. Aqueous phase containing
Artemether and Lumefantrine were filtered using Whattmann’s filter paper grade number 41.
The filtered aliquots were analyzed spectrophotometrically at 256 nm for Artemether and 342
nm for Lumefantrine.

1.2.3 Microscopic examination

1.2.4 Ultra-violet spectrophotometric analysis
1.2.5 Determination of λmax of Artemether
λmax of Artemether is determined in methanol, water, at buffer pH 6.8 and at pH 1.2 in the
given sample of drug.

1.3.1 Determination of λmax of Lumefantrine.

10 mg of Lumefantrine accurately weighed and transferred to 100 ml volumetric flask. It was
dissolved in 20 ml of methanol then sonicated for 15 min, and the volume was made up with
methanol. The solution was scanned between 200-400 nm and obtained results were

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compared with the reference values. [7]

1.3.2 Formulation and development

Liquid crystalline nanoparticles (cubosomes or hexosomes) containing Artemether and
Lumefantrine were formulated by Hydrotropic dilution method. In this method ethanolic
solution of GMO with drug and aqueous solution of poloxamer 407 were prepared by
vortexing. Ethanol used to dissolve monoolein, oleic acid, Artemether, Lumefantrine and
aqueous phase used to dissolve poloxamer 407. Water phase containing the poloxamer (10%
w/v) added to the ethanolic phase drop wise with continuously vortexing resulting in the
precipitation of the GMO. A milky suspension is formed which indicate the formation of
liquid crystalline as described in Fig. 4.1.35

1.3.3 MATERIALS AND METHODS

Table 1.1: List of drugs and excipients
S. No. Drugs and excipients
1 Artemether
2 Lumefantrine
3 Glyceryl monooleate
4 Poloxamer 407
5 Oleic Acid

Fig1.2: Formulation chart for LCs of Artemether & Lumefantrine

1.3.4 Characterization of Artemether & Lumefantrine liquid crystalline dispersion

1.3.5 Particle size analysis
The mean particle size and polydispersity index was measured using laser diffraction on a
Malvern Zetasizer Ver. 6.01r (Serial Number: MAL1027952, Malvern instruments Ltd.) at

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2oC considering a viscosity of pure water 0.8872. The particle size was analysed by diluting
the prepared formulations with distilled water.[8,9]

1.4.1 Entrapment efficiency[8,9]

Entrapment efficiency of Artemether and Lumefantrine was determined using the Nanosep
device. The liquid crystalline dispersion of Artemether and Lumefantrine were centrifuged in
cooling centrifuge (Remi India) using Nanosep device (MWCO: 2-3 KD, Pall Life Science;
India). 0.5 ml of prepared LCN formulation was taken in Nanosep device and then placed in
cooling centrifuge (Remi; India). The sample is then centrifuged at 13000 rpm for 30 min at
10ºC. Then aqueous phase was collected, and analysed for Artemether and Lumefantrine at
256 nm and 342 nm respectively by UV spectroscopy. The preliminary trials were performed
for the optimization of centrifugation speed and time. Time and speed 13000 rpm for 30 min
respectively were optimized to separate unetrapped drugs from the LCN .The encapsulation
efficiency (EE) was determined using the following equation:

1.4.2 Evaluation of optimized formulation

The optimized formulation was identified based on constraints using design expert software
(version 9.1.0, state ease Inc., Minneapolis, MN). The optimized formulation was formulated
according to method given in 5.3.2 and evaluated for particle size, entrapment efficiency, in-
vitro release, physical stability and chemical stability.

