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Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_6035_Библиотеки_им_академика_М_И_Перельмана.pdf
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15.8 CASE STUDY
Phytoestrogens have gained recognition as potential substitutes for traditional HRT in
mitigating symptoms related to menopause, owing to their ability to exert estrogenic
effects on target tissues (Warinsiriruk et al., 2022). One such phytoestrogen, P. candollei
var. mirifica, a plant with a deep-rooted reputation in Thai medicinal practice, contains
numerous potent constituents, including miroestrol, deoxymiroestrol, isomiroestrol, and
isoflavones, which exhibit strong estrogenic activity (Juengsanguanpornsuk et al., 2021;
Rani et al., 2022). In an effort to investigate this standardized plant extract’s potential as
a substitute for HRT, the author conducted recent, unpublished research examining the
effects of PMR extract when topically applied and encapsulated in niosomes. The study,
which was conducted in female ovariectomized Sprague‒Dawley rats to mimic menopause
status, demonstrated significant improvements in vaginal cornification and uterine weight.
The formulation was also well tolerated and did not result in any adverse effects. This case
study serves as a compelling example of the potential of phytoestrogens in drug discovery
and will be included in this chapter, which focuses on the efficacy and safety assessment
of phytoestrogens. Additionally, this chapter emphasizes the potential use of this plant as a
safe and effective alternative to traditional HRT.
In this particular case study, niosomes were utilized as a dermal delivery system for
phytoestrogens. They are similar in structure to liposomes, which are spherical structures composed of a lipid bilayer, but niosomes are composed of nonionic surfactants,
cholesterol, and sometimes other lipids. The nonionic surfactants used in niosomes have
hydrophilic (water-loving) and hydrophobic (water-fearing) regions, which allow them
to self-assemble into bilayer structures similar to cell membranes. These bilayers can
encapsulate hydrophilic and hydrophobic drugs, protecting them from degradation and
improving their bioavailability . Niosomes have several advantages over other drug delivery
systems. They are biodegradable, biocompatible, and nontoxic, making them safe for use
in the body. They can also be tailored to release drugs over a specic period, allowing for
sustained drug delivery. Additionally, niosomes can improve drug solubility and permeability , making them useful for drugs with poor solubility (Rungseevijitprapa et al., 2021).
The present study employed the thin-lm hydration method to prepare niosomal
formulations (sorbitan monolaurate:cholesterol:stearylamine, 4.5:4.5:1 molar ratio, 20
mM), followed by high-pressure homogenization to reduce particle sizes. To achieve the
desired concentration of plant extract in niosomes, lyophilization was chosen as a technique.
Trehalose, added at a ratio of 5:1 to the niosome formulations based on the weight of
trehalose and total lipid content, was selected as a cryoprotectant. This technique offers
several advantages, including preventing the loss of nonencapsulated actives from the
samples, which exert an initial effect upon release from the cream, followed by controlled
release of active ingredients from niosomes. Upon redispersion in aqueous solutions, the
mean particle sizes of the obtained lyophilized powders negligibly increased compared
with those of fresh preparations (P > 0.05).
The physical properties of all niosomes are presented in Figure 15.10, providing a detailed
analysis of their size distribution. The analysis showed a slight increase in the width of the
size distribution for both blank and active substance-loaded niosomes, which can be attributed
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to the aggregation of some nanoparticles during lyophilization. Despite this, the particles
remained within the optimal size range to enhance the permeation of active compounds
through the skin. These observations align with previous research, which suggests that
lyophilization can be used as an effective technique to increase nanoparticle concentration.
FIGURE 15.10 The physical appearance of niosomes after production in the upper panel and the size
distribution of the initial particles compared to their particles after redispersing the freeze-dried niosomal
powders in water in the lower panel. The figure highlights the morphology of three types of niosomes, including
empty niosomes (a), 1% w/v Pueraria candollei var. mirifica extract-loaded niosomes (b), and 0.1% w/v
17β-estradiol-loaded niosomes (c).
