1
1
In vitro skin permeation of sinigrin from its phytosome complex
2
Anisha Mazumdera, Anupma Dwivedia, Lizelle T. Foxa, Alicia Brümmera, Jan L. du Preeza,
3
Minja Gerbera, Jeanetta du Plessisa*
4
5
a
6
X6001, Potchefstroom 2520, South Africa.
7
*Author to whom correspondence should be addressed: [email protected];
8
Tel.: +2718 299 2274; Fax: +2787 231 5432
9
Centre of Excellence for Pharmaceutical Sciences, North-West University, Private Bag
2
10
Abstract
11
Objectives: Sinigrin is a major glucosinolate present in plants of the Brassicaceae family.
12
Recently sinigrin and its phytosome formulations have been investigated for its wound
13
healing actions, by our research group. The aim of this study was to demonstrate sinigrin
14
drug release from its phytosome complex and also to determine if the phytosome complex
15
enhances the delivery of sinigrin into the skin when compared to free sinigrin.
16
Methods: In vitro Franz cell diffusion studies were performed on human abdominal skin. The
17
morphology of the phytosome complex was examined by transmission electron microscopy.
18
The in vitro drug release was determined by using dialysis sacks.
19
Key findings: The in vitro drug release indicated a controlled and sustained release of
20
sinigrin from the phytosome complex. Tape stripping results showed that the sinigrin-
21
phytosome complex (0.5155 µg/ml) statistically significantly enhanced the delivery of sinigrin
22
into the stratum corneum-epidermis when compared to the free sinigrin (0.0730 µg/ml).
23
Conclusions: These results suggested the possibility of utilizing sinigrin-phytosome
24
complex, to optimally deliver sinigrin to the skin which can be further used for various skin
25
related diseases including wound healing.
26
27
Keywords: Sinigrin, Phytosome, Sinigrin-phytosome complex, Skin permeation, Tape
28
stripping
29
3
30
1. Introduction
31
The efficacy of employing the skin as the port of drug administration to the human body has
32
been known for many years. Since the skin constitutes an excellent natural and protective
33
barrier against the delivery of the therapeutic agents; topical and transdermal drug delivery is
34
very onerous. The skin is the largest organ in human body with a surface area of 1.8-2.0m2
35
comprising of three main layers; the epidermis, dermis and hypodermis (subcutaneous
36
layer)1. The epidermis contains an uppermost layer of cells called the stratum corneum,
37
which is the main hindrance of the skin and consists of corneocytes surrounded by lipid
38
regions. The viable epidermis is approximately 100–150 µm thick and consist of multiple
39
layers of keratinocytes and many other types of cells2. The stratum corneum lipids play a
40
crucial part in maintaining and structuring the lipid barrier which supplies protection against
41
external insults and water loss through the skin and is the cause of selective permeability3.
42
The penetration of drugs through the skin includes the diffusion through the entire epidermis
43
and the skin appendages (hair follicles and sweat glands). Topical drug delivery provides
44
advantages like controlled and sustained drug release, less fluctuations in plasma drug
45
levels, avoidance of first-pass metabolism, better patient compliance and enhanced local
46
(dermal) or systemic (transdermal) effects4.
47
Several methods have been investigated to enhance the permeation of therapeutic agents
48
through the skin and one such method is using a transdermal nanocarrier delivery system.
49
Many vehicle systems have been suggested to transport drugs through the skin, facilitating
50
drug retention and allowing a sustained release 5.One such vehicle, used recently for
51
transdermal drug delivery, is known as phytosomes or herbosomes.
52
Phytosomes are a vesicular drug delivery system in which phytoconstituents are surrounded,
53
bound and linked with a phospholipid molecule. Phytosomes have been renowned for
54
enhanced permeation of therapeutic agents through the skin during transdermal and dermal
55
delivery and when used as functional cosmetics, protect the skin against exogenous or
56
endogenous hazards in normal conditions as well as during a stressful environment, thus
4
57
providing synergistic effects6. Since phosphatidylcholine, being an essential part of the cell
58
membrane, is used in preparing phytosome formulations it acts as a carrier and also
59
nourishes the skin7,8.The transition of the phytoconstituents linked to phospholipids takes
60
place through interaction with the cutaneous structure of the skin, which helps in the release
61
of the bioactives. Phytosome complexes augment the rate of absorption of the
62
phytoconstituents through the skin to regulate the physiology of skin composition (without
63
damaging the epidermis), which highlights the potential utilization of phytosomes in
64
cosmetics as well as for systemic function via the skin9.
