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Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 74 No. 2 pp. 497ñ504, 2017
ISSN 0001-6837
Polish Pharmaceutical Society
FORMULATION AND ASSESSMENT OF SEMI-SOLID CARRIER
INCORPORATED WITH HERBAL EXTRACT OF LAWSONIA INERMIS
AYSHA ASLAM1,2*, FAISAL SAMEE3, MUHAMMAD ZAMAN1, ATTIQUE UR RAHMAN MUFTI4
and ATIQ UR REHMAN1
Faculty of Pharmacy, The University of Lahore, Lahore, Pakistan
Institute of Pharmaceutical Sciences, University of Veterinary and Animal Sciences, Lahore, Pakistan
3
College of Pharmacy, University of the Punjab, Lahore, Pakistan
4
Faculty of Pharmacy, University of Sargodha, Sargodha, Pakistan
1
2
Abstract: The development of an optimal microemulsion of lawsone for transdermal delivery was the core
objective of the study. Effects of formulation variable including oils, surfactants and co-surfactants on the percutaneous delivery of lawsone microemulsion have also been inspected. Pseudoternary phase diagrams with oil,
cosurfactant-surfactant mixtures (Smix) were constructed to recognize the microemulsion areas. Microemulsion
prepared was of 5%. The in vitro transdermal penetration of microemulsion of lawsone was determined by
Franz diffusion cell. These profiles indicated that the absorption and rate of penetration of lawsone microemulsion was far better than the saturated solution of lawsone. The formulation was characterized for pH, conductivity, viscosity and passed stability tests. A non-irritant formulation to skin was prepared in this way. The outcomes specify that the transdermal drug delivery ability and phase behavior of microemulsion is affected by the
type of cosurfactant and surfactant.
Keywords: cosurfactant, lawsone, surfactants, transdermal microemulsion
Natural Orange 6. Phase is solid. Chemically it is 2hydroxy-1,4-naphthoquinone. It is almost insoluble
in water, soluble in methanol and 95% ethanol.
Molecular weight is 174.153 g/mol and density is
1.46 g/cm3, melting point is 195-196OC with
decomposition, with an optical absorption maximum of 452 nm. Molecular formula is C10H6O3.
Aqueous lawsone extracts strongly absorb UV
light, and can act as effective sunscreens. The plant
has many activities including therapy for malignant
ulcers, jaundice and epilepsy, and for dyeing grey
hair (4). The leaves are used in obstinate skin diseases, bleeding disorder (5) diuretic, ophthalmia,
syphilitis, sores, amenorrhoea, scabies, headache,
bronchitis, boils, spleen diseases and favors the
growth of the hair. Flowers are refrigerant and used
in insomnia (6) and headache (7). The bark is used
in jaundice, spleen enlargement and leprosy. It is
used as medicinal plant because of its antibacterial,
antifungal, antihemorrhagic, hypotensive, sedative,
antiamoebiasis and astringent effects (8).
Many diseases are being treated with drug
obtained from plant herbal products (1). Knowledge
and benefits regarding medicinal drugs are available in the earliest literature. Nowadays, people are
using traditional medicines for their health care
needs (2). Lawsonia inermis generally known as
henna belongs to Lythraceae family nurtured in
warm dry climates. A source is a 2.6 m multibranched small tree which leaves are opposite,
elliptical, sub-sessile and broadly lanceolate
(1.5ñ5.0 cm ◊ 0.5ñ2 cm), with depressed veins on
the dorsal surface. Flowers have four sepals, petals
are obviate and a 2 mm calyx tube with white or red
stamens inserted in pairs on the rim. Ovary is four
celled style up to 5 mm long and erect; 4ñ8 mm
diameter brownish capsules fruits are small having
32ñ49 seeds, opening in four splits (3). It contains
tannins as active ingredients. Main chemical components include lawsone, hennadiol, scopoletin,
betulin, quinone and naphthoquinone. The primary
coloring matter of henna is lawsone, also known as
* Corresponding author:e-mail: [email protected]
497
498
AYSHA ASLAM et al.
The aim of the present study was to prepare
microemulsion using herbal plant extracts and evaluate their release from the topical preparations using
Franz cells. Nylon membrane was used for this purpose. Drug release from the topical preparation containing traditional remedy was investigated.