1.4.3 Physiochemical characterisation

1.4.4 Organoleptic evaluation
Drug studied on creaming, Discoloration.

b. Creaming
Optimised formulation “OF1” was analysed for creaming. Creaming involve the separation
ofdispersed phase form the liquid crystalline dispersion on storage under normal condition at
room temperature. The dispersion type OF1 of LCN was oil in water. The formed dispersion
was visually assessed for creaming during the storage period at 25oC/60% RH on weekly
basis up to three months.[10]

1.4.5 In-vitro release study[11]

In-vitro drug release study of Artemether and Lumefantrine was performed in simulated
gastric fluid pH 1.2 containing 1% w/v BKC and phosphate buffer of pH 6.8 containing 0.5%

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w/v SLS by using dialysis bag method, dialysis membrane having pore size 2.4 nm and a
molecular weight cut off 12000-14000 Dalton (HiMedia, India) were used. The dialysis bag
retains the nanoparticles and releases the free drug into the dissolution media. The dialysis
membrane was pre-treated with sodium bicarbonate and EDTA solution and kept in diluted
EDTA solution prior to use. The bag was washed with distilled water prior to use. 2 ml
formulation of Artemether and Lumefantrine was placed in dialysis bag. Separate dialysis
bags containing the formulation 2 ml in each were immersed in 200 ml simulated gastric fluid
for Lumefantrine and intestinal fluid (Artemether) maintained at 37±0.5oC and stirred at 100
rpm.

1.5.1 Melting point

Table 1.2: Melting point of Artemether and Lumefantrine
Parameter Drug Observed Reference
Melting point Artemether 85±2°C 86-88°C
Lumefantrine 130±2°C 128-132°C

1.5.2 Solubility studies

Solubility of ARTM and LMF was determined in different solvents including distilled water,
methanol, ethanol, ethyl acetate and chloroform..

Table 1.3: Solubility of Artemether in different solvents

Solvent Observed (mg/ml) Reference (mg/ml)
Ethanol 14 16
DMSO 9 10
DMF 17 20
Distilled water 1.5 2

Table 1.4: Solubility of Lumefantrine in different solvents

Solvent Observed (mg/ml) Reference (mg/ml)
Ethanol 2.2 2.8
DMSO 98 100
DMF 97 100
Oleic acid 156 158
Distilled water 0.05 0.1

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1.5.3 Partition coefficient

Partition coefficient of Artemether and Lumefantrine was determined by Shake flask method,
it was found that the values of partition coefficients of ARTM and LMF were complied with
the reference values represented in Table 1.5. Obtained result indicated that these drugs are
highly lipophilic in nature and may possess good permeability across the cellular membrane.

Table 1.5: Partition coefficient

Parameter Drug Observed Reference
log Po/w Artemether 3.26 3.06-3.53
log Po/w Lumefantrine 2.9 2.29-3.52

(a) Artemether at 40x b) Artemether at 100x

(c) Lumefantrine at 40x (d) Lumefantrine at 100x

Fig. 1.3: microscopicexamination of Artemether and Lumefantrine

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1.5.4 Infra red analysis of drugs

Fig. 1.4: Reference FTIR spectrum of Artemether

Fig. 1.5: FTIR spectrum of test sample of Artemether

sample spectra of Artemether respectively. On FTIR analysis, obtained spectra (Fig. 1.5) of
test samples of drug matched with the reference spectra given in USP 2009. The peaks
obtained in FTIR spectra of test sample were examined and found in accordance with the
functional groups present in reference spectra of Artemether. From this study it was
confirmed that procured drug sample was authentic.

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Lumefantrine

Fig. 1.6 Reference FTIR spectrum of Lumefantrine

Fig. 1.7: FTIR spectra of test sample of Lumefantrine

1.5.5 Determination of λmax of Artemether and Lumefantrine

 Determination of λmax of Artemether In buffer pH 1.2
λmax of Artemether in buffer pH 1.2 after derivatizing with 1N HCl was found 256 nm.

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Fig. 1.8: Absorption maxima of Artemether in buffer pH 1.2

λmax of Artemether in buffer after treating with 1N HCl was found 256

In phosphate buffer pH 6.8

λmax of Artemether in phosphate buffer pH 6.8 was found 256 nm

Fig. 1.9: Absorption maxima of Artemether in phosphate buffer pH 6.8

λmax of Artemether in phosphate buffer pH 6.8 after treating with 1N HCl was found 256
nm and which compiled with the literature value 256 nm .