65
Sinigrin is a type of glucosinolate mainly found in the seeds of Brassica nigra and other
66
members of the Brassicaceae family, such as Brussels sprouts, broccoli and cabbage.
67
Figure 1 represents chemical structure of sinigrin10. Glucosinolates appear as secondary
68
metabolites that are characteristic of plants of the Brassicaceae family11. It is well known that
69
glucosinolates are broken down enzymatically by myrosinase, mainly to isothiocyanates,
70
cyanides and thiocyanates which are the main known bioactives and are generally attributed
71
for its therapeutic benefits12. In a recent study conducted in our laboratory, we have
72
demonstrated the potential wound healing activity of sinigrin and its phytosome
73
formulations13. The aforementioned steered our interest in examining the in vitro topical
74
delivery of sinigrin and its phytosome complex. To date, there is no information concerning
75
the topical delivery of sinigrin and its phytosome complexes, making this research work
76
novel. We also endeavoured to demonstrate the in vitro drug release of the free sinigrin and
77
sinigrin-phytosome complexes by deploying a dialysis bag in our current study. Figure 2
78
depicts the graphical image of the in vitro skin permeation of the sinigrin from its phytosome
79
complex.
80
2. Materials and methods
81
2.1 Materials
82
Sodium hydroxide (NaOH) and potassium dihydrogen orthophosphate (KH2PO4) was
83
purchased from Merck, USA, sinigrin (purity≥98%) was purchased from Santa Cruz
5
84
Biotechnology, Germany. L-α- phosphatidylcholine hydrogenated (soya bean) (≥99%) was
85
procured from Sigma- Aldrich, USA and solvents, such as dichloromethane (DCM) and n-
86
hexane, were obtained from Sigma Aldrich, USA.
87
2.2 Preparation of sinigrin-phytosome complex
88
Sinigrin-phytosome complexes were prepared as per the previously described method13.
89
Phosphatidylcholine (PC) was combined with sinigrin, in a ratio of 1:1 (w/w), with 5ml of
90
DCM and stirred well while evaporating the DCM. When the DCM was completely removed,
91
5ml of n-hexane was added to the thin film, mixed well and then the solvent was allowed to
92
completely evaporate. After the n-hexane was completely evaporated, the thin film was
93
hydrated and probe sonicated for about few minutes (Hielscher UP 200ST ultrasonic device,
94
South Africa), to get the desired size phytosome vesicles.
95
2.3 Transmission electron microscopy
96
The samples were diluted with distilled water, and then a drop of the phospholipid vesicles
97
was placed onto a copper grid, leaving a thin liquid film. The films on the grid were then
98
stained with 1% osmium tetroxide and excess staining solution was removed using filter
99
paper, this was then allowed to dry for 15 mins. The stained films were observed and
100
photographed using transmission electron microscopy (TEM) (model FEI TecnaiTM G2,
101
USA), at an accelerating voltage of 120 kV, with images captured by a GATAN bottom
102
mount camera using digital micrographics.
103
2.4 In vitro drug release studies
104
The in vitro drug release studies were accomplished using the dialysis method at room
105
temperature. Dialysis sacks (Sigma Aldrich, USA), with a molecular weight cut-off ranging
106
from 12,000-14,000, were utilized during these experiments as the donor compartment.
107
Details for this method have been provided in supplementary data.
108
6
109
2.5 Preparation of phosphate buffer solution
110
The PBS (pH 7.4) used as the receptor solution during the in vitro permeation studies was
111
prepared according to the British Pharmacopoeia (British Pharmacopoeia, 2015)14. Details
112
for this method have been provided in supplementary data.
113
2.6 High performance liquid chromatography (HPLC) analysis of sinigrin
114
The HPLC method for the analysis of sinigrin was developed and validated according to ICH
115
guidelines15. The HPLC (Agilent 1100 series, Agilent, Palo Alto, USA) system consists of an
116
auto-sampler, variable wavelength detector (VWD) and an isocratic pump. A chromato-
117
graphic column (Venusil XBP C18, 5µm, 150x 4.6mm, Agela Technologies, Newark, USA)
118
was used for analytic separation. The mobile phase comprises of a mixture of tetrabutyl
119
ammonium bisulfate (TBASO4) and acetonitrile (80:20, v/v). TBASO4 was prepared by
120
dissolving it in deionized water (1.7g/l) and adjusting the pH to 7.3 ± 02. The flow rate was
121
kept at 1.0 ml/min, with a sample injection of 50µl volume and UV detection at 228 nm.