EXPERIMENTAL
Materials
Synthetic standard of lawsone was purchased
from Aldrich (Sigma-Aldrich, Germany). Olive oil,
seasame oil, oleic acid, Polysorbate 80, and sorbitan
monolaurate (were all from Daejung, Korea), propylene glycol (Merck, Pakistan), 2-propanol, ethanol
(BDH, England). All the chemicals used were of
analytical grade.
Plant material extraction
Sample of henna leaves were collected in June
2012. The leaves were air-dried and powdered. The
500 g powdered leaves were soaked in 1500 mL of
96% alcohol and 1500 mL of distilled water (alcohol : distilled water 1 : 1) (by maceration) in 5000
mL beaker for 14 days and filtered through Whatmann filter paper No. 41.
Solubility study
Addition method was used to check the solubility of L. inermis by adding ethanolic plant extract
in cosurfactants, oils and surfactants in stoppered
vial having 5 mL capacity through magnetic stirring
for 1 h at 25OC.
Preparation of microemulsion
The microemulsion was prepared as described
by Chen (9). A surfactant mixture was prepared by
manually mixing Tween 80 and ethanol as surfactant and cosurfactant, respectively, in a 1 : 1, v/v
ratio. Surfactant mixture was added to 0.5 g of oleic
acid (oil) and mixed vigorously with magnetic stirring. Plant extracts (0.5 g) were added to the surfactant mixture and then into oleic acid and stirred until
completely dissolved. In order to check transdermal
drug delivery potential, saturated aqueous solution
of drug was compared with the microemulsion formulation. Excess amount of L. inermis extract was
dissolved in distilled water to prepare the saturated
aqueous solution of the drug. Membrane filter was
used for filtration of solution.
Preparation of apparatus and sampling
Franz diffusion cells (PermeGear, Bethlehem,
PA) having receptor section with a size of 12.0 mL
and a surface area of 1.767 cm2 were used. Hydro
alcoholic solution 70 : 30, v/v was used as the receptor medium. Equilibration of the skin was done by
soaking it in hydro alcoholic solution for 12 h at 4OC
(10). Receptor medium was poured into the receptor
section and then the skin was placed in such a way
that stratum corneum side of the skin face the donor
section. A clamp was used to join two halves of the
cells. Trapping of air should be avoided beneath the
skin (11). In case of trapping of bubbles, sampling
port was slanted horizontally until the bubble
escaped. Water bath was used to maintain the temperature of the receptor solution at 32 ± 0.2OC with
a Teflon-coated magnetic bar for stirring of solution
at 600 rpm during the experiment. The donor section
was sealed with aluminum foil and contained 1.0
mL of test microemulsion (12). A long needle was
fixed to a syringe and was used to withdraw samples
at fixed intervals (0.5, 1, 2, 3, 4, 5, 6, 12 and 24 h).
Syringe filter (0.45 µm) was used to filter the solution analyzed by HPLC at 260 nm against a blank
(13). The same amount of fresh hydro alcoholic
solution was added to the receptor section after the
removal of the trial sample. The experiments were
performed three times.
Water titration method
Water titration method is mostly used to construct phase diagrams for microemulsions at room
temperature (14). The diagram was plotted by
ProSim Ternary Diagram software.
Characterization of microemulsions
HPLC analysis
HPLC system equipped with a column oven
(Varian Metathermô), an ultraviolet (UV) diode
array detector (Varian ProStarô) and a ternary
pump (Varian 9010) was used for chromatographic
analysis. Reverse-phase Supelcosil LC-PAH C18
chromatographic column (5 µm, 150 ◊ 3 mm) was
used for the separation of HNQ. Registered spectral
range was 190ñ400 nm with λmax at 260 nm. Mobile
phase: 1 mL acetic acid : methanol in volume ratio
20 : 80; flow rate: 0.8 mL/min; column and detector
temperature: 40OC. Autosampler injection was of 5
µL.
Preparation of stock solution:
Ten milligrams of lawsone was weighed and
dissolved in methanol and the final volume was
made up to 10 mL in a volumetric flask (stock solution 1 mg/mL). This prepared solution (100 µL) was
taken and the volume made up with methanol to
give 900 µL solution (0.1 mg/mL). Further dilution
499
Formulation and assessment of semi-solid carrier incorporated...
of the solution was done by adding 900 µL of
methanol in 100 µL of this solution to give concentration of 0.01 mg/mL. In the same way 0.001 and
0.0001 mg/mL solutions were prepared by serial
dilutions, respectively, and their peak areas were
determined by HPLC at 260 nm.