⏎
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The scanning electron microscopic images acquired revealed that the majority of the
prepared niosomes had sizes between 50 and 130 nm. The size distribution of vesicles for
the 1% w/v PME and 0.1% w/v 17β-estradiol-loaded niosomes was similar to that of their
respective blank formulations (Figure 15.11). These ndings indicate that the incorporation of active compounds had a negligible impact on the size of the niosomal particles.
Moreover, the optimal size range of the niosomes enhances the skin permeation of active
compounds. Hence, the study suggests that the developed niosomes are a promising
delivery system for transporting active compounds from the skin to the uterus and vagina
through systemic blood circulation.
FIGURE 15.11 Field emission scanning electron microscopy images of empty niosomes (a), niosomes
containing 1% w/v Pueraria candollei var. mirifica extract (b), and 0.1% w/v 17β-estradiol-loaded niosomes
(c). The magnification of the images is 30,000× original magnification.
⏎
In developing a topical cream for delivering active compounds, the incorporation
of niosomes into the base cream is a critical step. To conrm successful incorporation,
various oil-in-water (o/w) creams were prepared and veried using the Sudan III dye solu-
tion test. Optiphen, a preservative containing phenoxyethanol dissolved in caprylyl glycol
and a paraben, was used and is advantageous due to its formaldehyde-free composition,
avoiding the estrogenic activity commonly associated with parabens in topical products.
The blank, active compound-loaded niosomes, or free plant extract, were well dispersed
in the cream matrix, resulting in a homogeneous texture, indicating efcient delivery to
the skin. Furthermore, the physical characteristics of the incorporated niosomes remained
unchanged in the base cream. The yellowish color of creams containing phytoestrogenic
extracts can be attributed to the presence of plant extracts, which are known to possess
a yellowish color. Overall, the successful incorporation of niosomes into the base cream
provides a promising platform for developing a topical delivery system for active
compounds.
The aim of this case study was to evaluate the effect of topically applied creams
containing either 1% w/v PME-loaded niosomes or 1% w/v free PME on changes in
vaginal cornication and uterine weight. The study protocol received approval from the
Animal Welfare Institutional Review Boards of Ubon Ratchathani University (project
number 18). Before surgery, 4-month-old rats with normal estrus cycles were anesthetized
through an intramuscular injection of Zoletil 100 (tiletamine/zolazepam). The lumbar
dorsum was shaved, and the skin was scrubbed with a 10% w/v povidone-iodine solution,
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followed by wiping with a sterile saline solution. After surgery, the rats had a recovery
period of 7 days, and a diestrus phase conrmed the success of the ovariectomy. Five
groups of ve ovariectomized rats each were randomly distributed to be subjected to one
of the following formulations. Each received a dose of 200 µg of either phytoestrogenic
extract or 17β-estradiol per 9 cm2 of rat skin, applied twice daily over a span of 4 weeks:
(1) cream containing 1% w/v PME-loaded niosomes, (2) cream containing 1%w/v free
PME, (3) 0.1% w/v 17β-estradiol-loaded niosomes (positive control), (4) cream containing
empty niosomes, and (5) base cream (negative control). A xed amount of the test creams
was applied topically to the dorsal area of the skin twice daily at 07.00–08.00 a.m. and
05.00–06.00 p.m. for 4 weeks. One week before the application, the hair on each rat’s
back was trimmed. Before taking measurements, each rat was allowed to rest for at least
30 min in a regulated room, maintaining a temperature of 23 ± 2°C and a relative humidity
of 45 ± 5%. From each rat, a vaginal smear was collected and positioned on a microscope
slide, and a droplet of normal saline solution was added. The differentiation of cells was
examined using an automated microscope image-analysis system. Measurements were
recorded at baseline and during the 1st, 2nd, 3rd, and 4th weeks.