 Determination of λmax of Lumefantrine

Fi.g. 1.10 Absorption maxima of Lumefantrine in 0.1 N HCl

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λmax of Lumefantrine in 0.1N HCl was found 342 nm and obtained UV spectra of scanned
sample of pure drug was depicted in Fig. 1.10
 In buffer pH 1.2

Fig. 1.11: Calibration curve of Artemether in buffer pH 1.2

Fig. 6.12 representing the calibration curve of LMF. The calibration equation for straight line

was observed to be Y= 0.021X-0.0044 correlation coefficient (R2) of 0.9997 which was used
to calcu late concentration of samp les for dissolution study and other analytical purposes.

Fig. 1.12: Calibration curve of Artemether in phosphate buffer pH 6.8

In phosphate buffer pH 6.8

Fig.1.13 Calibration curve of Lumefantrine in 0.1 N HCl

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1.2.1Formulation and Development

Table 1.6: Formulation variables of LCs of Artemether/Lumefantrine
S. NO. Code Independent
variables
1 X1 Artemether
2 X2 Lumefantrine
3 X3 Oleic acid

Table 1.7: Response variables LCs of Artemether/Lumefantrine

S. NO. Code Dependent variables

1 Y1 Particle size
2 Y2 Entrapment efficiency of
Artemether

3 Y3 Entrapment efficiency of
Lumefantrine

Table 1.7: Actual and coded values of independent factors

*Every formulation contained 5 ml of liquid dispersion

1.2.1 Evaluation of Artemether and Lumefantrine LCN

Table 1.8: Compositions and particle size of liquid crystalline nanoparticles of
Artemether and Lumefantrine
S. No. Trial code Composition Particle size analysis
ARTM
LMF (mg) Oleic acid Z-average
(mg) PdI
(mg) (d.nm)
1 F1 20 100 125 158 0.60
2 F2 15 75 125 176 0.50
3 F3 15 75 125 184 0.30
4 F4 15 75 125 162 0.40
5 F5 20 50 125 195 0.20
6 F6 10 75 150 165 0.22
7 F7 15 50 150 187 0.58
8 F8 15 50 100 200 0.48
9 F9 10 50 125 193.5 0.10
10 F10 15 100 150 180 0.45
11 F11 20 75 100 192 0.38
12 F12 10 100 125 164 0.41
13 F13 10 100 125 157 0.25
15 F15 15 75 125 190 0.22
16 F16 15 100 100 156 0.38
17 F17 20 75 150 189 0.43

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1.2.2 Optical microscopic examination

I. Microscopic examination of F9 (LCs dispersion, PDI 0.6) at 40X

F9 dispersion after 2 days F9 dispersion after 7 days

Fig. 1.14: microscopic examination of F9 formulation

Table 1.9: Microscopic examination of LCN of Artemether and Lumefantrine
Microscopic examination
S. No. Formulation code Uniformity of Presence of oil drops
dispersion
1 F1 ++ ─
2 F2 ++ ─
3 F3 ++ ─
4 F4 ++ ─
5 F5 +++ ─
6 F6 +++ ─
7 F7 +++ +
8 F8 ++ +
9 F9 +++ ─
10 F10 +++ +
11 F11 ++ ─
12 F12 ++ ─
13 F13 ++ ─
14 F14 ++
15 F15 ++ ─
16 F16 ++ ─
─
17 F17 ++ ─
+++ = Uniformity of dispersion + = Oil drops present
++ = Less Uniform ─ = Oil drops not present

1.2.3Optimized formulation of LCN of Artemether and Lumefantrine

Based on the analysis of all the batches, it has been found that experimentally determined
results of “OF1” showed closeness between the predicted values and observed values for all
responses. This formulation considered as optimized and further evaluated for physical

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stability and in vitro drug release.