Microemulsions were inspected visually for
the following characteristics:
● Visual clarity
● Color
● Transparency
● Homogeneity
● Phase separation
Rheological measurements
RVDV III Brookfield viscometer was
employed to determine viscosity at various shear
rates and different torque values and results were
presented in cP. The Rheocalc V2.6 software was
used for calculation. The temperature was maintained at 25.2OC throughout the experiment.
Droplet size analysis
Zetasizer nano S (Malvern Instruments,
Worchestershire, UK) was used to conclude the
droplet size of the microemulsion employed. Light
scattering was observed at 25OC.
pH measurements
WTW inoLab, Germany pH meter was
employed to measure pH values of microemulsion at
25OC.
Table 1. In vitro physico-chemical parameters of formulation.
Table 2. Known concentrations of standard of lawsone with corresponding HPLC area values.
No.
Parameters
Observation
1.
Clarity
Clear
2.
pH
6.4
3.
Conductivity
0.25 µS/cm
4.
Viscosity
432-440 cp
5.
Flux
6.7 (µg/cm2/h)
Conductivity measurements
WTW Cond Multi 197i (Weilheim, Germany)
conductometer was employed to measure conductivity (σ) of microemulsion and results are presented in µS/cm (15).
Concentration
mg/mL
Area
a.u.
1
1
9844415
2
0.1
755025
3
0.01
60043
No.
Figure 1. Pseudoternary phase diagram of L. inermis based microemulsion
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AYSHA ASLAM et al.
Figure 2. Known concentrations of standard of lawsone with corresponding HPLC peak area values (a.u.)
Figure 3. HPLC chromatogram of lawsone
Figure 4. Average of percentage release vs. time interval
Formulation and assessment of semi-solid carrier incorporated...
Table 3. Average of percentage release vs. time interval.
No.
Time
h
Average drug
release %
1
0.5
8
2
1
15.5
3
2
27
4
3
37
5
4
50
6
5
66.5
7
6
70.5
8
12
81
9
24
93.5
501
Thermodynamic stability tests
The selected microemulsion formulation was
subjected to stress tests which are as follows (16):
???????
Heating/cooling cycle
The formulation was stored for 48 h at 4OC
(refrigerator temperature) and 40OC. The formulation was found to be stable at these temperatures.
Freeze thaw cycle
The formulation was stored for 48 h between 50OC and +25OC (room temperature). The formulation remained stable at these temperatures also.
Figure 5. Graphical representation of Korsmeyer Peppas model
Figure 6. Graphical representation of first order model
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AYSHA ASLAM et al.
Figure 7. Graphical representation of zero order model
Table 4. 'R2' and 'n' values for zero order, first order and Korsmeyer Peppas (KP) model.
Zero order
First order
KP model
R2
K
R2
K
R2
N
0.993
10.661
0.4746
0.076
0.9205
0.66
MS
F
Significance F
15.75831
0.005395
Table 5. One way ANOVA of permeation data of microemulsions.
Df
SS
Regression
1
4983.963
4983.963
Residual
7
2213.926
316.2752
Total
8
7197.889
Table 6. Regression statistics applied on the formulation data.
Regression statistics
Multiple R
0.832118
R2
0.69242
Adjusted R
2
0.64848
Standard error
17.78413
Observations
9
males/4 females. Sample (0.1 g) was retained on 1 ◊
1 cm2 gauze dressing and applied onto the skin of
inner arms directly with stretch adhesive tape
(Paragonô) to fix it. Erythema was measured with
Mexameterô (Courage and Khazaka, Germany)
after 8 h. Before use of the formulation, the skin of
inner arms was measured for control studies (17).
Stability studies
No phase separation occurred when centrifuged at 6000 rpm for 30 min.
Calculation of flux (J)
The rate of permeation per unit area or the rate
of transfer is called flux (J) and is represented by a
vector in space and the results are presented in
µg/cm2/h (18).
Skin irritation study
Ten human volunteers were selected and formulation was tested for 8 h. The volunteers were
within the age range of 22-29 years and include 6
Statistical analysis
The collected data were analyzed by appropriate statistical procedure - analysis of variance
(ANOVA) (19).
503
Formulation and assessment of semi-solid carrier incorporated...
Table 7. Summary of the statistical parameters of microemulsion.