The image depicted in Figure 15.12 presents the alterations in the physical appearance
of rats that underwent an ovariectomy procedure to simulate menopausal conditions. The
external characteristics of the ovariectomized rats were captured through photographs at
baseline and 8 months after ovariectomy. These were compared to a control group of rats
that underwent a sham operation. The ndings revealed that ovariectomy induced an elevation in dryness and wrinkles in comparison to the control group, which might be indicative
of a reduction in endogenous estrogens and aging of the rat skin.
Vaginal smear cytology is a widely accepted method for determining the stages of the
estrous cycle in rats. The cycle in female rats is typically short and lasts approximately
4–5 days. It is classied into four stages—proestrus, estrus, metestrus, and diestrus—each
distinguished by the primary cell types present in the vaginal smear. Analysis of the proportion ratio of three cellular types and cell characteristics in a vaginal smear is facilitated by
using an optical light microscope with 10× and 40 objective lenses. This cycle is inuenced
by uctuating hormone levels, with ovulation taking place from the start of proestrus
through the conclusion of the estrus stage. This is attributed to the secretion of hormones
such as prolactin, luteinizing hormone, and follicle-stimulating hormone from the anterior
pituitary gland, which peaks in the afternoon during the proestrus stage. Estrogen levels
gradually increase in the metestrus phase, peak at proestrus, and drop to a baseline level at
the estrus phase. Similar to estrogen, progesterone is secreted during metestrus and diestrus, followed by a drop and subsequent peak levels toward the end of proestrus. Therefore,
it is necessary to select virgin rats with a regular estrous cycle for the experiment, as only
60–70% of female rats have a normal estrous cycle, while others may present with longer
or irregular cycles.
Figure 15.13 presents microscopic images of distinct stages of the estrous cycle in
rats based on vaginal smear cytology. The three types of cells observed in the vaginal
smears were classied as epithelial cells, cornied cells, and leukocytes based on their
relative proportions. Epithelial cells are round and contain nuclei, while cornied cells are
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irregular in form and lack nuclei. Leukocytes are small, round cells. The proestrus stage is
characterized by a majority of nucleated epithelial cells, whereas the estrus stage comprises
nonnucleated cornied cells. The metestrus stage exhibits an equal mix of leukocytes,
cornied, and nucleated epithelial cells. In contrast, the diestrus stage is marked by a
predominance of leukocytes in vaginal smears. To ensure consistency in the results, only
rats with normal estrous cycles were selected for this study , while rats with irregular cycles
were excluded.
FIGURE 15.12 Comparison of skin features between sham-operated and ovariectomized (OVX) rats at
baseline and 8 months after the respective operation. The left image depicts a rat that underwent a sham
operation, while the right image illustrates a rat that experienced an ovariectomy.
⏎
Next, a study was undertaken to assess the effectiveness of topically applied PME
for hormonal replacement therapy on dorsal rat skin using a vaginal cornication assay.
Vaginal smear cytology was used as an internal indicator to evaluate the occurrence of
any systemic effects following cosmetic application. The vaginal cornication assay is a
widely adopted approach for assessing the effectiveness of topical hormonal replacement
therapy in rats. The study outcomes indicated that PME, when applied topically, has the
potential to be used as hormonal replacement therapy in rats. Below are the specics of the
effects of utilizing the chosen phytoestrogen as the HRT.
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FIGURE 15.13 Microscopic images of unstained vaginal samples from adult female Sprague‒Dawley rats,
captured during varying phases of the estrous cycle. Panels (a) and (b) correspond to proestrus, panels (c) and
(d) correspond to estrus, panels (e) and (f) correspond to metestrus, and panels g and h correspond to diestrus.
The images depict leukocytes (L), epithelial cells (E), and cornified cells (C). The magnification is 10 in the
left panel and 40× in the right panel, and the scale bar is 20 μm.