122
Fresh standard solutions of sinigrin dissolved in distilled water were prepared before each
123
study and injected into the HPLC to obtain calibration curves ranging from 0.60 to
124
12.00µg/ml in concentration. Limit of detection (LOD) was 0.015µg/ml and limit of
125
quantification (LOQ) was 0.07µg/ml.
126
2.7. In vitro skin permeation and tape stripping
127
2.7.1 Skin preparation
128
Permeation studies were performed using abdominal skin obtained from Caucasian female
129
patients after abdominal plastic surgery16. Prior to the surgery, informed consent was
130
obtained from all the patients, with donor identities kept confidential. Within 24 hrs after the
131
surgery, the skin was placed in plastic bags and frozen at -20°C until used. The research
132
ethics committee of the North-West University (Potchefstroom Campus) authorized ethical
133
approval for the procurement and use of the donated skin under reference number NWU-
134
00114-11-A5 (2011-08-25). Details for this method have been provided in supplementary
135
data.
7
136
2.7.2 Skin diffusion studies
137
Vertical Franz diffusion cells with an active diffusion area of 1.13 cm2 and receptor volume of
138
2 ml were used for the in vitro permeation studies. To maintain constant stirring of the PBS
139
(pH 7.4) receptor solution, a small magnetic stirrer bar was placed in the receptor
140
compartment of each Franz cell. Thereafter, the dermatomed skin was placed between the
141
receptor and donor compartments with the stratum corneum facing the donor compartment.
142
The two compartments were subsequently sealed with Dow Corning® high vacuum grease
143
and clamped together using a horseshoe clamp to prevent leakage.
144
The donor compartments were filled with 1ml of the donor solution, preheated to 32 °C in a
145
water bath to match normal human skin temperature. Parafilm® was used to cover the donor
146
compartments to prevent evaporation of the donor solutions during the diffusion. The
147
receptor compartments were filled with 2 ml PBS (pH 7.4), preheated to 37 °C, taking care
148
that no air bubbles formed underneath the skin. The receptor compartments were
149
submerged in water within a water bath set at 37°C to achieve a skin surface temperature of
150
32°C
151
HPLC analyzed. Details for this method have been provided in supplementary data.
152
2.7.3 Tape stripping
153
Tape stripping is a technique used to investigate the amount of active ingredient which
154
penetrated the outer layers of the skin after application of a formulation17. During this study
155
tape stripping was performed according to a method described previously18,19,20. After
156
completion of the 12 hrs permeation studies, the Franz cells were taken apart and the skin of
157
each cell pinned to Parafilm® supported by a steady surface. The skin area exposed during
158
the diffusion was distinctly indented by the Franz cells.
159
solution present on the surface of the skin was removed using dry paper towel. 3M Scotch ®
160
Magic™ tape was cut into pieces (16) large enough to cover the diffusion area. The first tape
161
strip applied on the skin was discarded as part of the cleaning process as it is may have
162
been contaminated with sinigrin adhering to the surface of the skin. In order to remove the
16
. After 12 hrs, the entire volume of the receptor compartment was withdrawn for
Some of the remaining donor
8
163
stratum corneum, the following 15 tape strips applied on the skin were placed in a vial filled
164
with PBS (pH 7.4) and stored at 4 °C overnight (referred to as stratum corneum-
165
epidermis18,19,20. After tape stripping, the left over skin (referred to epidermis-dermis) was cut
166
into smaller pieces and placed in a vial containing PBS (pH 7.4) for storage at 4 °C
167
overnight. Samples were withdrawn from each of the vials containing the tape strips and the
168
left over skin, filtered with 0.45 µm syringe filters and analyzed by HPLC.
169
2.8 Statistical data analysis
170
For the in vitro permeation studies, the mean amount per area (µg/cm2) of sinigrin which
171
diffused through the skin after 12 hrs was calculated. The concentration (µg/ml) sinigrin
172
delivered into the skin layers (stratum corneum-epidermis and epidermis-dermis) was
173
determined from the results obtained from the tape stripping studies. Statistica data analysis
174
software, version 12 21 (StatSoft, Inc., 2015) was used to examine the data obtained from the
175
skin diffusion and tape stripping studies.