Parameters
Coefficients
Standard error
t Stat
p-value
Lower 95%
Upper 95%
Intercept
28.480
8.014
3.55
0.009
9.530
47.431
Time
3.350
0.844
3.96
0.005
1.354
5.346
y = 28.48 + 3.35x
RESULTS
The solubility of lawsone in different oils,
cosurfactants and surfactants was determined.
Pseudoternary phase diagram 1 : 1 was constructed
in which oil phase is oleic acid, surfactant is Tween
80 and cosurfactant is ethanol. Instead of using pure
water, hydro alcoholic extract was used.
Pseudoternary phase diagram is given in Figure 1.
The microemulsion formulation was evaluated with respect to clarity, pH, viscosity, electrical
conductivity and flux. Viscosity was measured
using spindle no. 2 with speed of 20 rpm. Values of
viscosity, pH, conductivity and flux are given in
Table 1.
In vitro release studies of natural constituent of
L. inermis extract from microemulsion formulation
were done by combination of Franz cells and HPLC
determination technique using C18 column.
Standard curve was constructed against known concentration of lawsone standard vs. peak area of
HPLC chromatogram. Values for standard curve are
given in Table 2 and graphical representation is
given in Figure 2.
Regression value of standard curve is highly
applicable R2 = 0.9995.
Average percentage release of active constituent of formulation by using vertical diffusion
cells at different time intervals was given. Values of
graph are given in Table 3 along with the graphical
representation in Figure 4.
Different models were applied at time and average
percentage release. Following models were applied
and graphs were plotted.
1. Korsmeyer Peppas model
2. First order model
3. Zero order model
Graphical representation is given in Figures 57. The data of the formulation show results according to zero-order model.
Calculation of ëR2í and ëní value for each
model is given in Table 4.
The collected data were analyzed by appropriate statistical procedure which is analysis of variance (ANOVA) or by any suitable statistical procedure fit for data. The details are given in Table 5.
The data show that results are significant. The
results were analyzed by one-way analysis of variance (ANOVA). The value of p = 0.009295 (Table
5), therefore it showed significant result. The transdermal flux of microemulsion formulation significantly increases. Tables 5-7 summarize the statistical parameters of microemulsion.
The calculated value for Y intercept is 28.48 +
3.35 x. It shows that an increase in time will increase
the permeation rate of the extract.
DISCUSSION
Microemulsion was prepared by using Tween
80, ethanol and oleic acid. Four different formulations were prepared. Pseudoternary diagram was
constructed, in which shaded area represents the
microemulsion range whereas non shaded area represents unclear range. Phase diagrams were used to
determine concentrations required to prepare
microemulsion. All the four formulations were prepared at 1 : 1 ratio. Out of these four formulations
one was selected on the basis of clarity, pH, conductivity, flux and viscosity. The high conductivity
of microemulsion 0.25 µS/cm showed that it was
O/W type microemulsion. Microemulsion had a pH
of 6.4 which was the indication that it could be nonirritating to skin and will be best for topical application. The viscosity was found to be 432-440 cP. The
flux value 6.7 µg/cm2/h showed the highest permeation rate through skin. Surfactants are known to
enhance the permeability and flux. Same effect of
the non-ionic surfactant was noticed here in the current study that addition of non-ionic surfactant,
Tween 80 has increased the flux greatly. They
increase the permeation by reducing the barrier
characteristics and crystallinity of lipid bilayer thatís
why they are commonly and effectively used in topical formulations. Various formulated formulations
were evaluated by various stated parameters and
finally the best formulation was selected. The permeation profiles followed zero order kinetics. The
permeation experiments were analyzed by one-way
analysis of variance (ANOVA) at the level of p <
0.05. The value of p = 0.009295 which is smaller
than 0.05, therefore it showed significant result. The
504
AYSHA ASLAM et al.
micro emulsion formulation significantly increased
the transdermal flux in comparison to the saturated
aqueous drug solution which was used as the control.
CONCLUSION
Microemulsion containing lawsone was
formed effectively. Instead of simple aqueous solution of drug, prepared microemulsion is estimated to
boost the transdermal drug delivery potential of this
drug. Hazard of side effects can be overwhelmed in
this way and recurrent application will be possible.
Acknowledgment
To Mr. M. Nadeem Irfan Bukhari, for the technical support in the Laboratory of Pharmaceutics,
University College of Pharmacy, University of the
Punjab, Lahore, Pakistan.
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Received: 28. 03. 2016