⏎
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15.8.1 VAGINAL CELLULAR DIFFERENTIATION ASSAY
The effect of dermal administration of a cream containing 1% PME-loaded niosomes on
the vaginal tract was evaluated using vaginal smear cytology. Daily vaginal samples were
collected, and the predominant cell types were determined by calculating the cornification
of epithelial cells. Vaginal cornification is a term used to describe changes that occur in
the cells of the vaginal epithelium, which is the tissue that lines the inside of the vagina.
Normally , these cells are relatively flat and have a smooth appearance. However, when the
body is exposed to certain hormones, such as estrogen, the cells of the vaginal epithelium
can change in shape and become more tightly packed together. This process is known as
cornification. Vaginal cornification is often used as an indicator of estrogenic activity in
the body , as it is a well-known effect of estrogen. In research studies, vaginal cornification
is often measured to assess the effectiveness of treatments that aim to increase levels of
estrogen in the body, such as hormone replacement therapy. If phytoestrogenic molecules
penetrate from the upper skin into the bloodstream, they will reach the vaginal tract and
stimulate the differentiation of leukocytes into cornified cells. Before the experiments, the
baseline values of vaginal cells were checked for three consecutive days. The obtained data
were similar to the results obtained after a freshly ovariectomized procedure.
Figure 15.14 depicts changes in vaginal cellular differentiation induced by various
treatments. On day 4 post-topical application, the cream containing 1% plant extract-loaded
niosomes prompted leukocyte-to-nucleated-and-cornied-cell transformation, with cornied
cells reaching 35.23 ± 8.94% (Figure 15.15). By day 5, the percentage of cornied cells sharply
increased to 84.71 ± 5.47% and remained stable with minor uctuations, with complete
cornication achieved after 9 days of treatment. Treatment with cream containing free 1%
plant extract also caused leukocyte-to-nucleated-cell transformation, albeit with lower potency ,
observed after 7 days of treatment. Approximately 50% of the cornied cell populations were
seen in the vaginal smear until day 25, and differentiation to complete cornied cells occurred
after day 26. In the positive control group, where ovariectomized rats were given a cream
containing 0.1% 17β-estradiol-loaded niosomes, the transformation from leukocytes to fully
cornied cells took a mere 4 days, and this state persisted throughout the 28-day trial. However,
the negative control group, which received either cream containing empty niosomes or base
cream, did not experience any cell maturation beyond the leukocyte state.
Figure 15.15. Changes in the percentage of cornied cells, which are indicative of
vaginal cellular differentiation, were evaluated after a 28-day treatment with various creams
in ovariectomized rats. The creams used were (■) a cream containing niosomes loaded with
1% P. candollei var. mirica extract (PME), (▲) a cream containing free 1% P. candollei
var. mirica extract, (∆) a cream containing niosomes loaded with 0.1% 17β-estradiol, (□)
a cream containing blank niosomes, and (●) a base cream. The presented data represent the
mean ± standard deviation based on a sample group of ve (n = 5).
15.8.2 CHANGES IN RAT BODY WEIGHT
It is widely accepted that changes in body weight can serve as an indicator of the systemic
effects of dermally administered phytoestrogens, among other parameters. When these
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substances enter the systemic circulation, they are expected to trigger changes in body
weight loss or gain in animals. To evaluate the effect of PME on rat body weight changes,
the authors compared the results obtained with those of 17β-estradiol and placebo cream,
utilized as the respective benchmarks for positive and negative controls.
FIGURE 15.14 The alterations in vaginal cellular differentiation, captured on day 4 post-topical application of
various creams, as follows: (a) Cream comprising 1% P. mirifica extract-loaded niosomes, (b) cream comprising
free 1% P. mirifica extract, (c) cream comprising 0.1% 17β-estradiol-loaded niosomes, and (d) base cream. The
scale bar denotes 20 µm.