176
Firstly, data was analyzed by descriptive statistics, which involved the calculation of the
177
mean (with standard deviation) and median (measure of central location) values22,23
178
Secondly, the data was analyzed by comparative statistics, namely the independent sample
179
t-test, to determine whether there was a statistical significant difference22 between the
180
control study (sinigrin in water) and the phytosome-sinigrin complex. Statistical significant
181
differences were indicated when a p-value ˂ 0.05 was observed.
182
In vitro drug release study data were analyzed using GraphPad Prism™ Software (version
183
5.01, USA). Statistical significance was determined with two-way ANOVA statistics. Data are
184
presented as mean ± standard deviation (SD) and significance was set at p < 0.05.
185
3. Results and Discussion
186
3.1 Transmission electron microscopy
187
The sinigrin-phytosome complex was characterized with respect to size, zeta-potential,
188
structural morphology and complex formation efficiency previously by our research group13.
9
189
TEM, illustrated in Figure 3, provided the evidence of vesicle formation, displaying a uniform,
190
spherical shape with smooth outer surfaces and with little or no signs of agglomeration.
191
3.2 In vitro drug release
192
The in vitro drug release profiles of the free sinigrin and sinigrin-phytosome complex are
193
shown in Figure 4. The result revealed that the free sinigrin was completely released within 2
194
hrs, while the sinigrin-phytosome complex released 100% of sinigrin during a time span of 8
195
hrs. After 2 hrs, only 45.5% of sinigrin was released from the phytosome complex. The data
196
also demonstrated that the sinigrin release from phytosome complex was in a controlled and
197
sustained manner. The steady slow release of sinigrin from both phytosome complex
198
indicated that homogeneous phytosome complex formation of sinigrin had occurred.
199
similar study has been conducted where Ashwagandha phytosome complex and a
200
conventional formulation were subjected to an in vitro drug release study. It was found that
201
the phytosome complex had the highest cumulative percentage drug release compared to
202
the conventional formulation24. Sumathi and their research group formulated phytosomes
203
containing methanolic extracts of Nymphaea nouchali and trichosanthes dioica and also
204
demonstrated the in vitro drug release. Their findings proposed that the phytosome vesicles
205
containing Nymphaea nouchali and Trichosanthes dioica attained the maximum percentage
206
of extract release with longer duration of time, with enhanced in vitro release characteristics
207
in compared to the crude extracts25.
208
3.3 In vitro permeation studies and tape stripping
209
No sinigrin was observed in the receptor phase when sinigrin-phytosome complexes were
210
applied to the skin for 12 hrs (Figure 5). Nevertheless, the mean amount per area of sinigrin
211
which diffused through the skin after 12 hrs, during the control study (free sinigrin), was
212
2.102 ± 1.547µg/cm2. This may be due to the quick release of sinigrin (within 2 hrs) from the
213
free sinigrin solutions seen in the in vitro release studies. However, as the data set contains
214
extreme values, the median value is a more suitable measure of the central location than the
215
mean value23, consequently the median amount per area sinigrin that permeated through the
A
10
216
skin after 12 hrs is more representative of the true value, and was found to be 1.664 µg/cm 2
217
for the control study.
218
Successful research has been recognized indicating that the phytosome technology has the
219
therapeutic
220
phytoconstituents26
221
hours (over rat abdominal skin), of the flavonoid rutin, was enhanced when phytosome
222
complex was formed (compared to the uncomplexed rutin). After 24 hrs, the rutin content
223
found in the skin, via extraction, was also higher with the rutin-phytosome complexes than
224
the pure rutin. This indicated the rutin phytosomes were able to penetrate the stratum
225
corneum better than the free rutin. This higher amount of retention in the skin suggests that
226
phytosomes are able to deliver the rutin over an extended period of time. Another similar
227
study showed that resveratrol phytosomes also had higher skin permeation over 24 hrs and
228
retention at the dermal sites than the uncomplexed resveratrol29. The skin permeation of
229
curcumin with the phytosome curcumin was studied and it was found that complexed
230
curcumin showed 60% greater permeation of curcumin through rat skin, suggesting that
231
phytosome complex had better transdermal penetration than curcumin alone30.
232
The main aim of the current study was to determine whether the phytosome complex
233
enhanced the delivery of sinigrin into the skin layers by means of performing tape stripping
234
on the skin after the 12 hrs diffusion studies. Direct delivery to the wound site is desirable for
235
therapeutic agents, such as sinigrin, which are involved in the wound healing process13.