Fig. 1.15: Optimized formulation

1.2.4 Evaluation of optimized formulation “OF1”

1.2.5 Stability study of optimized formulation

a. Organoleptic evaluation
Optimized formulation was evaluated for phase separation, creaming and discoloration of
product. Biweekly basis visual assessment of optimized formulation was conducted for 3

months at storage condition 25oC/60% RH and obtained results were shown in table 1.11

Table 1.10: Result of physical stability study

1st month 2nd month 3rd month

S.no Parameters Weeks Weeks Weeks

2nd 4th 2nd 4th 2nd 4th

1 Phase separation ─ ─ ─ ─ + +
2 Creaming ─ ─ ─ ─ ─ ─
3 Discoloration ─ ─ ─ ─ ─ ─
Where:
+++ = unacceptable changes + = acceptable changes
++ = significant changes ─ = no change

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Table 1.11: Physical stability Study at 25 oC/60% RH

25oC/60% RH
S. no Parameters
Initial 1st month 2nd month 3rd month
1 Particle size (nm) 193.50±0.50 195.4±0.60 205.50±0.50 215.00±0.35

2 EE of ARTM 85.50±0.50 84.33±1.52 81.60±0.40 78.40±0.60

3 EE of LMF 88.50±0.50 87.93±0.87 85.50±0.50 80.50±0.50

Each value represents mean ± standard deviation (n= 3) EE= entrapment efficiency

1.3.1 In-vitro release study of optimized formulation (OF1)

In-vitro dissolution study for Artemether was conducted for 72 hrs. In similar way
Lumefantrine drug release was estimated for 72 hrs, separately in simulated gastric fluid using
the dialysis membrane.

Table 1.12: In-vitro drug release of Artemether from optimized formulation in

phosphate buffer pH 6.8
Time (Hrs) Cumulative drug release (%)
1 0.20±0.10
2 0.53± 0.27
4 20.00±2.50
8 30.00±2.10
12 35.60±2.20
24 60.0±2.50
48 95.23±3.20
72 98.08±3.54

Fig. 1.16: In-vitro dissolution profile of Artemether from optimized formulation

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Table 1.13: In-vitro drug release of Lumefantrine from optimized formulation in

simulated gastric fluid pH 1.2
Times (Hrs) Cumulative drug release (%)
1 0.20±0.10
2
4 0.50±0.30 5.946±1.50
8 12.17±2.20
12 20.60±2.50
24 40.50±2.65
48 75.64±2.46
72 89.20±2.80

CONCLUSION
An attempt to enhance the solubility of Artemether and Lumefantrine was achieved by
incorporating these drug candidates in a lipid carrier. Liquid crystalline nanoparticles of
Artemether and Lumefantrine were prepared by hydrotropic dilution method using glyceryl
monooleate, oleic acid, poloxamer, ethanol and water. The optimization of liquid
crystalline nanoparticles was achieved by response surface methodology (BBD).

 Pure sample of Artemether and Lumefantrine were supplied and used throughout the
experiments.
 Artemether and Lumefantrine were practically insoluble in water but soluble in
ethanol, DMSO and DMF.
 Partition coefficient for Artemether and Lumefantrine was found 3.26 and 2.90
respectively.
 λmax of Artemether and Lumefantrine was 256 nm and 342 nm, determined by UV
visible spectroscopy.

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 Out of 17 trials, trial F9 was suggested as an optimized formulation. The selection was
made on the basis of particle size and EE of ARTM and LMF.
 Entrapment efficiency of ARTM and LMF was found 85.00% and 88.50%
respectively.
 Drug release study of ARTM and LMF revealed that the drugs were released in a
remarkably controlled manner up to 72 hrs.
 Stability study revealed that the OF1 (optimized formulation) stable over a period of
three months.

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