⏎
The time course of changes in body weight of ovariectomized rats aged 12 months (8
months after ovariectomy) is shown in Figure 15.16. The initial average body weight for
all rats was 362.11 ± 22.49 g, showing no meaningful difference as per statistical analysis
(P > 0.05). However, after 2 weeks of dermal application of cream containing 1% w/v
PME-loaded niosomes prepared from sorbitan monolaurate and cholesterol in an aqueous
solution, the plant extract induced body weight loss. The values were 356.18 ± 33.69 g and
362.16 ± 24.14 g at baseline and 328.26 ± 20.88 g and 347.32 ± 25.38 g after 2 weeks for
cream containing 1% PME-loaded niosomes and cream containing free 1% PME, respectively. The decrease in body weight of rats exposed to phytoestrogenic extract following
topical application was directly related to the presence or absence of a delivery system,
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with niosomes yielding higher values than those from conventional cream. However, this
difference did not reach statistical signicance (P > 0.05).
FIGURE 15.15 Changes in the percentage of cornified cells, which are indicative of vaginal cellular
differentiation, were evaluated after a 28-day treatment with various creams in ovariectomized rats. The creams
used were (■) a cream containing niosomes loaded with 1% P. candollei var. mirifica extract (PME), (▲) a
cream containing free 1% P. candollei var. mirifica extract, (∆) a cream containing niosomes loaded with 0.1%
17β-estradiol, (□) a cream containing blank niosomes, and (●) a base cream. The presented data represent the
mean ± standard deviation based on a sample group of five (n = 5).
⏎
Within the group acting as the positive control, rats receiving cream containing 0.1%
17β-estradiol-loaded niosomes showed the most signicant weight loss, with values of
362.00 ± 15.04 g and 330.93 ± 7.12 g at baseline and 2 weeks after dermal application,
respectively (P < 0.05). These changes persisted throughout the study period (week 3 =
323.80 ± 7.05 g, week 4 = 309.50 ± 6.60 g). No uctuations in weight loss or gain were
noted in any rats treated with the base cream or cream containing empty niosomes. These
ndings suggest that the systemic effects of 17β-estradiol or the investigated plant extract
were caused by their dermal administration. The estrogen or phytoestrogenic molecules
that penetrated the bloodstream could disrupt energy homeostasis by inducing lower food
intake rates, which were mediated by peripheral factors released by white adipose tissue,
such as leptin and differential expression of hypothalamic neuropeptides.
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FIGURE 15.16 The impact of cream containing 1% P. candollei var. mirifica extract-loaded niosomes on
changes in the body weight of ovariectomized rats. The values present the average along with the standard
deviation (n = 5). The statistical examination indicates a significant difference between the test sites and their
respective baselines, indicated by *P < 0.05.
⏎
15.8.3 CHANGES IN RATS’ UTERUS WEIGHT
The use of a cream containing 1% PME-loaded niosomes can produce desired systemic
effects related to estrogenic activity, demonstrated by changes in body weight, vaginal
cellular differentiation, and gain of uterus weight. In ovariectomized rats, uterine weight
gain serves as an estrogenic biomarker for estrogen-related compounds, which was evaluated in Figures 15.17 and 15.18. After a 28-day administration period, the increase in uterine
weight among the negative controls did not show any notable differences. Both the base
cream and cream containing empty niosomes had similar relative uterine weights of 45.24
± 5.35 and 42.07 ± 4.90 mg/100 g body weight, respectively (P > 0.05). However, the
relative wet weights of the uterus significantly increased in rats treated with both free 1%
plant extract and 1% plant extract-loaded niosome groups compared to negative controls
(P < 0.001). Furthermore, the use of cream containing 1% PME-loaded niosomes resulted
in a higher uterine weight (313.46 ± 19.59 mg/100 g body weight) compared to cream
containing free 1% PME (115.46 ± 8.99 mg/100 g body weight, P < 0.001). The gain
in uterine weight of rats treated with PME-loaded niosomes was lower than that of rats
treated with cream containing 0.1% 17β-estradiol-loaded niosomes (P < 0.001). These
findings imply that phytoestrogenic compounds from the plant extract deeply penetrated