236
Tape stripping results showed neither the control nor the sinigrin phytosome complex
237
delivered sinigrin into the epidermis-dermis. However, as can be seen in Figure 5, the
238
sinigrin phytosome delivered a significantly (p ˂ 0.001) higher mean concentration sinigrin
239
(0.507 ± 0.041 µg/ml) into the stratum corneum-epidermis than the control solution (0.096 ±
240
0.116 µg/ml). However, as mentioned before, it is proposed to use the median concentration
241
values as these values are not influenced by a distortion in the data range31. Thus, when
242
comparing the median concentration (Figure 5) of sinigrin delivered to the stratum corneum-
243
epidermis, it was determined that the phytosome-sinigrin complex and the control solution
activities
to
be
used
transdermally
for
increasing
bioavailability
of
27
. A study by Das and Kalita28, showed the skin permeability, after 24
11
244
delivered sinigrin into this skin layer at concentrations of 0.5155 µg/ml and 0.0730 µg/ml,
245
respectively. The higher retention of sinigrin into this skin layer by the phytosome-sinigrin
246
complex may be explained by its gradual release of sinigrin from the phytosome over time
247
(as seen in Figure 4). Another explanation for the excellent delivery of sinigrin by
248
phytosomes into the stratum corneum-epidermis skin layer involves the formation of lipid-
249
compatible molecular complexes when the water soluble sinigrin are bound to
250
phospholipids32
251
diffuse through the skin layers, thereby causing only a small concentration of sinigrin to
252
accumulate in the stratum corneum-epidermis layer. Additionally, it is proposed that
253
phytosome complexes can potentially be used to directly deliver a higher concentration of
254
the phytoactive sinigrin, to the wound site, which is confirmed by our recent study13.
255
4. Conclusion
256
Sinigrin-phytosome complex exhibited no diffusion through the skin after 12 hrs, however,
257
results showed the phytosome complex were able to deliver a statistically significantly higher
258
concentration of sinigrin into the stratum corneum-epidermis when compared to the control
259
(free sinigrin). Therefore, it suggests that sinigrin phytosomes can be used to improve the
260
delivery of this phytoconstituent to the target site for wound healing, which is congruent with
261
our recent investigation13. It was also noteworthy that sinigrin was released in a slow and
262
controlled manner from the phytosome formulation. Furthermore, sinigrin-phytosome
263
complex has the potential to be used for various skin-related diseases and cosmetic
264
products. Further studies include investigation of the mechanism of action, permeation
265
through compromised skin, determination of the physiochemical properties of the complex.
266
Acknowledgements
267
This study was carried out with the financial support of the National Research Foundation
268
(NRF) of South Africa (Grants no. IFRR81178 and CPRR13091742482) and the Centre of
269
Excellence for Pharmaceutical Sciences (Pharmacen) at the North-West University,
270
Potchefstroom Campus, South Africa. The authors would like to acknowledge and are
33
.These results suggest the control solution caused the sinigrin to mostly
12
271
thankful to Dr. Anine Jordaan for her guidance and support for transmission Electron
272
Microscopy at Microscopy Department, North West University, Potchefstroom, South Africa.
273
Prof F. Steyn (Statistical Consultation Services of the North-West University, Potchefstroom
274
Campus, South Africa) is acknowledged for his assistance with the statistical data analysis.
275
Conflict of interest
276
The authors declare no conflict of interest.
277
Disclaimer
278
Any opinions, findings, conclusions, or recommendations expressed in this material are
279
those of the authors and therefore the NRF does not accept any liability in regard thereto.
280
13
281
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Figure legends
Figure 1. Chemical structure of sinigrin
Figure 2. Illustration of graphical image of the in vitro skin permeation of sinigrin phytosome
complex.
Figure 3. TEM pictures of sinigrin-phytosome complexes
Figure 4. In vitro release of free sinigrin and sinigrin-phytosome complex. Each value
represents the mean and SD of three experiments.
Figure 5. Box plots to illustrate the concentration (µg/ml) sinigrin present in the stratum
corneum-epidermis for the sinigrin-phytosome complex and free sinigrin. The mean and
median concentration values are shown as squares and plus signs, respectively.
17
Figure 1. Chemical structure of sinigrin
18
Figure 2. Illustration of graphical image of the in vitro skin permeation of sinigrin phytosome
complex.
19
Figure 3. TEM pictures of sinigrin-phytosome complexes
20
Cumulative % release
150
Free sinigrin
Phytosome complex
100
50
0.
5h
r
1h
r
1.
5h
r
2h
r
3h
r
4h
r
5h
r
6h
r
8h
r
0
Time (hrs)
Figure 4. In vitro release of free sinigrin and sinigrin-phytosome complex. Each value
represents the mean and SD of three experiments.
21
Figure 5. Box plots to illustrate the concentration (µg/ml) sinigrin present in the stratum
corneum-epidermis for the sinigrin-phytosome complex and free sinigrin. The mean and
median concentration values are shown as squares and plus signs, respectively
22
Supplementary Data
2.4 In vitro drug release studies
The in vitro drug release studies were accomplished using the dialysis method at room
temperature. Dialysis sacks (Sigma Aldrich, USA), with a molecular weight cut-off ranging
from 12,000-14,000, were utilized during these experiments as the donor compartment. The
dialysis bags were soaked in double-distilled water for 2 hrs before use, and then 4ml of
freshly prepared sinigrin-phytosome dispersion and sinigrin was poured respectively into
each bag with the two ends fixed by clamps. The bags were placed in a conical flask filled
with a receptor medium consisting 40ml phosphate buffer (PBS pH 7.4, PBS tablets was
dissolved in water). The conical flasks were placed on a magnetic stirrer plate at 37°C at 100
rpm. From each sample, 2ml were extracted at selected time intervals and the volume was
replaced with same amount of fresh PBS following each sampling. Samples were filtered
through 0.45 μm membrane filters and assayed by HPLC.
2.5 Preparation of phosphate buffer solution
The PBS (pH 7.4) used as the receptor solution during the in vitro permeation studies was
prepared according to the British Pharmacopoeia (British Pharmacopoeia, 2015)33,
incorporating 250.0ml of a 0.2M KH2PO4 solution to 393.4 ml of a 0.1M NaOH solution,
where-after the pH of the solution was adjusted to pH 7.4 using phosphoric acid (H3PO4).
2.7.1 Skin preparation
Permeation studies were performed using abdominal skin obtained from Caucasian female
patients after abdominal plastic surgery33. Prior to the surgery, informed consent was
obtained from all the patients, with donor identities kept confidential. Within 24 hrs after the
surgery, the skin was placed in plastic bags and frozen at -20°C until used. The research
ethics committee of the North-West University (Potchefstroom Campus) authorized ethical
approval for the procurement and use of the donated skin under reference number NWU00114-11-A5 (2011-08-25).
23
Prior to preparation, the skin was thawed at room temperature. Thereafter the skin was
removed from the plastic bags and cleaned using distilled water and paper towels to remove
any blood and other remaining fat tissue left on the skin surface. Using an electric
dermatome model 8821 (Zimmer, Inc., Warsaw, Ind. USA), a layer of skin with a thickness of
400µm and a width of 2.5cm was removed33. The excised skin was placed dermal side down
on filter paper and stored in aluminum foil at -20°C until used. One hour prior to the
permeation studies, the skin was thawed at room temperature and cut into circular pieces
before being placed on the diffusion apparatus.
2.7.2 Skin diffusion studies
Vertical Franz diffusion cells with an active diffusion area of 1.13 cm2 and receptor volume of
2 ml were used for the in vitro permeation studies. To maintain constant stirring of the PBS
(pH 7.4) receptor solution, a small magnetic stirrer bar was placed in the receptor
compartment of each Franz cell. Thereafter, the dermatomed skin was placed between the
receptor and donor compartments with the stratum corneum facing the donor compartment.
The two compartments were subsequently sealed with Dow Corning® high vacuum grease
and clamped together using a horseshoe clamp to prevent leakage.
The donor compartments were filled with 1ml of the donor solution, preheated to 32 °C in a
water bath to match normal human skin temperature. Parafilm® was used to cover the donor
compartments to prevent evaporation of the donor solutions during the diffusion. The
receptor compartments were filled with 2 ml PBS (pH 7.4), preheated to 37 °C, taking care
that no air bubbles formed underneath the skin. The receptor compartments were
submerged in water within a water bath set at 37°C to achieve a skin surface temperature of
32°C. After 12 hrs, the entire volume of the receptor compartment was withdrawn for HPLC
analyzed.
Two diffusion studies were performed using the method described above. For the first study,
the permeation of the sinigrin from sinigrin-phytosome complex was determined (n = 6). For
24
the second study, i.e. the control study, the donor phase consisted of sinigrin dissolved in
distilled water (n = 7).